Non-surgical, injectable sterilant for dogs and cats

Abstract

The present disclosure generally relates to methods and compositions for contraception. In embodiments of the invention, vector based vaccine approaches for dog and cat contraception are provided.

Description

[0001] NON-SURGICAL, INJECTABLE STERILANT FOR DOGS AND CATS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. Section 119(e) of copending and commonly-assigned U.S. Provisional Patent Application No. 63 / 664,386, filed June 26, 2024, entitled “THE INVENTION OF A NON-SURGICAL, INJECTABLE STERILANT FOR DOGS AND CATS”, which application is incorporated by reference herein.

[0004] TECHNICAL FIELD

[0005] The invention relates to methods and materials for the inhibition of fertility' in cats and dogs.

[0006] BACKGROUND OF THE INVENTION

[0007] There is an overpopulation of cats and dogs in various regions of the world which creates a health threat to people and pets. Rabid dogs are the main source of rabies deaths in people worldwide. In addition, free-roaming dogs and cats have a negative impact on wild species and the environment.

[0008] The conventional population control for dogs and cats is surgical sterilization via ovariohysterectomy and orchiectomy. However, many reasons why surgical sterilization may not be effective as the sole method for population control exist. It requires anesthesia, medical equipment, a suitable surgical facility, adequate recovery time, and the advanced training of a veterinarian. The cost of surgery is prohibitive for many owners, particularly in developing countries. In addition, many dogs and cats are not owned; thus, shelters and other animal organizations are responsible for financing the surgeries. Neutering these animals involves some method of capturing or trapping the animals, which can be labor-intensive. There is a need in the art for non-surgical methods and materials useful for inhibiting pregnancy in animals such as cats and dogs.

[0009] SUMMARY OF THE INVENTION

[0010] The present disclosure generally relates to methods and compositions for contraception. In embodiments of the invention, vector-based vaccine approaches for dog and cat contraception are provided. Embodiments of the invention disclosed herein include replication defective (E1 / E3 -deleted) human adenovirus type-5 vaccine vectors (or the like) that express the unique beta subunit of canine or feline luteinizing hormone (LH). When administered intramuscularly into the respective animals, this vector can induce an immune response against canine or feline LH that will result in LH beta specific antibodies. The antibodies block LH receptor binding, thereby preventing testosterone production in males and estrogen production and ovulation in females, resulting in infertility in both genders. In this way, embodiments of the invention can be used in methods of inhibiting pregnancy in dogs and cats.

[0011] The invention disclosed herein has a number of embodiments. For example, embodiments of the invention include compositions of matter including an adenoviral vector comprising a polynucleotide encoding a beta subunit of luteinizing hormone (LH), wherein the polynucleotide is disposed in the adenoviral vector in an arrangement of elements such that when the vector is transduced into a mammalian cell, exogenous beta subunit of luteinizing hormone polypeptides are expressed by the mammalian cell. In typical embodiments of the invention, the vector is a replication defective El / E3-deleted human adenovirus type-5 vaccine vector and the polynucleotide encodes a beta subunit of luteinizing hormone (LH) expressed by dogs, cats, horses, donkeys, camels, cows, pigs, sheep, goats, and deer. In certain embodiments of the invention, the composition comprises lipid nanoparticles.

[0012] Embodiments of the invention also include methods of inhibiting fertility in a mammal comprising immunizing the mammal with a composition disclosed herein, wherein immunizing the mammal results in the production of antibodies in the mammal that block LH receptor binding, thereby inhibiting testosterone production in males and estrogen production and ovulation in females such that fertility is inhibited. In certain embodiments of these methods, immunizing consists of a single intramuscular injection. In some embodiments of the invention, the polynucleotide encodes a beta subunit of luteinizing hormone (LH) expressed by dogs, for example, embodiments wherein the polynucleotide encodes at least two immunogenic epitopes present in the polypeptide sequence

[0013] LQGLLLWLLLSVGGVWASRGPLRPLCRPINATLAAENEACPVCITFTTTICAG YCPSMVRVLPAALPPVPQPVCTYHELHFASIRLPGCPPGVDPMVSFPVALSCR CGPCRLSNSDCGGPRAQSLACDRPLLPGLLFL (SEQ ID NO: 1). In other embodiments of the invention, the polynucleotide encodes a beta subunit of luteinizing hormone (LH) expressed by cats, for example embodiments wherein the polynucleotide encodes at least two immunogenic epitopes present in the polypeptide sequence MEMLQGLLLLWLLLLNVGGVWTSREPLRPLCRPINATLAAENEACPVCVTFT TTICAGYCPSMMRVLPAALPPVPQPVCTYRELRFASVRLPGCPPGVDPVVSFP VALSCRCGPCRLSSSDCGGPRAQPLACDRPPLPGLLFL (SEQ ID NO: 2). Typically in these embodiments, the mammal is immunized with the composition of claim 1 a single time, for example, embodiments wherein immunizing consists of a single intramuscular injection. Typically in these methods, the vector is a replication defective El / E3-deleted human adenovirus type-5 vaccine vector and is combined with lipid nanoparticles.

