Use of a synthetic peptide adjuvant for the immunization of baboons with denatured and deglycosylated pig zona pellucida glycoproteins

Baboons were immunized using a synthetic peptide adjuvant with two purified pig zona pellucida glycoproteins. The major zona pellucida glycoprotein (ZPI) was purified by preparative isoelectric focusing, and the 80 K deglycosylated zona pellucida protein (ZPIII) was purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis. The immunogenicity as well as the antigenicity of these proteins were evaluated by characterizing antibodies using the enzyme-linked immunoassay and by immunoblotting of zona pellucida proteins separated by high-resolution two-dimensional polyacrylamide gel electrophoresis. Both groups of animals developed antibodies that recognize the major zona pellucida glycoprotein, (ZPI) and immunoblotting procedures provide evidence that two of the major porcine zona pellucida glycoprotein families (ZPI and ZPIII) contain shared antigenic determinants. The animals immunized with ZPI showed decreased levels of estrogen throughout their menstrual cycles, and two of the animals ceased ovulation. All animals in the group immunized with ZPIII had a significant reduction in the numbers of antral follicles as compared with control animals. Although ovarian cyclicity was not altered significantly within a few months after immunization, two of the five animals in this group became amenorrheic by 8 months. Histologic analysis of ovarian tissue shows that follicles were absent or frequently abnormal in animals of both groups following long-term immunization. These studies demonstrate that the synthetic adjuvant is effective in inducing antibodies (to purified zona pellucida glycoproteins) that recognize antigenic determinants to either denatured or deglycosylated zona pellucida glycoproteins, and that some of these antibodies may interfere with normal ovarian function.     

The cellular immune response to immunization with zona pellucida antigens.

The cellular immune response of mice to porcine and rat zona pellucida and cynomolgus macaques to porcine zona pellucida antigens was evaluated. Mice mounted a vigorous cellular response to both antigens, as determined by the T cell proliferation response in vitro. There was poor cross-reactivity to murine zonae by T cells or serum antibodies from mice immunized with rat zona pellucida. Nevertheless, ovaries from the mice immunized with rat zona had significantly fewer antral follicles than adjuvant-treated controls, suggesting that the immune response to the zona antigen disrupted follicular development. T cells from two macaques that had been immunized with porcine zona pelludica proteins proliferated in vitro in response to this antigen. Both macaques also had strong antibody responses. The patterns of urinary steroid metabolites in these animals provided clear evidence of ovarian malfunction following immunization. The data indicate that a significant cellular immune response is generated upon immunization of animals with zona pellucida antigens regardless of whether the antigens are cross reactive with the host zona antigens. Whether impaired ovarian function and follicular development are related to the cellular response must be determined in future studies.     

Immunocontraceptive potential of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli and its corresponding synthetic peptide

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for development of immunocontraceptive vaccines. In this study, the efficacy to block fertility by immunization with recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C (r-bmZPC) expressed in Escherichia coli and its synthetic peptide (P(4): KGDCGTPSHSRRQPHVVSQWSRSA, aa residues 324-347) conjugated to diphtheria toxoid (DT) has been evaluated in a homologous system. Female bonnet monkeys, immunized with P(4)-DT conjugate showed better immunocontraceptive potential as compared to an r-bmZPC-DT immunized group. In spite of high anti-P(4) antibody titres, animals continued to have ovulatory cycles and showed no disturbance in cyclicity (except summer amenorrhoea). No ovarian pathology was observed in the P(4) immunized group. These results suggest that immunization with the P(4) may lead to block in fertility without obvious ovarian dysfunction. However, further inputs are required to identify additional ZP based B-cell epitopes to enhance the contraceptive efficacy.     

Use of immunoaffinity purified antibodies to zona pellucida to compare alloimmunization of male and female rabbits

