Canine zona pellucida glycoprotein-3: up-scaled production, immunization strategy and its outcome on fertility.

Zona pellucida (ZP) glycoproteins based contraceptive vaccines have been proposed for the management of wildlife population. In the present study, a fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by a dilysine linker and an ectodomain of dog ZP3 (ZP3; aa residues 23-348) without any affinity tag (TT-KK-ZP3) has been expressed in Escherichia coli. The recombinant protein was successfully produced in fed-batch fermentor and purified. The average yield of purified refolded protein was 12.20 ± 0.61 mg/2g wet cell pellet. Female FvB/J mice immunized with the varying doses of recombinant TT-KK-ZP3 supplemented with alum/PetGel A as adjuvants following a three injection schedule, showed dose dependent increase in serum IgG titer. Antibodies against TT-KK-ZP3 recognized native mouse/dog ZP and significantly inhibited mouse in-vitro fertilization (p=0.012). Immunized mice showed significant reduction in fertility (p<0.05). Higher antibody titers were associated with a decrease in the number of pups born to the immunized female mice. To reduce the number of injections, two injection schedule using various dose combinations of TT-KK-ZP3 supplemented with alum revealed lower immunogenicity and contraceptive efficacy as compared to the three injection schedule. To overcome this, CpG motif was included in addition to alum and both intraperitoneal and intranasal route of immunization following the two injection schedule was investigated. Inclusion of CpG significantly enhanced the antibody titer and improved contraceptive efficacy. In the mice immunized following intraperitoneal route, serum/vaginal IgG and in the mice immunized through intranasal route, vaginal IgA seemed to be important for curtailment in fertility. To conclude, the recombinant protein described herein may be a good candidate for developing contraceptive vaccine for the wildlife population management, in particular street dogs.

Milestones in contraceptive vaccines development and hurdles in their application.

Contraceptive vaccines have been proposed for controlling the growing human population and wildlife population management. Multiple targets such as gonadotropin releasing hormone (GnRH), luteinizing hormone, follicle stimulating hormone, gonadotropin receptors, sperm-specific proteins and zona pellucida glycoproteins have been exploited to develop contraceptive vaccine and their efficacy investigated and shown in various experimental animal models. Vaccines based on GnRH have found application in immuno-castration of male pigs for prevention of boar-taint. Vaccines based on zona pellucida glycoproteins have shown promising results for population management of wild horses and white-tailed deer. Phase II clinical trials in women with ß-human chorionic gonadotropin (ß-hCG)-based contraceptive vaccine established proof of principle that these can be developed for human application. Block in fertility by ß-hCG contraceptive vaccine was reversible. Further research inputs are required to establish the safety of contraceptive vaccines, improve their immunogenicity and to develop novel vaccine delivery platforms for providing long lasting immunity.

Recent progress toward development of vaccines against conception.

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.

Expression of a recombinant human sperm-agglutinating mini-antibody in tobacco (Nicotiana tabacum L.).

The murine monoclonal antibody (mAB) S19 recognizes an N-linked carbohydrate antigen designated sperm agglutination antigen-1 (SAGA1) located on the membrane protein CD52. This antigen is added to the sperm surface during epididymal maturation. Binding of the S19 mAB to SAGA-1 causes the rapid agglutination of sperm and blocks pre-fertilization events. Previous studies indicated that the S19 mAB may be a potential specific spermicidal agent (termed a spermistatic) capable of replacing current spermicidal products that contain harsh detergents with harmful side effects. The nucleotide sequences encoding the heavy (H) and light (L) chains of the S19 antibody were cloned. A chimeric gene was constructed using the nucleotide sequences encoding the variable regions of both the H and L chains, and this gene (scFv1 9) was expressed in transgenic tobacco (Nicotiana tabacum L.) to produce a recombinant anti-sperm antibody (RASA). Highest levels of RASA expression were observed in BY-2 plant cell suspension cultures and regenerated N. tabacum cv. Xanthi plants transformant in which the RASA coding sequences were expressed under the control of the Cauliflower Mosaic Virus 35S promoter containing a double-enhancer sequence (2X CaMV 35S). Subsequent modifications of the transgene including the addition of a 5'-untranslated sequence from the tobacco etch virus (TEV leader sequence), N-terminal fusion of the coding region with an endoplasmic reticulum targeting signal of patatin (pat) and C-terminal fusion with the endoplasmic reticulum retention signal peptide KDEL showed further enhancement of RASA expression. The plant-expressed RASA formed intrachain disulfide bonds and was primarily soluble in the cytoplasmic fraction of the cells. Introduction of a poly-histidine (6xHIS) tag in the recombinant RASA protein allowed for rapid purification of the recombinant protein using Ni-NTA chromatography. Optimization of scale-up production and purification of this plant-derived recombinant protein should provide large quantities of an inexpensive spermistatic plantibody.