Fig. 17.1
Schematic diagram showing various targets which are being investigated for contraceptive vaccine development. These can be divided broadly into three categories: targeting Gamete Production, Gamete Function, and Gamete Outcome. FSH Follicle stimulating hormone, GnRH Gonadotropin releasing hormone, hCG Human chorionic gonadotropin, LH Luteinizing hormone, LIF Leukemia inhibitory factor
17.2.1 Sperm Antigens
Various methodologies of genomics and proteomics have been used to delineate sperm antigens that have a role in fertilization/fertility and can be used for the contraceptive vaccine development. Recently, using gene knockout technology, >100 novel testis/sperm genes/proteins have been identified that have a crucial role in various aspects of fertility [50, 51]. Some of these gene knockouts cause a defect in testis development and endocrine milieu, some in spermatogenesis, some in mating behavior, some in sperm structure/function/motility, and others in fertilization. The majority of these knockouts also demonstrated an effect on nonreproductive organs concomitant with an effect on fertility. We did an extensive database analysis of these genes/proteins to examine how many of these have the characteristics required for the contraceptive vaccine development as discussed above. The knockouts of only a few genes/proteins induced a specific effect on fertility without a serious side effect. The majority of them are not expressed on the sperm surface, and thus not amenable to antibody binding. Although these can provide ideal targets for pharmacological inhibition for contraception, they are not suitable for contraceptive vaccine development. The gene knockout technology is a powerful approach to identify suitable novel targets and the list of gene knockout mice is ever growing.
17.2.1.1 Molecules Involved in Sperm-Oocyte Membrane Fusion
The molecules involved in sperm-oocyte membrane fusion have been actively examined for some time. Various candidates have been proposed that include DE, CD46, equatorin Sperad, and SAMP32 [78]. CD46 gene knockout mice do not show a defective sperm-oocyte fusion [23]. ADAM family proteins have drawn considerable attention because they have a putative fusion peptide (ADAM1) and disintegrin domains (ADAM2 and ADAM3) [60]. However, ADAM1, ADAM2, and ADAM3 gene knockout mice did not show a defect in sperm-oocyte membrane fusion, but show impairment in sperm-zona binding [10] CD9 present on the oocyte plasma membrane seems to be essential for fusion with the sperm cell [29]. It was thought that integrins α6 and β1 present on sperm are involved in binding to oocyte CD9 for sperm-oocyte fusion [3]. However, gene knockout of these molecules did not inhibit fertility [19].
Recently, a gene knockout was reported that is very interesting. The gene knockout mice of a sperm gene, designated as Izumo1, are healthy but all males are sterile [23, 24]. Izumo1 is named after a Japanese shrine dedicated to marriage. Male mice produce normal looking sperm that bind to and penetrate the zona pellucida but are incapable of fusing with the oocyte membrane. Izumo1 protein is a type-1, 377 amino acid long, transmembrane protein that belongs to the immunoglobulin super family (IgSF) and contains one extracellular immunoglobulin domain and one N-terminal domain. Izumo1 contains a glycosylation site that protects Izumo1 from fragmentation in the epididymis. This site, however, is not essential for normal function [25]. Mouse Izumo1 has a molecular identity of 56 kD and human Izumo1 is of 37 kD.
More recently, paralogues of Izumo1, the Izumo2, 3, and 4, have also been discovered in mammals [13]. Izumo2 and 3, along with 1, are testes-specific transmembrane proteins, while 4 is expressed in the testes and other nonreproductive tissues. They all have significant homology in the N-terminal domain [13]. All four Izumo1 genes have eight conserved cysteine residues within 144 amino acids with four alpha helices that exist between the residues. All Izumo1 genes, with the exception of Izumo3, are conserved across humans, mice, rats, bulls, dogs, and some species of monkeys [80]. Izumo1 protein can only be detected after the acrosome reaction. Recently, it was found that Izumo1 binds to Folr4, a folate receptor, on oocytes, designated as Juno [7].
Our laboratory was first to demonstrate that immunization with Izumo1 peptides causes long-term and reversible contraceptive effect in female mice [42]. We further demonstrated for the first time ever that immune infertile women and men have antibodies to Izumo1 [11, 44]. Subsequently, at least four studies have been published demonstrating the contraceptive effect after vaccination with Izumo1 [4, 73, 85, 86]. Izumo1 is an excellent candidate for antisperm contraceptive vaccine development.
