Scand. 7. Immunol. 36, Supp!. 11. 137-14:1. 1992
Future Perspectives for Vaccine Development V. C. STEVENS Ohio State University, Columhus. Ohio. USA
Stevens VC. Fulure Perspectives 1992;36(Suppl.10:137-43
for
Vaccine
E>evelopment.
Scand
J
Immunol
The successful development of an anti-fertility vaccine necessitates overcoming obstacles in eliciting an immune response to self species body constituents. The feasibility of accomplishing this task has been demonstrated for certain antigens described in this workshop: however, additional vaccine candidate antigens may yet be revealed from the application of recent advances in molecular biology. Improvements in vaccine design are likely to occur from discovery of more appropriate epitopes on targeting antigens, new carrier molecules for lerminaiing immunological tolerance, expression of vaccine antigens in suitable live vectors, ihe co-immunization with more than one antigen, the use of safer and/or more eiTective adjuvants and vehicles, more etTident immunization by targeting antigens to specific lymphoid cells, and the development of superior vaccine delivery syslems. Research directed to restricting the immune response to the gcniial tract and lo intentionally reverse the effects of immunization will likely ho pursued in the fulure. Ail of these areas need to be addressed if vaccines arc lo be developed that are nol only safe and effective but also highly acceptable as birth control methods. C. Slcivm. Collfgc of Medicine. Deptirirnvnl of OhsWlrics and Gynecohgy. /)/('/.V/(>H of Reproductive Biology. Ohio Slate Universily. Fifth Floor. Means Hall. 1654 Vpham Drive. Columbus, OH 43210-1228. USA
As shown by the presentations at this meeting, consideryble progress has beeti made in the past 20 years in exploring the potential of immunological methods for regulating human fertility. Some of the more promising leads for developing an anti-fertility vaccine formulation have been taken to the stage of clinical trials and others arc now reaching the stage of feasibility testing. The experience gained in developing the tirst vaccines, together with advances in molecular biology and immunochemistry, will tindoubtedly accelerate the rate of devising new and better formulations in the next decade. Outlined below are some personal views on advances expected to be made in this field considering what is known today and the special requirements for these vaccines to be effective, safe and acceptable to a wide population. No doubt these perspectives will change as time passes and new milestones are reached, but they may serve as a guide to investigators recently engaged in this area of research.
THE VACCINE DEVELOPMENT PROCESS The major steps required in developing a successful vaccine were described earlier at this meeting and the implementation of many of them illustrated by the scientists reporting progress on specific vaccine research. These steps will be briefly revisited here from the perspective of potential additions and/or improvements in future vaccines. New vaccine antigens Single molecule targets have been defined for the reproductive hormone-based vaccines described and some of the vaccines targeting sperm and ovum antigens. Also, in the case of the human chorionic gonadotropin (hCG) vaccine, a peptidc representing hormone-specitic epitopes has been employed for vaccine development [I], However, many opportunities remain to identify 137
138
V.C.Stevens
new epitope regions on these molecules for use in new vaccines [2]. One may question the need for defining individual epitopes when the entire molecule is available as a natural product or can be produced in bulk by recombinant DNA technology. Some arguments against this view include: (1) Few hormones or other defined molecules can be acquired from natural sources in sufficient quantity to manufacture adequate supplies of vaccine for millions of people. (2) The cost of recombinant DNA production of large, conformationally dependent, glycosylated molecules u.sing our existing knowledge would make vaccines unalTordablc to many. (3) Some desirable vaccine targets may share antigenic sites with non target molecules and. therefore, antigen-specific cpiiopes will be required to assure vaccine safety. In addition, while not likely to be a common problem, areas of antigens representing T-ce!I epitopes may need to be eliminated from imniunogcns to avoid irreversibility of vaccine effects [3|, (4) The focusing of the immune responses to the most desirable epitopes on target molecules can enhance the effectiveness of immunizations by eliminating antigenic competition by the less desirable epitopes. Thus, it is anticipated that most research will be directed towards defining antigen-specific epitopes in Follicle Stimulating Hormones (FSH). Gonadotropin Releasing Hormone (GnRH) and hCG, as well as the sperm antigens (SP-IO, PH20. LDH-Qand FA-l)andovumantigens(ZP-I, ZP-2 and ZP-3). Also, antigens contained on the membraneoftrophoblast cells may be included in this list should on-going studies indicate their efficacy and acceptability as vaccine immunogens. In addition to the currently defined vaccine targets, it is predictable that new targets will be revealed in future research. Examples of possible new molecules might be the elusive Early Pregnancy P'actor [4] and Lymphocyte Modulating Factor [5]. Studies in the field of reproductive immunology continue to suggest the presence of undefined molecules that regulate the immune system during pregnancy and participate in the maintenance of the conceptus as an allograft in the uterus. Studies during the next decade will likely yield new antigen targets for vaccine development.
