WO2019075893A1 - Placental chondroitin sulphate a immunogenic composition and application - Google Patents

Placental chondroitin sulphate a immunogenic composition and application Download PDF

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WO2019075893A1
WO2019075893A1 PCT/CN2017/116629 CN2017116629W WO2019075893A1 WO 2019075893 A1 WO2019075893 A1 WO 2019075893A1 CN 2017116629 W CN2017116629 W CN 2017116629W WO 2019075893 A1 WO2019075893 A1 WO 2019075893A1
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contraceptive
contraception
pregnancy
cell
csa
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PCT/CN2017/116629
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French (fr)
Chinese (zh)
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张居作
范秀军
张键
陈指龙
汪宝蓓
黄晨
陈杰
李梦霞
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中国科学院深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response

Definitions

  • the invention relates to the field of biopharmaceuticals, in particular to a placenta-like chondroitin sulfate A immunological composition and application.
  • contraceptive vaccines are mainly for certain components of hormones or reproductive fines. These hormones or germ cell-related components usually play a key role in the reproductive process, such as by inhibiting germ cell production (sperm or egg) or inhibiting germ cell function. And hinder normal pregnancy progression and maintenance [1] . Among these components, sperm became the first contraceptive vaccine target. In 1899, Nobel Prize winners Landsteiner and Metnikoff conducted antisperm antibody studies, respectively. Injection of antisperm antibodies can play a contraceptive effect; then surgeon Morris in 1937 In the year, the first US patent for anti-sperm contraceptive vaccine was applied. Although this vaccine can produce reversible contraceptive effects in pregnant women, its unclear side effects hinder its clinical application [2] . Currently, a variety of hormone and germ cell components have been used to conduct contraceptive vaccine studies (see Table 1).
  • a contraceptive vaccine that targets hormone molecules and their receptors.
  • Appropriate specific anti-hormone antibodies can block the normal function of hormones required for reproductive activities, and use hormone-immune mature individuals to stimulate the body to produce sufficient titers of bio-neutralizing antibodies, as long as there is sufficient antibody titer, vaccination
  • the person can maintain the state of contraception [5] .
  • GnRH has been validated in a variety of animal experiments, such as immune castration to control wild animal populations. In human clinical trials, GnRH is used to prolong the anovulatory period in postpartum women, and is used in male contraception and prostate. Treatment of cancer [3,10] .
  • the FSH contraceptive vaccine has been clinically tested in men, mainly to evaluate its immunogenicity and its impact on spermatogenesis, but the results obtained are not ideal. It is found to have only weak immunogenicity and can reduce sperm. Quantity and vitality, but no significant effect on other parameters of semen [4] .
  • the hCG vaccine can prevent maternal pregnancy recognition to terminate early pregnancy, and has successfully carried out Phase I and Phase II clinical trials in women and found it to be a very promising contraceptive vaccine [11,12] .
  • hCG-based contraceptive vaccines have been developed in a wide range of preclinical and clinical studies, such as the beta subunit hCG vaccine and the carboxy terminal peptide beta subunit hCG vaccine [13-15] , which promotes the neutralization of hCG.
  • the corpus luteum acts to block or interfere with peri-implantation embryos.
  • These vaccines are also used to inhibit excessive secretion of steroid hormones such as uterine fibroids, polycystic ovary syndrome, endometriosis and precocious puberty [11 ] .
  • the targeted hormone and its receptor contraceptive vaccine still have the risk of contraceptive failure, and it will cause secretion disorder in the body, disturbing ovarian function and affecting health.
  • Antigen molecules usually present on the surface of cells. These surface antigens are usually tissue-specific, immunogenic, and easily stimulate the body to produce antibodies.
  • the zona pellucida antigen is one of them. It is a non-cellular component, specific extracellular matrix glycoprotein, which covers the surface of female germ cells and peri-implantation embryos, in follicular formation, fertilization, early embryo development and other processes. It plays a key role and is considered to be a very promising candidate contraceptive vaccine [16] .
  • the zona pellucida vaccine may become an abortion vaccine by preventing the zona pellucida from being hydrolyzed by uterine proteases, affecting sperm penetration or embryo implantation [8,17] .
  • the zona pellucida vaccine needs to induce the body to produce high anti-Zona pellucida antibody titers to prevent fertilization, but too high anti-Zona pellucida antibodies are often difficult to maintain, and may cause ovarian damage, causing premature inactivation of primordial follicles, so It is difficult to get promoted and applied before solving the defect of ovarian injury.
  • sperm can become autoantigen and allogeneic antigens, can stimulate individuals of both sexes to produce antibodies, anti-sperm antibodies have been prevented from fertilization and embryo development in vitro and in vivo [7] .
  • Immunization of different species, including humans, with sperm or sperm extracts can stimulate the body to produce anti-sperm antibodies, leading to infertility [7] . Therefore, sperm can stimulate the body to produce an immune response, maintaining a certain anti-sperm antibody titer can promote the body to maintain infertility.
  • contraceptive vaccines that target sperm are often unable to maintain high antibody titers, fail to ensure that all sperm enter the conception, and cause contraceptive failure. At the same time, the source of sperm is seriously challenged, which hinders the promotion and application of sperm contraception vaccine.
  • Ferro VA Current advances in antifertility vaccines for fertility control and noncontraceptive applications. Expert review of vaccines 2002, 1(4): 443-452.
  • Perez-Lopez FR Pituitary repetitive stimulation with GnRH/TRH in women treated with three different oral steroid contraceptives. Acta endocrinologica 1990, 122(2): 163-167.
  • Manning PA Immunogenicity trial of a contraceptive vaccine based on human chorionic gonadotropin. Vaccine 1986, 4(4): 271.
  • Stevens VC Progress in the development of human chorionic gonadotropin antifertility vaccines. American journal of reproductive immunology 1996, 35(3): 148-155.
  • Saxena HM Predictions of Immunological Cross-Reactions of C-Terminal Peptide of Human Chorionic Gonadotropin beta-Chain Based Contraceptive Vaccine with Autoantigens.
  • Russian journal of immunology RJI:Official journal of Russian Society of Immunology 1999,4(2): 151-158.
  • the problems of the above-mentioned very promising target antigen vaccine include: the first targeted hormone and its receptor may cause the body's endocrine disorders, affecting the normal function of the body; second, targeting germ cells may cause tissue damage, irreversible infertility Infertility; the third target vaccine molecule does not produce sufficient antibody titers, affecting the contraceptive effect, resulting in contraceptive failure.
  • the present invention firstly proposes a contraceptive vaccine for a critical and sensitive period of embryonic attachment stage such as placental formation and embryo development, and targeting the attachment of cells to perform key cytotrophoblast cells can improve the sensitivity and effectiveness of contraception.
  • the pl-CSA secreted by the placenta trophoblast alone or in combination with the B cell dominant epitope of its core protein CSPG4 is used to prepare a contraceptive vaccine, and the B cell dominant epitope of the core protein CSPG4 is used to induce the antigenic reaction of the B cell. Immune action further increases the effect of contraception, thus preparing a safe and effective contraceptive vaccine.
  • One aspect of the present invention provides an immunological composition for contraception comprising at least one placental trophoblast cell-specifically expressing a polysaccharide and/or a protein antigen.
  • the embryo or placental trophoblast cell-specific expression polysaccharide is selected from the group consisting of placenta-like chondroitin sulfate A.
  • the immunological composition further comprises an immunopotentiating agent further comprising an antigenic reaction for inducing B cells, wherein the immunopotentiating agent is selected from a B cell of placenta-like chondroitin sulfate core protein CSPG4.
  • a dominant epitope preferably, the B cell dominant epitope of the placental-like chondroitin sulfate core protein CSPG4 is SEQ ID No. 2 or a homolog thereof.
  • the mass ratio of the embryonic or placental trophoblast cell-specific expression polysaccharide and/or protein antigen to the B cell dominant epitope of CSPG4 is 1-100:1, preferably 1-10:1.
  • the immunological composition for contraception further comprises an adjuvant.
  • the adjuvant is selected from Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum (aluminum phosphate or aluminum hydroxide), saponin, Quil A, RIBBI adjuvant, dimethyl double 18 Alkyl ammonium bromide (DDA), monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), trehalose dibehenate and muramyl dipeptide (MDP), poly I: C and so on.
  • Freund's complete adjuvant Freund's incomplete adjuvant
  • aluminum aluminum phosphate or aluminum hydroxide
  • saponin Quil A
  • RIBBI adjuvant dimethyl double 18 Alkyl ammonium bromide (DDA), monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), trehalose dibehenate and muramyl dipeptide (MDP), poly I: C and so on.
  • Another aspect of the invention provides a pharmaceutical composition comprising the immunological composition of the invention.
  • the pharmaceutical composition further comprises other active ingredients for contraception and/or termination of pregnancy.
  • active ingredients for contraception and/or termination of pregnancy may be desogestrel ethinyl estradiol, nonoxynol ether suppository, nonoxynol ether, mifepristone, levonorgestrel, levonorol Progesterone ethinyl estradiol.
  • Another aspect of the invention provides the use of an immunological composition of the invention in the manufacture of a medicament for preventing embryos from adhering to the endometrium.
  • Another aspect of the invention provides the use of an immunological composition of the invention for preventing embryos from being implanted in the endometrium.
  • Another aspect of the invention provides the use of an immunological composition of the invention in the manufacture of a medicament for the termination of pregnancy.
  • Another aspect of the invention provides the use of an immunological composition of the invention for the termination of pregnancy.
  • Another aspect of the invention provides the use of an immunological composition of the invention in the manufacture of a contraceptive.
  • Another aspect of the invention provides the use of an immunological composition of the invention for use in contraception.
  • the present invention provides a method of inhibiting human or non-human pregnancy in an immune human and non-human animal, comprising administering to the animal a therapeutically effective amount comprising one or more The immunological composition of the invention.
  • the embryo is attached to the endometrium, the subject of the contraception or the termination of pregnancy or the subject is a human or non-human animal, including domesticated animals and non-domestic animals.
  • the non-human animal is a domesticated animal.
  • a domesticated animal for example, but not limited to, dogs, cats, rats, mice, cows, horses, sheep, pigs, rabbits, and the like.
  • the non-human animal is a wild animal.
  • wild animals are dogs, cats, rats, mice, bats, foxes, raccoons, squirrels, possums, wolves, rabbits, and the like.
  • Still another aspect of the present invention provides a use of a placental trophoblast cell-specific expression of a polysaccharide and/or a protein antigen as a target for contraception or preventing embryos from attaching to the endometrium or terminating a pregnancy, or as a screening contraceptive or preventing embryos from being attached to Use of endometrial or drug targets for termination of pregnancy.
