Patent Application
20170216418
The present invention relates to an immunogenic LHRH composition, and in particular relates to an immunogenic LHRH composition containing LHRH comprising peptide constructs that leads to functional suppression of LHRH level in pigs resulting in effective immunocastration, removal of boar taint and growth enhancement in pigs.
Gonadotropin-releasing hormone (GnRH), also known as Luteinizing-hormone-releasing hormone (LHRH), is a trophic peptide hormone responsible for the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary. LHRH is synthesized and released from neurons within the hypothalamus.
Vaccination against the hypothalamic hormone Luteinizing-hormone-releasing-hormone (LHRH) has been demonstrated as an immunological method of controlling reproduction since the early 1970's. Eliciting an immune response to LHRH prevents the release from the anterior pituitary of the hormones LH and FSH, which are required for the development and maintenance of the gonads—the testes in the male and ovaries in the female. Thus reduction of LH and FSH levels leads to loss of reproductive function.
De-sexing (or neutering) operations are the most widely practiced surgical procedures in veterinary medicine and livestock animal management. A significant proportion of both sexes of domestic livestock and companion animals are routinely surgically de-sexed to prevent a variety of undesirable characteristics which accompany sexual maturity.
Immunological blocking of LHRH action can result in infertility in animals because LHRH controls testosterone production, which, in turn regulates the development of sperm and estrogen production, in turn causing the ripening of ova. Moreover, LHRH-based immunotherapy provides a means for contraception in male and female companion animals (e.g. dogs, cats, horses and rabbits) as well as mitigating undesirable androgen-driven behavior such as heat, territorial marking and aggression. Such process is reversible depending on the level of serum antibodies in treated animals upon immunization with LHRH-based immunotherapy. Lastly, immunological castration (e.g. antibody-based inhibition of LHRH action) has an additional application in the meat animal industry. Males are not processed into prime cuts of meat because of the offensive aroma and taste associated with their flesh as a result of circulating testosterone (e.g. boar taint). Since mechanical (e.g. surgical) castration of male food animals is no longer considered humane, immunological castration provides an acceptable alternative to this practice.
Several immunogenic forms of LHRH have been tested. For example, LHRH peptide has been conjugated to carrier protein(s) to enhance the peptide hormone's immunopotency. However, these protein carriers are too expensive for large scale use and the resultant peptide-protein conjugates are not as effective to (1) yield immunocastration over a long duration nor are they able to (2) generate anti-LHRH immune response in all animals, both conditions are required for an efficacious vaccine as a replacement for surgical castration.
Further, effective immunization with LHRH, a non-immunogenic 10-mer peptide, depends on the conjugation site between LHRH and the carrier protein(s). Moreover, protein linkage to LHRH is problematic as an immunogen because the majority of immune responses toward such an immunogen are directed to the large carrier protein(s) rather than to the LHRH peptide (the mass of the toxins or other carrier proteins are far larger than that of LHRH, a 10-mer peptide). This phenomenon frequently leads to carrier protein-induced epitopic immune suppression. Accordingly, an immune enhancer that is suitable for linkage to the LHRH peptide yielding inexpensive peptide construct for use as the key ingredient in a vaccine formulation capable of stimulating an early and strong immune response to the master hormone LHRH for multiple applications in immunocastration shall be sought. Likewise this immune enhancer also should avoid carrier-induced epitopic suppression. Therefore, an LHRH vaccine which has consistently high efficacy for immunocastration including (1) specific applications for removal of boar taint along with enhanced growth profile in pigs; and (2) for behavior modification to allow ease in animal management in cattle; is still highly desired to resolve current deficiencies in LHRH based immunocastration, in particular in pigs.
The invention provides a vaccine composition for pig castration, comprising a peptide immunogen and a veterinarily acceptable delivery vehicle or adjuvant, wherein the peptide immunogen comprises (a) an LHRH peptide of SEQ ID NO: 1, and (b) at least one T helper epitope or an immunostimulatory element selected from a group consisting of SEQ ID NOs: 2, 3, 4, 5, and 6, wherein the peptide of LHRH can be covalently linked through its N-terminus with the T helper epitope and/or an immunostimulatory element by a spacer sequence of Gly-Gly or εNLys. The spacer “εNLys” is a lysine residue present between two amino acids that is bound to (1) the C-terminus of the preceding amino acid at the ε-NH2 group of the lysine residue and (2) the N-terminus of the following amino acid at the C-terminus of the lysine residue. The terms “εNLys” and “εLys” and “εK” can be used interchangeably.
In another embodiment, the peptide immunogen of the present invention comprises SEQ ID NOs: 7, 8, 9, or 10, or a mixture thereof, and the veterinarily acceptable adjuvant is selected from a group consisting of ISA50V2 and Emulsigen D. The total amount of the peptide immunogen is more than 6.25 μg per dose, preferably, 50 μg to 200 μg.
Another aspect of the present invention relates to a method for inhibiting characteristics induced by the sexual maturation of pigs, comprising administering an effective amount of a vaccine composition of the present invention to pigs to reduce the production of testosterone and its derivatives such as dihydrotestosterone and estrogen in the immunized host. The characteristics include, but are not limited to, boar taint, sexual activity, fertility, and estrous behavior. The method of the present invention inhibits boar taint and the growth of testes or epididymides.
In one embodiment, the vaccine composition is administered by intramuscular injection into male pigs in a two-dose series with the first dose of the vaccine composition being applied to the pig at as early as 3 weeks of age with the second shot as a boost from 10 to 16 weeks or older, leading to effective immunocastration, enhanced growth, and removal of boar taint about two weeks after the boost.
Detailed description of the invention is given in the following embodiments with reference to the accompanying drawings.
The following description is of the best-contemplated mode to carry out the invention. This description is for purpose of illustrating the general principles of the invention and should not be taken in a limiting sense. The scope of the invention is best determined by reference to the appended claims.
