EP4630538A1 - Polynucleotide constructs and uses thereof - Google Patents

Abstract

The present disclosure relates generally to a polynucleotide construct comprising a Felid alphaherpesvirus 1 (FeHV-1) genome modified by insertion of one or more nucleic acid sequences into a non-coding region between two convergent FeHV-1 genes of the FeHV- 1 genome, wherein the one or more nucleic acid sequences encode for one or more feline reproductive protein antigens; capable of stimulating an immune response to reduce the occurrence of a pregnancy, and uses thereof.

Description

POLYNUCLEOTIDE CONSTRUCTS AND USES THEREOF

FIELD OF THE INVENTION

[0001] This disclosure relates generally to a polynucleotide construct encoding feline reproductive protein antigens capable of stimulating an immune response to reduce the occurrence of a pregnancy, and uses thereof.

BACKGROUND OF THE INVENTION

[0002] The impact of feral cats on native wildlife can be devastating, and controlling feral cat populations has proven to be difficult. In Australia and New Zealand, feral cats are responsible for killing millions of native animals every day. Feral cat populations are difficult to estimate as they are affected by factors such as land topology, environmental conditions, availability of prey and the difficulty of trapping enough feral cats to inform accurate estimates. Regardless of varied population estimates, feral cats undoubtedly pose one of the biggest threats to the future of native Australian wildlife.

[0003] Current control methods such as poisoning, baiting and trapping have not been particularly effective. This is largely because feral cats are highly wary animals and are not trapped easily. Any success made at trapping is quickly offset by immigration of cats from surrounding areas to re-establish populations. Baiting is also difficult as cats prefer live prey to scavenging from carcasses. More recently, the introduction of grooming traps has been a new tool aimed at exploiting the size of cats in comparison to other native species and their grooming habits. The technique involves the spraying of a sticky poison onto the surface of the feral cat that they then ingest as they groom the affected area. However, concern remains regarding the off-target poison effects on native species.

[0004] Feral cats are now the most expensive invasive species to manage in Australia with almost $19 billion spent on feral cats since the 1960s. There is urgent need for efficient and humane population control that is also non-toxic to native wildlife.

SUMMARY OF THE INVENTION

[0005] The present disclosure is predicated on the inventors' surprising discovery that polynucleotide constructs comprising a modified FeHV-1 genome that also encode for feline reproductive protein antigens are useful as virally vectored feline immunocontraceptives (WIC). Depending on the modifications, these polynucleotide constructs may be useful to manage feral cat populations and/or provide non-surgical contraceptive options for domestic or stray cats, avoiding the costs and potential risks associated with anesthesia and surgical desexing. In particular, in the context of feral cats, a self-disseminating WIC population control method may be a desirable tool for feral cat management. The polynucleotide constructs disclosed herein that utilise feline viruses as vectors also have the potential to induce protection against the feline virus, thus further benefiting the health and welfare of vaccinated cats.

[0006] According to a first aspect of the invention there is provided a polynucleotide construct comprising a Felid alphaherpesvirus 1 (FeHV-1) genome modified by insertion of one or more nucleic acid sequences into a non-coding region between two convergent FeHV- 1 genes of the FeHV-1 genome, wherein the one or more nucleic acid sequences encode for one or more feline reproductive protein antigens.

[0007] In one embodiment, the one or more feline reproductive protein antigens are selected from the group consisting of: a. gonadotrophin releasing hormone (GnRH); b. zona pellucida glycoprotein 3 (ZP3); c. follicle stimulating hormone; d. luteinising hormone; e. sperm adhesion molecule 1; and f. one or more fragments thereof.

[0008] In one embodiment, the one or more feline reproductive proteins are gonadotrophin releasing hormone and zona pellucida glycoprotein 3.

[0009] In one embodiment, the one or more nucleic acid sequences are inserted between the UL40 and UL-41 genes. In another embodiment, the one or more nucleic acid sequences are inserted between UL40 and UL-41 genes at a site selected from between position 25103 to 27077 of FeHV-1 GenBank Accession number KR296657. In a preferred embodiment, the one or more nucleic acid sequences are inserted between UL40 and UL-41 genes at a site selected from between position 26100 to 26109 of FeHV-1 GenBank Accession number KR296657. In another preferred embodiment, the one or more nucleic acid sequences are inserted between UL40 and UL-41 genes at position 26,104-26,105 of FeHV-1 GenBank Accession number KR296657.

[0010] In another embodiment, the modified FeHV-1 genome comprises a thymidine kinase (TK) gene that is modified by one or more amino acid substitution; and/or by replacing at least one codon of the TK gene with a codon that has a lower translational efficiency in a cell. In a particular embodiment, the TK gene is modified by replacing each codon with a codon that has a lower translational efficiency in a cell. In another embodiment, the codon is replaced with a codon that has a lower translational efficiency.

[0011] In one embodiment, the FeHV-1 genome is further modified to mutate or delete one or more FeHV-1 genes.

[0012] In another embodiment, the modified FeHV-1 genome is not inhibited for growth in feline cells.

[0013] In one embodiment, the modified FeHV-1 genome retains at least some of the horizontal transmission potential of an unmodified FeHV-1 genome. In yet another embodiment, the modified FeHV-1 genome has the same horizontal transmission potential of an unmodified FeHV-1 genome.

[0014] In one embodiment, the modified FeHV-1 genome has reduced horizontal transmission potential when compared to unmodified FeHV-1 genome. In another embodiment the modified FeHV-1 genome has little to no horizontal transmission potential when compared to unmodified FeHV-1 genome.

[0015] In one embodiment, there is provided a veterinary composition comprising the polynucleotide construct as described herein, and a veterinarily acceptable carrier, excipient or diluent.

[0016] In one embodiment, the is provided an immunocontraceptive vaccine comprising the polynucleotide construct or the veterinary composition as described herein. In another embodiment, the immunocontraceptive vaccine further comprises at least one adjuvant.

[0017] In one embodiment, there is provided a method of reducing the fertility of a feline, the method comprising administering to a feline in need thereof, the polynucleotide construct, the veterinary composition, or the immunocontraceptive vaccine as described herein. In a particular embodiment, the fertility of the feline is so reduced, the feline is reproductively sterile.

[0018] In another embodiment, there is provided a method of inducing an immune response against one or more feline reproductive protein antigens in a feline, the method comprising administering to a feline in need thereof, the polynucleotide construct, the veterinary composition, or the immunocontraceptive vaccine as described herein.

[0019] In one embodiment, there is provided a method of controlling a population of feral felines, the method comprising administering to a feral feline in need thereof, the polynucleotide construct, the veterinary composition, or the immunocontraceptive vaccine as described herein.

[0020] In one embodiment, in the methods described above, the polynucleotide construct, the veterinary composition, or the immunocontraceptive vaccine as described herein is administered to the subject orally, intranasally, intramuscularly or intraperitoneally.

[0021] In one embodiment, there is provided a use of the polynucleotide construct or the veterinary composition as described herein, in the manufacture of an immunocontraceptive vaccine medicament for reducing fertility in a feline subject.

[0022] In one embodiment, there is provided a use of the polynucleotide construct or the veterinary composition as described herein, in the manufacture of an immunocontraceptive vaccine medicament for inducing an immune response against one or more feline reproductive protein antigens in a feline subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the following drawings.

[0024] Figure 1 is a schematic of some examples of the FeHV-1 derived immunocontraceptives. (A) Homologous recombination between the FeHV-1 genome and a repair plasmid containing a CMV promoter, GnRH, ZP3, eGFP fluorescence genes and a BGH termination sequence to generate FHV-GZeG. (B) Generation of FHV-GZeGTmC: recombination between the WT TK region of the FHV-GZeG genome and a repair plasmid containing a deoptimized TK followed by a CMV promoter, mCherry gene and BGH gene. (C) Generation of Y N-GZeGTmC2: recombination between the WT TK region of FeHV- GZeG and a repair plasmid containing a codon-deoptimized TK fused to mCherry.

[0025] Figure 2 shows plaques of recombinant FeHV-1 containing three transgenes (two feline reproductive genes and eGFP) generated using CRISPR/Cas9 mediated transfection/infection in CRFK cells. Following CRISPR/Cas9 mediated transfection/infection viral plaques showing expression of eGFP were selected and plaque purified by three rounds of plaque picking under an overlay media of methyl cellulose, before viral amplification. Successful insertion of the transgenes, without disruption to the upstream or downstream regions of the FeHV-1 genome was confirmed using PCR and sanger sequencing. A & B show light field (A) and florescence dark field (B) photomicrographs of a viral plaque, scale bar = 200 pm. C and D show light field and florescence dark field photomicrographs of the same viral plaque, scale bar = 100 pm.

[0026] Figure 3 shows Crandell-Rees feline kidney cells inoculated with A)FHV-GZeG, B) FHV-GZeGTmC and C) FHV-GZeGTmC2. First column shows bright field view, second column shows GFP expression, while the third column shows mCherry expression. Scale bars represent 100pm.

[0027] Figure 4 shows growth curves of FeHV-1 wildtype viruses and FeHV-1 immunocontraceptive candidates in CRFK cells. A) One-step growth curve using a MOI of 5 and B) Multi-step growth curve using an MOI of 0.001. Virus titres at each time point were measured by TCID50/mL assay in CRFK cells. Each datapoint in the single step growth curve represents three replicates. Each datapoint in the multistep growth curve represents six replicates. Mean and standard deviation are shown.

[0028] Figure 5 shows plaque area, as a measure of cell-to-cell spread, of wildtype FeHV- 1 and the candidate immunocontraceptives in CRFK cells under a methyl-cellulose overlay media over 72 hours. Mean and standard deviation are shown. * p < 0.033, ** p < 0.02, *** p < 0.002, **** p < 0.0001.

[0029] Figure 6 shows cDNA transcript abundance measured by RT-qPCR. Zona Pellucida subunit 3 (ZP3) cDNA transcript (A) and thymidine kinase (TK) cDNA transcript (B). Each data point represents the mean cDNA value with standard deviation. Each data point encompasses the average of 6 replicates per virus per time point with standard deviation. Black bars indicate which FeHV-1 strains are significantly different to one another. * p < 0.033, ** p <0.02, *** p < 0.002, **** p < 0.0001.

[0030] Figure 7 shows detection of transgene expression. The predicted protein product equating to approximately 75 kDa (representing fused GnRH, ZP3 and eGFP) was detected in CRFK cells infected with the three different vaccine candidates but not in cells infected with the wildtype FeHV-1 strain using anti-eGFP antibodies. Smaller products, likely to be breakdown products, were also detected in all three cell cultures infected with the vaccine candidates, but not in wildtype FeHV infected cells.

[0031] Figure 8 shows the absence of growth of FeHV-GZeG in non-feline cells. A) RT- qPCR assessment of FeHV-GZeG mRNA abundance in the different cell lines 7 days post infection. B) qPCR assessment of FeHV-GZeG genome copy number in LA-4, Vero, JU56 and CRFK cellsO, 3 and 7 days post infection. Data points represent average score with ± one standard deviation.

[0032] Figure 9 is a schematic outlining study design for a 14-day assessment of FeHV-1 derived immunocontraceptives in a murine model. Inoculation with either mock, FeHV-1, FHV-GZeG, FHV-GZeGTmC or FHV-GZeGTmC2 occurred on day 0. Five mice per group were euthanised on days 1, 4, 8 and 14 post infection. Lung tissue and blood samples were taken from all mice on each of these days, and ovaries and testes were collected on day 14.

