Bulletin of the World Health Organization, 65 (6): 779-783 (1987)© World Health Organization 1987

Development of immunological methodsof fertility regulation*J. SPIELER1
A new approach to fertility regulation is the development of vaccines directedagainst human substances required for reproduction. Potential candidates for
immunological interference include reproductive hormones, ovum and sperm antigens, and antigens derivedfrom embryonic orfetal tissue. Several vaccines targetedat the beta chain of the human chorionic gonadotrophin molecule have reached theclinical trial stage in Australia, Finland, India and Sweden, and the preliminary
results are very encouraging. A prototype vaccine using the beta subunit of ovine
luteinizing hormone seems effective in female primates. Contradictory effects onspermatogenesis in male primates have been observed by different researchers usingfollicle-stimulating hormone as an antigen. Both this and gonadotrophin-releasing
hormone require further basic research. Immunological interference could also beaimed at sperm production or maturation, or at sperm-ovum interaction in thefemale reproductive tract. The best characterized sperm antigen to date is LDH-C4,
an isoenzyme of lactic dehydrogenase. The identification of new sperm antigens isbeing aided by monoclonal antibody techniques. Another approach is to detect, andthen mimic, the antisperm antibodiesfound in women and men with immunologicallymediated infertility. Research on ovum antigens is focusing on the zona pellucida(ZP); anti-ZP antibodies dramatically depress fertility in vitro and in vivo in avariety of species. Vaccines interfering with sperm function and fertilization could
be available for human testing by the early 1990s.

The world stands to gain immeasurably from the development of immunologicalmethods of fertility regulation. Birth control vaccines would not only enlarge the choice ofmethods open to men and women; they would offer specific advantages. For one thing, itshould prove possible to develop vaccines that are unlikely to disrupt the menstrual cycle
or cause the side-effects associated with hormonal contraceptives. Fertility-regulating
vaccines should be well accepted by users, given the general popularity of immunization.
Finally, there would be compelling advantages for service delivery because vaccines
would provide long-acting fertility regulation, could be administered by paramedical or
nonprofessional personnel, and could be integrated not only with family planning services
but with other health care programmes as well.
Fertility-regulating vaccines differ fundamentally from the vaccines long used in infectious disease control. The latter have the advantage of being directed against "foreign"and thus highly immunogenic pathogens. An antifertility vaccine must be capable of safely
and effectively inhibiting a human substance, which would need somehow to be renderedantigenic. A fertility-regulating vaccine, moreover, would have to produce and sustain
effective immunity in at least 95% of the vaccinated population, a level of protection rarelyachieved even with the most successful viral and bacterial vaccines. But while these challenges looked insuperable just a few years ago, recent advances in biotechnology-particularly in the fields of molecular biology, genetic engineering- and monoclonal antibody* A French translation of this article will appear in a later issue of the Bulletin.
Biologist, Research Division, Office of Population, United States Agency for International Development, Washington, DC
20523, USA. The views expressed in this article do not necessarily represent those of U.S.A.I.D.

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production- are bringing antifertility vaccines into the realm of the feasible.
In brief, a birth control vaccine acceptable for use in human beings must meet thefollowing criteria.
(1) The antigen must be unique to the reproductive target.
(2) The antigen must have a fertility-related function that can be blocked by antibody oris susceptible to cell-mediated immunity. Alternatively, the function should be locatedon a cell that can be lysed by complement.
(3) An acceptable level of effectiveness (,>95%) should be achieved by no more than
one or two injections for the primary immunization, with booster injections at intervals of
no less than six to 12 months.
(4) The vaccine must undergo sufficient testing in animals to ensure its safety for longterm use.
In addition, for the vaccine to be widely acceptable, its effects should be reversible, i.e.,the vaccine should not interfere with future childbearing at desired intervals.
Assuming that a suitable antigen is identified, a major obstacle to vaccine development
will be the lack of suitable animal models for evaluating efficacy and safety. Immunocontraception is a radical departure. No method of regulating fertility has ever before
rested on immunological principles, nor has any vaccine ever been directed towards theinhibition of a "self-like" component or secretion. Before a fertility-regulating vaccine
can be envisaged for widespread use in men or women, animal models must be found forthorough testing of toxicity, teratogenicity, effectiveness, duration and reversibility ofimmunity and specificity for the target.

POTENTIAL ANTIGENS FOR VACCINE DEVELOPMENT

The mammalian reproductive system is theoretically susceptible to immunological intervention at numerous points. Many reproductive hormones, as well as several antigens that
could be isolated from the ovum, sperm, embryonic tissue and fetal tissue, could becandidate targets.

