Search

Problem Various approaches have been developed to improve the antibody response of zona pellucida glycoprotein-3 (ZP3) vaccination. In this study, we investigated whether GM-CSF and IL-5 can be used as cytokine adjuvants to increase the humoral immune response generated by mouse ZP3 (mZP3) DNA vaccine. Method of study Mice in experimental group were injected by GM-CSF 4 days before the co-immunization of IL-5 and mZP3 DNA vaccine. The contraception and the correlation with humoral and cellular immune responses were analyzed after immunization and mating. The effect of cytokine adjuvant on the maturation of DCs was evaluated. Results Co-immunization of GM-CSF and IL-5 with mZP3 DNA vaccine induced the highest level of serum IgG and IL-4 expression in CD4+ T cells. Importantly, this strategy reduced mice fertility without disrupting normal ovarian morphology. GM-CSF enhanced the maturation of DCs evidenced by up-regulating the expression of MHC-II and CD86. Conclusion GM-CSF and IL-5 co-administration enhanced humoral immune responses to mZP3, and this may be a potential strategy for development of immunocontraceptive vaccine.

Sperm flagellar protein 2 (SFP2), which was earlier identified using a novel combinatorial approach, was evaluated for its contraceptive potential in mice.Male mice were actively immunized with two synthetic peptides of SFP2. Antipeptide antibody was characterized by Western blot and indirect immunofluorescence. Immune response was monitored, and mating studies were performed 6 and 22 weeks post-immunization.Antibodies to the SFP2 peptide 1 recognized a doublet at 220- to 230-kDa region only in the epididymal protein extract. Peptide 1 antibody recognized the cognate protein on spermatozoa from mouse, rat, and human. Histological analysis of testis and epididymis of the immunized mice indicated no deleterious effect. Incubation of sperm with the immune sera of peptide 1 caused significant reduction in motility and viability but did not agglutinate sperm. Only synthetic peptide 1 gave rise to high-level antibodies in all the immunized mice, which on mating resulted in reduced fertility rate (20%) when compared with PBS control animals (100%). The antibody levels in the immunized males declined by 22 weeks post-immunization, resulting in 100% reinstatement of fertility.These data provide an experimental basis for the development of effective contraceptive vaccine based on new epididymal target.

Free-roaming unowned stray and feral cats exist throughout the world, creating concerns regarding their welfare as well as their impact on the environment and on public health. Millions of healthy cats are culled each year in an attempt to control their numbers. Surgical sterilization followed by return to the environment is an effective non-lethal population control method but is limited in scope because of expense and logistical impediments. Immunocontraception has the potential to be a more practical and cost-effective method of control. This is a review of current research in immunocontraception in domestic cats. Functional characteristics of an ideal immunocontraceptive for community cats would include a wide margin of safety for target animals and the environment, rapid onset and long duration of activity following a single treatment in males and females of all ages, and sex hormone inhibition. In addition, product characteristics should include stability and ease of use under field conditions, efficient manufacturing process, and low cost to the user. Two reproductive antigens, zona pellucida and GnRH, have been identified as possible targets for fertility control in cats. Zona pellucida, which is used successfully in multiple wildlife species, has achieved little success in cats. In contrast, immunization against GnRH has resulted in long-term contraception in both male and female cats following a single dose. GnRH is an ideal contraceptive target because it regulates pituitary and gonadal hormone responses in both males and females, thus suppressing nuisance behaviors associated with sex hormones in addition to preventing pregnancy. The responsiveness of cats to fertility control via GnRH suppression should encourage researchers and cat control stakeholders to continue efforts to optimize vaccines that induce multiyear contraception following a single dose in a high proportion of treated cats.

The year 2011 marks the 84th year of the discovery of human chorionic gonadotropin (hCG) by Ascheim and Zondek. Originally considered and employed as a reliable diagnostic index for pregnancy, the multiple roles of hCG as an initiator and sustainer of pregnancy are now recognized. Besides pregnancy, the expression of hCG or its subunits is observed in a number of cancers of diverse type, in particular at advanced stage. Cancers expressing hCG/subunits have poor prognosis and adverse survival. Thus, immunological approaches against hCG have applications for control of fertility and for treatment of terminal cancers. Various mechanisms by which hCG exercises its action are discussed. These include its role as autocrine growth promoter, inhibitor of apoptosis, promotor of angiogenesis, invasiveness, and protection against rejection by the immune system. The article reviews various vaccines developed for control of fertility and for therapy of advanced-stage cancers expressing ectopically hCG/subunits. Also reviewed are the recombinant fully humanized and chimeric antibodies usable for emergency contraception, as vacation contraceptive, and as therapeutic antibodies for treatment of cancers.

