Immunocontraceptive Antigen Research Articles

Silkworm recombinant bovine zona pellucida protein 4 (bZP4) as a potential female immunocontraceptive antigen; impaired sperm-oocyte interaction and ovarian dysfunction.

Porcine zona pellucida proteins (ZPs) have been utilized as female immunocontraceptive antigens. The purpose of this study was to explore the potential use of silkworm recombinant bovine ZP4 as an alternative. When the protein was injected with monophosphoryl lipid A (MPL) – an immuno-stimulative agent – into two female goats, marked elevation of the anti-ZP4 titer was detected. Application of the purified specific IgG to a porcine in vitro fertilization system reduced the sperm penetration rate. In one goat, the cyclic profile of serum progesterone disappeared as the anti-ZP4 titer increased. Histological examination of the ovaries revealed degeneration of antral follicles with sparse infiltration of inflammatory cells in the theca, indicating that autoimmune oophoritis had been induced. Together, the present results suggest that recombinant ZP4 disturbs fertilization and exerts a pathogenic effect on follicle development in goats, thus indicating its potential as a female immunocontraceptive antigen.

Immunocontraceptive potential of the Ig‐like domain of Izumo

To investigate whether the Ig-like domain of sperm protein Izumo or the other part of the protein could be used as an immunocontraceptive antigen, three partially overlapping cDNA fragments (PA, PB, and PC), together covering entire mouse Izumo, were cloned, expressed, and purified. PB contains the whole Ig-like domain of mouse Izumo. The anti-PB antibody significantly inhibited the fusion of sperm with zona-free mouse eggs with no effect on sperm motility, while anti-PA and anti-PC antibodies virtually had no effect on sperm-egg fusion at the same concentration. Furthermore, in the presence of anti-PB antibody, the anti-sperm reactivity could be competitively inhibited by recombinant PB protein. The PB-specific antibody staining was restricted to the acrosome region in acrosome-reacted mouse spermatozoa by indirect immunofluorescence. Active immunization with the PB antigen sharply raised the antibody titers in mouse that were enough to cause a significant reduction in fertility compared to the PA and PC immunized groups. In conclusion, our data indicate that the Ig-like domain of Izumo plays an important role in the fertilization process, as verified by the dose-dependent reduction in fertilization rates in mouse IVF trials and mouse mating assay. These results indicate that the Ig-like domain of Izumo might be a new candidate for the development of a contraceptive vaccine.

ORIGINAL ARTICLE: Investigation of Recombinant Mouse Sperm Protein Izumo as a Potential Immunocontraceptive Antigen

ProblemTo determine if the recombinant mouse Izumo (mIzumo) could be used as a potential immunocontraceptive antigen.Method of studyThe 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.ResultsThe 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.ConclusionThe 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.

Immunocontraception in mice using repeated, multi‐antigen peptides: immunization with purified recombinant antigens

Two immunocontraceptive antigens (AgE and AgF) were constructed that included different combinations of highly species-specific peptides from the mouse reproductive antigens SP56, ZP3, ZP2, and ZP1 in the form of multi-antigen peptides (MAPs). Both AgE and AgF contained three tandem repeats each of ZP2 and ZP3 peptide epitopes and a single copy of a ZP1 peptide sequence all of which had previously been demonstrated to individually have immunodominant or contraceptive effects. In addition, AgF contained a single contraceptive peptide derived from SP56, the putative ZP3 receptor protein on sperm. The antigens were expressed and affinity purified as recombinant repeated multi-antigen (polyepitope) peptides using an Escherichia coli maltose binding protein (MBP) expression system. Female BALB/c mice actively immunized with these antigens in Freund's adjuvants produced variable serum antibody responses to the component peptides. Fertility rates for animals immunized with AgE (40%) and AgF (20%) were significantly reduced compared to MBP immunized mice (90%), but the reduction in fertility did not correlate with peptide-specific serum antibody levels. Ovaries from all immunized mice appeared histologically normal with no evidence of oophoritis. These results demonstrate that high levels of immunocontraception can be achieved in mice, without apparent side-effects, using species-specific immunogens that include repeated peptides from proteins involved in fertilization.

Evaluation of a DNA Vaccine for Immunocontraceptive Potential Against Zona Pellucida Glycoproteins in Cattle

Holstein cows were administered zona pellucida (ZP) DNA vaccine and used to determine the potential of recombinant rabbit ZP glycoproteins (rZP) as immunocontraceptive antigens. Zona pellucida proteins were purified and quantified. Cows were assigned to one of four treatment groups in which plasmids encoding rabbit ZP proteins were administered, i.d., using a gene gun (ZP55, n=2; ZP75, n=2; Hep55, n=2; and Control, n=3). Blood samples were taken before initial vaccination, once weekly for 5 wk and at 148 wk post-immunization. An ELISA was developed to assess anti-ZP titer levels in cow serum and ovarian function in cows was monitored using trans-rectal ultrasonography. Four of the six cows in ZP treatment groups developed antibody titer levels with similar linear responses over time. These cows also experienced reduced ovarian function as indicated by decreases in follicular and luteal activity. Estrous activity was observed in all cows and decreased in ZP treatment cows in comparison to Controls. Further research is needed to determine the relationship between ZP immunocontraception and ovarian function. Still, this study provides a basis for future researchers to use in developing a contraceptive vaccine for cattle.

