Contraceptive Vaccines Based on the Zona Pellucida Glycoproteins for Dogs and Other Wildlife Population Management


Satish Kumar Gupta, Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110 067, India.


Citation Gupta SK, Srinivasan VA, Suman P, Rajan S, Nagendrakumar SB, Gupta N, Shrestha A, Joshi P, Panda AK. Contraceptive vaccines based on the zona pellucida glycoproteins for dogs and other wildlife population management. Am J Reprod Immunol 2011; 66: 51–62

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.


Vaccines for contraception have been proposed as one of the options for fertility regulation with the aim to control growing human population as well as populations of several wild animals.1,2 Such a vaccine should elicit humoral- and/or cell-mediated immune responses against hormone(s)/protein(s) that are critical to accomplish successful conception; interference in their biological function will result in block of the fertility. There are several steps in the reproductive process that can be targeted for the immunological intervention to achieve contraception. The contraceptive vaccines based on gonadotropin-releasing hormone (GnRH), secreted by the hypothalamus, facilitating the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary, have been used to inhibit the fertility of various animal species.3–8 The contraceptive vaccine, GonaConTM, comprised of synthetic GnRH coupled to keyhole limpet hemocyanin (KLH) and a newly developed adjuvant, AdjuVacTM (diluted version of Johnes disease vaccine containing killed mycobacterium and oil), has been developed by the scientists at the US Department of Agriculture’s Wildlife Services, National Wildlife Research Center (NWRC). GonaConTM has shown variable efficacy to curtail fertility for varying period in several species.4,6,8 Contraceptive vaccine based on GnRH will be effective in both males and females by virtue of its presence and critical role during reproduction in both the sexes. To control human population, vaccine aiming to neutralize the bioactivity of human chorionic gonadotropin (hCG), which is essential for the establishment and maintenance of pregnancy, has also been explored. Phase-II clinical trials in women immunized with β-subunit of hCG annealed to the α-subunit of ovine LH (α-OLH), coupled to either tetanus toxoid (TT) or diphtheria toxoid (DT), revealed its efficacy to inhibit fertility, which was reversible.9 Attempts have been made and are being continued by several groups to evaluate the efficacy and safety of the contraceptive vaccines based on unique spermatozoon- and egg-associated proteins crucial for fertilization.1,2,10–20

The failure of various contraceptive vaccines proposed till date to achieve 100% contraceptive efficacy for a defined period is one of the major bottleneck for their use in controlling human population. In spite of this limitation, contraceptive vaccines can be used on the analogy of ‘herd immunization’ for controlling populations of various wild animal species. Wild animals may act as vectors or reservoirs for various diseases of zoonotic importance and may pose a major risk to the human health. Wildlife managers have often used lethal means such as shooting, trapping, and poisoning to control wildlife population. However, growing public concerns over animal welfare issues concomitant with the new legislations that forbid killing of these animals make such approaches increasingly unacceptable. For example, rabies is a fetal encephalomyelitis caused by a negative-stranded RNA virus (rabies virus) of Rhabdoviridae family. Stray dogs are the main vectors maintaining rabies virus circulation within human communities. Various agencies that are involved in controlling the population of the street dogs mainly use either spaying of female dogs or castration of male dogs. These measures have failed to effectively control the increase in their population. As a result, the incidence of rabies caused by the rabid dog bite has increased in recent years, which is prevalent in several developing countries.21 Human mortality from rabies has been estimated to be 55,000 deaths per year,22 and 99% of these deaths occur in the developing world. In India, approximately 20,000 people die from rabies, and 96% of these are infected by dogs.23,24 In this article, the prospects of zona pellucida (ZP) glycoproteins–based contraceptive vaccine for controlling wildlife population with the special reference to street dogs have been reviewed and analyzed.


Canine Oocyte and Composition of the Zona Pellucida Matrix

In female dogs, cluster of the primordial follicles is observed in the cortex of the ovary by third week after birth.25 Ovarian follicles in dogs may have either one or more than one egg. Ovulation is characterized by the rupture of several mature follicles. In contrast to other mammalian species, the released oocyte is immature and contains a germinal vesicle, which subsequently undergoes meiotic maturation in the oviduct.26 Dogs have one estrus cycle per season and on an average may have 2–3 estrus cycles per year. Each estrus cycle is followed by diestrus. Female dogs accept male dogs for mating only when they are in estrus. Subsequent to conception, increase in the progesterone levels is observed. However, if pregnancy does not ensue, bitches experience a pseudopregnancy of the similar duration as pregnancy.