The present invention provides peptides having LHRH, a 10-mer peptide linked through its N-terminus to a co-stimulatory or helper T cell epitope (Th epitope).
The term “LHRH” refers to Luteinizing-Hormone-Releasing Hormone (GenBank: AAB34379.1), which is a trophic peptide hormone responsible for the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary. The term LHRH used in this disclosure includes homologues of LHRH derived from different phyla including birds, fish, reptiles and invertebrates. The peptides of LHRH preferably comprise an amino acid sequence of SEQ ID NO: 1 as shown in Table 1.
The term “vaccine” is a general clinical term to describe an immunostimulatory composition that leads to the production of antibodies in a subject that has been exposed to the vaccine. From a clinical perspective, subjects that have been administered vaccine compositions will generate antibodies to the antigen present in the vaccine. Vaccines compositions described in the present application are not limited to biological preparations that improve immunity against a particular disease or pathogen.
The term “helper T cell epitope (Th epitope)” of the invention refers to Th epitopes derived from foreign pathogens including but not limited to, as examples, hepatitis B surface (HBsAg) and core antigen (HBc) helper T cell epitopes, pertussis toxin helper T cell epitopes (PT Th), tetanus toxin helper T cell epitopes (TT Th), measles virus F protein helper T cell epitopes (MVF Th), Chlamydia trachomatis major outer membrane protein helper T cell epitopes (CT Th), diphtheria toxin helper T cell epitopes (DT Th), Plasmodium falciparum circumsporozoite helper T cell epitopes (PF Th), Schistosoma mansoni triose phosphate isomerase helper T cell epitopes (SM Th), Escherichia coli TraT helper T cell epitopes (TraT Th). The pathogen-derived Th selected here as representative examples of promiscuous Th were listed as SEQ ID NOs: 2-9 and 42-52 in U.S. Pat. No. 5,759,551 and are incorporated herein by reference.
Useful Th epitopes may also include combinatorial Th epitopes. In Wang et al. (WO 95/11998), a particular class of combinatorial Th epitopes, a “Structured Synthetic Antigen Library” (SSAL) was described. Th SSAL epitopes comprise a multitude of Th epitopes with amino acid sequences organized around a structural framework of invariant residues with substitutions at specific positions. The sequences of the SSAL are determined by retaining relatively invariant residues while varying other residues to provide recognition of the diverse MHC restriction elements. This may be accomplished by aligning the primary amino acid sequence of a promiscuous Th, selecting and retaining as the skeletal framework, the residues responsible for the unique structure of the Th peptide, and varying the remaining residues in accordance with known MHC restriction elements. Lists of the invariant and variable positions with the preferred amino acids of MHC restriction elements are available to obtain MHC-binding motifs. These may be consulted in designing SSAL Th epitopes (Meister et al., Vaccine, 1995; 13:581-591). In one embodiment, the Th epitope includes SEQ ID NOs: 2, 3, 4, and/or 5 as shown in Table 1.
SEQ ID No: 6, refers to a peptide fragment derived from the invasin protein of the pathogenic bacteria Yersinia spp., a microbial outer membrane protein which mediates entry of the bacteria into mammalian cells Invasin of cultured mammalian cells by the bacterium was demonstrated to require interaction between the Yersinia invasin molecule and several species of the β1 family of integrins present on the cultured cells. Since T lymphocytes are rich in β1 integrins (especially activated immune or memory T cells), and the demonstrated T cell co-stimulatory properties associated with this invasin domain, it can be linked to promiscuous Th epitope comprising LHRH constructs to further enhance the immunogenicity of a designed peptide immunogen (U.S. Pat. No. 6,025,468).
The peptides of the invention include at least one LHRH, a Th epitope and optionally a co-stimulatory invasin peptide domain. The LHRH peptide (including homologues thereof) can be covalently linked through its N-terminal amino acids with a spacer to a peptide containing at least one sequence known to contain a Th epitope. The Th epitope can be covalently linked through its N-terminus to the co-stimulatory invasion peptide domain. The spacer includes, but is not limited to, Gly-Gly, Lys, εNLys, εNLys-(Lys)n where n=1 to 3, or Lys-Lys-Lys-εNLys (SEQ ID NO: 11), etc.
The peptides of the invention have from about 20 to about 100 amino acid residues, preferably from about 20 to about 70 amino acid residues and more preferably from about 25 to about 50 amino acid residues. In another preferred embodiment, the peptide has from about 27 to about 45 amino acid residues.
The number of Th epitope includes, but are not limited to, one, two, three, four, or more of the Th peptides as shown in Table 1 the invention. The peptides of the invention comprise at least one peptide of Th epitopes selected from SEQ ID NOs: 2, 3, 4, 5 which can be optionally linked to the N-terminus of the LHRH peptide of SEQ ID NO: 1. In one embodiment, Th epitopes may be linked to the N-terminus of LHRH to form a peptide of the invention. For example, the peptides of SEQ ID NOs: 2 and 3 may be sequentially linked to the N-terminus of LHRH by spacers (Gly-Gly, Lys, or εNLys). One skilled in the art would change the number of the Th epitope and kinds of spacers linked to LHRH peptide immunogen to obtain an optimal peptide of the invention, if necessary.
In another embodiment, the peptides of the invention comprise SEQ ID NOs: 7, 8, 9, and/or 10 as shown in Table 2, or having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity thereto.
