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There is an urgent need to develop a better method of contraception which is non-steroidal and reversible to control world population explosion and unintended pregnancies. Contraceptive vaccines (CV), especially targeting sperm-specific proteins, can provide an ideal contraceptive modality. Sperm-specific proteins can induce an immune response in women as well as men, thus can be used for CV development in both sexes. In this article, we will review two sperm-specific proteins, namely Izumo protein and YLP12 dodecamer peptide. Gene-knockout studies indicate that Izumo protein is essential for sperm-egg membrane fusion. Vaccination with Izumo protein or its cDNA causes a significant reduction in fertility of female mice. The antibodies to human Izumo inhibit human sperm penetration assay. Recently, our laboratory found that a significant percentage of infertile women have antibodies to Izumo protein. The second sperm-specific protein is YLP12 , a peptide mimetic sequence present on human sperm involved in recognition and binding to the human oocyte zona pellucida. Vaccination with YLP12 or its cDNA causes long-term, reversible contraception, without side effects, in female mice. Infertile, but not fertile, men and women have antibodies to YLP12 peptide. Our laboratory has isolated, cloned, and sequenced cDNA encoding human single chain variable fragment (scFv) antibody from infertile men which reacts with YLP12 peptide. The human YLP12 scFv antibody may provide a novel passive immunocontraceptive, the first of its kind. In conclusion, sperm-specific Izumo protein and YLP12 peptide can provide exciting candidates for antisperm CV development., (© 2014 The Protein Society.)

To investigate the effects of antitestosterone immunization, initiated during early puberty, on hypothalamic-pituitary-testicular feedback in rabbits, 16 early pubertal male rabbits were randomly allocated into 2 groups (n = 8), control or immunized against testosterone-3(O-carboxymethyl)oxime-BSA in Freund adjuvant at 4 mo of age (with a booster immunization 4 wk later). Blood samples (for antibody titers and hormone concentrations) were collected at 2- or 4-wk intervals after immunization. Compared with controls, antitestosterone immunization triggered: a substantial and sustained antibody response (P < 0.01); increases in serum concentrations of luteinizing hormone (LH) and testosterone and testis weight and volume (P < 0.05); hyperplasia of testicular interstitial tissue with clustered and hypertrophic Leydig cells; and greater (P < 0.05) enzyme protein and messenger RNA (mRNA) expression levels for testicular cholesterol side-chain cleavage cytochrome P-450, 17α-hydroxylase cytochrome P-450, and 3β-dydroxysteroid dehydrogenase. Furthermore, immunoneutralization of testosterone upregulated mRNA expressions for genes in sex steroid negative feedback loops, including androgen receptor (AR), estrogen receptor alpha (ER-α), kisspeptin encoded gene (kiss-1) and kisspeptin receptor (G-coupled receptor 54) and gonadotropin-releasing hormone (GnRH) in the hypothalamic arcuate nucleus, GnRH receptor and LH-β in pituitary, and AR, inhibin-α and βA subunits in testes (P < 0.05). However, immunization did not affect mRNA expressions for follicle-stimulating hormone β, AR, and ER-α in pituitary, or ER-α in testes. We concluded that antitestosterone immunization in male rabbits, initiated during early puberty, increased GnRH mRNA expression, and in turn LH synthesis by reducing testicular feedback signaling. Reduction of direct steroidal effects on the testis may also have increased testosterone secretion. Consequently, there was an accelerated testicular development during puberty and enhanced testicular function after puberty, which likely conferred prolonged reproductive advantages., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The study used a randomized complete block design (blocking factor was date of start on test) with 5 treatments: 1) physically castrated barrows (PC), 2) intact males (IM), 3) gilts (G), 4) immunologically castrated barrows (IC), and 5) immunologically castrated barrows fed ractopamine at 5 mg/kg (IC+RAC). The study used 192 pigs and was performed from the 16 wk of age (67.2 ± 2.52 kg BW) to a pen mean BW of 132.5 ± 3.60 kg. For IC+RAC, ractopamine was fed for the final 23 d of the study. Pigs were housed in groups of 4 (10 groups for PC, IM, G, and IC and 8 groups for IC+RAC) in a finishing building at a floor space of 1.18 m(2)/pig. Diets were formulated to meet requirements of IM except that the diet for the IC+RAC fed during the ractopamine feeding period was formulated to meet requirements of pigs on that treatment. Pigs had ad libitum access to feed and water throughout the study period and were individually weighed at the start, wk 2 and 4, and subsequently every week until the end of study. For the overall study period, IC had greater (P ≤ 0.05) ADG than the other genders (1,150, 1,024, 1,064, and 954 g/d for IC, PC, IM, and G, respectively; SEM = 25.8) and required fewer days to reach slaughter weight than the other genders (58.1, 61.6, 61.6, and 66.5 d for IC, PC, IM, and G, respectively; SEM = 1.26). Overall ADFI was less (P ≤ 0.05) for IM and G than IC and PC, which were similar (P > 0.05) in this respect (3.11, 3.06, 2.68, and 2.75 kg/d for IC, PC, IM, and G, respectively; SEM = 0.061). Overall G:F was greater (P ≤ 0.05) for IM than the other genders; IC had greater overall G:F than PC and G, which were similar in this respect (0.371, 0.335, 0.397, and 0.347 kg/kg for IC, PC, IM, and G, respectively; SEM = 0.0068). Immunologically castrated barrows had greater (P ≤ 0.05) ADG (30.7%) and ADFI (22.5%) than PC from the second week following the second Improvest dose to the end of the study. During the ractopamine feeding period, IC+RAC had greater (P ≤ 0.05) ADG (11.6%) and G:F (17.3%) than IC. The results of this study confirmed previously observed gender differences and effects of ractopamine on growth performance and that IC grew faster and had greater feed efficiency than PC during the study period.

The aim of this study was to evaluate safety (in terms of detecting possible adverse clinical effects attributable to vaccination), efficacy, and effects on growth performance of a gonadotropin releasing factor analog-diphtheria toxoid conjugate (commercially distributed as Improvac; Zoetis, Zaventem, Belgium) in male pigs raised in a commercial Greek farm. A total of 1,230 male pigs was enrolled in 16 weekly batches and allocated to 3 groups: barrows (castrated on the next day after birth [study Day 0]), pigs vaccinated with the above-mentioned product, and intact boars. Vaccinated pigs were injected subcutaneously with 2 mL of the anti-gonadotropin releasing factor (GnRF) vaccine at 9 to 11 wk of age (60-78 d) and 15 to 17 wk of age (102-120 d) and slaughtered at 22 to 25 wk of age (152-176 d). No clinical abnormalities or adverse events attributable to vaccination occurred. Mean BW of vaccinated pigs was 6% greater compared with barrows at slaughter (P < 0.0001). The vaccinated pigs had greater ADG than barrows from castration to slaughter (8%). In detail, a lower ADG from first to second vaccination (-12%; P < 0.0001) and a 27% greater ADG from second vaccination to slaughter (P < 0.0001) were observed. The ADG of vaccinated pigs and intact boars was not significantly different throughout the study, except from first to second vaccination (boars greater; P = 0.0059) and second vaccination to slaughter (vaccinates greater; P = 0.0390). Feed conversion ratio of barrows was 11 and 8% greater compared with vaccinated pigs (P = 0.0005) and boars (P = 0.0062) from first to second vaccination but was 23 to 26% lower compared with vaccinated pigs (P < 0.0001) and intact boars (P < 0.0001) from first vaccination to slaughter and 7 to 9.5% lower from the second vaccination to slaughter (P = 0.0029 and P = 0.0003 for vaccinates and intact boars, respectively). At slaughter, the belly fat androstenone concentration of all vaccinated pigs and 64% of intact boars was below 200 ng/g. Belly fat skatole concentration was below 20 ng/g in samples from all groups. In conclusion, vaccination against GnRF using the GnRF analog-diphtheria toxoid conjugate tested did not induce adverse clinical effects, proved effective, and compared with physical castration, resulted in greater BW at slaughter and improved ADG and feed conversion ratio from first vaccination to slaughter.