Prospects for fertility control in the European rabbit (Oryctolagus cuniculus) using myxoma virus-vectored immunocontraception

Research over the last 15 years has examined whether fertility control can reduce overabundant rabbit populations and whether an effective immunocontraceptive agent can be developed and delivered. The results of this research indicate that for fertility control to have an environmental impact at least 80% of females will need to be infertile and that this infertility will need to be permanent. Epidemiological studies suggest that this level of infertility may be very difficult to obtain with a recombinant myxoma virus because of competition with field strains of virus. Research with laboratory rabbits using recombinant myxoma virus to deliver an immunocontraceptive antigen demonstrated that it was possible to obtain the required level of infertility using rabbit zona pellucida C as an antigen. However, only ~50% of animals remained infertile in the medium term. Further research on delivery vector and antigen selection would be needed to produce a practical immunocontraceptive virus for laboratory testing. Such a virus would then need to be optimised for transmissibility before it would be suitable for field testing.

Prevalence of serum antibodies to canine adenovirus and canine herpesvirus in the European red fox (Vulpes vulpes) in Australia

To determine the seroprevalence and aspects of the epidemiology of canine adenovirus (CAdV) and canine herpesvirus (CaHV-1) in European red foxes (Vulpes vulpes) in Australia. Serum samples were collected opportunistically from foxes in 1991-1994 in Western Australia (WA) and South Australia (SA) and in 1980-1984 and 1990-1994 in New South Wales (NSW) and the Australian Capital Territory (ACT). The sera were examined for antibody to CAdV and CaHV-1 using ELISAs. Seroprevalence in the different regions was determined for both viruses and the CAdV data were analysed for interactions between decade of collection, age, season, region and gender using logistic regression. The overall prevalence of antibody to CAdV was 23.2% (308/1326) but was significantly higher in sera collected in the eastern states of Australia (47%: 233/498) than in WA (9%: 75/828). Overall, in NSW and the ACT, there was a significantly lower prevalence in juveniles than in adults and the prevalence in juveniles in the 1990s was significantly lower than in the 1980s. The prevalence was also significantly lower in the autumn than in the winter for juveniles but the reverse held for adults. The NSW and ACT data were subdivided into eastern (including the ACT) and western regions. This revealed a significantly higher prevalence in the winter than in the autumn for the west and the reverse in the east. In WA, the northern rangeland regions of WA had lower prevalence (1.9%) than the southern agriculture regions (10.7%). Seasonally, there was a peak prevalence in the spring dropping through the summer and autumn and rising again in the winter. This seasonal pattern was also found in the combined data for all sites in the 1990s. There was no gender difference in prevalence of CAdV either overall or in different regions. The overall prevalence of antibody to CaHV-1 was 2.2% (28/1300). The small number of positives allowed only limited statistical analysis that did not reveal any differences in decade of collection, age, season or region. CAdV infection is common in the Australian fox population whereas CaHV-1 infection is rare. For CAdV, the age and seasonal patterns of seroprevalence were generally consistent with the recruitment of young susceptible foxes into the population in the spring and the accumulation of infections with age. The differences in regional prevalences correlated with fox density. The low prevalence of antibody to CaHV-1 suggests that CaHV-1 may be a more suitable vector than CAdV for bait delivery of immunocontraceptive antigens to foxes in Australia.

Plant-based immunocontraceptive control of wildlife—“potentials, limitations, and possums”

Possums ( Trichosurus vulpecula), originally introduced from Australia, are spread over 90% of New Zealand and cause major economic and environmental damage. Immunocontraception has been suggested as a humane means to control them. Marsupial-specific reproductive antigens expressed at high levels in edible transgenic plant tissue might provide a safe, effective, and cheap oral delivery bait for immunocontraceptive control. As proof of concept, female possums vaccinated with immunocontraceptive antigens showed reduced fertility, and possums fed with potato-expressed heat labile toxin-B (LT-B) had mucosal and systemic immune responses to the antigen. This demonstrated that immunocontraception was effective in possums and that oral delivery in edible plant material might be possible. Nuclear transformation with reporter genes showed that transgenic carrot roots accumulate high levels of foreign protein in edible tissues, indicating their potential as a delivery vector. However, prior to attempts at large scale production, more effective immunocontraceptive antigen-adjuvant formulations are probably required before plant-based immunocontraception can become a major tool for immunocontraceptive control of overabundant vertebrate pests.

