1 Introduction

Criteria for ideal contraceptives
a. effectiveness
b. practicability
c. safety
d. acceptability
e. microbicide against infections

Nearly 100 years ago, scientists learned that spermatozoa could provoke an immune reaction if they were injected into the body. Efforts at translating this information into a contraceptive vaccine began in the mid-1960s and focused on inducing an immune reaction to sperm. In the mid-1970s, researchers realized that human chorionic gonadotropin (hCG) would also be a good candidate for a contraceptive vaccine, since this hormone is produced only during early pregnancy. After more than 20 years of research, though, the first small-scale human trial of an anti-hCG vaccine showed that nearly 20% of the women who received injections failed to develop an effective immune reaction^[1-2]. Research continues on two anti-hCG vaccines, one  created by India's National Institute of Immunology and one produced by WHO^[3].

Meanwhile, other researchers continue to explore the feasibility of developing vaccines targeting a variety of proteins on the surface of the egg or on the surface of the sperm, in the belief that if either gamete could be coated with antibodies, fertilization would not take place. Another potential vaccine is aimed at disrupting a chemical that assists in the fusion of the sperm and egg. Scientists have found it harder than expected to identify molecules peculiar to the gametes or the fertilization process, however, a necessary step so as not to provoke an autoimmune reaction among similar molecules elsewhere in the body^[4].

Although evidence for auto- or allo-immune response against sperm antigens that cause infertility in some men and women has become definite^[5], the terms of immunoprophylaxis and contraceptive vaccine have unambiguously been used in the development of immunocontraceptives^[3].This approach, of course, bring some best to science but we should be careful in any practical trials. First, conception was (and still is) not infection caused by microorganisms, like smallpox, and will never. Second, reproductive process is an evolutionary result of life, in which a highly coordinating mechanism between genetic system and immune system is involved. Third, one should be awareness of that teaching ones body to provoke an immune response against a self-like molecule is dangerous, especially in the era of HIV epidemic. In contrast, one should seek some beneficial methods to protect and improve our immune systems. In general, current vaccine approach to fertility regulation substantially contravene the evolutionary principles.

It is not surprising that there are controversies surrounding anti-fertility vaccines, focusing on the anti-hCG vaccine^[6]. It reflects on the role of women's health advocating in contraceptive technology development, and the responses of researchers to their actions. Such controversy is also held by some top leaders in the contraceptive research and development program. The immunocontraceptive area is one that we've really cut back on, Gabelnick says, Not knowing what we would get, we stopped doing new projects. Those skeptical words sound rational to most scientists and public but might be harmful to researches toward better understanding and manipulating our reproductive process. But others show their positive responses to immunocontraception and are looking for the window in which it will kill sperm and prevent infection without toxicity effects on vaginal tissue^[7].

The question may arise of what we are doing here, since we have never made a contraceptive vaccine and have no results to present. The reason is that two of us [Y.F. Wang and Y.F. Huang] proposed independently that if there be a method to interfere or eliminate the immunosuppressive factors in human semen, we might find out a good way toward ideal contraception^[8-9]. Importantly, one of us N.Q. L in early 1990s, had been thinking about hapten-carrier phenomena^[10,11], following, of course, Baruj Benacerrafs work^[12]. Central to Ls thinking in his hidden-epitope recognition model was that the nature of the immune T-cells memory is the recognition of the hidden internal structure of the same antigenic determinant as B-cell does, which reflects the restricted recognition of the immune system to a foreign molecule. The most important role of this thinking may be to interpret mysterious hapten-carrier phenomena in rather precise and sound terms. Since current immunological approaches to fertility regulation has been heavily based on the recognition of self-like molecules, and the classical hapten-carrier system is ogten employed, interactions of our thoughts about the development of immunocontraceptive vaccines and the advent of new techniques might open the door for reconsideration of our previous hypothesis and we want a place where we could look things up.

In general, whenever we thought of those complicated biological problems, we did remember what the genetist Theodosius Dobzhansky said, events in biology will be of significance only if one thinks it from an evolutionary view [Scientific American (1985) 253: 4, August].  We also appreciated what one of us [Y.F. Wang] put it some time ago, immunoinfertility in some men and women could be considered as the results of contraceptive experiments God conducts in human beings, if you do understand the implying mechanisms, you might find a good way to manage ones fertility process.