Wildlife, free-ranging and captive, poses and causes serious population problems not unlike those encountered with human overpopulation. Traditional lethal control programs, however, are not always legal, wise, safe, or publicly acceptable; thus, alternative approaches are necessary. Immunocontraception of free-ranging wildlife has reached the management level, with success across a large variety of species. Thus far, the immunocontraceptive research and management applications emphasis have been centered on porcine zona pellucida and gonadotropin-releasing hormone vaccines. Contraceptive success has been achieved in more than 85 different wildlife species, at the level of both the individual animal and the population. At the population management level with free-ranging species, the primary focus has been on wild horses, urban deer, bison, and African elephants. The challenges in the development and application of vaccine-based wildlife contraceptives are diverse and include differences in efficacy across species, safety of vaccines during pregnancy, the development of novel delivery systems for wild and wary free-ranging animals, and the constraints of certain non-contraceptive effects, such as effects on behavior. Beyond the constraints imposed by the public and a host of regulatory concerns, there exists a real limitation for funding of well-designed programs that apply this type of fertility control.

Current methods of contraception lack specificity and are accompanied with serious side effects. A more specific method of contraception is needed. Contraceptive vaccines can provide most, if not all, the desired characteristics of an ideal contraceptive. This article reviews several factors involved in the establishment of pregnancy, focusing on those that are essential for successful implantation. Factors that are both essential and pregnancy-specific can provide potential targets for contraception. Using database search, 76 factors (cytokines/chemokines/growth factors/others) were identified that are involved in various steps of the establishment of pregnancy. Among these factors, three, namely chorionic gonadotropin (CG), leukemia inhibitory factor (LIF), and pre-implantation factor (PIF), are found to be unique and exciting molecules. Human CG is a well-known pregnancy-specific protein that has undergone phase I and phase II clinical trials, in women, as a contraceptive vaccine with encouraging results. LIF and PIF are pregnancy-specific and essential for successful implantation. These molecules are intriguing and may provide viable targets for immunocontraception. A multiepitope vaccine combining factors/antigens involved in various steps of the fertilization cascade and pregnancy establishment may provide a highly immunogenic and efficacious modality for contraception in humans.

This is a review of current status and future perspectives on the development of antisperm contraceptive vaccines (CV) and immunocontraceptives. The development of antisperm CV is an exciting proposition. There is a strong rationale and recent data indicating that this proposition can translate into reality. The search for novel sperm-specific antigens/genes, that can be used for CV, continues using various recent developing technologies. Various approaches of proteomics, genomics, reproductive biology, mucosal immunity and vaccinology and several novel technologies such as gene knockout technology, phage display technology, antibody engineering, differential display technique, subtractive hybridization, and hybridoma technology are being used to delineate sperm-specific antigens and construct CV. Various sperm antigens/genes have been delineated, cloned, and sequenced from various laboratories. Vaccination with these sperm antigens (recombinant/synthetic peptide/DNA) causes a reversible contraceptive effect in females and males of various animal species, by inducing a systemic and local antisperm antibody response. The efficacy is enhanced by combination vaccination, including peptides based on various sperm antigens. Several human novel scFv antibodies with unique complementarity-determining regions (CDRs), that react with specific well-defined fertility-related sperm antigens, have been synthesized. These human infertility-related antibodies may find application in the development of novel immunocontraceptives. Besides finding the novel sperm antigens, the present and future focus is on enhancing the immunogenicity, bioefficacy, and on obliterating the inter-individual variability of the immune response, and proceeding for primate and human clinical trials. Multi-epitope vaccines combining sperm proteins involved in various steps of fertilization cascade have been found to enhance the immunogenicity and bioefficacy of the contraceptive effect. The in vitro synthesis of infertility-related human scFv antibodies may provide unique once-a-month immunocontraceptives, the first of its kind, for human use. The multi-epitope CV and preformed engineered human antibodies of defined specificity may obliterate the concern related to inter-individual variability of the immune response.