  • a pharmaceutically acceptable carrier can be included in the immunological composition or pharmaceutical composition including, but not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and immunological composition can be sterile and compatible with the mode of administration.
  • the immunological composition or pharmaceutical composition may also be other excipients commonly used in pharmaceutical preparations, for example containing a small amount of wetting agent, emulsifier or pH buffer.
  • the immunological composition or pharmaceutical composition of the invention may be a liquid solution, suspension, emulsion, sustained release dosage form or powder. Any conventional pharmaceutical carrier such as sterile saline solution or sesame oil can be used.
  • the medium may also contain conventional pharmaceutical auxiliary substances such as pharmaceutically acceptable salts for adjusting osmotic pressure, buffers, preservatives and the like.
  • the immunological composition or pharmaceutical composition of the present invention can be administered by any route including, but not limited to, by oral, transdermal, mucosal (e.g., vaginal, rectal, buccal or nasal mucosa), injection (e.g., muscle). , subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal) or by inhalation (eg, buccal or nasal).
  • the adjuvant of the immunological composition or vaccine or medicament of the present invention and the preparation method can be adjusted depending on the mode of administration.
  • attachment means that the embryonic trophoblast and the endometrium gradually associate with histology and physiology, so that the blastocyst is fixed in the endometrium, this physiological process become attached.
  • the immunological composition is a composition capable of eliciting an immune response
  • the pharmaceutical composition is capable of causing the test to be initiated.
  • the present invention firstly proposes a contraceptive vaccine for a critical and sensitive period of early placental formation and embryo development, targeting the attachment of cells to perform key cytotrophoblast cells, and improving the sensitivity and effectiveness of contraception.
  • the pl-CSA vaccine targets trophoblast cells, which are present in the blastocyst ectoderm and placenta, and have only a low cross with other normal cells of the body (such as thalamic-pituitary-ovarian tissue cells). Reactivity, it is not easy to damage the normal function of the body tissues and organs.
  • pl-CSA is a macromolecular polysaccharide with stable structure and strong immunogenicity.
  • the immunized animal can produce higher antibody titer.
  • pl-CSA can be induced by combining the B cell dominant epitope with its core protein CSPG4. B cell antigen response, enhance immune function, thereby increasing contraceptive effect;
  • pl-CSA is relatively easy to obtain, and the similarity of pl-CSA from different species sources is higher.
  • the pl-CSA content in the trophoblast cells is abundant, and the pl-CSA content in the discarded pig placenta is also very rich. Separating and purifying pl-CSA from the pig placenta can turn waste into treasure.
  • Figure 1 shows the working principle of the contraceptive vaccine.
  • FIG. 3 shows the expression of pl-CSA in human, mouse and porcine placental trophoblast cells and colocalization with CSPG4.
  • pl-CSA was fluorescently stained with anti-6 ⁇ His monoclonal antibody labeled with rVAR2 and Dylight650.
  • CSPG4 was fluorescently stained with rabbit anti-CSPG4 monoclonal antibody and FITC-labeled goat anti-rabbit secondary antibody.
  • DAPI labeled nuclei, Merge is a picture
  • the human placental trophoblast comes from the placenta of the delivery
  • the mouse placental trophoblast comes from the 15d placenta during pregnancy
  • the pig placenta and myometrium are from the 70s placenta and uterine muscle.
  • pl-CSA and CSPG4 were contained in the placental trophoblast of human, mouse and pig, while pu-CSA and CSPG4 were not expressed in pig uterus.
  • Figure 4 is a graph showing the results of pl-CSA immunization of BABL/c mice. Among them, A.pl-CSA sample and standard wave scanning surface, the absorption wavelength is consistent; B. experimental mouse anatomy; C. experimental mouse weight change; D. experimental mouse antibody level; E. experimental mouse pregnancy rate.
  • Figure 5 is a graph showing the results of BABL/c immunization of pl-CSA and its core protein B cell dominant epitope CBZ-1.
  • Figure 6 is a graph showing the results of changes in body weight of rats in acute toxicity.
  • Figure 7 is a graph showing the results of HE staining of tissue sections of infected rat viscera (including liver, spleen and kidney).
  • pl-CSA and CSPG4 are contained in the placental trophoblast of human, mouse and pig, while there is no expression of pl-CSA and CSPG4 in the pig uterus.
  • SEQ ID No. 1 is as follows
  • the successfully coupled agarose gel is a rVAR2 affinity chromatography column of pl-CSA;
  • the purified pl-CSA was configured as a solution pl-CSA immunogen particle.
  • the obtained pl-CSA immunogen particles and pl-CSA+CBZ-1 immunogen particles were mixed and emulsified with Freund's complete adjuvant and incomplete adjuvant, respectively, to prepare pl-CSA vaccine and pl-CSA+CBZ-1 vaccine. .
  • mice will be sacrificed after the 15th day of pregnancy, the embryo development will be dissected, serum will be collected for indirect ELISA to detect antibody levels; if not pregnant, 6 consecutive estrus cycles will be combined with male rats. In the cage, all females were tested for vaginal plugs, demonstrating that all females had completed mating. Six pregnant mice with estrus cycles were sacrificed and serum was collected for indirect ELISA to detect antibody production levels.
  • the contraceptive vaccine Ig1 used alone in pl-CSA reduced the pregnancy rate to 43%, and the blank group NcIg1 had a pregnancy rate of 75%; pl-CSA and its core protein B cell dominant epitope CBZ-1 (IVAVDEPTRPIYRFTQ) polypeptide
  • the Ig2 group prepared by the assembled contraceptive vaccine reduced the pregnancy rate of the mice to 35%, and the pregnancy rate of the blank group ncIg2 was 85%.
  • the experimental results showed that the pl-CSA immunizing agent and the pl-CSA+CBZ-1 immunizing agent can significantly reduce the conception rate compared with the blank control group, while the pl-CSA+CBZ-1 group has better contraceptive effect than the pl-CSA. .
  • Acute toxicity test 6-week female BALB/c mice were randomly divided into 8 groups, 10 in each group, and administered intramuscularly; 4 groups of pl-CSA immunizing agents, including pl-CSA-Tg1 group (100 ⁇ g/only) , pl-CSA-Tg2 group (200 ⁇ g/only), pl-CSA-Tg3 group (400 ⁇ g/only), pl-CSA-Tg4 group (800 ⁇ g/only); pl-CSA+CBZ-1 immunizing agent 4 groups, including CBZ-1-Tg1 group (100 ⁇ g/only), CBZ-1-Tg2 group (200 ⁇ g/only), CBZ-1-Tg3 group (400 ⁇ g/only), CBZ-1-Tg4 group (800 ⁇ g/only), single time 200 ⁇ L/mouse was administered, and 2 weeks were continuously observed, and the changes in signs and death were analyzed.
  • pl-CSA immunizing agents including pl-CSA-Tg1 group (100 ⁇ g/only) , pl-CSA-Tg2 group
  • mice were tested for serum, 100 ⁇ L/well, incubated at 37 ° C for 45 min; PBST was washed 3 times, each time 10 min 1% BSA was diluted 1:10000 HRP-labeled goat anti-mouse IgG, 100 ⁇ L/well, incubated at 37 ° C for 45 min; discarded secondary antibody PBST was thoroughly washed 5 times for 5 min each time; TMB coloring solution 50 ⁇ L/well was added, room temperature, color development in a dark room for 10 min; and 2 M H 2 SO 4 50 ⁇ L/well was added to terminate the reaction. The OD450 value was read in the microplate reader within 15 min.
  • the experimental results are shown in Figures 4D and 5C.
  • the results of the experiment showed that the antibody levels of pl-CSA+CBZ vaccine and pl-CSA vaccine were significantly higher than those of the blank group, and the antibody levels of the non-pregnant mice in the vaccine group or the blank group were higher than those in the pregnant group. There were significant differences in antibody levels in mice.
  • the experimental results also confirmed that the high antibody level affects the embryo attachment and enables the mice to successfully contraception.
  • mice obtained in Example 3 were prepared for liver, spleen and kidney, and the toxic reaction of each organ was observed by HE staining, and no obvious pathological changes were observed.
  • tissue samples were fixed in 4% neutral paraformaldehyde (Bouin's solution) for not less than 48h, and tapped in tap water overnight to make slices: (1) Ethanol gradient dehydration: 70% ethanol (2h) ⁇ 80% Ethanol (2h) ⁇ 90% ethanol (30min) ⁇ 95% ethanol (30min) ⁇ 100% ethanol (30min) ⁇ 100% ethanol (30min). (2) Xylene transparent: 50% ethanol xylene (12 min) ⁇ 100% xylene (10 min) ⁇ 100% xylene (10 min). (3) Dip wax: 50% xylene paraffin (2h) ⁇ 100% paraffin (1h) ⁇ 100% paraffin (1h). (4) Section: 5 ⁇ m serial section.
  • Exhibits and scraps 45 ° C distilled water, clean glass slides.
  • Baking sheet placed in an oven at 60 ° C for 20 min.
  • Dewaxing 100% xylene (1 min) ⁇ 100% xylene (1 min).
  • Hydration 100% ethanol (2min) ⁇ 95% ethanol (1min) ⁇ 85% ethanol (1min) ⁇ 75% ethanol (1min) ⁇ naturally dry (can be stored at -20°C).
  • HE staining Paraffin section prepared above ⁇ Hematoxylin staining solution (5 min) ⁇ Light water washing with distilled water (5 sec) ⁇ 1% hydrochloric acid ethanol (3 sec) ⁇ Light wash with distilled water (15 sec) ⁇ 0.5% eosin staining (45 sec ) ⁇ Distilled water, light wash (30 sec) ⁇ 80% ethanol (2 sec) ⁇ 90% ethanol (2 sec) ⁇ 95% ethanol (5 sec) ⁇ stone xylene carbonate (5 min) ⁇ xylene (1 min) ⁇ xylene (1 min) ⁇ Neutral gum seal (the gum is dried and observed to avoid bubbles). The dynamic changes of the placental tissue structure during each pregnancy were observed under a microscope and photographed.
  • the results of the experiment are shown in Fig. 7.
  • the results of HE staining showed that the tissue sections of the visceral organs (including liver, spleen and kidney) of the immunized mice showed no significant changes, which were basically consistent with the results of the PBS group, and the contraceptive vaccine containing pl-CSA was basically safe.

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Abstract

Provided is a placental chondroitin sulphate A-containing contraceptive immunogenic composition, the immunogenic composition also containing an immunopotentiator. Also provided is a use of the immunogenic composition in the preparation of a contraceptive drug for humans and non-human animals or a drug which prevents an embryo from attaching to an endometrium, or a drug or immunogenic preparation which terminates a pregnancy.

