Polynucleotide constructs and uses thereof

Abstract

The present disclosure generally relates to polynucleotide constructs comprising a feline alphaherpesvirus 1 (FeHV-1) genome modified by inserting one or more nucleic acid sequences into the non-coding region between two convergent FeHV-1 genes of the FeHV-1 genome, wherein the one or more nucleic acid sequences encode one or more feline reproductive protein antigens that can stimulate an immune response to reduce the incidence of pregnancy, and uses thereof.

Description

[Technical Field]

[0001] FIELD OF THE DISCLOSURE

[0001] This disclosure relates generally to polynucleotide constructs encoding feline reproductive protein antigens that can stimulate an immune response to reduce the incidence of pregnancy, and uses thereof. [Background technology]

[0002]

[0002] The impact of feral cats on native wildlife is devastating, yet controlling feral cat populations has proven difficult. In Australia and New Zealand, feral cats are responsible for the daily deaths of millions of native animals. Feral cat populations are difficult to estimate because they are influenced by factors such as terrain, environmental conditions, prey availability, and the difficulty of capturing enough feral cats to provide an accurate population estimate. While population estimates vary, feral cats are undoubtedly one of the greatest threats to the future of Australia's native wildlife.

[0003] Current control methods, including poisons, baits, and traps, are not very effective. This is primarily because feral cats are highly cautious animals and difficult to capture. Successful captures are quickly negated as cats move in from surrounding areas and repopulate the population. Cats are also difficult to feed because they prefer live prey to scavenging carcasses. Recently, grooming traps have been introduced as a novel approach that aims to take advantage of the size and grooming habits of cats compared to other native species. This technique involves spraying a sticky poison onto the skin of feral cats, which then ingest the poison while grooming the contaminated area. However, concerns remain about the impact of off-target poisoning on native species.

[0004]

[0004] Feral cats are currently the most expensive invasive species to manage in Australia, with approximately $19 billion spent on them since the 1960s. Efficient, humane population control that is harmless to native wildlife is urgently needed. Summary of the Invention [Problem to be solved by the invention]

[0005]

[0005] The present disclosure is based on the present inventors' surprising discovery that polynucleotide constructs containing modified FeHV-1 genomes that also encode feline reproductive protein antigens are useful as viral-vectored feline immunocontraceptives (VVICs). Depending on the modifications, these polynucleotide constructs may be useful for managing feral cat populations and / or providing non-surgical contraceptive options for domestic or feral cats, avoiding the costs and potential risks associated with anesthesia and surgical castration. In particular, for feral cats, population control methods using self-disseminated VVICs may be a desirable approach for feral cat management. The polynucleotide constructs disclosed herein, which utilize feline viruses as vectors, may also induce protection against feline viruses, thus further benefiting the health and welfare of vaccinated cats. [Means for solving the problem]

[0006]

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

[0007] In one embodiment, the one or more feline reproductive protein antigens are: a. Gonadotropin-releasing hormone (GnRH); b. Zona pellucida glycoprotein 3 (ZP3); C. Follicle-stimulating hormone; d. Luteinizing hormone; e. sperm adhesion molecule 1; and f. Selected from the group consisting of one or more fragments thereof.

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

[0009] In one embodiment, one or more nucleic acid sequences are inserted between the UL40 gene and the UL-41 gene. In another embodiment, one or more nucleic acid sequences are inserted between the UL40 gene and the UL-41 gene at a site selected from positions 25103 to 27077 of FeHV-1 GenBank Accession No. KR296657. In a preferred embodiment, one or more nucleic acid sequences are inserted between the UL40 gene and the UL-41 gene at a site selected from positions 26100 to 26109 of FeHV-1 GenBank Accession No. KR296657. In another preferred embodiment, one or more nucleic acid sequences are inserted between the UL40 gene and the UL-41 gene at positions 26104 to 26105 of FeHV-1 GenBank Accession No. KR296657.

[0009]

[0010] In another embodiment, the modified FeHV-1 genome comprises a thymidine kinase (TK) gene that has been modified by one or more amino acid substitutions and / or by replacing at least one codon in the TK gene with a codon that is less efficiently translated in the cell. In a specific embodiment, the TK gene has been modified by replacing each codon with a codon that is less efficiently translated in the cell. In another embodiment, the codons have been replaced with codons that are less efficiently translated.

[0010]

[0011] In one embodiment, the FeHV-1 genome is further modified by mutating or deleting one or more FeHV-1 genes.

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

[0011]

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

[0012]

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

[0013]

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

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

[0014]

[0017] In one embodiment, a method of reducing the fertility of a cat is provided, comprising administering to a cat in need thereof a polynucleotide construct, veterinary composition, or immunocontraceptive vaccine described herein. In certain embodiments, once the fertility of the cat is so reduced, the cat is sterile.

[0015]

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

[0016]

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

[0017]

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

[0018]

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

[0019]

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

[0020]

[0023] Embodiments of the present disclosure are herein described, by way of non-limiting example only, with reference to the following figures: [Brief explanation of the drawings]

[0021] [Figure 1]

[0024] Schematic diagram of several examples of FeHV-1-derived immunocontraceptives. (A) Homologous recombination between the FeHV-1 genome and a repair plasmid containing a CMV promoter, GnRH, ZP3, eGFP fluorescent gene, and BGH terminal sequences 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 deoptimized TK followed by a CMV promoter, mCherry gene, and BGH gene. (C) Generation of FHV-GZeGTmC2: Recombination between the WT TK region of FeHV-GZeG and a repair plasmid containing codon-deoptimized TK fused to mCherry. [Figure 2]

[0025] This figure shows recombinant FeHV-1 plaques containing three transgenes (two feline germline genes and eGFP) generated in CRFK cells using CRISPR / Cas9-mediated transfection / infection. After CRISPR / Cas9-mediated transfection / infection, viral plaques showing eGFP expression were selected and purified by plaque picking three times under a methylcellulose overlay medium before viral amplification. Successful insertion of the transgene without disrupting the upstream or downstream regions of the FeHV-1 genome was confirmed using PCR and Sanger sequencing. A and B show bright-field (A) and fluorescent dark-field (B) micrographs of viral plaques; scale bar = 200 μm. C and D show bright-field and fluorescent dark-field micrographs of the same viral plaques; scale bar = 100 μm. [Figure 3]

[0026] Crandell-Rees feline kidney cells inoculated with A) FHV-GZeG, B) FHV-GZeGTmC, and C) FHV-GZeGTmC2. The first column shows bright field images, the second column shows GFP expression, and the third column shows mCherry expression. Scale bars represent 100 μm. [Figure 4]

[0027] Figure 1 shows growth curves of FeHV-1 wild-type virus and FeHV-1 immunocontraceptive candidate in CRFK cells. A) Single-step growth curve using an MOI of 5; B) Multi-step growth curve using an MOI of 0.001. Virus titers at each time point were measured by TCID50 / mL assay in CRFK cells. Each data point in the single-step growth curve represents three replicates. Each data point in the multi-step growth curve represents six replicates. Means and standard deviations are shown. [Figure 5]

[0028] Figure 1 shows plaque areas of wild-type FeHV-1 and candidate immunocontraceptives as a measure of cell-to-cell spread over 72 hours in CRFK cells under methylcellulose overlay medium. Means and standard deviations are shown. *p<0.033, **p<0.02, ***p<0.002, ****p<0.0001. [Figure 6]

[0029] Figure 1 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 and standard deviation. Each data point contains the mean and standard deviation of six replicates per virus per time point. Black bars indicate FeHV-1 strains significantly different from each other. *p<0.033, **p<0.02, ***p<0.002, ****p<0.0001. [Figure 7]

[0030] Detection of transgene expression. A predicted protein product corresponding to approximately 75 kDa (representing fused GnRH, ZP3, and eGFP) was detected using an anti-eGFP antibody in CRFK cells infected with the three different vaccine candidates but not in cells infected with the wild-type FeHV-1 strain. A small product, likely a degradation product, was also detected in all three cultures of cells infected with the vaccine candidates but not in cells infected with wild-type FeHV. [Figure 8]

[0031] Figure 1 shows that FeHV-GZeG does not grow in non-feline cells. A) RT-qPCR assessment of FeHV-GZeG mRNA abundance in various cell lines at day 7 post-infection. B) qPCR assessment of FeHV-GZeG genome copy number in LA-4, Vero, JU56, and CRFK cells at days 0, 3, and 7 post-infection. Data points represent the mean score ± 1 standard deviation. [Figure 9]

[0032] Figure 1 shows a schematic diagram of the study design for the 14-day evaluation of FeHV-1-derived immunocontraceptives in a mouse model. Mock, FeHV-1, FHV-GZeG, FHV-GZeGTmC, or FHV-GZeGTmC2 were inoculated on day 0. Five mice per group were euthanized on days 1, 4, 8, and 14 postinfection. Lung tissue and blood samples were collected from all mice on each of these days, and ovaries and testes were collected on day 14. [Figure 10]

[0033] ELISA results for IgG / IgM antibodies against FeHV-1 and IgG antibodies against GnRH are shown. (A) Anti-GnRH IgG antibodies, (B) Anti-FeHV-1 IgG antibodies, and (C) Anti-FeHV-1 IgM antibodies. Individual absorbance values ​​are shown 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. [Figure 11]

[0034] Figure 1 shows the number of follicles in female mice for each inoculation group. Three ovaries per inoculation group (one ovary per mouse) were collected 14 days post-infection. Each data point represents the number of follicles found in the ovary of an individual mouse. The mean ± one standard deviation for each group is also shown. [Figure 12]

[0035] Ovaries and testes from mice inoculated with the FeHV-1 immunocontraceptive candidate FHV-GZeG. Panels A and B are photomicrographs of H&E-stained ovarian cross sections from female mice inoculated with (A) diluent only (negative control) or (B) FHV-GZeG. Oocytes (Oo) and corpora lutea (Cl) are marked. Scale bar = 200 μm. Panels C and D are photomicrographs of H&E-stained testicular cross sections from male mice inoculated with (C) diluent only (negative control) or (D) FHV-GZeG. Sperm-containing seminiferous tubules (ST) are surrounded by Leydig (L) cells. The lumen (Lu) and sperm (spz) within the seminiferous tubules are marked. Scale bar = 100 μm. [Figure 13-1]

[0036] FIG. 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-2]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-3]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-4]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-5]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-6]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-7]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-8]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-9]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-10]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-11]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-12]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-13]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-14]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-15]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-16]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. [Figure 13-17]

[0036] Figure 1 shows the amino acid and nucleotide sequences disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0022] 1.Definition

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0023]

[0038] Nucleotide and amino acid sequences are referred to by sequence identification numbers (SEQ ID NOs). <400> 1. <400> 2, etc. A summary of sequence identification numbers is provided herein.