In order to study the immunogenicity as well as tissue specificity of zona pellucida (ZP) antigens, the present studies have been designed to examine the effects of alloimmunization of male and female rabbits with rabbit zonae pellucidae. These studies are the first to demonstrate that high titers of antibodies to homologous ZP antigens are developed in male rabbits while no detectable antibodies are developed in females. As demonstrated using the ELISA assay, the antibodies from these males immunized with rabbit ZP, have a greater reactivity against rabbit ZP antigens than do antibodies from female rabbits heteroimmunized with porcine ZP. The antibodies from the male rabbits immunized with rabbit ZP also recognize antigenic determinants of porcine ZP. Methods for the immunoaffinity purification of antibodies from serum were developed to determine whether low levels of antibodies against ZP are present in sera of alloimmunized female rabbits. They also allow more detailed analysis of antibodies used to detect antigenic determinants which are cross-reactive between different mammalian species. Although this method was effective in isolating low levels of antibodies from male alloimmunized rabbits or from female rabbits heteroimmunized with porcine ZP proteins, no specific antibodies could be isolated from the serum of females alloimmunized with rabbit ZP. These studies more clearly demonstrate that zona pellucida antigens are specific to the ovary in that female rabbits do not develop significant antibody levels against rabbit ZP antigens, even following active immunization with adjuvant, while male rabbits develop high titers of antibodies.     

Antifertility effect of active immunization with ZP4 glycoprotein family of porcine zona pellucida in hamsters.

Female golden hamsters were immunized with solubilized porcine zona pellucida (s-PZP) or ZP4 glycoprotein family isolated from s-PZP by preparative SDS-PAGE. Both antigen preparations induced production of antibodies which reacted not only with porcine zona pellucida but also with the hamster zona pellucida. The hamsters immunized with solubilized porcine zona pellucida mainly produced antibodies reactive to ZP3, while the hamsters immunized with ZP4 mainly produced antibodies reactive to ZP4. The former animals became permanently infertile but the infertility in the latter animals was temporary and they became pregnant later. Histological studies revealed that the ovarian follicles in hamsters immunized with s-PZP were completely destroyed leaving only atrophic follicle-like cell clusters, while in the ovaries of hamsters immunized with ZP4 a number of small follicles with oocytes remained intact. These observations are encouraging for the further characterization of the ZP4 antigens as candidates for the development of a contraceptive vaccine.     

猪卵透明带抗原的放射免疫分析——卵透明带抗原的组织特异性和抗体效价的测定

本文报道用纯化的猪卵透明带抗原免疫家兔制得高效价的抗血清,并建立了灵敏度较高(4ng)的可靠的放射免疫测定方法。用于猪卵透明带抗原的组织特异性研究,发现与所测定的猪、猴十二种脏器组织提取液均无交叉反应,而与猪、猴、羊、免、大鼠和人的卵巢提取液均有明显的交叉反应,进一步证明猪卵透明带具有强的抗原性和组织特异性。同时还研究了家兔注射猪卵透明带抗原的免疫应答和十四个月抗生育有效期内抗体效价的动态变化。     

Update on zona pellucida glycoproteins based contraceptive vaccine

Zona pellucida (ZP) glycoproteins, due to their critical role in mammalian fertilization, have been proposed as candidate immunogens for development of a contraceptive vaccine. Active immunization studies in a variety of animal species, employing either native or recombinant zona proteins, has established their contraceptive potential. Hence, ZP glycoprotein-based contraceptive vaccines have a very good potential for controlling wild life population. To make it a realistic proposition, additional research inputs are required to develop new potent adjuvants and novel practical strategies for vaccine delivery. The observed ovarian dysfunction, often associated with immunization by ZP glycoproteins, is one of the major obstacles for their application in the control of human population. Ongoing studies to delineate epitopes of ZP glycoproteins that will generate an immune response capable of inhibiting fertility without any untoward effects on ovarian functions will help in determining their feasibility for human use.     

The immune response and fertility of koalas (Phascolarctos cinereus) immunised with porcine zonae pellucidae or recombinant brushtail possum ZP3 protein

To evaluate the potential contraceptive effect of immunisation with zona pellucida antigens, 50 free-ranging koalas were immunised with either porcine zonae pellucidae (PZP), recombinant brushtail possum ZP3 (recBP-ZP3) or buffer, in complete Freund's adjuvant. A single booster immunisation in incomplete Freund's adjuvant was administered 3–5 months later. Where possible animals were recaptured, reproductive status was assessed and blood was collected at 1–3-month intervals for the next 33 months. Forty-three koalas were recaptured at least three times allowing reliable assessments of their fertility. Fourteen animals were observed never to have a pouch young. Of the remaining 29 animals the reproductive productivity of PZP treated females was reduced compared with control and recBP-ZP3 treated females, in terms of both total number of young produced, and failure to produce further young in females of proven fertility. One month after the initial immunisation, serum antigen-specific antibody titres were higher in animals immunised with PZP or recBP-ZP3 compared to controls, and reached a plateau by 4 months. Antibody against the relevant immunising antigen was also detected in ovarian follicular fluid, uterine fluid and vaginal secretions. Epitope analysis suggested that immune responses other than antibodies directed against the ZP3 amino acid sequence were responsible for mediating infertility. The results demonstrate that the fertility of female koalas can be compromised by immunisation against zona pellucida antigens. However, unlike in the eastern grey kangaroo and the brushtail possum, immunisation with bacterial recombinant brushtail possum ZP3 did not compromise fertility in the koala.     