Study on feasibility of pCXN2-mIzumo as a potential immunocontraceptive antigen.Two groups of mice received 100 microg/mouse plasmids of pCXN2-mIzumo and pCXN2 respectively. RT-PCR Immunofluorescence assay and ELISA were performed to observe pCXN2-mIzumo expression and antibody response in the inoculated mice. Sperm penetration assay and animal mating were employed to detect differences of in vitro fertilization (IVF) rate and mean litter size between the experimental and control groups.Izumo cDNA positive bands were detected in sample from mice immunized with pCXN2-mIzumo. IgG response started to rise at 2 weeks after first boost and reached the highest antibody titers at 2 weeks after third boost of immunization with pCXN2-mIzumo in the experimental mice. In vitro fertilization rate in the experimental group (11.57%) was significantly lower than that in control (36.60%). Significant difference of mean litter size between female experimental and control groups was observed, and there was significant negative correlation between individual anti-serum titers and litter size (r = -0.308, P0.05).pCXN2-mIzumo plasmid possesses appreciable anti-fertility potential.

An effective, single-injection, multi-year, GnRH contraceptive agent is needed to control reproduction in overabundant white-tailed deer populations.Two GnRH conjugates, GonaCon (GnRH-KLH) and GonaCon-B (GnRH-blue protein), were prepared in emulsion form as one-injection and two-injection immunocontraceptive vaccine formulations. In addition, the GnRH-KLH protein conjugate was lyophilized and suspended in AdjuVac adjuvant to produce a fifth vaccine formulation. Each formulation was administered to a group of five captive adult female white-tailed deer. Reproductive performance of treated female deer was monitored for 5 years to determine the comparative efficacy of the various treatments.The longevity of the contraceptive response (2-5 years) was strongly influenced by the design of the conjugate antigen, the adjuvant used, and the delivery form of the vaccine.One-injection and two-injection formulations of GonaCon and GonaCon-B produced multi-year contraception in adult female white-tailed deer. GonaCon-B provided a longer lasting contraceptive effect.

To determine if the recombinant mouse Izumo (mIzumo) could be used as a potential immunocontraceptive antigen.The recombinant mIzumo fused with 6His tag (6His-mIzumo) was purified by immobilized Ni2+ affinity chromatography. Enzyme-linked immunosorbent assay and Western blot were used to detect anti-6His-mIzumo activities of serum from the mice immunized with 6His-mIzumo. Inhibition of the anti-6His-mIzumo antibody on mouse sperm-egg fusion in vitro was performed using the zona free oocytes and acrosome reacted sperm. Fertility of the 6His-mIzumo immunized male and female mice was compared with control mice.The recombinant mIzumo was successfully produced. Female and male mice inoculated with 6His-mIzumo developed a specific serum antibody and the highest antibody titer lasted at least 6 weeks. The serum anti-6His-mIzumo antibody almost completely blocked mouse sperm-egg fusion in vitro. However, there was no significant reduction in fertility for both male and female mice immunized with 6His-mIzumo compared with control mice.The circulated anti-mIzumo antibody can block mouse sperm-egg fusion in vitro but has no effect on fertility in vivo. It seems that application of Izumo as a candidate antigen in development of contraceptive vaccine needs further investigation.

Problem Methods to limit fertility of feral swine are needed to reduce transmission of diseases and agricultural and ecosystem damage. Method of Study We evaluated a single-shot GnRH immunocontraceptive vaccine in both male and female feral swine for its effect on fertility and functional status of the reproductive tissues. Captive feral pigs were randomly assigned to receive 1000 or 2000 μg GnRH-KLH vaccine treatments or no treatment. Results After 36 weeks, none of the 2000-μg-treated females and only 20% of the 1000-μg-treated females were pregnant. This corresponded to reduced serum progesterone, regressed tissues within the reproductive tract and lack of evidence for follicular development leading to ovulation. Males were less responsive to the vaccine than females, but more responsive to the lower dose of the vaccine than the higher dose. Conclusions The single-shot GnRH vaccine is effective in controlling fertility of female feral swine and may be useful for population reduction.