[0024]

[0039] Sequence reference numbers (eg, GenBank ID, EMBL-Bank ID, etc.) listed herein were current as of the date of submission.

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

[0025]

[0041] The articles "a" and "an" are used herein to refer to one or to more than one (i.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.

[0026]

[0042] As used herein, "and / or" means and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted as alternatives (or).

[0027]

[0043] Terms such as "administration concurrently" or "administering concurrently" or "co-administering" refer to the administration of a single composition containing two or more agents, or the administration of each agent as separate compositions and / or by separate routes, delivered contemporaneously or simultaneously, or sequentially within a sufficiently short period of time that the effective result is comparable to that obtained when all such agents are administered as a single composition. "Simultaneously" means that the agents are administered together at substantially the same time, preferably in the same formulation. "Contemporaneously" means that the agents are administered closely in time, e.g., one agent is administered within about one minute to about one day before or after another agent. Any contemporaneity is useful. However, when not administered simultaneously, the agents will often be administered within about one minute to about eight hours, suitably less than about one hour to about four hours. When administered contemporaneously, the agents are suitably administered to the same site on a subject. The term "same site" includes the exact location, but may be within about 0.5 to about 15 centimeters, and preferably within about 0.5 to about 5 centimeters. As used herein, the term "separately" means that the agents are administered at intervals, for example, at intervals of about one day to several weeks or months. The agents may be administered in any order. As used herein, the term "sequentially" means that the agents are administered in sequence, for example, at intervals of minutes, hours, days, or weeks. If appropriate, the agents may be administered in regular, repeated cycles.

[0028]

[0044] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to mean the inclusion of the specified step or element or group of steps or elements, but not the exclusion of other steps or elements or group of steps or elements. Thus, the use of terms such as "comprising" indicates that the listed elements are required or essential, while other elements are optional and may or may not be present. "Consisting of" means including and limited to whatever the phrase "consisting of" follows. Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and that no other elements may be present. "Consisting essentially of" means including any elements listed after this phrase, and is limited to including other elements that do not interfere with or contribute to the activity or activities specified in this disclosure because of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or activity of the listed elements.

[0029]

[0045] The term "construct" or "vector" typically refers to a DNA or RNA molecule used as a vehicle to transport recombinant genetic material, such as the 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 contains an insert (the heterologous nucleic acid sequence or transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector to transport genetic information into a host is typically to segregate, propagate, or express the insert in the target cell. A vector may be episomal, i.e., not integrated into the genome of the host cell, or it may be integrated into the host cell genome. A vector may also be replication-competent or replication-incompetent. Examples of polynucleotide vectors include, but are not limited to, plasmids, yeast artificial chromosomes (YACs), cosmids, transposons, and synthetic DNA fragments. Examples of viral vectors include, for example, AAV, lentivirus, retrovirus, adenovirus, herpesvirus, and hepatitis virus vectors. The choice of vector to be used will depend on the size of the insert, the host cell to be transfected, and the desired transformation efficiency or outcome, and will be readily apparent to those skilled in the art. Polynucleotide constructs of the present disclosure generally contain the elements necessary to drive expression of a nucleic acid sequence of interest also contained in the construct. Such elements can include regulatory elements such as a promoter operably linked to the nucleic acid sequence of interest, an internal ribosome entry site (IRES) (to drive transcription), and often also a polyadenylation sequence. In certain embodiments of the present disclosure, the construct may be contained within a vector. In addition to the components of the construct, the vector may contain, for example, one or more selectable markers, one or more origins of replication, such as those derived from prokaryotes and eukaryotes, at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the genome of a host cell.The two or more constructs may be contained within one nucleic acid molecule, such as a single vector, or may be contained 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 comprises at least one regulatory sequence operably linked to a nucleotide sequence of interest. Thus, for example, a promoter operably linked to a nucleotide sequence to be expressed is provided in the expression construct for expression in an organism or portion thereof, including a host cell. Conventional compositions and methods for preparing and using constructs and host cells to practice the present disclosure are well known to those of skill 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.

[0030]

[0046] As used herein, the term "corresponding" with reference to a particular gene is intended to mean a similar, equivalent, or comparable gene. For example, when referring to a corresponding endogenous gene, it is intended to mean a similar, equivalent, or comparable naturally occurring gene. When referring to a corresponding foreign gene, it is intended to mean a similar, equivalent, or comparable foreign gene. In some embodiments, corresponding genes have similar or equivalent functions or have sequence similarity. In one embodiment, corresponding genes may be identical in function and / or sequence. In another embodiment, corresponding genes may have approximately the same function or activity. In another embodiment, corresponding genes may have reduced function or activity. In some embodiments, the phrase "corresponds to" or "corresponding to" refers to a nucleic acid sequence that exhibits substantial sequence identity to a reference nucleic acid sequence. Generally, the nucleic acid sequence exhibits 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 up to 100% sequence identity to a reference nucleic acid sequence.

[0031]

[0047] As used herein, the terms "encode," "encoding," and the like refer to the ability of a nucleic acid to provide 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 both coding or non-coding sequences and non-coding sequences. Thus, the terms "encode," "encoding," and the like include an RNA product resulting from the transcription of a DNA molecule, a protein resulting from the translation of an RNA molecule, a protein resulting from the transcription of a DNA molecule to form an RNA product and subsequent translation of the RNA product, or a protein resulting from the transcription of a DNA molecule to provide an RNA product, the processing of the RNA product to provide a processed RNA product (e.g., mRNA), and subsequent translation of the processed RNA product.

[0032]

[0048] In the context of treating a disease or condition, an "effective amount" means that an amount of a drug or composition effective to prevent the appearance of symptoms of the condition, suppress such symptoms, and / or treat existing symptoms is administered to an individual in need of such treatment or prevention, either as a single dose or as part of a series of doses. The effective amount will vary depending on the age, health, and physical condition of the individual being treated, whether symptoms of the disease are evident, the taxonomic group of the individual being treated, the formulation of the composition, an evaluation of the medical condition, and other relevant factors. Optimal dosing regimens can be calculated by measuring drug accumulation in the subject's body. Optimal dosages vary depending on the relative efficacy in individual subjects and can generally be estimated based on, for example, EC50 values ​​found to be effective in in vitro and in vivo animal models. Those skilled in the art can easily determine optimal dosages, administration methods, and repetition rates. It is expected that this amount will fall within a relatively broad range that can be determined by routine testing.

[0033]

[0049] As used herein, the terms "expression," "expressed," or "to express" typically refer to any step involved in producing an RNA molecule or polypeptide, such as by transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0034]

[0050] "Isolated" means material that is substantially or essentially free from components that normally accompany it in its natural state.

[0051] As used herein, the terms "nucleic acid," "nucleic acid sequence," "polynucleotide," "oligonucleotide," and "nucleotide sequence" refer to mRNA, RNA, cRNA, rRNA, cDNA, or DNA, or combinations thereof. The term typically refers to a polymer of ribonucleotides, or deoxyribonucleotides, or modified forms of either type of nucleotide. The term includes single-, double-, or triple-stranded forms of DNA and RNA. The nucleic acids may be recombinant, of artificial and / or synthetic origin, and may contain modified nucleotides, for example, containing modified linkages, modified purine or pyrimidine bases, or modified sugars. The nucleic acids of the present disclosure may be in isolated or purified form, and may be formed, isolated, and / or manipulated by conventionally known techniques, such as cloning and expression of cDNA libraries, amplification, enzymatic synthesis, or recombinant techniques. Nucleic acids may also be synthesized in vitro by well-known chemical synthesis techniques, for example, as described in Belousov (1997) Nucleic Acids Res. 25:3440-3444.

[0035]

[0052] The terms "peptide," "polypeptide," and "protein" are understood to mean a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain. Amino acids are typically represented by one-letter or three-letter codes 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 (Ile); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val); W: tryptophan (Trp), and Y: tyrosine (Tyr).

[0036]

[0053] As used herein, the term "sequence identity" refers to the extent that sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over the comparison window (e.g., over 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or more nucleotides or amino acid residues). Thus, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which identical nucleobases (e.g., A, T, C, G) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. For purposes of this disclosure, "sequence identity" will be understood to mean "percentage of matches" calculated by an appropriate method. For example, sequence identity analysis can be performed using the DNASIS computer program (version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) using the standard default settings used in the reference manual that accompanies the software. Sequences may be aligned using a global alignment algorithm (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), which optimally aligns sequences over their entire length, although sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)).Alignment for determining percent amino acid sequence identity can be carried out by any means available to those skilled in the art, including publicly available computer software such as those available at (http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / ).Those skilled in the art can easily determine the appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared.As used herein, % sequence identity typically refers to the value generated by using pairwise sequence alignment (for example, using the Needleman-Wunsch algorithm) to generate the optimal global alignment of two sequences.

[0037]

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

[0038]

[0055] The present disclosure also extends to inexact identity (i.e., similarity) of sequences at the nucleotide or amino acid level, where any differences between sequences relate to amino acids (or, in the context of nucleotides, the amino acids encoded by said nucleotides), but 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 one embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity. For example, leucine may be substituted with an isoleucine or valine residue. This can be referred to as a conservative substitution. In one embodiment, an amino acid sequence may be modified by 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 compared to the unmodified polypeptide.

[0039]

[0056] The terms "subject," "patient," and "individual," as used interchangeably herein, refer to any subject, particularly a vertebrate subject, more particularly a mammalian subject, and even more particularly a feline subject.

[0040]

[0057] Each embodiment described herein may be used mutatis mutandis in any and all embodiments unless otherwise stated.