The peptides of this invention can be made by synthetic chemical methods which are well known to the ordinarily skilled artisan; See, for example, Grant, ed. (1992) Synthetic Peptides: A User's Guide, W.H. Freeman & Co., New York, N.Y., pp. 382. Hence, peptides can be synthesized using the automated solid phase synthesis with either t-butyloxycarbonyl (t-Boc) or 9-fluorenyl-methyloxycarbonyl (F-moc) chemistry on an Applied Biosystems Peptide Synthesizer Model 430A or 431. To synthesize a poly lysine core moiety, unprotected Di(t-Boc) or Di(F-moc)-Nα, Nε) lysine residues are used in place of t-Boc or F-moc with a protected ε-amino group. To improve the solubility of a designed peptide, the peptide can be elongated with additional serine or/and lysine residues at the N-terminus. After complete assembly of the desired peptide, the resin is treated according to standard procedures to cleave the peptide from the resin and deblock the protecting groups on the amino acid side chains. The free peptide is purified by HPLC and characterized biochemically. Alternatively, the longer linear peptides can be synthesized by well known recombinant DNA techniques. Any standard manual on DNA technology provides detailed protocols to produce the peptides of the invention.
The peptide of the invention when used as the key ingredient in a vaccine formulation can yield immunocastration including loss of the physical and/or chemical characteristics associated with sexual maturity such as sexuality, fighting, wandering, aggressive sexual behavior, unwanted organoleptic characteristics, tumors of reproductive organs and pregnancy, oestrous cycling, fertility, pregnancy, and tumors of the reproductive organs in an immunized animal. Accordingly, inhibiting the characteristics includes inhibiting sexual activity (for example, preventing male cattle mounting other male cattle), preventing or delaying ovulation, shrinking the testes and epididymides, reducing testosterone concentration, reducing aggressive behavior or reducing unwanted organoleptic characteristics such as boar taint.
The peptides of the invention are formulated for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as herein disclosed. A unit dosage form can, for example, contain the principal peptide antigen in amounts ranging from 0.5 μg to about 2,000 μg, generally, preferably, more than 6.25 μg, most preferably, 25 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100, 150 or 200 μg. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
This invention also provides compositions comprising pharmaceutically acceptable delivery systems for the administration of the peptide immunogens. The compositions comprise an immunologically effective amount of at least one peptide of this invention. The number of the peptide of the invention includes, but is not limited to, one, two, three, four, or more in a composition (vaccine) of the invention. For example, the composition of the invention comprises SEQ ID No: 7, 8, 9, 10, or a mixture thereof. In one embodiment, the composition of the invention includes a single peptide of SEQ ID No: 7, 8, 9, or 10. In another embodiment, the composition of the invention includes SEQ ID Nos: 7, 8, and 9 which is referred to LHRH3 in the following examples. In another embodiment, the composition of the invention includes SEQ ID No: 10 which is referred to LHRH1 in the following examples. When so formulated, the compositions of the present invention comprising LHRH or a homologue thereof as target antigenic site, are used for prevention/removal of boar taint, enhanced growth profile, immunocastration of pigs, and for contraception in males and females.
The peptide immunogens of the invention can be formulated as immunogenic compositions using adjuvants, emulsifiers, pharmaceutically-acceptable carriers or other ingredients routinely provided in vaccine compositions.
Adjuvants or emulsifiers which can be used in this invention include alum, incomplete Freund's adjuvant (IFA), liposyn, saponin, squalene, L121, emulsigen, monophosphoryl lipid A (MPL), QS21, and ISA 720, ISA 50, ISA 50V2, ISA 35 or ISA 206 as well as the other efficacious adjuvants and emulsifiers. The formulations are readily determined by one of ordinary skill in the art and also include formulations for immediate release and/or for sustained release. The present vaccines can be administered by any convenient route including subcutaneous, oral, intramuscular, intraperitoneal, or other parenteral or enteral route. Similarly the immunogens can be administered in a single dose or multiple doses. Immunization schedules are readily determined by the ordinarily skilled artisan.
In a particular embodiment, the delivery vehicle and adjuvant is Montanide™ ISA 50V2 (an oil vaccine adjuvant composition comprised of vegetable oil and mannide oleate for production of water-in oil (i.e. w/o) emulsions), Tween® 80 (also known as: Polysorbate 80 or Polyoxyethylene (20) sorbitan monooleate), a CpG oligonucleotide, and/or any combination thereof. In another embodiment, the pharmaceutical composition is a water-in-oil-in-water (i.e. w/o/w) emulsion with Emulsigen or Emulsigen D as the adjuvant. Also provided are other ingredients routinely incorporated with vaccine formulations, and instructions for dosage such that a balanced B and T cell immune response is generated.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption or delayed release of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate or/and gelatin.
The invention also provides a method for inducing the titer of anti-LHRH antibodies, decrease of testosterone concentration, prevention of boar taint, shrinkage of testes and epididymides, immunocastration of pigs, and for contraception by administering the subject peptide compositions to the mammals for a time.
Generally, the method of the invention can induce the titer of anti-LHRH antibodies in a pig, and the titer of anti-LHRH antibodies is more than 2.0, preferably, 2.5, or 3.0 (Log10) after one or two vaccinations. The method of the invention also can suppress the testosterone concentration in pigs, and the testosterone concentration is less than 2.5 nmol/L, preferably, 2.0, 1.87, or 1.0 nmol/L after one or two vaccinations. Further, the method of the invention can increase the body weight of pigs, preferably with an increase by 10% or more.
The compositions of the invention may be administered by any suitable method known in the art, including, but not limited to, oral administration or by injection, preferably, intramuscular injection.
The composition of the instant invention contains an effective amount of one or more of the peptide immunogens of the present invention and a pharmaceutically acceptable carrier. Such a composition in a suitable dosage unit form generally contains about 0.5 μg to about 1 mg of the peptide immunogen per kg body weight. When delivered in multiple doses, it may be conveniently divided into an appropriate amount per dose. For example, the dose, e.g. 6.25 μg to 200 μg; preferably 50 μg, may be administered by injection, preferably intramuscularly. This may be followed by repeat (booster) doses. Dosage will depend on the age, weight and general health of the subject as is well known in the vaccine and therapeutic arts.