[0033] Figure 10 shows ELISA results for IgG/ IgM antibodies against FeHV-1 and IgG antibodies against GnRH (A) Anti-GnRH IgG antibodies (B) anti-FeHV-1 IgG antibodies (C) and anti-FeHV-1 IgM antibodies. Shown are individual absorbance values for each mouse inoculated with either mock, FHV-1, FHV-GZeG, FHV-GZeGTmC or FHV- GZeGTmC2. Positive and negative control values for each ELISA are shown next to each data set.

[0034] Figure 11 shows the number of ovarian follicles in female mice in each inoculation group. Three ovaries (one ovary per mouse) per inoculation group was collected on day 14 post-infection. Each data point indicates the number of follicles found in an ovary of an individual mouse. The mean ± one standard deviation for each group is also shown.

[0035] Figure 12 shows ovaries and testes from mice inoculated with FeHV-1 immunocontraceptive candidate FHV-GZeG. Panels A and B show photomicrographs of H&E stained sections of ovaries of female mice inoculated with (A) diluent only (negative control) or (B) FHV-GZeG. Oocytes (Oo) and corpus luteum (Cl) are marked. Scale bar = 200 . Panels C and D show photomicrographs of H&E stained sections of testes of male mice inoculated with (C) diluent only (negative control) or (D) FHV-GZeG. Seminiferous tubules (ST) containing sperm are surrounded by Leydig (L) cells. Within the seminiferous tubules the lumen (Lu) and spermatozoa (spz) are marked. Scale bar = 100 pm.

[0036] Figure 13 shows the amino acid sequences and nucleotide sequences disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the 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, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0038] Nucleotide and amino acid sequences are referred to by sequence identifier numbers (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>l, <400>2, etc. A summary of sequence identifiers is provided herein.

[0039] All sequence reference numbers (e.g., GeneBank ID, EMBL-Bank ID, etc.) provided herein were current as at the filing date.

[0040] For the purposes of the present invention, the following terms are defined below.

[0041] The articles “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0042] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0043] The terms “administration concurrently” or “administering concurrently” or “coadministering” and the like refer to the administration of a single composition containing two or more agents, or the administration of each agent as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such agents are administered as a single composition. By “simultaneously” is meant that the agents are administered at substantially the same time, and desirably together in the same formulation. By “contemporaneously” it is meant that the agents are administered closely in time, e.g., one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the agents will be administered within about one minute to within about eight hours and suitably within less than about one to about four hours. When administered contemporaneously, the agents are suitably administered at the same site on the subject. The term “same site” includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters. The term “separately” as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months. The agents may be administered in either order. The term “sequentially” as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the agents may be administered in a regular repeating cycle.

[0044] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term “comprising” and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements. [0045] The term "construct" or “vector” typically refers to a DNA or RNA molecule used as a vehicle to transfer recombinant genetic material, such as a heterologous nucleic acid construct of the present disclosure, into a host cell. The construct or vector may be a linear or circular double stranded nucleic acid molecule. Suitable vectors include plasmids, bacteriophages, viruses, fosmids, cosmids, and artificial chromosomes. A vector typically comprises an insert (a heterologous nucleic acid sequence or transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector which transfers genetic information to the host is typically to isolate, multiply, or express the insert in the target cell. Vectors can be episomal, i.e., do not integrate into the genome of a host cell, or can integrate into the host cell genome. The vectors may also be replication competent or replication-deficient. Exemplary polynucleotide vectors include, but are not limited to, plasmids, yeast artificial chromosomes (YACs), cosmids, transposons, synthetic DNA fragments. Exemplary viral vectors include, for example, AAV, lentiviral, retroviral, adenoviral, herpes viral and hepatitis viral vectors. Selection of the vectors to be used will take into consideration the size of the insert, the host cell to be transfected and the desired transformation efficiency or outcome, and would be readily known to the persons skilled in the art. Polynucleotide constructs of the present disclosure will generally include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct. Such elements may include control elements such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, internal ribosome entry sites (IRES) and often includes a polyadenylation sequence as well. In certain embodiments of the disclosure, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and/or elements to facilitate stable integration of the construct into the genome of a host cell. Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors. An “expression construct” (also referred to herein as an “expression cassette”) generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0046] The term “corresponding” as used herein in reference to a particular gene is intended to mean an analogous or equivalent or comparable gene. For example, where reference is made to a corresponding endogenous gene, it is intended to mean the analogous, equivalent or comparable naturally-occurring gene. Where reference is made to a corresponding exogenous gene, it is intended to mean an analogous, equivalent or comparable exogenous gene. In some embodiments, the corresponding gene has analogous or equivalent function or having sequence similarity. In one embodiment, the corresponding gene may be identical in function and/or sequence. In another embodiment, the corresponding gene may have about the same function or activity. In another embodiment, the corresponding gene may have reduced function or activity. In some embodiments, the phrase “corresponds to” or “corresponding to” is meant a nucleic acid sequence that displays substantial sequence identity to a reference nucleic acid sequence. In general the nucleic acid sequence will display at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence identity to the reference nucleic acid sequence.

[0047] As used herein, the terms "encode," "encoding" and the like refer to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to "encode" a polypeptide if it can be transcribed and/or translated, typically in a host cell, to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide. Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non-coding sequence. Thus, the terms "encode," "encoding" and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide an RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.

[0048] By “effective amount”, in the context of treating a disease or condition is meant the administration of an amount of an agent or composition to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition. The effective amount will vary depending upon the age, health and physical condition of the individual to be treated and whether symptoms of disease are apparent, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the subject. Optimum dosages may vary depending on the relative potency in an individual subject, and can generally be estimated based on, e.g., EC50 values found to be effective in in vitro and in vivo animal models. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[0049] The term “expression”, "expressed" or "expressing", as used herein, typically refers to any step involved in the production of an RNA molecule or a polypeptide, such as by transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0050] By “isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state.

[0051] As used herein, the term “nucleic acid”, “nucleic sequence”, “polynucleotide”, “oligonucleotide” and “nucleotide sequence” as used herein refers to mRNA, RNA, cRNA, rRNA, cDNA, or DNA, or a combination thereof. The term typically refers to polymeric form of nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single-, double- or triple- stranded forms of DNA and RNA. It can be of recombinant, artificial and /or synthetic origin and it can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar. The nucleic acids of the present disclosure can be in isolated or purified form, and made, isolated and /or manipulated by techniques known per se in the art, e.g., cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant technology. The nucleic acids can also be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444.

[0052] The terms “peptide”, “polypeptide” and “protein” are to be understood as referring to a chain of amino acids linked by peptide bonds, irrespective of the number of amino acids forming said chain. Amino acids are typically represented by their one-letter or three-letters code, according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (He); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Vai); W: tryptophan (Trp) and Y: tyrosine (Tyr).

[0053] The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison (e.g., over 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200 or more nucleotides or amino acids residues). Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present disclosure, “sequence identity” will be understood to mean the “match percentage” calculated by an appropriate method. For example, sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Sequences may be aligned using a global alignment algorithms (e.g., Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for the purposes of determining percent amino acid sequence identity can be achieved by any means available to persons skilled in the art, illustrative examples of which include publicly available computer software, such as is available at http://blast.ncbi.nlm.nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Persons skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. As used herein, % sequence identity typically refers to values generated using pair wise sequence alignment that creates an optimal global alignment of two sequences (e.g., using the Needleman-Wunsch algorithm).

[0054] The term "sequence identity", as used herein, includes exact identity between compared sequences at the nucleotide or amino acid level. Sequence identity, as herein described, typically relates to the percentage of amino acid residues in the candidate sequence that are identical with the residues of the corresponding peptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C- terminal extensions, nor insertions shall be construed as reducing sequence identity or homology.

[0055] The present disclosure also extends to non-exact identity (i.e., similarity) of sequences at the nucleotide or amino acid level where any difference(s) between sequences are in relation to amino acids (or in the context of nucleotides, amino acids encoded by said nucleotides) that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. For example, where there is non-identity (similarity) at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In an embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity. For example, leucine may be substituted for an isoleucine or valine residue. This may be referred to as a conservative substitution. In an embodiment, the amino acid sequences may be modified by way of conservative substitution of any of the amino acid residues contained therein, such that the modification has no or negligible effect on the functional activity of the modified polypeptide when compared to the unmodified polypeptide. [0056] The terms "subject", “patient” and “individual” used interchangeably herein, refer to any subject, particularly a vertebrate subject, more particularly a mammalian subject, and even more particularly, a feline subject.

[0057] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

Table 1. Brief Description of the Sequences

2. Polynucleotides

[0058] As noted elsewhere herein, the present disclosure provides a polynucleotide construct comprising a FeHVFelid alphaherpesvirus 1 (FeHV-1) genome modified by insertion of one or more nucleic acid sequences into a non-coding region between two convergent FeHV-1 genes, wherein the one of more nucleic acid sequences encode for one or more feline reproductive protein antigens.

[0059] Felid alphaherpesvirus 1 (FeHV-1) is a double-stranded DNA alphaherpesvirus and is a common cause of feline viral rhinotracheitis and is characterised by signs of upper respiratory tract disease. Felid alphaherpesvirus 1 is also known as Felid herpesvirus 1 (FHV-l) and feline rhinotracheitis virus, terms which are used interchangeably herein.The virus is transmissible and primarily shed from nasal mucosa, upper trachea and nasopharynx. Primary FeHV-1 infection frequently results in lethargy, decreased appetite and sneezing, which may progress to pyrexia, ocular and nasal discharge and conjunctivitis. In more severe cases, infected cats have laboured breathing. Primary infection may induce an immune response that protects against disease but not against re-infection. Reactivation of FeHV-1 can cause cytolytic disease, including nasal discharge and sneezing.

[0060] Feline herpesvirus-1 is considered restricted to the Felidae family. Felid species that have been confirmed to have been infected with FeHV-1 include cheetah (Acinonyx jubatus), lion (Panthera leo), cougar (Puma concolor) and tiger (Panthera tigris).

[0061] FeHV-1 has a large dsDNA genome of approximately 134 kb. The FeHV-1 genome contains 78 open reading frames (ORFs) that are either essential or non-essential for viral replication. Many FeHV-1 ORFs have been functionally characterised and some effect the virulence of the virus. FeHV-1 strains vary in virulence, and some strains have been modified to generate attenuated FeHV-1 strains, which reduce or avoid altogether, inducing clinical signs of disease that are associated with virulent, wildtype FeHV-1 strains. The transmissible nature of FeHV-1 also means FeHV-1 based WICs could be selfdisseminating and capable of spreading through feral cat populations, removing the substantial human input currently needed to manage feral cat populations in remote locations. FeHV-1 can achieve lifelong latency with periodic reactivation in hosts. This may be favourable for WIC as reactivation in host cells could also re-stimulate production of any vectored reproductive protein antigens and periodically re-stimulate a host immune responses.

[0062] In one embodiment, the polynucleotide construct disclosed herein comprises a FeHV-1 genome that is a wildtype (WT) FeHV-1 strain. In one embodiment, the FeHV-1 genome is selected from FeHV-1 strains UT88, G2620, 25B3B1, C7301 and FeHV-Z. In one embodiment, the FeHV-1 genome is GenBank Accession number KR296657. In another embodiment, the FeHV-1 genome is a FeHV-1 genome as disclosed in Vaz et al. 2016 BMC Genomics 17:704. In another embodiment, the FeHV-1 genome is GenBank Accession number KR381779, KR381780, KR381781, KR381782, KR381783, KR381784, KR381785, KR381786, KR381787, KR381788, KR381789, KR381790, KR381791, KR381792, KR381793, KR381794, KR381795, KR381796, KR381797, KR381798, KR381799, KR381800, KR381801, KR381802 or KR381803. Other strains of FeHV-1 suitable for the generation of the polynucleotide disclosed herein would be known to the persons skilled in the art.