The 80 kDa human sperm antigen (HSA) is a sperm-specific and conserved antigen, capable of inducing immunological infertility. Partial N-terminal amino acid sequences of 80 kDa HSA (Peptide NT) and its peptides obtained by digestion with endoproteinase Lys-C (peptides 1-4) and endoproteinase Glu-C (peptides 5-6) did not show any sequence homology with reported known proteins deposited in the Gen-Bank. These sequenced peptides were synthesized and conjugated to key hole limpet haemocyanin (KLH) and evaluated for its antifertility effects. The present communication describes the characterization of these peptides and their antibodies.Peptides NT, 1, 2, 3 and 4 were synthesized and conjugated to KLH. Antibodies to KLH conjugated peptides were raised in rabbits by active immunization and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) using sperm extract coated wells. The binding specificity of the synthetic peptides or purified 80 kDa HSA to their antibodies was assessed in the presence of various doses of respective synthetic peptides or 80 kDa HSA. The binding specificity was further confirmed by Western blot analysis. Antipeptide antibodies were also checked for sperm agglutinating activity, in-vitro.Active immunization of rabbits elicited significant antibody titers against the synthetic peptides, except for peptide 3. Antipeptide antibodies specifically recognized the native protein in an ELISA and induced in-vitro agglutination of human, rat and monkey sperm. In addition, Western blot analysis showed that these antipeptide antibodies specifically bind to the 80 kDa HSA band of the sperm extract.Synthetic peptides of 80 kDa HSA are immunogenic and antibodies raised against these peptides recognize the native protein detected by ELISA, Western blot analysis. In addition, they possess sperm agglutinating activity. These findings suggest that they are promising candidates in the development of immunocontraceptives.

The zona pellucida is a feasible candidate for contraceptive vaccine. Our previous study showed that a human zona pellucida (ZPA) recombinant protein produced antiserum that inhibited human in vitro fertilization in rabbits. The objective of this study is to examine whether the human recombinant protein produce fertilization-blocking antiserum in a non-human primate.The amino terminal regions consisting of pig (198 amino acids) and human (206 amino acids) zona pellucida proteins were prepared by Escherichia coli to produce antisera in bonnet monkeys (Macaca radiata). Antibody production was assessed by immunofluorescent staining and enzyme-linked immunosorbent assay (ELISA). Fertilization-blocking test was carried out using human oocytes that had failed in intracytoplasmic sperm injection (ICSI) treatment under patient's informed consent.Both antisera against pig and human recombinant proteins recognized native human zona pellucida by immunofluorescent staining. However, anti-recombinant human ZPA reacted to native pig zona protein more strongly than pig zona pellucida by ELISA. When human oocytes were treated with antisera before insemination, anti-human ZPA antiserum inhibited human sperm binding to human zona pellucida, but anti-pig ZPA antiserum did not.Bonnet monkey produced fertilization-blocking antibody by immunization with human recombinant ZPA. The homologous or highly similar sequence is effective for developing a contraceptive vaccine when using recombinant peptides.

We recently cloned and sequenced a sperm-specific antigen, designated as testis-specific antigen-1 (TSA-1), from human testis. The present study was conducted to examine its expression and function in murine sperm, in order to find out whether or not the mouse can provide a suitable model for examining its immunocontraceptive effects.The antibodies (Ab) were raised against purified human rTSA-1 in virgin female rabbits. The rTSA-1 was run in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the gel containing the approximately 18 kDa band was cut, minced and used for immunization to obtain the specific Ab. The immunoglobulins from preimmune bleed and from animals injected with adjuvant alone served as control. These Ab were analysed in enzyme-linked immunosorbent assay (ELISA), Western blot procedure, immunoprecipitation procedure, immunocytochemical technique (ICT), immunobead binding technique (IBT), acrosome reaction and sperm-zona binding assay.Active immunization of female rabbits with purified rTSA-1 protein of 18 kDa, produced high titer Ab against the recombinant antigen. These Ab to rTSA-1 were used in the present study. In Western blot procedure, rTSA-1 Ab recognized a specific protein band of approximately 24 +/- 3 kDa in murine sperm extract, the band similar to found in human sperm extract. In the immunoprecipitation procedure, rTSA-1 Ab immunoprecipitated the protein band of similar size from extracts of murine sperm and murine testis. The ICT and the IBT studies revealed the subcellular localization of TSA-1 on the surface of acrosome and tail regions of the non-capacitated and capacitated murine sperm cells. In functional bioassays, rTSA-1 Ab inhibited the acrosome reaction and sperm-egg binding in vitro.These data indicate that the TSA-1 is expressed in murine sperm and may have a biological role in sperm function and sperm-egg binding. In vitro inhibition of capacitation/acrosome reaction and sperm-zona binding suggests that the mouse can provide a suitable model to examine the immunocontraceptive effects of TSA-1 in actively immunized animals.