To investigate the effects of active immunisation against pregnenolone on reproductive traits in rabbits, 16 early pubertal male rabbits (4mo old) were randomly and equally allocated into two groups, control or immunised against pregnenolone-hemisuccinate-BSA in Freund's adjuvant (with a booster 4wk later). Blood samples (for antibody titres and hormone concentrations) were collected at 2 or 4wk-intervals after immunisation until rabbits were killed, 24wk after the primary immunisation. Compared to controls, rabbits immunised against pregnenolone had increased serum antibody titres (P<0.01) and decreased serum concentrations of both testosterone and LH (P<0.01 for each). At 24wk after the primary immunisation, testes were severely atrophied, spermatogenesis was arrested and steroidogenesis was suppressed, as evidenced by lesser amounts of testicular cholesterol side-chain cleavage cytochrome P-450 and 17α-hydroxylase cytochrome P-450 mRNA (P<0.05). Furthermore, the amounts of mRNA for GnRH in the arcuate nucleus (Arc), of the hypothalamus and GnRH receptor and LH-β in the pituitary and genes in sex-hormone negative feedback loops (androgen receptor, oestrogen alpha receptor, kisspeptin encoded gene and kisspeptin receptor) in the Arc were decreased in pregnenolone-immunised rabbits compared to controls (P<0.05). It was concluded that immunisation against pregnenolone directly blocked testicular steroidogenesis, which reduced synthesis of hypothalamic GnRH and subsequently synthesis of pituitary LH by abolishing the permissive action of sex steroids on hypothalamic GnRH neurons, thereby disrupting spermatogenesis. This was apparently the first report that active immunisation against pregnenolone was a means of immunological castration., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Although there are several methods of contraception available, the population explosion and unintended pregnancies continue to cause major health issues and constrain resources worldwide. A better method of contraception that is acceptable in developed as well as developing countries is urgently needed. Contraceptive vaccines (CVs) can fulfil most of the properties of an ideal contraceptive. The development of CVs is an exciting proposition and has drawn significant attention lately. The purpose of this article is to review the present status and future perspectives of CVs for human use, and for control of farm, feral, stray and domestic animal populations.

To elicit potent humoral immunity and produce adequate neutralizing antibody especially in the genital tract and eventually to promote its immunogenicity, we designed an Eppin B-cell-dominant-epitope-based mimovirus vaccine with an RGD motif which can be nasally inoculated into male mice. Our results indicate that this immune strategy successfully generated a high antibody response with significantly higher anti-Eppin IgA in the genital tract, and eventually achieve significant inhibition of fertility without any interference with testis function and alteration in structural integrity. The fertility rate of the females mating with the vaccinated males declined and the progeny size was greatly reduced, but the contraceptive efficacy was still far from that of immunocontraceptives for human use. However, the research showed a new contraceptive vaccine construction and inoculation avenue, that is, mimovirus vaccine delivered nasally. Further investigation geared toward improving fertility inhibition efficacy using this inoculation strategy still remains to be explored.

Native porcine zona pellucida (PZP) immunocontraception has been used to inhibit fertility in more than 80 species of ungulates, although the duration of contraception efficacy varies among species in both Perissodactyla and Artiodactyla. This study examined anti-PZP antibody titers in Dall sheep and domestic goats at the Milwaukee County Zoo, and also Himalayan tahr and Armenian Mouflon sheep at the San Diego Zoo Safari Park, and, for comparison, Altai wapiti, lowland wisent, Javan banteng, and southern pudu at the San Diego Zoo Safari Park, all were given a primer dose and booster dose of PZP. Of the San Diego Zoo Safari Park animals, the 4 comparison species demonstrated the typical 1-yr pattern of anti-PZP antibodies, whereas the Armenian sheep and Himalayan tahr showed prolonged (2-3 yr) antibody responses after a single primer and booster dose. The Dall sheep and domestic goats had significantly longer durations of antibody titers (3 yr) from a single year's treatment (primer plus booster). Analysis of the data indicates that Armenian sheep, Himalayan tahr, Dall sheep, and domestic goats have prolonged responses, and are more sensitive to PZP in that they produce a protracted antibody response.

The National Wildlife Research Center (NWRC) began immunocontraception vaccine research by testing porcine zona pellucida (PZP) on white-tailed deer (Odocoileus virginianus). Early PZP research demonstrated that PZP induced infertility; however, increased length of the rut was observed in PZP-treated deer. An alternative vaccine using a keyhole limpet hemocyanin-gonadotropin-releasing hormone (KLH-GnRH) conjugate formulated with modified Freund's adjuvant was developed at NWRC. Suppression of GnRH has reduced reproduction in both sexes but is most effective in females. This vaccine was effective in preventing contraception in female deer for several years after a prime and boost. Due to adverse side effects of Freund's adjuvant, NWRC developed a new adjuvant called AdjuVac, a mineral oil/surfactant adjuvant with the addition of Mycobacterium avium as an immunostimulant. The price of KLH prompted a search for a more economical hemocyanin carrier protein for the GnRH peptide. Blue protein, derived from the mollusk Concholepas concholepas, proved to be a successful option. Formulation improvements resulted in a vaccine that can be effective as a single injection for multiple years, now called GonaCon. GonaCon is registered with the Environmental Protection Agency (EPA) for use in white-tailed deer in urban/suburban areas and for wild horses (Equus caballus) and burros (Equus asinus). Future GonaCon applications may include reducing reproduction to manage populations of other wildlife species, such as prairie dogs (Cynomys ludovicianus) in urban areas and suppressing reproduction to reduce the spread of venereal diseases such as brucellosis. Research is being conducted to develop a GnRH vaccine used in combination with the rabies vaccine to control population growth in free-roaming dogs, with the secondary effect of managing the spread of rabies. The EPA would regulate all these uses. Research is also ongoing on a GnRH vaccine to delay the onset of adrenocortical disease in pet ferrets (Mustela putorius), a use regulated by the United States Department of Agriculture.