[0041] [Table 1-1]

[0042] [Table 1-2]

[0043] 2. Polynucleotides

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

[0044]

[0059] Feline alphaherpesvirus 1 (FeHV-1) is a double-stranded DNA alphaherpesvirus and a common cause of feline viral rhinotracheitis, characterized by symptoms of upper respiratory tract disease. Feline alphaherpesvirus 1 is also known as feline herpesvirus 1 (FHV-1) and feline rhinotracheitis virus, terms used interchangeably herein. The virus is contagious and is primarily shed from the nasal mucosa, upper trachea, and nasopharynx. Primary FeHV-1 infection often causes lethargy, loss of appetite, and sneezing, and may progress to fever, ocular and nasal discharge, and conjunctivitis. In more severe cases, infected cats may exhibit labored breathing. Primary infection can induce an immune response that protects against disease but not against reinfection. Reactivation of FeHV-1 can cause cytolytic disease, including nasal discharge and sneezing.

[0045]

[0060] Feline herpesvirus-1 is thought to be restricted to the Felidae family. Cat species confirmed to be infected with FeHV-1 include cheetahs (Acinonyx jubatus), lions (Panthera leo), cougars (Puma concolor), and tigers (Panthera tigris).

[0046]

[0061] FeHV-1 has a large dsDNA genome of approximately 134 kb. The FeHV-1 genome contains 78 open reading frames (ORFs), some of which are essential or non-essential for viral replication. Many FeHV-1 ORFs have been functionally characterized, and some confer viral virulence. Virulence varies among FeHV-1 strains, and some strains have been modified to create attenuated FeHV-1 strains that induce reduced or no clinical signs of disease associated with virulent wild-type FeHV-1 strains. The transmissibility of FeHV-1 also means that FeHV-1-based VVICs are self-disseminating and can spread through feral cat populations, eliminating the need for extensive human intervention currently required to manage feral cat populations in remote areas. FeHV-1 can remain latent for life, periodically reactivating within the host. This may be favorable for VVICs, as reactivation in host cells can also restimulate the production of vectored germ protein antigens, periodically restimulating the host immune response.

[0047]

[0062] In one embodiment, the polynucleotide construct disclosed herein comprises a FeHV-1 genome that is a wild-type (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 has GenBank accession number KR296657. In another embodiment, the FeHV-1 genome is the FeHV-1 genome 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 producing the polynucleotides disclosed herein will be known to those of skill in the art.

[0048]

[0063] The term "reproductive protein" as used herein includes proteins involved in reproduction and / or fertility of a subject. These include, but are not limited to, sex hormones, proteins acting after mating, proteins mediating gamete production, utilization, storage, signaling, and proteins regulating fertilization and post-fertilization pathways. Reproductive proteins may be proteins involved in female-specific reproductive functions, such as egg proteins or proteins involved in placental / uterine function or proteins involved in embryonic development. Reproductive proteins may also be proteins involved in male-specific reproductive functions, such as sperm and seminal fluid proteins, sperm-egg and sperm-reproductive organ interaction proteins. Reproductive proteins may also be proteins that affect reproductive functions in both sexes. In one embodiment, the one or more feline reproductive protein antigens are: a. Gonadotropin-releasing hormone (GnRH); b. Zona pellucida glycoprotein 3 (ZP3); C. Follicle-stimulating hormone; d. Luteinizing hormone; e. sperm adhesion molecule 1; and f. one or more fragments thereof is selected from the group consisting of:

[0049]

[0064] In one embodiment, the one or more feline reproductive protein antigens comprise gonadotropin-releasing hormone (GnRH). Gonadotropin-releasing hormone (GnRH) is a peptide involved in inducing the release of follicle-stimulating hormone (FSH, essential for regulating puberty and sexual maturation) and luteinizing hormone (LH, essential for the release of testosterone in males and the initiation of ovulation in females). Due to its essential nature in both males and females, gonadotropin-releasing hormone is an attractive immunocontraceptive target. Because it is a small peptide (SEQ ID NO: 5), it is relatively easy to incorporate into a viral vector.

[0050]

[0065] In another embodiment, the one or more feline reproductive protein antigens comprise follicle-stimulating hormone (FSH). FSH is essential for pubertal development in both sexes. FSH is secreted by the anterior pituitary gland and induces the development of ovaries in females and testes in males. Mutations in the beta FSH subunit in mice caused infertility and impaired follicular development in females and oligozoospermia and subfertility in males (Sairam et al., (2001) Arch. Med. Res. 32, 601-608). Feline follicle-stimulating hormone consists of two subunit chains. The alpha chain consists of 96 amino acids (SEQ ID NO: 19), while the beta chain is 111 amino acids long (SEQ ID NO: 21).

[0051]

[0066] In another embodiment, the one or more feline reproductive protein antigens comprise luteinizing hormone (LH), which (SEQ ID NO: 23 and SEQ ID NO: 25) is involved in inducing the secretion of sex steroids, such as testosterone in males and estrogen in females.

[0052]

[0067] In another embodiment, the one or more feline reproductive protein antigens include zona pellucida glycoprotein 3 (ZP3). Zona pellucida (ZP) is a gene that induces the synthesis of the ZP layer, a glycoprotein matrix that surrounds the outer layer of the oocyte. It plays an important role in promoting sperm binding through the acrosome reaction, thereby fusing sperm with the egg. If the ZP layer is damaged or missing, fertilization is unlikely and females become infertile (Wassarman, PM Mammalian Fertilization. (1999) Cell 96, 175-183). The evolution of ZP genes is complex and shows significant differences between species. Cats express three ZP subunits, designated ZP2, ZP3 (SEQ ID NO: 3), and ZP4.

[0053]

[0068] As an example of a male reproductive protein, sperm adhesion molecule 1 (SPAM1) is involved in several processes responsible for successful fertilization of an egg. Its most important function is to promote sperm binding to the ZP layer and acrosomal exocytosis of sperm. Sperm adhesion molecule 1 transcripts are expressed in the testis and epididymis, and the SPAM1 protein is highly conserved among mammalian species. Mice carrying a SPAM1 mutation have reduced fertility as measured by litter size and penetrating efficiency (Zheng et al., (2001) Mamm. Genome 12, 822-829), and inoculation of male guinea pigs with SPAM1 protein caused infertility in all male guinea pigs as measured by their ability to impregnate fertile females (Primakoff et al., (1997) Biol. Reprod. 56, 1142-1146). In one embodiment, the one or more feline reproductive protein antigens comprise sperm adhesion molecule 1 (SPAM1).

[0054]

[0069] In one embodiment, the one or more feline reproductive protein antigens are selected from the group consisting of gonadotropin-releasing hormone (GnRH), zona pellucida glycoprotein 3 (ZP3), follicle-stimulating hormone, luteinizing hormone, sperm adhesion molecule 1, and one or more fragments thereof. In a preferred embodiment, the one or more feline reproductive proteins comprise gonadotropin-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 a fusion protein of gonadotropin-releasing hormone of SEQ ID NO: 29 and zona pellucida glycoprotein 3 of SEQ ID NO: 31.

[0055]

[0070] In some embodiments, one or more nucleic acid sequences encoding one or more feline reproductive protein antigens may be modified to regulate expression (i.e., to improve expression or ability to induce an immune response). By way of example, one or more nucleic acid sequences encoding one or more feline reproductive protein antigens may be operably linked to a strong promoter that improves expression of the nucleic acid, and / or the nucleic acid sequences encoding the feline reproductive protein antigens may be codon-optimized.

[0056]

[0071] As used herein, the term "codon-optimized" or "codon optimization" means that one or more codons are replaced with synonymous codons that improve expression of the resulting polypeptide or protein, but leave the amino acid sequence of the translated protein unchanged. Conversely, as used herein, "codon-deoptimized" or "codon-deoptimized" means that one or more codons are replaced with synonymous codons that result in lower translation efficiency in cells, resulting in reduced expression of the resulting polypeptide or protein, but leave the amino acid sequence of the translated protein unchanged. This is based on the discovery that although there are 64 different codons (61 codons that code for amino acids and 3 that code for stop codons), only 20 different amino acids are translated, and many amino acids can be coded for by multiple codons. The frequency of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA can be biased in different species. The codon usage bias of various organisms is known, and therefore, a particular nucleotide sequence may be codon-optimized for expression in a host cell. Such codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. Codon-optimized / codon-deoptimized coding regions can be designed in a variety of different ways, and can be performed using methods available online, published methods, or companies that provide codon optimization / deoptimization services. One codon optimization method is described, for example, in International Publication No. 2015 / 012924, which is incorporated herein by reference. Briefly, a nucleic acid sequence encoding a product is altered with synonymous codon sequences. Suitably, the entire length of the product open reading frame (ORF) may be altered. 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 other embodiments, only one codon in the open reading frame is altered.By using one of these methods, frequencies can be used for any given polypeptide sequence to generate nucleic acid fragments of codon-optimized / codon-deoptimized coding regions that encode the polypeptide.

[0057]

[0072] As used herein, the term "germ protein antigen" refers to a full-length germ protein or a variant thereof, or a derivative of a portion or fragment of a germ protein, capable of inducing an immune response in a subject, including a humoral (antibody) and / or cellular immune response in vivo. A full-length germ protein or a variant thereof, or a derivative of a portion or fragment of a germ protein, may suitably comprise a single epitope or may comprise multiple epitopes, including B-cell and T-cell epitopes, or mimotopes thereof.

[0058]

[0073] "One or more feline germ protein antigens" refers to one, two, three, four, five, six, seven, eight, nine, ten, eleven, or more feline germ protein antigens that are capable of eliciting a humoral (antibody) and / or cellular immune response in vivo when administered to a subject. In one embodiment, a polynucleotide construct comprising the FeHV feline alphaherpesvirus 1 (FeHV-1) genome has been modified to include one or more nucleic acid sequences encoding at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, or more preferably at least nine feline germ protein antigens.