[0063] The term "reproductive protein" as used herein include proteins involved in the reproduction and/or reproductive potential of a subject. These include and are not limited to sex hormones, proteins that act after copulation, proteins that mediate gamete production, usage, storage, signal transduction and proteins that control fertilization and post-fertilisation pathways. The reproductive proteins can be proteins involved in female-specific reproductive functions, such as egg proteins or proteins involved in placental I uterine function or proteins involved in embryo development. The reproductive proteins can be proteins involved in male-specific reproductive functions, such as sperm and seminal fluid proteins, and sperm-egg and sperm-reproductive tract interaction proteins. The reproductive protein can be a protein that affects both male and female reproductive function. In an embodiment, the one or more feline reproductive protein antigens are selected from the group consisting of: a. gonadotrophin releasing hormone (GnRH); b. zona pellucida glycoprotein 3 (ZP3); c. follicle stimulating hormone; d. luteinising hormone; e. sperm adhesion molecule 1; and f. one or more fragments thereof.

[0064] In an embodiment, the one or more feline reproductive protein antigens comprises Gonadotropin Releasing Hormone (GnRH). Gonadotropin Releasing Hormone (GnRH) is a peptide hormone responsible for directing the release of follicle stimulating hormone (FSH, essential for moderating puberty and sexual maturation) and luteinising hormone (LH, essential for the release of testosterone in males and a trigger of ovulation in females). Gonadotropin releasing hormone is an attractive immunocontraceptive target due to its essential nature in males and females. It is small peptide (SEQ ID NO: 5), making its incorporation into viral vectors relatively easy.

[0065] In another embodiment, the one or more feline reproductive protein antigens comprises follicle stimulating hormone (FSH). FSH is essential for the development of puberty in males and females. Secreted by the anterior pituitary gland, FSH directs the development of ovaries in females and testes in males. Mutations in beta FSH subunit of mice resulted in infertility and impaired ovarian follicle development in females and oligospermia and reduced fertility in males (Sairarn et al. (2001) Arch. Med. Res. 32, 601- 608). Feline Follicle stimulating hormone is composed of two subunit chains. The a-chain consists of 96 amino acids (SEQ ID NO: 19) while the P-chain is 111 amino acids in length (SEQ ID NO:21).

[0066] In another embodiment, the one or more feline reproductive protein antigens comprises Luteinising hormone (LH). Luteinising hormone (LH) (SEQ ID NO: 23 and SEQ ID NO:25) is responsible for directing the secretion of sex steroids such as testosterone in males and oestrogen in females.

[0067] In another embodiment, the one or more feline reproductive protein antigens comprises zona pellucida glycoprotein 3 (ZP3). Zona pellucida (ZP) is a gene directing the synthesis of the ZP layer, a glycoprotein matrix surrounding the outer layer of the oocyte. It has essential roles in facilitating sperm binding through the acrosome reaction, whereby sperm fuse with the egg. If the ZP layer is damaged or missing then fertilisation is unlikely, rendering the female infertile (Wassarman, P. M. Mammalian Fertilization. (1999) Cell 96, 175-183). The evolution of ZP genes is complex and significant divergence has occurred between species. Felids express three ZP subunits termed ZP2, ZP3 (SEQ ID NO:3) and ZP4.

[0068] By the way of an example of a male reproductive protein, Sperm adhesion molecule 1 (SPAM1) is involved with several processes responsible for successful fertilisation of the egg. The most important function is to facilitate binding of the sperm to the ZP layer and sperm acrosomal exocytosis. Sperm adhesion molecule 1 transcripts are expressed in the testis and epididymis and the SPAM1 protein is highly conserved amongst mammalian species. In mice harbouring SPAM1 mutations, there was a reduction in fertility as measured by litter size and penetration efficiency (Zheng et al. (2001) Mamm. Genome 12, 822-829), while in male guinea pigs, inoculation with the SPAM1 protein resulted in all male guinea pigs become infertile as measured by their ability to impregnate fertile females (Primakoff et al. (1997) Biol. Reprod. 56, 1142-1146. In an embodiment, the one or more feline reproductive protein antigens comprises Sperm adhesion molecule 1 (SPAM1).

[0069] In one embodiment, the one or more feline reproductive protein antigens are selected from the group consisting of gonadotrophin releasing hormone (GnRH); zona pellucida glycoprotein 3 (ZP3); follicle stimulating hormone; luteinising hormone; sperm adhesion molecule 1; and one or more fragments thereof. In a preferred embodiment, the one or more feline reproductive proteins comprise gonadotrophin releasing hormone and zona pellucida glycoprotein 3. In a preferred embodiment, the nucleic acid sequence encoding the one or more feline reproductive protein antigens comprises SEQ ID NO: 30 or SEQ ID NO:32, which encode for fusion proteins of gonadotrophin releasing hormone and zona pellucida glycoprotein 3 of SEQ ID NO: 29 or SEQ ID NO: 31 respectively.

[0070] In some embodiments, the one or more nucleic acid sequences that encodes for the one or more feline reproductive protein antigens can be modified to modulate their expression (z.e., to improve their expression or capacity to induce an immune response). By the way of example, the one or more nucleic acid sequences that encode for the one or more feline reproductive protein antigens can be operatively linked to a strong promoter that improves expression of the nucleic acids, and/or nucleic acid sequences that encode for the feline reproductive protein antigens can be codon optimized.

[0071] The term ’’codon optimized" or "codon optimization" as used herein refers to the replacement of one or more codons with synonymous codons which allows improved expression of the resultant polypeptide or protein, while keeping the amino acid sequence of a translated protein unchanged. Conversely, "codon deoptimized" "codon deoptimisation" as used herein refers to the replacement of one or more codons with synonymous codons that have a lower translational efficiency in a cell, which results in reduced expression of the resultant polypeptide or protein, while keeping the amino acid sequence of a translated protein unchanged. This is based on the discovery that although there are 64 different codons (61 codons encoding for amino acids and 3 stop codons) but only 20 different translated amino acids, that is many amino acids can be encoded for by more than one codon. The frequency of occurrence of synonymous codons (z.e., codons that code for the same amino acid) in coding DNA can be biased in different species. Codon usage bias for a variety of organisms is known, such that a particular nucleotide sequence can be codon-optimised for expression in a host cell. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. Codon-optimized I codon-deoptimized coding regions can be designed by various different methods and may be performed using methods which are available on-line, published methods, or a company which provides codon optimizing / deoptimizing services. One codon optimizing method is described, e.g., in International Patent Publication No. WO 2015/012924, which is incorporated by reference herein. Briefly, the nucleic acid sequence encoding the product is modified with synonymous codon sequences. Suitably, the entire length of the open reading frame (ORF) for the product can be modified. However, in some embodiments, only a fragment of the ORF may be altered. In some embodiments, only a few codons in the open reading frame are altered. In another embodiment, only one codon in the open reading frame is altered. By using one of these methods, one can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized I codon-deoptimized coding region which encodes the polypeptide.

[0072] The term "reproductive protein antigen" here refers to a full length reproductive protein or variant thereof, or to a derivative a part or fragment of the reproductive protein, which is capable of inducing an immune response in a subject, including a humoral (antibody) and I or cellular immune response, in vivo. The full length reproductive protein or variant thereof, or to a derivative a part or fragment of the reproductive protein may suitably comprise a single epitope or it may comprise a plurality of epitopes, including B cell and T cell epitopes or mimotopes thereof.

[0073] By “one or more feline reproductive protein antigens”, it is meant 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more feline reproductive protein antigens that are capable of raising a humoral (antibody) and / or cellular immune response in vivo when administered to the subject. In an embodiment, polynucleotide construct comprising a FeHV Felid alphaherpesvirus 1 (FeHV-1) genome is modified to comprise one or more nucleic acid sequences that encodes for at least 1, preferably at least 2, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, or more preferably at least 9 feline reproductive protein antigens.

[0074] Where the polynucleotide construct comprises one or more nucleic acid sequences that encode for at least two feline reproductive protein antigens, the feline reproductive protein antigens may be expressed as separate proteins or as a fusion protein. As used herein, the term “fusion protein” typically refers to a polypeptide composed of two or more peptide sequences linked to one another. In one embodiment, the polynucleotide comprise the one or more nucleic acid sequences that encode for the one or more feline reproductive protein antigens expressed as a fusion protein. In an embodiment, the fusion protein comprises two or more peptide sequences linked to one another end-to-end. In an embodiment, the fusion protein comprises two or more peptide sequences linked to one another in a linear configuration via a suitable linking moiety, also referred to herein as a linker. Suitable methods of linking peptide sequences will be familiar to persons skilled in the art, illustrative examples of which include peptide (amide) bonds. As used herein, the term “linker” refers to a short polypeptide sequence interposed between any two neighboring peptide sequences as herein described. In an embodiment, the linker is a polypeptide linker of 1 to 10 amino acids, preferably 1, 2, 3, 4 or 5 naturally or non-naturally occurring amino acids. In the event that a polypeptidic linker is used to join two respective peptide sequences, the linker will be advantageously incorporated such that its N-terminal end is bound via a peptide bond to the C-terminal end of the one peptide sequence, and its C-terminal end via a peptide bond to the N-terminal end of the other peptide sequence. The individual peptide sequences within the fusion protein may also have one or more amino acids added to either or both ends, preferably to the C-terminal end. Thus, for example, linker or spacer amino acids may be added to the N- or C-terminus of the peptides or both, to link the peptides and to allow for convenient coupling of the peptides to each other and/or to a delivery system such as a carrier molecule. In one embodiment, an internal ribosome entry sites (IRES) sequence may be used as a linker. In an embodiment, the IRES sequence comprises, consists, or consists essentially of an amino acid of SEQ ID NO:37, or any amino acid sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto. In another embodiment, the linker or spacer peptide may be a flexible linker, such as a "GS" linker comprising stretches of glycine and/or serine residues. The linker or spacer peptide may be a rigid linker, such as an (EAAAK)_n or a proline-rich linker. In another example, the linker or spacer peptides may comprise a cleavage site, that allows for cleavage of the linker peptide. In another example, the linker or spacer peptides may comprise a selfcleaving peptide, which induce ribosome skipping during protein translation and failure of peptide bond formation. In one embodiment, the linker or spacer peptide is a 2A selfcleaving peptide. In one embodiment, the linker or spacer peptide is a P2A self-cleaving peptide (SEQ ID NO: 34), T2A self-cleaving peptide (SEQ ID NO: 35), or a E2A selfcleaving peptide (SEQ ID NO: 36).

[0075] The FeHV-1 genome comprises total of 78 predicted open reading frames I genes, encoding 74 distinct proteins. In another embodiment, the one or more nucleic acid sequences that encodes for one or more feline reproductive protein antigens is inserted into a non-coding region between two divergent FeHV-1 genes. In one embodiment, the one or more nucleic acid sequences that encodes for one or more feline reproductive protein antigens is inserted into a non-coding region between two convergent FeHV-1 genes. In one embodiment, the one or more nucleic acid sequences that encodes for the one or more feline reproductive protein antigens is inserted with minimal disruption to the upstream or downstream regions of the FeHV-1 genome. In one embodiment, the one or more nucleic acid sequences that encodes for the one or more feline reproductive protein antigens is inserted without disruption to the upstream or downstream regions of the FeHV-1 genome.

