Zona pellucida-based contraceptive vaccines for human and animal utility

Journal of Reproductive Immunology 88 (2011) 240–246

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Zona pellucida-based contraceptive vaccines for human and animal utility Satish K. Gupta a,∗ , N. Gupta a , P. Suman a , S. Choudhury a , K. Prakash a , T. Gupta a , R. Sriraman b , S.B. Nagendrakumar b , V.A. Srinivasan b a b

Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India R&D Center, Indian Immunologicals Limited, Gachibowli, Hyderabad 500 032, India

a r t i c l e

i n f o

Article history: Received 29 September 2010 Received in revised form 26 December 2010 Accepted 16 January 2011

Keywords: Contraceptive vaccine DNA vaccine Recombinant protein Synthetic peptide Zona pellucida glycoproteins

a b s t r a c t 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. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Several clinical studies of men and women with unexplained infertility have documented the presence of antibodies against antigens associated with the sperm and the oocyte (egg) (Isojima et al., 1968; Hovav et al., 1994). These observations suggest that immunological block of fertility is prevalent in humans and led to the idea of developing vaccines for fertility regulation, which otherwise have been used successfully to combat infection. Hypothetically, contraceptive vaccines can be designed to inhibit the reproductive process at multiple steps. During mammalian reproduction, the spermatozoa bind to the oocyte leading to fertilization. There are unique spermatozoa

∗ Corresponding author. Tel.: +91 11 26741249; fax: +91 11 26742125. E-mail address: [email protected] (S.K. Gupta). 0165-0378/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2011.01.011

and oocyte-associated proteins against which an immune response can be elicited thereby leading to the inhibition of gamete functions. The development of gametes is under the control of gonadotropins namely follicle stimulating hormone (FSH) and luteinizing hormone (LH) that are secreted from the pituitary under the influence of gonadotropin releasing hormone (GnRH) secreted by the hypothalamus. Contraceptive vaccines based on GnRH have been used successfully to inhibit fertility in various animal species (Hodges and Hearn, 1977; Oonk et al., 1998; Miller et al., 2000; Walker et al., 2007). In addition to the control of wild animal populations, GnRH-based vaccines have been used to eliminate the taint and thus improve the quality of boar and ram meat (Dunshea et al., 2001). Its use in humans for the treatment of hormone-dependent prostate cancer has also been proposed (Parkinson et al., 2004). After fertilization, the developing embryo secretes human chorionic gonadotropin (hCG) which is critical for establishment

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and maintenance of pregnancy. The proof of principle that immunocontraceptive vaccine can block fertility in human has also been established using a vaccine composed of the ␤-subunit of hCG annealed to the ␣-subunit of ovine LH (␣-OLH) which was coupled to either tetanus toxoid (TT) or diphtheria toxoid (DT). Immunization of fertile women with ␤-hCG–␣-OLH–TT/DT led to generation of bioneutralizing anti-hCG antibodies; immunized women showing antibody titres above 50 ng/ml were protected against conception, and the block in fertility was reversible (Talwar et al., 1994). However, the vaccine failed to elicit protective antibody titres in 100% of the immunized women, which is one of the major deterrents to using this vaccine in routine family planning program. The ␤-hCG-based vaccines, which were initially designed for protection against conception may however find utility as therapeutic vaccine in human subjects suffering from hCG secreting cancers (Delves et al., 2007). This review will primarily focus on the utility of the zona pellucida (ZP) glycoproteins based contraceptive vaccines for the management of wildlife population and their limitations for human contraception. 2. Zona pellucida glycoproteins ZP is an extra cellular matrix that surrounds the mammalian oocyte and plays an important role during fertilization. It is responsible for the binding of the spermatozoa to the oocyte and induction of the acrosome reaction in the ZP-bound spermatozoon. In mammals, ZP is composed of either 3 or 4 glycoproteins (Goudet et al., 2008; Gupta et al., 2009). Murine ZP matrix is composed of 3 glycoproteins designated as ZP1 [623 amino acid (aa)], ZP2 (713 aa) and ZP3 (424 aa). The ZP matrix of pig, cow and dog is also composed of 3 glycoproteins but, instead of ZP1, ZP4 (∼540 aa) is present. The ZP matrix of rat, hamster, bonnet monkey (Macaca radiata) and human has complement of all the 4 zona proteins: ZP1, ZP2, ZP3 and ZP4. The ZP glycoproteins are heavily glycosylated and have both N- and O-linked glycans. Analysis of the aa sequence of the four ZP glycoproteins from various species revealed that across species, a given zona protein shares a certain degree of sequence homology. For example, human ZP3 at the amino acid level exhibits an identity of 67% with mouse, 69% with rabbit, 74% with pig and 93.9% with bonnet monkey ZP3. Comparison of the amino acid sequence of the zona proteins within a given species also revealed variable sequence identity. At the amino acid level, human ZP1 has 47% identity with human ZP4 suggesting that these may have evolved from a common ancestral gene either by gene duplication or exon swapping (Swanson et al., 2001). The sequence identity of human ZP1 with ZP2 is 33% at the amino acid level. Human ZP3 has the least sequence identity with human ZP1, ZP2 or ZP4. 3. Contraceptive vaccines based on native ZP glycoproteins The ZP glycoproteins, due to their critical role in the fertilization process, tissue specificity and accessibility to