PROBLEM: The present study was conducted to investigate the oocyte membrane protein(s) that is involved in sperm binding in the mouse, and whether or not it can be used for the development of a contraceptive vaccine. METHOD OF STUDY: The zona-free oocytes were treated with Triton X-100 and the extract was analyzed for homogeneity in the sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) after staining with silver nitrate. The appropriate band of 50 ± 4 kD, designated as OM antigen, was excised from the gel and used for the immunization. The female rabbits were immunized with the excised band per se and the female mice were immunized with the OM antigen after conjugation to keyhole limpet hemocyanin (KLH). The affinity-purified antibodies were analyzed in the enzyme-linked immunosorbent assay (ELISA), immunoprecipitation procedure, western blot procedure, indirect immunofluorescence technique (IFT), and sperm-oolemma binding assay. Actively-immunized mice were analyzed for in vivo fertility. RESULTS: The Triton X-100 extract of zona-free oocytes predominantly showed a single protein band of 50 ± 4 kD in the SDS-PAGE. Active immunization of female rabbits and of female mice with OM antigen raised high antibody titers (ELISA titer > 1:4096) that specifically recognized the OM antigen in the immunoprecipitation and Western blot procedures, and reacted with the oocyte in the IFT. These antibodies demonstrated a significant (P

PROBLEM: An 80 kDa human sperm antigen (80 kDa HSA) has been identified by western blot technique using serum of an immunoinfertile woman as a probe. The 80 kDa HSA has been subsequently purified from sperm extract and investigated for antifertility effects. METHOD OF STUDY: The purified 80 kDa HSA was used to immunize adult male and female rats. Rabbit anti 80 kDa HSA antibodies were used for immunofluorescent and immunohistochemical staining to demonstrate the presence of 80 kDa HSA on the sperm and to investigate its tissue distribution. The N-terminal sequence of native 80 kDa HSA and the peptides obtained by its endoproteinase Lys-C was determined. RESULTS: Active immunization of male and female rats with 80 kDa HSA caused infertility in all the immunized animals. Immunofluorescent staining showed its localization on the head region of the human and rat spermatozoa. While immunohistochemical studies showed its localization in the testes and epididymis but not in other somatic tissues. Partial amino-acid sequence analysis showed no sequence homology with any of the known protein in the database. CONCLUSIONS: 80 kDa HSA is a promising candidate antigen for immunocontraception as it is characterized and found to cause infertility upon active immunization, specific to spermatozoa and is conserved.

Reduction of excess numbers of white-tailed deer (Odocoileus virginianus) is an example of a potential use for immunocontraception as a means of wildlife population management.A 4 year multifaceted study was conducted to determine the long term effects of gonadotropin releasing hormone (GnRH) contraceptive vaccine on the fertility and behavior of female and male white-tailed deer. Deer were monitored for breeding behavior, hormone levels, pregnancy, fawning and GnRH specific antibody levels.Treatment lead to reduced fawning rates, altered estrus behavior, reduced concentrations of progesterone, contraception and failure to maintain pregnancy following conception. GnRH immunized does bred to untreated bucks had an 88% reduction in fawning caused by either immunocontraception or immunocontragestion. The vaccine effect is reversible, directly related to the antibody titer. Infertility lasted up to two years without boosting. GnRH immunized bucks demonstrated no interest in sexual activity when paired with control females. Depending on the immunization schedule, antlers either dropped early or remained in velvet.The results of this study demonstrate that GnRH vaccine is effective in inducing a reversible infertility in white-tailed deer, the infertility lasting up to two years without boosting.

