Contraceptive vaccines: a journey with few milestones and some hurdles

SK Gupta1, A Shrestha1, V Minhas1, N Gupta1, S Srichandan2, AK Panda2

1Reproductive Cell Biology Laboratory; 2Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India

To control growing global human population, contraceptive vaccines have been proposed. These will also have utility in the management of wildlife population of some species that may have direct relevance to humans, in particular those involved in spread of zoonotic diseases. The current challenges in developing contraceptive vaccines for human application involves, (i) achieving 100% contraceptive efficacy, (ii) protective antibody titres in all the recipients for a definite duration, and (iii) reversibility. In spite of these shortcomings, these vaccines can be used in the population management of wildlife. Keeping this in view, recently our group has cloned and expressed in E. coli recombinant dog zona pellucida glycoprotein-3 as a fusion protein with promiscuous T cell epitope of tetanus toxoid (TT) with a dilysine spacer (TT-KK-dZP3) to avoid chemical conjugation with the carrier. The protein was expressed without any tag and the procedure to upscale its expression in fermentor and purification optimized. Immunization of female mice (FvB/J) with the above recombinant protein given along with the synthetic polymer Montanide™ PetGel A (adjuvant approved for animal use) or alum led to a significant reduction in fertility. To reduce the number of injections and generate long lasting immune response TT-KK-dZP3 has been entrapped in the microsphere particles made up of polylactide (PLA). Active immunization studies with microsphere particles are ongoing. Recombinant dog ZP3 as a fusion protein along with dog Izumo has also been expressed, its contraceptive efficacy tested in mice. Further porcine ZP3 with or without T cell epitope of TT and ZP4 with or without promiscuous T cell epitope of bovine RNase have also been expressed in E. coli. Immunization of female mice with all the above 4 recombinant proteins led to generation of high antibody titres as well as T cell responses. The polyclonal antibodies against recombinant porcine ZP3/ZP4 also inhibited in vitro fertilization using mouse eggs and sperm. Significant curtailment of fertility was observed in female mice immunized with recombinant porcine zona proteins. In addition, recombinant fusion protein comprising of mouse Sp17 and equatorin as well as individually have been produced in E. coli and their contraceptive efficacy will be presented at the conference. To conclude, it is desirable to develop immunization strategies using permissible potent adjuvants that can generate protective antibody titres in 100% of the recipient, if contraceptive vaccines have to be used for human fertility regulation. Easy use of vaccine delivery approaches will be one of the key factors for using contraceptive vaccine for the management of wildlife population.

Developing a polyvalent chimeric peptide contraceptive vaccine

F Xue1,2,3, Y Liu1,2, L Wang1,2, H Tang4, W Xu4, C Xu1,2

1Department of Histology and Embryology, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2Shanghai Key Laboratory of Reproductive Medicine, Shanghai, China; 3Department of Dermatology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China; 4Department of Reproductive Biology, Shanghai Institute of Planned Parenthood Research, Shanghai, China

The current methods of contraception (e.g. surgery and oral drugs) lack specificity and have various side effects. A more specific and safe approach of contraception needs to be developed. Contraceptive vaccines can provide the most desired characteristics of an ideal contraceptive. The concept of vaccine is widely accepted, for its effect can last for a long time and is easy to reverse. Unlike other contraception drugs, contraceptive vaccine consists of non-pharmacological substances and does not affect the endocrine system, thus having no interference with sexual response or sexual functionality. In addition, it costs reasonably and is suitable for mass production. As early as 1999, therefore, Human Reproduction Programme (HRP) of the World Health Organization (WHO) recommended this method as a high priority for research and development. Now, it has become an international hot-spot and cutting-edge of research, which is expected to become the safest, most effective, and reversible approach to contraception.

Briefly, the aim of the current research was to target factors involved in the establishment of pregnancy. Among those essential and pregnancy-specific factors, our group has identified several targets on sperms required for egg-sperm interaction and fertilization. After systemic and long-term investigation, we found that infection of Ureaplasma urealyticum (Uu) was closely associated with male infertility. We revealed that Uu and human sperm membrane shared cross-reactivity antigen and that the cross-reactivity antigen was the UreG protein of Uu and testicular type human nuclear autoantigenic sperm protein (tNASP). Two sperm specific proteins, i.e. Izumo and SPESP (sperm equatorial segment the protein) were identified to show cross-reactivity using bioinformatics screen of Swiss-Prot database. Consequently, antibodies targeting the N-terminal of SPESP, Ig-like domains of Izumo, and the 6th exon of tNASP produced obvious and reversible anti-fertility effect without any side effects. Our findings proved that tNASP, Izumo and SPESP are unique and exciting molecules on the sperm for developing contraceptive vaccine.