[0059]

[0074] When a polynucleotide construct contains one or more nucleic acid sequences encoding 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 consisting of two or more peptide sequences linked together. In one embodiment, a polynucleotide contains one or more nucleic acid sequences encoding one or more feline reproductive protein antigens expressed as a fusion protein. In one embodiment, a fusion protein contains two or more peptide sequences linked end-to-end. In one embodiment, a fusion protein contains two or more peptide sequences linked together in a linear configuration via a suitable linking moiety, also referred to herein as a linker. Suitable methods for linking peptide sequences are well known to those skilled in the art, and examples include peptide (amide) bonds. As used herein, the term "linker" refers to a short polypeptide sequence inserted between any two adjacent peptide sequences as described herein. In one embodiment, the linker is a polypeptide linker of 1 to 10 amino acids, preferably 1, 2, 3, 4, or 5 naturally occurring or non-naturally occurring amino acids. When a polypeptide linker is used to join two respective peptide sequences, the linker is advantageously incorporated so that its N-terminus is linked to the C-terminus of one peptide sequence via a peptide bond, and its C-terminus is linked to the N-terminus of the other peptide sequence via a peptide bond. Individual peptide sequences within the fusion protein may also have one or more amino acids added to one or both ends, preferably the C-terminus. Thus, for example, linker or spacer amino acids may be added to the N- or C-terminus or both ends of the peptides to link the peptides and conveniently link them to each other and / or to a delivery system such as a carrier molecule. In one embodiment, an internal ribosome entry site (IRES) sequence may be used as a linker.In one embodiment, the IRES sequence comprises, consists of, or consists essentially of the amino acids 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 a chain of glycine and / or serine residues. The linker or spacer peptide may be (EAAAK). n Alternatively, the linker or spacer peptide may be a rigid linker, such as a proline-rich linker. In another example, the linker or spacer peptide may comprise a cleavage site that allows for cleavage of the linker peptide. In another example, the linker or spacer peptide may comprise a self-cleaving peptide that induces ribosome skipping during protein translation and prevents peptide bond formation. In one embodiment, the linker or spacer peptide is a P2A self-cleaving peptide (SEQ ID NO: 34), a T2A self-cleaving peptide (SEQ ID NO: 35), or an E2A self-cleaving peptide (SEQ ID NO: 36).

[0060]

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

[0061]

[0076] In one embodiment, one or more nucleic acid sequences are inserted between UL56 and V1, between CIRC and UL55, between UL51 and UL50, between UL46 and UL45, between UL40 and UL41, between UL36 and UL35, between UL31 and UL30, between V32 and UL26, between UL11 and Ul10, between UL8 and UL7, and / or between US8 and FH1-1.

[0062]

[0077] In a preferred embodiment, one or more nucleic acid sequences are inserted between the UL40 and UL-41 genes. In another preferred embodiment, the nucleic acid sequence is inserted between the UL40 and UL-41 genes at a site selected from between 25103 and 27077 of FeHV-1 GenBank Accession No. KR296657. In another preferred embodiment, the nucleic acid sequence is inserted between the UL40 and UL-41 genes at a site selected from between 26100 and 26109 of FeHV-1 GenBank Accession No. KR296657. In another preferred embodiment, the nucleic acid sequence encoding one or more feline reproductive protein antigens is inserted between the UL40 and UL-41 genes at between 26104 and 26105 of FeHV-1 GenBank Accession No. KR296657.

[0063]

[0078] The insertion of one or more nucleic acids into the FeHV-1 genome backbone can be targeted to a predetermined or designated genome locus. Methods for targeted, site-specific genome integration include the use of homologous recombination and CRISPR-Cas9, zinc finger nuclease, and TALEN genome editing technologies, the applications of which are known to those skilled in the art. In one embodiment, homologous recombination technology is used to insert one or more nucleic acid sequences encoding one or more feline reproductive protein antigens into the FeHV-1 genome backbone. In one embodiment, CRISPR / Cas9 is used to insert one or more nucleic acid sequences encoding one or more feline reproductive protein antigens. This can be achieved using a CRISPR / Cas9 plasmid carrying one or more guide RNAs specific to the targeted insertion site in the FeHV-1 genome.

[0064]

[0079] In certain embodiments, the thymidine kinase gene of the FeHV-1 genome is modified to attenuate 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 are known to those skilled in the art and include the ORF2 locus, the UL region, the gI / E region, DNA polymerase, ICP6, gE, gI, and gG.

[0065]

[0080] Thymidine kinase (TK) is a nonessential viral virulence gene involved in viral replication (Nishiyama, Y. (1996) Nagoya J. Med. Sci. 59, 107-119). Because TK is not essential for viral replication, it is possible to modify TK to attenuate the virus. Indeed, TK manipulation (including the creation of TK-deficient viruses) has been studied in several herpesviruses (Schroder et al. (2019) J. Gen. Virol. 100, 642-655; Cornick et al. (1990) Can. J. Vet. Res. 54, 260-266; Kit et al. (1985) Arch. Virol. 86, 63-83). An alternative approach is to reduce TK expression.

[0066]

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

[0067]

[0082] In some embodiments, the thymidine kinase gene of the FeHV-1 genome is modified to reduce or disrupt TK gene expression. Reducing TK gene expression can be achieved in many ways. For example, TK gene expression can be reduced by reducing the transcription and / or translation efficiency of the gene. In one embodiment, expression of the thymidine kinase (TK) gene of FeHV-1 is reduced by 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 is translated less efficiently in the cell than the replaced codon; and / or adding at least one codon to the coding sequence of the TK gene that is translated less efficiently than other codons; and disrupting the TK gene; modifying the TK gene to include a nucleotide sequence encoding an RNA destabilization element; and expressing a nucleic acid in a cell at a reduced level of the expression product of the TK gene. In one embodiment, the TK gene is modified by one or more amino acid substitutions; and / or replacing at least one codon of the TK gene with a codon that is translated less efficiently in the cell. In certain embodiments, the TK gene is modified by replacing each codon with a codon that is translated less efficiently in the cell, hi other embodiments, the codons are replaced with codons that are translated less efficiently or attenuated.

[0068] 3. Composition

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

[0069]

[0084] The polynucleotide constructs described herein can be formulated for administration with any number of carriers, excipients, or diluents. For example, various 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 sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, and lyophilized preparations may be combined with a sterile solution prior to administration. The compositions may further contain pharmaceutically acceptable auxiliary substances, such as pH-adjusting and buffering agents, osmolality adjusting agents, wetting agents, and the like, as needed to achieve near-physiological conditions, including, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, sucrose, or other carbohydrates, among others. Suitable methods for preparing compounds and compositions suitable for oral, topical, rectal, or parenteral administration (including subcutaneous injection, intradermal, intravenous, intramuscular, intrathecal, intrasternal injection and infusion techniques), or for administration by inhalation, intranasal, or spray, will be known or apparent to those skilled in the art and are described in more detail, for example, in A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th ed., Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Cansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., 3rd ed., Amer. Pharmaceutical Assoc.

[0070]

[0085] The polynucleotide constructs described herein can be used without further adjuvants or carriers. The polynucleotide constructs described herein can be used in combination with additional carriers or adjuvants to enhance immune responses in a subject. Accordingly, the present disclosure also extends to compositions further comprising an immunostimulant or adjuvant. Preferably, the immunostimulant or adjuvant is administered simultaneously with the polynucleotide construct, as described herein. As described herein, the immunostimulant or adjuvant may be administered before or after the polynucleotide construct, as appropriate, as can be appropriately determined by one skilled in the art. As used herein, the term "immunostimulant" or "adjuvant" refers to a substance that, when mixed with an immunogen or antigen molecule, elicits a stronger immune response than the immunogen or antigen molecule alone. For example, an adjuvant can enhance immunogenicity and provide a superior immune response. Suitable immunostimulants or adjuvants are well known to those skilled in the art, and examples include aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate (also known 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, for example, saponins or saponin derivatives derived from Quillaja saponaria, Panax ginseng, Panax notoginseng, Panax quinquefolium, Platycodon grandiflorum, Polygala senega, Polygala tenuifolia, Quillaja brasiliensis, Astragalus membranaceus, and Achyranthes bidentata. Examples of saponin-based adjuvants include immune stimulating complex, immune stimulating complex matrix, ISCOMATRIX™ adjuvant, Matrix M™ adjuvant, Matrix C™ adjuvant, Matrix Q™ adjuvant, AbISCO®-100 adjuvant, AbISCO®-300 adjuvant, ISCOPREP™, ISCOPREP™ derivatives, adjuvants containing ISCOPREP™ or ISCOPREP™ derivatives, QS-21, QS-21 derivatives, and adjuvants containing QS-21 or QS21 derivatives. The compositions and vaccines described herein may also be associated with immunomodulators, including, for example, cytokines, chemokines, and growth factors. Mixtures of two or more adjuvants within the same vaccine composition are also contemplated herein.

[0071]

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

[0072]

[0087] The term "immunocontraceptive" refers to a substance, molecule, or composition capable of stimulating the host immune system to reduce the chances of pregnancy occurring or continuing. This can occur by interfering with gamete production or interfering with fertilization. This immune response can cause temporary infertility in animals or long-term infertility, including permanent infertility.

[0073] 4.How to use

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

[0074]

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

[0075]

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

[0076]

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

[0077]

[0092] The terms "immunization" and "vaccination" are used interchangeably herein and refer to administering to a subject a polynucleotide construct, composition, or immunocontraceptive vaccine described herein to generate an immune response, which can have a prophylactic effect, a therapeutic effect, or a combination thereof. For example, administration of a polynucleotide construct, composition, or immunocontraceptive vaccine described herein can have a prophylactic / protective effect or a therapeutic effect by reducing the severity of clinical symptoms of FeHV-1 infection / exposure. This prophylactic and / or therapeutic effect occurs in conjunction with the contraceptive effect of administration of a polynucleotide construct, composition, or immunocontraceptive vaccine described herein, but it is understood that this prophylactic and / or therapeutic effect is distinct from the contraceptive effect.

[0078]

[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 cellular immune response against a target. A "humoral immune response" typically refers to an immune response mediated by antibody molecules, while a "cellular immune response" typically is mediated by T lymphocytes and / or other white blood cells. As a non-limiting example, a polynucleotide construct, composition, or immunocontraceptive vaccine 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 an innate immune response against a target antigen.