17.2.1.2 Molecules Involved in Sperm-Zona Pellucida Interaction and Function
Several sperm genes/antigens have been delineated, cloned, and sequenced. Although, the antibodies to some of these antigens affect sperm function/fertilization in vitro, the immunization with only a few of them have shown to cause a contraceptive effect in vivo in any animal model. Notable among these are lactate dehydrogenase-C4 (LDH-C4) [15], PH-20 [67], SP-17 [28], SP-10 [21], FA-1 [34, 55, 56, 94], and YLP12 [45, 58]. Most of these active immunization studies, except related to PH-20 antigen, were carried out in the mouse model. At the present time, no sperm antigen has undergone Phase I/II clinical trial in humans. Two studies have examined the effect of sperm antigen vaccination in nonhuman primate model. One study reported reduced fertility of female baboons after immunization with LDH-C4 [62]. However, a study by another group found no effect on fertility in female monkeys after vaccination with LDH-C4 [82]. The reason for this discrepancy is not clear at the present time. Male monkeys were immunized with an epididymal protein, designated as epididymal protein inhibitor (Eppin) [64]. After immunization, 78 % monkeys that developed high anti-Eppin antibody titers became infertile, and 71 % of them recovered fertility after immunization was stopped. To maintain high antibody titers, booster injections with Freund’s adjuvant have to be given every 3 weeks for almost the whole duration of study of 691 days. The potential immunopathological effects of immunization were not examined. This interesting study indicates that antisperm CV can also be developed for men.
Antibodies to several sperm antigens inhibit sperm-oocyte interaction/fertilization in vitro. However, the active immunization with many of these molecules does not inhibit fertility in vivo. Also, the gene knockouts of many of these molecules do not inhibit fertility. For example, although antibodies to fertilin/PH-30 inhibit fertilization in vitro [78], active immunization with fertilin/PH-30 does not affect fertility in vivo [17]. Similarly, although antibodies to sperm integrins α6 and β1 inhibit sperm-oocyte fusion in vitro [3], the gene knockouts of these molecules do not affect fertility in vivo [19]. These differences in in vitro and in vivo effects may be because: (a) the class/subclass, valency, affinity, and kinetics of the antibodies generated in vivo and in vitro vary; (b) antibodies have to be present in time and space to bind to the appropriate molecules; and/or (c) there may be redundancy of some of these molecules.
Another problem that the sperm vaccinologists are facing at the present time is to find an appropriate animal model to examine the efficacy of a sperm antigen. The most used animal model is the mouse. However, up until now, no one has reported 100 % block in fertility after immunization with any single antigen in the mouse model. Even immunizations with the whole sperm or their solubilized preparations do not cause a total block in fertility in mice, male or female. The maximum reduction in fertility after immunization with any antigen/sperm preparation is up to 70–75 %. Very few, if any, knockout of a single gene has made mice totally infertile. The recently reported Izumo gene knockout did make the male mice almost totally infertile [23]. It needs to be seen whether or not the 70–75 % reduction in fertility in the mouse model translates to 100 % reduction in humans. The female mouse ovulates several eggs every cycle and a woman ovulates mostly one egg every cycle. So there are differences between the man and mouse. Over 70–75 % reduction in fertility in the mouse model may translate to 100 % block in humans. Maybe that is the inherent nature of the mouse model in which it is difficult to make mice completely infertile. However, after active immunization or deleting a single gene, one does find a few mice that are totally infertile. Vaccination with multiple sperm epitopes (peptide and DNA) enhances the efficacy but still does not cause 100 % contraceptive effect [40–42].
The phage display technology is a novel and innovative tool for delineating specific binding peptide sequences to various ligands and antibodies. It was first reported by George Smith in 1985 [77]. This technology is being widely used in several laboratories at the present time. The peptide sequences are presented on the surface of filamentous phage to examine their interaction with specific ligands/antibodies. The DNA encoding any peptide sequence gets incorporated into genome of the phage capsid protein and the encoded peptide is expressed and displayed on phage surface as fusion protein. We used this technology and the 2-D gel electrophoresis/matrix-assisted laser desorption mass spectrometry (MALDI MS) to delineate the peptide sequences that are involved in human immunoinfertility [5, 39, 66, 72] and the peptide sequences present in human sperm cell that are involved in binding to human zona pellucida [58].
Besides antibodies, various cytokines can also affect sperm function and fertility either positively or negatively. For example, interferon-gamma and tumor necrosis factor-alpha can negatively affect sperm motility and function [47] and interleukin-6 can enhance sperm capacitation and acrosome reaction [46]. A sperm cell has receptors for many of these cytokines such as interferon-gamma and interferon-alpha [57]. These factors are present in the seminal plasma and the levels are modulated to various degrees in infertility. Immunization with the whole sperm preparation or specific sperm antigens can raise many cytokines besides antibodies that can affect sperm function [48].
17.2.2 Passive Immunization and scFv Antibodies
The progress in the development of contraceptive vaccines against various targets including sperm has been hampered by the following facts: (1) Delineating the appropriate fertility-related antigen(s), (2) variability of the immune response among the vaccinated individuals, (3) attainment and maintenance of high titer of antibodies for bioefficacy, (4) time lag to achieve reasonably good antibody titers after the first injection, and (5) uncertainty regarding how long the antibody titer will remain in the circulation to exercise the contraceptive effects. The last four concerns are associated with the active immunization studies involving contraceptive vaccines. It is envisaged that these four concerns may be taken care of using the passive immunization approach [49]. The passive immunization approach has been successful for protection against various immunological and infectious diseases [9, 91].
