Immunogenicity and contraceptive efficacy of recombinant fusion protein encompassing Sp17 spermatozoa-specific protein and GnRH: Relevance of adjuvants and microparticles based delivery to minimize number of injections.

PROBLEM: Requirement of multiple injections of contraceptive vaccines to achieve infertility is one of the important impediments for their application. In the present study, attempts have been made to reduce the number of injections of contraceptive vaccine. METHOD OF STUDY: Fusion protein encompassing C-terminus fragment of sperm protein Sp17 (aa residues 76-126) and two copies of gonadotropin-releasing hormone along with T-cell epitopes and dilysine linkers (abbreviated as Sp17C -GnRH2 ) was expressed in Escherichia coli. Its immunogenicity and contraceptive efficacy have been evaluated in female FVB/J mice using different adjuvants and delivery platforms. RESULTS: Immunization of female mice with recombinant Sp17C -GnRH2 (25 µg/injection/mouse) emulsified with squalene-arlacel A following two injections schedule led to failure of 88.8% immunized animals to conceive, which was not significantly different from mice immunized with same protein along with alum following three injections schedule. To make single-dose vaccine, poly d,l-lactic acid-based microparticles (PLA-MPs) entrapping Sp17C -GnRH2 were prepared. Immunization of female mice with a combination of soluble Sp17C -GnRH2 (12.5 µg/injection/mouse) along with Sp17C -GnRH2 entrapped in PLA-MPs (12.5 µg/injection/mouse) in alum showed higher antibody titres and contraceptive efficacy as compared to mice immunized with Sp17C -GnRH2 entrapped in PLA-MPs alone in alum. Immunization with recombinant Sp17C -GnRH2 led to long-term infertility as second mating (150 days after immunization) of various groups of immunized mice showed similar infertility as observed during first mating. CONCLUSION: Single-dose immunization with PLA-MPs entrapping Sp17C -GnRH2 along with soluble recombinant protein in alum generated long-lasting infertility in female mice.

Antisperm contraceptive vaccines: where we are and where we are going?

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.

Contraceptive vaccines targeting factors involved in establishment of pregnancy.

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.

Contraceptive vaccines for the humane control of community cat populations.

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.

Immunization with a DNA vaccine of testis-specific sodium-hydrogen exchanger by oral feeding or nasal instillation reduces fertility in female mice.

OBJECTIVE: To investigate the effect of immunization with a DNA vaccine of testis-specific sodium-hydrogen exchanger (tsNHE) via oral feeding or nasal instillation on fertility in female mice and to look at its potential mechanism. DESIGN: Prospective, research study. SETTING: Institution-affiliated research laboratory. ANIMAL(S): Sexual mature BALB/c mice. INTERVENTION(S): Female mice immunized orally or nasally with the DNA vaccine at 2-week' intervals. MAIN OUTCOME MEASURE(S): Number of newborns and fertility rate of the vaccinated female mice were scored. RESULT(S): We identified a novel testis-specific sodium-hydrogen exchanger, tsNHE, which is localized to the principal piece of sperm flagellum. Immunization of female mice with the tsNHE DNA vaccine via oral feeding or nasal instillation statistically significantly decreased fertility rate and the newborn numbers compared with the controls. The antiserum or vaginal fluid from the tsNHE cDNA vaccinated female mice could specifically recognize the principal piece of sperm tail and triggered sperm agglutination. The antibodies also showed a statistically significant inhibitory effect on in vitro sperm motility and fertilization. CONCLUSION(S): The sodium-hydrogen exchanger might be an excellent target molecule for developing a new contraceptive.

Experimental immunological infertility effect of anti-GAPDH-2 antibodies on the fertility of female mice.

OBJECTIVE: To examine the relationship between an antibody against GAPDH-2, a sperm-specific protein, and infertility of female mice. DESIGN: Basic research. SETTING: National Research Institute for Family Planning Beijing, World Health Organization Collaboration Center of Human Reproduction. ANIMAL(S): New Zealand rabbit, NIH and ICR mice. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Enzyme-linked immunoabsorbent assay, Western blot and indirect immunostaining assays, standard fertility assay, and sperm agglutination assay. RESULT(S): Antibodies against the full-length GAPDH-2 were raised. Its specificity was assessed by immunoblotting and indirect immunostaining assays. The antibody immunoreacted with human sperm GAPDH-2 and the mouse homolog GAPDS but did not cross-react with GAPDH. Treatment of female mice with IP injection of anti-GAPDH-2 serum significantly reduced their fertility. Anti-GAPDH-2 serum caused the agglutination of normal mice sperm in vitro. The anti-GAPDH-2 antibody was detectable in the sera and uterine fluid of the mice immunized with GAPDH-2. CONCLUSION(S): These results show that GAPDH-2 should be further evaluated as a promising candidate in the development of an antifertility immunogen. Detecting anti-GAPDH-2 antibodies in the bodily fluid of subjects afflicted with indeterminate infertility may be a new diagnostic index.