As in other mammals, canine oocyte is also surrounded by a glycoproteinaceous matrix, termed as ZP. The ZP matrix plays a crucial role during fertilization by mediating species-specific spermatozoa binding to the oocyte, induction of the acrosome reaction in the zona-bound spermatozoon, and subsequent to sperm membrane fusion with the egg oolemma helps in the avoidance of polyspermy.27 It also acts as a protective barrier for the growing oocyte and for the early stages of the embryonic development until implantation of the blastocyst into the endometrium. Canine ZP matrix is composed of three glycoproteins designated as ZP glycoprotein-2 (ZP2; 715 aa), ZP glycoprotein-3 (ZP3; 426 aa), and ZP glycoprotein-4 (ZP4; 493 aa).28,29 Canine ZP3 has a signal peptide (aa residues 1–22), which drives it into the secretory pathway and is cleaved off from the mature protein (Fig. 1). Toward the C-terminus, there is a consensus pro-protein convertase (furin) cleavage site (aa residues 349–352; RNRR), followed by trans-membrane domain (aa residues 386–407) and cytoplasmic domain (aa residues 407–426). Furin cleavage site helps in ZP3 secretion and their assembly into the ZP matrix.27 Computational analysis of the canine ZP3 for the secondary structure prediction revealed the presence of 5.9% alpha helices, 31.0% beta sheets, and 63.1% random coils. It has 16 cysteine residues of which 14 are conserved in ZP3 from mouse, rabbit, cat, pig, and human. Canine ZP3 has three potential sites for N-linked glycosylation at positions 123, 145, and 244. In addition, there are three O-linked glycosylation sites at positions 154, 160, and 161. Murine ZP matrix as in dogs is also composed of 3 glycoproteins, but instead of ZP4, ZP glycoprotein-1 (ZP 1; 623 aa) is present.30 The ZP matrix of humans has complement for all the four zona proteins.31 Based on the theoretical pI, the canine ZP proteins are basic in nature, unlike mouse and human ZP proteins, which are acidic in nature.

Figure 1.

 Multiple alignment of the deduced amino acids (aa) sequence of dog ZP glycoprotein-3 (ZP3) with the cat, pig, human, rabbit, and mouse ZP3: The amino acid sequences of dog, cat, pig, human, rabbit, and mouse ZP3 were obtained from uniprot protein database and multiple alignment was performed using Clustal W program. Asterisks represent conserved aa among various species taken for the comparison. Various domains/regions in dog ZP3 are highlighted in different colors. The signal sequence (aa residues 1–22) is highlighted in red, furin cleavage site (aa residues 349–352) in green, trans-membrane domain (aa residues 386–407) in blue, and cytoplasmic domain (aa residues 407–426) in orange.

Analysis of the aa sequence of the canine ZP glycoproteins with the ZP glycoproteins of other species revealed various degrees of sequence identity. For example, canine ZP3 at aa level exhibited an identity of 65% with mouse, 63% with rabbit, 76% with pig, 79% with cat, and 70% with human ZP3 (Fig. 1). Canine ZP2 showed an identity of 57% with mouse, 66% with rabbit, 70% with pig, 82% with cat, and 68% with human ZP2. Canine ZP4 has an aa identity of 67% with rabbit, 70% with pig, 74% with cat, and 67% with human ZP4. The orthologue of the canine Zp4 gene is present in the mouse genome as a pseudogene.29

Contraceptive Efficacy of the Native ZP Glycoproteins in Dogs

ZP glycoproteins, by virtue of their critical role during fertilization, have emerged as potential target antigens for the inhibition of fertility. Immunization with the zona proteins elicits humoral- and/or cell-mediated immune responses (Fig. 2). Antibodies against the ZP proteins have the potential to inhibit binding of the spermatozoa to the egg, thereby preventing fertilization. Alternatively, the immune responses generated against the ZP proteins may inhibit folliculogenesis, which may be primarily mediated by oophoritogenic T-cell epitopes, thereby leading to the curtailment of fertility (Fig. 2). Initially, various active immunization studies to evaluate the contraceptive potential of the ZP glycoproteins have employed porcine ZP glycoproteins as pig ovaries were easily accessible from the abattoirs. Upon immunization, antibodies generated against porcine ZP glycoproteins react with ZP isolated from various species including humans.32

Figure 2.