On present evidence, therefore, it would be worthwhile among others to search for future contraceptive candidates in immunoglobulins rather than vaccines. Since vaccines are usually directed against foreign microorganisms or their products, antifertility vaccines will be directed against self-like molecules^[3]. Therefore, vaccine approaches to fertility regulation will certainly increase the difficulties in all aspects the vaccine would raise, such as the social, ethical, legal and regulatory issues. We do not intend to argue the feasibility of such antifertility vaccines approaches. What we want to do is just to discover the applications of anti-semen antibodies and to use them. That will simplify the problems.

2 Immunoglobulin-based passive immunocontraception

3 Advantages of passive immunocontraception

Passive immunization, the administration of exogenously produced antibodies raised against a reproductive substance, has until recently been limited to the use of sera derived from non-human species^[14]. In the case of such application to birth control in humans, the repetitive use of animal sera has been considered hazardous because of possible reactions to foreign proteins. However, the recent development of new technology for producing human immunoglobulins in vitro from transgenic technology and genetic vaccine techonology^[15] opens the door for reconsideration of passive immunization as a safe method of immunological birth control.

Passive immunization procedures have the advantage of allowing control over the nature of antibodies employed and duration of use of the method. The major disadvantage is that the duration of effectiveness from a single application is usually only a few days. However, following advantages will make it most attractive to both scientists and individuals.

3.1 Immunoglobulins against a sperm component of fertility specific can be shown to completely block the fertility of experimental animals, e.g, fertilization protein-1 (FA-1)

Advances in sperm-based-immunocontraceptive approaches are critically reviewed by Naz recently^[5]. FA-1 has been purified and characterized from murine and human sperm and testis using an MCA that completely blocks murine IVF and human SPA. FA-1 is a glycoprotein. It exists as both a dimer (51.2 kDa) as well as monomer (23 kDa). FA-1 develops in the testis during later stages (secondary spermatocyte onward) of spermatogenesis. FA-1 is an evolutionarily conserved antigen present in sperm of various mammalian species including mouse, rabbit, bull, rhesus monkey, and humans. FA-1 seems to have the similar function in these species. Anti-FA-1 MCA completely blocked IVF in these species. Also, the active immunization of female rabbits with purified FA-1 caused a significant reduction (up to complete block) in fertility.

The mechanism by which the anti-FA-1 antibodies inhibit fertilization is by affecting sperm-zona interaction. The human FA-1 binds to purified ZP3 of porcine zona pellucida in enzyme-linked immunosorbent assay (ELISA) and Western blotting, and completely neutralizes its sperm ligand activity in boar sperm-porcine zonapellucida attachment bioassay. This is an interesting finding because antibodies to porcine ZP3 antibodies have been shown to cross-react with human zona pellucida. The antibodies to FA-1 inhibit human sperm-human zona interaction, reinforcing the concept that FA-1 is involved in sperm-zona pellucida-ligand interaction. However, in the heterologous assay, the FA-1 MCA completely blocks SPA. The SPA has been reported to be an indirect measure of capacitation. Thus it is conceivable that these antibodies are directed to an important sperm component (enzymatic or non-enzymatic) that is vital to capacitation. In fact, the immunoaffinity-purified FA-1 MCA inhibits acrosome reaction of human sperm cells in solution (but not on the zona pellucida surface), suggesting a mechanism through which the antibody can inhibit the human sperm penetration of zona-free hamster oocytes. Recently, Naz and colleagues found that the purified FA-1 antigen completely blocks human sperm-human zona binding in the hemizona assay. Taken together, these findings suggest that FA-1 antigen may be a human sperm receptor (ligand) for the zona pellucida, that also has a role in sperm cell capacitation and/or acrosome reaction. It is interesting to find that FA-1 antigen has an autophosphorylating activity and is tyrosine phosphorylated during human sperm capacitation/acrosome reaction. Tyrosine phosphorylation of FA-1 antigen seems to have a vital role in capacitation/acrosome reaction and in zona pellucida binding.