Synthesis of toll-like receptor-2 targeting lipopeptides as self-adjuvanting vaccines.

Effective Th1- and Th2-type immune responses that result in protective immunity against pathogens can be induced by self-adjuvanting lipopeptides containing the lipid moiety dipalmitoyl-S-glyceryl cysteine (Pam2Cys). The potent immunogenicity of these lipopeptides is due to their ability to activate dendritic cells by targeting and signaling through Toll-like receptor-2 (TLR-2). In addition, the simplicity and flexibility in their design as well as their ease of chemical definition and characterisation makes them highly attractive vaccine candidates for humans and animals. We describe in this chapter the techniques involved in the synthesis of an immunocontraceptive lipopeptide vaccine as well as the experimental assays carried out to evaluate its efficiency.

Immunocontraceptive properties of recombinant sperm protein DE: implications for the development of novel contraceptives.

OBJECTIVE: To evaluate the immunocontraceptive properties of recombinant DE, a sperm epididymal protein involved in fertilization, via an experimental study in rats as a critical step toward the development of a human immunocontraceptive. DESIGN: In vivo study in rats. SETTING: Animal care facility of an academic research center. ANIMAL(S): Seventy-four 90-day-old Wistar male and female rats distributed into three groups. INTERVENTION(S): Animals received five injections (intramuscular and subcutaneous) of recombinant DE (recDE), native DE (nDE), or MBP (maltose-binding protein). At various times, animals were anesthetized and bled. MAIN OUTCOME MEASURE(S): Anti-DE levels and tissue specificity of sera were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot, respectively. Fertility was analyzed by natural mating. The testes and epididymides were analyzed by histology. RESULT(S): Recombinant DE raised an immune response with the same kinetics and higher anti-DE levels than that elicited by nDE. Sera against recDE recognized epitopes of DE that were different from those recognized by anti-nDE sera but specifically reacted with DE in epididymis and sperm without cross-reacting with other tissues tested. Male and female recDE-injected animals presented a statistically significant reduction in their fertility with no evidence of pathologic effects. CONCLUSION(S): Recombinant DE is able to both elicit a specific immune response and inhibit male and female fertility, supporting the use of this sperm epididymal protein for the development of an immunocontraceptive approach.

Mapping of B cell epitopes on the zona pellucida 2 protein of a marsupial, the brushtail possum (Trichosurus vulpecula).

Immunocontraceptive vaccines against zona pellucida (ZP) proteins are being developed for brushtail possum (Trichosurus vulpecula) management in New Zealand. Mapping of B cell epitopes on the ZP2 protein of possums was undertaken in this study to define the antigenic regions that may be crucial to sperm-egg binding. The amino acid sequence of the full-length possum ZP2 protein (712 amino acids) was used to synthesize a complete set of 71 (15-mer) biotinylated peptides with an offset of five amino acids. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with recombinant possum ZP2 (rZP2) constructs. Seventeen continuous epitopes were located on possum ZP2 protein. Comparisons of the peptide binding pattern of antibodies in individual sera with the fertility status of the same immunized possums revealed three significant infertility-relevant peptide epitopes (amino acids 111-125, 301-315, and 431-445). One of these (amino acids 431-445) bound to possum spermatozoa from the caudal epididymis. The implications of these findings for developing immunocontraceptive vaccines for possum control are discussed.

A novel retro-inverso gonadotropin-releasing hormone (GnRH) immunogen elicits antibodies that neutralize the activity of native GnRH.

GnRH vaccines have been successfully used for the inhibition of gonadotropin secretion and gonadal function. As an alternative to native GnRH, retro-inverso (RI) GnRH might be an improved immunogen. The RI peptides are composed of D-amino acids assembled in the reverse order (C to N terminus) in relation to the parent L peptide. These peptides are immunogenic and can produce high titers of antibodies that bind the parent peptide with high affinity and specificity. We show that RI-GnRH peptides conjugated to ovalbumin as well as unconjugated RI-GnRH elicit high titers of anti-GnRH antibodies in rabbits and mice. Antibodies were affinity purified and shown by ELISA to be selective for mammalian GnRH compared with GnRH II and [Gln(8)]GnRH. The binding kinetics of antibody-peptide interactions was determined using biosensor technology (BIACORE). The purified anti-GnRH antibodies inhibited GnRH-stimulated signal transduction in COS-1 cells expressing the human GnRH receptor. Immunization of mice with unconjugated and conjugated RI-GnRH peptide, in the absence of complete Freund's adjuvant, produced antisera that cross-reacted with mammalian GnRH. As RI peptides are resistant to cleavage by proteolytic enzymes, they are potentially orally active. The ability of RI-GnRH peptides to produce antibodies to GnRH without conjugation and without Freund's complete adjuvant constitutes a novel vaccine with improved properties of potential application in animal management and sex hormone-dependent cancers.