[0076] In one embodiment, the one or more nucleic acid sequences are inserted between UL56 and VI; CIRC andUL55; UL51 and UL50; UL46 and UL45; UL40 and UL41; UL36 and UL35; UL31 and UL30; V32 and UL26; ULI 1 and U110; UL8 and UL7; and/or US8a and FHl-1.

[0077] In one preferred embodiment, the one or more nucleic acid sequences are inserted between UL40 and UL41. In another preferred embodiment, the nucleic acid sequences are inserted between UL40 and UL-41 genes at a site selected from between 25103 to 27077 of FeHV-1 GenBank Accession number KR296657. In another preferred embodiment, the nucleic acid sequences are inserted between UL40 and UL-41 genes at a site selected from between 26100 to 26109 of FeHV-1 GenBank Accession number KR296657. In another preferred embodiment, the nucleic acid sequences encoding the one or more feline reproductive protein antigens are inserted between UL40 and UL-41 genes at position 26,104-26,105 of FeHV-1 GenBank Accession number KR296657.

[0078] The insertion of the one or more nucleic acids into the FeHV-1 genome backbone can be targeted to a pre-determined, or a specified genome locus. Methods of targeted, sitespecific genome integration include using homologous recombination and CRISPR-Cas9, Zinc Finger nucleases and TALEN genome editing techniques, application of which would be known to the person skilled in the art. In one embodiment, homologous recombination techniques are used to insert the one or more nucleic acid sequences that encodes for one or more feline reproductive protein antigens into the FeHV-1 genome backbone. In one embodiment, CRISPR/Cas9 is used to insert the one or more nucleic acid sequences that encodes for one or more feline reproductive protein antigens. This can be performed using CRISPR/Cas9 plasmid carrying one or more guide RNAs specific to the targeted insertion site in the FeHV-1 genome. [0079] In specific embodiments, the thymidine kinase gene of the FeHV-1 genome is modified to attenuate the FeHV-1. In other embodiments, other virulence genes of FeHV-1 are further modified to further attenuate FeHV-1. Other genes that can be modified to attenuate FeHV-1 will be known to persons skilled in the art, including the ORF2 locus, UL region, gl/E region, DNA polymerase, ICP6, gE, gl and gG.

[0080] Thymidine kinase (TK) is a non-essential viral virulence gene involved in viral proliferation (Nishiyama, Y. (1996) Nagoya J. Med. Sci. 59, 107-119). Due to the non- essential nature of TK in viral replication it may be possible to modify TK, with the goal of virus attenuation, and in fact TK manipulation (including generating TK deficient viruses), has been studied in several herpesviruses (Schroder et al. (2019) J. Gen. Virol. 100, 642- 655; Comick et al. (1990) Can. J. Vet. Res. 54, 260-266; Kit et al. (1985) Arch. Virol. 86, 63-83.) An alternative method is to reduce the expression of TK.

[0081] In some embodiments, the thymidine kinase gene of the FeHV-1 genome is modified to increase TK gene expression by codon optimisation, or by inserting a heterologous promoter that is stronger than the native TK promoter.

[0082] In some embodiments, the thymidine kinase gene of the FeHV-1 genome is modified to decrease or disrupt TK gene expression. Reducing the expression of the TK gene can be achieved in many ways. For example, expression of the TK gene can be reduced by reducing the transcription and/or translational efficiency of the gene. In an embodiment, the expression of thymidine kinase (TK) gene of the FeHV-1 is reduced by any one or more of the following: replacing the endogenous promoter of the TK gene with a weaker promoter; replacing at least one codon of the TK gene with a codon that has a lower translational efficiency in the cell than the codon it replaces and/or; adding at least one codon into the coding sequence of the TK gene wherein the codon has a lower translational efficiency than other codons; and disrupting the TK gene; modifying the TK gene to include a nucleotide sequence encoding an RNA destabilizing element; and expressing a nucleic acid molecule in the cell, which reduces the level of an expression product of the TK gene. In one embodiment, the TK gene is modified by one or more amino acid substitution; and/or replacing at least one codon of the TK gene with a codon that has a lower translational efficiency in a cell. In a particular embodiment, the TK gene is modified by replacing each codon with a codon that has a lower translational efficiency in a cell. In another embodiment, the codon is replaced with a codon that has a lower or reduced translational efficiency. 3. Compositions

[0083] In one embodiment, the is provided a veterinary composition comprising the polynucleotide construct as described herein, and a veterinarily acceptable carrier, excipient or diluent.

[0084] The polynucleotide construct as described herein, can be formulated for administration with any number of carriers, excipients or diluents. For example, a variety of aqueous (veterinarily acceptable) carriers, excipients or diluents may be used, such as buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques or may be sterile- filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may further comprise pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity-adjusting agents, wetting agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, sucrose or other carbohydrates, among many others. Suitable methods for preparing compounds and compositions suitable for oral, topical, rectal or parenteral (which includes subcutaneous injections, intradermal, intravenous, intramuscular, intrathecal, intrastemal injection and infusion techniques) administration or for administration by inhalation, intranasally or spray, will be known or apparent to those skilled in the art and are described in more detail in, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7.sup.th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3.sup.rd ed. Amer. Pharmaceutical Assoc.

[0085] The polynucleotide construct as described herein, can be used without additional adjuvants or carriers. The polynucleotide construct as described herein, can be used in combination an additional carriers or adjuvant to enhance an immune response in the subject. The present disclosure therefore extends to compositions further comprising an immunopotentiator or adjuvants. Preferably, the immunopotentiator or adjuvant is administered concomitantly with the polynucleotide construct, as described herein. The immunopotentiator or adjuvant can be administered prior or subsequently to the polynucleotide construct, as described herein, depending on the need as can be suitably determined by persons skilled in the art. The term "immunopotentiator," or "adjuvant" as used herein, is intended to mean a substance that, when mixed with an immunogen or antigenic molecule, elicits a greater immune response than the immunogen or antigenic molecule alone. For example, an adjuvant can enhance immunogenicity and provide a superior immune response. Suitable immunopotentiators or adjuvants will be familiar to persons skilled in the art, illustrative examples of which include aluminium salts (e.g. aluminium hydroxide, aluminium phosphate and potassium aluminium sulfate (also referred to as Alum)), liposomes, virosomes, water-in-oil or oil-in-water emulsions (e.g. Freund's adjuvant, Montanide®, MF59® and AS03), 3-O-desacyl-4’-monophosphoryl lipid A (MPL) and adjuvants containing MPL (e.g. AS01, AS02 and AS04) and saponin-based adjuvants. Saponin-based adjuvants include saponins or saponin derivatives from, for example, Quillaja saponaria, Panax ginseng Panax notoginseng, Panax quinquefolium, Platycodon grandiflorum, Polygala senega, Polygala tenuifolia, Quillaja brasiliensis, Astragalus membranaceus and Achyranthes bidentata. Exemplary saponin-based adjuvants include iscoms, iscom matrix, ISCOMATRIX™ adjuvant, Matrix M™ adjuvant, Matrix C™ adjuvant, Matrix Q™ adjuvant, AbISCO®-100 adjuvant, AbISCO®-300 adjuvant, ISCOPREP™, an ISCOPREP™ derivative, adjuvant containing ISCOPREP™ or an ISCOPREP™ derivative, QS-21, a QS-21 derivative, and an adjuvant containing QS-21 or a QS21 derivative. The compositions and vaccines as herein described can also be associated with immumodulatory agents, including, for example, cytokines, chemokines and growth factors. Mixtures of two or more adjuvants within the same vaccine composition are also contemplated herein.

[0086] There is provided, the is provided an immunocontraceptive vaccine comprising the polynucleotide construct or the veterinary composition as described herein. In another embodiment, the immunocontraceptive vaccine further comprises at least one adjuvant.

[0087] The term "immunocontraceptive" refers to a substance, molecule or composition that is capable of stimulation of the host immune system to reduce the chance of pregnancy occurring or continuing. This can occur by preventing the production of gametes, or preventing fertilization. The immune response may cause the animal to be temporarily infertile, or may cause a longer term infertility, including permanent sterility. 4. Methods of use

[0088] The present disclosure also extends to a method of reducing the fertility of a feline, the method comprising administering to a feline in need thereof, the polynucleotide construct, the veterinary composition, or the immunocontraceptive vaccine described herein.

[0089] In some embodiments, the fertility of the feline is reduced by at least about 1%, by at least 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, preferably by at least about 50%, preferably by at least about 60%, preferably by at least about 70%, preferably by at least about 80%, preferably by at least about 90%, preferably by at least about 100%. In a particular embodiment, the fertility of the feline is so reduced, the feline is sterile. Methods of measuring the fertility of a subject will be known to persons skilled in the art (see for example Levy et al. (2011) Theriogenology 76:1516; Looper et al. (2001) Zoo Biology 20:407).

[0090] The present disclosure also extends to a method of inducing an immune response against one or more feline reproductive protein antigens in a feline, the method comprising administering to a feline in need thereof, the polynucleotide construct, the veterinary composition, or the immunocontraceptive vaccine described herein.

[0091] In some embodiments, the reduction in fertility and/or induction of an immune response against the one or more feline reproductive protein antigens in the feline subject is temporary. In some embodiments, the reduction in fertility and/or induction of an immune response persists for at least 1 month, for at least 2 months, for at least 3 months, for at least 4 months, for at least 5 months, for at least 6 months, for at least 7 months, for at least 8 months, for at least 9 months, for at least 10 months, for at least 11 months, for at least 12 months, for at least 13 months, for at least 14 months, for at least 15 months, for at least 16 months, for at least 17 months, for at least 18 months, for at least 19 months, for at least 20 months, for at least 24 months, for at least 26 months, for at least 28 months, for at least 30 months, for at least 36 months, for at least 58 months. In some embodiments, the reduction in fertility and/or induction of an immune response against one or more feline reproductive protein antigens in the feline subject is permanent.

[0092] The terms "immunisation" and "vaccination" are used interchangeably herein to refer to the administration of the polynucleotide constructs, compositions or immunocontraceptive vaccines as described herein, to a subject for the purposes of raising an immune response and can have a prophylactic effect, a therapeutic effect, or a combination thereof. For example, administration of the polynucleotide constructs, compositions or immunocontraceptive vaccines as described herein can have prophylactic I protective effect or therapeutic effect by decreasing the severity of clinical symptoms of FeHV-1 infection I challenge. While this prophylactic and/or therapeutic effect occurs in conjunction with the contraceptive effect of the administration of the polynucleotide constructs, compositions or immunocontraceptive vaccines as described herein, it is to be understood that this prophylactic and/or therapeutic effect is distinct from the contraceptive effect.

[0093] As described elsewhere herein, the terms "immune response"”, "immunological response" and the like are typically used herein to refer to the development in a subject of a humoral and/or a cellular immune response to the target. A "humoral immune response" typically refers to an immune response mediated by antibody molecules, while a "cellular immune response" is typically mediated by T-lymphocytes and/or other white blood cells. In a non-limiting example, the polynucleotide construct, composition or immunocontraceptive vaccine as described herein, when administered to a subject induces an immune response selected from one or more of a neutralizing antibody response, a cytotoxic T lymphocyte (CTL) response, a natural killer T cell response and / or a helper T lymphocyte (e.g., CD4+ T cell) response and innate immune response to the target antigen.