New vaccine design In recent years, 'modern vaccines' against infectious diseases have employed "subunit" fractions of bacterial or viral particles for improving safety and efficacy of immunizations. The first vaccines using recombinant fusion proteins have now emerged. These advances normally utilize Tcell and B-ccl! epitopes, as responses to both are needed to prevent infection. As anti-fertility vaccines normally employ only human B-cell epitopes, sources of T helper function must be acquired by conjugating a foreign (non-human) molecule to the reproductive antigen. Many examples of this approach have been described at this meeting. However, current technology now permits us to link specific portions of reproductive molecules (representing B-ccll epitopes) to specific portions of foreign molecules (representing T-cell epitopes to huinan lymphocytes) by recombinant DNA methods or by pcplide synthesis procedures. Whilst certain fusion proteins containing reproductive antigens expressed with bacterial foreign proteins and synthetic hCG peptides coupled to toxoid carriers have been successfully used for vaccine development [6], no immunogens have been prepared to date in which the sequences represented do not elicit antibody responses to carrier molecules or exhibit hypersensitivity reactions following repeated immunizations. Although today immunologists cannot assure that it is feasible to design a vaccine whieh will elicit high antibody levels without some degree of immediate or delayed hypersensitivity, careful restriction of the immune response to define T- and B-cell epitopes may offer an opportunity to minimize side-eftccts in future vaceines. Several examples of T epitope regions of bacterial and virtil antigens have been described [7,8]. some with minimal MHC restriction, which might be suitable for providing T help to reproductive antigens in outbred populations (promiscuous T sites). The combination of these with specific Bcell epitope regions of reproductive molecules as vaccine immunogens will constitute an exciting area of research in future years. Another new means to provide an imniunogen containing B-cell epitopes together with foreign T-ceil epitopes is the insertion of nucieotide message into the genome of live vectors such as Vaccinia virus [9]. The application of this technique to modern vaccine development has been shown to be feasible, but considerable debate persists regarding the overall safety of adminis-
Perspectives for Vaccine Development tering live micro-organisms and the selection of the most appropriate vectors. The advantages of this approach are that an excellent immune response can be triggered by viral and bacterial antigens and that long-term immunity can readily be established. Potential drawbacks to this means of immunization are the possibility of inducing infectionsand/or permanent sterility. Clearly, the potential for applying this technology to antifertility vaccine development is siiflficiently high to stimulate research for answering ihese critical questions. Although il is not a new idea, the use of a combination of antigens from several reproductive molecules in a single "cocktail" vaccine will likely be a consequence of future research. Theoretically, a vaccine providing the simultaneous presence of antibodies directed to sperm, ova and conceptus antigens in the reproductive tract of women would have a higher eflicaey than one providing antibodies to only a single antigen. Likewise, antibodies to both GnRH and FSH in the circulation of men might offer a "back-up' mechanism to ensure a state of infertility. Of course, any combination of antigens in such a vaccine would be possible only if vaccines against the single antigens were separately demonstrated to be safe and efficacious. While not technically a part of new vaccine design, the probable use of anti-fertility vaccines in combination with other means of birth control will surely be a reality and is worthy of mention in regard to new vaccine development. It is well known and widely accepted that significant antibody production following successful immunization does not occur until 7-10 days following exposure of the lymphoid system to an immunogen. In practice, responses to weak antigens usually require 14-21 days. Thus, we cannot expect, as we do with certain other contraceptives, to offer immediate protection (to men or women) from initiating a pregnancy by administering an anti-fertility vaccine. An acceptable alternative method must be offered or co-administered upon primary immunization during at least the first month of vaccine use. These methods could be ones currently available (steriod injections or pills, lUDs, barrier methods) or new drugs or devices might yet be developed for specific use with anti-fertility vaccines. Depending upon the predictability of the duration of vaccine effectiveness amongst individuals, such alternative methods might also be prescribed for
139
use at a defined time following immunizations in lieu of ascertaining whether antibodies to a reproductive antigen remain at a protective level. The precise requirements for "mixed contraceptive' applications and their feasibility will need to await the results of further clinical trials of proposed vaccines. At the point in time when immunological birth control methods are ready to enter family planning programmes, this issue will need to be seriously addressed.
