Special Programme of Research, Development and Research Training in Human Reproduction,
World Health Organization, 1211 Geneva 27, Switzerland
The methods of birth control currently available can be broadly classified into steroid hormone methods (oral pills, injectable preparations and implants), inert and medicated devices (IUDs), barrier methods (condoms and caps), chemical methods (spermicidal foams and gels), surgical methods (salpingectomy and vasectomy), and various combinations of these approaches. Whilst these methods have found wide acceptance in many parts of the world and have had a major impact in the family planning arena, the range of options is limited. In addition, many of the methods have drawbacks associated with them, such as concerns about the safety of their long-term use, inconvenience of use, unreliability, and permanence of effect, which limit their acceptance and availability in the widely differing social, cultural, religious, service and personal settings of users in many countries, particularly in the developing world.
Safe, effective and acceptable contraceptive vaccines may be an attractive addition to the currently available range of family planning methods in that they would:
- Confer long-term (but not permanent) protection following a single course of immunization.
- Be free of overt pharmacological activity and the metabolic and endocrine disturbances that often accompany other methods of birth control.
- Not require insertion of a device or implant.
- Remain effective without continuous conscious action by the user.
- Be inexpensive to manufacture.
Strictly speaking, the term contraceptive vaccines should be used only in connection with those vaccines which have an effect prior to fertilization. Over the years, however, this term has been used to describe all vaccines which have an antifertility effect, irrespective of whether this occurs prior to fertilization or after fertilization and prior to the completion of implantation of the blastocyst. In an attempt to avoid the confusion caused by this incorrect usage, the term fertility regulating vaccines (FRVs) has been introduced. This latter definition will be used from now on in this paper.
Options for FRV development
The early stages of mammalian reproduction comprise a complex series of events which, for convenience, can be divided into three stages.
- The production and transport of the male and female gametes (sperm and ova).
- The interaction of the gametes leading to fertilization.
- The process of implantation of the developing embryo into the uterine endometrium.
It has been clearly demonstrated, in a large number of studies carried out over the past two decades or more, that varying types and degrees of antifertility affects result from active and/or passive immunization against many of the functionally and structurally important molecules that are present during these three stages of the reproductive process (1,2,10,20). The antifertility effect can be produced through antibody-mediated neutralization of the biological actions of hypothalamic and pituitary hormones and inhibition of sperm enzyme activities, and through a variety of tissue-specific reactions involving both antibodies and immune cells.
However, many of these approaches would not be acceptable for clinical use since the immune responses elicited to some of these molecules, whilst having an antifertility effect, can produce endocrine and other metabolic disturbances and/or have the potential for eliciting immunopathology. Molecules for development into FRVs suitable for eventual use by men and women need, therefore, to be carefully selected.
Selection of molecules for FRV development
The most attractive candidates for FRV development are molecules which:
- When eliminated or neutralized by immunological means will result in a safe, effective and acceptable means of fertility regulation.
- Are restricted to the intended target cell or tissue.
- Are present in a site where a controlled immune response will not lead to immunopathology.
- Are present transiently and/or at low levels compared to the anticipated immune response.
- Will not elicit other undesirable immune responses.
- Can be chemically characterized and easily manufactured in large quantities and at low cost.
The molecules most closely satisfying these criteria are tissue-specific proteins expressed on, or produced by, the mature gametes and the trophectoderm of the preimplantation embryo. A large number of laboratory experiments and clinical studies have been conducted over the past two decades to evaluate the safety and efficacy of a number of prototype FRVs based on these molecules (1,2,10,20).
Current status of FRV development
Research on anti-sperm vaccines has focused, primarily, on two types of sperm antigens:
- Functional antigens such as the enzymes known (or suspected) to be required for sperm metabolism (lactic dehydrogenase-X) or involved in sperm-egg interactions and the processes leading to fertilization (acrosin and hyaluronidase).
- Structural antigens such as the molecules expressed on the sperm cell membrane and which may be involved in gamete interaction and fusion.
Varying degrees of fertility reduction have been observed in several animal species (mice and rats ~ 55%, rabbits ~ 70%, and baboons ~ 30%) actively immunized with mouse lactic dehydrogenase-X (LDH-X or LDH-C4) and with a synthetic peptide based on a portion of the molecule (7). Recent work on this antigen has progressed to the point at which a cDNA expression library, derived from human testis, has been screened with polyclonal and monoclonal antibodies raised to murine LDH-C4 (6). As a result of this research, the nucleotide sequence coding for human LDH-X has been deduced and engineered into an expression vector system (12). A vaccine containing the LDH-X antigen prepared in this way is currently being evaluated in a preliminary efficacy trial in baboons.