Prior to 2010, the introduced population of American bison (Bison bison) on Santa Catalina Island, California, was managed through the shipment of surplus bison to private ranches, Native American reservations, and livestock auctions on the mainland. In response to escalating costs, transport-induced stress to the animals, and ecologic impacts associated with high bison numbers on-island between shipments, the use of the immunocontraceptive vaccine porcine zona pellucida (PZP) as a fertility control option for managing the population was investigated. Between 2009 and 2012, a total of 64 bison cows (> or =1 yr old) received primer inoculations of 100 microg PZP emulsified with 0.5 ml Freund's modified adjuvant (FMA) delivered through a combination of intramuscular injections by hand (50 bison cows) during roundups and via field darting (14 bison cows). Pre-rut booster inoculations of 100 microg PZP emulsified with 0.5 ml Freund's incomplete adjuvant (FIA) were administered exclusively via field darting in 2010, 2011, and 2012 to 45, 48, and 61 bison cows (> or =1 yr old), respectively. During the present study, 38 adult cows (marked and unmarked) received one or more PZP inoculations during their first, second, or third trimesters of pregnancy, and of these individuals, 35 successfully produced calves. Low pregnancy values detected in the remaining three cows have been attributed to residual progesterone associated with unsuccessful fertilization. The 2010 pretreatment calving rate (calves born per cow) determined via direct observation was 67.4% (29 calves from 43 cows). Through the use of PZP, the calving rate was reduced to 10.4% by 2011 and to 3.3% by 2012. Considering the annual mortality rate of 2-5% documented during this study, the results demonstrate the potential of PZP use as an effective nonlethal tool for controlling population growth in free-ranging bison.

Opinions are divided as to whether human intervention to control elephant (Loxodonta africana) population growth is desirable, partly because of elephant welfare concerns. Female contraception through immunization with porcine zona pellucida (PZP) proteins is viable. The effects of sustained use and application of the PZP vaccine on elephant behavioral and spatial responses were examined by evaluating herd ranging, fission-fusion dynamics, association patterns, and reproductive and sexual behaviors. Minimal change was anticipated as a result of long calf dependence on and association with cows, a reduced but not indefinite 0% growth rate and the known mechanism of action of PZP vaccines, and minimal expected change in resource requirements necessitating behavioral or spatial use adaptations. Although behavioral effects identified in previous hormonal contraceptive trials were evident, it was demonstrated that immunocontraception caused no prolonged behavioral, social, or spatial changes over the 11-yr study period. Individually identified elephants were monitored from 1999 to 2011. Minimal, short-term social disruption, with temporary changes to the herds' core ranges, was observed during the annual treatment events, particularly in the first three treatment years, when vaccinations were conducted exclusively from the ground. Thereafter, when vaccinations were conducted aerially, minor disruptions were confined to the morning of administration only. Despite sustained treatments resulting in demographic changes of fewer calves being born, treatments did not alter spatial range use, and no adverse interherd-intraherd relations were observed. Similarly, resource requirements did not change as calving still occurred, although in fewer numbers. It was concluded that PZP immunocontraception has no detectable behavioral or social consequences in elephants over the course of 11 yr, providing a convincing argument for the use of sustained immunocontraception in the medium to long term as an important tool for elephant management. Behavioral consequences of alternative management approaches should all receive similar scrutiny to enable managers to make informed decisions when weighing management interventions.

Previous reports have demonstrated gradual reductions of white-tailed deer (Odocoileus virginianus) populations through immunocontraception, with stabilization occurring after 2-4 yr of treatment, and subsequent reductions of 6-10% annually. These studies employed porcine zona pellucida (PZP) vaccines that required two initial treatments and annual retreatments. From 2005 to 2010, 258 adult and yearling female deer on Fripp Island, South Carolina, were treated with one of several PZP preparations designed to produce 2+ yr of effective contraception with a single treatment. These included several preparations of SpayVac and of native PZP-adjuvant emulsion plus PZP and QA-21 in timed-release pellets. Deer were chemically immobilized, ear-tagged, and administered initial treatments by hand in February-March. Some treated deer were boosted remotely with PZP-adjuvant emulsion 1.5 - 4.5 yr after initial treatments. Ground-based distance sampling was used to estimate deer population density at Fripp Island, a resort community, and at a relatively undeveloped neighboring control site, Hunting Island. Most vaccine preparations tested reduced fawning rates by 75% to 95% for at least 1 yr. From 2005 to 2011, deer density on Fripp Island declined by 50%, from 72 deer/km(2) to 36 deer/km(2), an average annual reduction of 11%. In contrast, population density on the Hunting Island control site fluctuated between 2005 and 2011, averaging 23 deer/km(2) (range, 19-28 deer/km(2)). Population declines on Fripp Island were associated with an increase in the proportion of treated females and with a progressive decrease in winter fawn:doe ratios, from 1.21 fawns/doe in 2005 to 0.19 fawns/doe in 2010. Winter fawn:doe ratios averaged 1.36 fawns/doe (range, 0.84 - 1.62 fawns/doe) at the Hunting Island control site. Annual survivorship averaged approximately 79% among ear-tagged females. The rate at which deer populations diminished in association with PZP treatments on Fripp Island was higher than that seen at other study sites, although the reasons for the more rapid decline on Fripp Island are not well understood.