The invention also provides a method for inhibiting characteristics induced by the sexual maturation of pigs, comprising administering an effective amount of a vaccine composition of the invention.
The number and timing of doses are not limited in the method of the invention. Generally, the method of the invention includes at least one dose, preferably, two, three, four, five doses or more, preferably, two doses or more. One skilled in the art would easily know to increase the number of does to induce the efficiency. The first dose can be delivered to pigs at any age (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks old). The time points of vaccination can be changed or modified depending on different types of pigs, dosage, adjuvant, and administration method.
Generally, prime vaccination (first dose) is done after 3, preferably 3 to 8, weeks of age. A boost vaccination (second dose) is preferably given between 3 and 13 weeks after the priming. The standard scheme would have 3 to 13 weeks between priming and boosting.
In one embodiment, a preferred vaccination schedule would be the following: the priming would be given about 8 weeks of age, the booster would be given about 16 weeks of age, and in cases where pigs are slaughtered after 6 to 8 weeks of booster.
The prime/boost vaccination schedule of the invention is suitable for pigs. Depending on the production methods used in different countries, in many cases pigs may not reach the age of 21 weeks.
Specific embodiments of the present invention include, but are not limited to, the following:
(1) A vaccine composition for castrating pigs, comprising a peptide immunogen and a veterinarily acceptable delivery vehicle or adjuvant, wherein the peptide immunogen comprises
(a) a LHRH peptide of SEQ ID NO: 1, and
(b) at least one T helper epitope selected from a group consisting of SEQ ID NOs: 2, 3, 4, and 5, and, optionally, an immunostimulatory peptide of SEQ IN NO:6, wherein the LHRH peptide is covalently linked through its N-terminus residue to the T helper epitope.
(2) The vaccine composition according to (1), wherein the peptide of LHRH, T helper epitope, and, optionally, an immunostimulatory peptide, is linked by Gly-Gly, εNLys, Lys-εNLys, Lys-Lys-εNLys, Lys-Lys-Lys-εNLys (SEQ ID NO: 11), εNLys-Lys-Lys, or εNLys-Lys-Lys*-Lys (SEQ ID NO: 12).
(3) The vaccine composition according to (1), wherein the peptide immunogen comprises SEQ ID NOs: 7, 8, 9, and/or 10, or a mixture thereof.
(4) The vaccine composition according to (1), wherein the veterinarily acceptable adjuvant comprises ISA50, ISA50V2, or Emulsigen D.
(5) The vaccine composition according to (1), wherein the total amount of the peptide immunogen is about 12.5 μg to 200 μg per dose.
(6) A method for inhibiting characteristics induced by the sexual maturation of a pig, comprising administering an effective amount of a vaccine composition of claim 1 to a pig.
(7) The method according to (6), wherein the characteristic comprises boar taint, sexual activity, sexuality, fertility, and estrous behavior.
(8) The method according to (6), wherein the vaccine composition is administered by intramuscular or subcutaneous injection.
(9) The method according to (6), wherein first dose of the vaccine composition is applied to the pig at the age of 3 to 8 weeks old.
(10) The method according to (6), wherein the second dose of the vaccine composition is applied to the pig at the age of 6 to 16 weeks old.
Additional specific embodiments of the present invention include, but are not limited to the following:
(1) A veterinary composition comprising:
(a) a peptide immunogen selected from the group consisting of
(b) a veterinarily acceptable delivery vehicle or adjuvant.
(2) The veterinary composition according to (1), wherein the peptide immunogen in (a) is (i) a mixture of SEQ ID NOs: 7, 8, and 9.
(3) The composition according to (2), wherein the veterinarily acceptable adjuvant comprises ISA50, ISA50V2 or Emulsigen D.
(4) The composition according to (2), wherein the total amount of the peptide immunogen is about 12.5 μg to 200 μg per dose.
(5) A method for inhibiting characteristics induced by the sexual maturation of a pig, comprising administering an effective amount of the composition of (2) to a pig.
(6) The method according to (5), wherein the characteristics comprise boar taint, sexual activity, sexuality, fertility, and estrous behavior.
(7) The method according to (5), wherein the composition is administered by intramuscular or subcutaneous injection.
(8) The method according to (5), wherein first dose of the composition is applied to the pig at the age of 3 to 8 weeks old.
(9) The method according to (5), wherein the second dose of the composition is applied to the pig at the age of 6 to 16 weeks old.
(10) A method for reducing the production of testosterone and its derivatives in an animal comprising administering an effective amount of the composition of (2) to the animal.
(11) The veterinary composition according to (1), wherein the peptide immunogen in (a) is (ii) SEQ ID NO: 10.
(12) The composition according to (11), wherein the veterinarily acceptable adjuvant comprises ISA50, ISA50V2 or Emulsigen D.
(13) The composition according to (11), wherein the total amount of the peptide immunogen is about 12.5 μg to 200 μg per dose.
(14) A method for inhibiting characteristics induced by the sexual maturation of a pig, comprising administering an effective amount of the composition of (11) to a pig.
(15) The method according to (14), wherein the characteristics comprise boar taint, sexual activity, sexuality, fertility, and estrous behavior.
(16) The method according to (14), wherein the composition is administered by intramuscular or subcutaneous injection.
(17) The method according to (14), wherein first dose of the composition is applied to the pig at the age of 3 to 8 weeks old.
(18) The method according to (14), wherein the second dose of the composition is applied to the pig at the age of 6 to 16 weeks old.
(19) A method for reducing the production of testosterone and its derivatives in an animal comprising administering an effective amount of the composition of (11) to the animal.
Methods for synthesizing LHRH related peptide constructs that were included in the development effort for an efficacious targeting LHRH vaccine design and formulation are described below. The peptides can be synthesized in small-scale amounts, which are useful for laboratory pilot and field studies, as well as large-scale (kilogram) amounts, which are useful for industrial/commercial production of vaccine formulations and serological assays.