Although anti-acrosin antibodies have been shown to inhibit the action of this enzyme on substrates in vitro and that anti-hyaluronidase antibodies could inhibit rabbit in vitro fertilization, active immunization of rabbits and sheep with these two enzymes, either alone or in combination, did not result in a significant reduction in fertility (14). No major vaccine research programme involving these particular antigens is currently in progress.
Sperm membrane antigens.
The recent advances that have occurred in the field of molecular genetics and in the production of monoclonal antibodies (MAbs) now provide an opportunity to identify, isolate, and synthesize peptides, representing part or all of the primary structure of selected protein molecules, which can be subsequently evaluated as antifertility immunogens. Two sperm antigens identified in this way, SP-10 and PH-20, have been shown to have promising antifertility effects when injected into laboratory animals (3). Further antifertility studies with these antigens, in primates, are underway or planned.
Because of its crucial role in the gamete interactions leading to fertilization, the majority of the research directed at developing an anti-ovum vaccine has focused on the zona pellucida (ZP), the jelly-like glycoprotein coat surrounding the egg, as a source of potential candidate antigens.
Active or passive immunization with crude preparations of solubilized whole porcine ZP has been shown to reduce the fertility of the females of several species of laboratory animals including baboons (9). However, amenorrhea, of variable duration, was observed in the majority of the immunized baboons, suggesting possible intraovarian complications. Efforts are now underway to isolate antigens expressed on the surface of the ZP only during the peri- and post-ovulatory period and that are involved in sperm-ZP interactions. In addition, amino acid sequence data on some of the protein components of the ZP have also been obtained and mice immunized with a 16 amino acid peptide of one of the ZP proteins have produced anti-ZP antibodies and exhibited reversible infertility with no evidence of ovarian damage (13). To date, however, no convincing data have been presented to indicate that a zona-specific antigen of defined chemical structure can inhibit fertility without causing an inflammatory reaction in the ovary which might be indicative of a risk of acute ovarian disturbances or long-term immunopathology.
A number of placenta-specific antigens have been investigated as vaccine candidates. As with studies on sperm antigens, both structural antigens, forming part of the trophoblast cell membrane, and functional antigens, such as placental hormones, have been evaluated.
Structural placental antigens.
Pregnancy-specific ß1 glycoprotein (SP-1) is a placental protein which is rapidly secreted by the syncytiotrophoblast and can be detected by immunofluorescence in the cytoplasm and on the plasma membrane of this tissue (4). Although an antifertility effect was observed when female baboons and cynomolgus monkeys were actively immunized with human SP-1, in the majority of cases (50-80%), this effect was manifested as a late abortion. In contrast, no antifertility effect was observed in similarly treated rhesus monkeys, nor in rhesus monkeys immunized with rhesus SP-1, even in the presence of high titres of anti-SP-1 antibodies. Similar studies in baboons indicated that the rate of secretion of SP-1 in early pregnancy was probably too high to be neutralized by the antibody in the maternal circulation. Another placental antigen PP-5, unlike SP-1, is not found in the cytoplasm of trophoblast cells but appears to be an integral component of the cell membrane. It is present in a low concentration in trophoblast tissue, is not secreted into the maternal circulation and is very difficult to isolate from placental tissue. However, a small group of female monkeys was actively immunized with human PP-5 and a substantial reduction in fertility was shown. Attempts to extend these studies, by immunizing rhesus monkeys with rhesus PP-5, were thwarted by the major difficulties experienced in obtaining a sufficient number of rhesus placentae from which the protein is isolated (5).
To facilitate and accelerate definition of the chemical structure of the more promising candidate antigens, trophoblast-derived gene libraries are being screened with appropriate MAbs and PAbs to isolate and subsequently sequence the genes coding for these molecules. From the nucleotide sequence thus obtained, the primary structure of the antigen can be deduced and corresponding peptides synthesised for evaluation as candidates for vaccine development. Several candidate molecules have been identified in this way and are currently being evaluated for tissue-specificity and antifertility efficacy (3).
Hormonal placental antigens.
