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Development of vaccines targeting the zona pellucida
Development
MRC Reproductive
of vaccines targeting the zona pellucida M. Paterson
and R.J. Aitken
Biology Unit, Centre
for Reproductive
Current
Opinion
in immunology
Introduction The idea of a contraceptive vaccine which would provide a safe and reliable form of contraception that would be long lasting and reversible, with a mechanism of action that inhibits fertilization rather than disrupts early development, is an attractive one, particularly given the increasing rate of world population growth. The mammalian zona pellucida (ZP) has provoked a great deal of interest as a potential target for the immunological disruption of fertilization. Studies carried out more than 15 years ago (Shivers et d, Science 1972, 1781211-1213) showed that an antiserum raised against an aqueous extract of hamster ovarian tissue could block the fertilization of hamster ova in vitro, It was rapidly established that the antibodies responsible for this contraceptive effect were directed against antigens on the ZP. This was subsequently coniirmed in a variety of species includlng rabbits (Sacco and Shivers, J Reprod Fertil 1972, 32:415-420), hamsters (Oikawa and Yanigamachi, J Re prod B&Z 1975,45:487+4) and humans (Sacco, Biol Re pod 1977,16:164-173). Since these studies, great efforts have been made to identify and isolate those epitopes on the surface of the ZP that are responsible for this contraceptive elfect. The two most extensively studied systems are those of the mouse and the pig. The porcine ZP, in particular, has been the focus of such research because of its cross-reactivity with human ZP and the ability of anti-porcine ZP antibodies to block sperm-egg interactions in the human. The porcine ZP is highly glycosylated and can be resolved by two-dimensional electrophoresis into four major glyin order of decoproteln families identified as ml-4 creasing molecular mass. The marked microheterogeneity shown by each glycoprotein family can be attributed to the sequential addition of carbohydrate structures to the peptide core. Iectin-binding studies have indicated that the outermost surface of the ZP is rich in carbohydrate residues and that the sperm receptor sites are in this domain (Oikavva et al, Nature 1973, 241~256-259; Oikawa et al, J Rtpod Fertill975, 43~33136). These have been shown to contain carbohydrate and to be lo-
Biology, Edinburgh,
UK
1990, 2:743-747
calized to the O-linked oligosaccharide side chains of the major ZP glycoprotein, ZP3. Anti-ZP antibodies are thought to exert their contraceptive effect in one of two ways: by direct interaction with the sperm receptor site, or by binding to antigens that are close to the receptor site, masking the sperm receptor by steric hindrance. In heterologous systems, the latter mechanism is thought to be predominant, as the formation of an immunoprecipitate through the crosslinking activity of anti-ZP antibodies is a prerequisite for the inhibition of sperm binding. Univalent antibodies (Fab), which cannot cross-link, have no contraceptive effect (A&ken et al, J Rqtn-od Fertill982,66:327-334). Understanding the precise mechanism by which cross-linking porcine antibodies are able to disrupt the fertilization of human ova in vitro is critical to the development of a contraceptive vaccine. A number of studies of active immunization have been carried out in the past few years in a variety of species, including rabbits (Skinner et d, Enkurinob~ 1984, dogs (hIah&Brown el d, Biol Reprod 115:2418-24321, 1985, 32:761-772) and squirrel monkeys (Sacco et al,
FertiI Sterill983, 39:350-358). In all these studies, antibodies raked against the ZP were found to reduce fertility. However, in many of the immunized animals, hormonal and histological analysis suggested that ovarian function had been disrupted; this could, in part, have contributed to the animals infertility. zany of the recent active immunization studies covered in this review have addressed this problem.
Active immunization Some of the ovarian dysfunction observed in immunization progmmmes may be partly attributed to the use of crude or whole ZP preparations as the immunogens. Mahi-Brown et al. [ll used both crude and partially puriiied porcine ZP to examine the cause of abnormal oestrus cycles in bitches. All the animals became infertile but those bitches that developed high tines of anti-ZP
Abbreviations alum-aluminium hydroxide; EBCD-endo B-galactosidase digestion; FCA-Freund’s complete adjuvant; BlMeyhole limpet haemocyanin; NUFA-non-ulcerative Freund’s adjuvant; SPLPS-sodium phthalylated lipopolysaccharide; ZP-zona pellucida.