非哺乳动物卵透明带的生殖免疫学研究

从淡水鱼类(鲤、草、鲢、鳙)卵中显微分离出的透明带,经聚丙烯酰胺凝胶圆盘电泳,对蛋白染料氨基黑10B 着染,呈单条蓝黑色带,它和大白鼠卵透明带、细菌粘多糖及唾液粘蛋白有类似的电泳谱及迁移率。用鱼卵透明带经福氏佐剂乳化后免疫家兔,可诱发家兔产生抗鱼卵透明带抗体。被动血凝试验测得抗体滴度为1000,玻片沉淀反应及琼脂凝胶平板双扩散试验表明,此抗血清可以和不同种鱼透明带及大白鼠透明带起交叉反应,猴抗鱼透明带抗血清可以和猪卵透明带起交叉反应。鱼卵透明带可以成功地用放射性同位素~(131)Ⅰ进行标记。用鱼卵透明带主动免疫家兔所诱发抗血清的电泳分析表明,丙种球蛋白区带随免疫时间加长而逐渐加深加宽.~(131)Ⅰ标记的鱼透明带与兔抗鱼卵透明带的抗血清一起混合后再电泳,丙种球蛋白区带的放射性计数率最高。初步认为鱼卵透明带的化学成分及免疫学特异性可能与哺乳动物存在着一定关系,有一定的共同抗原性,这为寻找丰富的异种卵透明带抗原提供了一定的线索。     

Autoimmunity and premature ovarian failure

PURPOSE OF REVIEW: The different patterns of autoreactivity that may account for the premature infertility observed in patients with premature ovarian failure are described. RECENT FINDINGS: Animal model studies have detailed fundamental immune dysregulatory patterns that induce ovarian failure in the context of global polyglandular involvement, as well as autoimmune mechanisms that induce ovarian failure in the context of targeted ovarian pathology. Recent studies on premature ovarian failure patients implicate the ubiquitously expressed glycolytic enzyme, alpha-enolase, as a potential antigenic target, particularly in those patients with polyglandular involvement; and the ovarian-specific maternal-effect protein, Mater, whose expression is essential for fertility. SUMMARY: Several fundamentally distinct mechanisms may account for premature ovarian failure, including global immune dysregulation, particularly in patients with polyglandular autoimmunity. Premature ovarian failure may also be due to inflammatory autoimmunity targeted to ovarian-specific germline antigens (e.g., zona pellucida proteins or Mater) or differentiation/regulatory factors (e.g., inhibin-alpha). Moreover, the ovarian autoimmunity may be mediated by T cells (e.g., those targeting zona pellucida proteins) or B cells/antibodies (e.g., those targeting inhibin-alpha). Thus premature ovarian failure appears to be a complex disease entity with multiple underlying etiopathogenic contributions including the possibility of several distinctly different autoimmune mechanisms.     

鳙鱼卵透明带物质抗家兔生育效应的初步研究

鳙鱼卵透明带类物质加弗氏佐剂乳化后,对雌性家兔进行免疫注射。以被动血凝试验测定家兔抗鳙鱼卵透明带热溶性物质的血清效价。当雌兔血清抗体达一定水平时,与有生育力的雄兔进行交配,交配后于不同时间,将雌兔处死解剖,计算胎兔数及卵巢中黄体数,以精、卵结合抑制率为评价抗生育效应的指标。结果表明,家兔经鳙鱼卵透明带类物质免疫以后,血清抗体效价上升非常显著,于此同时,抗生育效应也显著增加。文章就鱼卵透明带类物质用于控制哺乳动物生育的可能性进行了讨论。     