We investigated the effect of immunization against gonadotropin releasing hormone (GnRH) using a commercial canine GnRH vaccine on estrus suppression and unwanted estrous behavior in mares. In experiment 1, mares were immunized (n = 6) twice with vaccine (5 mL) given intramuscularly 4 weeks apart or received a control diluent (n = 5). Transrectal ultrasonographic examination of the reproductive tracts was performed three days a week for 40 weeks after initial vaccination. Blood samples were collected weekly for GnRH antibody titer and progesterone concentration determination. In experiment 2, privately-owned mares (n = 12) were immunized twice with vaccine (1 mL) given intramuscularly 4 weeks apart. Blood samples were collected prior to each vaccination as well as 12 and 20 weeks after initial treatment, and transrectal ultrasonographic examinations of the reproductive tracts were performed 12 weeks after the first vaccination. Vaccinated mares in experiment 1 responded with a GnRH antibody titer, progesterone concentrations significantly lower than controls, and cessation of ovarian activity. Vaccinated mares in experiment 2 also responded with a GnRH antibody titer, progesterone concentrations that remained basal for the duration of the study, and cessation of ovarian activity. Owners of vaccinated mares in experiment 2 reported that the number of unwanted estrous behaviors present before vaccination significantly decreased following vaccination. In conclusion, GnRH immunization using a canine GnRH vaccine is an effective method for suppressing estrus and unwanted estrous behavior., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Immunization against GnRH reduces the occurrence of boar-taint-causing compounds in entire male pigs and is likely to alter growth performance, including whole-body protein deposition (PD; N retention × 6.25). Thirty-six male pigs were used to determine the effects of immunization against GnRH with GnRH analog on N retention and plasma urea nitrogen (PUN; a measure of AA catabolism). Four treatments were used: 1) conventional, early castrates (EC), 2) entire male pigs (EM), 3) entire male pigs immunized with GnRH analog (IM), and 4) entire male pigs surgically castrated after 6 wk of age (between 25 and 40 kg BW; late castrates; LC). The GnRH analog was injected at 30 and 70 kg BW. Within each of 9 litters, 4 males were randomly assigned to the 4 treatments. Pigs were fed corn- and soybean-meal-based diets that were not limiting in essential nutrients for high PD. Five consecutive N balances were conducted between d -9 and -4, 1 and 7, 9 and 16, 20 and 26, and 30 and 36, relative to administration of the booster dose of GnRH analog at d 0. Blood was sampled on d -4, -1, 2, 5, 8, 11, 14, 19, 28, and 37. There was an interactive effect of treatment and time on N retention (P < 0.001). Across periods, N retention for EC and LC were similar (32.7 vs. 33.6 g/d) and lower than EM (39.2 g/d, P < 0.001). The N retention in EM and IM were similar up to d 7 (37.8 vs. 38.5 g/d), tended to be greater for EM than IM between d 9 and 16 (38.4 vs. 34.9 g/d, P = 0.07) and was greater for EM than IM after d 20 (40.9 vs. 34.9 g/d, P < 0.05). Between d 9 and 36, N retention in IM was similar to EC and LC. The PUN concentrations were similar in EC and LC across sampling times (15.50 vs. 15.86 mg/dL) and greater than EM (9.33 mg/dL, P < 0.05). The PUN concentrations were similar in EM and IM up to d 5 (9.88 vs. 9.59 mg/dL), tended to be less in EM than IM on d 8 (9.08 vs. 11.85 mg/dL, P < 0.10), and were lower in EM than IM from d 11 to 37 (8.94 vs. 14.80 mg/dL, P < 0.05). After d 8, PUN concentrations were similar for IM, EC, and LC (14.31, 15.13, and 15.55 mg/dL, respectively). In conclusion, the results of the current study show that N retention and PUN patterns in EC and LC are very similar and lower than those in EM. Between d 7 and 16 after administration of the booster dose of GnRH analog, N retention and PUN in IM changed gradually from EM levels to approach levels in EC and LC, which should be considered when developing feeding programs for IM.

Problem: To overcome availability of the purified native zona pellucida (ZP) glycoproteins for immunocontraception, porcine ZP3, and ZP4 were expressed in E. coli., Method of Study: Purified recombinant proteins were characterized by SDS-PAGE and Western blot, and immunogenicity and contraceptive efficacy determined in FvB/J female mice., Results: Purified ZP3, ZP3 with promiscuous T-cell epitope of tetanus toxoid, ZP4 and ZP4 incorporating promiscuous T-cell epitope of bovine RNase revealed ~44-, ~49-, ~53-, and ~55-kDa bands by SDS-PAGE and Western blot, respectively. Immunization of female mice with recombinant proteins elicited high antibody titers as well as T-cell responses. Immune sera recognized mouse oocyte ZP and also inhibited in vitro fertilization. Immunized mice showed significant decrease in fertility. Recombinant proteins were able to recall memory antibody response in female mice primed with porcine native ZP., Conclusion: Availability of recombinant porcine proteins will be useful in the development of contraceptive vaccine., (© 2013 John Wiley & Sons A/S.)

Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine.

Problem: This study was undertaken to evaluate the efficacy and safety of recombinant SP-10 protein for male contraception., Methods of Study: Adult male mice were divided into five groups. Group I was placebo-treated control while Groups II-V were immunized with recombinant SP-10 protein on day 0 and 21 with different doses (range 25-100 μg). From each Group, animals were put for mating fertility test. Histological and hematological parameters, sperm characteristics, serum clinical biochemistry and testosterone levels were investigated., Results: Group I showed normal fertility. Group II-V showed dose dependent reduction in fertility. In contrast, at higher dose (75 and 100 μg), all animals were sterile for three months. Further, all parameters under investigation in experimental groups were comparable to those of control animals., Conclusion: Our study has put forth a proof of concept for male contraception for the first time, which may be considered suitable for contraceptive vaccine development., (© 2013 John Wiley & Sons A/S.)

Reasons for Performing Study: Active immunisation against gonadotrophin-releasing hormone (GnRH) provides a reversible method for control of oestrous behaviour and fertility in mares. Previous reports failed to demonstrate the interval to resumption of cyclic ovarian activity after GnRH-vaccination., Hypothesis: Administration of the GnRH-vaccine Improvac in a large group of mares of various ages will result in effective, reliably reversible suppression of ovarian activity within a 2 year period., Methods: The mares, subdivided into 3 age categories, were vaccinated twice (with a 35 day interval) using 400 µg Improvac and monitored via blood samples until Day 720 after initial vaccination for serum progesterone concentration determination by radioimmune assay and anti-GnRH antibody titre by enzyme immunoassay. Samples were collected until individuals resumed cyclic ovarian activity., Results: All mares showed suppression of cyclic ovarian activity by clinical examination and serum progesterone concentration (SPC) ≤ 1 nmol/l by Day 70 and 92.2% resumed cyclic activity by SPC at Day 720 with a mean interval = 417.8 days (s.d. ± 23.9; range 232-488 days, median 344 days). A significant age effect (P = 0.028) on the interval, but not on GnRH-antibody titre response, was observed between the youngest (≤ 4 years) and oldest (≥ 11 years) categories., Conclusions: Immunising adult mares of all ages with Improvac resulted in a reversible suppression of cyclic ovarian activity in most mares. An age effect, with the youngest mares showing a longer interval to reversibility, was observed., (© 2012 EVJ Ltd.)

OBJECTIVE. The objective of this study was to prepare, characterize, and determine immunological activities of specific transfer factor (STF) specific to human sperm antigen (HSA) for the preparation of antisperm contraceptive vaccine that can be used as an immunocontraceptive. METHODS. HSA-STF was prepared using the spleens of rabbits vaccinated with HSA. The specific immunological activities were examined by lymphocyte proliferation test (LPT), leukocyte adhesion inhibition test (LAIT), and by determining the concentrations of IL-4, γ -IFN, and IL-21. HSA-STF was a helveolous substance, having a pH value of 7.0 ± 0.4 and UV absorption maxima at 258 ± 6 nm. It contained seventeen amino acids; glycine and glutamic acids were the highest in terms of concentrations (38.8 μ g/mL and 36.3 μ g/mL, resp.). RESULTS. The concentration of polypeptide was 2.34 ± 0.31 mg/mL, and ribose was 0.717 ± 0.043 mg/mL. The stimulation index for lymphocyte proliferation test was 1.84, and the leukocyte adhesion inhibition rate was 37.7%. There was a statistically significant difference between the cultural lymphocytes with HSA-STF and non-HSA-STF for γ -IFN and IL-21 (P < 0.05), but there was no statistical significance for IL-4 (P > 0.05). CONCLUSION. HSA-STF was prepared and characterized successfully. It had immunological activity which could transfer the immune response specific to HSA and prove to be a potential candidate for the development of male immunocontraceptive agents.