 Schematic diagram to show the possible mode of action followed by Zona pellucida (ZP)-based contraceptive vaccine: Immunization of the animals with various forms of ZP proteins generates both humoral and cellular immune responses. Antibodies (Ig) primarily manifest their contraceptive effect by inhibiting sperm–egg interaction. The cell-mediated immune responses generated by ‘oophoritogenic’ T-cell epitope(s) present within the zona proteins are primarily responsible for the disturbances in ovarian follicular development. However, the role of anti-zona antibodies in affecting folliculogenesis has not been elucidated. PrF, primordial follicle; PF, primary follicle; SF, secondary follicle; AF, antral follicle.

Immunization of female dogs with the solubilized zonae pellucidae prepared from either the pig or the dog oocytes revealed higher antibody titers in the pig ZP immunized group as compared with the dog ZP immunized group.33 Immune sera from the pig ZP immunized bitches led to the precipitation of the zona surface both in vivo and in vitro and also inhibited in vitro penetration by sperm across the zonae. Bitches immunized with the porcine zonae failed to conceive when mated with the male dogs and exhibited abnormal cycles.33 At this juncture, it was suggested that the observed side-effects might be owing to the impurities of other ovary-associated proteins, which may be present in the solubilized zonae pellucidae employed in these studies. Subsequently, the procedure to purify ZP glycoproteins, including individual porcine ZP glycoproteins, from native source that are devoid of other contaminating ovary-associated proteins was established.34 Further, bitches were immunized with the crude porcine ZP as well as the purified porcine ZP employing three different adjuvants namely alum, Freund’s, and CP-20,961.35 Immunization with the crude porcine ZP produced moderate to high antibody titers and resulted in infertility. Bitches with high anti-ZP antibodies experienced prolonged pro-estrus and estrus cycles with abnormal estradiol and progesterone levels. Animals immunized with the purified porcine ZP led to moderate antibody titers, remained infertile, and had lesser side-effects. As compared with alum, the animals immunized using Freund’s and CP-20,961 adjuvants generated higher antibody titers. The ovaries showed disturbances in the follicular development and the granulosa cell nest in the bitches immunized with the crude porcine ZP, and the luteal cysts in those immunized with the purified porcine ZP3.36 These studies demonstrated that the use of purified porcine ZP glycoproteins decreased considerably the side-effects on ovarian functions; though, other mechanisms causing minimal damage to the ovaries could not be averted.

Contraceptive Efficacy of the Recombinant Zona Proteins in Dogs

Use of the purified ZP proteins obtained from native source does not completely rule out the possibility of minor contaminants from other ovary/egg-associated or zona proteins. Keeping this in view, our group cloned and expressed dog ZP2 and ZP3 in Escherichia coli.37,38 Recombinant dog ZP3, excluding signal sequence and transmembrane-like domain after furin-like cleavage site corresponding to aa residues 23–348, was expressed in E. coli.37 In SDS–PAGE and Western blot, the recombinant dog ZP3 revealed a major band of 42 kDa and a minor band of 32 kDa. Using similar strategy, recombinant dog ZP2, excluding the N-terminal signal sequence and C-terminal transmembrane-like domain, was also expressed in E. coli. The expressed protein revealed a major band of 70 kDa both in SDS–PAGE and Western blot.38 To produce recombinant protein in large amount, batch fermentation with glycerol as carbon source was optimized for the expression of recombinant dog ZP3, which yielded ∼30 mg of protein from one liter of the culture.37 Polyclonal antibodies against the E. coli-expressed recombinant dog ZP2 and ZP3 were raised in rabbits. These antibodies showed strong reactivity with the ZP matrix in an indirect immunofluorescence assay employing dog ovarian cryosections.37,38