Localization of species conserved zona pellucida antigens in mammalian ovaries

The mammalian zona pellucida (ZP) consists of three glycoproteins (ZP1, ZP2 and ZP3), which are variably conserved among species at the genomic and amino acid levels. In order to evaluate the expression of ZP during ovarian development, a population of antibodies was selected that recognize species conserved antigenic domains of the three ZP proteins. Domain specific antibodies were selected from sera of rabbits immunized with all three native pig ZP proteins by elution of antibodies bound to each of the three human ZP recombinant proteins expressed from cDNAs, using the baculovirus expression system in insect cells. Immunoblot analysis was used to characterize the specificity of the antibodies and immunohistochemistry was used to evaluate the stage specific expression of ZP proteins during ovarian follicular development of the mouse, baboon and human. This study demonstrates that the conserved domains of all three ZP proteins are localized in the oocyte extracellular ZP matrix as well as in a subset of granulosa cells. However, this expression does vary among species with respect to the stage and cell type during early stages of ovarian follicular development. These antibodies should serve as excellent markers for evaluating early stages of human ovarian follicular development and in the development of contraceptive agents.     

Effects on fertility of immunizing mice with anti-idiotypic antibodies to porcine zona pellucida antigen

OBJECTIVE: To evaluate the effects on fertility by immunization with anti-idiotypic antibodies to porcine zona pellucida (PZP) antigen. METHOD: Anti-idiotypic antibodies (Ab(2)) were produced in New Zealand rabbits immunized with 17D3 monoclonal antibodies (mAbs) (IgG, Ab(1)) to PZP antigen. The antisera were first passed through immuno-affinity chromatography column linked to normal mouse IgG so as to remove the antibody bound to normal mouse IgG The passing elute was then purified by immuno-affinity chromatography using 17D3 mAbs to get the Ab(2). Female BALB/c mice, 5-week-old, were grouped and immunized with the Ab(2), PZP antigen, target antigen of the Ab(1) and normal rabbit IgG, respectively. The treated female mice were mated with male BALB/c mice and sacrificed at gestation day 10. Analyses included ELISA measurement of anti-ZP antibody titer, fetal number determination and evaluation of ovarian histomorphology. RESULTS: The Ab(2) appeared as a single protein band by SDS-PAGE. Shown by a competitive inhibition ELISA, the Ab(2) specifically bound to the variable region of the 17D3 Ab(1). Compared with controls, the female mice immunized with Ab(2) showed a decreased pregnancy rate and a statistically significant reduction in fetal numbers. Histological examination of ovaries demonstrated that Ab(2) exposure interfered less with follicular development than did exposure to PZP. CONCLUSION: Immunization of female mice with Ab(2) to PZP mAbs suppresses fertility and fetal numbers with minimal ovarian pathology.     

不同病因对卵母细胞透明带、胚胎发育和妊娠结局的影响

目的:探讨女性不同病因对卵母细胞透明带、胚胎发育和妊娠结局等的影响。方法:回顾性分析因单因素不孕体外受精治疗的197个周期,以输卵管因素为对照组(n=145),比较多囊卵巢综合征(polycystic ovarian syndrome,PCOS)(n=25)、子宫内膜异位症(endometriosis,EMs)(n=19)和不明原因不孕症(n=8)等各组的受精率、种植率、妊娠率以及透明带双折射性和透明带厚度。结果:平均获卵数PCOS组(17.9±1.9枚)显著高于其它各组(对照组:11.3±0.6枚,EMs组:9.4±1.2枚和不明原因组:13.1±2.9)(P<0.01),其它各组无统计学差异(P>0.05);PCOS组、EMs组和不明原因组受精率(83.66%、75.40%、77.12%)、优质胚胎率(57.26%、51.14%、59.30%)、种植率(25.53%、17.39%、29.40%)和妊娠率(52.38%、41.18%、42.86%)与对照组(80.13%、58.99%、28.76%、43.70%)间无统计学差异(P>0.05),但EMs组各项指标均低于对照组。透明带双折射值各组间差异极显著(对照组:7.13±0.28,PCOS组:4.97±0.55,EMs组:3.67±0.75,不明原因组:14.19±1.41)(P<0.01),其中EMs组透明带双折射值最低。受精当日透明带厚度EMs组显著高于其它各组(对照组:18.84±0.07,PCOS组:18.48±0.17,EMs组:19.75±0.24,不明原因组:18.98±0.26)。结论:EMs对受精率、妊娠率和透明带双折射值等的影响最明显,透明带的双折射值和厚度可在一定程度上反映不同病因对卵母细胞发育潜力的影响。     