Description

一种胎盘样硫酸软骨素A免疫组合物及应用Placenta-like chondroitin sulfate A immunological composition and application thereof 技术领域Technical field
本发明涉及生物制药领域，具体涉及一种胎盘样硫酸软骨素A免疫组合物及应用。The invention relates to the field of biopharmaceuticals, in particular to a placenta-like chondroitin sulfate A immunological composition and application.
背景技术Background technique
目前，还没有一种避孕疫苗应用于临床实践。避孕疫苗的靶标主要针对激素或者生殖细的某些组份，这些激素或者和生殖细胞相关组分通常在生殖过程发挥关键作用，如通过抑制生殖细胞产生(精子或者卵子)或者抑制生殖细胞的功能，以及阻碍正常妊娠进展和维持[1]。在这些组分中，精子成为第一个避孕疫苗靶标，1899年诺贝尔奖获得者Landsteiner和Metnikoff分别开展了抗精子抗体研究，注射抗精子抗体能够起到避孕的效果；随后外科医生Morris在1937年申请了第一个抗精子避孕疫苗的美国专利，尽管这一疫苗能够在生育妇女中产生可逆的避孕效果，其不明确的副作用阻碍了其临床应用[2]。目前，有多种激素和生殖细胞组分被用来开展避孕疫苗的研究(如表1)。Currently, there is no contraceptive vaccine for clinical practice. The target of contraceptive vaccines is mainly for certain components of hormones or reproductive fines. These hormones or germ cell-related components usually play a key role in the reproductive process, such as by inhibiting germ cell production (sperm or egg) or inhibiting germ cell function. And hinder normal pregnancy progression and maintenance [1] . Among these components, sperm became the first contraceptive vaccine target. In 1899, Nobel Prize winners Landsteiner and Metnikoff conducted antisperm antibody studies, respectively. Injection of antisperm antibodies can play a contraceptive effect; then surgeon Morris in 1937 In the year, the first US patent for anti-sperm contraceptive vaccine was applied. Although this vaccine can produce reversible contraceptive effects in pregnant women, its unclear side effects hinder its clinical application [2] . Currently, a variety of hormone and germ cell components have been used to conduct contraceptive vaccine studies (see Table 1).
表1.已有研究避孕疫苗的靶向分子Table 1. Targeting molecules for research on contraceptive vaccines
Figure PCTCN2017116629-appb-000001
Figure PCTCN2017116629-appb-000001
第一，靶向激素分子及其受体的避孕疫苗。合适的特异性抗激素抗体能够阻断生殖活动所需激素正常功能的行使，用激素免疫成熟个体刺激机体产生足够滴度的具有生物中和性的抗体，只要有足够的抗体滴度，接种疫苗者就能保持避孕状态[5]。目前，GnRH已经通过多种动物实验进行验证，如通过免疫去势来控制野生动物种群，在人的临床实验中，GnRH被用来延长产后妇女的无排卵期，同 时被用于男性避孕和前列腺癌的治疗[3,10]。FSH避孕疫苗已经在男性上开展了I期临床实验，主要评估其免疫原性以及对精子发生的影响，但是得到的结果并不理想，发现其仅有很弱的免疫原性，能够减少精子的数量和活力，但对精液的其他参数无显著影响[4]。hCG疫苗能够阻止母体妊娠识别来终止早期妊娠，目前已经成功的在女性身上开展了I期和II期临床实验，发现其是一种非常有前景的避孕疫苗[11,12]。几种以hCG为基础的避孕疫苗已经开展了广泛的临床前期和临床期研究，如β亚单位hCG疫苗和羧基末端肽β亚单位hCG疫苗[13-15]，这些疫苗通过中和hCG的促黄体作用来阻止或者干扰围植入期胚胎附植，这类疫苗也被用来抑制类固醇激素的过量分泌，如子宫肌瘤、多囊卵巢综合症、子宫内膜异位和性早熟等[11]。但是，靶向激素及其受体类避孕疫苗仍然存在避孕失败的风险，且会造成机体内分泌紊乱，扰乱卵巢功能，影响健康。First, a contraceptive vaccine that targets hormone molecules and their receptors. Appropriate specific anti-hormone antibodies can block the normal function of hormones required for reproductive activities, and use hormone-immune mature individuals to stimulate the body to produce sufficient titers of bio-neutralizing antibodies, as long as there is sufficient antibody titer, vaccination The person can maintain the state of contraception [5] . Currently, GnRH has been validated in a variety of animal experiments, such as immune castration to control wild animal populations. In human clinical trials, GnRH is used to prolong the anovulatory period in postpartum women, and is used in male contraception and prostate. Treatment of cancer [3,10] . The FSH contraceptive vaccine has been clinically tested in men, mainly to evaluate its immunogenicity and its impact on spermatogenesis, but the results obtained are not ideal. It is found to have only weak immunogenicity and can reduce sperm. Quantity and vitality, but no significant effect on other parameters of semen [4] . The hCG vaccine can prevent maternal pregnancy recognition to terminate early pregnancy, and has successfully carried out Phase I and Phase II clinical trials in women and found it to be a very promising contraceptive vaccine [11,12] . Several hCG-based contraceptive vaccines have been developed in a wide range of preclinical and clinical studies, such as the beta subunit hCG vaccine and the carboxy terminal peptide beta subunit hCG vaccine [13-15] , which promotes the neutralization of hCG. The corpus luteum acts to block or interfere with peri-implantation embryos. These vaccines are also used to inhibit excessive secretion of steroid hormones such as uterine fibroids, polycystic ovary syndrome, endometriosis and precocious puberty [11 ] . However, the targeted hormone and its receptor contraceptive vaccine still have the risk of contraceptive failure, and it will cause secretion disorder in the body, disturbing ovarian function and affecting health.
第二，靶向生殖细胞相关分子。细胞表面通常会存在的抗原分子，这些表面抗原通常具有组织特异性，免疫原性强，容易刺激机体产生相应的抗体。透明带抗原是其中之一，这是一种非细胞成份、特异性的细胞外基质糖蛋白，覆盖在雌性生殖细胞卵子和围植入期胚胎表面，在卵泡形成、受精、早期胚胎发育等过程中起关键作用，被认为是非常有前途的候选避孕疫苗[16]。透明带避孕疫苗可能通过阻止透明带被子宫蛋白酶水解，影响精子穿透或者胚胎植入，成为一种堕胎疫苗[8,17]。透明带避孕疫苗需要诱导机体产生很高的抗透明带抗体滴度才能阻止受精，但过高的抗透明带抗体通常很难维持，而且可能造成卵巢损伤，引起原始卵泡过早失活，因此在解决损伤卵巢这一缺陷之前很难得到推广应用。Second, targeting germ cell related molecules. Antigen molecules usually present on the surface of cells. These surface antigens are usually tissue-specific, immunogenic, and easily stimulate the body to produce antibodies. The zona pellucida antigen is one of them. It is a non-cellular component, specific extracellular matrix glycoprotein, which covers the surface of female germ cells and peri-implantation embryos, in follicular formation, fertilization, early embryo development and other processes. It plays a key role and is considered to be a very promising candidate contraceptive vaccine [16] . The zona pellucida vaccine may become an abortion vaccine by preventing the zona pellucida from being hydrolyzed by uterine proteases, affecting sperm penetration or embryo implantation [8,17] . The zona pellucida vaccine needs to induce the body to produce high anti-Zona pellucida antibody titers to prevent fertilization, but too high anti-Zona pellucida antibodies are often difficult to maintain, and may cause ovarian damage, causing premature inactivation of primordial follicles, so It is difficult to get promoted and applied before solving the defect of ovarian injury.
雄性生殖细胞精子表面也存在一些特异性的蛋白，因此靶向精子的避孕疫苗可能非常有前景[18]。精子能够成为自身抗原和异体抗原，能够刺激两种性别的个体产生抗体，抗精子抗体已被在体内外阻止受精和胚胎发育[7]。用精子或者精子提取物免疫不同物种，包括人，能够刺激机体产生抗精子抗体，从而导致不孕不育[7]。因此精子能够刺激机体产生免疫反应，维持一定的抗精子抗体滴度能够促使机体保持不孕不育状态。但是靶向精子的避孕疫苗通常因不能维持高抗体滴度，不能确保阻止所有精子进入受孕状体而造成避孕失败，同时精子的来源受到了严重挑战，这些情况阻碍了精子避孕疫苗的推广应用。There are also some specific proteins on the surface of sperm cells in male germ cells, so contraceptive vaccines targeting sperm may be very promising [18] . Sperm can become autoantigen and allogeneic antigens, can stimulate individuals of both sexes to produce antibodies, anti-sperm antibodies have been prevented from fertilization and embryo development in vitro and in vivo [7] . Immunization of different species, including humans, with sperm or sperm extracts can stimulate the body to produce anti-sperm antibodies, leading to infertility [7] . Therefore, sperm can stimulate the body to produce an immune response, maintaining a certain anti-sperm antibody titer can promote the body to maintain infertility. However, contraceptive vaccines that target sperm are often unable to maintain high antibody titers, fail to ensure that all sperm enter the conception, and cause contraceptive failure. At the same time, the source of sperm is seriously challenged, which hinders the promotion and application of sperm contraception vaccine.
【参考文献】【references】
1.Gupta SK,Shrestha A,Minhas V:Milestones in contraceptive vaccines  development and hurdles in their application.Human vaccines &immunotherapeutics 2014,10(4):911-925.1.Gupta SK, Shrestha A, Minhas V: Milestones in contraceptive vaccines Development and hurdles in their application.Human vaccines &immunotherapeutics 2014,10(4):911-925.
2.Lekhwani S,Vaswani N,Ghalaut VS,Shanker V,Singh R:Immunocontraceptives:How far from reality？Advanced biomedical research 2014,3:247.2.Lekhwani S, Vaswani N, Ghalaut VS, Shanker V, Singh R: Immunocontraceptives: How far from reality? Advanced biomedical research 2014, 3:247.
3.Barmat LI,Chantilis SJ,Hurst BS,Dickey RP:A randomized prospective trial comparing gonadotropin-releasing hormone (GnRH)antagonist/recombinant follicle-stimulating hormone (rFSH) versus GnRH-agonist/rFSH in women pretreated with oral contraceptives before in vitro fertilization.Fertility and sterility 2005,83(2):321-330.3.Barmat LI, Chantilis SJ, Hurst BS, Dickey RP: A randomized prospective trial comparing gonadotropin-releasing hormone (GnRH)antagonist/recombinant follicle-stimulating hormone (rFSH) versus GnRH-agonist/rFSH in women pretreated with oral contraceptives before in Vitro fertilization.Fertility and sterility 2005,83(2):321-330.