A large repertoire of LHRH related antigenic peptides having sequences with lengths from approximately 10 to 40 amino acids were designed for the screening and selection of the most optimal peptide constructs for use in an efficacious LHRH vaccine. Each construct contains an LHRH peptide (SEQ ID NO: 1) synthetically links to a carefully designed helper T cell (Th) epitope or an immunostimulatory peptide, identified in Table 1 (SEQ ID NOs: 2 to 6). The LHRH peptides used in the LHRH vaccine of the invention are SEQ ID NOs: 7 to 10.
All peptides used for immunogenicity studies or related serological tests for detection and/or measurement of anti-LHRH antibodies were synthesized on a small scale using Fmoc chemistry by peptide synthesizers of Applied BioSystems Models 430A, 431 and/or 433. Each peptide was produced by an independent synthesis on a solid-phase support, with Fmoc protection at the N-terminus and side chain protecting groups of trifunctional amino acids. Completed peptides were cleaved from the solid support and side chain protecting groups were removed by 90% Trifluoroacetic acid (TFA). Synthetic peptide preparations were evaluated by Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) Mass Spectrometry to ensure correct amino acid content. Each synthetic peptide was also evaluated by Reverse Phase HPLC (RP-HPLC) to confirm the synthesis profile and concentration of the preparation.
Despite rigorous control of the synthesis process (including stepwise monitoring the coupling efficiency), peptide analogues were also produced due to unintended events during elongation cycles, including amino acid insertion, deletion, substitution, and premature termination. Thus, synthesized preparations typically included multiple peptide analogues along with the targeted peptide. Despite the inclusion of such unintended peptide analogues, the resulting synthesized peptide preparations were nevertheless suitable for use in immunological applications including immunodiagnosis (as antibody capture antigens) and vaccination (as peptide immunogens). Typically, such peptide analogues, either intentionally designed or generated through synthetic process as a mixture of byproducts, are frequently as effective as a purified preparation of the desired peptide, as long as a discerning QC procedure is developed to monitor both the manufacturing process and the product evaluation process to guarantee the reproducibility and efficacy of the final product employing these peptides. Large scale peptide syntheses in the multi-hundred to kilo gram quantities were conducted on a customized automated peptide synthesizer UBI 2003 at 15 mmole to 50 mmole scale according to the same solid phase peptide synthesis principle.
For active ingredients used in the final vaccine formulations for field trials, LHRH peptide constructs were purified by preparative RP-HPLC under a shallow elution gradient and characterized by MALDI-TOF mass spectrometry, amino acid analysis and RP-HPLC for purity and identity.
A mixture of three LHRH peptide immunogens (LHRH3: SEQ ID NOs: 7, 8 and 9) and a single peptide immunogen (LHRH1: SEQ ID NO: 10) were formulated respectively in an water in oil (W/O) emulsion delivery system using an ISA adjuvant from Seppic (France) or in a water in oil in water (W/O/W) Emulsigen D from MVP (USA). Briefly, peptides in saline solution (20% w/v NaCl solution) were combined in equal molar ratios, filtered aseptically (with 0.22 micron filter) and then mixed with delivery vehicle ISA50V2 or Emulsigen D adjuvant through homogenization. The formulation processes were monitored throughout for viscosity. Final products were characterized by identity test, physical test, and sterility test. All formulation, filling and packaging procedures were performed in a clean room to maintain the sterile condition.
A total of 40 boars at 8 weeks of age and 8 surgical castrated pigs were used for the study. These pigs were divided into groups with 8 pigs per group as shown in Tables 3 and 4 with the LHRH3 (SEQ ID NOs: 7, 8, and 9) formulated with oil based adjuvant (Montanide™ ISA50V or ISA50V2) to form an water-in-oil (W/O) emulsion as shown in Table 3 (Groups 1 to 4), or with oil based adjuvant Emulsigen D to form the water in oil in water (W/O/W) emulsion as shown in Table 4 (Groups 8 to 11), to enhance the immunogenicity of the finished vaccine products. Three control groups included pigs receiving saline as the negative controls (Groups 5 and 12), pigs having been surgically castrated as the positive controls (Groups 6 and 13), and pigs receiving the state-of-the-art LHRH vaccine Improvac® for direct efficacy comparison (Groups 7 and 14).
Blood samples were collected at 0, 4, 8, 10, 12, 14, and 16 weeks post initial immunization (or WPI) for measurement of the serum titers of anti-LHRH antibodies and the serum concentrations of testosterone, respectively in pigs.
a. Titration of Serum Anti-LHRH Antibodies by Enzyme-Linked Immunosorbent Assay (ELISA).
ELISA assay used for evaluating serum titers of anti-LHRH antibodies were developed and described below.
The wells of 96-well plates were coated individually for 1 hour at 37° C. with 100 μL of individual target peptides, at 2 μg/mL individually or as an equal molar mixture, unless specifically mentioned, in 10 mM NaHCO3 buffer, pH 9.5 unless noted otherwise.
The peptide-coated wells were incubated with 250 μL of 3% by weight of gelatin in PBS in 37° C. for 1 hour to block non-specific protein binding sites, followed by three washes with PBS containing 0.05% by volume of Tween® 20 and dried. 100 μL of the diluted sera samples were added to each of the wells and allowed to react for 60 minutes at 37° C. The wells were then washed six times with 0.05% by volume Tween® 20 in PBS to remove unbound antibodies. 100 μL of the peroxidase-labeled goat anti-swine IgG at a pretitered optimal dilution and in 1% by volume normal goat serum with 0.05% by volume Tween® 20 in PBS, was added to each well and incubated at 37° C. for another 30 minutes. The wells were washed six times with 0.05% by volume Tween® 20 in PBS to remove unbound antibody and reacted with 100 μL of the substrate mixture containing 0.04% by weight 3′,3′,5′,5′-Tetramethylbenzidine (TMB) and 0.12% by volume hydrogen peroxide in sodium citrate buffer for another 15 minutes. This substrate mixture was used to detect the peroxidase label by forming a colored product. Reactions were stopped by the addition of 100 μL of 1.0 M H2SO4 and absorbance at 450 nm (A450) determined.