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systemic antibodies, have emerged as potential candidates for regulation of fertility through immunological intervention. Antibodies generated against a given ZP glycoprotein showed a variable degree of immunological cross-reactivity among various species, which has allowed heterologous immunization. Initial studies have employed porcine ZP proteins due to easy accessibility of the pig ovaries from abattoirs and the antibodies against porcine ZP proteins showed immunological reactivity with ZP from various species including humans. Immunization of female rabbits with the heat solubilized isolated zona pellucida (SIZP) generated antibodies that cross-reacted with the rabbit ZP and immunized animals failed to conceive (Wood et al., 1981). However, the infertility was irreversible and administration of exogenous gonadotropins failed to restore ovulation. Histology of the ovaries revealed destruction of oocytes in all the growing follicles and severe depletion of the pool of resting follicles (Skinner et al., 1984). Thus, it became obvious that the infertility was a consequence of ovarian dystrophy and not due to the inhibition of sperm–oocyte interaction. Immunization of female dogs with porcine ZP also resulted in prolonged proestrus or estrus cycles (Mahi-Brown et al., 1982). Histological examination of ovaries from animals that generated high titres of antibodies against the immunogen revealed depletion of oocytes. Immunization of squirrel monkeys (Saimiri sciureus) with porcine ZP3 (complex of ZP3 and ZP4) induced infertility and also led to a decrease in the number of ovulated oocytes (Sacco et al., 1987). The immunized animals, in spite of high anti-porcine ZP3 antibodies, showed a recovery in ovarian functions after 10–15 months of immunization. Immunization of female bonnet monkey with purified porcine ZP3 or porcine ZP3 conjugated to ␤-hCG along with adjuvants permissible for human use, led to generation of high anti-porcine ZP3 antibody titres (Bagavant et al., 1994). The animals remained infertile during the presence of high antibody titres, and continued to have ovulatory cycles. Laproscopic examination revealed normal ovaries with developing follicles. Following the decline in antibody titres, 50% of the animals became pregnant. Ovarian histology of the animals that failed to regain fertility did not reveal any signs of inflammation or lymphocytic infiltration. There was also no significant increase in the number of atretic or degenerated follicles. Hence, the observed variations in the extent of ovarian dysfunction in the studies described above may be due to various factors such as (i) the animal model used – some species are more susceptible than others upon immunization with ZP-based contraceptive vaccine; (ii) purity of the ZP glycoproteins – minor contamination with other ovary or oocyte associated proteins may be responsible for ovarian dysfunction, and (iii) the adjuvant used – Freunds’ complete adjuvant (FCA) generates high antibody titres and also results in ovarian atrophy, compared to other adjuvants such as alum or synthetic muramyl dipeptide derivative (MDP) (Sacco et al., 1989; Mahi-Brown et al., 1985; Upadhyay et al., 1989; Bagavant et al., 1994). The observed ovarian pathology, subsequent to immunization with the ZP-based contraceptive vaccines, is one of the major hurdles for its application to human contracep-