@ Current Biology Ltd ISSN0952-7915
743
744
Reproduction
antibodies had abnormal oestrus cycles and their hormonal levels indicated that ovulation had failed. Histopathological examination of their ovaries revealed the presence of follicular cysts lined by a thin layer of granulosa cells; a concomitant loss of oocytes was another major finding. The authors speculated that a layer of anti-ZP antibodies precipitated on the surface of the ZP may have blocked the normal intercellular communication between the oocyte and granulosa cells. Although electron microscopy revealed an electron-dense layer of immunoprecipitate, no changes were seen in the junctional complexes between the granulosa cells and the oocyte surface. However, the ovaries used for this histological examina tion were removed shortly after immunization, and it may be that long-term immunization is required for these effects to manifest themselves. In animals with lower antibody tines, oestradiol and progesterone levels indicated little, if any, impairment of ovarian cyclic& and their infertility was attributed to the presence of anti-ZP antibodies on the surface of the ovulated oocyte. SigniiicantIy, when the ZP immunogen was purified by size exclusion and ion-exchange chromatography, only one of the immunized animals showed any sign of ovarian dysfunction. Experiments with highly purified antigens containing a restricted number of epitopes could lead to an understanding of the underlying mechanisms of these pathological effects. In this context, a large-scale study involving 50 squirrel monkeys was carried out by Sacco et al. (Biol Reprod 1987, 36:481-490) using the highly pur&d porcine glycoprotein ZP3 as immunogen. There seemed to be initial disturbances to ovarian function, as judged by laparoscopic observation and oocyte production data, although these effects were not as severe as those reported by other groups (Gulyas et al, Gamete Res 1983,4:299--307; Skinner et al, 1984; Mahi-Brown et al, 1985). At 6-7 months post-immunization, histological examination revealed a disruption in folliculogenesis but, by 10-15 months, ovarian function was recovering. These initial disturbances are therefore reversible, despite the consistently high titres of anti-ZP antibodies throughout this period. This would suggest that anti-ZP antibodies can cause infertility by disturbing two major reproductive processes: folliculogenesis and sperm-egg interaction. Before a viable vaccine can be produced, it is essential that the antigens responsible for each of these effects are identified and separated.
location of the sperm receptor In this context, several groups have investigated the role of ZP3, in which the sperm attachment and species specificity functions are thought to reside (Sacco et al, J Reprod Immunol 1984, 6:8!+103). Biochemical investigations have shown that ZP3 is composed of two structumlly and immunologically distinct molecules, ZP3a (deglycosylated molecular weight, 37 kD) and ZP3p (deglycosylated molecular weight, 32 kD; Yurewicz et al, J Biol C?wm 1987, 262:567-571). The two components
ZP3-EBGDol and ZP3-EBGDP have been isolated after partial deglycosylation by endo g-galactosidase digestion (EBGD) [2] and their relative immunogenicity and antigenicity studied. Five preparations of the ZP3 glycoprotein family were investigated, namely ZP3, three enzymically deglycosylated preparations (ZP3-EBGD, ZP3EBGDu and ZP3EBGDp) and a chemically deglycosylated preparation, ZP3-DG. Antisera raised in both mice and rabbits revealed a distinct order of immunogenicity for the different preparations: ZP3 and ZP3-EBGDP > ZP3EBGD and ZP3-EBGM > ZP3-DG. Therefore, when carbohydrate is removed, either enzymically or chemically, ZP3 becomes less immunogenic, suggesting that carbohydrate side chains are important in conferring immunogenic&y. Although all the antisera formed a dense immunoprecipitate on the surface of porcine ZP, antisera to ZP3-EBGDa, ZP3-EBGDB and ZP3DG only formed a discontinuous precipitation layer, possibly because the epitopes recognized by these antisera are less numerous and are spatially distant. Pretreatment of ZPs with antisera to ZP3,ZP3EBGD and ZP3EBGDu inhibited boar sperm binding in a dose-dependent manner but pretreatment with antisera to ZP3-EBGDP and ZP3-DG did not inhibit binding. The authors concluded that sperm receptor activity is associated with ZP3EBGDu. These results contrast with those of Henderson et al. (Gamete Res 1987, 18:251-265), who showed that antisera to DGZP32/36 (similar to ZP3-DG) inhibited the binding of human sperm to salt-stored human ZPs by approximately 90% and that a surface immunoprecipitate was formed (Fig. 1). In addition, antisera against DGZP-32 (equivalent to chemically deglycosylated ZP3-EBGDP) inhibited sperm binding by almost 95%. Whether these discrepancies are due to differences in the method of isolation of the immunogen or are species-related has yet to be ascertained.
Influence of carbohydrate The carbohydrate moieties of porcine ZP3 are clearly important because of their immunogenic&y and involvement in sperm receptor functions. However, a contraceptive vaccine based on carbohydrate residues may be technically d&At to achieve at present because of the problems associated with synthesizing precisely deiined carbohydrate sequences, either chemically or as part of a recombinant glycoprotein molecule. Consequently, several groups have tried to determine whether antigens with contraceptive potential reside within the polypeptide backbone. when porcine ZP is chemically deglycosylated using the reagent trifluoromethane sulphonic acid, it resolves into iive major bands on sodium dodecyl sulphate polyacrylamide gel electrophoresis; these have molecular weights of 66, 52, 36, 32 and 16kD. Antisera against the 32kD peptide are capable of suppressing human sperm-human egg interactions in vittr, (Henderson et al, 1987). Aitken et al. (In CONR4DETZO Znternutionul Work.dh@ on Gamete Interaction eded by N.