Multiple phage-peptide constructs, where the peptides mimic sperm epitopes that bind to zona pellucida (ZP) proteins, were generated via selection from a phage display library using a novel approach. Selections were designed to allow for identification of ZP-binding phage clones with potential species-specific properties, an important feature for wildlife oral vaccines as the goal is to control overpopulation of a target species while not affecting non-target species' reproduction. Six phage-peptide antigens were injected intramuscularly into pigs and corresponding immune responses evaluated. Administration of the antigens into pigs stimulated production of anti-peptide antibodies, which were shown to act as anti-sperm antibodies. Potentially, such anti-sperm antibodies could interfere with sperm delivery or function in the male or female genital tract, leading to contraceptive effects. Staining of semen samples collected from different mammalian species, including pig, cat, dog, bull, and mouse, with anti-sera from pigs immunized with ZP-binding phage allowed identification of phage-peptide constructs with different levels of species specificity. Based on the intensity of the immune responses and specificity of these responses in different species, two of the antigens with fusion peptide sequences GEGGYGSHD and GQQGLNGDS were recognized as the most promising candidates for development of contraceptive vaccines for wild pigs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We investigated the use of a commercial gonadotropin-releasing hormone (GnRH) vaccine as a method of temporary and reversible immunocastration in intact male dogs. Four privately owned dogs were vaccinated twice at 4-week intervals. Blood samples were collected at 0, 4, 12 and 20 weeks following the initial vaccination. These samples were analysed for GnRH antibody titres, luteinizing hormone (LH) and testosterone concentrations. Scrotal measurements were made at the time of sample collection, and testicular volume was calculated using the formula of an ellipsoid. As a result of vaccination, dogs displayed an elevated GnRH antibody titre, decreased LH and testosterone concentrations and decreased testicular volume, which reversed by the end of the study period. Therefore, these results suggest that immunizing against GnRH may be a possible choice for temporary and reversible immunocastration., (© 2012 Blackwell Verlag GmbH.)

Overpopulation of cats and dogs is a serious worldwide problem that demands novel, safe and cost-effective solutions. The objective of this study was to generate and characterize phage-peptide conjugates with gonadotropin-releasing hormone (GnRH) for potential use as an immunocontraceptive. A filamentous phage vector f5-8 with wild-type phage coat proteins was used as a carrier for construction of chemical conjugates with GnRH, a peptide that acts as a master reproductive hormone. In such conjugates, the phage body plays the role of a carrier protein, while multiple copies of GnRH peptide stimulate production of neutralizing anti-GnRH antibodies potentially leading to contraceptive effects. To generate the constructs, four different GnRH-based peptides were synthesized and conjugated to phage particles in a two-step procedure: (i) peptides were reacted with phage to form a conjugate using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride chemistry (EDC) and (ii) the conjugates were separated from remaining free peptides by dialysis. Formation and specificity of phage-GnRH conjugates were confirmed by three independent methods: spectrophotometry, electron microscopy and ELISA. When the conjugates were tested for interaction with sera collected from cats and dogs immunized with GnRH-based vaccines in independent studies, strong specific ELISA signals were obtained, suggesting the potential use of the conjugates for cat and dog immunosterilization. The ability of the conjugates to stimulate production of anti-GnRH antibodies in vivo was evaluated in mice. While optimization of dose, immunization route and adjuvant still requires investigation, our preliminary results demonstrated the presence of anti-GnRH antibodies in sera of mice immunized with such conjugates. Fertility trials in cats and dogs will be needed to evaluate contraceptive potentials of the phage-GnRH peptide chemical conjugates., (© 2012 Blackwell Verlag GmbH.)

The effect of immunization against gonadotrophin releasing hormone (GnRH) on sexual behavior, total scrotal size, semen characteristics and serum concentrations of testosterone, was evaluated for 24 wks in sexually mature camels (Camelus dromedarius). Eight bull camels were randomly divided into a treatment and control group. Four male camels were immunized using 2 mg GnRH - tandem-dimer conjugated to ovalbumin, (Pepscan Systems, the Netherlands) administered subcutaneously, 4 wks apart. Control male camels received the same amount of saline solution. Significant decline in serum testosterone level was observed in three immunized camels out of four, whereas one camel showed no effect. The testosterone levels reached to <1.0 ng/mL serum by week 4 after booster injection and remained suppressed through the course of the study. The total testicular volume was not affected until the end of the experiment. In treated animals, the sexual behavior negatively affected after the booster injection. Anti-GnRH vaccine had a seriously detrimental effect on the acrosin amidase activity and normal acrosome percentages in treated male camels. It is concluded that the vaccine was effective in reducing serum testosterone levels and libido, and it had a serious harmful effect on the acrosin amidase activity and percentages of spermatozoa with normal acrosome. The immunogen did not affect the total testicular volume., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The objective was to investigate the effects of a novel DNA vaccine (pcISI) harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats. Female Wistar rats (n=18 per group) were immunized (twice, 4 wk apart) with 10, 50, or 100 μg (T1, T2 and T3, respectively), of the pcISI plasmid. At 4 wk after the second immunization, plasma antibody titers were higher (P<0.05) in T3 than in either T1 or T2 (0.341±0.123, 0.236±0.068, and 0.251±0.077, respectively, mean±SD). Concurrrently, plasma concentrations of FSH and estradiol were highest (P<0.05) in T3, and were higher (P<0.05) in T1 and T2 than in control groups. For antibody-positive rats, there was a correlation (P<0.01) between antibody titer and FSH concentrations after two pcISI immunizations. The number of mature follicles in the T3 group (46.00±4.65) was higher (P<0.05) than in two control groups (29.25±3.72 and 27.92±3.48), and also higher (P<0.05) than in T1 and T2 (37.17±4.99 and 38.75±7.09). Antibody-positive rats had more mature ovarian follicles than negative rats (46.75±4.23 vs. 35.60±3.38, P<0.05). Moreover, litter size and number of placentas were increased (P<0.05) in the pcISI immunization groups, except for the T1 group, compared to the control groups. In conclusion, the pcISI DNA vaccine successfully induced a humoral immune response, improved reproductive hormone concentrations, stimulated follicular development, and increased number of placentas and litter size. Furthermore, 100 μg yielded the best immune response., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Problem: Evaluation of proacrosin/acrosin ability to induce an immune response in male mice after genetic immunization and assessment of animal fertility., Method of Study: Mice received 50 μg per animal of a plasmid containing the human proacrosin cDNA (pSF2-Acro) (control: empty plasmid, pSF2). The humoral response was evaluated by ELISA and immunocytochemistry. In vivo fertility was assessed by mating immunized males with control females. The effect of antibodies upon Ca(+2)-ionophore-induced acrosomal exocytosis (AE) and in vitro sperm-zona pellucida (ZP) binding was also studied., Results: pSF2-Acro-immunized mice developed high levels of specific antibodies (P < 0.05) that recognized the sperm acrosomal cap. The number of fertile mice was lower (P = 0.027) in pSF2-Acro-immunized animals than in controls. Litter size was smaller (P < 0.05) in the pSF2-Acro group compared with controls. A negative correlation (P < 0.05) between antibody levels and litter size was found. Antiproacrosin/acrosin antibodies inhibited sperm-ZP binding (P < 0.0001) and Ca(+2)-ionophore-induced AE (P < 0.05)., Conclusion: DNA immunization against proacrosin elicits an immune response in male mice associated with abnormal sperm functions and reduced fertility., (© 2012 John Wiley & Sons A/S.)