To evaluate their immunocontraceptive potential, the recombinant dog ZP2 and ZP3 were coupled with DT. Three groups of female dogs (n = 4) were immunized with the recombinant dog ZP2-DT, ZP3-DT, and DT using Squalene and Arlacel-A as adjuvants.38 Sodium phthalyl derivative of lipopolysaccharide (SPLPS; 1 mg), prepared as described by Elin et al.,39 was also included in the first injection as an additional adjuvant. Immunization of the animals led to the generation of antibodies against the respective ZP proteins as well as against DT. Mating studies revealed that all the four female dogs immunized with the recombinant dog ZP2–DT became pregnant, whereas three of four animals immunized with DT conceived, suggesting that ZP2 is not a good candidate for developing contraceptive vaccine for female dogs. On the other hand, three of four female dogs immunized with the recombinant dog ZP3-DT failed to conceive. Ovarian histopathology revealed that the block in fertility in the group of female dogs immunized with the recombinant dog ZP3 was as a result of follicular atresia and atretic changes in the ZP. These preliminary studies suggested that the dog ZP3 can be a potential candidate for the development of the contraceptive vaccine for controlling dog population.

Utility of the Promiscuous T non-B-cell Epitopes as ‘Carrier’ in the Recombinant Dog ZP3-Based Contraceptive Vaccine

To elicit high antibody titers against E. coli-expressed recombinant dog ZP3 in female dogs, conjugation with ‘carrier’ protein such as DT has been proposed.37,38 Use of glutaraldehyde as a coupling agent to prepare conjugate of the recombinant dog ZP3 with DT in the above studies has a risk of formation of homopolymers and heterogeneous population of the resulting conjugate. Hence, to prepare protein–protein conjugate with defined stoichiometry, several hetero-bifunctional coupling reagents such as 1-ethyl-3-(3diethylaminopropyl)-carbodiimide (EDCI) and m-maleimidobenzyoyl-N-hydroxysuccinate ester (MBS) have been successfully used. To control street dog population, it is desirable to have contraceptive vaccine, which is cost-effective. Additional conjugation step during the vaccine production is likely to increase its cost. Further, use of such vaccines also led to the generation of high antibody titers against the ‘carrier’ protein, which sometime led to the suppression of antibodies against the protein/peptide critical for the success of reproduction.40 Alternately, ‘promiscuous’ T non-B-cell epitopes from a variety of antigens have a potential to provide the T-cell help41,42 that can be employed to generate humoral immune response against self-proteins.11,20,43‘Promiscuous’ T non-B-cell epitope binds to the various MHC molecules and therefore elicits immune response in larger proportion of the immunized outbred population. Further, antibodies will not be generated against the ‘promiscuous’ T non-B-cell epitope and thus ruling out the possibility of ‘carrier-’mediated suppression of the humoral immune response.

To avoid the conjugation of recombinant dog ZP3 with DT, dog ZP3 was expressed as a fusion protein with T non-B-cell epitope of TT (aa residues 830–844) in E. coli (unpublished observations). Two groups of female dogs (n = 4) were immunized with the purified recombinant fusion protein adsorbed on aluminum hydroxide either at 200 μg or 1 mg per injection on days 0, 21, and 35. Immunization led to the generation of high antibody titers against recombinant dog ZP3 in both the groups. Subsequently, immunized animals were mated with male dogs of proven fertility along with control unvaccinated group of female dogs (n = 4). All female dogs immunized with the recombinant fusion protein failed to conceive, whereas unvaccinated female controls became pregnant and delivered normal pups. The immunized animals were followed up, and additional booster of 1 mg recombinant fusion protein/animal was given on day 273 in both the above groups. In the subsequent breeding season, none of the immunized animals from both the groups accepted the males for mating and thus did not conceive (unpublished observations). These initial observations are encouraging and needs validation by undertaking large field trials to ascertain the utility of recombinant dog ZP3-based contraceptive vaccine for the management of the population of street dogs.