[0014] Another related embodiment of the invention comprises methods of inhibiting fertility in a dog comprising immunizing the dog with a composition of matter including lipid nanoparticles combined with an El / E3-deleted human adenovirus type-5 vaccine vector comprising a polynucleotide encoding an immunogenic epitope in SEQ ID NO: 1, wherein when the vector is transduced into a dog cell, immunogenic peptides of exogenous beta subunit of luteinizing hormone polypeptides are expressed by the cell, thereby creating an immunogen to which an immune response in the dog is generated, thereby inhibiting fertility.

[0015] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1. Cartoon schematic showing parts of the brain, testis and ovaries and Luteinizing hormone (LH), Follicle-stimulating hormone (FSH) and Gonadotropin-releasing hormone (GnRH) release.

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. In the description of the preferred embodiment, reference may be made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and reagent substitutions may be made without departing from the scope of the present invention.

[0020] Fertility' is regulated by a series of hormones. Once released by the hypothalamus, gonadotropin-releasing hormone (GnRH) binds to receptors in the pituitary gland and activates gonadotropic (endocrine) cells to release luteinizing hormone (LH) and follicle stimulating hormone (FSH). FSH and LH then travel through the general circulatory system from the brain to the gonads, where they stimulate the production of gametes and the secretion of sex steroid hormones. In males, LH binding to its receptor in the testes is essential for gonadal testosterone production, a prerequisite for spermatogenesis. In females, LH receptor binding in the ovaries induces estrogen production in the follicles. LH is also responsible for ovulation and for maintenance of pregnancy. Structurally, luteinizing hormone (LH) belongs to a family of glycoprotein hormones that are all composed of an identical a- subunit and a unique P-subunit, coupled by non-covalent interactions. The unique beta subunit endows each hormone with the ability to bind its own receptor. The polypeptide sequences that comprises the unique luteinizing hormone beta subunit of dogs, cats, horses, donkeys, camels, cows, pigs, sheep, goats, deer and others is known in the art and described, for example, in UniProt databases.

[0021] Immune-contraception is the concept of inducing immunity' against an essential component of the reproductive system by vaccination. LH-beta is an essential component of the reproductive system in both males and females. Vaccinating against LH-beta in a way7that results in a strong Th2 antibody response, would block LH by antibody neutralization as soon as it is released into the circulation by the pituitary gland in the brain and uncouple LH-receptor signaling.

[0022] Despite of the induction of long-lasting memory, the strength of an immune response and therefore the effectiveness of any given vaccine (the presence of antibodies in the serum) decreases over time until it ceases to be protective, unless reinfection occurs or booster vaccinations are administered that constitute renewed exposure to antigen. So how can artisans ensure continued antibody presence without a booster vaccination?

[0023] The biology of LH provides a unique solution to this challenge. Both testosterone and estrogen constitute a negative feedback loop that prevents more LH from being secreted by the pituitary. Like vaccinating, spaying and neutering uncouples LH from its receptor, which in turn interrupts the negative feedback loop that prevents more LH from being released into the circulation. In the absence of the gonads, LH is continuously secreted, resulting in high serum LH. In fact, spayed or neutered dogs often develop lymphoma, which has been attributed to their continuous high serum LH levels compared to intact animals (up to 20 times higher). Similar to spaying or neutering, immunization will block LH from reaching its receptor. This continued antigen presence functions as an internal booster that induces secondary immune responses and maintains antibodies at a high level.

[0024] In 1974, Lunnen and colleagues vaccinated 8 male beagle dogs with bovine LH mixed with Freud’s complete adjuvant (FCA) (PMID: 4462837). Four dogs were immunized for a 15-week short-term study and 4 dogs for a 52-week long-term study. The analysis included antibody titers, androgen hormone levels, ejaculate volume, and histology and weight of testes, prostate gland and epididymides. All dogs induced a primary antibody response after vaccination. The antibody response dipped slightly in the weeks following the last booster injection, but then recovered and climbed steadily upward for the duration of the study. Antibody development in individual dogs showed high variability, however all dogs but one maintained high antibody responses.

