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Induction of high titer mouse--antihuman spermatozoal antibodies by liposome incorporation of spermatozoal membrane antigens.

This is the first report of induction of high titer mouse-antihuman spermatozoal antibodies using a spermatozoal antigen incorporated in liposomes. The peptide antigens that were incorporated into pure phosphatidylcholine liposomes are known to react with the naturally occurring antibodies in sera of sterile patients. Electron microscopy and the complement-dependent glucose release tests indicated that the peptides were entrapped in the aqueous phase between the lipid bilayers in the liposome vehicles. This is in contrast to most other liposomal antigens that are expressed on the outer lipid surface. In view of the nontoxic nature of the liposomes these results could be of some interest in immunological fertility regulation trials with appropriate antigens in an allogeneic system.

Animals↗
PubMed2004

Reversible immunocontraception in male monkeys immunized with eppin.

Various forms of birth control have been developed for women; however, there are currently few options for men. The development of male contraceptives that are effective, safe, and reversible is desired for family planning throughout the world. We now report contraception of male nonhuman primates (Macaca radiata) immunized with Eppin, a testis/epididymis-specific protein. Seven out of nine males (78%) developed high titers to Eppin, and all of these high-titer monkeys were infertile. Five out of seven (71%) high-anti-Eppin titer males recovered fertility when immunization was stopped. This study demonstrates that effective and reversible male immunocontraception is an attainable goal. This method of immunocontraception may be extended to humans.

Animals↗
PubMed2002

Differences in immunocontraceptive responses in white-tailed deer (Odocoileus virginianus) and goats (Capra hircus).

Thirteen deer (Odocoileus virginianus) and three goats (Capra hircus) were given an initial immunization of 300 microg porcine zona pellucida (PZP) combined with 50 mg synthetic trehalose dicorynomycolate ml(-1) (STDCM) in drakeol i.m. in the semitendinosus and semimembranosus muscles. This immunization was followed by two booster injections of 300 microg PZP, 2 weeks apart. The vaccinations were made from the same purified batch of PZP and lot of adjuvant. All the doses were made at the same time and injected on the same day. The immune response was quantified by measuring serum anti-PZP IgG antibody concentration by ELISA. The results showed that the mean serum IgG concentration of the deer increased from 0.000 +/- 0.003 to 0.083 +/- 0.023 absorbance units, whereas in the goats the mean IgG concentration increase was from 0.044 +/- 0.019 to 1.245 +/- 0.774 absorbance units. Both the goats and deer showed a significant increase in IgG concentration (P < 0.05) after the final booster injection compared with preimmune serum. The final goat IgG concentration was significantly (P < 0.05) higher than that of the deer. If PZP and Freund's adjuvant are administered to deer, immunocontraception is achieved, but the IgG concentration in the deer in this study did not appear to be compatible with immunocontraception.

Animals↗
PubMed2004

Development of mouse-specific contraceptive vaccines: infertility in mice immunized with peptide and polyepitope antigens.

Mouse-specific immunocontraceptive peptides have been identified in mouse proteins with key roles in reproduction from sequence comparisons to other species and tested for efficacy as immunocontraceptive antigens. Peptides were derived from granulocyte-macrophage colony-stimulating factor (GMCSF), the placental 27 kDa heat-shock protein (HSP), leukemia inhibitory factor receptor (LIFR), oviduct glycoprotein (OGP), proliferin (PLF), prolactin (PRL), sperm protein SP56 and mouse zona pellucida subunits 1 and 3 (ZP1, ZP3). Fertility of female BALB/c mice was reduced after immunization with several peptides either conjugated to a carrier protein or in the form of recombinant polyepitopes. The most effective conjugated peptides (SP56, GMCSF and PRL) induced peptide-specific serum antibodies and reduced fertility by 50%. Fertility of mice was also reduced after immunization with polyepitope antigens containing up to five different peptides fused to maltose-binding protein, but antibodies were not produced against all the encoded peptides. The most effective polyepitope antigen (containing PLF, SP56, ZP1 and ZP3 peptides) reduced fertility by 50% but induced only SP56 and ZP1 antibodies. We demonstrate that lack of antibody response to a given peptide epitope (ZP3) can be overcome if repeated copies are used in the polyepitope antigen construct, but the effect varies between mouse strains. We conclude that infertility induced in mice with a range of peptide-based vaccines is dependent on antigen formulation and genetic factors but does not necessarily correlate with peptide-specific antibody levels. In light of these results, strategies to improve the efficacy of peptide-based antifertility vaccines are discussed.