Utility of the ZP-Based Contraceptive Vaccine to Control Population of Other Animal Species

While studies on the efficacy of recombinant dog ZP3 to control population of street dogs are in progress, ZP-based contraceptive vaccines have been successfully used to control population of various other animal species. Immunization of feral horses (Equus caballus),44 white-tailed deers (Odocoileus virginianus),45 African elephants (Loxodonta affricana),46 koalas (Phascolarctos cinereus),47 and grey seals (Halichoerus grypus)48 with the native porcine ZP-based vaccine decreases the fertility in immunized animals. Long-term follow-up studies of the vaccinated feral horses and white-tailed deers revealed no significant deleterious effects on the health of the immunized animals, except oophoritis.49–51 In addition to porcine ZP, the efficacy of E. coli-expressed recombinant brushtail possum (Trichosurus vulpecula) ZP3 to curtail fertility of marsupials, which are considered as pests in Australia and New Zealand, has also been demonstrated.52,53 The above studies are suggestive of enormous potential of the ZP-based contraceptive vaccines for wildlife management, where observed oophoritis may not be a deterrent factor. In fact, for wildlife population management, long-term infertility or even permanent sterility is often desirable.

Strategies to Enhance the Contraceptive Efficacy of the ZP-Based Contraceptive Vaccine

It is imperative to increase the contraceptive efficacy of the ZP-based contraceptive vaccines and devise novel vaccine delivery systems for their utility in wildlife population management. It would require the generation of long-lasting high antibody responses in larger proportion of the immunized animals, which is relevant for interference in the functions of the gametes. To achieve the above goal, the following strategies have been explored.

Immunogen design  Immunization of the female dogs with DT-conjugated 18 mer synthetic peptide corresponding to the canine ZP2 (aa residues 50–67; CTSILDPEKLTLKAPYET) in complete Freund’s adjuvant, followed by booster in incomplete Freund’s adjuvant, led to the generation of antibodies reactive to dog ZP.54 Immunization with synthetic peptide inhibited the follicular development, which was accompanied by oocyte degeneration.54 Immunized dogs failed to show any estrus period and thus remained infertile. To increase the contraceptive efficacy, it has been proposed to use either multiple B-cell epitopes of a given zona protein or epitopes from multiple zona proteins. It can be achieved by using a physical mixture of multiple immunogens. As an example, female bonnet monkeys were immunized with a physical cocktail of bonnet monkey ZP3 peptides, individually conjugated to DT. Antibodies generated by following the immunization with the ZP3 peptide cocktail had higher efficacy to inhibit in vitro human sperm–oocyte binding as compared with those immunized with the individual ZP3 peptide–DT conjugate.55 This may be owing to the cooperative effect among the antibodies pertaining to different domains of ZP3. Chimeric synthetic peptides and recombinant proteins encompassing multiple epitopes of different zona proteins have also been evaluated as an alternative to the physical mixture. Antibodies generated against chimeric synthetic peptides encompassing epitopes of bonnet monkey ZP4 (previously designated as ZP1; aa residue 251–273) and bonnet monkey ZP3 (aa residue 324–347) separated by a triglycine spacer elicited higher inhibitory effect on human sperm–oocyte binding in a hemizona assay as compared with antibodies against the individual peptides.56 On the same analogy, antibodies against chimeric E. coli-expressed recombinant protein encompassing epitopes of bonnet monkey ZP2 (aa residues 86–113), ZP3 (aa residues 324–347), and ZP4 (aa residues 132–147) also showed significant inhibition in the binding of the human sperm to the human zona in a hemizona assay.57 To increase the contraceptive efficacy, chimeric synthetic peptides/recombinant proteins encompassing epitopes of ZP glycoprotein(s) and spermatozoa specific proteins have also been evaluated with the premise that simultaneous generation of antibodies against both the gametes will be more effective.58–60

Presentation of ZP proteins (a) Microsphere: Polymeric particle–based delivery system improves the immunogenicity of entrapped antigen.61 The most commonly studied polymers are poly (d, L – lactide-co-glycolide) (PLGA) and polylactide (PLA). These biodegradable, biocompatible polymers have been extensively used for the delivery of antigens as well as for the development of single-dose vaccines. PLA/PLGA polymer particle–based vaccine delivery systems offer the following advantages:

  • • Single-point immunization by the polymer particle–entrapped antigen elicits long-lasting antibody response.
  • • The quality of immune response depends on the particle size. Nanoparticles favor cellular response, whereas microparticles favor humoral response. Immunization with different sized particles thus provides a tool to modulate immune response.62
  • • Polymer particle–entrapped vaccines have the capacity to induce the memory antibody response from single-point immunization.63