Serum dilutions were carried out in accordance with the purpose for measuring serum titers of LHRH antibodies. For the determination of antibody titers in pigs that received peptide-based LHRH vaccine formulations, a 10-fold serial dilution of sera from 1:10 to 1:10,000 was performed on the ELISA and the titer of a tested serum, expressed as Log10, was calculated by linear regression analysis of the A450.
According to
b. Assessment of Serum Testosterone Concentration
Serum Testosterone (TT) concentration was measured by commercial ELISA or RIA kits. The kits included the DRG® Testosterone ELISA Kit (EIA-5179), BioVendor ELISA kit, and the Seamans RIA kit. Testosterone concentrations in serum of Groups 1 to 4 and Groups 8 to 11 formulated in ISA50V2 or Emulsigen D respectively were measured in this particular example by DRG® Testosterone ELISA Kit which correlated reversely to the corresponding serum antibody titers in pigs with the concentrations falling below the castration cut-off value (1.87 nmol/L in general or 1.0 nmol/L in more strict criteria) two weeks after the boost (i.e. 10 WPI or pigs at 18 weeks of age). Pigs in all other groups formulated in ISA50V2 or Emulsigen D maintained a castration level of testosterone, as shown in
Compared to pigs receiving the vaccine formulations, pigs in the negative and castration control groups always maintained a background level of low anti-LHRH antibody titers with the serum testosterone concentrations fluctuating in the negative controls (Groups 5 and 12) as the uncastrated animals; for pigs in the surgically castrated positive controls (Groups 6 and 13), the serum testosterone concentrations all remained at the low castrating level.
Those pigs receiving LHRH vaccine Improvac® maintained a high level of serum testosterone concentration until 12 WPI when the boost was given and such level was decreased after 14 WPI, i.e. two weeks after the boost. The results showed clearly that Improvac® is not as potent as the vaccine formulations of the invention (Groups 1 to 4 and Groups 8 to 11, containing peptide SEQ ID NOs: 7, 8, and 9 formulated in ISA50V2 or Emulsigen D) for immunocastration of pigs.
c. Body Weight
According to
In this Example, the protocol was similar to the protocol of Example 3, except that the peptide immunogen in the current vaccine formulation was changed to SEQ ID NO: 10, and the dosage was changed according to Tables 5 and 6. After immunization, sera from all animals were collected at multiple time points for determination of the titers of LHRH antibodies and the corresponding concentrations of serum testosterone. Additionally, the sexual organs (testes and epididymides) of pigs at 24 weeks of age were also collected for weight measurement at the time of sacrifice.
a. Titration of Serum Anti-LHRH Antibodies by ELISA
According to
b. Assessment of Serum Testosterone Concentration
According to
c. Body Weight
According to
In this Example, the protocol was similar to the protocol of Example 3, except the peptide immunogen was changed to SEQ ID NO: 10, with dosage shown according to Table 7. After immunization, sera were collected at multiple time points for determination of titers of LHRH antibodies and the corresponding serum testosterone concentrations. Additionally, the sexual organs (testes and epididymides) of pigs were also collected upon sacrifice at 32 weeks of age for weight measurement.
The objective of this study was to extend the field trial to 24WPI, or 32 weeks of age, with serum samples collected for measurement of titers of LHRH antibodies and corresponding serum testosterone concentrations for assessment of the duration of the immunocastration efficiency of the vaccine formulations of the invention employing a standard immunization protocol with the prime at 8 weeks of age (0 WPI) and a boost at 16 weeks of age (8 WPI).
a. Titration of Serum Anti-LHRH Antibodies by ELISA
As shown in
b. Assessment of Serum Testosterone Concentration
As shown in
c. Shrinkage of Sexual Organs (Testes and Epididymides) Upon Immunocastration by LHRH1 Vaccine Formulations
At the end of the extended study at 24 WPI (i.e. pigs at 32 weeks of age), the pigs were sacrificed and their sexual organs including testes and epididymides were weighed for assessment of immunocastration efficiency. The testes and epididymides of the pigs from Groups 1 to 4 were found to shrink to malfunctioned sizes as shown in
d. Body Weight
Pigs from Groups 1 to 4 had a better performance in body weight gain when compared to those pigs in the control groups (Groups 5 and 6) as shown in
e. Boar-Taint Removal as Measured by Concentrations of Androstenone and Skatole in Belly Fat
In this duration study, quantitation of boar-taint factors at the end of the study demonstrated that effective removal of boar taint in belly fat could prolong the pig sale window schedule to 24 WPI (i.e. 32 weeks of age). In this study, the androstenone and skatole were extracted from belly fat when the pigs were sacrificed and then detected by HPLC. According to
In addition, individual androstenone and skatole concentrations of a particular sample are plotted for boar-taint risk factor assessment and separated into four sectors as low, medium/low, medium/high, and high for all samples collected from this study as shown in
In this Example, the protocol was similar to the protocol of Example 3, except that the peptide immunogen used in the vaccine formulations was changed to SEQ ID NO: 10 with adjuvant ISA50V2, and the time points of prime and boost immunization were modified according to Table 8. Sera were collected at various time points for measurement of titers of anti-LHRH antibodies and serum concentrations of testosterone.