3.2 Immunoglobulins against sperm developed in the majority of men after vasectomy can not affect the health of the individual

FA-1 antigen is involved in human immunoinfertility (both men and women)^[5]. The available data indicate that the anti-FA-1 antibodies that are present in vasectomized men and in infertile patients may play a causal rather than an associated casual role in infertility. Involvement of an antigen in human immunoinfertility indicates: a) its immunogenicity (auto- as well as iso-) in humans, and b) potential of its antibodies in causing infertility if a sufficient antibody titer is present. Since most infertile men are healthy individuals without any disease concomitant with infertility, the presence of antibodies to an antigen, is indicative, though not confirmative of its sperm specificity in humans. Thus, the involvement of FA-1 in immunoinfertility indirectly indicates its sperm-specificity, and auto- as well as isoantigenic potentials in humans. Thus, if an antigen, such as FA-1 is involved in human immunoinfertility, the extensive phase I clinical trials to investigate its toxicity in actively-immunized subjects may not be absolutely necessary.

3.3 Immunoglobulins per se do not prepare host to provoke both humoral and cellular immune response against target molecules

It has been demonstrated by those classical experiments that passive immunization of animals with antibodies does not prepare the animals to provoke both humoral and cellular immune response against the corresponding antigen^[10-11]. Therefore, the use of immunoglobulin-based contraceptives will not stimulate individuals immune response against sperm antigens at all.

3.4 Immunoglobulins could neutralize sperms ejaculated into virgina without activating complement system if the immunoglobulins are chicken immunoglobulins

A major concern is then focused on if the immune complex of sperm-antibody could activate females complement factors. Recent data show that chicken antibodies in their reactions with mammalian constituents do not activate the mammalian complement system [http://www.immunsystem.se/Advantages.html: IgY does not activate complement factors. Updated 13 August 1998].

3.5 Immunoglobulins against HIV/STDs infectious organisms could be produced by hens and involved in the contraceptive formula

As an extra advantage, immunoglobulins against HIV/STDs could be produced by hens in large amount and cost-effectively. In addition, chicken is one of the most infection resistant animals as far as we know. Therefore, use of the combination of immunglobulins against both semen and HIV/STDs as contraceptives will provide women and men with good protection for birth control and infection.

All women, by virtue of their cycles, face some level of immunosuppression in reproductive tract^[16-17]. Data suggest that repeated exposure to semen will facilitate the transmission of virus and establishement of infection when all depositions of semen occur, but systemic absorption would also be possible by vaginal intercourse if lesions or abrasions acused for example by other STD's^[18]. Although some population of women may experience a higher risk of infection than others, it is almost impossible to predict, on an individual basis, who will develop a life-threatening complication. It is therefore critical that all women have access to passive immunocontraceptives if and when the needs arise.

3.6 Immunoglobulins when orally taken up will survive through the gastrointestinal tract in healthy adult volunteers, implying their potential applications for human therapy of immunoinfertility and HIV/STDs infection

Concerns that if the orally uptake of immunoglobulins can reach the local site of reproductive tract to play their roles are reasonable. Knowledges about the two functions of the immune system in the gut are now available^[19]: (1) to protect the host from enteric infections by viruses, bacteria and parasites; and (2) to minimize the induction of immune responses (both antibody and cell-mediated) to the immense and varied load of food antigens, the vast majority of which do not present a threat to the integrity of the host. Indeed, the induction of such immune responses could pose a greater risk to the host than any of the antigens themselves.

The results of many experiments have shown that a state of tolerance develops after ingesting a wide dose range of a protein such as ovalbumin (OVA) so that a subsequent parenteral challenge with OVA results in a 90%-100% reduction in the systemic immune response compared with that of control animals. Even though very low doses of OVA may induce immunity, this tolerant state can persist for up to two years after the initial feeding.

Recent data show that immunoglobulins from different species can survive through the gastrointestinal tract in healthy adult volunteers, suggesting their potential implications for human therapy^[20]. 

3.7 Immunoglobulin formula could be most easily accessible to vagina by female herself before sex

Since immunoglobulins like those produced by ourselves immune system are virtually harmless and can protect individuals from conception and infection, therefore, will be generally acceptable by individuals worldwide. We consulted many colleagues both males and females and got rather positive response to accept such formula as a contraceptive choice.

3.8 Immunoglobulins from chicken will be most easily produced in large amount cost effectively

4 Global perspectives

4.1 Urgent needs

Overpopulation is a global problem of significant magnitude, with grave implications for the future^[22]. It is now generally accepted that the currently available methods of fertility regulation are inadequate to meet the varied and changing personal needs of couples at different times in their reproductive lives and in the widely different geographical, cultural, religious, and service settings that exist around the world^[23]. On the other hand, the global HIV epidemic continues to expand at an alarming rate. The current epidemics of AIDS and other sexually transmitted diseases (STDs) have created an urgent need for a new type of contraceptive: one that is both a spermicide and a microbicide^[7].