Amino Acid Sequence↗
PubMed2002

Molecular approaches for the evaluation of immune responses to zona pellucida (ZP) and development of second-generation ZP vaccines.

It has long been established that there are major variations in both the immunogenicity and antigenicity of native zona pellucida (ZP) proteins. These differences appear to be more pronounced with respect to genetically engineered ZP proteins, which do not have native post-translational modifications (for example glycosylation and sulphation). As the number of animal species that are now included in population management programmes using native porcine zona pellucida (PZP) proteins expands, it is increasingly important to carry out studies to evaluate the immune response variations among different species as well as the individual variation within a species. In an attempt to compare these complex immune responses, we have evaluated antibodies from numerous species immunized with native, genetically engineered ZP and synthetic ZP peptides. Such an immunocontraceptive method could have great potential. These studies are critical not only for the development of predictable immune responses that result in permanent sterilization versus reversible contraceptive effects, but also for predicting which vaccinogens (native ZP protein versus genetically engineered ZP proteins) might have detrimental effects on animal and human populations.

Animals↗
PubMed2002

Comparative effects of GnRH and porcine zona pellucida (PZP) immunocontraceptive vaccines for controlling reproduction in white-tailed deer (Odocoileus virginianus).

Fawning rates and mating behaviour were compared between white-tailed deer (Odocoileus virginianus) treated with GnRH and porcine zona pellucida (PZP) immunocontraceptive vaccines from 1997 to 2000. Female deer from a herd of 102 deer at Seneca Army Depot, near Romulus, New York, were treated with prime and booster injections of PZP (n = 22) or GnRH vaccine (n = 32), or remained untreated as controls (n = 34). During the summers after booster treatment, observed fawning rates for adult female deer were similar for both PZP-treated (0.10-0.11 fawns per female) and GnRH-treated (0.13-0.22 fawns per female) female deer, and were significantly lower (t = -8.93 and t = -9.73; P < or = 0.0005, respectively) than those observed for control female deer (1.22-1.38 fawns per female). During the second (0.36 fawns per female) and third summers (0.61 fawns per female) after the last booster injection, GnRH-treated female deer still produced significantly fewer fawns than did the controls (1.38 and 1.31 fawns per female, respectively). In one breeding season after treatment, five of 18 (28%) females vaccinated with PZP produced fawns, similar to the rate for GnRH-treated females (29%). In addition, females treated with GnRH had fewer oestrous cycles per female (0.06, P < or = 0.05) than did either control (0.22 cycles per female) or PZP-treated deer (0.36 cycles per female). Initial PZP treatment followed by a booster dose 5-7 months later reduced fawn production and prolonged the breeding season as females repeatedly returned to oestrus, similar to results reported in other studies.

Animals↗
PubMed2001

Cloning and sequencing of cDNA encoding for a novel human testis-specific contraceptive vaccinogen: role in immunocontraception.