It is expected that entrapment of ZP proteins in polymer particles and optimization of immunization conditions will help in improving the immunogenicity of the candidate vaccine and may provide long-term protection from a single-point immunization. A single injection of the porcine ZP in liposomes generated long-lasting (5 years) antibody titers with concomitant reduction in fertility in grey seals.48

(b) Virus-like particles (VLPs) and bacterial ghosts: Virus-like particles (VLPs) are essentially non-infective viruses, comprised of self-assembled viral envelop proteins without the genetic material, and have size and conformation similar to that of intact virion. VLPs have shown great potential to efficiently present foreign peptides for generating antibody response in the recipients.64 One such efficient method is based on Johnson grass mosaic virus (JGMV) coat protein (CP), which can be engineered for presenting the foreign peptides, and self-assembles to form rod-shaped VLPs.65 Mouse ZP3 peptide (QAQIHGPR), spermatozoa-specific YLP12 peptide (YLPVGGLRRIGG), and fusion peptide comprising YLP12 and ZP3 epitopes separated by a diglycine spacer (YLP12-GG-ZP3) have been presented on JGMV VLPs. Immunization of female mice with the VLPs presenting YLP12-GG-ZP3 fusion peptide and a physical mixture of the VLPs presenting either YLP12 or ZP3 epitope led to the generation of specific antibody responses and induced subfertility in the immunized animals.66

Bacterial ghost, made up of non-living bacterial cell without the genetic component, has also been used effectively to deliver antigen for eliciting immune response. Immunization of female brushtail possums with bacterial ghost expressing either N-terminal (aa residues 41–316) or C-terminal (aa residues 308–636) fragments of possum ZP2 fused to maltose-binding protein led to the generation of both humoral- and cell-mediated immune responses.67 A reduction in the fertility of possum immunized with C-terminal fragment of the possum ZP2 was also observed.67 Bacterial ghost can be used to formulate the contraceptive vaccines for oral bait delivery to manage the wildlife population.

(c) Live vectors: Live bacteria and viruses have also been used as vectors to present ZP proteins to the immune system. Female mice immunized through oral route by attenuated Salmonella typhimurium-expressing mouse ZP3 led to the generation of antibodies against ZP3 in reproductive tract as well as in the systemic circulation.68 The immunized animals showed reduced fertility. Host-specific live vectors such as ectromelia virus (a natural pathogen that causes mouse pox) and cytomegalovirus (mouse-specific beta herpes virus) have also been used to express mouse ZP3.69,70 Female mice infected with the recombinant ectromelia virus led to the generation of anti-ZP3 antibodies, and immunized mice were infertile for 5–9 months after infection.69 Infertility was associated with the disruption of the ovarian follicular development. Regain of fertility was observed with a decline in the circulating anti-ZP3 antibody levels. Reinfection with the recombinant virus boosted the anti-ZP3 antibody levels and once again resulted in infertility. Female mice infected with the engineered cytomegalovirus expressing mouse ZP3 also led to total sterility after 21 days post-infection.70 Infected animals exhibited profound ovarian pathology with mild focal oophoritis associated with the infiltration of CD4+ and CD8+ T cells. Infection of the female rabbits with the engineered myxoma virus expressing rabbit ZP3 and ZP4 (ZPB) also led to the reduction in fertility with concomitant ovarian dysfunction.71,72 However, engineered myxoma virus expressing rabbit ZP2 failed to elicit contraceptive effect.72 In spite of promising results obtained using this approach, concerns about the practical utility of host-specific live vectors for wildlife population management have been expressed by various groups because of their safety, in case of engineered virus losing host specificity.