Immunocontraception

The immunological contraceptive methods whose development is described in this work appear to inhibit the action of antigenic molecules necessary for fertilization. Antigens in the gametes or their envelopes that intervene in reconnaissance or fusion of the gametes appear to be more promising targets than those at the level of gamete production in the gonads. Clinical examples show that infertility may be spontaneously acquired in both sexes through active immunization. Contraceptive action can only be sought if the gametes carry specific antigens, so that other physiological functions will not be disturbed, and if the antigens play a determining role in fertilization. Research currently is oriented toward 3 complementary targets, the sperm and the 2 envelopes of the oocyte, the zona pellucida and the cumulus oophorus. Possibilities appear to exist of preventing the intervention of several different molecules in fertilization, although most of them are still poorly understood. In the past 10 years, various monoclonal antibodies have been produced against sperm of different animal species, some of which are capable of inhibiting fertilization in the same species and also in human beings. But in vivo effects of these antibodies have not been valuated, even in the same species. Active immunization of male or female guinea pigs with a sperm surface antigen has led to sterility, through inhibition of attachment of sperm to the zona pellucida, but the monoclonal antibody against the protein does not recognize the human sperm. Polyclonal antibodies against the same protein might be possible for human contraception. The biochemical and physiological study of monoclonal antibodies against human sperm is facilitated if the antibodies cross with rodent sperm. 2 such antibodies directed against proteins secreted by the human testicles are capable of inhibiting murine and human fertilization in vitro. Attempts to achieve active or passive immunization by targeting antigens of the zona pellucida have been underway for 2 decades in different animal species using ever more selective antigenic material. But in vivo animal studies caused serious ovarian disorders that would be unacceptable in contraception. A polyclonal antibody against the intercellular matrix of the human cumulus oophorus is capable of inhibiting fertilization in vitro, with the action resulting from a strong reduction in the number of sperm attached to the zona pellucida. Numerous aspects of immunocontraception are still at the research stage. Apart from the choice of the moist appropriate antigens, active immunization in human subjects must be preceded by massive production of purified antigens. Research is needed on the adjuvant, the possibility of maintaining high levels of antibodies, and the return of fertility. Despite the obvious public health need, few laboratories are engaged in this type of research.     

Regulation of fertility by anti-hCG vaccines

Immunointerruption of pregnancy consists of preventing pregnancy or terminating it at an early stage through antibodies. The antibodies may be obtained after administration of vaccine to induce their formation through active immunization, or by direct injection through passive immunization. Antigens that could potentially be used are found in sperm, the zona pellucida, and reproductive hormones, especially the chorionic gonadotropins. The sperm antigens are basically enzymes such as hyaluronidase and accrosine. 3 glucoproteins have been identified in the zona pellucida of mice and pigs. In vitro studies have shown that fertilization can be prevented if eggs are exposed to antizona-pellucida antibodies along with sperm. Active immunization could give longer term results, but ovarian function could also be affected if the antigens weren't purified. Much research has been devoted to identifying human embryonic antigens through analysis of the proteins of the cells of embryonic teratocarcinoma. Among placental antigens, the glucoprotein SP1 synthesized by the trophoblast is under study. Anti-SP1 antibodies appear to cause abortion in monkeys, but knowledge of these antigens is still fragmentary. Various reproductive hormones have been studied, but too many undesirable effects could result from the use of luteinizing hormone, luteinizing hormone releasing hormone, follicle stimulation hormone, or steroids. Human chorionic gonadotropin (hCG), however, is more promising. It is a glucoprotein formed of alpha and beta subunits, both of which are needed for hCG to interact with its receptor. The alpha subunit has the same sequence as that of other hormones of the same species, but the beta subunit is specific to each hormone. Studies are underway to determine the site of amino acids and peptide sequences capable of inducing an anti-hCG response which would inactivate the biological activity of hCG. Different teams have used synthetic peptides analogous to sequences of beta-hCG or fragments obtained by enzymatic cleavage of natural hCG to block pregnancy in rats and baboons. The presence of antibodies can block pregnancy without disturbing ovulation or modifying menstrual regularity. No toxic or secondary effect has been observed in animals. A multicenter phase 1 test using beta-hCG coupled with tetanus antitoxin caused almost all the women participating to develop antibeta-hCG and antitetanus anatoxin antibodies, but titres of antibodies varied greatly between different women, required 5-6 months to develop, and declined rapidly thereafter. Several pregnancies were observed, especially in women with low titres of antibodies. The approach of passive immunization through direct injection of antibodies has met with numerous obstacles, including lack of success in producing human monoclonal antibodies. Although a 2nd generation of vaccines in under study, the potential role of immunointerruption of pregnancy in fertility regulation remains to be clarified.     