Sperm-specific antigens are attractive candidates for the development of a contraceptive vaccine. Using the subtractive cDNA hybridization technology, the present study was conducted to obtain a human sperm-specific antigen. The 32P-labeled single stranded cDNA of human testis, subtracted with poly(A)+ RNA of human peripheral white blood cells, was used to screen the human testis cDNA-ZAP II library. The putative positive clones were further screened for binding with the solubilized human oocyte zona pellucida preparation (HZP). After screening 10(7) colonies, one positive clone, designated contraceptive vaccinogen (CV), was obtained. It had an insert of approximately 1.3 kb, that was cloned and sequenced. The sense strand was identified by using the in vitro transcription and translation procedures, and the full-length sequence was obtained by using the 5' rapid amplification of 5' -cDNA ends (5'-RACE) procedure. The full-length CV cDNA has an ORF of 312 amino acids (aa) with the first ATG Met start codon at nucleotide (nt) 35 and the stop codon TAA, at nt 959. The translated protein has a calculated molecular mass of 35.3 kD and four potential N-linked glycosylation and several phosphorylation sites. Hydropathy plot generated from the deduced aa sequence showed it to be a membrane-anchored peptide. Extensive computer search in the database did not find any homology of existing sequences with CV both for nt and aa. Northern blot analysis indicated the human testis-specific expression of CV antigen. The coding region of CV cDNA was subcloned into pET22b(+) vector and expressed. The expressed recombinant (r)CV protein had a molecular size of approximately 44 kD, and it specifically reacted with the ZP3 component of HZP. Rabbit rCV antibodies recognized the rCV, and a cognate antigen of approximately 64 kD in the human sperm extract. The antibodies showed binding with the live and methanol-fixed human sperm, and significantly (P < 0.001) inhibited human sperm penetration of zona-free hamster oocytes, as well as human sperm binding to human oocyte zona pellucida. These findings indicate that the testis/sperm- specific CV antigen has a role in human sperm function and may find clinical applications in the contraceptive vaccine development and in the specific diagnosis and treatment of male infertility.

Adult↗
PubMed2003

Contraceptive responses of mice immunized with purified recombinant mouse zona pellucida subunit 3 (mZP3) proteins.

Mouse zona pellucida subunit 3 (mZP3) was tested for efficacy as an immunocontraceptive antigen by comparing the fertility of mice immunized with recombinant mZP3 proteins. Recombinant protein was expressed using either the vaccinia virus T7 mammalian (vmZP3 protein) or baculovirus insect cell (bmZP3 protein)-expression systems. Female BALB/c or wild mice were immunized by i.p. injection using Freund's complete adjuvant and boosted three times with affinity purified recombinant proteins in Freund's incomplete adjuvant. Most mice developed antibodies that crossreacted to the respective mZP3 antigens by ELISA or western blot. In BALB/c mice immunized with vmZP3, fertility and mean litter size were reduced transiently to 25% and 10%, respectively, of those of control mice. However, immunization with bmZP3 did not affect either the fertility or mean litter sizes in BALB/c or wild mice immunized with bmZP3. The results demonstrate that reduction in fertility can be achieved in female BALB/c mice immunized using Freund's adjuvants and recombinant mZP3 protein produced in a mammalian, but not an insect, cell-expression system. Arguments are presented for the likely role of glycosylation of the mZP3 antigen in inducing contraceptive immune responses.

Animals↗
PubMed2005

Induction of castration by immunization of male dogs with recombinant gonadotropin-releasing hormone (GnRH)-canine distemper virus (CDV) T helper cell epitope p35.

Immunocastration is a considerable alternative to a surgical castration method especially in male animal species for alleviating unwanted male behaviors and characteristics. Induction of high titer of antibody specific for gonadotropin-releasing hormone (GnRH) correlates with the regression of testes. Fusion proteins composed of canine GnRH and T helper (Th) cell epitope p35 originated from canine distemper virus (CDV) F protein and goat rotavirus VP6 protein were produced in E. coli. When these fusion proteins were injected to male dogs which were previously immunized with CDV vaccine, the fusion protein of GnRH-CDV Th cell epitope p35 induced much higher antibody than that of GnRH-rotavirus VP6 protein or GnRH alone. The degeneration of spermatogenesis was also verified in the male dogs immunized with the fusion protein of GnRH-CDV Th cell epitope p35. These results indicate that canine GnRH conjugated to CDV Th cell epitope p35 acted as a strong immunogen and the antibody to GnRH specifically neutralized GnRH in the testes. This study also implies a potential application of GnRH-based vaccines for immunocastration of male pets.

Amino Acid Sequence↗
PubMed2006

Mucosal inoculation of Lactobacillus expressing hCGbeta induces an anti-hCGbeta antibody response in mice of different strains.