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Table 1 Effectiveness of ZP based contraceptive vaccines for controlling wildlife population. Target species

Immunogen

Status of fertility

Reference

Feral horse (Equus caballus)

Native porcine ZP

Kirkpatrick et al. (1990, 1996) Kirkpatrick and Turner (2002, 2003)

White-tailed deer (Odocoilus virginianus)

Native porcine ZP

African elephant (Loxodonta africana)

Native porcine ZP

Grey Seal (Halichoerus grypus)

Native porcine ZP

Marsupials (i) Koalas (Phascolarctos cinereus) (ii) Eastern Grey Kangaroos (Macropus giganteus) (iii) Brushtail possum (Trichosurus vulpecula) Dog (Canis lupus familaris)

Native porcine ZP Recombinant brushtail possum ZP3

Successfully used to control wild horses population at Assateague Island National Seashore, MD, USA; long term immunization studies revealed no significant debilitating effect on health. Successfully used to manage white-tailed deer population inhabiting Fire Island National Seashore, NY, USA; eosinophilic oophoritis observed in immunized animals. Field trials in Kruger National Park, Skukuza, RSA and other South African National Parks by remotely administered vaccine with drop-out darts; reduced pregnancy in immunized animals; no deleterious effect on the ovary and cyclicity. Decreased fertility over a long period (5 years). Efficacy trials done in New Zealand and Australia revealed the utility of ZP based contraceptive vaccine to curtail fertility in the immunized animals.

Native Porcine ZP Recombinant dog ZP3

tion. However in several countries, growing populations of wild animals that act as vector or reservoir for various diseases pose a major risk to human health as well as agriculture. Wildlife managers have often used lethal means such as shooting, trapping and poisoning to control these populations. However, growing public concern about animal welfare issues make such approaches increasingly unacceptable to society. For wildlife management, the contraceptive vaccines based on native porcine ZP have been used successfully to control population of feral horses (Equus caballus; Kirkpatrick et al., 1990), whitetailed deer (Odocoileus virginianus; McShea et al., 1997), African elephant (Loxodonta africana; Fayrer-Hosken et al., 2000) and koalas (Phascolarctos cinereus; Kitchener et al., 2009b) (Table 1). In grey seals (Halichoerus grypus), a single immunization with porcine ZP delivered in liposomes, had up to 90% contraceptive efficacy for 5 years (Brown et al., 1997). The long term field studies with porcine ZP-based contraceptive vaccines in wild horses and whitetailed deers have revealed that the vaccine is safe as no significant debilitating short- or long-term health effects were observed in the vaccinated animals (Kirkpatrick and Turner, 2002, 2003; Curtis et al., 2007). For controlling wildlife population, the oophoritis associated with ZPbased contraceptive vaccines may not be a major concern as long term infertility or permanent sterility is often desirable. In addition to the application of controlling wildlife population, contraceptive vaccines based on porcine ZP have also shown very promising results in inhibiting fertility of various captive zoo animals (Kirkpatrick et al., 1996).

Successfully curtail fertility in the immunized females; Oophoritis observed in the immunized animals.

McShea et al. (1997) Curtis et al. (2007)

Fayrer-Hosken et al. (2000)

Brown et al. (1997) Kitchener et al. (2009a,b) Cui et al. (2010)

Mahi-Brown et al. (1982) Srivastava et al. (2002)

4. Recombinant zona protein-based contraceptive vaccines Instead of native ZP proteins, use of recombinant proteins ensures no contamination by other ovarian or oocyte-associated proteins. Immunization of marmosets (Callithrix jacchus) with the recombinant human ZP3 expressed in Chinese Hamster Ovary (CHO) cells generated antibodies that led to long-term infertility, which was associated with ovarian pathology characterized by depletion of primordial follicles (Paterson et al., 1998). Female baboons (Papio anubis) immunized with Escherichia coli-expressed recombinant bonnet monkey ZP4 (bmZP4; previously designated as ZP1) conjugated to DT showed high antibody titres against bmZP4. The immunized animals remained infertile following mating with males of proven fertility in the presence of high antibody titres and later conceived upon decline in the antibody titres (Govind and Gupta, 2000). Encouraged with these observations, with a view to developing contraceptive vaccines for humans, the efficacy and safety of the E. coli-expressed recombinant bonnet monkey ZP proteins were then assessed in the homologous animal model (Govind et al., 2002). Female bonnet monkeys were immunized with recombinant bonnet monkey ZP2 (bmZP2) and bmZP4 conjugated with DT. Immunization with the self proteins conjugated to DT resulted in generation of high antibody titres against the respective proteins. The immunized animals remained infertile when mated with males of proven fertility in the presence of high circulating antibody titres, and failed to conceive even