4.Schally AV,Parlow AF,Carter WH,Saito M,Bowers CY,Arimura A:Studies on the site of action of oral contraceptive steroids.II.Plasma LH and FSH levels after administration of antifertility steroids and LH-releasing hormone(LH-RH).Endocrinology 1970,86(3):530-541.4.Schally AV, Parlow AF, Carter WH, Saito M, Bowers CY, Arimura A: Studies on the site of action of oral contraceptive steroids. II. Plasma LH and FSH levels after administration of antifertility steroids and LH-releasing hormone (LH -RH).Endocrinology 1970,86(3):530-541.
5.Delves PJ:How far from a hormone-based contraceptive vaccine？Journal of reproductive immunology 2004,62(1-2):69-78.5.Delves PJ: How far from a hormone-based contraceptive vaccine? Journal of reproductive immunology 2004, 62 (1-2): 69-78.
6.Talwar GP,Gupta JC,Rulli SB,Sharma RS,Nand KN,Bandivdekar AH,Atrey N,Singh P:Advances in development of a contraceptive vaccine against human chorionic gonadotropin.Expert opinion on biological therapy 2015,15(8):1183-1190.6. Talwar GP, Gupta JC, Rulli SB, Sharma RS, Nand KN, Bandivdekar AH, Atrey N, Singh P: Advances in development of a contraceptive vaccine against human chorionic gonadotropin. Expert opinion on biological therapy 2015, 15(8): 1183-1190.
7.Shi J,Yang Z,Wang M,Cheng G,Li D,Wang Y,Zhou Y,Liu X,Xu C:Screening of an antigen target for immunocontraceptives from cross-reactive antigens between human sperm and Ureaplasma urealyticum.Infection and immunity 2007,75(4):2004-2011.7.Shi J,Yang Z,Wang M,Cheng G,Li D,Wang Y,Zhou Y,Liu X,Xu C:Screening of an antigen target for immunocontraceptives from cross-reactive antigens between human sperm and Ureaplasma urealyticum.Infection and Immunity 2007,75(4):2004-2011.
8.Gupta SK,Gupta N,Suman P,Choudhury S,Prakash K,Gupta T,Sriraman R,Nagendrakumar SB,Srinivasan VA:Zona pellucida-based contraceptive vaccines for human and animal utility.Journal of reproductive immunology 2011,88(2):240-246.8. Gupta SK, Gupta N, Suman P, Choudhury S, Prakash K, Gupta T, Sriraman R, Nagendrakumar SB, Srinivasan VA: Zona pellucida-based contraceptive vaccines for human and animal utility. Journal of reproductive immunology 2011, 88 (2 ): 240-246.
9.Ferro VA:Current advances in antifertility vaccines for fertility control and noncontraceptive applications.Expert review of vaccines 2002,1(4):443-452.9. Ferro VA: Current advances in antifertility vaccines for fertility control and noncontraceptive applications. Expert review of vaccines 2002, 1(4): 443-452.
10.Perez-Lopez FR:Pituitary repetitive stimulation with GnRH/TRH in women treated with three different oral steroid contraceptives.Acta endocrinologica 1990,122(2):163-167. 10. Perez-Lopez FR: Pituitary repetitive stimulation with GnRH/TRH in women treated with three different oral steroid contraceptives. Acta endocrinologica 1990, 122(2): 163-167.
11.Talwar GP,Singh OM,Gupta SK,Hasnain SE,Pal R,Majumbar SS,Vrati S,Mukhopadhay A,Srinivasan J,Deshmukh U et al:The HSD-hCG vaccine prevents pregnancy in women:feasibility study of a reversible safe contraceptive vaccine.American journal of reproductive immunology 1997,37(2):153-160.11.Talwar GP, Singh OM, Gupta SK, Hasnain SE, Pal R, Majumbar SS, Vrati S, Mukhopadhay A, Srinivasan J, Deshmukh U et al: The HSD-hCG vaccine opposite pregnancy in women: feasibility study of a reversible safe Contraceptive vaccine. American journal of reproductive immunology 1997, 37(2): 153-160.
12.Manning PA:Immunogenicity trial of a contraceptive vaccine based on human chorionic gonadotropin.Vaccine 1986,4(4):271.12. Manning PA: Immunogenicity trial of a contraceptive vaccine based on human chorionic gonadotropin. Vaccine 1986, 4(4): 271.
13.Stevens VC:Progress in the development of human chorionic gonadotropin antifertility vaccines.American journal of reproductive immunology 1996,35(3):148-155.13. Stevens VC: Progress in the development of human chorionic gonadotropin antifertility vaccines. American journal of reproductive immunology 1996, 35(3): 148-155.
14.Saxena HM:Predictions of Immunological Cross-Reactions of C-Terminal Peptide of Human Chorionic Gonadotropin beta-Chain Based Contraceptive Vaccine with Autoantigens.Russian journal of immunology:RJI:official journal of Russian Society of Immunology 1999,4(2):151-158.14. Saxena HM: Predictions of Immunological Cross-Reactions of C-Terminal Peptide of Human Chorionic Gonadotropin beta-Chain Based Contraceptive Vaccine with Autoantigens.Russian journal of immunology: RJI:Official journal of Russian Society of Immunology 1999,4(2): 151-158.
15.Zhang FC,Wang N,Liu DM,Jian Y,Chen YZ,Shen XZ,Cao YQ,Wang B:Development of Female Contraceptive Vaccine Through DNA Inoculation of Human Chorionic Gonadotropin Beta Subunit(hCGss).Methods in molecular medicine 2000,29:439-449.15.Zhang FC, Wang N, Liu DM, Jian Y, Chen YZ, Shen XZ, Cao YQ, Wang B: Development of Female Contraceptive Vaccine Through DNA Inoculation of Human Chorionic Gonadotropin Beta Subunit (hCGss). Methods in molecular medicine 2000, 29:439-449.
16.Gupta SK,Srivastava N,Choudhury S,Rath A,Sivapurapu N,Gahlay GK,Batra D:Update on zona pellucida glycoproteins based contraceptive vaccine.Journal of reproductive immunology 2004,62(1-2):79-89.16. Gupta SK, Srivastava N, Choudhury S, Rath A, Sivapurapu N, Gahlay GK, Batra D: Update on zona pellucida glycoproteins based contraceptive vaccine. Journal of reproductive immunology 2004, 62(1-2): 79-89.
17.Gupta SK,Jethanandani P,Afzalpurkar A,Kaul R,Santhanam R:Prospects of zona pellucida glycoproteins as immunogens for contraceptive vaccine.Human reproduction update 1997,3(4):311-324.17. Gupta SK, Jethanandani P, Afzalpurkar A, Kaul R, Santhanam R: Prospects of zona pellucida glycoproteins as immunogens for contraceptive vaccine. Human reproduction update 1997, 3(4): 311-324.
18.Naz RK:Development of genetically engineered human sperm immunocontraceptives.Journal of reproductive immunology 2009,83(1-2):145-150.18. Naz RK: Development of genetically engineered human sperm immunocontraceptives. Journal of reproductive immunology 2009, 83(1-2): 145-150.
尽管避孕的疫苗的研究在不同的方面取得了长足的进步，目前还没有一种避孕疫苗应用于临床实践，开发避孕疫苗一直处在研究阶段。上述非常有前景的靶标抗原疫苗存在的问题包括：第一靶向激素及其受体可能造成机体内分泌失调，影响机体正常功能；第二，靶向生殖细胞可能造成组织损伤，发生不可逆性不孕不育；第三靶标疫苗分子不能产生足够的抗体滴度，影响避孕效果，造成避孕失败。因此避孕疫苗的研究、开发和临床应用具有广阔的前景，迫切需要一种高效、 价格合适、使用舒适、能够推广应用、物种选择性低的避孕疫苗来满足人类避孕、外来动物入侵和野生动物种群失衡防控的需求。Although the research on contraceptive vaccines has made great progress in different aspects, there is currently no contraceptive vaccine used in clinical practice, and the development of contraceptive vaccines has been in the research stage. The problems of the above-mentioned very promising target antigen vaccine include: the first targeted hormone and its receptor may cause the body's endocrine disorders, affecting the normal function of the body; second, targeting germ cells may cause tissue damage, irreversible infertility Infertility; the third target vaccine molecule does not produce sufficient antibody titers, affecting the contraceptive effect, resulting in contraceptive failure. Therefore, the research, development and clinical application of contraceptive vaccines have broad prospects, and there is an urgent need for an efficient Contraceptive vaccines that are affordable, comfortable to use, can be applied, and have low species selectivity to meet the needs of human contraception, foreign animal invasion and wildlife population imbalance control.
发明内容Summary of the invention
本发明首次提出避孕疫苗针对胚胎附植期这样一个胎盘形成和胚胎发育的关键、敏感的时期，靶向附植执行关键细胞滋养层细胞的观点，可以提高避孕的敏感性和有效性。将胎盘滋养层特异性分泌的pl-CSA单独或者与其核心蛋白CSPG4的B细胞优势抗原表位组合制备避孕疫苗，利用其核心蛋白CSPG4的B细胞优势抗原表位来诱导B细胞的抗原反应，增强免疫作用，进一步增加避孕效果，从而制备一种安全、有效的避孕疫苗。The present invention firstly proposes a contraceptive vaccine for a critical and sensitive period of embryonic attachment stage such as placental formation and embryo development, and targeting the attachment of cells to perform key cytotrophoblast cells can improve the sensitivity and effectiveness of contraception. The pl-CSA secreted by the placenta trophoblast alone or in combination with the B cell dominant epitope of its core protein CSPG4 is used to prepare a contraceptive vaccine, and the B cell dominant epitope of the core protein CSPG4 is used to induce the antigenic reaction of the B cell. Immune action further increases the effect of contraception, thus preparing a safe and effective contraceptive vaccine.
本发明一个方面提供了一种避孕用免疫组合物，其中包含至少一种胎盘滋养层细胞特异表达多糖和/或蛋白抗原。One aspect of the present invention provides an immunological composition for contraception comprising at least one placental trophoblast cell-specifically expressing a polysaccharide and/or a protein antigen.
在本发明的技术方案中，所述的胚胎或胎盘滋养层细胞特异表达多糖选自胎盘样硫酸软骨素A。In the technical solution of the present invention, the embryo or placental trophoblast cell-specific expression polysaccharide is selected from the group consisting of placenta-like chondroitin sulfate A.