[0025] The authors recognized that high levels of anti-LH antibodies 271 days after the last booster dose and a year after first immunization in 3 out of 4 dogs in the longterm study may be an indication "that endogenous LH stimulates a secondary, anamnestic production of antibody.£' With other words the observed responses have characteristics of a secondary immune response, where antibody titers begin to decline but then reappear stronger upon renewed antigen encounter (similar to what would be expected after a booster immunization).

[0026] Average androgen levels (measured as testosterone equivalents) in the immunized dogs were similar to those in neutered dogs. Prostate gland, testes and epididymides had reduced weight in immunized animals compared to control animals as well as marked atrophy and fibrosis visible on the histology. The most profound phenotype was a lack of spermatogenesis in the dogs that maintained antibodies.

[0027] In summary, immunization against bovine LH led to sustained high levels of antibodies and caused severe impairment of reproductive function for 52 weeks in three out of four dogs, without any adverse side effects. Similar results were observed in farm animals. Five Heifers, immunized with bovine LH mixed with adjuvant remained acyclic for the entire 2 year-duration of the study, whereas all five control-animals conceived after placement with a bull (PMID: 2262426). Similarly, immunization of adult female sheep (ewe) with LH prohibited pregnancy in 9 / 10 animals during two breeding seasons (PMID: 2513399). Encouraging data were also obtained in male farm animals following LH immunization. Immunizing bull calves with purified LH resulted in the development of LH neutralizing antibodies, lack of masculine development, markedly diminished testosterone concentrations in serum, and testicular repression. Reduced weight gain and lack of development of secondary sexual characteristics were at levels expected of surgical castrates (PMID: 16726059). In summary', preliminary' data in animals suggest that immunizing against LH causes infertility for as long as antibodies are present in both male and female animals.

[0028] The data provides evidence that endogenous LH likely serves as a continuous internal booster to keep antibody titers high over long periods of time by inducing a secondary' memory' immune response. Further aspects of embodiments of the invention are discussed in the sections below.

[0029] The invention disclosed herein has a number of embodiments. For example, embodiments of the invention include compositions of matter including an adenoviral vector comprising a polynucleotide encoding a beta subunit of luteinizing hormone (LH). wherein the polynucleotide is disposed in the adenoviral vector in an arrangement of elements such that when the vector is transduced into a mammalian cell, exogenous beta subunit of luteinizing hormone polypeptides are expressed by the mammalian cell. In ty pical embodiments of the invention, the vector is a replication defective El / E3-deleted human adenovirus type-5 vaccine vector (see, e.g., Small et al., Hum Gene Ther. 2013 Dec 24;25(4):328-338. doi: 10.1089 / hum.2013.216; and US Patent publication US20230022109A1) and the polynucleotide encodes a beta subunit of luteinizing hormone (LH) expressed by dogs, cats, horses, donkeys, camels, cows, pigs, sheep, goats, and deer. In certain embodiments of the invention, the composition comprises lipid nanoparticles.

[0030] Embodiments of the invention also include methods of inhibiting fertility in a mammal comprising immunizing the mammal with a composition disclosed herein, wherein immunizing the mammal results in the production of antibodies in the mammal that block LH receptor binding, thereby inhibiting testosterone production in males and estrogen production and ovulation in females such that ferti li ty is inhibited. In certain embodiments of these methods, immunizing consists of a single intramuscular injection. In some embodiments of the invention, the polynucleotide encodes a beta subunit of luteinizing hormone (LH) expressed by dogs, for example, embodiments wherein the polynucleotide encodes at least two immunogenic epitopes present in the polypeptide sequence

[0031] LQGLLLWLLLSVGGVWASRGPLRPLCRPINATLAAENEACPVC1TFTTTICAG YCPSMVRVLPAALPPVPQPVCTYHELHFASIRLPGCPPGVDPMVSFPVALSCR CGPCRLSNSDCGGPRAQSLACDRPLLPGLLFL (SEQ ID NO: 1). In other embodiments of the invention, the polynucleotide encodes a beta subunit of luteinizing hormone (LH) expressed by cats, for example embodiments wherein the polynucleotide encodes at least two immunogenic epitopes present in the polypeptide sequence MEMLQGLLLLWLLLLNVGGVWTSREPLRPLCRPINATLAAENEACPVCVTFT TTICAGYCPSMMRVLPAALPPVPQPVCTYRELRFASVRLPGCPPGVDPVVSFP VALSCRCGPCRLSSSDCGGPRAQPLACDRPPLPGLLFL (SEQ ID NO: 2). Typically in these embodiments, the mammal is immunized with the composition of claim 1 a single time, for example, embodiments wherein immunizing consists of a single intramuscular injection. Typically in these methods, the vector is a replication defective El / E3-deleted human adenovirus type-5 vaccine vector and is combined with lipid nanoparticles.