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after decline in the antibody titres. Ovarian histopathology of the immunized animals revealed the presence of atretic follicles with degenerated oocytes, which may have been the principle cause for the block in fertility (Govind et al., 2002). The contraceptive efficacy of the recombinant human ZP glycoproteins expressed in mammalian cell has also been evaluated in cynomolgus monkeys (Martinez and Harris, 2000) where groups of animal were immunized with recombinant human ZP2, ZP3 and ZP4 (previously designated as ZP1). The animals immunized with recombinant ZP4 remained infertile for 9–35 months. Animals immunized with ZP2 and ZP3, as well as the unimmunized controls, all became pregnant well before those immunized with ZP4. During the time of high antibody titres, some animals experienced disruption of the menstrual cycle, which eventually returned to normal. These studies suggest that of the 3 human zona proteins evaluated, ZP4 has the best contraceptive efficacy. The active immunization studies performed in nonhuman primates using recombinant non-human primate or human zona proteins revealed contraceptive efficacy which were invariably associated with either transient or permanent ovarian dysfunction. These studies have also shown that the observed ovarian pathology using native ZP proteins may not be associated with contamination by other ovary associated proteins, but may be an inherent property of the zona proteins per se. To propose the ZP-based contraceptive vaccine for human application, the issue of ovarian pathology has to be resolved, which is described in detail later in this article. Nonetheless, with this shortcoming, we have explored the potential of recombinant zona protein-based contraceptive vaccine for controlling street dog populations (Table 1). In many developing countries, rabies is a major zoonosis of significant public health concern, which mainly spreads to humans by rabid dog bites (Meslin et al., 1994). Increasing street dog population in India is associated with a high incidence of rabies. The canine ZP2 and ZP3 were expressed in E. coli. Purified E. coli-expressed recombinant dog ZP2 (rdZP2; ∼70 kDa) and ZP3 (rdZP3; ∼42 kDa) were conjugated with DT (Srivastava et al., 2002). Three groups of female dogs (n = 4 per group) were immunized with rdZP2DT, rdZP3-DT and DT alone. Immunization with rdZP2-DT and rdZP3-DT led to generation of antibodies against the respective zona proteins as well as against DT. Mating of the immunized female dogs during the estrous period with males of proven fertility resulted in conception in all the 4 animals immunized with rdZP2-DT, suggesting that antibodies against ZP2 failed to prevent fertility in dogs. In the group of dogs immunized with rdZP3-DT, 3 out of 4 animals did not conceive whereas, in DT immunized group 3 out of 4 animals conceived, suggesting that antibodies against ZP3 have the potential to prevent conception in female dogs. The block in fertility was associated with antidZP3 antibody titres in the immunized animals. Ovarian histopathology revealed that the block in fertility in the group of female dogs immunized with rdZP3-DT is probably manifested by inhibition in the development of follicles as well as atretic changes in the ZP. To generate antibody titres against self antigens, which is usually the aim with contraceptive vaccines, conjuga-