[0094] Methods for measuring an immune response will be known to persons skilled in the art, illustrative examples of which include measurement of antibody titres, plaque-reduction neutralization assay, micro-neutralization assay, solid-phase heterogeneous assays (e.g., enzyme-linked immunosorbent assay), solution phase assays (e.g., electrochemiluminescence assay), Western immunoblot, amplified luminescent proximity homogeneous assays, flow cytometry, intracellular cytokine staining, functional T-cell assays including suppressor T-cell assays, functional B-cell assays, functional monocytemacrophage assays, dendritic and reticular endothelial cell assays, measurement of NK or NKT cell responses, oxidative burst assays, cytotoxic-specific cell lysis assays, pentamer binding assays, and phagocytosis and apoptosis evaluation.

[0095] The present disclosure also extends to a method of controlling a population of feral felines, the method comprising administering to a feral feline in need thereof, the polynucleotide construct, the veterinary composition, or the immunocontraceptive vaccine as described herein.

[0096] By the way of example, the population of feral felines maybe controlled by administration of the polynucleotide construct, the veterinary composition, or the immunocontraceptive vaccine as described herein to feral felines, to result in a decrease in feral cat population by at least about 1%, by at least 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, preferably by at least about 60%, preferably by at least about 70%, preferably by at least about 80%, preferably by at least about 90%, preferably by at least about 100%, over a period of time; for example, the decrease in feral cat population can be observed over about 12 months, over about 18 months, over about 24 months, over about 30 months, over about 36 months, over about 42 months, over about 48 months, over about 52 months or over about 60 months.

[0097] In one embodiment, there is provided a use of the polynucleotide construct or the veterinary composition as described herein, in the manufacture of an immunocontraceptive vaccine medicament for reducing fertility in a feline subject.

[0098] In one embodiment, there is provided a use of the polynucleotide construct or the veterinary composition as described herein, in the manufacture of an immunocontraceptive vaccine medicament for inducing an immune response against one or more feline reproductive protein antigens in a feline.

[0099] For administration to a host or subject (e.g., a feline), the polynucleotide constructs, compositions and/or vaccines described herein can be formulated for administration by a variety of routes. For example, the polynucleotide constructs, compositions and/or vaccines described herein can be formulated for oral, topical, rectal or parenteral administration or for administration by inhalation, intranasally or spray. The term "parenteral", as used herein, includes subcutaneous injections, intradermal, intravenous, intramuscular, intrathecal, intrastemal injection and infusion techniques. In an embodiment, the polynucleotide construct, the composition or immunocontraceptive vaccine described herein is administered to the subject by intramuscular injection. In an embodiment, the polynucleotide constructs, compositions and/or vaccines described herein is administered to the subject by intraperitoneal injection. In another embodiment, the polynucleotide constructs, compositions and/or vaccines described herein can be administered to a subject through contact with one or more subjects that has been inoculated or infected with a transmissible polynucleotide construct comprising a FeHVFelid alphaherpesvirus 1 (FeHV-1) genome modified by insertion of one or more nucleic acid sequences into a non-coding region between two convergent FeHV-1 genes of the FeHV-1 genome, wherein the one or more nucleic acid sequences encode for one or more feline reproductive protein antigens, in an example of horizontal transmission.

[0100] In one embodiment, in the methods described above, the polynucleotide constructs, compositions and/or vaccines described herein is administered to the subject orally, intranasally, intramuscularly or intraperitoneally.

[0101] The polynucleotide constructs, compositions and/or vaccines described herein will suitably comprise a therapeutically effective amount of the polynucleotide. The phrase "therapeutically effective amount" typically means an amount of the polynucleotide, as described herein, necessary to attain the desired response, for example, the inducement of an immune response to the target (z.e., the reproductive antigen). Typically, the appropriate dosage of the polynucleotide as described herein, may depend on a variety of factors including, but not limited to, a subject’s physical characteristics (e.g., age, weight, sex), whether the polynucleotide described herein, is being used as single agent or administered with other therapeutic or prophylactic agents, the progression (i.e., pathological state) of the condition, or the schedule of administration e.g., weekly, monthly, biannually, annually etc.) and other factors that may be recognized by persons skilled in the art. Various general considerations that may be considered when determining, for example, an appropriate dosage of the vaccine composition (see, e.g., in Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; and Gilman et al., (Eds), (1990), "Goodman And Gilman's: The Pharmacological Bases of Therapeutics", Pergamon Press). It is expected that the amount will fall in a relatively broad range that can be determined through methods known to persons skilled in the art. Illustrative examples of a suitable therapeutically effective amount of polynucleotide for administration to a subject include from about 1 mL of 10³ to 1ml of 10⁸ TCID50, 1 mL of 10⁴to 1ml of 10⁷ TCID50, 1 mL of 10⁵ to 1ml of 10⁶ TCID50. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals, or the dose may be proportionally reduced as indicated by the exigencies of the situation. [0102] The polynucleotide constructs, compositions or vaccines as described herein, may be administered to a recipient in isolation or in combination with other additional therapeutic agent(s) or contraceptive agents. In embodiments where a veterinary composition or immunocontraceptive vaccine comprising the polynucleotide as described herein, is formulated for administration with additional therapeutic agent(s), the administration may be simultaneous or sequential (i.e., administration of the polynucleotide, composition or vaccine is followed by administration of the additional agent(s) or vice versa). Thus, here two or more entities are administered to a subject "in conjunction", they may be administered in a single composition at the same time, or in separate compositions at the same time, or in separate compositions separated in time.

[0103] In a non-limiting example, the polynucleotides, compositions and/or vaccines, as described herein, may be administered in conjunction with another contraceptive agent. An "antiviral agent" typically means an agent which, when administered to a subject, is capable of significantly reducing the virus titer in the blood or serum either directly (e.g., by inhibiting a viral enzyme activity) or indirectly (e.g., via modulation of the antiviral responses of a host cell), either transiently or in a sustained way. A contraceptive agent typically means an agent, which, when administered to a subject, is capable of significantly preventing pregnancy.

[0104] In an embodiment, the polynucleotide, compositions or immunocontraceptive vaccine as described herein, may suitably be given in an appropriate single dosage in order to elicit an immune response. In other embodiments, the initial dose may be followed by boosting dose. The boosting dose may comprise the same polynucleotide, composition or immunocontraceptive vaccine as the initial (priming) dose, whether at an equivalent dose (e.g., the same or similar dose), a lower dose or a higher dose as compared to the initial dose.

[0105] The administration regime need not differ from any other generally accepted vaccination programs. For instance, a single administration in an amount sufficient to elicit an effective immune response may be used. Alternatively, as noted above, other regimes of initial administration of the complex followed by boosting, including as described above. Boosting may occur at times that take place well after the initial administration if the immune response (as measured, e.g., by antibody titres) falls below acceptable levels. [0106] Alternatively, or in addition, the polynucleotides, compositions and/or vaccines, as described herein, can be used in combination an additional immunopotentiator or adjuvant to enhance an immune response in the subject. The present disclosure therefore extends to compositions further comprising an immunopotentiator or adjuvant. Preferably, the immunopotentiator or adjuvant is administered concomitantly with the polynucleotide, composition or immunocontraceptive vaccine as described herein. The immunopotentiator or adjuvant can be administered prior or subsequently to the polynucleotide, composition or immunocontraceptive vaccine as described herein, depending on the need as can be suitably determined by persons skilled in the art. The term "immunopotentiator," as used herein, is intended to mean a substance that, when mixed with an immunogen, elicits a greater immune response than the immunogen alone. For example, an immunopotentiator can enhance immunogenicity and provide a superior immune response. Suitable immunopotentiators or adjuvants will be familiar to persons skilled in the art.

[0107] As used herein, the terms "treat," "treated," or "treating" when used with respect to a disease or pathogen refers to a treatment which increases the resistance of a subject to the disease or to infection with a pathogen (i.e., decreases the likelihood that the subject will contract the disease or become infected with the pathogen), as well as a treatment after the subject has contracted the disease or become infected with the pathogen in order to fight a disease or infection (e.g., to reduce, eliminate, ameliorate or otherwise stabilise a disease or infection). In an embodiment, the polynucleotide, composition or immunocontraceptive vaccines as described herein, is capable of providing protective immunity to a host. The term "immunity", as used herein, is intended to mean the ability of a host (e.g., feline), to induce an immune response to the reproductive proteins and/or have reduced capacity for pregnancy, as a result of its exposure to the polynucleotide, composition or immunocontraceptive, as described herein, disease or death that would otherwise follow exposure to a pathogen. Protective immunity is typically achieved by one or more of mucosal, humoral, or cellular immunity.

[0108] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following nonlimiting example. EXAMPLES

MATERIALS AND METHODS

Viruses and cell lines

[0109] This study used the F2 strain of FeHV-1 from the Feligen vaccine (Virbac, Genbank accession KR296657).

[0110] Crandell-Rees feline kidney (CRFK) cells were used. The CRFK cell monolayers were maintained in culture containing Dulbecco’s Modified Eagle medium (DMEM, Sigma Aldrich) with 5% v/v foetal bovine serum (FBS, Gibco), 10 mM HEPES (N-2- hydroxyethylpiperazine-N’-2-ethanesulfonic acid, pH 7.7), 50 pg/ml ampicillin and of 50 pg/ml gentamycin. Maintenance media contained the same reagent concentrations, except the percentage of FBS was reduced to 1% v/v. Cell cultures were maintained in open systems at 37°C in a humidified atmosphere of 5% v/v CO2 in air.

Transfection and Infection

[0111] CRISPR/Cas9 manipulation of herpesvirus genomes was first reported in 2015 and was applied to human herpesvirus-1 (HHV-1) (Russell et al. (2015) J. Virol. Methods 213, 18-25). The addition of a CRISPR/Cas9 construct during transfection/infection allows for selective targeting of insertion sites in the herpesvirus genome. The double-stranded breaks induced by the CRISPR/Cas9 system is targeted to the herpesvirus genomes that have not undergone recombination. This technique increases the proportion of recombinant genomes following transfection/infection and in HHV-1 resulted in a third of the viral progeny being recombinants. Using this method, ten different HHV-1 mutants were generated by targeting insertion sites of converging genes.

[0112] A similar CRISPR/Cas9 transfection/infection protocol was used to insert of three transgenes (two feline reproductive-related genes as well as a green fluorescence gene). This was performed by the addition of a CRISPR/Cas9 plasmid carrying a guide RNA (gRNA) (SEQ ID NO:1 and SEQ ID NO:2) specific to an insertion site in the FeHV-1 genome (GenBank Accession number KR296657). In addition to facilitating selection for recombinant virus, the addition of CRISPR/Cas9 may also enhance the efficiency of FeHV- 1 recombination with the repair plasmid, as the induction of double stranded breaks by the CRISPR/Cas9 gRNA may increase the rate at which the insert site carried by the repair plasmid becomes incorporated into the FeHV-1 genome via homology directed repair. Growth / kinetics assay

[0113] For Cell-to-cell spread assays, the area of individual plaques was measured to assess the cell-to-cell spread of FeHV-1 and the FeHV-1 immunocontraceptive candidates. Crandell-Rees feline kidney cells at 90% confluency were infected with dilutions of the different viruses. The area of approximately 20 individual plaques per virus were measured 24, 48 and 72 hours after infection by capturing photomicrographs using a Leica inverted light microscope and a Leica DMC4500 camera. The area of the plaques was measured in the photomicrographs using ImageJ software (Fiji).