[0079]

[0094] Methods for measuring immune responses are known to those skilled in the art and include, for example, measurement of antibody titers, plaque reduction neutralization assays, microneutralization assays, solid-phase heterogeneous assays (e.g., enzyme-linked immunosorbent assays), solution-phase assays (e.g., electrochemiluminescence assays), Western immunoblots, amplified luminescence proximity homogeneous assays, flow cytometry, intracellular cytokine staining, functional T cell assays including suppressor T cell assays, functional B cell assays, functional monocyte-macrophage assays, dendritic and reticuloendothelial cell assays, measurement of NK or NKT cell responses, oxidative burst assays, cytotoxic specific cytolysis assays, pentamer binding assays, and assessment of phagocytosis and apoptosis.

[0080]

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

[0081]

[0096] By way of example, feral cat populations may be controlled by administering a polynucleotide construct, veterinary composition, or immunocontraceptive vaccine described herein to feral cats to cause a reduction in the feral cat population of at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, preferably at least about 60%, preferably at least about 70%, preferably at least about 80%, preferably at least about 90%, preferably at least about 100%, over a period of time, for example, such that a reduction in the feral cat population may be observed over about 12 months, about 18 months, about 24 months, about 30 months, about 36 months, about 42 months, about 48 months, about 52 months, or about 60 months.

[0082]

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

[0083]

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

[0084]

[0099] For administration to a host or subject (e.g., a cat), the polynucleotide constructs, compositions, and / or vaccines described herein can be formulated for administration by various 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, intranasal, or spray. As used herein, the term "parenteral" includes subcutaneous injection, intradermal, intravenous, intramuscular, intrathecal, and intrasternal injection and infusion techniques. In one embodiment, the polynucleotide constructs, compositions, or immunocontraceptive vaccines described herein are administered to a subject by intramuscular injection. In one embodiment, the polynucleotide constructs, compositions, and / or vaccines described herein are administered to a subject by intraperitoneal injection. In another embodiment, the polynucleotide constructs, compositions, and / or vaccines described herein can be administered to a subject by contacting the subject with one or more subjects that have been vaccinated or infected with an infectious polynucleotide construct comprising an FeHV feline alphaherpesvirus 1 (FeHV-1) genome that has been modified by inserting one or more nucleic acid sequences into the non-coding region between two convergent FeHV-1 genes of the FeHV-1 genome, wherein in one example of horizontal transmission, the one or more nucleic acid sequences encode one or more feline reproductive protein antigens.

[0085]

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

[0086]

[0101] The polynucleotide constructs, compositions, and / or vaccines described herein suitably comprise a therapeutically effective amount of the polynucleotide. The phrase "therapeutically effective amount" typically refers to the amount of the polynucleotide described herein necessary to achieve a desired response, e.g., induction of an immune response against a target (i.e., a reproductive antigen). Typically, the appropriate dosage of the polynucleotide described herein may depend on various factors, including, but not limited to, the subject's physical characteristics (e.g., age, weight, sex), whether the polynucleotide described herein is used as the sole agent or is administered in conjunction with other therapeutic or prophylactic agents, the progression of the condition (i.e., pathological condition), or the administration regimen (e.g., weekly, monthly, semi-annually, yearly, etc.), and other factors that would be recognized by one of skill in the art. For example, various general considerations may be taken into account when determining the appropriate dosage of a vaccine composition (see, e.g., Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th ed., Lippincott, Williams, & Wilkins; and Gilman et al. (eds.), (1990) "Goodman and Gilman's: The Pharmacological Bases of Therapeutics," Pergamon Press). It is expected that this amount will fall in a relatively broad range that can be determined by methods known to those skilled in the art. Examples of appropriate therapeutically effective amounts of polynucleotides to administer to a subject include about 10 3 TCID50 1mL to 10 8 TCID50 1ml, 10 4 TCID50 1mL to 10 7 TCID50 1mL, 10 5 TCID50 1mL to 10 6 The dose contains 1 mL of TCID50. The dosing regimen may be adjusted to provide the optimal therapeutic response. For example, multiple divided doses may be administered daily, weekly, monthly, or at other appropriate time intervals, or the dose may be proportionally reduced as indicated by the exigencies of the situation.

[0087]

[0102] The polynucleotide constructs, compositions, or vaccines described herein may be administered to a recipient alone or in combination with other additional therapeutic or contraceptive agents. In embodiments in which a veterinary composition or immunocontraceptive vaccine comprising a polynucleotide described herein is formulated for administration with an additional therapeutic agent, administration may be simultaneous or sequential (i.e., administration of the polynucleotide, composition, or vaccine followed by administration of the additional agent, or vice versa). Thus, when two or more elements are administered to a subject "in combination," they may be administered simultaneously in a single composition, or simultaneously in separate compositions, or separated in time in separate compositions.

[0088]

[0103] In a non-limiting example, the polynucleotides, compositions, and / or vaccines described herein may be administered in combination with another contraceptive. An "antiviral agent" typically refers to an agent that, when administered to a subject, can significantly reduce the viral titer in the blood or serum, either directly (e.g., by inhibiting viral enzymatic activity) or indirectly (e.g., through modulation of the host cell's antiviral response), either temporarily or persistently. A contraceptive typically refers to an agent that, when administered to a subject, can significantly prevent pregnancy.

[0089]

[0104] In one embodiment, the polynucleotides, compositions, or immunocontraceptive vaccines described herein may be suitably administered in a single dose adequate to elicit an immune response. In other embodiments, an initial dose may be administered followed by a booster dose. The booster 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 compared to the initial dose.

[0090]

[0105] The dosing regimen need not differ from other commonly accepted vaccination programs. For example, a single dose may be used in an amount sufficient to elicit an effective immune response. Alternatively, as noted above, other regimens of initial administration of the conjugate, including those described above, may be followed by booster doses. Booster doses may be administered long after the initial dose if the immune response (e.g., as measured by antibody titers) falls below acceptable levels.

[0091]

[0106] Alternatively, or in addition, the polynucleotides, compositions, and / or vaccines described herein may be used in combination with an additional immunostimulant or adjuvant to enhance the immune response in a subject. Accordingly, the present disclosure also extends to compositions further comprising an immunostimulant or adjuvant. Preferably, the immunostimulant or adjuvant is administered simultaneously with the polynucleotides, compositions, or immunocontraceptive vaccines described herein. The immunostimulant or adjuvant may be administered before or after the polynucleotides, compositions, or immunocontraceptive vaccines described herein, as appropriate, as can be determined by one of skill in the art. As used herein, the term "immunostimulant" refers to a substance that, when mixed with an immunogen, elicits a stronger immune response than the immunogen alone. For example, an immunostimulant can enhance immunogenicity and result in a superior immune response. Suitable immunostimulants or adjuvants are well known to those skilled in the art.

[0092]

[0107] As used herein, the terms "treat," "treated," or "treating," when used in reference to a disease or pathogen, refer to treatments that increase a subject's resistance to infection by a disease or pathogen (i.e., reduce the likelihood that a subject will contract the disease or become infected with a pathogen), as well as treatments to combat a disease or infection after a subject has contracted the disease or become infected with a pathogen (e.g., reduce, eliminate, ameliorate, or otherwise stabilize the disease or infection). In one embodiment, the polynucleotides, compositions, or immunocontraceptive vaccines described herein can provide protective immunity to a host. The term "immunity," as used herein, refers to the ability of a host (i.e., a cat) to mount an immune response against reproductive proteins and / or reduce the likelihood of pregnancy or otherwise becoming ill or dying from exposure to a pathogen as a result of exposure to a polynucleotide, composition, or immunocontraceptive described herein. Protective immunity is typically achieved through one or more of mucosal, humoral, or cellular immunity.

[0093]

[0108] In order that the invention may be more readily understood and put into practice, particularly preferred embodiments are described below by way of non-limiting examples. [Example]

[0094] material and method Viruses and cell lines

[0109] In this study, the F2 strain of FeHV-1 from the Feligen vaccine (Virbac, GenBank accession number KR296657) was used.

[0095]

[0110] Crandell-Rees feline kidney (CRFK) cells were used. CRFK cell monolayers were maintained in a medium containing 5% v / v fetal bovine serum (FBS, Gibco), 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.7), Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich) supplemented with 50 μg / ml ampicillin and 50 μg / ml gentamicin. The maintenance medium contained the same reagent concentrations except that the FBS was reduced to 1% v / v. Cell cultures were maintained in an open system at 37°C in a humidified atmosphere with 5% v / v CO2 in air.

[0096] Transfection and infection

[0111] CRISPR / Cas9 manipulation of herpesvirus genomes was first reported in 2015 and used on human herpesvirus-1 (HHV-1) (Russell et al. (2015) J. Virol. Methods 213, 18-25). Addition of CRISPR / Cas9 constructs during transfection / infection allows selective targeting of insertion sites in the herpesvirus genome. Double-strand breaks induced by the CRISPR / Cas9 system are targeted to unrecombined herpesvirus genomes. This technique increased the proportion of recombinant genomes after transfection / infection, with one-third of viral progeny being recombinant in HHV-1. Using this method, 10 different HHV-1 mutants were generated by targeting insertion sites in the converging gene.

[0097]

[0112] Using a similar CRISPR / Cas9 transfection / infection method, we inserted three transgenes (two feline reproductive-related genes and a green fluorescent gene) into the FeHV-1 genome (GenBank accession number KR296657) by adding a CRISPR / Cas9 plasmid carrying guide RNAs (gRNAs) (SEQ ID NO: 1 and SEQ ID NO: 2) specific for the insertion site in the FeHV-1 genome. In addition to facilitating the selection of recombinant viruses, the addition of CRISPR / Cas9 may also increase the efficiency of FeHV-1 recombination with the repair plasmid, because induction of a double-strand break by the CRISPR / Cas9 gRNA may increase the rate at which the repair plasmid's insertion site is integrated into the FeHV-1 genome by homologous recombination repair.

[0098] Proliferation / Kinetic Assays

[0113] In the cell-to-cell spread assay, the area of ​​individual plaques was measured to assess the cell-to-cell spread of FeHV-1 and the FeHV-1 immunocontraceptive candidate. Crandell-Rees feline kidney cells at 90% confluency were infected with different virus dilutions. The area of ​​approximately 20 individual plaques per virus was measured at 24, 48, and 72 hours post-infection by taking photomicrographs using a Leica inverted light microscope and a Leica DMC4500 camera. Plaque area was measured on the photomicrographs using ImageJ software (Fiji).