在本发明的技术方案中，免疫组合物中还包含免疫组合物中还包含诱导B细胞的抗原反应的免疫增强剂，所述免疫增强剂选自胎盘样硫酸软骨素A核心蛋白CSPG4的B细胞优势抗原表位，优选地，所述胎盘样硫酸软骨素A核心蛋白CSPG4的B细胞优势抗原表位如序列SEQ ID No.2或其同源物。In the technical solution of the present invention, the immunological composition further comprises an immunopotentiating agent further comprising an antigenic reaction for inducing B cells, wherein the immunopotentiating agent is selected from a B cell of placenta-like chondroitin sulfate core protein CSPG4. A dominant epitope, preferably, the B cell dominant epitope of the placental-like chondroitin sulfate core protein CSPG4 is SEQ ID No. 2 or a homolog thereof.
IVAVDEPTRPIYRFTQ SEQ ID No.2IVAVDEPTRPIYRFTQ SEQ ID No. 2
在本发明的技术方案中，胚胎或胎盘滋养层细胞特异表达多糖和/或蛋白抗原与CSPG4的B细胞优势抗原表位的质量比为1-100：1，优选为1-10：1。In the embodiment of the present invention, the mass ratio of the embryonic or placental trophoblast cell-specific expression polysaccharide and/or protein antigen to the B cell dominant epitope of CSPG4 is 1-100:1, preferably 1-10:1.
在本发明的技术方案中，避孕用免疫组合物中还包含佐剂。In the technical solution of the present invention, the immunological composition for contraception further comprises an adjuvant.
在本发明的技术方案中，佐剂选自弗氏完全佐剂、弗氏不完全佐剂、铝(磷酸铝或氢氧化铝)、皂苷、Quil A、RIBBI佐剂、二甲基双十八烷基溴化铵(DDA)、单磷酰脂质A(MPL)、海藻糖二霉菌酸酯(TDM)、海藻糖二山嵛酸酯(dibehenate)和胞壁酰二肽(MDP)、聚I:C等。In the technical solution of the present invention, the adjuvant is selected from Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum (aluminum phosphate or aluminum hydroxide), saponin, Quil A, RIBBI adjuvant, dimethyl double 18 Alkyl ammonium bromide (DDA), monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), trehalose dibehenate and muramyl dipeptide (MDP), poly I: C and so on.
本发明另一个方面提供一种药物组合物，其中包含了本发明的所述的免疫组合物。 Another aspect of the invention provides a pharmaceutical composition comprising the immunological composition of the invention.
在本发明的技术方案中，所述的药物组合物中还包含其它避孕和/或中止妊娠的活性成分。所述的避孕和/或中止妊娠的活性成分示例性的可以为去氧孕烯炔雌醇、壬苯醇醚栓、壬苯醇醚、米非司酮、左炔诺孕酮、左炔诺孕酮炔雌醚。In the technical solution of the present invention, the pharmaceutical composition further comprises other active ingredients for contraception and/or termination of pregnancy. Exemplary active ingredients for contraception and/or termination of pregnancy may be desogestrel ethinyl estradiol, nonoxynol ether suppository, nonoxynol ether, mifepristone, levonorgestrel, levonorol Progesterone ethinyl estradiol.
本发明另一个方面提供了本发明所述的免疫组合物在制备阻止胚胎附植于子宫内膜的药物中的用途。Another aspect of the invention provides the use of an immunological composition of the invention in the manufacture of a medicament for preventing embryos from adhering to the endometrium.
本发明另一个方面提供了本发明所述的免疫组合物用于阻止胚胎附植于子宫内膜中的用途。Another aspect of the invention provides the use of an immunological composition of the invention for preventing embryos from being implanted in the endometrium.
本发明另一个方面提供了本发明所述的免疫组合物在制备中止妊娠的药物中的用途。Another aspect of the invention provides the use of an immunological composition of the invention in the manufacture of a medicament for the termination of pregnancy.
本发明另一个方面提供了本发明所述的免疫组合物在用于中止妊娠中的用途。Another aspect of the invention provides the use of an immunological composition of the invention for the termination of pregnancy.
本发明另一个方面提供了本发明所述的免疫组合物在制备避孕药物中的用途。Another aspect of the invention provides the use of an immunological composition of the invention in the manufacture of a contraceptive.
本发明另一个方面提供了本发明所述的免疫组合物在用于避孕中的用途。Another aspect of the invention provides the use of an immunological composition of the invention for use in contraception.
本发明再一个方面提供了本发明所述的免疫组合物在免疫人类及非人类动物抑制人类或非人类怀孕的方法，所述方法包含向动物给药治疗有效量的包含一种或多种本发明所述的免疫组合物。In a further aspect, the present invention provides a method of inhibiting human or non-human pregnancy in an immune human and non-human animal, comprising administering to the animal a therapeutically effective amount comprising one or more The immunological composition of the invention.
在本发明的技术方案中，所述的胚胎附植于子宫内膜、避孕或中止妊娠的对象或受试者为人类或者非人类的动物，其中包括驯养动物和非驯养动物。In the technical solution of the present invention, the embryo is attached to the endometrium, the subject of the contraception or the termination of pregnancy or the subject is a human or non-human animal, including domesticated animals and non-domestic animals.
在本发明的技术方案中，所述的非人类动物是驯养动物。例如，但不限于，狗、猫、大鼠、小鼠、牛、马、羊、猪、兔等。In the technical solution of the present invention, the non-human animal is a domesticated animal. For example, but not limited to, dogs, cats, rats, mice, cows, horses, sheep, pigs, rabbits, and the like.
在本发明的技术方案中，所述的非人类动物是野生动物。例如，但不限于，野生动物是狗、猫、大鼠、小鼠、蝙蝠、狐狸、浣熊、松鼠、负鼠、狼、兔等。In the technical solution of the present invention, the non-human animal is a wild animal. For example, but not limited to, wild animals are dogs, cats, rats, mice, bats, foxes, raccoons, squirrels, possums, wolves, rabbits, and the like.
本发明再一个方面提供了胎盘滋养层细胞特异表达多糖和/或蛋白抗原作为避孕或阻止胚胎附植于子宫内膜或中止妊娠的作用靶点的用途，或者作为筛选避孕或阻止胚胎附植于子宫内膜或中止妊娠的药物靶点的用途。Still another aspect of the present invention provides a use of a placental trophoblast cell-specific expression of a polysaccharide and/or a protein antigen as a target for contraception or preventing embryos from attaching to the endometrium or terminating a pregnancy, or as a screening contraceptive or preventing embryos from being attached to Use of endometrial or drug targets for termination of pregnancy.
在本发明中，免疫组合物或药物组合物中可以包含可药用载体，包括但不限于盐水、缓冲盐水、葡萄糖、水、甘油、乙醇及其组合。载体和免疫组合物可以是无菌的，并且与给药方式相适合。免疫组合物或药物组合物也可以药物制剂中常用的其它辅料，例如含有少量润湿剂、乳化剂或pH缓冲剂。 In the present invention, a pharmaceutically acceptable carrier can be included in the immunological composition or pharmaceutical composition including, but not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and immunological composition can be sterile and compatible with the mode of administration. The immunological composition or pharmaceutical composition may also be other excipients commonly used in pharmaceutical preparations, for example containing a small amount of wetting agent, emulsifier or pH buffer.
本发明的免疫组合物或药物组合物可以是液体溶液、悬液、乳液、持续释放剂型或粉剂。可以使用任何常用药物载体例如无菌盐水溶液或芝麻油。介质也可以包含常规的药用辅助物质例如调整渗透压的可药用盐、缓冲剂、防腐剂等。The immunological composition or pharmaceutical composition of the invention may be a liquid solution, suspension, emulsion, sustained release dosage form or powder. Any conventional pharmaceutical carrier such as sterile saline solution or sesame oil can be used. The medium may also contain conventional pharmaceutical auxiliary substances such as pharmaceutically acceptable salts for adjusting osmotic pressure, buffers, preservatives and the like.
本发明的免疫组合物或药物组合物的给药方式可以通过任何途径，包括但不局限于，通过口腔、经皮、粘膜(例如，阴道、直肠、口腔或鼻粘膜)、注射(例如，肌肉、皮下的、静脉内、肠胃外的、腹膜内的、鞘内的)或通过吸入(例如，口腔的或鼻的)。根据给药方式的不同，可以调整本发明免疫组合物或疫苗或药物中的辅料以及制备方法。The immunological composition or pharmaceutical composition of the present invention can be administered by any route including, but not limited to, by oral, transdermal, mucosal (e.g., vaginal, rectal, buccal or nasal mucosa), injection (e.g., muscle). , subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal) or by inhalation (eg, buccal or nasal). The adjuvant of the immunological composition or vaccine or medicament of the present invention and the preparation method can be adjusted depending on the mode of administration.
在本发明中，所述“附植”与“着床”含义相同，是指胚胎滋养层与子宫内膜逐渐发生组织学和生理学的联系，使胚泡固着于子宫内膜，这一生理过程成为附植。In the present invention, the meaning of "attachment" and "implantation" means that the embryonic trophoblast and the endometrium gradually associate with histology and physiology, so that the blastocyst is fixed in the endometrium, this physiological process Become attached.
在本发明中，所述“药物组合物”和“免疫组合物”均按其最宽含义理解，例如，免疫组合物为能够引发免疫反应的组合物，药物组合物为能够起到引发受试者机体向预期的健康方向发展的组合物。In the present invention, both the "pharmaceutical composition" and the "immunological composition" are understood in their broadest sense, for example, the immunological composition is a composition capable of eliciting an immune response, and the pharmaceutical composition is capable of causing the test to be initiated. A composition that develops in the direction of expected health.
有益效果Beneficial effect
1、本发明首次提出避孕疫苗针对附植早期这样一个胎盘形成和胚胎发育的关键、敏感的时期，靶向附植执行关键细胞滋养层细胞的观点，提高避孕的敏感性和有效性。1. The present invention firstly proposes a contraceptive vaccine for a critical and sensitive period of early placental formation and embryo development, targeting the attachment of cells to perform key cytotrophoblast cells, and improving the sensitivity and effectiveness of contraception.
2、pl-CSA疫苗靶向的是滋养层细胞，这些细胞存在于囊胚滋养外胚层和胎盘中，与机体其他的正常细胞(如下丘脑-垂体-卵巢等组织细胞)仅有较低的交叉反应性，不容易损害机体组织器官正常功能的行使。2. The pl-CSA vaccine targets trophoblast cells, which are present in the blastocyst ectoderm and placenta, and have only a low cross with other normal cells of the body (such as thalamic-pituitary-ovarian tissue cells). Reactivity, it is not easy to damage the normal function of the body tissues and organs.