New means to potentlute vaccines The nature of the immune response required for an anti-fertility vaccine is synonymous with immunization with a weak antigen. Ideal target antigens are those normally exposed to the lymphoid system that are not recognized by human T lymphocytes and are bound by many fewer B cells than arc foreign antigens. Thus, a state of total Tcell tolerance and partial B-ccll tolerance must be overcome to render self antigens sufficiently imniunogenic to temporarily neutralize reproductive function. Techniques used to bypass Tcell tolerance by associating self molecules with foreign substances were discussed in the previous paragraphs, but enhancement of B-ccll activity is also essential to the prtxiuction of high levels of antibodies. Activated B cells are normally stimulated to produce antibodies by cyiokine secretions from T cells which have been activated by presentation of the immunogen to them. Materials or compounds capable of boosting the sensitivity of this chain of events result in increased antibody production by non-specific 'adjuvant' stimulation. Perhaps the best example of adjuvant activity is represented by F-reund's C:omplete Adjuvant. This substance is a mixture of mineral oil (an attractant for macrophages to the injection site) and bacterial cells (that evoke activation of T cells). The immunogen to be used is dissolved or suspended in an aqueous solution and emulsified with the oil. which results in a depot formulation allowing a slow release of the immunogen to the tymphoid system. This adjuvant has been used for decades to enhance antibody levels in experimental animals, but use in humans has been precluded because of the severe reactions produced at the injection site. However, the understanding of its mechanism of action has provided a basis for developing other formulations that
140
I; C. Sierens
will enhance antibody production with sufficiently less severe sidc-ctTects acceptable for human use. Currently, there is a plethora of experimenial compound.s. particles, oilii and biological extracts under evaluation for enhancing immune responses to vaccines intended for human application [10]. It is not appropriale here to review the status of this field of research, but suffice it to say that several approaches olTcr promise that one or more adjuvant tbrmulalions will soon be available for application In the anii-renilily vaccine field. This area of research is vital to ihe successor providing suitable methods in the future. Experimentally, il is possible to raise the sensiliviiy of ihe immune response lo immunogens without hyperstiniulaiion of cytokine production. One method ihal has demonstrated utility in this regard is the targeting ofantigens to B cells by linking them iO4i monoelonal antibody directed to a B-cell surrace marker. This technique allows a higher proportion of antigen molecules lo bind B cells and provides a larger population of cells capable of specific antibody production- The application ol" this approach to vaccine development has been demonstrated and further relinements will likely be employed in new vaccines |] I]. New experimental compounds have also been developed thai arc intracellular B-cell stimulants [I2|. These substances have been shown to elicit antibody production by antigen-iictivatcd B cells in vitro without exposure to T cells or T-eell cytokines. Thus, it seems highly probable that future vaccines against self antigens might become available that incorporate components capable of high anlibtidy produclion without antigen-speeifie T-ccll helper function. Such vaccines would have ihc tiislincl advantage of reducing or eliminating hypersensiiivity reactions elicited byT-cellaclivalion. Such an advance would offer major opportunities for application to birth control vaccines. New vaccine delivery systems Currently approved vaccines are delivered by ihe injection of antigens dissolved in physiological saline or absorbed to aluminium hydroxide precipitates. These vehicles arc siiiiablc lor many vaccines that employ particulatc antigens or those that degrade slowly in the body. However, most soluble antigens are cleared rapidly from the
injection site and the immune response induced Tollowing immunization isshori-lived. The major reason for this abbreviated response has been shown lo be the lack of antigen persistence. Means of slowing ihe release olaniigen from the injection site have heen devised by injecting immunogens in water oil emulsions, as in Frcund's Complete Adjuvant, using oils not producing unacceptably severe rcaciions. This approach has yielded procedures suitable for testing anti-fcrtiliiy vaccines bul has lallen short of meeting the needs lor eliciting high levels of antibodies for a protracted period Irom a single injection. Research aimed al solving this problem has evaluated liposomes. synthetic polymer panicles. ISCOMs and a variety of emulsions of oil and water for providing noi only enhanced lymphocyte activation, but also slow release of the immunogen from the sile of injection. Perhaps niosi promise for achieving ihc goal of a sustained antibody response to a soluble antigen has come from research evaluating biodegradable microsphere systems for controlled antigen release, These systems employ synthetic polymers of polylactic and or polyglycolic acids to entrap immunogens and to release them gradually over lime as the polymers degrade at the silc of injection (13]. These materials have been shown lo be safe from loxicity and can be manutaclured on a large scale- Data presented al this meeting have demonstrated that ihe immunogen in an hCG vaccine, delivered by these microsphcres. can sustain high antihndy levels in rabbits lor more Ihun one year from single intramuscular injection (Pig. 1). It is expected that this approach will be utilized widely in future vaccine applications. Almost all the vaccine research discu.ssed here has mvolved systemic immunization to achieve immune responses. Certainly, vaccines eliciting safe and effective systemic immunity would be acceptable to birth conlrol applications. Nonetheless, should it be shown to be feasible, a means to restrict ihc immune response lo the genital tract (particularly in females) would probably provide a safer means to regulate human fertility. As conception normally occurs in the oviduct of women, antibodies present only in that environment against sperm, ova or conceplus antigens would etTcctivcIy block lerliiily ami would minimize the risk of side-eflecis Irom a generalized state of immunity against self antigens. If local antibody produflion (IgA antibodies) ean be
Perspectives for Vaccine Developmeni
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X)
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i
1
i
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1
10
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40
50
50
70
80
90
100
Weeks Post Immunization FIG. I. Meananiibody levels(fi = 4t in rabbilsrciiclive lo liCG during a Iwo-ycar period following injections of an hCG pepiide immunogen rncorporiiled into biodegradable microspheres al 0 and 52 weeks.