Because of the steady decline in both industry and foundation support for contraceptive research^[24], it seems the right time to integrate very limited resources but great pool of wisdom to tackle this urgent problem and initiate a pilot study on this approach, since those techniques are already sophisticated and scientific rationales are sound. Supplemented to traditional immunobiology and immunochemistry, both transgenic techniques and genetic vaccine techniques will find their most potentials in this area.

Specially engineered hens for this purpose are encouraged in order to further decline the cost. First, IgY should not direct against non-reproductive specific components. Second, IgY should direct against sperm specific antigens, like FA-1, and plasma immunosuppressive factors. Third, if applicable, IgY should arm its ability to neutralize HIV envelop protein, like gp120 and other STDs infection.

The cDNA encoding for the FA-1 antigen has been cloned and sequenced from murine testis. Naz and colleagues also have isolated two putative clones from the human testis C gt11 cDNA expression library which react with FA-1 MCA^[25]. The functional epitope of FA-1 antigen, recognizing the FA-1 MCA, has been isolated, sequenced and synthesized. Interestingly, the sera from immunoinfertile patients (men and women) and not from fertile humans show a strong reaction with this synthetic decapeptide epitope, and the decapeptide-reactive immunoaffinity-purified antibodies inhibit human SPA. Northern blot analysis of mRNA isolated from various human tissues, confirmed the testis-specific expression of the FA-1 antigen in humans. Active immunization trials using the synthetic decapeptide epitope as well as purified recombinant antigen obtained by expression using the pGEX system are being planned in a non-human primate model.

4.2 AIDS-related challenges

Scientists have understood more about the HIV than any other virus. However, development of vaccine capable of anti-HIV infections remains severe challenge as it faced when the virus was discoved^[26]. The problem is, unlike the human immune response against most acute virus infections, natural immune response does not destroy HIV. This defeat makes researchers unable to understand that an effective vaccine should initiate what immune activity.

One of the greatest concerns to such research has been the increasing awareness of possible contributions of human seminal plasma immunosuppressive factors to immunoinfertility and infections. Evidence for human immunosuppression status during insemination has been well documented over the years. The immunosuppressive contributions to fertilization are mainly made by males seminal plasma immunosuppressive factors although female also prepare herself with lowest level of immune response against sperms during ovulation.

Recent findings of the presence of a CD₄-binding glycoprotein, namely gp17 (apparent MW=17 500 Da) in human seminal plasma may be relevant to the control of sexual transmission of HIV-1. Since its binding to CD₄ was inhibited by anti-CD₄ mAbs directed against V1, a region of CD₄ implicated in the binding to MHC class II antigens and to the HIV-1 envelope protein gp120, but not by mAbs directed against other CD₄ determinants^[27].

Furthermore, this 17-kDa CD₄-binding glycoprotein is also expressed in  mammary tumor cells upon hormone treatment and in biopsies from breast cancer patients. The finding that breast cancer cells express a protein able to interact with the CD₄ domains involved in the recognition of class II major histocompatibility antigens suggests a possible mechanism by which a tumor may affect the activity of tumor-infiltrated CD₄⁺ T-cells^[28].

Unfortunately, researches of fertilization and HIV infection have been separated as two relatively independent fields although some authors have expressed their concerns on the possible mechanisms by which human immunosuppression status during insemination might contribute to infections by microorganisms. From the analyses of the accumulated data, the possible links between human seminal plasma immunosuppressive factors, HIV infection and tumor genesis are emerging. A better understanding of those complicated phenomena is needed for future therapy development.

It is not overestimated that overpopulation and rapid prevelance of HIV infections are threatening our future. We claim and draw public attention to this matter, probably because there is a mythic force that govern and balance between human beings reproductive system and immune system, and no currently available methods can manipulate it without any unacceptable complications. Of course, currently available methods of fertility regulation can not meet the practical demands for fertility regulation and prevention of infection. Therefore, development of successful strategies for contraception and primary prevention of HIV infection in women must be a top public health priority.

5 Acknowledgements

References

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