Human chorionic gonadotrophin
A major candidate for vaccine development, and one on which research is the mostadvanced to date, is human chorionic gonadotrophin (hCG). This hormone, produced by
the embryo, is essential for the maintenance of early pregnancy. It is a glycoprotein witha relative molecular mass of approximately 38 000 and 30% carbohydrate content. ThehCG molecule consists of two chains, designated alpha and beta, whose structures havebeen established. The alpha chain of hCG is similar to the alpha chain of pituitaryhormones that control ovarian and testicular function. However, part of the beta chain ofhCG is unique to this hormone, and this has formed the basis of an attempt to develop avaccine for use in women. Two basic approaches are being followed. WHO's Special
Programme of Research, Development and Research Training in Human Reproduction(HRP), through its Task Force on Vaccines for Fertility Regulation, has been supporting
research on a synthetic vaccine directed against the last 37 amino acids of the C-terminalend of the beta hCG molecule. This is the end of the molecule that is unique to beta hCG.
The first phase of human testing (phaseI -safety, dose and immune response) of thisvaccine is currently under way at the Flinders Medical Centre in Australia with support
from WHO. Preliminary results indicate that antibody levels several-fold higher than thoseestimated to confer efficacy have been elicited with few or no side-effects.

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A second approach is being followed by the National Institute of Immunology, New
Delhi, and the Population Council, New York. This involves developing a vaccine against
the entire beta chain of the hCG molecule. Phase I clinical trials are currently being
conducted in five centres in India using three different beta hCG preparations, and asimilar phase I trial will begin shortly in Finland and Sweden with support from thePopulation Council. Eminent scientists from both the National Institute of Immunology
and the Population Council participate in meetings of the WHO Task Force Steering
Committee to help ensure interagency coordination of research in this area.
One important aspect of both approaches to developing an anti-hCG vaccine is the useof a carrier protein. Such a protein may be, for example, tetanus toxoid, diphtheria toxoid
or the beta chain of cholera toxin, and serves two purposes. The carrier enhances theimmunogenicity of the hCG antigen. At the same time, it confers immunoprophylaxis
against a disease of public health importance, which is particularly critical in lessdeveloped countries.

Reproductive hormones
A prototype vaccine using the beta subunit of ovine luteinizing hormone (oLH-(3)emulsified with Freund's complete adjuvant has been studied extensively in female primates by the Population Council. No evidence of acute or chronic health hazards wasobserved in this heterologous immunization model, although the vaccine did cause ashortened luteal phase with reduced progesterone levels. There was initial concern about
potential immunopathology as a result of cross-reactivity between circulating antibodies
and pituitary tissue, and immune-complex disease. These problems did not materialize inthis model; however, further studies using both homologous and heterologous immunization models are needed to dispel this concern. While the mechanism of action of thisapparently efficacious vaccine in animals has not been fully established, there is noevidence that immunized non-human primates become pregnant after mating. The Population Council has received approval from the US Food and Drug Administration toconduct phase I clinical trials for testing the immunogenicity of this vaccine in women. Inaddition, two of the beta hCG vaccine preparations currently undergoing phase I trials inIndia also include oLH antigens.
Other candidate hormones for vaccine development include follicle-stimulating hormone(FSH) and gonadotrophin-releasing hormone (GnRH). A vaccine against GnRH would
block ovulation in the female and spermatogenesis in the male. However, it may have tobe accompanied by estrogen/progesterone or androgen replacement therapy in the femaleand male, respectively. As for the anti-FSH vaccine, the underlying principle rests on theclassical concept according to which FSH alone is responsible for the control of spermatogenesis in man, while LH controls Leydig cell function and hormone production. Thus,suppression or inactivation of FSH should lead to azoospermia and infertility without
affecting testosterone levels and libido. Research being carried out in bonnet monkeys at
the Indian Institute of Science in Bangalore continues to provide encouraging results along
these lines. However, similar experiments conducted in rhesus monkeys at the Max Planck
Institute in Munster (Federal Republic of Germany) showed that animals rendered infertile
for several years experienced a spontaneous qualitative recovery of spermatogenesis evenin the presence of high FSH antibody titres. One possible explanation is that testosterone
alone can reinitiate spermatogenesis in the absence of FSH. Before vaccines of this nature
can be fully exploited, further basic information is needed on the precise role of FSH in
human spermatogenesis and on the possible immunopathological effects of anti-FSH andanti-GnRH antibodies.
As for vaccines against steroid hormones, there is no immediate role envisaged forthem in human fertility regulation. However, research on such antigens is likely to be of

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immense value in the fields of animal husbandry and veterinary medicine. It should also
provide much needed data on reproductive biology and basic immunology.