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Flo. 2. Mean antibody levels in=4) in rabbits reactive lo hCG followmg immunizalions with an hCG immunogen incorporaled into microsphcres and subsequently injected daily for 4 days with !dy titre information, an individual subject will not know exactly when he or she has returned to a fertile state and method failures and/or anxiety could compromise meth(K1 acceptability, hollowing the extensive clinical testing of a particular vaccine, Ihe definition of a "safe period' for most subjects might be apparent. However, there will inevitably be vaccine users who wish to become parents prior to the natural waning of antibodies even though they may have been informed of probable vaccine duration before immunization. For these individuals, our current state of knowledge offers no safe means of terminating the eflccts ol' immunization upon
demand and returning their fertility immediately. There are theoretical possibilities to purge the body of specific immune cells and antibodies, but such procedures have never been tested in humans. One method, demonstrated in rodents, is based on targeting T or B cells with a toxic compound linked to the antigen to which the cells have been sensitized. This prtxedure could eliminate B cells producing antibtxlies to a reproductive antigen and. following antibody clearance, fertility would he re-established. However, the safety of administering toxic compounds for the purpose of fertility enhancement is unlikely ever to be justified. Another possihle approach, involving absorption oi effective antibodies from the circulation, might offer a safer method. As most reproductive antigens will not trigger an immune response unless linked to a foreign carrier, the administration of the reproductive haptens in great molar excess to the circulating antibodies might be capable of removing antibodies for a sufficient period to allow conception to occur (Fig. 2). Should the presence of sensitized cells in the latter stages of pregnancy present no health risk, gestation could proceed in the presence of B memory cells sensitized to the reproductive antigen. A potential risk to this approach is the intentional prodtiction of antigen antibody complexes during antibody absorption. Careful studies in animals for evaluating glomerular nephritis would need to be conducted prior to any clinical trial of this procedure. Notwithstanding the manifold problems surely to be encountered in developing methtxls of vaccine reversal, its importance to method acceptability will likely stimulate research in this area in the years ahead.
ETHICAL C O N S I D E R A T I O N S Any method of birth control that will induce long-term infertility without any means by the user to reverse this state, such as an anti-fertility vaccine, provides opport unities for ethical abuse. Such a method could be provided without or with inadequate informed consent or imposed by coercive governments or organizations. As control over reproductive function is a basic human right, stringent adherence to high ethical standards in the provision and promotion of vaccines must accompany their use. Potential problems with the provision of contraceptive vaccines
Perspectives for Vaccine Development could occur unless 'vaccination' against pregnancy was clearly distinguished from vaccination against infectious diseases. Despite the remoteness of these practical problems, consideration of the ethics of applying new technology to human birth control must be made during the vaccine development process.
8
9 10
REFERENCES 1 Stevens VC. Cinader B. Powell JE ei ul. Preparalion and fomiuialion of a hCG anlifertilily vaccine: selection ol" peptide immunogen. Am J Reprod Immunol 1981:6:307-14. 2 Jemmerson R. Patcrson Y. Mapping antigenic sites of proteins: implications Tor [he design of synihetic vaccines. Bio Tech I986:4:IS 31. 3 Millar SE. Chamow SM. Balir AW ,•/ aL Vaccination with a synthetic zona pellucida pepiide produces long-term conlraception in female mice. Science 1989:246:935-8. 4 Morion H. Rolfe BE, McNeil L, Clarke P. Clarke FM, Clunie GJA. Early pregnancy Factor: tissues involved In its produclion m Ihe mouse. J Repro Immunol 1980:2:73-82. 5 Szekeres-Burtho J. Chaouat G. Lymphocyle-derived progesterone-induced blocking factor corrects resorplion in a murino abortion system. Am J Reprod Immunol I99();23:26 «. 6 Herr JC. Wrighl RM. John E. Fosler J, Kays T, Flickinger CJ. Identilication of human acrosomal antigen Sp-IO in primates and pigs. Bio Repro 1990:42:377 82. 7 Ho PC. Mutch DA. Winkel KD. Saul AI J, Jones GL. Doran TJ. Rzepczyk. Identification of two
11
12
13
14
15
16
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promiscuousT cell epitopes from tetanus toxin. Eur J Immunol 1990:20:477-83. Partidos CD, Steward MW. Prediction and identification of a T cell epitope in the fusion protein of measles virus immunodominant in mice and humans. J Gen Virol 1990:71:2099-105. Moss B. Flexner C. Vaccinia virus expression veclors. Ann Rev Immunol 1987:5:305 24. Warren HS. Vogel FR. Chedid LA. Current status of immunologicai adjuvants. Ann Rev Immunol 1986:4:369 88Barber BH. Carayanniotis G. Antigen delivery by immtino-largeting: a possibilily Tor adjuvant-free vaccines? In: Technological Advances in Vaccine Development. New York: Alan R. Liss. 19X8:471. Golding B. Chang SP, Golding H. Jones RE. Pratt KL. Burger DR. Riitenbcrg MB. Human lymphocytes can generate thymus-indc|>endent as well as thymus-depeiident anti-hapien plaque-forming cell responses in rilro. J Immunol I98l:!27:22O-4. Slevens VC. Powell JE, Rickey M. Lee AC. Lewis DH. Studies of various delivery systems for an liCG immunocontraceplion. In: Gamete Interaction. Prospects for Immunocontraception. New York: Wilcy-Liss. 1990:549-63. Mestecky J, The common mucosal immune system and current strategies for induction of immune responses in external secretions. J Clin Immunol 1987:7:265-76. Eldridge JH, Gilley RM. Staas JK. Moldoveanu Z. Meulbroek JA, Tice TR. Biodegradable microspheres: vaccine delivery system for oral immunization. Curr Topics Micro Immunol 1988:146:59-66. O'Hagan DT. Palin K. Davis SS, Arthursson P. Sjoholm I. Microparticles as potentially orally active immunological adjuvants. Vaccine 1989:7:421-4.