To show that an anti-human chorionic gonadotrophin-beta (hCGbeta) antibody response can be induced by inoculating Lb. expressing hCGbeta through different mucosal pathways in mice of two strains, female BALB/c and C57BL/6 mice were immunized via vaginal, oral or nasal routes with 10(8), 10(9), and 10(10)Lb.hCGbeta (a recombinant Lactobacillus expressing hCGbeta). The mice were immunized twice with a booster in study week 3. An indirect ELISA was used to determine anti-hCGbeta IgG and IgA antibodies in vaginal lavage and serum, obtained from the 2nd to 8th week after the primary immunization. Flow cytometry was used to analyze the lymphocyte proliferation from these tissues, 1 week after the primary immunization. The hCGbeta antigen-specific antibody-secreting cells of spleen, uterus, and vagina were evaluated by enzyme-linked immunospot assay (ELISpot), 2 weeks after the booster. The analysis showed that 10(9) and 10(10)Lb.hCGbeta inoculations induced similar anti-hCGbeta antibody responses, while the three mucosal pathways induced similar antibody responses. The antiserum obtained after boosters with 10(9) and 10(10)Lb. hCGbeta was able to neutralize more than 100 ng/ml hCG antigen, both in BALB/c and C57BL/6 mice. The highest antibody titer induced by vaginal mucosal immunization was stronger than that obtained via the other mucosal pathways. The B cells in the vagina appeared to proliferate after vaginal immunization (P<0.05). The numbers of anti-hCGbeta IgG and IgA antibody-secreting cells in the uterus and vagina were greater than in the spleen. Therefore, the vaginal mucosal route appears to be a better immunization pathway to induce higher anti-hCGbeta antibody levels in the reproductive tract.

Administration, Intranasal↗
PubMed1998

Antibody response of elk immunized with porcine zona pellucida.

Immunocontraception using porcine zona pellucida (PZP) vaccines is being explored as a nonlethal method of solving the problems of locally overabundant wildlife populations. This study characterized the immunological response of captive elk (Cervus elaphus nelsoni) to PZP challenge using 18 3-yr-old cows and was conducted from 14 September 1994 to 13 December 1995. All animals were given a single PZP inoculation and 1 mo later six of these animals were randomly chosen and received a booster inoculation. Blood samples were drawn from all animals at the time of the initial inoculation and 1, 2, 4, 6, 10, and 15 mo later. Immunological response was assessed by measuring anti-PZP antibody levels in serum. All animals demonstrated a strong immune response with no evidence that the booster enhanced antibody levels. Antibody levels rose from between 0 and 4 at the time of the initial injection to peak levels of 85 to 163 within 2 to 6 mo, followed by a noticeable decline by 15 mo post-vaccination. Limited data suggest that antibody levels > 100 may be required to effect contraception. High individual variability in immune response observed in this study suggests it may be difficult to predict the proportion of animals effectively treated. Disruption of seasonal synchrony in calving also could occur if antibody levels in individuals fall below effective levels while animals are still cycling. These results indicate that immunocontraception using PZP vaccines is possible for elk. However, carefully controlled population experiments will be required in order to assess the potential and limitations for management applications of this technique.

Animals↗
PubMed1986

Pathways to immunocontraception: biochemical and immunological properties of glycoprotein antigens of the porcine zona pellucida.

The zona pellucida (ZP) is an extracellular coat which surrounds the mammalian oocyte and whose macromolecular composition is unique for each species. Two-dimensional gel electrophoresis of porcine ZP under reducing conditions resolves four major acidic, charge-heterogeneous glycoproteins with apparent molecular weights of 82,000 (ZP1), 61,000 (ZP2), 55,000 (ZP3) and 21,000 (ZP4). Fractionation of heat-solubilized porcine ZP under nondissociative, nonreducing conditions resulted in a preparation, termed purified pig zona antigen (PPZA), substantially enriched in ZP3 and lacking ZP1 and ZP2. PPZA was highly immunogenic in both rabbit and squirrel monkey. Anti-PPZA sera exhibited contraceptive potential as assessed by in vitro inhibition of homologous sperm-zona binding following antibody treatment of human, pig and squirrel monkey oocytes. Alternatively, fractionation of sodium dodecyl sulfate (SDS)-dissociated porcine ZP under nonreducing conditions resulted in isolation of two electrophoretically homogeneous antigens with apparent molecular weights of 82,000 (82K antigen) and 55,000 (55K antigen) as judged by nonreducing SDS-polyacrylamide gel electrophoresis. The 82K antigen exhibited an estimated molecular weight of 345,000 by gel filtration chromatography in nondissociative solvent and was comprised of ZP1 and disulfide-linked heterodimers of ZP2+ZP4 and ZP3+ZP4. The 55K antigen was comprised of ZP3 which was highly aggregated in nondissociative solvent. Each antigen had a characteristic carbohydrate composition and proved to be immunogenic in the rabbit. The effects of active heteroimmunization of squirrel monkeys with 55K antigen on fertility are under investigation.