[0114] For the One step growth kinetics of FeHV-1 immunocontraceptives in cell culture, Crandell-Rees feline kidney cell cultures at 90% confluency were prepared in 6-well cell culture plates. The cells were infected (MOI = 5) in triplicate with FeHV-1 or the FeHV-1 immunocontraceptive candidates (excluding FeHV-GZeGTmC2 which did not reach sufficient titre for the target MOI). Cells were infected for 1 hour before inoculum was removed and cells were washed 5 times with sterile DMEM media. Infected cells were harvested at 1, 8, 18, 24, 32 and 48 hours post infection. Three wells per virus, per time point were collected for this growth analysis. Each well collected from the 6-well plates was titrated twice, and the average TCID50 titre calculated. This resulted in three data points per virus per time point. The harvested samples were spun at 5,000 * g for 5 minutes to separate the CRFK cells from the supernatant. The cells were then resuspended in DMEM cell culture media as described previously, freeze-thawed (-80°C) and a TCID50 assay was then used to titrate the different viruses/timepoints.

[0115] For the Multi-step growth kinetics of FeHV-1 immunocontraceptives in cell culture Crandell-Rees feline kidney cells at 90% confluency were prepared in a 48-well tray. Inoculation of 6 wells per virus per timepoint was conducted at an MOI of 0.001 with FeHV- 1 or the FeHV-1 immunocontraceptive. Cells were infected for 1 hour before being washed 5 times with sterile DMEM media. Inoculated wells were harvested at 1, 24, 48, 72 and 96 hours post infection. This resulted in six data points per virus per time point. The samples were then freeze-thawed (-70 DC) and titrated on CRFK cells using a TCID50 assay.

Transcript analyses

[0116] The transcript abundance of ZP3 and TK was compared in CRFK cells infected with FeHV-1 or FeHV-1 immunocontraceptives. The mRNA levels of ZP3 and TK were measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR). For this, CRFK cells at 100% confluency were infected with 2 MOI of FeHV-1, FHV-GZeG, FHV-GZeGTmC or FHV-GZeGTmC2. Infected cells were collected in RLT buffer (from RNeasy® kit, Qiagen) at 2, 4 and 6 hours post inoculation. Total RNA was extracted using the RNeasy® kit (Qiagen) according to manufacturer’s instructions. Samples were then treated to remove DNA contamination using the Turbo DNA-free™ kit (Ambion). The DNA-free samples were then reverse transcribed with SuperScript™ III (Thermo Fischer) according to manufacturer’s instructions.

Western blots

[0117] Crandell-Rees feline kidney cells were separately infected with either FeHV-1 or FeHV-1 immunocontraceptives at an MOI of approximately 5 and incubated at 37°C in 5% v/v CO2 in air for 48 hours. Cells and supernatant fractions were then separated, and infected cells were resuspended in DMEM following separation from the supernatant. Before loading onto a SDS-PAGE gel, samples were incubated at 100°C for 5 minutes in a reducing buffer containing a final concentration of 25 mM Tris HCL (pH 6.8), 1% w/v SDS, 5% v/v glycerol, 50 mM b-mercaptoethanol and 0.00005% w/v bromophenol blue.

[0118] Samples were then separated by electrophoresis through an SDS-PAGE at 200 V for 45 minutes. Proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Trans-Blot Turbo Transfer System, Bio-Rad) according to manufacturer’s instructions.

[0119] The PVDF membrane was blocked for an hour in 5% w/v skim milk powder in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KC1, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4) and washed three times with PBS-T (PBS containing 0.05% v/v Tween 20). The membrane was incubated with primary antibody (rabbit anti-eGFP, Thermo Fisher, at a dilution of 1:1000 for 1 hour) followed by washing four times with PBS-T and incubation with secondary antibody (swine anti-Rabbit IgG HRP-conjugated antibodies, Agilent DAKO, at a dilution of 1:1000 for 1 hour). The membrane was washed three times in PBS-T and one final time in PBS before being developed (Clarity Western ECL substrate, BioRad, 5-minute development time) and imaged (ChemiDoc, Bio-Rad) using the chemiluminescent function.

[0120] For the extraction /detection of vRNA and vDNA from cell cultures inoculated with the FeHV-1 derived constructs, RNA was extracted and reverse transcribed using random hexamers (Thermo Fischer Scientific) and SuperScript™ III Reverse Transcriptase (Thermo Fischer Scientific) to produce cDNA. For the detection of vDNA, the extracts were stored at -20°C without DNase treatment for use as template in qPCR.

[0121] The cDNA were used as template in qPCRs targeting the FeHV-1 infected-cell polypeptide 4 (ICP4), ZP3 or thymidine kinase (TK) genesr

[0122] The stored vDNA extracts originating from mouse lung homogenates were used as template in conventional PCRs targeting the FeHV-1 ICP4 and TK genes, and the GAPDH gene (using feline GAPDH primers that also amplify murine GAPDH) (Table 2). Positive control samples included mock infected mouse lung homogenate spiked with laboratory stocks ofFeHV-1, and also samples containing 1:300 dilution ofpGEM-T vectors separately carrying ZP3, ICP4, TK and GAPDH inserts that were generated for qPCR analyses.

ELISA detection of GnRH and FeHV-1 antibodies

[0123] To detect IgG antibodies, purified GnRH and FeHV-1 were coated at 5 pg per well in coating buffer (32 mM Na2CO3, 38 mM NaHCO3, pH 9.6) onto 96 well plates (Maxisorb, Nunc). Detection of IgM antibodies against FeHV-1 was conducted on plates coated with 2.5 pg of FeHV-1 antigen per well. Once coated with antigen, the plates were wrapped in cling film and incubated overnight at 4°C. Excess coating antigen was removed by aspiration and wells were washed with PBS containing 0.05% tween 20 (PBS-T) at a pH of 7. Unoccupied sites were blocked with 100 pl of bovine serum albumin (BSA) blocking buffer in PBS-T (1% w/v BSA Fraction V (Roche), 5% v/v normal sheep serum, 10% v/v PBS-T) for a minimum of 2 hours at 37°C. Serum samples were prepared at a 1 :20 dilution in a BSA diluent buffer (0.5 % w/v BSA Fraction V, 2.5% v/v normal sheep serum in PBS) and 50 pl added to wells as the primary antibody. Polyclonal antibodies to GnRH (ThermoFisher Cat # PAI-121) at 1:500 dilution in PBS-T were used as a positive control primary antibody for the GnRH ELISA. The positive control primary antibody for the FeHV-1 ELISA was serum diluted 1 : 1000 in PBS-T from a domestic/owned cat that had a neutralising antibody titre to FeHV-1 of 16024 (reciprocal of the highest dilution of the serum capable of neutralising FeHV-1). The primary antibodies were incubated for 2 hours at room temperature followed by four washes with PBS-T. The secondary antibodies were then added at a 1:500 dilution in BSA diluent and incubated for 45 minutes. The secondary antibodies for detection of IgG antibodies to FeHV-1 and GnRH in the mouse serum was horse radish peroxidase (HRP) labelled sheep anti-mouse IgG (Cytiva Cat NA931V). Detection of IgM antibodies against FeHV-1 used HRP labelled anti-mouse IgM antibodies raised in goat (Sigma-Aldrich Cat #A8786) as the secondary antibody. For the GnRH antibody positive control, the secondary antibody was HRP conjugated anti-rabbit IgG raised in pig (Agilent DAKO Cat #PO217). The secondary antibody used in the FeHV-1 positive control was anti-feline IgG antibodies raised in goat (Thermo Fisher Scientific Cat #A18757).

[0124] After incubation, the solutions containing secondary antibody were aspirated and wells washed four times with PBS-T. One hundred microlitres of substrate, 1-Step™ ABTS Substrate Solution (Thermo Fisher Scientific Cat #37615), was allowed to develop for 20 minutes. Wells were then read in an FLUOstar Omega Microplate Reader (BMG LABTECH) at an absorbance of 410 nm.

[0125] FeHV-1 antigen was purified by infecting CRFK cells at 100% confluency with FeHV-1. Viral supernatant was removed and cleared of cell debris by centrifugation at 5000 x g for 5 minutes at 4°C. The virus was then pelleted from the supernatant at 40,000 * g for 1 hour at 4°C. The supernatant was removed, and the pellet was resuspended in TNE (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA) buffer. Virus in suspension was then overlayed on a continuous gradient of 5 - 15% w/v Ficoll in TNE. The tube was centrifuged at 15,000 x g for 2 hours at 4°C with no break. A band of virus was visualised, and an 18- gauge needle inserted just below the band in order to collect the band. The fraction was diluted with TNE buffer and further pelleted at 40,000 x g for 1 hour at 4°C. Supernatant was removed and the pellet finally resuspended in 100 ul of TNE buffer to form the purified FeHV-1 antigen.

EXAMPLE 1: CONSTRUCTION OF CANDIDATE FEHV-1 DERIVED POLYNUCLEOTIDE CONSTRUCTS

Transfection/infection -with CRISPR/Cas9

Generation of repair plasmids

[0126] Repair plasmids were generated for use in CRISPR-Cas9 assisted homologous recombination between the repair plasmid and the virus genome in a transfection-infection system, as previously described for herpes simplex virus- 1 (HSV-1) (Russell et al. (2015) J. Virol. Methods 213, 18-25). [0127] To generate the first immunocontraceptive candidate, FHV-GZeG, a repair plasmid containing the ZP3 (having the nucleotide sequence of SEQ ID NO:4) and GnRH (having the nucleotide sequence of SEQ ID NO:6) sequences fused to the enhanced green fluorescence (eGFP) gene, flanked by sequences homologous to the FeHV-1 genome upstream and downstream of the target insertion site was constructed.

[0128] The homologous sequences that allowed for the insertion of GnRH, ZP3 and eGFP into the genome of the wildtype (WT) FeHV-1 strain, flanked an insert site between the converging UL40 and UL41 genes (Figure 1A). The FeHV-1 homologous flanking regions (upstream 25,103 - 26,100 bp and downstream region 26,109 - 27,077 bp using GenBank Accession number KR296657 as the reference sequence) were amplified from DNA extracted from cultured FeHV-1 using the primers in Table 2. The insert sequence containing the genes for a CMV promoter, GnRH, ZP3, eGFP and a BGH termination region was synthesised by Genscript (Piscataway, USA) and provided as a 3,068 bp insert in the pUC57 vector.

Table 2: Primer sequences used in construction and confirmation of FHV-GZeG

[0129] The insert sequence was then assembled with the FeHV-1 homologous flanking regions by splicing by overlap extension (SOE) PCR. This fragment was then ligated into the pGEM-T vector and electroporated in JM109 electrocompetent Escherichia coli (E. coli) cells. Plasmid DNA was extracted and sequenced to confirm the clone contained the correct insert sequence.

[0130] To construct the immunocontraceptive candidates FHV-GZeG mC and FHV- GZeGTmC2 (Fig IB, 1C), two repair plasmids ("pGEM-T.TmC” for FeHV.GZeGTmC and “pGEM-T.TmC2” for FeHV.GZeGTmC2) containing codon deoptimized TK (SEQ ID NO: 12) and mCherry sequences (SEQ ID NO: 16) flanked by sequences homologous to the FeHV-1 genome upstream and downstream of the TK gene were generated allowing for replacement of wildtype TK gene with the deoptimized TK. The regions of homology upstream and downstream of TK (65,436 - 66-387 bp and 67,434 - 68,525 bp, respectively, using GenBank Accession number KR296657 as the reference sequence) were chosen. Codon deoptimization of the WT TK sequence was generated using the online IDT codon optimisation tool (https://sg.idtdna.com/CodonOpt). The wildtype TK sequence (SEQ ID NO: 10) was used as the basis for the codon-deoptimisation, and the least favoured variant was selected for each codon using gallus gallus as the reference organism.