Future Perspectives for Vaccine Development V. C. STEVENS Ohio State University, Columhus. Ohio. USA
Stevens VC. Fulure Perspectives 1992;36(Suppl.10:137-43
for
Vaccine
E>evelopment.
Scand
J
Immunol
The successful development of an anti-fertility vaccine necessitates overcoming obstacles in eliciting an immune response to self species body constituents. The feasibility of accomplishing this task has been demonstrated for certain antigens described in this workshop: however, additional vaccine candidate antigens may yet be revealed from the application of recent advances in molecular biology. Improvements in vaccine design are likely to occur from discovery of more appropriate epitopes on targeting antigens, new carrier molecules for lerminaiing immunological tolerance, expression of vaccine antigens in suitable live vectors, ihe co-immunization with more than one antigen, the use of safer and/or more eiTective adjuvants and vehicles, more etTident immunization by targeting antigens to specific lymphoid cells, and the development of superior vaccine delivery syslems. Research directed to restricting the immune response to the gcniial tract and lo intentionally reverse the effects of immunization will likely ho pursued in the fulure. Ail of these areas need to be addressed if vaccines arc lo be developed that are nol only safe and effective but also highly acceptable as birth control methods. C. Slcivm. Collfgc of Medicine. Deptirirnvnl of OhsWlrics and Gynecohgy. /)/('/.V/(>H of Reproductive Biology. Ohio Slate Universily. Fifth Floor. Means Hall. 1654 Vpham Drive. Columbus, OH 43210-1228. USA
As shown by the presentations at this meeting, consideryble progress has beeti made in the past 20 years in exploring the potential of immunological methods for regulating human fertility. Some of the more promising leads for developing an anti-fertility vaccine formulation have been taken to the stage of clinical trials and others arc now reaching the stage of feasibility testing. The experience gained in developing the tirst vaccines, together with advances in molecular biology and immunochemistry, will tindoubtedly accelerate the rate of devising new and better formulations in the next decade. Outlined below are some personal views on advances expected to be made in this field considering what is known today and the special requirements for these vaccines to be effective, safe and acceptable to a wide population. No doubt these perspectives will change as time passes and new milestones are reached, but they may serve as a guide to investigators recently engaged in this area of research.
THE VACCINE DEVELOPMENT PROCESS The major steps required in developing a successful vaccine were described earlier at this meeting and the implementation of many of them illustrated by the scientists reporting progress on specific vaccine research. These steps will be briefly revisited here from the perspective of potential additions and/or improvements in future vaccines. New vaccine antigens Single molecule targets have been defined for the reproductive hormone-based vaccines described and some of the vaccines targeting sperm and ovum antigens. Also, in the case of the human chorionic gonadotropin (hCG) vaccine, a peptidc representing hormone-specitic epitopes has been employed for vaccine development [I], However, many opportunities remain to identify 137
138
V.C.Stevens
new epitope regions on these molecules for use in new vaccines [2]. One may question the need for defining individual epitopes when the entire molecule is available as a natural product or can be produced in bulk by recombinant DNA technology. Some arguments against this view include: (1) Few hormones or other defined molecules can be acquired from natural sources in sufficient quantity to manufacture adequate supplies of vaccine for millions of people. (2) The cost of recombinant DNA production of large, conformationally dependent, glycosylated molecules u.sing our existing knowledge would make vaccines unalTordablc to many. (3) Some desirable vaccine targets may share antigenic sites with non target molecules and. therefore, antigen-specific cpiiopes will be required to assure vaccine safety. In addition, while not likely to be a common problem, areas of antigens representing T-ce!I epitopes may need to be eliminated from imniunogcns to avoid irreversibility of vaccine effects [3|, (4) The focusing of the immune responses to the most desirable epitopes on target molecules can enhance the effectiveness of immunizations by eliminating antigenic competition by the less desirable epitopes. Thus, it is anticipated that most research will be directed towards defining antigen-specific epitopes in Follicle Stimulating Hormones (FSH). Gonadotropin Releasing Hormone (GnRH) and hCG, as well as the sperm antigens (SP-IO, PH20. LDH-Qand FA-l)andovumantigens(ZP-I, ZP-2 and ZP-3). Also, antigens contained on the membraneoftrophoblast cells may be included in this list should on-going studies indicate their efficacy and acceptability as vaccine immunogens. In addition to the currently defined vaccine targets, it is predictable that new targets will be revealed in future research. Examples of possible new molecules might be the elusive Early Pregnancy P'actor [4] and Lymphocyte Modulating Factor [5]. Studies in the field of reproductive immunology continue to suggest the presence of undefined molecules that regulate the immune system during pregnancy and participate in the maintenance of the conceptus as an allograft in the uterus. Studies during the next decade will likely yield new antigen targets for vaccine development.