Earlier studies have demonstrated that antibodies directed towards the N-terminal (residues 10-17) and C-terminal (residues 200-207) regions on chicken riboflavin carrier protein (RCP; 219 AA) are effective in pregnancy termination in rodents and sub-human primates. In the present study, the immunocontraceptive potential of three additional immunodominant sequences comprising of residues 33-49, 64 83 and 130-147 (CYA, CED and CGE peptides, respectively) of chicken RCP was investigated.The three antigenic peptides were synthesized by using Fmoc chemistry. Oligoclonal antibodies were generated in rabbits. Bioneutralizing capacity of these peptides was assessed by passive and active immunoneutralization studies.All the three peptides-specific antisera recognized their cognate epitopes on native RCP. When the affinity purified peptide IgG were administered on three consecutive days to pregnant rats (on days 10, 11 and 12), it was observed that the rats injected with CED and CGE-IgG failed to deliver any pups whereas the animals which received CYA IgG delivered normal pups. Active immunization of fertile female rats with CED or CGE peptide conferred protection from pregnancy.These results demonstrate the presence of two additional stretches in chicken RCP which can serve as mini-vaccines.

Vaccine(s) for contraception will provide an attractive alternative to contraception. There has been a renewed interest in defining fertilization-related sperm antigens that can be used for the development of a contraceptive vaccine. The sperm-zona pellucida (ZP) recognition and binding constitutes the most important event in the fertilization process, and the molecules involved at this site are attractive candidates for immunocontraception. Extensive research indicates that there are four sperm surface proteins that bind to oocyte ZP3 in humans and they belong to the four molecular regions of 95, 63, 51, and 14-18 kDa, respectively. Our laboratory is actively engaged in cloning and sequencing of cDNAs encoding for these sperm proteins and investigating the applications of recombinant (r) proteins in immunocontraception and infertility. Presently, the cDNAs encoding for the 14-18-kDa proteins, designated the NZ-1 and NZ-2, respectively, and the 51-kDa protein, designated fertilization antigen, have been cloned and sequenced. Active immunization with the rFA-1 antigen caused a reversible block/inhibition in fertility of female mice by raising a sperm/testis-specific immune response. FA-1 antigen is also involved in human immunoinfertility, and a recent clinical trial indicates that it will have a clinical application in the treatment of immunoinfertile men. The long-term goal is to employ various recombinant fertilization antigens and/or their bioeffective peptide epitopes in a single formulation, to generate an anti-sperm vaccine that will be strongly immunogenic and highly efficacious for regulation of fertility.

PROBLEM: Reduction of excess numbers of white-tailed deer (Odocoileus virginianus) is a prime example of a potential use for immunocontraception as a means of wildlife population management. Oral vaccination appears to be the most pragmatic way to deliver immunocontraceptive vaccines to free-roaming populations of deer, but there was little, if any, prior evidence that oral vaccination is a viable concept in deer. METHOD OF STUDY: We used live Bacillus Calmette-Guerin (BCG) in a recombinant form (rBCG), which expressed Borrelia burgdorferi outer surface protein A, to test whether deer vaccinated orally with a specific antigen expressed in a live vector produce detectable antibody titers. RESULTS: The data indicate that oral vaccination of deer with an expressed antigen is feasible, as demonstrated by peak antibody titers to the expressed antigen. Also, peak titers measured by enzyme-linked immunosorbent assay were highest in orally vaccinated deer: 1600 in deer vaccinated by injection and 6400 in those vaccinated orally. CONCLUSIONS: The results of this study demonstrate that it is feasible to vaccinate deer orally with a live vector.

PROBLEM: The unique recognition events that result in the avid binding of mammalian spermatozoa to the surface of the zona pellucida (ZP) are being exploited in the development of contraceptive vaccines. In this study, the safety and efficacy of a vaccination strategy based on the induction of active immunity against purified, glycosylated, recombinant human ZP3 (rhZP3) has been evaluated in a primate model, Callithrix jacchus. METHOD OF STUDY: Long-term infertility was established after immunization with rhZP3 and the resulting immune sera reacted with rhZP3 on an enzyme-linked immunosorbent assay (ELISA) and immunolocalized exclusively to the outer surface of native ZP on marmoset ovarian sections. However, this contraceptive effect was inevitably associated with the eventual appearance of an ovarian pathology characterized by a depletion of primordial follicles. In an attempt to circumvent this side effect, human ZP3 (hZP3) was epitope mapped and four continuous, immunodominant B-cell epitopes (hZP3 45-64 , hZP3 93-110 , hZP3 172-190 and hZP3 341-360 ) were evaluated for contraceptive efficacy in vivo. Using peptide-tetanus toxoid (TT) conjugates to enhance immunogenicity, antipeptide antibodies were raised against these immunogens, which also cross-reacted with rhZP3 on ELISA. In addition, antibodies against hZP3 45-64 and hZP3 172-190 recognized native ZP on marmoset ovarian sections when a microwave technique was used to enhance epitope presentation. RESULTS: No ovarian pathology was observed after the long-term administration of these peptide immunogens, and fertility was suppressed when compared with TT controls but could not be correlated to the antibody titer. CONCLUSION: Clearly, further research is required to identify optimal B-cell epitopes that will reliably induce infertility, free from any ovarian pathology.