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.

Effectiveness of zona pellucida protein ZPB as an immunocontraceptive antigen

Immunization of female mammals with native zona pellucida (ZP) proteins is known to cause infertility. Since each human ZP protein is now available as a purified recombinant protein, is it possible to compare the immunocontraceptive potential of each ZP protein. A breeding study was conducted in cynomolgus monkeys (Macaca fasicularis) after immunization with recombinant human ZP (rhZP) proteins (ZPA, ZPB, ZPC) separately and in combinations. This study demonstrated that immunization with recombinant human ZPB (rhZPB) protein caused cynomolgus monkeys to become infertile for 9-35 months. A second study was conducted in baboons (Papio cynocephalus), which yielded a similar result. The baboons immunized with rhZPB became infertile for 9 to > 20 months. During the time of maximum antibody titre, some animals experienced disruption of the menstrual cycle, but eventually all of the animals resumed normal menstrual cycles. Control animals and animals immunized with other rhZP proteins all became pregnant before any of the rhZPB-treated animals. This is the first study in which a recombinant ZP protein has consistently induced infertility in a primate without permanent disruption of the normal menstrual cycle.

Examination of the immunocontraceptive potential of recombinant rabbit fertilin subunits in rabbit.

Recombinant fertilin subunits produced in a bacterial expression systems were used to test fertilin as an immunocontraceptive antigen in the European rabbit (Oryctolagus cuniculus). Wild female rabbits (n = 40) were immunized with either recombinant rabbit fertilin alpha or beta subunits by the s.c. or intra-Peyer's patch route. High titers of serum anti-fertilin polyclonal IgG antibodies were achieved in all rabbits after repeated boosts, with fertilin-specific IgG but not IgA antibodies detected in vaginal lavages of all animals. The serum IgG antibodies recognized polypeptides in detergent extracts of rabbit sperm with relative molecular masses of 48, 53, and 85 kDa on reducing SDS-PAGE gels and were shown to bind to the head region of methanol-fixed and live caudal rabbit sperm. Preincubation of rabbit sperm with these anti-fertilin IgG antibodies at concentrations of 400 micrograms/ml blocked sperm binding to zona-intact oocytes and inhibited fertilization in vitro by 60-80%. However, despite the levels of circulating and vaginal IgG antibodies achieved, only 4 immunized does failed to become pregnant out of 33 that ovulated. The remaining animals either showed no effect on fertility (n = 29) relative to control animals or failed to ovulate (n = 7). All control animals ovulated and were either fully fertile (n = 15) or were mated to infertile males (n = 4). In addition, proven-fertile male domestic rabbits (n = 3) were immunized s.c. and boosted three times with fertilin beta. Only one animal subsequently showed impaired fertility. These results show that in the rabbit, high levels of circulating sperm-reactive anti-fertilin antibodies and the presence of vaginal IgG does not ensure infertility.

Inhibitory effect of antibodies to synthetic sperm antigen(YAL-198) on development of embryo

Possums (Trichosurus vulpecula), originally introduced from Australia, are spread over 90% of New Zealand and cause major economic and environmental damage. Immunocontraception has been suggested as a humane means to control them. Marsupial-specific reproductive antigens expressed at high levels in edible transgenic plant tissue might provide a safe, effective, and cheap oral delivery bait for immunocontraceptive control. As proof of concept, female possums vaccinated with immunocontraceptive antigens showed reduced fertility, and possums fed with potato-expressed heat labile toxin-B (LT-B) had mucosal and systemic immune responses to the antigen. This demonstrated that immunocontraception was effective in possums and that oral delivery in edible plant material might be possible. Nuclear transformation with reporter genes showed that transgenic carrot roots accumulate high levels of foreign protein in edible tissues, indicating their potential as a delivery vector. However, prior to attempts at large scale production, more effective immunocontraceptive antigen-adjuvant formulations are probably required before plant-based immunocontraception can become a major tool for immunocontraceptive control of overabundant vertebrate pests.

Initial evaluation of fertilin as an immunocontraceptive antigen and molecular cloning of the cynomolgus monkey fertilin β subunit

Fertilin (PH-30) is a sperm surface protein that functions in sperm adhesion and fusion with the egg plasma membrane. Because of its essential function in fertilization, fertilin is a potential target for novel contraceptive approaches. In a pilot fertility trial, immunization of male guinea pigs with purified guinea pig fertilin resulted in complete infertility. The contraceptive effect was partial (two out of six animals were infertile) when female guinea pigs were immunized with the antigen. These results suggest that fertilin or domains of fertilin may be effective as immunocontraceptive antigens. As a step toward achieving this goal, we communicate the cDNA and deduced amino acid sequence of the monkey fertilin β subunit. © 1996 Wiley Liss, Inc.