New vaccine design In recent years, 'modern vaccines' against infectious diseases have employed "subunit" fractions of bacterial or viral particles for improving safety and efficacy of immunizations. The first vaccines using recombinant fusion proteins have now emerged. These advances normally utilize Tcell and B-ccl! epitopes, as responses to both are needed to prevent infection. As anti-fertility vaccines normally employ only human B-cell epitopes, sources of T helper function must be acquired by conjugating a foreign (non-human) molecule to the reproductive antigen. Many examples of this approach have been described at this meeting. However, current technology now permits us to link specific portions of reproductive molecules (representing B-ccll epitopes) to specific portions of foreign molecules (representing T-cell epitopes to huinan lymphocytes) by recombinant DNA methods or by pcplide synthesis procedures. Whilst certain fusion proteins containing reproductive antigens expressed with bacterial foreign proteins and synthetic hCG peptides coupled to toxoid carriers have been successfully used for vaccine development [6], no immunogens have been prepared to date in which the sequences represented do not elicit antibody responses to carrier molecules or exhibit hypersensitivity reactions following repeated immunizations. Although today immunologists cannot assure that it is feasible to design a vaccine whieh will elicit high antibody levels without some degree of immediate or delayed hypersensitivity, careful restriction of the immune response to define T- and B-cell epitopes may offer an opportunity to minimize side-eftccts in future vaceines. Several examples of T epitope regions of bacterial and virtil antigens have been described [7,8]. some with minimal MHC restriction, which might be suitable for providing T help to reproductive antigens in outbred populations (promiscuous T sites). The combination of these with specific Bcell epitope regions of reproductive molecules as vaccine immunogens will constitute an exciting area of research in future years. Another new means to provide an imniunogen containing B-cell epitopes together with foreign T-ceil epitopes is the insertion of nucieotide message into the genome of live vectors such as Vaccinia virus [9]. The application of this technique to modern vaccine development has been shown to be feasible, but considerable debate persists regarding the overall safety of adminis-
Perspectives for Vaccine Development tering live micro-organisms and the selection of the most appropriate vectors. The advantages of this approach are that an excellent immune response can be triggered by viral and bacterial antigens and that long-term immunity can readily be established. Potential drawbacks to this means of immunization are the possibility of inducing infectionsand/or permanent sterility. Clearly, the potential for applying this technology to antifertility vaccine development is siiflficiently high to stimulate research for answering ihese critical questions. Although il is not a new idea, the use of a combination of antigens from several reproductive molecules in a single "cocktail" vaccine will likely be a consequence of future research. Theoretically, a vaccine providing the simultaneous presence of antibodies directed to sperm, ova and conceptus antigens in the reproductive tract of women would have a higher eflicaey than one providing antibodies to only a single antigen. Likewise, antibodies to both GnRH and FSH in the circulation of men might offer a "back-up' mechanism to ensure a state of infertility. Of course, any combination of antigens in such a vaccine would be possible only if vaccines against the single antigens were separately demonstrated to be safe and efficacious. While not technically a part of new vaccine design, the probable use of anti-fertility vaccines in combination with other means of birth control will surely be a reality and is worthy of mention in regard to new vaccine development. It is well known and widely accepted that significant antibody production following successful immunization does not occur until 7-10 days following exposure of the lymphoid system to an immunogen. In practice, responses to weak antigens usually require 14-21 days. Thus, we cannot expect, as we do with certain other contraceptives, to offer immediate protection (to men or women) from initiating a pregnancy by administering an anti-fertility vaccine. An acceptable alternative method must be offered or co-administered upon primary immunization during at least the first month of vaccine use. These methods could be ones currently available (steriod injections or pills, lUDs, barrier methods) or new drugs or devices might yet be developed for specific use with anti-fertility vaccines. Depending upon the predictability of the duration of vaccine effectiveness amongst individuals, such alternative methods might also be prescribed for
139
use at a defined time following immunizations in lieu of ascertaining whether antibodies to a reproductive antigen remain at a protective level. The precise requirements for "mixed contraceptive' applications and their feasibility will need to await the results of further clinical trials of proposed vaccines. At the point in time when immunological birth control methods are ready to enter family planning programmes, this issue will need to be seriously addressed.