[0032] Another related embodiment of the invention comprises methods of inhibiting fertility in a dog comprising immunizing the dog with a composition of matter including lipid nanoparticles combined with an El / E3-deleted human adenovirus type-5 vaccine vector comprising a polynucleotide encoding an immunogenic epitope in SEQ ID NO: 1, wherein when the vector is transduced into a dog cell, immunogenic peptides of exogenous beta subunit of luteinizing hormone polypeptides are expressed by the cell, thereby creating an immunogen to which an immune response in the dog is generated, thereby inhibiting fertility.

[0033] BETA SUBUNIT OF LUTEINIZING HORMONE (LH)

[0034] Luteinizing hormone subunit beta also known as lutropin subunit beta or LHP is a polypeptide that in association with an alpha subunit common to all gonadotropin hormones forms the reproductive signaling molecule luteinizing hormone. See, e.g., OMIM: 152780 MGL 96782; HomoloGene: 81806; and GeneCards: LHB.

[0035] The polypeptide sequences that comprise the immunogenic epitopes of the unique luteinizing hormone beta subunit of dogs, cats, horses, donkeys, camels, cows, pigs, sheep, goats, deer and others is known in the art and described, for example, in UniProt databases. Illustrating this, the unique luteinizing hormone beta subunit polypeptide sequence in dogs has UniProt designation “Pl 8842 • LSHB CANLF'’ (LQGLLLWLLLSVGGVWASRGPLRPLCRPINATLAAENEACPVCITFTTTICAG YCPSMVRVLPAALPPVPQPVCTYHELHFASIRLPGCPPGVDPMVSFPVALSCR CGPCRLSNSDCGGPRAQSLACDRPLLPGLLFL (SEQ ID NO: 1)), while the unique luteinizing hormone beta subunit of cats has UniProt designation “077805 • LSHB FELCA ’’(MEMLQGLLLLWLLLLNVGGVWTSREPLRPLCRP1NATLAAE NEACPVCVTFTTTICAGYCPSMMRVLPAALPPVPQPVCTYRELRFASVRLPGC PPGVDPVVSFPVALSCRCGPCRLSSSDCGGPRAQPLACDRPPLPGLLFL (SEQ ID NO: 2)).

[0036] ADENOVIRUS VACCINE VECTORS

[0037] Subunit vaccines that consist of purified protein mixed with adjuvant are expensive to produce and less effective and immunogenic compared to vaccine vectors. Virus vectors based on El-deleted / E3-deficient adenoviruses such as human serotype 5 (hAd5) are highly immunogenic. Adenovirus vectors are also efficient in expressing encoded transgenes and induce strong humoral and cellular immune responses after intramuscular injection. There is no pre-existing human adenovirus immunity’ in dogs and cats. They are easily up-scaled to high titers, cost-effective to produce due to their stability, and have been FDA approved.

[0038] Adenovirus vaccine vectors have been successfully used or tested in primates, humans, farm animals, and in dogs and cats. A few examples are summarized in the following section.

[0039] Adenovirus Vaccine Vectors in Animals and Humans

[0040] Primates vaccinated intramuscularly with a single moderate dose of a chimpanzee adenovirus vector (AdC68rab.gp) expressing the rabies virus glycoprotein all survived a rabies challenge 22 months later (PMID: 24503087). The Johnson & Johnson coronavirus vaccine for use in humans is based on a human adenovirus vaccine vector. A single dose protects against death related to covid- 19 98-100% of the time. A chimpanzee adenovirus was used as a vaccine against rift valley fever virus (RVFV). A single intramuscular dose of the virus without adjuvant elicited high titer RVFV neutralizing antibody that solidly protected sheep, goats, and cattle against lethal RVFV challenge 4 weeks after immunization, while all sheep goats and cattle in the placebo group developed viremia (PMID: 26847478).

[0041] A chimpanzee Ad vector expressing rabies virus glycoprotein was used to immunize beagle dogs. All dogs that were immunized intramuscularly developed high neutralizing and total anti rabies IgG antibody two weeks after immunization, for both the high and the low dose groups. Two low dose animals and two control animals were challenged with rabies virus at 12 weeks (4 weeks after the booster immunization). The boost effect increased for 2 weeks and then declined to similar levels compared to the prime group at the time of the challenge. However, vaccinated animals survived the challenge with no signs of rabies, whereas control animals became symptomatic and had to be euthanized (PMID:31394410).