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tion with carrier proteins is often used (Talwar et al., 1994; Govind and Gupta, 2000; Govind et al., 2002; Srivastava et al., 2002). Sometimes, due to generation of high antibody titres against immunodominant carrier proteins, there is a possibility of carrier-mediated suppression of antibody generation against the target antigen (Sad et al., 1991). To overcome this issue, ‘promiscuous’ T non-B cell epitopes from a variety of antigens have been proposed, which bind to various MHC molecules thereby enabling the generation of antibody response in an outbred population with different MHC backgrounds (Ho et al., 1990; Lairmore et al., 1995). Further, the probability of carrier-mediated suppression of the immune response is reduced as antibodies will not be generated against the ‘promiscuous’ T non-B cell epitopes. Keeping this in view, our group has expressed canine ZP3 (23–348 aa residues) as a recombinant fusion protein in E. coli with ‘promiscuous’ T non-B cell epitope of TT (831–844 aa residues). Female dogs were immunized with recombinant protein (rTT-dZP3) either at 200 ␮g or 1.0 mg per injection/animal on days 0, 21 and 35 using aluminum hydroxide as an adjuvant (unpublished observations). In the group of animals immunized with 200 ␮g rTT-dZP3, 2 female dogs showed estrus during the following breeding season and 2 failed to come into heat and all the 4 immunized animals failed to conceive on mating. Following immunization with 1.0 mg recombinant protein, all the female dogs failed to come into heat and so could not accept the male during mating and did not conceive. In contrast to this, all the 4 unvaccinated female controls became pregnant and delivered normal pups. Animals from both the groups were given a booster of 1 mg recombinant protein/animal on day 273. In the subsequent breeding season, none of the immunized animals from either group accepted the male for mating and thus did not conceive (unpublished observations). These studies suggest that street dog populations can be controlled in a humane way using recombinant dog ZP3 fused with ‘promiscuous’ T non-B cell epitope of TT. In Australia and New Zealand, extensive studies have been done to explore the potential of recombinant zona proteins to control the population of marsupials that are considered pests (Table 1). Immunization of eastern grey kangaroos (Macropus giganteus) and brushtail possums (Trichosurus vulpecula) with E. coli-expressed recombinant possum ZP3 led to a significant curtailment of fertility in the immunized female animals (Kitchener et al., 2009a; Cui et al., 2010). Female possums immunized with synthetic peptide corresponding to possum ZP2 (aa residues 431–445) coupled to keyhole limpet hemocyanin also led to a significant reduction in the production of embryos (Duckworth et al., 2007). To control the population of rodents which are also considered as pests in several countries, the possibility of using host-specific live vectors such as ectromalia virus (mouse pox virus) and cytomegalovirus (mouse beta herpes virus) have been explored to deliver mouse ZP3. Use of such immunization regimens led to decrease in fertility in the immunized mice (Jackson et al., 1998; Lloyd et al., 2003). However by and large, the recombinant viruses have low infectivity of host tissues compared to wild type viruses, thereby resulting in reduced contraceptive effi-

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cacy. Another concern regarding use of live viral vectors in the wild is the impact on other species, if the recombinant virus should lose its host specificity. 5. Strategies to avoid oophoritis associated with ZP-based contraceptive vaccines To develop a contraceptive vaccine for humans, it is essential to avoid the oophoritis associated with immunization by ZP-based contraceptive vaccines. Using a mouse model, it was shown that the observed oophoritis is due to the presence of ‘oophoritogenic’ T cell epitopes in the ZP-based immunogens (Luo et al., 1993). Immunization with the minimal B cell epitope of mouse ZP3 and the phenylalanine substituted with glutamine of the overlapping ‘oophoritogenic’ T cell epitope (QAQIHGPR), collinearly synthesized with the ‘promiscuous’ T cell epitope of bovine RNase (VCAYKTTQANK) led to generation of antibodies in inbred mice of eight different haplotypes and B6AF1 mice without activation of oophoritogenic T cells (Lou et al., 1995). When mated, the litter size of the immunized group was considerably lower compared to the control group and reduction in the litter size correlated with the antibody titres. This showed a means by which MHC-driven non-responsiveness to a self antigen can be overcome and the self pathogenic T cell responses can be avoided. Immunization of female marmoset with either human ZP3 synthetic peptides conjugated to TT or marmoset ZP3 synthetic peptide incorporating a promiscuous T cell epitope of TT generated an antibody response against the respective peptides (Paterson et al., 1998, 1999). The antibodies recognized native marmoset/human ZP and also inhibited in vitro human sperm–zona binding. No loss of ovarian function/pathology was observed in the immunized animals. However, a consistent reduction in fertility correlated to the antibody titres was not observed (Paterson et al., 1998, 1999). Studies based on delineation of the B cell epitope of bonnet monkey zona proteins, revealed that synthetic peptides corresponding to 324–347 aa residues of ZP3 and 251–273 aa residues of ZP4 (previously designated as ZP1) showed promising in vitro contraceptive efficacy with human gametes (Afzalpurkar et al., 1997; Sivapurapu et al., 2002). Synthetic peptide encompassing the above B cell epitopes of bmZP3 and bmZP4 separated by a triglycine (GGG) spacer resulted in production of antibodies with higher contraceptive potential compared to individual epitopes as analyzed by the hemizona assay (Sivapurapu et al., 2005). Female bonnet monkeys immunized with the above bmZP3 synthetic peptide conjugated to DT failed to conceive when mated with males of proven fertility, despite having ovulatory cycles as judged by serum progesterone profile (Kaul et al., 2001). There was no ovarian pathology observed in the immunized animals. Reduction in fertility without concomitant ovarian pathology was also observed in white-tailed deer immunized with porcine ZP4 peptide corresponding to 79–130 aa residues and mice immunized with mouse ZP3 peptide corresponding to 328–442 aa residues (Miller and Killian, 2002; Hardy et al., 2002). In another approach to adjuvant-free immunization, we expressed mouse ZP3 and