New means to potentlute vaccines The nature of the immune response required for an anti-fertility vaccine is synonymous with immunization with a weak antigen. Ideal target antigens are those normally exposed to the lymphoid system that are not recognized by human T lymphocytes and are bound by many fewer B cells than arc foreign antigens. Thus, a state of total Tcell tolerance and partial B-ccll tolerance must be overcome to render self antigens sufficiently imniunogenic to temporarily neutralize reproductive function. Techniques used to bypass Tcell tolerance by associating self molecules with foreign substances were discussed in the previous paragraphs, but enhancement of B-ccll activity is also essential to the prtxiuction of high levels of antibodies. Activated B cells are normally stimulated to produce antibodies by cyiokine secretions from T cells which have been activated by presentation of the immunogen to them. Materials or compounds capable of boosting the sensitivity of this chain of events result in increased antibody production by non-specific 'adjuvant' stimulation. Perhaps the best example of adjuvant activity is represented by F-reund's C:omplete Adjuvant. This substance is a mixture of mineral oil (an attractant for macrophages to the injection site) and bacterial cells (that evoke activation of T cells). The immunogen to be used is dissolved or suspended in an aqueous solution and emulsified with the oil. which results in a depot formulation allowing a slow release of the immunogen to the tymphoid system. This adjuvant has been used for decades to enhance antibody levels in experimental animals, but use in humans has been precluded because of the severe reactions produced at the injection site. However, the understanding of its mechanism of action has provided a basis for developing other formulations that
140
I; C. Sierens
will enhance antibody production with sufficiently less severe sidc-ctTects acceptable for human use. Currently, there is a plethora of experimenial compound.s. particles, oilii and biological extracts under evaluation for enhancing immune responses to vaccines intended for human application [10]. It is not appropriale here to review the status of this field of research, but suffice it to say that several approaches olTcr promise that one or more adjuvant tbrmulalions will soon be available for application In the anii-renilily vaccine field. This area of research is vital to ihe successor providing suitable methods in the future. Experimentally, il is possible to raise the sensiliviiy of ihe immune response lo immunogens without hyperstiniulaiion of cytokine production. One method ihal has demonstrated utility in this regard is the targeting ofantigens to B cells by linking them iO4i monoelonal antibody directed to a B-cell surrace marker. This technique allows a higher proportion of antigen molecules lo bind B cells and provides a larger population of cells capable of specific antibody production- The application ol" this approach to vaccine development has been demonstrated and further relinements will likely be employed in new vaccines |] I]. New experimental compounds have also been developed thai arc intracellular B-cell stimulants [I2|. These substances have been shown to elicit antibody production by antigen-iictivatcd B cells in vitro without exposure to T cells or T-eell cytokines. Thus, it seems highly probable that future vaccines against self antigens might become available that incorporate components capable of high anlibtidy produclion without antigen-speeifie T-ccll helper function. Such vaccines would have ihc tiislincl advantage of reducing or eliminating hypersensiiivity reactions elicited byT-cellaclivalion. Such an advance would offer major opportunities for application to birth control vaccines. New vaccine delivery systems Currently approved vaccines are delivered by ihe injection of antigens dissolved in physiological saline or absorbed to aluminium hydroxide precipitates. These vehicles arc siiiiablc lor many vaccines that employ particulatc antigens or those that degrade slowly in the body. However, most soluble antigens are cleared rapidly from the
injection site and the immune response induced Tollowing immunization isshori-lived. The major reason for this abbreviated response has been shown lo be the lack of antigen persistence. Means of slowing ihe release olaniigen from the injection site have heen devised by injecting immunogens in water oil emulsions, as in Frcund's Complete Adjuvant, using oils not producing unacceptably severe rcaciions. This approach has yielded procedures suitable for testing anti-fcrtiliiy vaccines bul has lallen short of meeting the needs lor eliciting high levels of antibodies for a protracted period Irom a single injection. Research aimed al solving this problem has evaluated liposomes. synthetic polymer panicles. ISCOMs and a variety of emulsions of oil and water for providing noi only enhanced lymphocyte activation, but also slow release of the immunogen from the sile of injection. Perhaps niosi promise for achieving ihc goal of a sustained antibody response to a soluble antigen has come from research evaluating biodegradable microsphere systems for controlled antigen release, These systems employ synthetic polymers of polylactic and or polyglycolic acids to entrap immunogens and to release them gradually over lime as the polymers degrade at the silc of injection (13]. These materials have been shown lo be safe from loxicity and can be manutaclured on a large scale- Data presented al this meeting have demonstrated that ihe immunogen in an hCG vaccine, delivered by these microsphcres. can sustain high antihndy levels in rabbits lor more Ihun one year from single intramuscular injection (Pig. 1). It is expected that this approach will be utilized widely in future vaccine applications. Almost all the vaccine research discu.ssed here has mvolved systemic immunization to achieve immune responses. Certainly, vaccines eliciting safe and effective systemic immunity would be acceptable to birth conlrol applications. Nonetheless, should it be shown to be feasible, a means to restrict ihc immune response lo the genital tract (particularly in females) would probably provide a safer means to regulate human fertility. As conception normally occurs in the oviduct of women, antibodies present only in that environment against sperm, ova or conceplus antigens would etTcctivcIy block lerliiily ami would minimize the risk of side-eflecis Irom a generalized state of immunity against self antigens. If local antibody produflion (IgA antibodies) ean be
Perspectives for Vaccine Developmeni
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Weeks Post Immunization FIG. I. Meananiibody levels(fi = 4t in rabbilsrciiclive lo liCG during a Iwo-ycar period following injections of an hCG pepiide immunogen rncorporiiled into biodegradable microspheres al 0 and 52 weeks.