Detection and significance of serum zona pellucida antibodies in sterile females

Sera of 100 infertile women and of 100 patients with early pregnancy as control group were investigated for antibodies to zona pellucida by the indirect immunofluorescence. Zonae of oocytes from pigs were used for the procedure. A positive control serum with a high titer was produced by immunization of rabbits with an antigen from pig zonae. The frequency of antibodies to zona pellucida was 29% for infertile patients and 18% in the control group. The titers of these autoantibodies were higher in infertile than in fertile women. There was no increase of these antibodies if the age increased and no relation to the menarche and the ovulation. Antibodies to zona pellucida were demonstrated frequently in patients with disturbances of cycle, free Fallopian tubes, secondary infertility, and ovarian and unknown origin of infertility.     

家兔、恒河猴抗猪卵透明带血清的免疫荧光研究

以间接免疫荧光法检测用热溶猪卵透明带抗原(HSPZ)和分离的猪卵透明带抗原(IPZA)免疫家兔与恒河猴所获得的抗血清,结果显示:兔抗HSPZ 血清的阳性滴度是1:3800,兔和恒河猴抗IPZA 的血清的阳性滴度分别是1:3400和1:1800。经猪肝粉及猪固相血浆吸收后,抗HSPZ 血清的滴度明显下降:但抗IPZA 血清的滴度仅轻度下降。HSPZ 及IPZA 均具有较好的免疫原性,然而HSPZ 含有较多的非特异性物质。实验结果还表明:猪卵透明带抗原和鱼卵透明带抗原之间仅有部分交叉。     

A new look at antifertility vaccines

This article reviews new advances in biochemistry, biotechnology, and immunology relevant to antifertility vaccine development and evaluates the current status and future prospects of contraceptive vaccines and other immunologic approaches to fertility regulation. Contraceptive vaccine candidates include human chorionic gonadotropin, human luteinizing hormone and luteinizing hormone releasing hormone, and reproductive steroid hormones. Sperm enzymes are attractive for a contraceptive vaccine; among the sperm antigens studied are antibodies to hyaluronidase, acrosin, and lactate dehydrogenase-C4. Several laboratories have developed monoclonal antibodies to a variety of sperm antigens and are using them to identify and characterize new sperm proteins and their roles in fertility. Considerable progress has been made toward biochemical characterization of unique glycoproteins constituting the zona pellucida. Zona pellucida antigens are good candidates because antizona antibodies may block both fertilization and implantation, and low amounts of antibody would be sufficient because of the small number of mature eggs with zona present at any time. Studies are underway to identify human embryonic antigens through examination of the protein profile of human teratocarcinoma cell lines at various stages of differentiation and through analysis of antibodies in human pregnancy and infertility sera. Placental and extraembryonic membranes produce several tissue-specific antigens that have been considered for antifertility vaccines, but concern that they could produce late or incomplete abortion has prevented their serioud consideration. Because of possibly serious systemic side effects, presence of the blood-testis barrier, and large number of sperm produced daily, it is unlikely that sperm vaccines can be safely administered to men. Nautural protective mechanisms will probably render some immunocontraceptive approaches ineffective. The possibility of serious pathogenic side effects of contraceptive vaccines demands vaccines demands a cautious approach to their development.     

Analysis of B cell epitopes of a glycoprotein porcine zona pellucida (pZP1)

The zona pellucida (ZP) of mammalian oocytes forms an extracellular matrix composed of three major glycoproteins and plays an important role in sperm-zona interactions. As ZP had a strong organ-specific but species-cross-reactive antigenicity and passive or active immunization with ZP antigens could impair fertilization, the possibility of developing a immunocontraceptive vaccine has been extensively studied. Studies on active immunization with porcine ZP (pZP) that contain B cell epitopes and T cell epitopes demonstrated that a temporary infertility could be induced along with the elevation of antibody titers, but it was always associated with ovarian failure. This could be due to the oophoritis by activation of pathogenic T cell immunity. It is the general consideration that any adverse effects by vaccination should be avoided for an immunocontraception. From this point of view, the analysis of B cell epitopes of pZP protein would be helpful for construction of a safe immunocontraceptive vaccine with zona antigens. We determined the amino acid sequence of the B epitope in the pZP1 protein by using a monoclonal antibody (MAb-5H4) that possesses a fertilization blocking ability. In addition, antiserum raised to the epitope sequence was revealed to block in vitro fertilization of homologous animal species.