Since contraceptive effect can be significantly improved by polyvalent chimera peptide vaccine by combination of different epitopes, we sought to establish a polyvalent chimeric peptide vaccine consisting of Izumo, SPESP and tNASP. Therefore, further work is required to characterize the B-cell epitope (BCE) and T-cell epitope (TCE) of each protein to develop a chimeric peptide vaccine. In the current study, we found that one of five natural B-cell epitopes (BCE) of Izumo Ig-like domain developed an outstanding anti-fertility effect. Additionally, it presented specific fusion ability to the egg membrane in vivo and in vitro. This particular BCE showed unique expression in sperm and was highly conserved across the species. Meanwhile, we combined different epitopes of Izumo, tNASP and SPESP to design a chimeric peptide vaccine, and evaluated its contraceptive effect. Collectively, these studies will provide a solid foundation for developing a successful polyvalent sperm chimera peptide immunocontraceptive vaccine in the near future.

Developing a contraceptive vaccine based on epididymal protease inhibitor (Eppin): Immunological contraception has become an important study topic due to its good social and economic values. Up to now, there is still no ideal contraceptive vaccine, which may be probably due to the lack of ideal target antigen. Eppin was secreted by the epididymidis/testis, associated with the sperm surface during epididymal transit, and bound to semenogelin in semen. re-Eppin shows the capacity to block fertility. In order to further develop contraceptive vaccine based on Eppin, several researches are performing, such as (1) Using the bioinformation method to predict the possible B-cell epitopes, screening and identifying the dominant epitopes by anti-fertility assay in vivo and sperm-egg binding assay in vitro, eight peptides can elicit similar specific antibody titer, but only two peptides significantly decrease the rate of pregnancy in mice. (2) Investigating the crystal structure of Eppin by expression, purification the soluble protein in prokaryotic and eukaryotic system, crystallization for X ray diffraction. (3) Trying the new self-assembled peptide nanofibers delivery system and microneedle-based transcutaneous immunization in mice with E-Q11 self-assembled peptide was performed. (4) Optimal designing the peptide vaccine and the anti-fertility test in primates animal will start in the further.

Induction of endogenous miR223 expression by sperm in the female reproductive tract following mating in mice

JE Schjenken, SA Robertson

Robinson Institute and School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, SA, Australia

Seminal fluid interacts with epithelial cells lining the female reproductive tract to induce pro-inflammatory cytokines and chemokines, in an inflammation-like response that initiates the maternal immune adaptations required for tolerance of paternal antigens and pregnancy progression. Soluble factors in seminal plasma including TGFB are identified as key signalling agents, but these don't fully account for the female response, since our recent microarray studies show substantial differences in female tract gene expression depending on the presence of sperm in the ejaculate. Various bioactive molecules regulate the progression and quality of the female immune response, potentially including microRNAs (miRNA). We therefore hypothesized that miRNAs induced by sperm contribute to regulating gene expression in the female tract following insemination. CBAF1 female mice were mated with intact or vasectomized Balb/c males, and sacrificed at 8 h post-coitum (pc) and compared with unmated estrus mice (n = 16 mice per group). Total RNA (including miRNA) was extracted using the miRNeasy kit (Qiagen) and miRNA or gene expression profiles were examined by microarray and qRT-PCR. Analysis of endometrial miRNA array data showed that 34 miRNAs were differentially regulated (>1.5 fold) in endometrial tissue after mating with intact males compared to vasectomized males. Amongst these miRNAs we focused on miR223, which is involved in the regulation of inflammatory cytokine expression and controlling macrophage and neutrophil recruitment and activation. Taqman miRNA assay confirmed the differential expression of this miRNA in endometrium at 8 h pc, showing a 3.38-fold increase (P < 0.01) after intact compared to vasectomized mating. Analysis of miR223 expression over the course of the peri-conceptional period showed that miR223 expression was maximal at 8 h pc following exposure to sperm, while induction was delayed and diminished in females exposed to seminal plasma alone (n ≥ 4 mice per group, at 8, 16, 36, 60 and 84 h pc). In situ hybridization revealed that luminal and glandular epithelial cells are the primary site of miR223 expression. As sperm carry miR223, we then used mice with a null mutation in miR223 to examine whether the observed increase in miR223 was due to deposition of sperm-borne miR223. No miR223 expression was detected in either the unmated controls or miR223 null females mated with Balb/c males, while miR223 null males were comparable to wild-type C57Bl/6 males in their capacity to induce endometrial miR223 expression in CBAF1 females (n ≥ 4 mice per group). This indicates that the observed increase in miR223 is not due to transmission of sperm-derived miR223 but induction of endogenous expression in the female tract. Additionally, analysis of putative immune targets of miR223 including Nfat5, Acvr2a and Zxdc in the endometrium showed that these were regulated in the expected manner, suggesting that these genes are targeted by miR223 to regulate inflammatory cytokines and leukocytes during the peri-conceptional period. These data suggest that exposure to sperm upregulates miR223 in the endometrium, and that miR223 may act as a novel regulator of the female immune response required for embryo implantation. Future studies will examine the effect of miR223 deficiency on immune cell profiles during the peri-conceptional period and implantation success.