3、pl-CSA是大分子多糖，结构较为稳定，免疫原性强，免疫动物能够产生较高的抗体滴度；同时将pl-CSA与其核心蛋白CSPG4的B细胞优势抗原表位组合，可以诱导B细胞的抗原反应，增强免疫作用，从而增加避孕效果；3. pl-CSA is a macromolecular polysaccharide with stable structure and strong immunogenicity. The immunized animal can produce higher antibody titer. At the same time, pl-CSA can be induced by combining the B cell dominant epitope with its core protein CSPG4. B cell antigen response, enhance immune function, thereby increasing contraceptive effect;
4、pl-CSA比较容易获得，且不同物种来源pl-CSA相似性较高。滋养层细胞中pl-CSA含量比较丰富，废弃的猪胎盘中pl-CSA含量也非常丰富，从猪胎盘中分离纯化pl-CSA可以变废为宝。4, pl-CSA is relatively easy to obtain, and the similarity of pl-CSA from different species sources is higher. The pl-CSA content in the trophoblast cells is abundant, and the pl-CSA content in the discarded pig placenta is also very rich. Separating and purifying pl-CSA from the pig placenta can turn waste into treasure.
附图说明DRAWINGS
图1为避孕疫苗工作原理图。Figure 1 shows the working principle of the contraceptive vaccine.
图2为本发明的技术路线图。 2 is a technical roadmap of the present invention.
图3为pl-CSA在人、小鼠和猪胎盘滋养层细胞的表达及与CSPG4的共定位图。pl-CSA是以rVAR2和Dylight650标记的抗6×His单克隆抗体进行荧光染色，CSPG4是以兔抗CSPG4单克隆抗体和FITC标记的山羊抗兔二抗进行荧光染色，DAPI标记细胞核，Merge是图片组合，人胎盘滋养层来自分娩胎盘，小鼠胎盘滋养层来自妊娠15d胎盘，猪胎盘和子宫肌层来自妊娠70d胎盘和子宫肌。由共聚焦结果图可知，在人类、小鼠和猪的胎盘滋养层中均包含了pl-CSA和CSPG4，而猪子宫肌内无pl-CSA和CSPG4表达。Figure 3 shows the expression of pl-CSA in human, mouse and porcine placental trophoblast cells and colocalization with CSPG4. pl-CSA was fluorescently stained with anti-6×His monoclonal antibody labeled with rVAR2 and Dylight650. CSPG4 was fluorescently stained with rabbit anti-CSPG4 monoclonal antibody and FITC-labeled goat anti-rabbit secondary antibody. DAPI labeled nuclei, Merge is a picture In combination, the human placental trophoblast comes from the placenta of the delivery, the mouse placental trophoblast comes from the 15d placenta during pregnancy, and the pig placenta and myometrium are from the 70s placenta and uterine muscle. From the confocal result map, pl-CSA and CSPG4 were contained in the placental trophoblast of human, mouse and pig, while pu-CSA and CSPG4 were not expressed in pig uterus.
图4为pl-CSA免疫BABL/c小鼠结果图。其中，A.pl-CSA样品和标准品波普扫面，吸收波长一致；B.实验鼠解剖情况；C.实验鼠体重变化情况；D.实验鼠抗体水平；E.实验鼠受孕率。Figure 4 is a graph showing the results of pl-CSA immunization of BABL/c mice. Among them, A.pl-CSA sample and standard wave scanning surface, the absorption wavelength is consistent; B. experimental mouse anatomy; C. experimental mouse weight change; D. experimental mouse antibody level; E. experimental mouse pregnancy rate.
图5为pl-CSA与其核心蛋白B细胞优势抗原表位CBZ-1组装疫苗免疫BABL/c小鼠结果图。A.实验鼠体重变化情况；B.实验鼠受孕率；C.实验鼠抗体水平。Figure 5 is a graph showing the results of BABL/c immunization of pl-CSA and its core protein B cell dominant epitope CBZ-1. A. Changes in body weight of experimental rats; B. Pregnancy rate of experimental mice; C. Antibody level of experimental mice.
图6为为急性毒性实验鼠体重变化结果图。Figure 6 is a graph showing the results of changes in body weight of rats in acute toxicity.
图7为免疫鼠内脏(包括肝、脾和肾)组织切片、HE染色结果图。Figure 7 is a graph showing the results of HE staining of tissue sections of infected rat viscera (including liver, spleen and kidney).
具体实施方式Detailed ways
以下列实施例说明某些具体的特点和/或实施方案。这些实施例不应该被解释为将本公开限制于所描述的具体特征或实施方案。Certain specific features and/or embodiments are described in the following examples. These examples should not be construed as limiting the disclosure to the particular features or embodiments described.
实施例1pl-CSA及其核心蛋白CSPG4的表达和定位Example 1 Expression and localization of pl-CSA and its core protein CSPG4
此部分采用免疫荧光的方式进行。首先，收集人的分娩胎盘、小鼠妊娠15d胎盘和猪妊娠70d子宫胎盘，4％的多聚甲醛固定充分；按着实施例5的方式制片；3％H2O2(10min)灭活内源性过氧化物酶；pH6.0的柠檬酸盐缓冲液95℃水浴保持10min进行抗原修复；兔抗CSPG4抗体(1:200)和自制的rVAR2-His-tag(1:100)试剂4℃孵育过夜；PBST(含0.1％Triton X-100)浸洗3次，每次5min；FITC标记的羊抗兔二抗(1:1000)和Dylight650标记的抗6×His单克隆抗体(1:1000)，室温孵育40min；PBST浸洗3次，每次5min；DAPI复染核；PBST(含0.1％Triton X-100)浸洗3次，每次5min→防荧光猝灭封片剂封片，荧光共聚焦显微镜观察拍照。实验结果参见图3。从图3结果可以看出 在人类、小鼠和猪的胎盘滋养层中均包含了pl-CSA和CSPG4，而猪子宫肌内无pl-CSA和CSPG4表达。This part is carried out by immunofluorescence. First, the human placenta of the child, the 15d placenta of the pregnancy and the 70t of the gestation of the pig were collected, and 4% of the paraformaldehyde was fixed; the film was prepared in the same manner as in Example 5; 3% H 2 O 2 (10 min) was inactivated. Endogenous peroxidase; pH 6.0 citrate buffer 95 ° C water bath for 10 min for antigen retrieval; rabbit anti-CSPG4 antibody (1:200) and homemade rVAR2-His-tag (1:100) reagent 4 Incubate overnight at °C; PBST (containing 0.1% Triton X-100) was immersed 3 times for 5 min each; FITC-labeled goat anti-rabbit secondary antibody (1:1000) and Dylight650-labeled anti-6×His monoclonal antibody (1: 1000), incubate for 40 min at room temperature; immersed in PBST 3 times for 5 min each; DAPI counterstained nuclei; PBST (containing 0.1% Triton X-100) was washed 3 times for 5 min each time → anti-fluorescence quenching and sealing tablets Photographing was performed by fluorescence confocal microscopy. See Figure 3 for the experimental results. It can be seen from the results in Fig. 3 that pl-CSA and CSPG4 are contained in the placental trophoblast of human, mouse and pig, while there is no expression of pl-CSA and CSPG4 in the pig uterus.
实施例2免疫剂的制备Example 2 Preparation of an immunizing agent
制备并纯化胎盘样硫酸软骨素A：Preparation and purification of placenta-like chondroitin sulfate A:
(1)猪胎盘破碎或者连续培养的滋养层细胞系半机械分离，得到组织细胞浆液；(1) The semi-mechanical separation of the trophoblast cell line with broken or continuous culture of pig placenta, obtaining tissue cell serum;
(2)将步骤(1)获得的组织细胞浆液与裂解液混合(V细胞浆液:V裂解液＝1:10)，裂解液中含RNase、DNase I、胰酶和protease K，得到裂解的组织细胞浆液；(2) mixing the tissue cell slurry obtained in the step (1) with the lysate (V cell slurry: V lysate = 1:10), and the lysate contains RNase, DNase I, trypsin and protease K to obtain a lysed tissue. Cell serum
(3)将步骤(2)获得的裂解的组织细胞浆液灭活裂解液中的酶；加入1/4体积的Sevag液除蛋白，得到pl-CSA粗品；(3) inactivating the enzyme in the lysate by the lysed tissue cell slurry obtained in the step (2); adding a 1/4 volume of the Sevag solution to remove the protein to obtain a crude pl-CSA;
(4)对疟原虫感染红细胞表面抗原VAR2CSA基因序列SEQ ID No.1的5’端加入BamH I酶切位点，3’端加入Sal I酶切位点；将基因片段SEQ ID No.1与pET28a(+)原核表达载体相连，转化大肠杆菌BL21感受态细胞，进行原核表达，利用rVAR2末端6×His标签进行Ni2+亲和层析，获得高纯度的rVAR2(684.1μg/mL)。(4) Adding the BamH I restriction site to the 5' end of the VAR2CSA gene sequence of Plasmodium erythrocyte surface antigen SEQ ID No. 1, and adding the Sal I restriction site at the 3'end; the gene fragment SEQ ID No. 1 and The pET28a(+) prokaryotic expression vector was ligated and transformed into E. coli BL21 competent cells for prokaryotic expression. Ni2 + affinity chromatography was performed using rVAR2 terminal 6×His tag to obtain high purity rVAR2 (684.1 μg/mL).
SEQ ID No.1如下所示SEQ ID No. 1 is as follows
Figure PCTCN2017116629-appb-000002
Figure PCTCN2017116629-appb-000002
Figure PCTCN2017116629-appb-000003
Figure PCTCN2017116629-appb-000003
Figure PCTCN2017116629-appb-000004
Figure PCTCN2017116629-appb-000004
(5)将步骤(4)中获得的高纯度的rVAR2与NHS活化琼脂糖(浓度4％)偶联：成功偶联的琼脂糖凝胶既为pl-CSA的rVAR2亲和层析柱；连接储液瓶、管道、电极三通、恒流泵、层析柱、管道、恒流泵、电极二通、样品或废液收集瓶，完成亲和层析纯化系统的组建。(5) Coupling the high-purity rVAR2 obtained in the step (4) with the NHS-activated agarose (concentration: 4%): the successfully coupled agarose gel is a rVAR2 affinity chromatography column of pl-CSA; The liquid storage bottle, the pipeline, the electrode tee, the constant current pump, the chromatography column, the pipeline, the constant current pump, the electrode two-pass, the sample or the waste liquid collection bottle, complete the establishment of the affinity chromatography purification system.
(6)将步骤(3)中获得的pl-CSA粗品步骤5)所得的层析柱进行分离，收集高纯度pl-CSA，以苯酚硫酸法检测pl-CSA浓度。(6) The column obtained in the step (p) of the crude pl-CSA obtained in the step (3) was separated, and high-purity pl-CSA was collected, and the concentration of pl-CSA was measured by a phenol sulfuric acid method.