Several of these antibodies have become treatment modalities in the clinics [9, 12, 69]. Phage display technology has been widely used to obtain a variety of engineered antibodies, including single chain variable fragments (scFv) antibodies against several antigens [65, 68, 89, 92, 93]. ScFv is an antibody fragment that plays a major role in the antigen-binding activity, and is composed of variable heavy (VH) and variable light (VL) chains connected by a peptide linker. The most widely used peptide linker is a repeat of a 15-residue sequence of glycine and serine (Gly4Ser)3. The affinity and stability of the scFv antibodies produced in bacteria are comparable with those of the native antibodies and are maintained by a strong disulfide bond. ScFv antibodies can be produced on a large scale using specially modified bacterial hosts and have an advantage over the whole immunoglobulin (Ig) molecule. ScFv antibodies lack the Fc portion that eliminates unwanted secondary effects associated with Fc, and due to its small size can be easily absorbed into tissues and gene manipulated [90]. The mouse monoclonal antibody can elicit strong anti-mouse antibody reaction, chimeric antibody can cause antichimeric response, and xenogenic complementarity-determining regions (CDRs) of humanized antibodies can also evoke an anti-idiotypic response, when injected into humans [27, 36, 74]. Antibodies must be of human origin to be used in humans. The potential poor immunogenicity and toxicity of an antigen, and ethical issues, limit immunizing humans to obtain human antibodies. However, the phage display technology can be used to obtain these antibodies against target antigens if they exist involuntarily in humans, such as ASA in immunoinfertile men and women, and vasectomized men.
We recently did a study to obtain fertility-related scFv human antibodies that can be used for CV immunoinfertility. Peripheral blood leukocytes (PBL) were obtained from antisperm antibody-positive immunoinfertile and vasectomized men, activated with human sperm antigens in vitro, and cDNA was prepared from their RNA and PCR-amplified using several primers based on all the available variable regions of VH and VL chains [44, 70]. The amplified VH and VL chains were ligated and the scFv repertoire was cloned into pCANTAB5E vector to create a human scFv antibody library. Panning of the library against specific antigens yielded several clones, and the four strongest reactive (designated as AFA-1, FAB-7, YLP20, and AS16) were selected for further analysis. These clones were shown to have novel sequences with unique complementarity determining regions (CDRs) when a search was performed in the immunogenetic database. ScFv antibodies were expressed, purified, and analyzed for human sperm reactivity and effects on human sperm function. AFA-1 and FAB-7 scFv antibodies, having IgG3 heavy and IgK3 light chains, recognized human sperm FA-1 antigen, which is involved in human sperm function and fertilization. The third, YLP20 scFv antibody, reacted with a sperm protein of 48 ± 5 kD, which contains the dodecamer sequence, YLPVGGLRIGG. The fourth antibody, AS16, reacted with a 18 kD sperm protein (major band) and was found to be a human homolog of the mouse monoclonal recombinant antisperm antibody (RASA) [61]. These antibodies inhibited human sperm capacitation/acrosome reaction in a concentration-dependent manner. This is the first study to report the use of phage display technology to obtain human antisperm scFv antibodies of defined antigen specificities from immune infertile/vasectomized men. These antibodies will find clinical applications in the development of novel immunocontraceptives and specific diagnostics for immunoinfertility in humans. The contraceptive effect of these antibodies in vivo is currently being investigated.
Conclusion
In conclusion, development of CV targeting sperm is an exciting proposition, and may provide a valuable alternative to the presently available methods. As limitation with other vaccines, the progress in CV development has been delayed due to variability of immune response after vaccination. The multi-epitope vaccines may enhance the efficacy and obliterate the concern of the interindividual variability of the response. Also, this concern may be addressed by the passive immunization approach using preformed human antibodies. Several antibodies are being tried as therapeutic agents. At the present time, >100 antibodies are in clinical trials and ~20 FDA-approved monoclonal antibodies are available in the market for various clinical conditions, including cancer and infectious diseases. Over 80 % of these antibodies are genetically engineered [22, 35]. The scFv antibodies that we have synthesized in vitro using cDNAs from antisperm antibody-positive immune infertile and vasectomized men may provide useful, once-a-month immunocontraceptive. These human antibodies are sperm-specific and inhibit sperm function in vitro. Their immunocontraceptive potential in vivo is presently being investigated. In a WHO meeting on contraception in Geneva, Switzerland, November 13–14, 2012, development of CVs was enlisted as one of the highest priorities in the contraceptive field. An International Task Force has been set for CV development [43]. This has heightened the interest in immunocontraception.
Acknowledgments
This work is supported by the NIH grant HD24425. The excellent typing assistance provided by Carly Lasure, B.S., is gratefully acknowledged.
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