Sperm antibodies in rat models of male hormonal contraception and vasectomy.

The presence of sperm antibodies correlates with nearly every pathological condition of the male reproductive tract. In the seasonal breeder, mink, a decrease in gonadotrophin secretion and testicular regression also induces sperm antibodies. Because the Sertoli cells and the principal cells of the epididymis (i.e. the cells mainly responsible for protection of germ cells from autoimmune destruction) are dependent on androgens, and because the androgen concentration decreases in both the testis and epididymis during male hormonal contraception, the presence of IgG class sperm antibodies in serum was studied in rats during the suppression and recovery phases of testosterone contraception and after vasectomy. Five-centimetre long testosterone implants were placed under the dorsal skin of rats under pentobarbitone anaesthesia. The control rats received empty implants. All implants were left in the rats for 27 or 53 days. The total number of testicular antigens detected by sera from the vasectomized rats increased significantly until 66 days post-operation, and then decreased to the levels of intact rats. The number of testicular antigens detected by sera from rats receiving contraceptive doses of testosterone did not increase before the testosterone capsules were removed, but at 40 days post removal of the silastic capsules, the number of antigens detected by the sera was significantly higher than in intact rats and at 77 days post removal of the silastic capsules, the number of antigens detected by the sera was significantly higher than at 27 days after starting testosterone administration. No significant changes in the number of antigens detected by the sera could be observed after the implanting of empty capsules or after their removal. Vasectomy mostly induced antibodies against testicular antigens in the molecular ratio ranges of 70-82, 25-33 and 21-24.5 kD. Antibodies against antigens in these molecular ratio ranges were not significantly induced during or after treatment with contraceptive doses of testosterone. Cell nuclei with apoptotic morphology could be observed in the seminiferous tubules of the vasectomized rats, but DNA in situ 3'-end labelling of testes could not confirm any differences between the testes of vasectomized and sham-operated rats or between testosterone-treated and empty implant-treated rats. CD3+ T cells could not be observed in the testes of any of the treatment groups. These results suggest that the immunological conditions remain stable in the testes after vasectomy and during testosterone treatment, but that the animals are more prone to develop autoantibodies after vasectomy and during recovery from treatment with exogenous testosterone.

Identification of bioneutralization epitopes of human follicle stimulating hormone in the regions 31-52 and 66-75 of its beta-subunit.

The crucial role played by follicle stimulating hormone (FSH) in regulating both male and female reproduction and the possibilities of developing contraceptive methods for males by blocking the function of the hormone, makes it important to delineate the hormone-specific bioneutralization epitopes of human follicle stimulating hormone (hFSH) on its beta-subunit. Predictive methods were used to identify the potential surface-oriented regions of hFSH-beta. Peptides corresponding to these regions, i.e. 31-52, 66-75 and 86-95 hFSH-beta, were synthesized, anti-peptide antibodies were elicited in rabbits and the properties of these antisera to bind hFSH and neutralize its biological activity were assessed. Anti-31-52 hFSH-beta antisera bound hFSH specifically, whereas anti-66-75 and anti-86-95 hFSH-beta antisera did not show any detectable binding, proving the region 31-52 hFSH-beta to be a specific antigenic determinant of hFSH. The bioneutralizing abilities of the anti-peptide antibodies were assessed by measuring the hFSH-induced progesterone secretion by rat granulosa cells in vitro. Antibodies to 31-52 and 66-75 hFSH-beta neutralized the bioactivity of hFSH, but anti-86-95 hFSH-beta antibodies did not. Furthermore, the three linear peptides and two disulphide looped peptides of 31-52 hFSH-beta and 86-95 hFSH-beta were also subjected to the in-vitro granulosa cell assay. The linear peptides 31-52 hFSH-beta and 66-75 hFSH-beta and the cyclic 31-52 hFSH-beta disulphide loop peptide significantly inhibited the hFSH-induced progesterone secretion by rat granulosa cells, but the linear 86-95 hFSH-beta peptide and the corresponding cyclic disulphide loop peptide did not. The results clearly show that the regions 31-52 and 66-75 of hFSH-beta harbor bioneutralization epitopes of the hormone. The studies also indicate that cyclization of the linear 31-52 hFSH-beta peptide greatly enhances receptor recognition and that the region 66-75 hFSH-beta may also be involved in hormone-receptor interaction.

A new look at antifertility vaccines.

PIP: 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.^ieng