[0099]

[0114] For the one-step growth kinetics of the FeHV-1 immunocontraceptive in cell culture, 90% confluent Crandell-Rees feline kidney cell cultures were prepared in six-well cell culture plates. Cells were infected in triplicate with FeHV-1 or the FeHV-1 immunocontraceptive candidate (MOI = 5) (except for FeHV-GZeGTmC2, which did not reach a sufficient MOI for the target). Cells were infected for 1 hour, after which the inoculum was removed and the cells were washed five times with sterile DMEM medium. Infected cells were harvested at 1, 8, 18, 24, 32, and 48 hours postinfection. Three wells per virus per time point were harvested for this growth analysis. Titers were measured in duplicate for each harvested well from the six-well plate, and the average TCID50 titer was calculated. This resulted in three data points per virus per time point. The harvested samples were spun at 5,000 x g for 5 minutes to separate the CRFK cells from the supernatant. Cells were then resuspended in DMEM cell culture medium as described above, frozen and thawed (-80°C), and then the different viruses / time points were titrated using the TCID50 assay.

[0100]

[0115] For multistep growth kinetics of the FeHV-1 immunocontraceptive in cell culture, Crandell-Rees feline kidney cells were prepared in 48-well trays at 90% confluency. Six wells per virus per time point were inoculated at an MOI of 0.001 for FeHV-1 or FeHV-1 immunocontraceptive. Cells were infected for 1 hour and then washed five times with sterile DMEM medium. Inoculated wells were harvested at 1, 24, 48, 72, and 96 hours postinfection. This resulted in six data points per virus per time point. Samples were then frozen and thawed (-70°C) and titered on CRFK cells using a TCID50 assay.

[0101] Transcript analysis

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

[0102] Western blot

[0117] Crandell-Rees feline kidney cells were separately infected with FeHV-1 or FeHV-1 immunocontraceptive at an MOI of approximately 5 and incubated at 37°C in 5% v / v CO2 in air for 48 hours. Cell and supernatant fractions were then separated, and after separation from the supernatant, infected cells were resuspended in DMEM. Before loading onto an SDS-PAGE gel, samples were incubated for 5 minutes at 100°C in 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.

[0103]

[0118] Samples were then separated by electrophoresis on SDS-PAGE for 45 min at 200 V. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Trans-Blot Turbo Transfer System, Bio-Rad) according to the manufacturer's instructions.

[0104]

[0119] PVDF membranes were blocked for 1 hour with 5% w / v skim milk powder in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, and 1.8 mM KHPO, pH 7.4) and washed three times with PBS-T (PBS containing 0.05% v / v Tween 20). The membranes were incubated with a primary antibody (rabbit anti-eGFP, Thermo Fisher Scientific, 1:1000 dilution) for 1 hour, washed four times with PBS-T, and then incubated with a secondary antibody (swine anti-rabbit IgG HRP-conjugated antibody, Agilent DAKO, 1:1000 dilution) for 1 hour. The membranes were washed three times with PBS-T and one final time with PBS before being developed (Clarity Western ECL substrate, BioRad, 5-minute development time) and imaged using the chemiluminescence function (ChemiDoc, BioRad).

[0105]

[0120] To extract and detect vRNA and vDNA from cell cultures inoculated with FeHV-1-derived constructs, RNA was extracted and reverse transcribed to generate cDNA using random hexamers (Thermo Fischer Scientific) and SuperScript™ III reverse transcriptase (Thermo Fischer Scientific). For vDNA detection, extracts were stored at -20°C without DNase treatment for use as templates in qPCR.

[0106]

[0121] The cDNA was used as a template for qPCR targeting the FeHV-1 infected cell polypeptide 4 (ICP4), ZP3, or thymidine kinase (TK) genes.

[0122] Archived vDNA extracts from mouse lung homogenates were used as templates for conventional PCR targeting the FeHV-1 ICP4 and TK genes, as well as the GAPDH gene (using feline GAPDH primers, which also amplify mouse GAPDH) (Table 2). Positive control samples included mock-infected mouse lung homogenates spiked with a laboratory stock of FeHV-1, and samples also contained 1:300 dilutions of pGEM-T vectors carrying the ZP3, ICP4, TK, and GAPDH inserts separately, generated for qPCR analysis.

[0107] ELISA detection of GnRH and FeHV-1 antibodies

[0123] To detect IgG antibodies, purified GnRH and FeHV-1 were dissolved in coating buffer (32 mM Na2CO3, 38 mM NaHCO3, pH 9.6) and coated onto 96-well plates (Maxisorb, Nunc) at 5 μg per well. Detection of IgM antibodies against FeHV-1 was performed on plates coated with 2.5 μg of FeHV-1 antigen per well. After antigen coating, plates were wrapped in plastic wrap and incubated overnight at 4°C. Excess coating antigen was removed by aspiration, and wells were washed with PBS containing 0.05% Tween 20, pH 7 (PBS-T). Empty sites were blocked with 100 μl 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 h at 37°C. Serum samples were diluted 1:20 in BSA dilution buffer (0.5% w / v BSA fraction V, 2.5% v / v normal sheep serum in PBS), and 50 μl was added to the wells as the primary antibody. A polyclonal antibody against GnRH (ThermoFisher catalog no. PA1-121) diluted 1:500 in PBS-T was used as the positive control primary antibody for the GnRH ELISA. The positive control primary antibody for the FeHV-1 ELISA was a domestic cat serum with a neutralizing antibody titer of 16024 (the reciprocal of the highest dilution of serum capable of neutralizing FeHV-1) diluted 1:1000 in PBS-T. The primary antibody was incubated for 2 hours at room temperature, followed by four washes with PBS-T. The secondary antibody was then added at a dilution of 1:500 in BSA dilution buffer and incubated for 45 minutes. The secondary antibody for detecting IgG antibodies against FeHV-1 and GnRH in mouse serum was horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (Cytiva catalog number NA931V). For detecting IgM antibodies against FeHV-1, goat-produced HRP-conjugated anti-mouse IgM antibody (Sigma-Aldrich catalog number A8786) was used as the secondary antibody.For the GnRH antibody positive control, the secondary antibody was an HRP-conjugated anti-rabbit IgG (Agilent DAKO catalog number PO217) produced in pigs. The secondary antibody used for the FeHV-1 positive subjects was an anti-cat IgG antibody (Thermo Fisher Scientific catalog number A18757) produced in goats.

[0108]

[0124] After incubation, the solution containing the secondary antibody was aspirated, and the wells were washed four times with PBS-T. The color was developed with 100 μl of 1-Step™ ABTS substrate solution (Thermo Fisher Scientific catalog number 37615) for 20 minutes. The wells were then read at an absorbance of 410 nm on a FLUOstar Omega microplate reader (BMG Labtech).

[0109]

[0125] FeHV-1 antigen was purified by infecting 100% confluent CRFK cells with FeHV-1. The viral supernatant was removed by centrifugation at 5,000 × g for 5 minutes at 4°C to remove cellular debris. 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. The virus in suspension was then layered onto a continuous gradient of 5–15% w / v Ficoll in TNE. The tube was centrifuged uninterrupted at 15,000 × g for 2 hours at 4°C. The viral band was visualized and collected by inserting an 18-gauge needle just below the band. This fraction was diluted with TNE buffer and further pelleted at 40,000 × g for 1 hour at 4°C. The supernatant was removed and the pellet was finally resuspended in 100 μl of TNE buffer to form purified FeHV-1 antigen.

[0110] Example 1: Construction of candidate FeHV-1-derived polynucleotide constructs CRISPR / Cas9 transfection / infection Preparation of repair plasmid

[0126] A repair plasmid was generated for use in CRISPR-Cas9-assisted homologous recombination between the repair plasmid and the viral 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).

[0111]

[0127] To generate the first immunocontraceptive candidate, a repair plasmid, FHV-GZeG, was constructed containing 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 fluorescent (eGFP) gene, flanked by sequences homologous to the FeHV-1 genome upstream and downstream of the targeted insertion site.

[0112]

[0128] Homologous sequences that allowed the insertion of GnRH, ZP3, and eGFP into the genome of the wild-type (WT) FeHV-1 strain flanked the insertion 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 listed in Table 2. The insert sequence containing the genes for the CMV promoter, GnRH, ZP3, eGFP, and BGH transcription termination region was synthesized by Genscript (Piscataway, USA) and provided as a 3,068-bp insert in the pUC57 vector.

[0113] [Table 2-1]

[0114] [Table 2-2]

[0115]

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

[0116]

[0130] To construct the immunocontraceptive candidates FHV-GZeGTmC and FHV-GZeGTmC2 (Figures 1B and 1C), two repair plasmids (pGEM-T.TmC for FeHV.GZeGTmC and pGEM-T.TmC2 for FeHV.GZeGTmC2) containing the codon-deoptimized TK (SEQ ID NO: 12) and mCherry sequence (SEQ ID NO: 16) flanked by sequences homologous to the FeHV-1 genome upstream and downstream of the TK gene were generated to allow replacement of the wild-type TK gene with the deoptimized TK. The homologous regions 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 selected. Codon de-optimization of the WT TK sequence was generated using the online IDT codon optimization tool (https: / / sg.idtdna.com / CodonOpt). Using the wild-type TK sequence (SEQ ID NO: 10) as the basis for codon de-optimization, the least favored variant was selected for each codon using the red junglefowl (Gallus gallus) as the reference organism.

[0117]

[0131] FHV-GZeGTmC and FHV-GZeGTmC2 differed in the location of mCherry in the sequences. The plasmid used to generate FHV-GZeGTmC contained a deoptimized TK gene followed by a CMV promoter, mCherry, and a BGH transcription termination sequence (see Figure 1B). FHV-GZeGTmC2 contained a deoptimized TK gene fused to mCherry flanked on both sides by regions homologous to the FeHV-1 genome (Figure 1C). The homologous regions were generated by amplification using the primers listed in Table 1. The deoptimized TK gene fused to the mCherry segment was artificially synthesized and joined on both sides with the homologous flanking regions using single-stranded excision PCR as previously described.