PROBLEM: Phase II clinical trials with the heterospecies dimer of βhCG and α-subunit of ovine luteinizing hormone (HSD)-human chorionic gonadotropin (hCG) vaccine showed that pregnancy was prevented at and above 50 ng/ml titers, whereas conceptions occurred below 35 ng/ml of hCG bioneutralization capacity. The effect of below-protective threshold anti-hCG antibodies on the progression of pregnancy and the normality of progeny was studied. METHOD OF STUDY: Four women enrolled by informed consent in the Phase II trials. The women studied were immunized with the HCG vaccine and did not receive booster injections in consideration of their desire to have another child. They were examined clinically at least once every month until delivery. The babies were followed up from 2 to 3.5 years, and their anthropometric indices were compared with elder siblings. RESULTS: The pregnancies progressed to 35 to 38 weeks with the usual obstetric problems, and the children developed normally. CONCLUSIONS: The anti-fertility effect of the HSD-hCG vaccine is reversible and low titers of antibodies below the protective threshold have no apparent side effects on the progression of pregnancy and on the early development of the progeny.

To develop a vaccine for reversible control of fertility in women. MATERIALS AND PROTOCOLS: Purified beta subunit of hCG annealed to purified alpha subunit of ovine LH linked chemically to tetanus toxoid (TT) and diphtheria (DT); vaccine employed at 300 micrograms gonadotropin equivalent per injection adsorbed on alhydrogel with 1 mg SPLPS added in the first injection; Phase I safety trials in 47 women with elective tubal ligation; Phase II efficacy studies in 148 proven fertile women (2 children), sexually active, desirous of family planning using IUD; IUD removed when anti-hCG titres exceed 50 ng/ml hCG bioneutralization capacity; boosters given to maintain above threshold antibody levels; post coital tests conducted in 8 volunteers; sera of protected women analysed for immuno-determinants recognized by competitive enzyme immunoassays employing a panel of monoclonal antibodies and by direct binding to synthetic peptides; recombinant vaccines expressing beta hCG as a secreted product or as a fused protein anchored on membrane.Immunization was well tolerated with no significant changes in endocrine, metabolic and hematological indices. Normal ovulatory cycles were maintained as indicated by menstrual regulation. The vaccine was highly effective in preventing pregnancy (1 pregnancy in 1224 cycles ) at and above antibody titres of 50 ng/ml. Antibodies declined in course of time in absence of boosters, with conceptions occurring below 35 ng/ml titres indicating regain of fertility. Ability of antibodies to prevent pregnancy was confirmed by post coital tests. High avidity (10(10) M-1) and other characteristics of antibodies generated by the vaccine are described and compared with those induced by two other hCG vaccines having undergone Phase I trials. The antibody response of the HSD vaccine in humans is characterized predominantly to an epitope recognized by the monoclonals 206 and P3W80. The antibodies had low or no reactivity with the carboxy terminal peptide and 38-57 region peptide. Live recombinant vaccines expressing beta hCG as a membrane anchored peptide generated antibody response to hCG in all animals following a single injection.Reversible fertility control is feasible with the HSD-hCG vaccine without impairment of ovulation or disturbance of menstrual regularity. Suggestions have been made for further optimization of the vaccine, which include replacement of TT and DT by a panel of T non B determinants communicating with the entire MHC spectrum and development of recombinant vaccine expressing beta hCG along with membrane anchored carrier.