DNA vaccine  The efficacy of DNA vaccines, as an alternate to conventional immunization with protein-based vaccines, has been extensively investigated.73 Several investigators have used prime-boost strategy, which involves priming with the DNA vaccine followed by boosting with the protein. Immunization of mice with the DNA vaccine encoding rabbit ZP3 (aa residue 263–415) led to reduction in the fertility without any disturbance in the ovarian follicle development.74 In another study, mice were immunized with the DNA vaccine encoding mouse ZP3 along with the recombinant ZP3.75 Immunized animals showed higher reduction in the fertility as compared with the groups of mice immunized with either DNA vaccine or recombinant protein. A significant correlation between the normal follicular development and the inhibition of T-cell response in the animals immunized with a combination of ZP3 DNA vaccine and protein was observed.75 Using prime-boost strategy, our group showed that antibodies generated by the DNA vaccine encoding chimeric protein encompassing epitopes of human ZP3 and ZP4 significantly decreased the ZP3- and ZP4-induced acrosomal exocytosis in the capacitated human sperm.76 In context to design contraceptive vaccine for street dog population control, cDNA encoding dog ZP3 was cloned in a mammalian expression vector VR1020 downstream of the tissue plasminogen activator signal sequence under cytomegalovirus promoter. The antibodies generated by the DNA vaccine encoding dog ZP3 recognized the native dog ZP.77 Subsequently, the plasmid DNA encoding dog ZP3 was entrapped in the PLG microspheres as described previously.62 Two groups of mice (n = 5) were either immunized with the plasmid DNA or the plasmid DNA entrapped in microspheres. Mice immunized with the plasmid DNA received two injections of 100 μg DNA each on days 0 and 15 followed by E. coli-expressed recombinant dog ZP3 (20 μg per injection) booster on day 30. In contrast, mice immunized with the plasmid DNA entrapped in microsphere received a single injection of equivalent amount of the plasmid DNA (100 μg per animal) followed by protein boost on day 30. Immunization of mice with the microsphere led to the generation of antibodies against ZP3, which progressively increased till 182 days post-immunization (Fig. 3), whereas mice immunized with the plasmid DNA showed an increase in the antibody titer till day 45 post-immunization, which progressively declined (Fig. 3). The progressive increase in the antibody titer till 182 days following immunization with the plasmid DNA entrapped in microsphere could be owing to the slow release of the plasmid DNA, its improved stability inside the particle, and the adjuvant effect of the polymeric particles.

Figure 3.

 Immunogenicity of DNA vaccine encoding dog ZP glycoprotein-3 (ZP3) entrapped in microsphere: The construction of plasmid DNA encoding dog ZP3 has been previously described.77 A group of female BALB/c mice (n = 5) was immunized with 100 μg of plasmid DNA/injection/animal on days 0 and 15 followed by booster of E. coli-expressed recombinant dog ZP3 (20 μg per animal) on day 30. To study the role of microsphere in enhancing the antibody response, the above plasmid DNA was entrapped in poly d, L – lactide-co-glycolide microspheres, and another group (n = 5) of female BALB/c mice was immunized with a single dose of 100 μg DNA/mice followed by booster with the E. coli-expressed recombinant dog ZP3 (20 μg per animal) on day 30. The immunized animals were followed up for antibody titers against dog ZP3 by ELISA on days 28, 45, 75, 120, and 182. The values are expressed as mean ± S.E.M. of the antibody titer, which represent the reciprocal of dilution giving an absorbance of 1.0.


The efficacy of contraceptive vaccines based on ZP glycoproteins/proteins to curtail fertility in various animal species has been established beyond doubt by several investigators. For wildlife management, with special reference to the control of street dog population, the ovarian pathology accompanied by ZP immunization may not be a concern as spaying is the currently approved humane method to control their population. In fact, permanent sterility may be desirable to control street dog population. In that situation, even a vaccine not having 100% contraceptive efficacy may find utility in the management of street dog population. However, the most appropriate mode of vaccine delivery to target dog population remains to be addressed. This can be achieved either by formulating appropriate bait for oral vaccine delivery or by using a dart gun for remote vaccine delivery. For effective management of vaccine delivery systems, it would be highly desirable to reduce the number of injections to achieve contraception. Further, an additional interesting proposition would be to deliver ZP-based contraceptive vaccine along with the rabies vaccine in street dogs.

Corresponding Author

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Dr. Satish Kumar Gupta
Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110 067, India


Financial support from the National Institute of Immunology, New Delhi; Indian Immunologicals Limited, Hyderabad; Department of Biotechnology, Government of India; Indian Council of Medical Research, Government of India; and The Humane Society of United States, Washington DC, USA, is gratefully acknowledged.