[0118] Construction of CRISPR / Cas9 plasmids for use in transfection / infection methods

[0132] For the FHV-GZeG immunocontraceptive, CRISPR / Cas9 guide RNAs (gRNAs) (SEQ ID NO: 1 and SEQ ID NO: 2) were constructed based on the targeted insertion site between UL40 and UL41. For both TK deoptimized variants, gRNAs within the WT TK sequence were selected that were sufficiently different from the deoptimized TK sequence (SEQ ID NO: 17 and SEQ ID NO: 18). CRISPR / Cas9 with guide RNAs specific to the insertion site was incorporated to increase the likelihood that viruses that had not undergone homologous recombination insertion (parental strains) would be targeted for cleavage by CRISPR / Cas9, thereby selecting recombinant FeHV-1 virus particles.

[0119]

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

[0120]

[0134] To generate FHV-GZeG, transfection / infection was first performed using the F2 strain of FeHV-1 from the Feligen vaccine. The immunocontraceptive candidates FHV-GZeGTmC and FHV-GZeGTmC2 were then generated by transfection / infection using FHV-GZeG as the infecting strain. The transfection / infection method described below was performed as described in Russell et al. (2015).

[0121] Development of a feline immunocontraceptive vaccine candidate

[0135] Fluorescence microscopy revealed the expected fluorescence patterns for each of the different viruses (Figures 2 and 3). PCR amplification of the predicted recombination regions of the plaque-purified viruses followed by DNA sequencing demonstrated that sequential plaque purification successfully yielded pure recombinant virus stocks free of wild-type FeHV-1 virus contamination. In all cases, sequencing results showed the sequence expected from successful recombination between the repair plasmid and the FeHV-1 genome, except for a single point mutation in the TK gene of FHV-GZeGTmC2, containing a C-to-T change at nucleotide 69,554, when using KR296657 (SEQ ID NO: 14) as the reference sequence. This resulted in the predicted amino acid change from threonine to methionine (SEQ ID NO: 13).

[0122] Example 2: Growth kinetics of FeHV-1 derived polynucleotide constructs

[0136] One-step growth curves comparing the kinetics of FeHV-1, FHV-GZeG, and FHV-GZeGTmC showed that for all viruses, the virus titer was approximately 1 × 10 at 24–32 h postinoculation. 7 TCID 50 The results showed that the virus titer peaked at 10 / mL and then began to decrease (Fig. 4A). Statistically significant differences in virus titers were observed at 8 and 24 hours after inoculation. At 8 hours after inoculation, the mean FeHV-1 titer (10 2.63 TCID 50 / mL) is the FHVGZeGTmC titer (10 3.6 TCID 50 / mL, P = 0.0379). At 24 hours after inoculation, the FeHV-1 titer (10 8.5 TCID 50 / mL) is the FHV-GZeG titer (10 7.3 TCID 50 / mL, P = 0.0124) and FHV-GZeGTmC titers (10 7.13 TCID 50 / mL, P=0.0038).

[0123]

[0137] In the multi-step growth curve, the highest titers were recorded at 72 or 96 hours after inoculation (Fig. 4B). Significant differences were observed at 48, 72, and 96 hours after inoculation. The mean titers of FHV-GZeGTmC at 48 hours after inoculation (10 2.8 TCID 50 / mL) is FeHV-1(10 4.5 TCID 50 / mL) and FeHV-GZeG (10 4.5 TCID 50 / mL) (p = 0.0057 for both comparisons). The mean titer of FeHV-GZeGTmC2 at 72 hours (10 4 TCID 50 / mL) is FeHV-1(10 5.3 TCID 50 / mL, P = 0.0329) and FHV-GZeG (10 6.3 TCID 50 / mL, P<0.0001), but was significantly lower than both FHV-GZeGTmC (10 4.16 TCID 50 / mL) was significantly lower than that of FHV-GZeG (P = 0.0001). The mean titer of FHV-GZeGTmC at 96 hours after inoculation (10 4.6 TCID 50 / mL) was FHV-GZeG (10 6.5 TCID 50 / mL, P=0.0042).

[0124]

[0138] Differences in cell-to-cell spread among FHV-1, FHV-GZeG, FHV-GZeGTmC, and FHV-GZeGTmC2 were assessed by measuring the area of ​​viral plaques in infected CRFK cells over 3 days (Figure 5). No significant differences were detected among the different virus strains during the first 48 hours. However, at 72 hours postinoculation, the average size of plaques induced by FHV-GZeGTmC2 (0.25 mm2) was 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 of plaques induced by FHV-GZeGTmC (0.27 mm2) was also significantly smaller than those induced by FHV-GZeG at this time point (P = 0.0011).

[0125] Example 3: Transgene and thymidine kinase expression of FHV-1 derived polynucleotide constructs

[0139] Assessment of ZP3 transcript levels in infected cell cultures (Fig. 6A) compared with FeHV-1 immunocontraceptive-infected cells at 2, 4, and 6 h postinoculation. At 4 h postinfection, ZP3 transcript levels in FHV-GZeG-infected cells (103.35 copies / reaction) were significantly lower than those in FHV-GZeGTmC-infected cells (104.14 copies / reaction, P = 0.047) and FHV-GZeGTmC2-infected cells (104.67 copies / reaction, P < 0.001).

[0126]

[0140] Comparison of TK transcript levels in infected cell cultures (Fig. 6B) showed that the transcript level in FHV-GZeGTmC2-infected cells (102.03 copies / reaction) was significantly lower than that in FeHV-1-infected cells (103.19 copies / reaction, P = 0.0425) at 2 h postinoculation. At 4 h postinoculation, the transcript level in FHV-GZeGTmC-infected cells (103.75 copies / reaction) was significantly lower than that in FeHV-1-infected cells (105.05 copies / reaction, P = 0.0078) and FHV-GZeGTmC2-infected cells (104.9 copies / reaction, P = 0.0223).

[0127]

[0141] The protein product of the transgene insertion in each of these viruses was predicted to contain the ZP3-GnRH-eGFP fusion protein. Antibodies to eGFP detected a protein of the expected size (75 kDa) in the cells and supernatant fractions of CRFK cells infected with FHV-GZeG, FHV-GZeGTmC, and FHV-GZeGTmC2 viruses, but this protein was not detected in the cells or supernatant fractions of CRFK cells infected with wild-type FeHV-1 (Fig. 7). Other smaller products reacting with the GFP antibody (likely cleavage products of the ZP3-GnRH-eGFP protein) were also detected in samples of FHV-GZeG, FHV-GZeGTmC, or FHV-GZeGTmC2-infected cells.

[0128] Example 4: Host specificity of FeHV-1 derived polynucleotide constructs

[0142] FeHV-1 has a narrow host range and is thought to be restricted to the feline family. Here, we investigate the species specificity of FeHV-1 and three modified FeHV-1 variants containing antigens targeted by immunocontraceptives. Their ability to replicate in respiratory tissues, cause clinical signs, and induce destruction of reproductive tissues was studied in vivo in a mouse model. Non-feline cell types from a variety of species, including livestock, wildlife, and nonhuman primates, were also investigated for their ability to support FeHV-1 infection.

[0129]

[0143] Non-feline cell lines MDBK (bovine), MDCK (canine), JU56 (wallaby), Ptk1 (rat kangaroo), Vero (African green monkey), LA-4 (mouse), and feline CRFK cells were infected with the immunocontraceptive candidate FHV-GZeG at an MOI of 10 (4-hour incubation) and examined for their ability to support infection by measuring the presence of viral RNA (vRNA), GFP expression, and cytopathic effect (CPE). Cells were observed every 48 hours under an inverted microscope using both light and fluorescence microscopy, and viral growth was detected as evidenced by green fluorescence or CPE compared with uninfected controls. Seven days after inoculation, cells were harvested for extraction and vRNA detection. A second experiment was performed using the cell lines Vero, LA-4, JU56, and CRFK inoculated with FHV-GZeG to quantify viral DNA (vDNA) by qPCR at 0, 3, and 7 days after inoculation.

[0130]

[0144] No evidence of viral infection was detected in any of the non-feline cells inoculated with FHV-GZeG, as determined by the absence of CPE and the absence of cDNA by RT-qPCR using primers amplifying the wild-type (WT) TK gene. No evidence of viral transcripts was detectable 7 days post-infection in any of the non-feline cell lines (Figure 8A). The progression of FHV-GZeG infection in these cell lines, as well as in JU56 and CRFK cells, was analyzed by qPCR. No evidence of FHV-GZeG infection was detected by qPCR using primers amplifying the WT TK gene, and a consistent decline in vDNA was observed from day 0 to day 7 in JU56, Vero, and LA-4 cells (Figure 8B).

[0131] Mouse in vivo study design

[0145] Mice were divided into five groups of 20 mice (10 males and 10 females). On day 0, mice were anesthetized by inhalation of 5% v / v isoflurane in oxygen and inoculated with 50 μl of either FeHV-1, FHV-GZeG, FHV-GZeGTmC, or FHV-GZeGTmC2 virus inoculum at a concentration of 10 TCID / mL or sterile DMEM (mock-infected group). Mice were returned to their respective cages and monitored until they fully recovered from anesthesia. Five mice from each group were euthanized 1, 4, 8, and 14 days after inoculation (Figure 9). After inoculation, mice were observed twice daily for signs of illness during the first week, then once daily during the second week.

[0132]

[0146] An unused portion of the virus inoculum was retained and stored at -70°C. The inoculum was thawed and the TCID 50 Assays were used to titrate and ensure that the inoculation was given at the correct dose.

[0133]

[0147] At 1, 4, 8, and 14 days after inoculation, mice were euthanized by cervical dislocation under deep anesthesia. At 1, 4, 8, and 14 days, postmortem blood and lung samples were collected. Blood samples were centrifuged at 4000 × g for 5 minutes at room temperature to collect serum. Serum was stored at -70°C until needed. Lung samples were minced with a sterile scalpel and then stored in DMEM at -70°C. At 14 days after inoculation, ovaries and testes were collected in paraformaldehyde (4% w / v in PBS).

[0134]

[0148] Lungs from mice inoculated with FeHV-1 and FeHV-1-derived immunocontraceptives were harvested at several time points up to 2 weeks postinfection. 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-GZeGTmC and FHV-GZeGTmC2-inoculated groups), no vDNA was detected in lung homogenates at any time point postinfection in any group (Table 3). A product of the correct size was amplified from mock-infected lung samples spiked with FeHV-1 and from positive control samples. Primers targeting feline GAPDH but capable of amplifying mouse GAPDH (Table 3) amplified an 80-bp product, confirming successful DNA extraction.