Contraception has been practiced for thousands of years. Nevertheless, it took until the late 1950s and early 1960s before a major breakthrough in contraceptive technology was achieved by the introduction of oral hormonal contraceptives. However, we have not succeeded in the development of a non-hormonal contraceptive that is comparable to the pill regarding its efficacy, safety, and acceptance. The immunological interference with the complex fertilization process is a very attractive target in this respect, whereby the zona pellucida, a non-cellular surrounding of all mammalian eggs, represents a potentially ideal target. Another interesting target are sperm : either for the development of a female contraceptive or for a male contraceptive, although the latter approach does not look very promising so far. In conclusion, given the enormous impact on mankind of a growing world population and given the very individual needs for contraceptive methods of different women in one and the same country and in different cultures we should make widely available a whole set of suitable, adjusted methods of fertility control and this includes the search for an effective method of male fertility control.

Autoantibodies to ZP3, a major glycoprotein of the zona pellucida (ZP) with sperm receptor function, can block sperm/oocyte interaction. However, only mice of certain major histocompatibility complex (MHC) haplotype respond to the ZP3 peptide. Moreover, ZP3-specific T cells can mediate ovarian autoimmune disease. A chimeric peptide has been designed that induces antibody to native ZP3 regardless of the MHC haplotype of the inbred mice tested. This results in reduction in fertility that is reversible. Infertility correlates well with ZP antibody titer, and the mice do not develop concomitant autoimmune oophoritis. The vaccine contains (1) a promiscuous foreign T-cell peptide capable of eliciting a T-cell response regardless of the animals' MHC haplotype, and (2) a modified native B-cell peptide of ZP3.

Problem Immunization with beta subunit of gonadotropin may elicit antibody formation to endogenous heterodimeric gonadotropin and result in reproductive unresponsiveness. The objectives of this study were to determine if antibodies produced in rats following immunization with human follicle stimulating hormone beta-subunit (hFSH-beta) could bind to and immunoneutralize heterodimeric FSH, and to elucidate the immunoneutralizing epitope. Method Mature female Sprague-Dawley rats received subcutaneous injections of 10 micrograms of hFSH-beta emulsified in complete Freund's adjuvant, while control animals received only adjuvant. Animals received 10 micrograms hFSH-beta booster injections emulsified in incomplete Freund's adjuvant 2, 4, 11, and 21.5 wk after the initial immunization. Results Immunization with hFSH-beta produced appreciable antibody titers against human FSH (hFSH) as measured in enzyme-linked immunosorbent assays (ELISA) of immunized rat sera. A more modest titer to rat (rFSH) and no antibody response to rat Luteinizing Hormone (rLH) was observed, thus confirming the specificity of the immune response. Titers against hFSH increased throughout the study. Rat anti-hFSH-beta sera was tested to determine its ability to inhibit binding (immunoneutralizing) of 125I-hFSH to the FSH receptor. Continued immunization resulted in all animals producing immunoneutralizing antibodies. Immunization of rats also resulted in disrupted estrous cycles, but only animals with subsequent titers high enough to completely block binding of FSH to its receptor failed to conceive. In order to assess the immunoneutralizing epitope, antisera were tested in a peptide ELISA. Peptides used in the ELISA corresponded to amino acids that spanned the entire hFSH beta sequence. It was found that antibodies from all rats immunized with hFSH beta bound to amino acids within hFSH-beta 33-53. Conclusions Collectively, these data suggest that amino acids within hFSH-beta 33-53 are necessary but not sufficient to confer immunocontraception. Amino acids within this linear sequence appear in a variety of epitopes of hFSH-beta and hFSH, only some of which are immunoneutralizing. (Am J Reprod Immunol. 1993; 29:000-000.)

The goals of this study were to determine the site at which fertility is impaired in mice immunized with LDH-C4 and to determine whether immunization of both males and females would have a greater antifertility effect than immunization of one sex alone. Mice were immunized with LDH-C4 in two systemic doses in Freund's adjuvants and two gastric doses in bicarbonate buffer. The presence of anti-LDH-C4 antibodies in uterine fluid was confirmed. Male and female mice were assigned to four blocks in which either the males, the females, both, or neither were immunized. Oviducts were viewed directly 1 h after mating for the presence of sperm. No significant effect of immunization on sperm transport to the oviduct could be demonstrated. Fertilization was evaluated 4 h after mating. It was found that immunization of males, but not females, impaired fertilization (19.6% versus 50.8% of the oocytes penetrated in 17 and 19 females, respectively). Orchitis was found histologically in 43.8% of the immunized males and 10% of the control males.