Problem: To examine the immunocontraceptive properties of the plasmid pcDNA-mCRISP1 and compare them to the corresponding recombinant mCRISP1 (r-mCRISP1)., Method of Study: RT-PCR and indirect immunofluorescence were performed to observe the mCRISP1 protein expression in COS-7 cells. Three groups of mice received three injections of r-mCRISP1, pcDNA-mCRISP1 or pcDNA vector, respectively. ELISA and Western blot were used to examine the immune responses and immunoreactivity of antisera. Sperm-egg penetration assay was performed to examine the effect of anti-mCRISP1 antibodies in vitro fertilization of mouse oocytes. Fertility and mean litter size were analysed by natural mating. Histological analysis was carried out to look for potential immunopathologic effects of the antibodies., Results: COS-7 cells transfected with pcDNA-mCRISP1 present the expression of mCRISP1. Both r-mCRISP1 and pcDNA-mCRISP1 raised an immune response against r-mCRISP1 protein and native CRISP1 in mouse sperm. The titres of anti-mCRISP1 antibodies from DNA immunized mice were significantly lower than that of r-mCRISP1 immunized mice, but it lasted relatively longer. Male and female pcDNA-mCRISP1 injected animals presented a statistically significant reduction in their fertility with no signs of immunopathologic effects., Conclusion: These studies demonstrated the feasibility of generating an immune response to mCRISP1 protein by DNA vaccine and pcDNA-mCRISP1 plasmid causing significant anti-fertility potential., (© 2012 John Wiley & Sons A/S.)

The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76°C for 3h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Three experiments were conducted to evaluate methods of immunization against GnRH on antibody titer, luteal activity, and pregnancy in beef heifers. Experiment 1 evaluated the efficacy of adjuvants with 30 heifers. Control heifers were immunized against human serum albumin (HSA) emulsified in Freund's complete adjuvant (FCA). The other 4 treatments contained GnRH conjugated to HSA (HSA-GnRH) emulsified in FCA, Freund's incomplete adjuvant (FIA), DEAE dextran (DD) + mineral oil (MO), or DD+FIA. Treatment was in the mammary gland for all experiments. Titers against GnRH for heifers immunized against HSA-GnRH with FCA, DD+MO, or DD+FIA were greater than titers for HSA-GnRH with FIA or control heifers (P < 0.01). Body weight was reduced (P < 0.05) in control and FCA heifers compared with FIA, DD+MO, and DD+FIA heifers. Heifers immunized with DD+MO and DD+FIA had fewer granulomas in mammary glands than heifers treated with FCA (P < 0.01). In Exp. 2, 36 heifers were used to determine the effect of the protein conjugated to GnRH on titers against GnRH. Heifers (6/treatment) received a primary immunization against GnRH conjugated to HSA (HSA-GnRH), ovalbumin (OA-GnRH), or keyhole limpet hemocyanin (KL-GnRH), or heifers were immunized against each carrier protein. Antigens were emulsified in DD+FIA. Immunization of heifers against OA-GnRH, KL-GnRH, or HSA-GnRH suppressed luteal activity (P < 0.01) for 23, 16, and 12 wk, respectively, and antibody titers against GnRH were greater (P < 0.01) for 19, 5, and 7 wk, respectively, compared with heifers immunized against the carrier proteins. In Exp. 3, 90 heifers were used to determine the effect of immunization against GnRH on ovarian activity and pregnancy rate. Heifers (30/treatment) received a primary and 2 or 3 booster immunizations against GnRH conjugated to OA, and controls received a primary and 2 booster immunizations against OA. All antigens were emulsified in DD+FIA. At 8 wk after primary immunization, heifers were exposed to fertile bulls for 24 wk. Pregnancy rate was less (P < 0.01) for 3-booster heifers (13%) compared with control (83%) and 2-booster (62%) heifers. We conclude that immunization against GnRH, conjugated to OA and emulsified in DD+FIA, does not influence ADG and produces sufficient titers against GnRH to prevent estrous cycles with few mammary granulomas. Immunization against GnRH with 3 booster immunizations prevented luteal activity and pregnancy in most beef heifers for more than 4 mo.

The transforming growth factor β (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.

Objective: To evaluate the contraceptive ability of two B-cell epitopes in CatSper1., Design: In vivo study with mice., Setting: University laboratory animal service center., Animal(s): BALB/c mice., Intervention(s): Two predicted B-cell epitopes in the extracellular part of transmembrane domains and pore region of CatSper1 were synthesized to immunize male mice., Main Outcome Measure(s): Fertility, epididymal sperm function, and the presence of specific antibody in immunized males were investigated., Result(s): Significant reduction of fertility was observed in mating trial with no evident systemic illness or abnormal mating behavior. Epididymal sperm of epitope-immunized males exhibited impaired ability to fertilize eggs in vitro, and showed sperm agglutination in some animals, while presenting no changes in sperm viability or progressive motility. High titer of antibodies was induced in the sera, and the antibodies' specificity was confirmed. The binding of the antibodies to epididymal sperm of epitope-immunized males was observed., Conclusion(s): CatSper members could be the effective and viable targets for immunocontraception. These two epitopes in CatSper1 share high identity between mouse and human and may be effective for fertility regulation in humans., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

SPINLW1 (previously known as eppin (epididymal protease inhibitor)) is a target under intense scrutiny in the study of male contraceptive vaccines. B-cell-dominant epitopes are now recognized as key parts of the induction of humoral immune responses against target antigens. The generation of robust humoral responses in vivo has become a crucial problem in the development of modern vaccines. In this study, we developed a completely novel B-cell-dominant-epitope-based mimovirus vaccine, which is a kind of virus-size particulate antigen delivery system. The mimovirus successfully self-assembled from a cationic peptide containing a cell-penetrating peptide of TAT49-57 and a plasmid DNA encoding both three SPINLW1 (103-115) copies and adjuvant C3d3. The male mice were immunized with the epitope-based mimovirus vaccine, which resulted in a gradual elevation of specific serum IgG antibody levels. These reached a peak at week 4. Mating for the fertility assay showed that the mimovirus vaccine had accomplished a moderate fertility inhibition effect and investigation into the mechanism of action showed that it did so by interfering with the reproductive function of the sperm but that it did not damage the structures of the testes or cause serum testosterone to decline. Our results suggest an ideal protocol for suppressing fertility in mice by an engineered mimovirus vaccine.