[0131] The YHV-GZeGTmC and YHM -GZeGTmC2 differed with respect to the mCherry position in the sequence. The plasmid for generation of FHV-GZeG mC contained the deoptimized TK gene, followed by a CMV promoter, mCherry and BGH termination sequence (See Figure IB). FHV-GZeG mC2 contained deoptimized TK gene fused to mCherry flanked either side by regions of homology to the FeHV-1 genome (Fig 1C). The regions of homology were generated by amplification using primers in Table 1. The deoptimized TK gene fused to mCherry segment was artificially synthesised and joined either side with the homologous flanking regions using SOE PCR as previously described. Construction of CRISPR/Cas9 plasmids for use in transfection/infection procedure

[0132] For the FHV-GZeG immunocontraceptive, CRISPR/Cas9 guide RNA (gRNA) (SEQ ID NO:1 and SEQ ID NO:2) was constructed based on the targeted insertion site between UL40 and UL41. For both TK deoptimized variants, a gRNA site within the WT TK sequence was chosen that was sufficiently diverged from the deoptimized TK sequence (SEQ ID NO: 17 and SEQ ID NO: 18). The CRISPR/Cas9 carrying a guide RNA specific to the insertion site was included to increase the chance that any virus that had not undergone homologous recombination insertion (parental strain) would be targeted for cleaving by CRISPR/Cas9, therefore selecting for recombinant FeHV-1 virions.

[0133] Sense and antisense oligos were constructed to contain the protospacer adjacent motif (PAM) site and target sequence and were subsequently synthesised (see Table 2).

[0134] Transfection/infection was first conducted using the F2 strain of FeHV-1 from the Feligen vaccine to generate FHV-GZeG. Immunocontraceptive candidates FHV-GZeGTmC and FHV-GZeGTmC2 were then generated by transfection/infection using FHV-GZeG as the infecting strain. The transfection/infection methodology described below was performed as described Russel et al., (2015).

Generation of candidate feline immunocontraceptive vaccines

[0135] The fluorescence microscopy showed the expected patterns of fluorescence for each of the different viruses (Figures 2 & 3). PCR amplification and subsequent DNA sequencing of the expected recombination regions of the viruses after plaque purification showed that sequential plaque purification was successful in deriving stocks of pure recombinant viruses, without any contaminating wildtype FeHV-1 virus. In all cases the sequencing results showed the sequence expected from successful recombination between the repair plasmid and the FeHV-1 genome, with the exception of a single point mutation in the TK gene of FHV-GZeG7mC2 which contained a C to T change at nucleotide position 69,554 using KR296657 as a reference sequence (SEQ ID NO: 14). This resulted in a predicted amino acid change from a threonine to a methionine (SEQ ID NO: 13).

EXAMPLE 2: GROWTH KINETICS OF FEHV-1 DERIVED POLYNUCLEOTIDE CONSTRUCTS

[0136] The single step growth curve comparing the kinetics of FeHV-1, FHV-GZeG and FHV-GZeGTmC showed viral titres peaked at approximately 1 x 10⁷ TCIDso/mL 24-32 hours after inoculation for all viruses, before starting to decline (Figure 4A). Statistically significant differences in viral titres were observed at 8 and 24 hours post inoculation. At 8 hours post inoculation, the mean FeHV-1 titre (10²-⁶³ TCIDso/mL) was significantly lower than the FHV GZeGTmC titre (10^{3 6} TCIDso/mL, P = 0.0379). At 24 hours post inoculation, the FeHV-1 titre (10⁸-⁵ TCIDso/mL) was significantly greater than both the FHV-GZeG titre (10⁷³ TCIDso/mL, P = 0.0124) and the FHV -GZeGTmC titre (10^{7 13} TCIDso/mL, P = 0.0038).

[0137] In the multi-step growth curve, the highest titres were recorded at 72 or 96 hours after inoculation (Figure 4B). Significant differences were observed at 48, 72 and 96 hours post inoculation. At 48 hours after inoculation the mean titre of F f\ -GZeGTmC (10²⁸ TCIDso/mL) was significantly lower than the mean titres of both FeHV-1 (10⁴-⁵ TCIDso/mL) and FeHV-GZeG (10⁴-⁵ TCIDso/mL) (p = 0.0057 for both comparisons). At 72 hours, the mean titre of Fo -GZeGTmC2 (10⁴ TCIDso/mL) was significantly lower than both FeHV- 1 (10⁵³ TCIDso/mL, P = 0.0329) and FHV-GZeG (10⁶³ TCIDso/mL, P < 0.0001), whilst the titre of FFW -GZeGTmC (10^{4 16} TCIDso/mL) was significantly lower than FHV-GZeG (P = 0.0001). At 96 hours post inoculation the mean titre of YHM -GZeGTmC (10⁴⁶ TCIDso/mL) was significantly lower than FHV-GZeG (10⁶-⁵ TCIDso/mL, P = 0.0042).

[0138] Differences in cell-to-cell spread between FeHV-1, FHV-GZeG, FFFV -GZeGTmC and FFW-GZeGTmC2 were assessed by measuring the areas of viral plaques in infected CRFK cells over three days (Figure 5). No significant differences were detected between the different virus strains in the first 48 hours. However, at 72 hours after inoculation, the average size of plaques induced by FFW-GZeGTmC2 (0.25 mm2) were significantly smaller than those induced by FHV-1 (0.34 mm2, P =0.047) and FHV-GZeG (0.39 mm2, P = 0.0002). The average size plaques induced by FFFV -GZeGTmC (0.27 mm2) were also significantly smaller (P = 0.0011) than those induced by FHV-GZeG at this timepoint.

EXAMPLE 3: EXPRESSION OF TRANSGENE AND THYMIDINE KINASE OF FHV-1 DERIVED POLYNUCLEOTIDE CONSTRUCTS

[0139] Assessment of ZP3 transcript levels in infected cell cultures (Fig 6A) compared FeHV-1 immunocontraceptive infected cells at 2, 4 and 6 hours post infection. At 4 hours post infection the level of ZP3 transcript in FHV-GZeG infected cells (103.35 copies/reaction) was significantly lower than in FHV- GZeGTmC infected cells (104.14 copies/reaction, P = 0.047) and in FHV-GZeGTmC2 (104.67 copies/reaction, P < 0.001). [0140] Comparison of TK transcript levels in infected cell cultures (Fig 6B) showed that the level of transcript in ¥HV-GZeGTmC2 infected cells (102.03 copies/reaction) was significantly lower than in FeHV-1 infected cells (103.19 copies/reaction, P = 0.0425) at 2 hours post inoculation. At 4 hours post inoculation the level of transcript in FHV-GZeGTmC infected cells (103.75 copies/reaction) was significantly lower than in FeHV-1 infected cells (105.05 copies/reaction, P = 0.0078) and ¥HV-GZeGTmC2 infected cells (104.9 copies/reaction, P = 0.0223).

[0141] The protein product from the insertion of the transgene in each of these viruses is expected to contain a ZP3-GnRH-eGFP fused protein. An antibody to eGFP detected protein of the expected size (75 kDa) in the cell and supernatant fractions of CRFK cells infected with the FHV-GZeG, FHV-GZeGTmC and FHV -GZeGTmC2 viruses, but this protein was not detected in the cell or supernatant fractions of CRFK cells infected with wildtype FeHV- 1 (Fig 7). Additional smaller products reactive to the GFP antibody (likely break down products of the ZP3-GnRH-eGFP protein) were also detected in the samples from FHV- GZeG, FHV-GZeGTmC or FHV-GZeGTmC2 infected cells.

EXAMPLE 4: HOST SPECIFICITY OF FEHV-1 DERIVED POLYNUCLEOTIDE CONSTRUCTS

[0142] FeHV-1 is considered to have a narrow host range restricted to the Felidae family. Here, the species-specificity of FeHV-1 and three modified FeHV-1 variants containing antigens intended as immunocontraceptive targets is investigated. Their ability to replicate in respiratory tissue, cause clinical signs and induce disruptions in reproductive tissues was studied in an in vivo murine model. Non-feline cell types from diverse species including domestic animals, wildlife species and a non-human primate were also investigated for their ability to support FeHV-1 infection.

[0143] Non-feline cell lines MDBK (bovine), MDCK (canine), JU56 (wallaby), Ptkl (Rat Kangaroo), Vero (African Green Monkey), LA-4 (mouse) and feline CRFK cells were infected at MOI of 10 (4 hour incubation) with the immunocontraceptive candidate FHV- GZeG and examined for their ability to support infection as measured by the presence of viral RNA (vRNA), expression of GFP and cytopathic effect (CPE). Cells were viewed with an inverted microscope using both light and fluorescence microscopy every 48 hours to detect any viral growth, as evidenced by green fluorescence or CPE compared to the uninfected controls. At 7 days post inoculation, cells were harvested for extraction and detection of vRNA. A second experiment using cell lines Vero, LA-4, JU56 and CRFK inoculated with FHV-GZeG was conducted to quantify viral DNA (vDNA) by qPCR at 0, 3 and 7 days post inoculation.

[0144] No evidence of viral infection was detected in any of the non-feline cells inoculated with FHV-GZeG as determined by lack of CPE and absence of cDNA by RT-qPCR using primers that amplify the wildtype (WT) TK gene. No evidence of viral transcripts could be detected at 7 days post infection in any of the non-feline cell lines (Figure 8A). The progress of FHV-GZeG infection in these cell lines, and in JU56 and CRFK cells, was analysed by qPCR. There was no evidence of FHV-GZeG infection detected by qPCR using primers to amplify the WT TK gene and a consistent decline in vDNA was observed in JU56, Vero and LA-4 cells from day 0 to 7 (Fig 8B).

Murine in vivo study design

[0145] Mice were divided into five groups of 20 mice (10 male and 10 female). On day 0, mice were anesthetised by inhalation with 5% v/v isoflurane in oxygen and inoculated with 50 l of virus inoculum at a concentration of 106.15 TCID50/mL of either FeHV-1, FHV- GZeG, FHV -GZeGTmC, FHV -GZeGTmC2 or sterile DMEM (mock infected group). Mice were returned to their boxes and monitored until fully recovered from anaesthesia. Five mice from each group were euthanised on day 1, 4, 8 and 14 post-inoculation (Fig 9). Following inoculation, mice were observed for any signs of illness twice per day in the first week, dropping to once per day in the second week.

[0146] The unused portion of the virus inoculums were retained and stored at -70°C. Inoculums were thawed and titrated using a TCID50 assay on CRFK cells to confirm inoculation had occurred with the correct dosage.

[0147] On days 1, 4, 8 and 14 post inoculation mice were euthanised via cervical dislocation under deep anaesthesia. At days 1, 4, 8 and 14, blood and lung samples were collected postmortem. Blood samples were centrifuged at 4000 * g for 5 minutes at room temperature to allow for collection of serum. The serum was stored at -70°C until required. Lung samples were minced with a sterile scalpel blade prior to storage in DMEM at -70°C. On day 14 post inoculation, ovaries and testes were collected in paraformaldehyde (4% w/v in PBS). [0148] The lungs from mice inoculated with FeHV-1 and FeHV-1 derived immunocontraceptives were removed at several time points up until 2 weeks post infection. No vDNA was detected in lung homogenates at any time point post infection from any of the groups in this study using FeHV-1 specific primers targeting ICP4, WT TK (mock, FeHV-1 and FHV-GZeG inoculated groups) and deoptimized TK primers (FHV-GZeG mC and FFW-GZeGTmC2 inoculated groups) (Table 3). A product of the correct size was amplified from the mock-infected lung sample spiked with FeHV-1, and from positive control samples. Primers targeting feline GAPDH but capable of amplifying murine GAPDH (Table 3) amplified an 80 bp product and confirmed that DNA extraction had been successful.