As illustrated in
Additionally, as illustrated in
A conservative and consumer-friendly approach was established in the pork industry to set the cut-off value for androstenone at 0.5 μg/g fat for boar taint risk factor assessment. The low risk for tainted meat could be obtained either with a medium androstenone level (0.5-1.0 μg/g fat) combined with a low skatole level (<0.1 μg/g fat) or with a low level of androstenone (<0.5 μg/g fat) combined with a medium level of skatole (0.1-0.22 μg/g fat). A medium risk would occur when both parameters (biomarkers) for boar taint occur in medium levels (androstenone 0.5-1.0 μg/g fat; skatole 0.1-0.22 μg/g fat). When both biomarkers (androstenone and skatole) exceed these cut off values at the same time it would imply a high risk for the meat to be regarded as tainted (Table 9).
In this Example, the protocol was about the same as that of Example 3. Pigs were immunized with LHRH3 (SEQ ID NOs: 7, 8 and 9) formulated in adjuvant ISA50V2 to form the water-in-oil (W/O) emulsion as shown in Table 10. After immunization, sera were collected at multiple time points for measurement of serum testosterone concentrations and the titers of anti-LHRH antibodies. Additionally, the tissues and organs of pigs were collected for measurement of the weight of testes and epididymides, and for measurement of amount of skatole and androstenone in belly fat by HPLC.
a. Shrinkage of Testes and Epididymides
The shrinkage of testes and epididymides was observed with the isolated organs weighted when the pigs were sacrificed at various time points, i.e. 10, 12, 14, and 16 WPI, respectively, of the study (
b. Measurement of Boar-Taint
5 g of back fat collected from vaccinated pigs or intact boars was individually added to 1.5 mL of 100% methanol. The fat was then grounded with sand texture slides until the tissues were completely homogenized. The fat extract was transferred into 15 mL centrifuge tubes and sonicated for 5 minutes at room temperature. The fat extract was cooled on ice for 15 minutes, and then centrifuged at 4,000 rpm for 20 minutes at 5° C. to separate from the tissue debris. The supernatant was transferred to another 1.5 ml centrifuge tube.
After immunization of pigs with the vaccine formulations, peak titer of anti-LHRH antibodies appeared at 10 WPI, i.e. 2 weeks after the boost, with the testosterone level being suppressed to near castration level around or lower than 1.0 nmol/L which yielded decreasing concentrations of the androstenone in fat as illustrated in
Moreover, the skatole concentration of pigs receiving the LHRH3 vaccine formulations of the invention was from about 0.004 to 0.2 μg/g fat (
In this Example, the protocol was similar to the protocol of Example 3, with pigs immunized with the LHRH1 (SEQ ID NO: 10) peptide formulated in adjuvant ISA50V2 to form a water-in-oil (W/O) emulsion as shown in Table 11 except that the dosage was at 25 ug/2 mL/dose, and the time points of vaccination were modified according to Table 11.
a. Titration of Serum Anti-LHRH Antibodies by ELISA
The titer of anti-LHRH antibodies at the initial stage of this study was 1.458±0.007 (Log10)) (Mean±SD), which was the background of the assay. After prime and boost immunizations (Groups 1 to 3), the antibody titers increased gradually and all reached the levels at 2 weeks post boost. The mean antibody titer of Group 1 declined from 3.636±0.577 (Log10) at the peak level at 10 WPI to 2.418±0.742 (Log10) at the end of the study. The mean antibody titer of Group 2 declined from 3.868±0.221 (Log10) at the highest level among these 4 groups to 2.806±0.213 (Log10) at the end of the study. The immune responses of Groups 3 and 4 were similar in antibody profiles. The results indicated that the 1st immunization could be administered from 8 weeks of age to 3 weeks of age, preferably (
b. Assessment of Serum Testosterone Concentration
As illustrated in
For assessment of duration of immunity, pigs in Group 3 indicated the best performance (i.e. lowest testosterone concentrations) whose average serum testosterone concentration at 24 weeks of age was 0.896±0.386 nmol/L. There was no significant difference (P=0.835) between Groups 3 and 4. As the schedule advanced, the testosterone concentration increased to 1.913±0.930 nmol/L (Group 2) and 4.149±3.603 nmol/L (Group 1). There was a significant difference between Groups 1 and 3 (P=0.010) as analyzed by Dunn's method.
c. Measurement of Sexual Organs
The in-life testis sizes (length and width) were measured from 12 weeks of age on and were calculated into volume, using the formulae as follows:
Testis volume=1/2×a×b2, where
The sexual organ measurements included (1) the testis in length and width in life, and (2) the testis and epididymis weights, length and width at necropsy. According to
The distribution of androstenone and skatole concentrations at 24 weeks of age was shown in
d. Body Weight
The body weight of each pig was also measured in this study. Each group of pigs grew in a similar pattern. The average weight for pig sales on the market in Taiwan is about 115 kg, which could be achieved by 26 weeks of age.
In conclusion, in comparison to the regular immunization program with prime and boost conducted at 8 and 16 weeks of age respectively, the prime schedule of this invention could be flexibly changed to the pig age of 3 weeks old. Furthermore, the boost schedule could be given as late as from age of 14 to 16 weeks as shown in this study.