The uncontrolled reproduction of free-roaming feral cats contributes to overpopulation and associated concerns regarding their welfare and impact on public health and the environment. Nonsurgical fertility control that could be administered to feral cats in the field would be a powerful tool for cat population control. The objective was to test the efficacy and duration of activity of a single-dose GnRH immunocontraceptive vaccine (GonaCon™) on the fertility of adult female laboratory cats. Vaccinated cats (n = 15) received a single injection of vaccine containing a GnRH-KLH conjugate (200 μg) emulsified in a mycobacterial and oil adjuvant on study Day 0. Sham-treated cats (n = 5) received a single injection containing all vaccine components except the GnRH-KLH conjugate. A breeding trial started on study Day 120. Vaccinated cats had a longer time to conception (median 39.7 mo) compared to sham-treated cats (4.4 mo; P < 0.001). A total of 93% of vaccinated cats remained infertile for the first year following vaccination, whereas 73, 53, and 40% were infertile for 2, 3, and 4 y, respectively. At study termination (5 y after a single GnRH vaccine was administered), four cats (27%) remained infertile. The GnRH antibody titers declined more rapidly in short-term responding cats with < 2 y of infertility (n = 4), compared to long-term responding cats that experienced fertility control for >2 y (n = 11) (P < 0.05). Non-painful but persistent late-onset granulomatous injection site masses appeared 2 y after initial vaccination in five cats. We concluded that GnRH immunocontraception is an ideal candidate for further development for feral cat control., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The zona pellucida 3 (ZP3), an autoantigen, once used to develop contraceptive vaccine has been faced a safety issue. Avoiding its pathogenic T cell activation, we intranasally co-delivered the mZP3 DNA- and protein-based vaccines in mice and observed that a higher level of sIgA and IgG antibodies in vaginal washes, bronchoalveolar lavages and serum and yielded a lower level of fertility and mean litter size. Importantly, histological analysis showed that normal follicular developments of the infertile mice were not disrupted in the co-delivered group. Thus, the intranasal co-delivery may present a safe strategy for the development of contraceptive vaccine., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Aim: To enhance the immunocontraceptive effect of Lagurus lagurus zona pellucida 3 DNA vaccine, and to achieve the prospect of application through the pVAX1-sig-LTB-lZP3-C3d3 different immunity pathway., Methods: Two adjuvant molecules were constructed into the recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 as DNA vaccine which contains Escherichia coli heat-labile enterotoxin B subunit and the molecular adjuvant 3 copies of C3d. The results of RT-PCR and western blot showed that the DNA vaccine was expressed in mRNA and protein level. The female C57BL/6 mice were immunized by three ways: intramuscular injection, intranasal or oral route.Antibody levels and types were detected by ELISA., Results: ELISA results showed that recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 immunization induced specific IgG, IgA levels were significantly different comparing with control (P<0.01). Antifertility experiment showed that the experimental group reduced the average fertility significantly different compared with the control group (P<0.01)., Conclusion: Restriction analysis, RT-PCR and Western blot showed that the recombinant plasmid constructed correctly and can be the expression of mRNA and protein levels.It resulted that the recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 can induce the specific immune response efficiently and enhance the immunocontraceptive effects.

Previously we have found that DNA vaccine, pCMV4-rZPC' can generate specific antibodies against rabbit ZPC (amino acid 263-415, rZPC'), which binds to ovarian ZP and leads to a significant reduction of fertility in vivo. The purpose of this study was to evaluate the effect of antisera from pCMV4-rZPC(')-immunized mice on sperm-oocyte interaction in vitro. The effect of antisera from DNA vaccine-immunized mice on fertilization and early embryonic development was studied using an in vitro fertilization system. The results showed that the antisera supplemented in fertilization medium (10%, v/v) significantly decreased the rate of fertilization compared to that of control groups (P<0.05); whereas the antisera showed no significant effect on the rate of fertilization when ZP-free eggs were used. Moreover, the antisera pre-neutralized with mouse soluble zona pellucida lost the capacity to inhibit fertilization when compared with that of control groups. In addition, the antisera showed no detrimental effect on early developmental potential of mouse embryos in vitro. Taken together, our study provided herein direct evidence showing that antisera generated by DNA vaccine can block sperm-egg recognition during fertilization via targeting the oocyte ZP proteins., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (© 2011 John Wiley & Sons A/S.)

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., (© 2011 John Wiley & Sons A/S.)

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., (© 2011 John Wiley & Sons A/S.)

Zona pellucida (ZP) glycoproteins, by virtue of their critical role in fertilization, have been proposed as candidate antigens for the development of contraceptive vaccines. In this review, the potential of a ZP-based contraceptive vaccine for the management of wildlife population, with special reference to street dogs, is discussed. Immunization of various animal species, including female dogs, with native porcine ZP led to inhibition of fertility, which was associated with the ovarian dysfunction. Immunization of female dogs with Escherichia coli-expressed recombinant dog ZP glycoprotein-3 (ZP3) either coupled to diphtheria toxoid or expressed as fusion protein with 'promiscuous' T non-B-cell epitope of tetanus toxoid also led to inhibition of fertility. To improve the contraceptive efficacy of ZP-based contraceptive vaccine, various groups are working on improving the immunogen, use of DNA vaccine as prime-boost strategy, and delivering the zona proteins/peptides presented on either virus-like particles or entrapped in microsphere. Host-specific live vectors such as ectromelia virus and cytomegalovirus have also been used to deliver mouse ZP3 in mice. Various studies show the enormous potential of the ZP-based vaccine for the management of wildlife population, where permanent sterilization may be desirable., (© 2011 John Wiley & Sons A/S.)

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., (© 2011 John Wiley & Sons A/S.)

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., (© 2011 John Wiley & Sons A/S.)