[0135]

[0149] Serum samples from mice collected 1, 4, 8, and 14 days later were evaluated for the presence of antibodies to GnRH and FeHV-1. When comparing groups for IgG antibodies to GnRH or FeHV-1, there were no differences in absorbance values ​​between the inoculated group compared to the mock-infected group, as assessed by the Mann-Whitney U test (Figures 10A and 10B). Similarly, there were no detectable differences in absorbance values ​​detecting IgM to FeHV-1 between the inoculated and mock-infected groups, except between FeHV-1-infected and mock-infected mice (Figure 10C).

[0136]

[0150] No evidence of FeHV-1 replication was detected in non-feline cell lines in vivo or in vitro, and no evidence of disruption of reproductive tissues or processes was detected in a mouse model. This finding supports previous findings that FeHV-1 cannot infect outside its host range of cats and further supports its use as a feline-specific viral vector for immunocontraception.

[0137]

[0151] Evaluation of the ovaries of infected mice revealed no significant difference in the total number of follicles in the ovaries of mice vaccinated with the FeHV-1-derived immunocontraceptive compared to mock-infected mice (Figure 11, Figures 12A and 12B). Examination of the testes revealed no obvious morphological changes between groups (Figures 12C and 12D), and the levels of sperm and apoptotic cells were similar; however, the small number of male mice per group at the final time point (n = 2) precluded statistical analysis.

[0138] Example 5: FeHV-1 derived polynucleotide constructs as immunocontraceptive vaccines in cats

[0152] An advantage of the polynucleotide constructs described herein as immunocontraceptive vaccines is their potential ability to modulate transmissibility.

[0139]

[0153] Using the FeHV-1 genome for the insertion of reproductive-related genes, without further modification, the immunocontraceptive candidate FHV-GZeG is expected to retain the potential for horizontal transmission (i.e., infected animals can transmit the FeHV-1 vectored immunocontraceptive to other animals). This is an advantageous feature of a self-disseminating immunocontraceptive in terms of wildlife population control. The non-attenuated immunocontraceptive candidate (FHV-GZeG) can be transmitted from cat to cat and induce a contraceptive effect, providing a self-perpetuating means of population reduction. As shown herein, the modified FeHV-1 exhibits strong feline host specificity and is expected to have no effect on non-feline cells.

[0140] [Table 3]

[0141]

[0154] The transmissibility of FeHV-1 vectored immunocontraceptives is advantageous in remote areas where feral cat control is important, but is undesirable for use in domestic / captive cats. The FHV-GZeGTmC and FHV-GZeGTmC2 candidates, which contain additional disruptions in TK virus virulence genes and optionally other virulence genes, may have reduced or absent horizontal transmission and may be more suitable for use in domestic / captive cats.

[0142]

[0155] A breeding study will be conducted involving cats vaccinated with one of the three polynucleotide constructs, compositions, or immunocontraceptives described herein and sham-treated control cats. Vaccinated and unvaccinated female cats will be housed with fertile males for a period of 4 months to 2 years. Cats will also be examined for adaptive immune responses to reproductive antigens. In females, the ability of the immunocontraceptive to prevent pregnancy will be assessed by the absence or reduced number of offspring. In males, the inability to impregnate unvaccinated females or reduced sperm counts in ejaculates indicates immunocontraceptive efficacy. The infectivity of the FeHV-1 immunocontraceptive spread to "contact" cats in a free-roaming environment can also be examined.

[0143] Example 6: Seroprevalence of FeHV-1 in feral and domestic cats in Victoria

[0156] Understanding FeHV-1 exposure in both wild and domestic cats may help understand the status and risks / benefits of vaccines utilizing FeHV-1 as a vector, such as FeHV-1-vectored immunocontraceptive vaccines.

[0144]

[0157] Serum samples from three separate populations of feral cats in Victoria, Australia, were evaluated for FeHV-1 neutralizing antibodies. Serum was collected from cats in Hatteras (12 cats), Point Cook (69 cats), and Phillip Island (66 cats). Samples were collected from Phillip Island and Hatteras. All serum samples were collected postmortem from feral cats after the animals were humanely euthanized for unrelated reasons between mid-2016 and mid-2021. In addition to the feral cat samples, serum samples from 44 domestic pet cats were obtained from the ASAP Laboratory in Mulgrave, Victoria. These samples were previously sent to the ASAP Laboratory in 2019 from veterinary clinics around Victoria and on the Victoria-New South Wales border.

[0145]

[0158] Briefly, serum was diluted to a final dilution of 1 / 640 and incubated with 100 50% tissue culture infectious doses (100TCID50) of FeHV-1. 96-well trays were incubated at 37°C for 1 hour, after which CRFK cells were added and incubated for an additional 3 days at 37°C in a humidified atmosphere of 5% v / v CO2 in air. Serum titers were recorded as the reciprocal of the highest serum dilution that neutralized the virus. Samples that showed no virus neutralization at any dilution were recorded as having an antibody titer of <5.

[0146]

[0159] Table 1 shows the seropositivity results for various cat populations. Among wild cats, seropositivity results showed that no feral cats in Hatteras had any FeHV-1 VNAb present, whereas feral cats in Point Cook had the highest seropositivity, with FeHV-1 VNAb detectable in 17 of 69 samples (24.6%). Among domestic cats, FeHV-1 VNAb was detectable in 84.1% (37 / 44) of cats (Table 1). There was no significant difference in the proportion of seropositive cats among the three feral cat areas, but all three feral cat populations had significantly lower seropositivity rates compared with domestic cats (P < 0.0001, Fisher's exact test). Neutralizing antibody titers were not significantly different between domestic and wild cats when compared by Mann-Whitney U test (P > 0.05). The weight of Phillip Island cats and the age of domestic cats were examined for correlation with VNAb titers using Pearson's correlation and nonparametric Spearman's correlation. In both cases, no correlation was observed (P = 0.12, P = 0.54). Overall, neutralizing antibody titers were consistently lower than those induced by several other alphaherpesviruses in their respective mammalian hosts.

[0147] [Table 4]

[0148]

[0160] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entireties.

[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.

[0149]

[0162] Throughout this specification, it is the intent to describe preferred embodiments of the present invention without limiting the invention to any one embodiment or particular collection of features. Accordingly, those skilled in the art will appreciate, in light of this disclosure, that various modifications and changes can be made to 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

1. A polynucleotide construct comprising a FeHV feline alphaherpesvirus 1 (FeHV-1) genome modified by inserting 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 one or more feline germ protein antigens.

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

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

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

5. 5. The polynucleotide construct of claim 4, wherein 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. 6. The polynucleotide construct of claim 4 or claim 5, wherein one or more nucleic acid sequences are inserted at a site selected from nucleotides 26100 to 26109 of FeHV-1 GenBank Accession Number KR296657.

7. The modified FeHV-1 genome a. one or more amino acid substitutions; and / or b. Replacing at least one codon in the thymidine kinase (TK) gene with a codon with low translation efficiency in cells 7. The polynucleotide construct according to claim 1, comprising a TK gene modified by

8. 8. The polynucleotide construct of claim 7, wherein the TK gene is modified by replacing each codon with a cellular codon that has low translation efficiency in the cell.

9. 9. The polynucleotide construct according to claim 7 or claim 8, wherein the codon has been replaced with a cellular codon that has low translation efficiency in chicken cells.

10. The polynucleotide construct of 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.

11. The polynucleotide construct according to any one of claims 1 to 10, wherein the modified FeHV-1 genome is not inhibited for growth in feline cells.

12. 12. The polynucleotide construct of claim 11, wherein the modified FeHV-1 genome retains at least some of the horizontal transmission potential of the unmodified FeHV-1 genome.

13. 13. The polynucleotide construct of claim 12, wherein the modified FeHV-1 genome has the same potential for horizontal transmission as the unmodified FeHV-1 genome.

14. 11. The polynucleotide construct of any one of claims 1 to 10, wherein the modified FeHV-1 genome has a reduced likelihood of horizontal transmission when compared to an unmodified FeHV-1 genome.

15. 15. The polynucleotide construct of claim 14, wherein the modified FeHV-1 genome has little or no potential for horizontal transmission when compared to an unmodified FeHV-1 genome.

16. A veterinary composition comprising the polynucleotide construct of any one of claims 1 to 15 and a veterinarily acceptable carrier, excipient, or diluent.

17. An immunocontraceptive vaccine comprising a polynucleotide construct according to any one of claims 1 to 15 or a veterinary composition according to claim 16.

18. 18. The immunocontraceptive vaccine of claim 17, further comprising at least one adjuvant.

19. 19. A method for reducing the reproductive performance of a cat, the method comprising administering to a cat in need thereof a polynucleotide construct according to any one of claims 1 to 15, a veterinary composition according to claim 16, or an immunocontraceptive vaccine according to claim 17 or claim 18.

20. 20. The method of claim 19, wherein the cat is sterile if its fertility is reduced.

21. 19. A method for inducing an immune response to one or more feline reproductive protein antigens in a cat, the method comprising administering to a cat in need thereof a polynucleotide construct according to any one of claims 1 to 15, a veterinary composition according to claim 16, or an immunocontraceptive vaccine according to claim 17 or 18.

22. 19. A method for controlling the population of feral cats, comprising administering a polynucleotide construct according to any one of claims 1 to 15, a veterinary composition according to claim 16, or an immunocontraceptive vaccine according to claim 17 or claim 18 to a feral cat in need thereof.

23. 23. The method of any one of claims 19 to 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 intranasally, intramuscularly, or intraperitoneally to the cat.

24. Use of a polynucleotide construct according to any one of claims 1 to 15, a veterinary composition according to claim 16 in the manufacture of an immunocontraceptive vaccine drug for reducing the fertility of a feline subject.

25. Use of a polynucleotide construct according to any one of claims 1 to 15, or a veterinary composition according to claim 16, in the manufacture of an immunocontraceptive vaccine drug for inducing an immune response against one or more feline reproductive protein antigens in a feline subject.

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