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Flo. 2. Mean antibody levels in=4) in rabbits reactive lo hCG followmg immunizalions with an hCG immunogen incorporaled into microsphcres and subsequently injected daily for 4 days with !dy titre information, an individual subject will not know exactly when he or she has returned to a fertile state and method failures and/or anxiety could compromise meth(K1 acceptability, hollowing the extensive clinical testing of a particular vaccine, Ihe definition of a "safe period' for most subjects might be apparent. However, there will inevitably be vaccine users who wish to become parents prior to the natural waning of antibodies even though they may have been informed of probable vaccine duration before immunization. For these individuals, our current state of knowledge offers no safe means of terminating the eflccts ol' immunization upon
demand and returning their fertility immediately. There are theoretical possibilities to purge the body of specific immune cells and antibodies, but such procedures have never been tested in humans. One method, demonstrated in rodents, is based on targeting T or B cells with a toxic compound linked to the antigen to which the cells have been sensitized. This prtxedure could eliminate B cells producing antibtxlies to a reproductive antigen and. following antibody clearance, fertility would he re-established. However, the safety of administering toxic compounds for the purpose of fertility enhancement is unlikely ever to be justified. Another possihle approach, involving absorption oi effective antibodies from the circulation, might offer a safer method. As most reproductive antigens will not trigger an immune response unless linked to a foreign carrier, the administration of the reproductive haptens in great molar excess to the circulating antibodies might be capable of removing antibodies for a sufficient period to allow conception to occur (Fig. 2). Should the presence of sensitized cells in the latter stages of pregnancy present no health risk, gestation could proceed in the presence of B memory cells sensitized to the reproductive antigen. A potential risk to this approach is the intentional prodtiction of antigen antibody complexes during antibody absorption. Careful studies in animals for evaluating glomerular nephritis would need to be conducted prior to any clinical trial of this procedure. Notwithstanding the manifold problems surely to be encountered in developing methtxls of vaccine reversal, its importance to method acceptability will likely stimulate research in this area in the years ahead.
ETHICAL C O N S I D E R A T I O N S Any method of birth control that will induce long-term infertility without any means by the user to reverse this state, such as an anti-fertility vaccine, provides opport unities for ethical abuse. Such a method could be provided without or with inadequate informed consent or imposed by coercive governments or organizations. As control over reproductive function is a basic human right, stringent adherence to high ethical standards in the provision and promotion of vaccines must accompany their use. Potential problems with the provision of contraceptive vaccines
Perspectives for Vaccine Development could occur unless 'vaccination' against pregnancy was clearly distinguished from vaccination against infectious diseases. Despite the remoteness of these practical problems, consideration of the ethics of applying new technology to human birth control must be made during the vaccine development process.
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REFERENCES 1 Stevens VC. Cinader B. Powell JE ei ul. Preparalion and fomiuialion of a hCG anlifertilily vaccine: selection ol" peptide immunogen. Am J Reprod Immunol 1981:6:307-14. 2 Jemmerson R. Patcrson Y. Mapping antigenic sites of proteins: implications Tor [he design of synihetic vaccines. Bio Tech I986:4:IS 31. 3 Millar SE. Chamow SM. Balir AW ,•/ aL Vaccination with a synthetic zona pellucida pepiide produces long-term conlraception in female mice. Science 1989:246:935-8. 4 Morion H. Rolfe BE, McNeil L, Clarke P. Clarke FM, Clunie GJA. Early pregnancy Factor: tissues involved In its produclion m Ihe mouse. J Repro Immunol 1980:2:73-82. 5 Szekeres-Burtho J. Chaouat G. Lymphocyle-derived progesterone-induced blocking factor corrects resorplion in a murino abortion system. Am J Reprod Immunol I99();23:26 «. 6 Herr JC. Wrighl RM. John E. Fosler J, Kays T, Flickinger CJ. Identilication of human acrosomal antigen Sp-IO in primates and pigs. Bio Repro 1990:42:377 82. 7 Ho PC. Mutch DA. Winkel KD. Saul AI J, Jones GL. Doran TJ. Rzepczyk. Identification of two
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promiscuousT cell epitopes from tetanus toxin. Eur J Immunol 1990:20:477-83. Partidos CD, Steward MW. Prediction and identification of a T cell epitope in the fusion protein of measles virus immunodominant in mice and humans. J Gen Virol 1990:71:2099-105. Moss B. Flexner C. Vaccinia virus expression veclors. Ann Rev Immunol 1987:5:305 24. Warren HS. Vogel FR. Chedid LA. Current status of immunologicai adjuvants. Ann Rev Immunol 1986:4:369 88Barber BH. Carayanniotis G. Antigen delivery by immtino-largeting: a possibilily Tor adjuvant-free vaccines? In: Technological Advances in Vaccine Development. New York: Alan R. Liss. 19X8:471. Golding B. Chang SP, Golding H. Jones RE. Pratt KL. Burger DR. Riitenbcrg MB. Human lymphocytes can generate thymus-indc|>endent as well as thymus-depeiident anti-hapien plaque-forming cell responses in rilro. J Immunol I98l:!27:22O-4. Slevens VC. Powell JE, Rickey M. Lee AC. Lewis DH. Studies of various delivery systems for an liCG immunocontraceplion. In: Gamete Interaction. Prospects for Immunocontraception. New York: Wilcy-Liss. 1990:549-63. Mestecky J, The common mucosal immune system and current strategies for induction of immune responses in external secretions. J Clin Immunol 1987:7:265-76. Eldridge JH, Gilley RM. Staas JK. Moldoveanu Z. Meulbroek JA, Tice TR. Biodegradable microspheres: vaccine delivery system for oral immunization. Curr Topics Micro Immunol 1988:146:59-66. O'Hagan DT. Palin K. Davis SS, Arthursson P. Sjoholm I. Microparticles as potentially orally active immunological adjuvants. Vaccine 1989:7:421-4.