The world's population is continuing to grow at an alarming rate and yet no novel methods of contraception have been introduced since 1960s. The paucity of our current contraceptive armoury is indicated by the 46 million abortions that are performed each year, largely in developing countries where population growth is greatest. Thus, whatever new forms of fertility control we develop for the next millennium, the particular needs of developing countries should be borne in mind. Contraceptive vaccines have the potential to provide safe, effective, prolonged, reversible protection against pregnancy in a form that can be easily administered in the Third World. In this review we consider the contraceptive targets that might be pursued, how vaccines might be engineered and the problems generated by inter-individual variations in antibody titre. We conclude that the specifications for a safe, effective, reversible vaccine are more likely to be met in animals than man., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Human chorionic gonadotropin (hCG) is synthesized soon after fertilization and is essential for embryonic implantation. A vaccine targeting hCG would be an ideal choice for immuno-contraception; an anti-hCG vaccine developed by Talwar et al., has previously undergone Phase II efficacy trials, providing proof of principle. These trials established the threshold levels of bio-neutralizing anti-hCG antibody titers required to prevent pregnancy; however, these titers (>50 ng/ml) were achieved in only 80% of immunized women. In this communication, we report a novel recombinant anti-hCG vaccine which demonstrates improved immunogenicity. hCGβ was genetically fused at C-terminal to the B-subunit of E. coli heat-labile enterotoxin. The recombinant fusion protein (hCGβ-LTB) was expressed in Pichia pastoris and, upon adsorption on Alhydrogel along with Mycobacterium indicus pranii (MIP) as an immuno-modulator, evoked a very high anti-hCG immune response in 100% of immunized BALB/c mice. This recombinant vaccine is expected to reduce cost as well as facilitate production of a molecularly consistent conjugate on a large scale., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Contraceptive vaccines can be designed to inhibit (i) production of the gametes (sperm and oocyte), (ii) functions of gametes leading to block in fertilization, and (iii) the gamete outcome (pregnancy). The zona pellucida (ZP) glycoproteins have been proposed as candidates for developing contraceptive vaccines by virtue of their critical role in fertilization. Immunization of non-human primates with either native or recombinant ZP proteins leads to curtailment of fertility, which however is invariably associated with ovarian pathology. To avoid oophoritis, immunogens corresponding to mapped B cell epitopes of ZP proteins that are devoid of 'oophoritogenic' T cell epitopes have been proposed. However, ways to overcome the observed oophoritis associated with the ZP-based contraceptive vaccines are yet to be fully defined. This is essential if their use for control of human fertility is to be considered. Nonetheless, contraceptive vaccines based on ZP proteins have shown very promising results in controlling wildlife population such as wild horses, white-tailed deers, elephants, marsupials, grey seals and dogs, where long term infertility or even permanent sterility is desirable., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Management of prairie dogs in the past has included poisoning, fumigants, barriers, and relocation. Because of the diverse attitudes related to prairie dog management, nonlethal methods that allow the existence of prairie dogs but help minimize damage related to population growth need to be developed. GonaCon™ is an immunocontraceptive vaccine that elicits antibodies to native GnRH; this prevents the secretion of reproductive hormones necessary for sperm and oocyte production. Prairie dogs were vaccinated with 0.1, 0.2, or 0.4 mL of the GonaCon™ emulsion intramuscularly in the upper thigh containing 100, 200, or 400 μg GnRH conjugate, respectively. Control animals were vaccinated with 0.4 mL saline emulsion in the upper thigh. Blood samples (≤1 mL) were taken from the femoral vein once pretreatment and at 1, 2, 3, 4, 6, and 15 months post-vaccination. Age (adult or juvenile) did not affect immune response to GonaCon™. Antibody titers were higher in the 200 and 400 μL GonaCon™ groups than in the 100 μL group, and there was no difference between the 200 and 400 μL GonaCon™ groups. No adverse effects of GonaCon™ were noted on weight or blood chemistry parameters during the study. GonaCon™ will likely contracept prairie dogs for ≥1 year in the field using either 200 or 400 μg conjugate. GonaCon™ could be incorporated into management plans to help maintain prairie dog populations while reducing habitat degradation due to overpopulation., (Published by Elsevier Ltd.)

Previous studies have reported on the development of a recombinant murine cytomegalovirus (rMCMV) containing the mouse zona pellucida 3 (mZP3) gene for use as a virally vectored immunocontraceptive (VVIC). This study aimed to alter promoter control over foreign antigen expression and cellular localisation of the antigen expressed in order to overcome virus attenuation previously encountered. Early studies reported on the mZP3 gene expressed by a strong constitutive human cytomegalovirus immediate-early 1 promoter (pHCMV IE1). This virus was able to induce >90% infertility in BALB/c mice despite being heavily attenuated in vivo. In this study the mZP3 was placed under the control of the MCMV early 1 (pMCMV E1) promoter and the inducible tetracycline promoter (Tet-On). In both instances the recombinant virus was able to induce infertility in directly infected mice. However, the viruses remained attenuated. This study demonstrated the capacity to manipulate the nature of the immune response by altering promoter control over foreign antigen expression and cellular localisation of the expressed antigen. We were able to demonstrate that by using the MCMV E1 promoter it was still possible to sterilize female BALB/c mice with an MCMV vector expressing mZP3. The use of the MCMV E1 promoter provides an added level of safety to any MCMV based VVIC approach as it only allows for transgene expression in MCMV permissive cells., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The world's population is nearing 6.8 billion, and we are in need of a male contraceptive that is safe, effective, reversible and affordable. Hormonal approaches, which employ different formulations of testosterone administered in combination with other hormones, have shown considerable promise in clinical trials, and they are currently at the forefront of research and development. However, the long-term effects of using hormones throughout a male's reproductive life for contraception are unknown, and it may take decades before this information becomes available. Because of this, many investigators are aiming to bring a nonhormonal male contraceptive to the consumer market. Indeed, there are several distinct but feasible avenues in which fertility can be regulated without affecting the hypothalamus-pituitary-testis axis. In this review, we discuss several approaches for fertility control involving the testis that one day may lead to the development of a nonhormonal male contraceptive., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In ewes, immunization against GnRH blocks LH pulses but mean serum FSH concentrations are only partly reduced; the fate of the FSH peaks that precede ovarian follicular waves has not been studied. In this study, we used immunization against GnRH to examine the need for pulsed GnRH secretion in the genesis of FSH peaks in the anestrous ewe. Six anestrous ewes were given a GnRH immunogen on Day 0 and a booster injection on Day 28. Control ewes (n=6) received adjuvant only. Transrectal ultrasonography was performed daily for 2 days prior to and 10 days following both the primary (Days -2 to 10) and booster (Days 26-38) injections and for a 13-day period beginning 26 days after booster injection (Days 54-66). Blood samples were collected daily. Intensive bleeding (every 12min for 7h) was performed on Days 9, 37, and 65 of the experimental period to characterize the pulsatile pattern of LH secretion. GnRH antibody titers were increased and LH pulses were abolished immediately after booster immunization (P<0.05). The number of FSH peaks, FSH peak concentration and amplitude and basal FSH concentrations were only decreased in immunized ewes in the period of observations starting 26 days after booster immunization (P<0.05); however, some peaks were still seen. The number of follicular waves was decreased in the period around booster immunization and no follicular waves were seen during the period starting 26 days after booster immunization in immunized ewes (P<0.05). In summary, in anestrous ewes, when pulsed LH secretion was abolished by immunization against GnRH, the peaks in serum concentrations of FSH that trigger ovarian follicular waves continued for a period of time. We concluded that although blocking the effects of GnRH gradually causes a diminution of FSH secretion, there is no acute requirement for GnRH in the regulation of FSH peaks. The existence of FSH peaks in the absence of follicular waves, and pulsed LH secretion, suggests that some endogenous rhythm may drive the occurrence of FSH peaks, independent of both changes in negative feedback by secretory products from ovarian antral follicles and GnRH., (Copyright 2010 Elsevier B.V. All rights reserved.)