Animals↗
PubMed1989

Reproductive and endocrine changes in ewes actively immunized against luteinizing hormone.

The objective of this study was to examine reproductive and endocrine changes in ewes actively immunized against a conjugation of luteinizing hormone (LH) and ovalbumin (LH immunized n = 10) or ovalbumin alone (control; n = 10). Ewes were immunized at weeks 0, 3 and 5 of the 93-week study and exposed to a fertile ram during weeks 7-25 (first breeding season) and weeks 60-67 (second breeding season). Immunization against LH abolished estrus and prevented pregnancy in all but one LH immunized ewe during the two breeding seasons. Uterine weights of LH immunized ewes were lighter than those observed in control ewes while ovarian weights did not differ between the treatment groups. No differences were observed between basal circulating concentrations of LH measured by an in vitro bioassay for the two treatment groups. However, serum concentrations of immunological follicle stimulating hormone, and the number of medium sized ovarian follicles (4-6 mm) were increased in LH immunized ewes. It was concluded from this study that immunization against LH prevents ovulation presumably by blocking the preovulatory surge of LH and not altering basal concentration of LH.

Animals↗
PubMed2004

Assessment of the immunological and biological efficacy of two different doses of a recombinant GnRH vaccine in domestic male and female cats (Felis catus).

This study has assessed the immunological and biological efficacy of two different doses of a recombinant GnRH vaccine administered to intact domestic cats. Fifteen kittens, 8-9 weeks of age, were allocated to three unequal treatment groups: group 1, 1 male control cat; group 2, 5 females administered 10 microg of GnRH antigen; and group 3, 4 males and 5 females administered 100 microg of GnRH antigen. Animals in groups 2 and 3 were immunized at study days 0, 28 and 643. One of the four males (AJZ3) in the high dose group showed a more rapid decline in GnRH antibody titers and received an additional immunization at day 461. Blood samples were collected on study days 28, 35, 56, 97, 157, 213, 270, 325, 377, 433, 496, 549, 605, and 685. The injection sites were monitored for tissue reactivity on study days 5, 7, 12, and 28. The animals' general health and demeanor was monitored on a daily basis. Sera obtained from 11 animals on day 549 were submitted for biochemistry analysis. Two males and two females were necropsied at the completion of the study and histopathological examination of the gonads, hypothalamus, pituitary, kidneys and uterus was performed. All 14 immunized animals developed immunoneutralizing titers to GnRH. GnRH titers peaked at day 56 and 13 of 14 cats maintained these titers for >20 months. Except for AJZ3, the immunized males' serum testosterone concentrations were below the assay's level of detection after the second immunization. None of the 10 immunized females showed signs of estrous behavior or became pregnant. Testicular and ovarian histology was consistent with suppression of LH and FSH activity. The majority of tissue reactions resolved by 28 days post-vaccination. Serum biochemistry and tissue histopathology revealed no evidence of tissue or organ damage. This study was unique in that a recombinant GnRH antigen was used to stimulate and maintain biologically relevant titers in very young male and female cats for at least 20 months.

Animals↗
PubMed1996

Possum biocontrol: prospects for fertility regulation.

Research has begun recently into biocontrol of brushtail possums as the only long-term, cost-effective solution to the possum problem in New Zealand, where possums cause significant damage to native forests, threaten populations of native plants and animals, and infect cattle and deer with bovine tuberculosis. Fertility regulation as a means of biocontrol has the support of major animal welfare and conservation groups in New Zealand. Systems are being investigated, mostly in reproduction and development, with the ultimate aim of developing immunologically-based fertility regulation (immunocontraception), but much basic information essential to such an approach for possums is lacking. The key components for the success of this approach--suitable vectors expressing possum-specific reproductive antigens sufficiently to block reproduction--are reviewed. The social and political issues of local, national and international risk and acceptability arising from such an approach are also discussed.

Animal Welfare↗