制备pl-CSA疫苗和pl-CSA+CBZ-1疫苗Preparation of pl-CSA vaccine and pl-CSA+CBZ-1 vaccine
根据生物信息学分析(http://crdd.osdd.net/raghava/abcpred/)CSPG4蛋白(ID：NP_001888.2)B细胞优势抗原表位，选择参数较高B细胞优势抗原表位的CBZ-1，序列如SEQ ID No.2所示(IVAVDEPTRPIYRFTQ SEQ ID No.2)，化学合成多肽SEQ ID No.2；将多肽与纯化的pl-CSA按质量比1:1-1:10混合，超声条件完成自组装，获得pl-CSA+CBZ-1免疫原颗粒。According to the bioinformatics analysis (http://crdd.osdd.net/raghava/abcpred/) CSPG4 protein (ID: NP_001888.2) B cell dominant epitope, CBZ- with higher B cell dominant epitope was selected. 1, the sequence is shown in SEQ ID No. 2 (IVAVDEPTRPIYRFTQ SEQ ID No. 2), chemically synthesized polypeptide SEQ ID No. 2; the polypeptide and purified pl-CSA mixed at a mass ratio of 1:1-1:10, ultrasound The self-assembly was completed under conditions to obtain pl-CSA+CBZ-1 immunogen particles.
将纯化的pl-CSA配置成溶液pl-CSA免疫原颗粒。将得到的pl-CSA免疫原颗粒和pl-CSA+CBZ-1免疫原颗粒与弗氏完全佐剂和不完全佐剂分别混合、乳化，制备pl-CSA疫苗和pl-CSA+CBZ-1疫苗。The purified pl-CSA was configured as a solution pl-CSA immunogen particle. The obtained pl-CSA immunogen particles and pl-CSA+CBZ-1 immunogen particles were mixed and emulsified with Freund's complete adjuvant and incomplete adjuvant, respectively, to prepare pl-CSA vaccine and pl-CSA+CBZ-1 vaccine. .
实施例3pl-CSA的免疫原性和避孕效果评价Example 3 Evaluation of immunogenicity and contraceptive effect of pl-CSA
表2.pl-CSA疫苗免疫分组Table 2. pl-CSA vaccine immunization group
Figure PCTCN2017116629-appb-000005
Figure PCTCN2017116629-appb-000005
Figure PCTCN2017116629-appb-000006
Figure PCTCN2017116629-appb-000006
①免疫分组：6周龄雌性BALB/c小鼠，随机分成4组，以PBS为阴性对照，每组20只(表2)，另外饲养40只10周龄BALB/c雄鼠。1 Immunization group: 6-week-old female BALB/c mice were randomly divided into 4 groups, with PBS as a negative control, 20 rats in each group (Table 2), and another 40 10-week-old BALB/c male rats.
②免疫程序：各组雌性小鼠自由采食和饮水，每笼2只，接种pl-CSA以及pl-CSA+CBZ-1疫苗的时间分别为0、1、2周(购入时设置为0周)，共免疫3次，第一次使用弗氏完全佐剂与免疫原混合，第二、三次使用弗氏不完全佐剂与免疫原混合。第4周开始将各组雌鼠与雄鼠合笼，查到阴栓后第9天采用B超监测是否怀孕。如果怀孕，即胚胎已经附植成功，则在妊娠第15天后将小鼠处死，解剖观察胚胎发育，收集血清，用于间接ELISA检测抗体水平；如果不怀孕，连续6个发情周期与雄鼠合笼，所有雌鼠均以检测到阴栓，证明所有雌鼠均已完成交配。处死6个发情周期未怀孕小鼠，收集血清，用于间接ELISA检测抗体的产生水平。2 Immunization procedure: Female mice in each group were fed ad libitum and drinking water, 2 per cage, and the time of inoculation of pl-CSA and pl-CSA+CBZ-1 vaccine was 0, 1, 2 weeks respectively (set to 0 when purchased) Week), a total of 3 immunizations, the first use of Freund's complete adjuvant mixed with the immunogen, and the second and third use of Freund's incomplete adjuvant mixed with the immunogen. At the 4th week, the female rats of each group were caged together with the male rats. On the 9th day after the detection of the female emboli, B-ultrasound was used to monitor pregnancy. If pregnant, that is, the embryo has been successfully implanted, the mice will be sacrificed after the 15th day of pregnancy, the embryo development will be dissected, serum will be collected for indirect ELISA to detect antibody levels; if not pregnant, 6 consecutive estrus cycles will be combined with male rats. In the cage, all females were tested for vaginal plugs, demonstrating that all females had completed mating. Six pregnant mice with estrus cycles were sacrificed and serum was collected for indirect ELISA to detect antibody production levels.
以pl-CSA单独使用的避孕疫苗Ig1组将小鼠受孕率降低至43％，空白组NcIg1受孕率为75％；以pl-CSA与其核心蛋白B细胞优势抗原表位CBZ-1(IVAVDEPTRPIYRFTQ)多肽组装制备的避孕疫苗Ig2组将小鼠受孕率降低至35％，空白组ncIg2受孕率为85％。实验结果显示，pl-CSA免疫剂和pl-CSA+CBZ-1免疫剂相比空白对照组能显著降低受孕率，而pl-CSA+CBZ-1组相比于pl-CSA的避孕效果更佳。The contraceptive vaccine Ig1 used alone in pl-CSA reduced the pregnancy rate to 43%, and the blank group NcIg1 had a pregnancy rate of 75%; pl-CSA and its core protein B cell dominant epitope CBZ-1 (IVAVDEPTRPIYRFTQ) polypeptide The Ig2 group prepared by the assembled contraceptive vaccine reduced the pregnancy rate of the mice to 35%, and the pregnancy rate of the blank group ncIg2 was 85%. The experimental results showed that the pl-CSA immunizing agent and the pl-CSA+CBZ-1 immunizing agent can significantly reduce the conception rate compared with the blank control group, while the pl-CSA+CBZ-1 group has better contraceptive effect than the pl-CSA. .
实施例4pl-CSA避孕疫苗的安全性评价Example 4 Safety Evaluation of pl-CSA Contraceptive Vaccine
急性毒性实验：将6周雌性BALB/c小鼠随机分成8组，每组10只，采取肌肉注射给药；其中pl-CSA免疫剂4组，包括pl-CSA-Tg1组(100μg/只)、pl-CSA-Tg2组(200μg/只)、pl-CSA-Tg3组(400μg/只)、pl-CSA-Tg4组(800μg/只)；pl-CSA+CBZ-1免疫剂4组，包括CBZ-1-Tg1组(100μg/只)、CBZ-1-Tg2组(200μg/只)、CBZ-1-Tg3组(400μg/只)、CBZ-1-Tg4组(800μg/只)，单次给药200μL/只，连续观察2w，分析体征变化和死亡情况。Acute toxicity test: 6-week female BALB/c mice were randomly divided into 8 groups, 10 in each group, and administered intramuscularly; 4 groups of pl-CSA immunizing agents, including pl-CSA-Tg1 group (100 μg/only) , pl-CSA-Tg2 group (200μg/only), pl-CSA-Tg3 group (400μg/only), pl-CSA-Tg4 group (800μg/only); pl-CSA+CBZ-1 immunizing agent 4 groups, including CBZ-1-Tg1 group (100μg/only), CBZ-1-Tg2 group (200μg/only), CBZ-1-Tg3 group (400μg/only), CBZ-1-Tg4 group (800μg/only), single time 200 μL/mouse was administered, and 2 weeks were continuously observed, and the changes in signs and death were analyzed.
实验结果显示，除CBZ-1-Tg4组1只小鼠注射给药后10min内死亡外，其余小鼠体质量无显著性差异(图6)，给药后连续观2w，均未见明显异常，第14d脱颈处死动物剖检，心、肝、脾、肺、肾、胃肠道、脑部大体解剖无明显 病理变化(图7)。The results of the experiment showed that there was no significant difference in the body weight of the mice in the CBZ-1-Tg4 group except for 10 minutes after the injection (Fig. 6). After the administration, the body was continuously observed for 2 weeks, and no obvious abnormalities were observed. On the 14th day, the animal was removed from the neck and the anatomy of the heart, liver, spleen, lung, kidney, gastrointestinal tract and brain was not obvious. Pathological changes (Figure 7).
实施例5间接ELISA评价抗体水平Example 5 Evaluation of antibody levels by indirect ELISA
针对实施例3中的PBS组、pl-CSA组和pl-CSA+CBZ-1组小鼠，分别检测怀孕小鼠与未孕小鼠的血清中抗体水平。For the PBS group, the pl-CSA group, and the pl-CSA+CBZ-1 group mice in Example 3, serum antibody levels in pregnant mice and non-pregnant mice were measured, respectively.
分别以纯化后pl-CSA多糖2μg作为抗原，稀释于0.05M碳酸盐中包被于96孔板，200μL/well，4℃过夜；弃掉包被液，200μL PBST缓冲液清洗3次，每次5分钟；加入200μL封闭液(5％脱脂奶粉溶于PBST,pH＝7.2)，37℃，孵育2.5h；PBST清洗3次，每次5min；1％BSA(溶于PBST)按1:1000稀释小鼠待检血清，100μL/well，37℃孵育45min；PBST清洗3次，每次10min1％BSA按1:10000稀释HRP标记羊抗鼠IgG,100μL/well，37℃孵育45min；弃掉二抗，PBST彻底清洗5次，每次5min；加入TMB显色液50μL/well，室温，暗室中显色10min；加入2M H2SO4 50μL/well终止反应。15min内于酶标仪中读取OD450数值。2μg of purified pl-CSA polysaccharide was used as antigen, diluted in 0.05M carbonate and coated in 96-well plate, 200μL/well, 4°C overnight; discard the coating solution and wash 3 times with 200μL PBST buffer. 5 minutes; add 200 μL blocking solution (5% skim milk powder dissolved in PBST, pH=7.2), incubate at 37 ° C for 2.5 h; wash with PBST 3 times for 5 min each; 1% BSA (dissolved in PBST) diluted 1:1000 The mice were tested for serum, 100 μL/well, incubated at 37 ° C for 45 min; PBST was washed 3 times, each time 10 min 1% BSA was diluted 1:10000 HRP-labeled goat anti-mouse IgG, 100 μL/well, incubated at 37 ° C for 45 min; discarded secondary antibody PBST was thoroughly washed 5 times for 5 min each time; TMB coloring solution 50 μL/well was added, room temperature, color development in a dark room for 10 min; and 2 M H 2 SO 4 50 μL/well was added to terminate the reaction. The OD450 value was read in the microplate reader within 15 min.