B-cell epitope map of human zona pellucida glycoprotein-4

W Xu1, J Wang1, Y He1, H Tang1, SK Gupta2, S Gu3, C-N Ji3, Y Xie3

1National Population and Family Planning Key Laboratory of Contraceptives Drugstore & Devices, Shanghai Institute of Planned Parenthood Research, Shanghai, China; 2Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India; 3State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai, China

Background: An ideal synthetic peptide immunogen should encompass relevant antigenic epitopes of target protein to enhance its efficacy. Thus, it is imperative to map all linear B cell epitopes (BCE) on a given target antigen. The aim of the current study was to report the fine epitope map of human zona pellucida glycoprotein-4 (huZP4) mapped using sera to huZP4N58–234 and huZP4C227–463.

Methods: Recombinant huZP4N and huZP4C were expressed in E. coli and polyclonal antisera rose against the purified respective recombinant protein in rabbits. Two sets of 18/8mer peptides fused with truncated GST188 protein as carrier were expressed in E. coli, which overlapped each other by 10/7 residues and spanning huZP4 protein sequence58–463. Each bacterial total protein containing 18 or 8mer peptide was used as antigen in Western blotting for epitope mapping.

Results: Eighteen of fine BCEs on huZP4 protein were mapped, which were HYIM102–105, APDTDW137–142, WCDSI142–146, SIPARDR145–151, YYGN180–183, RAVYEN252–257, LVAT259–262, FTLPPP301–306, PLTLELQ314–320, DKNYGSY324–330, YYGVGDYP330–337, HRTDPYL354–360, TDPLS373–377, DNYQTQ393–398, QLIPV398–402, LDLPF406–410, SFVNPTVE423–430 and PDLS460–463, but did not include an epitope ENELV256–260 mapped using a monoclonal antibody MA-1662. Sixteen epitopes in the epitome of huZP4 were found to be 100% conservative, and two epitopes were highly conservative in bonnet monkey by aligning the sequence of human and bonnet monkey ZP4. In mouse, rabbit and pig the number of 100% conservative epitopes were one, three and four epitopes, respectively.

Conclusions: Decoding of the whole epitome on huZP4 will provide more candidate epitopes without ZP-specific oophoritogenic T cell epitopes, thus facilitating the designing of polyvalent-peptide-based contraceptive vaccine. The conservation analysis of whole huZP4 epitome and six identified fine epitopes on huZP3 among their homologous proteins provides further evidence at epitope level to choose bonnet monkey as an animal model for testing efficacy of ZP vaccine.

The current status of contraceptive vaccines

RK Naz

Reproductive Immunology and Molecular Biology Laboratories, Department of Obstetrics and Gynecology, West Virginia University, School of Medicine, Morgantown, West Virginia, USA

In spite of availability of several methods of contraception, the population explosion and unintended pregnancies continue to pose major health issues worldwide. A better method of contraception that is acceptable in developed as well as developing countries is urgently needed. Contraceptive vaccines (CVs) can fulfill most of the properties of an ideal contraceptive. We will review the targets that are being explored for immunocontraception. The status of CVs targeting gamete production/function/outcome for control of farm/feral/stray/domestic animal populations, and for human use will be described. The presentation will specifically focus on the current status and future perspective of vaccines based on leukemia inhibitory factor (LIF) and hCG. Also, it will be discussed how novel and emerging technologies can expedite the development of CVs.