In this Example, the protocol was similar to the protocol of Example 3, except that the peptide immunogen employed in the vaccine formulation was changed to SEQ ID NO: 10, and the time points of vaccination were modified according to Table 12. The purpose of this Example is to assess the duration of castration if the vaccine formulation is administrated to very young piglets and the immunological treatment results in sexual organ atrophy. This experiment is designed to evaluate very early “Flexible Treatment Regimens” and to determine the earliest prime time in the immunization regimen.
a. Titration of Serum Anti-LHRH Antibodies
As illustrated in
Of these groups, only pigs in Group 3 with the prime immunization at three weeks old and boost at 6 weeks of age mounted a high titer of anti-LHRH antibodies of 2.767±0.476 (Log10) at eight weeks of age. For pigs in Groups 1 to 3, after anti LHRH antibodies achieving the highest titers two weeks after the boost, the antibody titers were maintained at 1.718±0.297, 1.765±0.340 and 1.770±0.283 (Log10) for Group 1, 2, and 3, respectively. Pigs in Groups 3 and 4 also received prime immunization conducted at three weeks of age but boosted at 16 weeks of age. Pigs in Groups 4 and 5 could mount higher antibody titers, 3.249±0.346 and 2.893±0.786 at 18 weeks old respectively, two weeks after boost at the age of 16 weeks. As for pigs receiving the commercial vaccine Improvac® (Group 6), the titers of anti-LHRH antibodies were induced to 1.724±0.339 (Log10) at 20 weeks old, i.e. two weeks after the boost. Neither the positive control, i.e. pigs receiving surgical castration (Group 7), nor the negative control, i.e. pigs receiving no vaccination (Group 8), could induce the anti-LHRH antibodies titers beyond the background level, which was assayed with the value of 1.451±0.006 and 1.445±0.010 (Log10) respectively.
b. Assessment of Serum Testosterone Concentration
As illustrated in
When the boost immunization was delayed to age of 16 weeks in Groups 4 and 5, regardless of whether the first dose was administrated at 3 or 8 weeks of age, the testosterone concentration was suppressed to low levels at around 0.586±0.184 and 0.893±1.192 nmol/L for Groups 4 and 5 at 20 weeks of age, respectively. In contrast, although pigs in Group 6 followed the immunization instruction of Improvac®, the testosterone level in pigs remained at high concentration (about 9.415±7.560 nmol/L) in serum at 20 weeks of age. For pigs in Group 7 receiving surgical castration, the testosterone concentrations for each of the pigs always were maintained at background level (about 0.559±0.372 nmol/L). As for pigs in Group 8 receiving no vaccination and served as negative controls, the mean testosterone concentration was maintained at normal concentration (between 3.546±2.409 nmol/L and 7.380±3.269 nmol/L) during the trial.
In conclusion, the anti-LHRH antibody titers could be induced efficiently with the prime immunization at as early as 3 weeks of age and a boost thereafter which maintained high titers of such antibodies at the end of the trial giving a similar antibody profile to those following a regular immunization scheme with prime immunization at 8 weeks of age and boost at 16 weeks of age. Therefore, the serum testosterone concentration could be suppressed to low levels efficiently when the prime immunization was administered at 3 weeks to 8 weeks of age while the boost was administered at 16 weeks of age. It is convenient for the farmers to conduct immunocastration with the flexible early stage immunization scheme for pigs at 3 weeks of age when many other vaccines in general are being administered.
A large-scale field study was conducted in a pig farm located in southern Taiwan to assess the immunocastration efficacy in a field practice with two GMP batches of LHRH3 vaccine formulations. In this study, thirty-six crossbred male pigs and eight castrated pigs at 8 to 9 weeks of age were prepared in the farm.
Pigs were classified into 4 groups, and weighed at 0, 8, 10, 12, and 14 WPI. All pigs were immunized at 0 and 8 WPI and bled at 0, 8, 10, 12, and 14 WPI for serum titers of anti-LHRH antibodies and serum testosterone concentrations as shown in Table 13. After slaughter, weights of sexual organs (testes, epididymis, seminal vesicles, and prostate glands) with back fat thickness and loin-eye area (logissimus muscle) measured.
a. Titration of Serum Anti-LHRH Antibodies by ELISA
As illustrated in
b. Assessment of Serum Testosterone Concentration for Immunocastration
As illustrated in
c. Increase in Body Weights in Vaccinated Pigs
The body weight of each pig was measured as listed in Table 16. As observed, body weights of pigs in Groups 1 and 2 are heavier than that of Group 3.
As illustrated in
d. Shrinkage of Genital Track Sexual Organs
At the end of this study at 14 WPI (i.e. 22 weeks of age), the pigs were sacrificed and the testes, epididymides, seminal vesicles, and prostate gland were weighted for the evaluation of the immunocastration efficiency. For pigs in Groups 1 and 2, the weights of genital tract sexual organs including testes, epididymides, seminal vesicles, and prostate gland were found to be significantly decreased (Table 18), with these genital tract sexual organ shrinking to nonfunctional sizes.
In summary, the LHRH peptide immunogen based vaccine formulations of the present invention has a higher immunocastration effect than currently available commercial product (Improvac®). In addition to the humane factor for conversion of conventional surgical castration into the upcoming immunocastration in the animal husbandry field, the significant weight gains as a result of such immunocastration practice will generate significant economic benefits which would push the trend of animal castration to the front line after decades of exploration.
While the invention has been described by way of examples and in terms of the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments. To the contrary, it is intended to cover various modifications and similar arrangements (as would be apparent to those skilled in the art). Therefore, the scope of the appended claims should be accorded the broadest interpretation so as to encompass all such modifications and similar arrangements.
#WPI: Weeks Post (first) Immunization
§LHRH3 contains the peptides of SEQ ID NOs: 7, 8 and 9.
#WPI: Weeks Post (first) Immunization
§LHRH3 contains the peptides of SEQ ID NOs: 7, 8, and 9.
0 μg/2 mL/dose
#WPI: Weeks Post (first) Immunization
§LHRH1 contains the peptide of SEQ ID NO: 10
0 μg/2 mL/dose
#WPI: Weeks Post (first) Immunization
§LHRH1 contains the peptide of SEQ ID NO. 10
0 μg/2 mL/dose
#WPI: Weeks Post (first) Immunization
§LHRH1 contains the peptide of SEQ ID NO: 10
§LHRH3 contains the peptides of SEQ ID NOs: 7, 8, and 9.
†D: Days of age;
#WPI: Weeks Post (first) Immunization.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/48164 | 7/25/2014 | WO | 00 |