实验结果参见图4D和图5C。实验结果说明给予pl-CSA+CBZ疫苗和pl-CSA疫苗后抗体水平相比于空白组均有显著上升，而无论是给予疫苗组还是在空白组中未孕小鼠的抗体水平均高于怀孕小鼠的抗体水平，且均存在显著性差异。实验结果也证实了，高抗体的水平影响了胚胎附植进而使小鼠能够成功避孕。The experimental results are shown in Figures 4D and 5C. The results of the experiment showed that the antibody levels of pl-CSA+CBZ vaccine and pl-CSA vaccine were significantly higher than those of the blank group, and the antibody levels of the non-pregnant mice in the vaccine group or the blank group were higher than those in the pregnant group. There were significant differences in antibody levels in mice. The experimental results also confirmed that the high antibody level affects the embryo attachment and enables the mice to successfully contraception.
实施例6组织HE和免疫组化染色Example 6 Tissue HE and immunohistochemical staining
对实施例3获得的小鼠取肝脏、脾脏和肾脏分别制片并通过HE染色观察各器官是否有毒性反应，无明显病理变化。The mice obtained in Example 3 were prepared for liver, spleen and kidney, and the toxic reaction of each organ was observed by HE staining, and no obvious pathological changes were observed.
制片：组织样品经4％的中性多聚甲醛(Bouin's液)固定不少于48h，自来水中流水浸洗过夜后制作切片：(1)乙醇梯度脱水：70％乙醇(2h)→80％乙醇(2h)→90％乙醇(30min)→95％乙醇(30min)→100％乙醇(30min)→100％乙醇(30min)。(2)二甲苯透明：50％乙醇二甲苯(12min)→100％二甲苯(10min)→100％二甲苯(10min)。(3)浸蜡：50％二甲苯石蜡(2h)→100％石蜡(1h)→100％石蜡(1h)。(4)切片：5μm连选切片。(5)展片和捞片：45℃蒸馏水展片，洁净载玻片捞片。(6)烤片：60℃烤箱放置20min。(7)脱蜡：100％二甲苯(1min)→100％二甲苯(1min)。(8)水化：100％乙醇(2min)→95％乙醇(1min)→85％乙醇(1min)→75％乙醇(1min)→自然凉干(可-20℃保存备用)。 Production: tissue samples were fixed in 4% neutral paraformaldehyde (Bouin's solution) for not less than 48h, and tapped in tap water overnight to make slices: (1) Ethanol gradient dehydration: 70% ethanol (2h) → 80% Ethanol (2h)→90% ethanol (30min)→95% ethanol (30min)→100% ethanol (30min)→100% ethanol (30min). (2) Xylene transparent: 50% ethanol xylene (12 min) → 100% xylene (10 min) → 100% xylene (10 min). (3) Dip wax: 50% xylene paraffin (2h) → 100% paraffin (1h) → 100% paraffin (1h). (4) Section: 5 μm serial section. (5) Exhibits and scraps: 45 ° C distilled water, clean glass slides. (6) Baking sheet: placed in an oven at 60 ° C for 20 min. (7) Dewaxing: 100% xylene (1 min) → 100% xylene (1 min). (8) Hydration: 100% ethanol (2min)→95% ethanol (1min)→85% ethanol (1min)→75% ethanol (1min)→naturally dry (can be stored at -20°C).
HE染色：上述制定的石蜡切片→苏木精染色液(5min)→蒸馏水流水轻洗(5sec)→1％盐酸乙醇(3sec)→蒸馏水流水轻洗(15sec)→0.5％伊红液染色(45sec)→蒸馏水流水轻洗(30sec)→80％乙醇(2sec)→90％乙醇(2sec)→95％乙醇(5sec)→石碳酸二甲苯(5min)→二甲苯(1min)→二甲苯(1min)→中性树胶封固(树胶干后才移动观察，避免产生气泡)。显微镜下观察观察胎盘组织结构在各妊娠时期的动态变化，拍照。HE staining: Paraffin section prepared above → Hematoxylin staining solution (5 min) → Light water washing with distilled water (5 sec) → 1% hydrochloric acid ethanol (3 sec) → Light wash with distilled water (15 sec) → 0.5% eosin staining (45 sec )→Distilled water, light wash (30 sec)→80% ethanol (2 sec)→90% ethanol (2 sec)→95% ethanol (5 sec)→stone xylene carbonate (5 min)→xylene (1 min)→xylene (1 min) → Neutral gum seal (the gum is dried and observed to avoid bubbles). The dynamic changes of the placental tissue structure during each pregnancy were observed under a microscope and photographed.
实验结果参见附图7，HE染色结果显示免疫鼠内脏(包括肝、脾和肾)组织切片未见明显改变与PBS组结果基本一致，证明包含pl-CSA的避孕疫苗使用基本安全。 The results of the experiment are shown in Fig. 7. The results of HE staining showed that the tissue sections of the visceral organs (including liver, spleen and kidney) of the immunized mice showed no significant changes, which were basically consistent with the results of the PBS group, and the contraceptive vaccine containing pl-CSA was basically safe.

Claims (10)

  1. 一种避孕用免疫组合物，其中包含至少一种胎盘滋养层细胞特异表达多糖和/或蛋白抗原。An immunological composition for contraception comprising at least one placental trophoblast cell-specific expression of a polysaccharide and/or a protein antigen.
  2. 根据权利要求1所述的避孕用免疫组合物，所述的胚胎或胎盘滋养层细胞特异表达多糖选自胎盘样硫酸软骨素A。The contraceptive immunological composition according to claim 1, wherein the embryo or placental trophoblast cell-specific expression polysaccharide is selected from the group consisting of placental-like chondroitin sulfate A.
  3. 根据权利要求1-2任一项所述的避孕用免疫组合物，免疫组合物中还包含诱导B细胞的抗原反应的免疫增强剂，优选地，所述免疫增强剂选自胎盘样硫酸软骨素A核心蛋白CSPG4的B细胞优势抗原表位，更优选地，所述胎盘样硫酸软骨素A核心蛋白CSPG4的B细胞优势抗原表位如序列SEQ ID No.2或其同源物。The immunological composition for contraception according to any one of claims 1 to 2, wherein the immunological composition further comprises an immunopotentiator for inducing an antigenic reaction of the B cell, preferably, the immunopotentiator is selected from the group consisting of placenta-like chondroitin sulfate The B cell dominant epitope of the A core protein CSPG4, more preferably, the B cell dominant epitope of the placental-like chondroitin sulfate core protein CSPG4 is SEQ ID No. 2 or a homolog thereof.
  4. 根据权利要求3所述的避孕用免疫组合物，胚胎或胎盘滋养层细胞特异表达多糖和/或蛋白抗原与CSPG4的B细胞优势抗原表位的质量比为1-100：1，优选为1-10：1。The immunological composition for contraception according to claim 3, wherein the mass ratio of the embryonic or placental trophoblast cell-specific expression polysaccharide and/or protein antigen to the B cell dominant epitope of CSPG4 is 1-100:1, preferably 1- 10:1.
  5. 根据权利要求1-4任一项所述的避孕用免疫组合物避孕用免疫组合物中还包含佐剂；优选地，佐剂选自弗氏完全佐剂、弗氏不完全佐剂、铝(磷酸铝或氢氧化铝)、皂苷、Quil A、RIBBI佐剂、二甲基双十八烷基溴化铵(DDA)、单磷酰脂质A(MPL)、海藻糖二霉菌酸酯(TDM)、海藻糖二山嵛酸酯(dibehenate)和胞壁酰二肽(MDP)、聚I:C。The contraceptive immune composition according to any one of claims 1 to 4, further comprising an adjuvant in the contraceptive immunological composition; preferably, the adjuvant is selected from the group consisting of Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum ( Aluminum phosphate or aluminum hydroxide), saponin, Quil A, RIBBI adjuvant, dimethyldioctadecyl ammonium bromide (DDA), monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) ), trehalose dibehenate and muramyl dipeptide (MDP), poly I: C.
  6. 一种药物组合物，其中包含了权利要求1-5任一项所述的一种或多种免疫组合物，优选地，药物组合物中包含了一组以上独立包装的免疫组合物。A pharmaceutical composition comprising the one or more immunological compositions of any of claims 1-5, preferably a pharmaceutical composition comprising more than one set of individually packaged immunological compositions.
  7. 根据权利要求6所述的药物组合物，药物组合物中还包含其它避孕和/或中止妊娠的活性成分。The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition further comprises other active ingredients for contraception and/or termination of pregnancy.
  8. 胎盘滋养层细胞特异表达多糖和/或蛋白抗原作为避孕或阻止胚胎附植于子宫内膜或中止妊娠的作用靶点的用途，或者作为筛选避孕或阻止胚胎附植于子宫内膜或中止妊娠的药物靶点的用途。 Placental trophoblast cells specifically express polysaccharides and/or protein antigens as a target for contraception or to prevent embryos from attaching to the endometrium or halting pregnancy, or as screening for contraception or preventing embryos from attaching to the endometrium or terminating pregnancy. The use of drug targets.
  9. 权利要求1-5任一项所述的免疫组合物或权利要求6-7任一项所述的药物组合物在用于人类及非人类动物进行避孕或阻止胚胎附植于子宫内膜或中止妊娠的中的用途，优选地，所述用途用于非诊断和治疗目的。The immunological composition according to any one of claims 1 to 5 or the pharmaceutical composition according to any one of claims 6 to 7 for contraception or to prevent embryos from being attached to the endometrium or to stop in humans and non-human animals. Use in pregnancy, preferably, the use is for non-diagnostic and therapeutic purposes.
  10. 权利要求1-5任一项所述的免疫组合物或权利要求6-7任一项所述的药物组合物在制备用于人类及非人类动物避孕药物或阻止胚胎附植于子宫内膜的药物或中止妊娠的药物或免疫制剂中的用途。 The immunological composition according to any one of claims 1 to 5 or the pharmaceutical composition according to any one of claims 6 to 7 for preparing a contraceptive for human and non-human animals or preventing embryos from being attached to the endometrium. The use of drugs or drugs or immunization agents that terminate pregnancy.
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Publication number Priority date Publication date Assignee Title
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* Cited by examiner, † Cited by third party
Title
ZHANG, BAOZHEN ET AL.: "Medical Abortion by Trophoblast-Targeted Nanopartciles Loaded with Methotrexate", T HE 4TH SKLRB SYMPOSIUM ON REPRODUCTIVE BIOLOGY, 27 October 2016 (2016-10-27) *
ZHANG, YINGCHEN: "Anti-fertile Potential and Humoral Immune Reaction of Polypeptide Vaccine Based on Fusogenic Trophoblast Epitope", CHINA MASTER S THESES FULL-TEXT DATABASE (ELECTRONIC JOURNALS), 15 March 2008 (2008-03-15) *

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