Sperm antigens
The development of a vaccine based on sperm antigens will require a great deal of basic
research. Interference with fertility could occur at several points: during sperm productionin the testes, during sperm maturation in the epididymis, or during interaction with the egg
in the female reproductive tract. Selective inactivation of those sperm components that
actually participate in the process of fertilization would appear to be the best approach,thus providing a truly contraceptive vaccine. Whether vaccines involving sperm antigens
would be more appropriately used in women or men remains an open question at this time.
However, as it appears most likely that their use in women would involve the fewest complications and potential hazards, the best approach would probably be active immunizationof the female or a passive local delivery such as vaginal administration of the anti-spermantibody.
It is estimated that only about 1 % of the proteins in sperm have been identified thus far.
Among these there are very few sperm surface proteins, and not all of them are yet known
to act as potential contraceptive antigens. The defined antigens in sperm include LDH-C4,protamine, acrosin, hyaluronidase, and some plasma membrane antigens and differentiation products. With the exception of LDH-C4, they are not readily accessible to antibody attack because they are not expressed on the surface of the sperm. Thus, unless hightitres of genital tract immunoglobulin can be achieved, these antigens are not goodcandidates for vaccine development.
The best characterized sperm antigen is LDH-C4, an isoenzyme of lactic dehydrogenase
found only in male germ cells. Although this enzyme primarily occurs intracellularly,
antibodies to it react with the surface of intact sperm. Antibodies to LDH-C4 have been
produced in a variety of species and active immunization has reduced but not completely
blocked fertility in mice, rabbits and baboons. Antibodies probably inhibit fertility bycausing sperm agglutination. The structure of LDH-C4 has been determined, and vaccines
based on polypeptide fragments of the molecule have likewise been shown to inhibit
fertility. Recent studies have been directed towards the development of a synthetic peptidethat would elicit an immune response to the native protein. Research has also focusedon stimulating the secretory IgG system in primates to ensure high antibody titres in thegenital tract.
.The identification of new sperm-specific antigens has been aided tremendously by theuse of monoclonal antibody (MAb) techniques. Some MAbs to specific regions of sperm
have been prepared that are being used in turn to identify new antigens. This work could
lead to a breakthrough in the sperm vaccine field by facilitating the characterization ofsperm surface antigens that would be readily accessible to antibodies present in the female
reproductive tract.
In June 1986 WHO and Family Health International, North Carolina, USA, cosponsored a workshop that involved the prior worldwide distribution and testing of a codedpanel of 67 mouse MAbs alleged to react with human sperm. The workshop, attended byinvestigators active in the field, led to a consensus on the feasibility of using MAbs toidentify reproduction-specific antigens that might represent suitable candidates for thedevelopment of antifertility vaccines. Five anti-sperm MAbs demonstrated sufficient
specificity to warrant further investigation as reagents for identifying potential vaccines.
Another approach to the identification of sperm antigens is the evaluation of serum
samples obtained from women and men considered to have immunologically mediatedinfertility. This approach provides information concerning naturally (spontaneously)
occurring antibodies that could perhaps be intentionally mimicked in fertile individuals.

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Aside from their association with infertility, circulating antibodies in immunologicallyinfertile individuals do not appear to cause any health hazards.
From 1974 to 1981 the WHO Task Force supported a reference serum bank for reproductive immunology at the University of Aarhus, Denmark. Collaborating investigatorsfrom around the world submitted sera from infertile couples in the hope of better characterizing the immunological factors contributing to infertility. Unfortunately, this approachto antigen identification for selecting potential vaccine candidates has not been as rewarding as anticipated; many of the test procedures used for characterizing and categorizing thesera gave inconsistent results. Recent advances in biotechnology may begin to render thisapproach more promising.

Ovum antigens
While the initial stages of oogenesis take place during fetal life, oocytes only undergo
final maturation in women shortly before ovulation. These final maturation stages may be
associated with the production of specialized antigenic substances that could be suitabletargets for immunocontraception in women. Antibodies directed to the non-fertilized egg
in the ovary or in the reproductive tract could prevent fertilization. Although there may be
specific antigens associated with the oocyte itself, no such antigens have yet been isolated
and purified.
Most research on ovum antigens is directed towards the zona pellucida (ZP), theacellular, gelatinous layer surrounding the ovum. Several research groups have recently
made considerable progress in the biochemical characterization of unique glycoproteins
constituting the ZP. Both in vitro and in vivo tests following active and passive immunization have shown that antibodies to ZP can have dramatic effects on fertility in a varietyof species. The immunological response to ZP antigens is remarkably consistent between
individual animals and between species. However, there have been indications in somespecies that anti-ZP antibodies also alter ovarian function and ovum development.
Whether this will occur in all primates and in women is not known; more detailed studies
using purified zona antigens are clearly needed to determine the respective mechanism(s)of action of immunization with different ZP antigens.
CONCLUSIONS

Multidisciplinary research and collaboration between interested investigators, institutions and funding agencies are essential if the formidable challenge of developing birthcontrol vaccines is to be met successfully in the near future. Research is needed to identify
additional antigens, particularly those that will exert an immunocontraceptive effect, andto develop methods of making available large quantities of the purified antigens selected
for further study.
Given the predictable variation in individual responses to immunization with fertilityregulating vaccines, research is also needed in the field of "basic vaccinology", to findthe best carrier proteins, adjuvants, vehicles and delivery systems. Such research will also
have spin-off benefits for the development of vaccines against communicable diseases.
Within the next 12 months information will be available on the results of current andplanned clinical trials of several different vaccines directed against human chorionicgonadotrophin. It is estimated that vaccines that interfere with sperm function and fertilization should be available for human testing by the early 1990s.