[0042] Five cats injected with a simian adenovirus serotype-23 vector expressing the spike (S) protein of SARS CoV-2 to immunize them against COVID-19, were analyzed for neutralizing antibody titers in cat serum over the course of 10 weeks. The neutralization of a pseudo virus by cat sera at week 10 was ~7-fold higher than the highest titers from immunized rhesus macaques, ~5- fold higher than neutralizing titers from COVID patients and ~60-fold higher than sera from vaccinated humans who received a COVID vaccine 2 or 4 weeks earlier (PMID:35756078).

[0043] In summary, vaccinating against endogenous luteinizing hormone (LH) with a single intramuscular injection of a replication-defective adenovirus vector expressing the beta subunit of feline or canine LH, will result in long lasting infertility in both male and female cats and dogs. In this context, embodiments of the injection provide an injectable sterilant comprising a vector expressing immunogenic polypeptides of a canine or feline LH beta subunit. Conventional safety and efficacy studies can be used to analyze the immunologic responses, reproductive functions and potential side effects (toxicity) in cats and dogs.

[0044] All publications mentioned herein are incorporated by reference to disclose and describe aspects, methods and / or materials in connection with the cited publications. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art.

[0045] Unless otherwise defined, all terms of art. notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. CONCLUSION

[0046] This concludes the description of the preferred embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.

Claims

CLAIMS:

1. A composition of matter including an adenoviral vector comprising a polynucleotide encoding a beta subunit of luteinizing hormone (LH), wherein the polynucleotide is disposed in the adenoviral vector in an arrangement of elements such that when the vector is transduced into a mammalian cell, exogenous beta subunit of luteinizing hormone polypeptides are expressed by the mammalian cell.

2. The composition of claim 1, wherein the vector is a replication defective El / E3-deleted human adenovirus type-5 vaccine vector.

3. The composition of claim 1, wherein the polynucleotide encodes a beta subunit of luteinizing hormone (LH) expressed by dogs, cats, horses, donkeys, camels, cows, pigs, sheep, goats, and deer.

4. The composition of claim 1, wherein the composition comprises lipid nanoparticles.

5. A method of inhibiting fertility in a mammal comprising immunizing the mammal with a composition of claim 1, wherein immunizing the mammal results in the production of antibodies in the mammal that block LH receptor binding, thereby inhibiting testosterone production in males and estrogen production and ovulation in females such that fertility is inhibited.

6. The method of claim 5. wherein immunizing consists of a single intramuscular injection.

7. The method of claim 5, wherein the polynucleotide encodes a beta subunit of luteinizing hormone (LH) expressed by dogs.

8. The method of claim 5, wherein the polynucleotide encodes at least two immunogenic epitopes present in the polypeptide sequence LQGLLLWLLLSVGGVWASRGPLRPLCRPINATLAAENEACPVCITFTTTICAG YCPSMVRVLPAALPPVPQPVCTYHELHFASIRLPGCPPGVDPMVSFPVALSCR CGPCRLSNSDCGGPRAQSLACDRPLLPGLLFL (SEQ ID NO: 1).

9. The method of claim 5, wherein the polynucleotide encodes a beta subunit of luteinizing hormone (LH) expressed by cats.

10. The method of claim 5, wherein the polynucleotide encodes at least two immunogenic epitopes present in the polypeptide sequence MEMLQGLLLLWLLLLNVGGVWTSREPLRPLCRP1NATLAAENEACPVCVTFT TTICAGYCPSMMRVLPAALPPVPQPVCTYRELRFASVRLPGCPPGVDPVVSFP VALSCRCGPCRLSSSDCGGPRAQPLACDRPPLPGLLFL (SEQ ID NO: 2).

11. The method of claim 8. wherein the mammal is immunized with the composition of claim 1 a single time.

12. The method of claim 5, wherein immunizing consists of a single intramuscular injection.

13. The method of claim 11, wherein the vector is a replication defective E1 / E3- deleted human adenovirus type-5 vaccine vector.

14. The method of claim 13, wherein the composition comprises lipid nanoparticles.

15. A method of inhibiting fertility in a dog comprising immunizing the mammal with a composition of matter including lipid nanoparticles combined wi th an E1 / E3- deleted human adenovirus type-5 vaccine vector comprising a polynucleotide encoding an immunogenic epitope in SEQ ID NO: 1, wherein when the vector is transduced into a dog cell, immunogenic peptides of exogenous beta subunit of luteinizing hormone polypeptides are expressed by the cell, thereby creating an immunogen to which an immune response in the dog is generated, thereby inhibiting fertility.

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