sperm specific (YLP12 ) epitopes with Johnson grass mosaic virus coat protein to present these antigens as virus-like particles (VLPs). Immunization of female mice with such VLPs without any adjuvant led to a significant reduction of fertility in the immunized animals (Choudhury et al., 2009). These observations suggest that immunization with the synthetic peptides encompassing B cell epitopes that are devoid of ‘oophoritogenic’ T cell epitopes corresponding to ZP glycoproteins have the potential to curtail fertility without eliciting any kind of ovarian pathology. In addition to synthetic peptides, recent studies suggest that genetic immunization with a DNA vaccine can obviate ovarian pathology. Immunization of mice with DNA vaccine encoding rabbit ZP3 corresponding to 263–415 aa residues resulted in a significant reduction in the number of new born pups compared to the control. Histology of the ovaries from the immunized mice revealed morphologically normal follicles at different stages of development (Xiang et al., 2003). In another study, co-immunization of mice with DNA vaccine encoding mouse ZP3 and recombinant ZP3 protein also led to a significant decrease in fertility (Li et al., 2007). Histological analysis showed normal follicular development in infertile mice which was associated with a decrease in T cell proliferation. These immunized animals show a decrease in the production of inflammatory cytokines and IFN␥ and an increase in the production of IL10. There was also an increase in the production of Foxp3+ regulatory T (Treg) cells in the immunized group. These observations need to be further validated before proposing that immunization with a DNA vaccine encoding ZP proteins may decrease or eliminate the ovarian pathology often observed in animals immunized with native or recombinant zona proteins. 6. Concluding comments The proof of concept to prevent conception in women has been established with the ␤-hCG-based contraceptive vaccine; however there are several issues that remained to be resolved before contraceptive vaccines can be used for controlling human fertility. These are (i) failure to generate 100% contraceptive efficacy, (ii) the requirement for repeated injections, (iii) variation in the antibody titres among the immunized subjects thus making it difficult to predict the duration of contraceptive efficacy, and (iv) the safety, in particular the oophoritis induced by ZP-based contraceptive vaccines. However despite these shortcomings, the currently available contraceptive vaccines have utility in controlling wildlife population, where 100% contraceptive efficacy is not an absolute requirement and essentially a ‘herd’ immunization approach is likely to be effective. The oophoritis observed in animals subsequent to immunization with ZP-based contraceptive vaccine may not be a concern as current methods of wildlife management involves spaying. In fact, for wildlife management long term infertility or permanent sterility may be desirable. Nonetheless, further scientific inputs are required to improve vaccine design to enhance immunogenicity and contraceptive efficacy, development of more potent and safe adjuvants and novel vaccine delivery systems to generate long lasting immunity and thereby reduce the number

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