By far the greatest amount of work carried out over the past two decades has been concerned with the development and clinical testing of vaccines directed against the glycoprotein hormone, human chorionic gonadotrophin (hCG). The principal function of hCG, which is produced by the trophectoderm of the pre-implantation embryo within a few days of fertilization, appears to be the maintenance of the corpus luteum in the ovary thus ensuring its continued production of progesterone. Since progesterone is needed for the successful completion of implantation of the blastocyst, if the production or function of hCG can be inhibited immunologically, the corpus luteum would regress, its production of progesterone would decline and menstruation would occur at or about the expected time, thus mimicking the events that occur naturally in a non-conceptual cycle.
One type of anti-hCG vaccine, developed by the Population Council in New York and by the National Institute of Immunology (NII) in New Delhi, is based on the whole beta subunit of the hormone (ß-hCG) (21,22). The other type of anti-hCG vaccine, developed with support from the WHO Task Force on Vaccines for Fertility Regulation, is based on a portion (the carboxyterminal peptide or CTP) of the beta subunit of the hormone (ß-hCG-CTP) (15-18). The reason for these two different approaches to the development of anti-hCG vaccines is that in 1974 WHO opted for the theoretically safer approach of the CTP vaccine following demonstration of the crossreactivity of antibodies raised to the whole ß-subunit with human luteinizing hormone (hLH) and the concerns that this raised about possible ovulation inhibition, menstrual cycle disturbances, and potential immunopathology. Such antibodies are not raised by the CTP vaccine. Although recent data from the Population Council and NII clinical trials with their respective ß-hCG vaccines indicate no menstrual cycle disturbances and no affect on ovulation, the question of long-term immunopathological or other sequelae, if any, of the elicited crossreactive immunity to hLH, is still unresolved.
The Population Council ß-hCG vaccine has been tested in a Phase I clinical trial and has been reported to be immunogenic and free of short-term side-effects (22). The NII ß-hCG vaccine has been tested in both Phase I and Phase II clinical trials and has also been reported to be free of short-term side-effects as well as being effective in preventing pregnancies (21).
A Phase I clinical trial has been conducted with the ß-hCG-CTP vaccine (11). The antibody titres raised in this Phase I trial were estimated to be in excess of the level needed to neutralize the hCG in the maternal circulation at the time of implantation. A Phase II trial with this vaccine is scheduled to start in 1993 (8).
All of these anti-hCG vaccines require multiple injections to achieve and maintain levels of immunity that are considered effective. Studies are underway to develop various formulations of these vaccines that will provide long-acting protection following a single injection. Data obtained in preliminary studies with the ß-hCG-CTP vaccine incorporated in biodegradable polymers indicate that it may be possible, using this technology, to produce a vaccine offering one year of protection from a single administration (19).
Future prospects and needs
The single-injection version of the anti-hCG vaccine is likely to be the first FRV vaccine to enter wide-scale clinical use. However, further studies are underway to develop an optimized anti-hCG vaccine incorporating alternative hCG peptides, T-helper cell epitopes, new immunostimulants and delivery systems.
Other studies are addressing ways in which the variation in the magnitude and duration of individual immune responses elicited by vaccines can be controlled, and strategies are being developed to provide protection during the lag period following injection of the vaccine and the attainment of an effective level of immunity.
Although FRVs are designed to have a comparatively short duration of effect, of approximately 12 months, some individuals may use them repeatedly to receive protection for a period of several years. It is imperative, therefore, that long-term safety studies are carried out to determine the nature, extent and consequences of such long-term use. Appropriate tests of both structural and functional effects need to be conducted if crossreactive immunity is detected in non-target cells or tissues.
In addition, careful examination in a relevant animal model is needed of the effect, if any, on the mother and fetus/offspring if a pregnancy occurs in the presence of sub-effective immunity.
Mechanism of action, reversibility and choice
FRVs could act by preventing sperm production, by interfering with ovulation, by inhibiting fertilization, or by preventing implantation of the blastocyst. It is important that studies are carried out to clearly determine how each FRV works. By understanding their mechanisms of action, more efficient and predictable FRVs can be prepared and rational intervention strategies can be developed to reverse the effects of the FRVs on demand. In addition, the user would be able to be fully informed of the known or suspected mechanisms of action of FRVs so that he or she can choose a FRV that is compatible with their personal beliefs and needs.
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