[0149] Serum samples from mice collected on days 1, 4, 8 and 14 were assessed for the presence of antibodies to GnRH and FeHV-1. When comparing groups for IgG antibodies to GnRH or FeHV-1, there was no difference in absorbance values between inoculated groups when compared to the mock infected group as assessed by Mann- Whitney U test (Fig 10A & 10B). Similarly, there was no detectable difference in absorbance values detecting IgM to FeHV-1 between inoculated groups and the mock infected group, with the exception between FeHV-1 and mock infected mice. (Fig 10C).

[0150] No evidence of FeHV-1 replication was detected in vivo or in vitro in the non-feline cell lines, nor was any evidence of disruption to reproductive tissues or processes detected in the murine model. The findings support previous descriptions of FeHV-1 inability to infect outside of the feline host range and adds support for its use as a feline-specific viral vector for immunocontraception.

[0151] Assessment of the ovaries of infected mice revealed no significant differences by Mann- Whitney U test in the total number of follicles in the ovaries of mice inoculated with the FeHV-1 derived immunocontraceptives compared to mock infected mice (Figure 11, Figure 12A & 12B). Examination of the testes revealed no apparent morphological changes between groups (Figure 12C & 12D) and similar levels of sperm and apoptotic cells, however statistical analyses were precluded by the small number (n = 2) of male mice per group at the final time point. EXAMPLE 5: FEHV-1 DERIVED POLYNUCLEOTIDE CONSTRUCTS AS IMMUNOCONTRACEPTIVE VACCINES IN FELINES

[0152] The advantage of the polynucleotide constructs described herein, as an immunocontraceptive vaccine is the potential ability to modulate their transmissibility.

[0153] The immunocontraceptive candidate FHV-GZeG using the FeHV-1 genome for insertion of reproductive-related genes without further modifications, is expected to retain horizontal transmission potential (i.e., an infected animal transmitting the FeHV-1 vectored immunocontraceptive to another animal). This is an advantageous feature of an immunocontraceptive in the context of feral animal population control is self-dissemination. The non-attenuated immunocontraceptive candidate (FHV-GZeG) should be able to be transmitted from cat to cat, and induce a contraceptive effect, resulting in a self-perpetuating means of population decline. As demonstrated herein, the modified FeHV-1 demonstrate strong feline host specificity and do not appear to affect non-feline cells.

Table 3: The primers displayed in this table were used for reverse transcription quantitative polymerase chain reaction (RT-qPCR), qPCR and conventional PCR. [0154] While FeHV-1 vectored immunocontraceptive transmissibility is advantageous in remote areas where feral cat control is critical, however it would be undesirable for use in domestic/owned cats. The FHV-GZeG mC and FHV -GZeGTmC2 candidates with additional disruption to the TK viral virulence gene, and optionally other virulence genes, are likely to have reduced or lack horizontally transmission, to make it more suitable for use in domestic/owned cats.

[0155] A breeding trial involving cats vaccinated with either one of the three polynucleotide constructs, compositions or immunocontraceptives described herein, and control sham treated cats will be conducted. Vaccinated and unvaccinated female cats will be housed with fertile males over a period between 4 months - 2 years. The cats will also be investigated for an adaptive immune response towards reproductive antigens For females, the ability of the immunocontraceptives to protect against pregnancy would be assessed by an absence or reduction in offspring numbers. For males, inability to impregnate unvaccinated females or a reduction in sperm numbers in ejaculate would be indicative of an immunocontraceptive effect. A free roaming environment would also allow for investigation of infectivity of the FeHV-1 immunocontraceptives to spread to ‘in contact’ cats.

EXAMPLE 6: FEHV-1 SEROPREVALENCE IN VICTORIAN FERAL AND OWNED CATS

[0156] Understanding FeHV-1 exposure in both feral and owned cats may be helpful in understanding the context and the risks/benefits of vaccines that utilise FeHV-1 as vector, including FeHV-l-vectored immunocontraceptive vaccines.

[0157] Serum samples from 3 separate populations of feral cats in Victoria, Australia were assessed for FeHV-l-neutralising antibodies. Serums were collected from cats in Hattah (12 cats), Point Cook (69 cats) and Phillip Island (66 cats). Samples from Phillip Island and Hattah were collected. All serum samples were collected between mid-2016 and mid-2021 from feral cats post-mortem after the animals were humanely culled for unrelated reasons. In addition to samples from feral cats, serum samples from 44 owned (pet) cats were obtained from ASAP laboratories in Mulgrave, Victoria. These samples had been previously sent to ASAP laboratories from veterinary clinics around Victoria and on the Victorian-NSW border in 2019. [0158] Briefly, serums were diluted to a final dilution of 1/640 and incubated with 100 50% tissue culture infective dose (100 TCID50) of FeHV-1. The 96-well trays were incubated for one hour at 37°C prior to the addition of CRFK cells and incubation for a further 3 days at 37°C in a humidified atmosphere of 5% v/v CO2 in air. Antibody titres were recorded as the reciprocal of the highest dilution of the serum to neutralise virus. The antibody titres for samples where no viral neutralisation was observed at any dilution were recorded as < 5.

[0159] Table 1 shows the seroprevalence results in the different groups of cats. In feral cats the seroprevalence results ranged from no evidence of FeHV-1 VNAb in feral cats from Hattah, to the highest level of seroprevalence in feral cats from Point Cook, where 17 out of 69 samples (24.6%) had detectable FeHV-1 VNAb. In owned cats, 84.1% (37/44) of cats had detectable FeHV-1 VNAb (Table 1). Whilst there was no significant difference between the proportion of seropositive cats between the three feral cat locations, all three feral cat groups showed significantly lower seroprevalence compared to the owned cats (P < 0.0001, Fisher’s exact test). Neutralising antibody titres were not significantly different between owned and feral cats when compared by Mann- Whitney U test (P > 0.05). The weights of the cats from Phillip Island and the age of the owned cats were tested for any correlation with VNAb titre using Pearson’s correlation and nonparametric Spearman’s correlation, respectively. No correlation was observed in either instance (P = 0.12, P = 0.54). In general, the titres of neutralising antibodies were consistently low compared to the neutralising antibody titres than can be induced by some other alphaherpesviruses in their respective mammalian hosts.

Table 4: Serum-virus neutralisation assay results from testing samples from feral and owned cats in Victoria

[0160] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0161] The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

[0162] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide construct comprising a FeHV Felid alphaherpesvirus 1 (FeHV- 1) genome modified by insertion of one or more nucleic acid sequences into a non-coding region between two convergent FeHV-1 genes of the FeHV-1 genome, wherein the one or more nucleic acid sequences encode for one or more feline reproductive protein antigens.

2. The polynucleotide construct of claim 1, wherein the one or more feline reproductive protein antigens are selected from the group consisting of: a. gonadotrophin releasing hormone (GnRH); b. zona pellucida glycoprotein 3 (ZP3); c. follicle stimulating hormone; d. luteinising hormone; e. sperm adhesion molecule 1; and f. one or more fragments thereof.

3. The polynucleotide construct of claim 1 or claim 2, wherein the one or more feline reproductive protein antigens comprise gonadotrophin releasing hormone and zona pellucida glycoprotein 3.

4. The polynucleotide construct of any one of claims 1 to 3, wherein the one or more nucleic acid sequences are inserted between the UL40 and UL-41 genes of the FeHV-1 genome.

5. The polynucleotide construct of claim 4, wherein the one or more nucleic acid sequences are inserted at a site selected from nucleotides 25103 to 27077 of FeHV-1 GenBank Accession number KR296657.

6. The polynucleotide construct of claim 4 or claim 5, wherein the one or more nucleic acid sequences are inserted at a site selected from nucleotides 26100 to The polynucleotide construct of any one of claims 1 to 6, wherein the modified FeHV-1 genome comprises a thymidine kinase (TK) gene that is modified by a. one or more amino acid substitution; and/or b. replacing at least one codon of the TK gene with a codon that has a lower translational efficiency in a cell The polynucleotide construct of claim 7, wherein the TK gene is modified by replacing each codon with a codon that has a lower translational efficiency in a cell. The polynucleotide construct of claim 7 or claim 8, wherein the codon is replaced with a codon that has a lower translational efficiency in a chicken cell. The polynucleotide construct of claim any one of claims 1 to 9, wherein the modified FeHV-1 genome comprises a mutation or deletion of one or more FeHV- 1 genes other than the TK gene. The polynucleotide construct of claim any one of claims 1 to 10, wherein the modified FeHV-1 genome is not inhibited for growth in feline cells. The polynucleotide construct of claim 11, wherein the modified FeHV-1 genome retains at least some of the horizontal transmission potential of an unmodified FeHV-1 genome. The polynucleotide construct of claim 12, wherein the modified FeHV-1 genome has the same horizontal transmission potential of an unmodified FeHV-1 genome. The polynucleotide construct of any one of claims 1 to 10, wherein the modified FeHV-1 genome has reduced horizontal transmission potential when compared to unmodified FeHV-1 genome. The polynucleotide construct of claim 14, wherein the modified FeHV-1 genome has little to no horizontal transmission potential when compared to unmodified FeHV-1 genome. A veterinary composition comprising the polynucleotide construct of any one of claims 1 to 15, and a veterinarily acceptable carrier, excipient or diluent. An immunocontraceptive vaccine comprising the polynucleotide construct of any one of claims 1 to 15 or the veterinary composition of claim 16. The immunocontraceptive vaccine of claim 17, further comprising at least one adjuvant. A method of reducing the fertility of a feline, the method comprising administering to a feline in need thereof, the polynucleotide construct of any one of claims 1 to 15, the veterinary composition of claim 16, or the immunocontraceptive vaccine of claim 17 or claim 18. The method of claim 19, wherein when the fertility of the feline is reduced, the feline is reproductively sterile. A method of inducing an immune response against one or more feline reproductive protein antigens in a feline, the method comprising administering to a feline in need thereof the polynucleotide construct of any one of claims 1 to 15, the veterinary composition of claim 16 or the immunocontraceptive vaccine of claim 17 or claim 18. A method of controlling a population of feral felines, the method comprising administering to a feral feline in need thereof, the polynucleotide construct of any one of claims 1 to 15, the veterinary composition of claim 16 or the immunocontraceptive vaccine of claim 17 or claim 18. The method of any one of claims 19-22, wherein the polynucleotide construct of any one of claims 1 to 15, the veterinary composition of claim 16 or the immunocontraceptive vaccine of claim 17 or claim 18 is administered to the feline intranasally, intramuscularly or intraperitoneally. Use of the polynucleotide construct of any one of claims 1 to 15, the veterinary composition of claim 16, in the manufacture of an immunocontraceptive vaccine medicament for reducing fertility in a feline subject. Use of the polynucleotide construct of any one of claims 1 to 15, the veterinary composition of claim 16, in the manufacture of an immunocontraceptive vaccine medicament for inducing an immune response against one or more feline reproductive protein antigens in a feline subject.