Development of vaccines targeting the zona pellucida Paterson and Aitken
juvant used, the difference in immunogenkity between ZP3 and DGZP-32 was immediately apparent. A primary injection of 100 clg glycosylated ZP3 induced an immediate elevated antibody response whereas primary administration of DGZP-32 gave a weak antibody response or no response at all. The poor immunogeniclty of DGZP32 combined with the mild adjuvant induced low antibody titres, and three out of the four marmosets conceived shortly after the introduction of a male. In these animals, the induction of immunity was associated with a reversible disruption of ovarian cyclicity. The fourth animal, which developed the highest antibody titres, continued to cycle normally and remalned infertile throughout the course of the study ( > 12 months). The animals immunized with DGZP-32 emuls&d in NUFA had higher antibody tines; this was directly related to their infertility as three of the four marmosets with the highest titres remained infertile. Sporadic minor disruptions in the normal ovulatory pattern were reported but the pattern quickly returned to normal. Only one out of the five animals immunized with ZP3 showed any sign of ovarian dysfunction, but this group of animals had the highest antibody levels of all, quickly attaming titres of about 1: 20480 (Fig. 2). It is therefore difficult to ascribe these disruptions in ovarian function directly to the presence of anti-ZP antibodies. These disturbances do, however, seem to coincide with the administration of antigen. It has been suggested that the type of adjuvant used in an immunization programme may alkt ovarian folliculogenesis. In a study by Upadhyay et al [3], bonnet monkeys were immunized with ZP3 using either Freund’s complete adjuvant (FCA) or sodium phthalylated lipopolysaccharide (SPLITS) as adjuvants. Whereas immunlzation with SPLPS elicited an antibody response without affecting ovarian follicular development, immunization with FCA resulted in high antibody titres and ovarian follicu lar atrophy. This suggests that FCA is not the adjuvant of choice when immunizing with ZP antigens. However, in the study by Aitken et al. (1989), control animals given NUFA alone continued to cycle normally.
Fig. 1. Cross-reactivity of human and pig zonae pellucidae demonstrated by dark-field and indirect immunofluorescence studies. Salt-stored human ova were incubated for 1 h at 37°C in a poiyclonal antiserum raised against porcine DCZP-32. (a) Examined under dark-field illumination. (b) Incubated for a further Mmin in a second, fluorescein-labelled antibody raised against rabbit IgG and observed using a fluorescence microscope.
Alexander. Alan R I&s, 1989) compared this deglycosylated polypeptide with ZP~, its fully glycosylated coun terpart, using an active immunization protocol in a primate model, the common marmoset. Two different adjuvants were used to enhance the immune response: a mlld adjuvant composed of peanut oil, muramyl dipeptide and aluminuim hydroxide (alum), or, alternatively, one composed of non-ulcerative Freund’s adjuvant (NUFA), bacille Calmette-G&in and alum. Irrespective of the ad-
Dunbar et al. [4] investigated the use of a synthetic peptide adjuvant (muramyl dipeptide) in the immunization of baboons with glycosylated and deglycosylated antigens. Using ZP3 and an SOkD deglycosylated porcine peptide (equivalent to deglycosylated ZPl) as immunogens, they found that the glycosylated molecule elicited a higher immunological response. In two of the live animals that received ZP3, cyclic&y was disrupted; they become amenorrhoic by the eighth month. No visible changes in Q~licity were observed in the group receiving the SOkD peptide. Histopathologkal examination of the ovaries of both groups after nine cycles (or their equivalent), indicated that long-term immunization had altered the development of primary follicles, but these effects were more apparent in the ZP3-immunized group. Future research should, therefore, focus on identifying individual antigenie epitopes that inhibit fertilization but do not alter ovarian function.
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Fig.2. (a)Antibody titre and plasma progesterone values for a marmoset immunized with the 32 kD deglycosylated porcine zona peptide.
Note the temporary impairment of ovarian cyclicity following the primary, first and second booster injections. fb) Antibody titres and progesterone levels in a marmoset immunized with ZP3 in an adjuvant composed of non-ulcerative Freund’s adjuvant, bacille CalmetteGuerin and aluminium hydroxide Note the rapid rise in antibody titres and the normal patterns of ovarian cyclicity. Titres have no units as such and are taken to be the highest dilution of antisera at which an antibody/antigen reaction still occurs.
Syntheticantigens Of 4 the mammal& ZPs studied so far, that of the mouse is the best characterized. Using information gained from the cloning of the mouse ZP3 gene (Ringuette et al, Dev Bid 1988, 127:287-2951, hdlar et al. [5] have developed an alternative approach to contraceptive vaccination. Using a monoclonal antibody to ZP3 that blocked fertilization in the mouse, they were able to locate the epitopes responsible for this contraceptive effect A 1.Okilobase complementary DNA encoding these epitopes was cut into random fragments and cloned into the expression vector kgtl 1. This library was then screened using the monoclonal antibody and eight positive clones were obtained. A nucleic acid sequence common to all the positive clones was identified. This encoded a seven-amino-acid peptide (residues 336-342) recognized by the antibody. A 16-amino-acid peptide representing the sequence 328-343 was synthesized and conjugated to keyhole limpet haemocyanin (KLH) via the
N-terminal cysteine residue. Active immunization of mi&e with this conjugate elicited varying antibody titres, separating the mice into two groups, high and low responders. When mating trials were carried out, the control animals (given KLH with adjuvant) gave birth to litters within 3r/2weeks of the introduction of a male. Three animals, found to be low responders, also gave birth within the first month. A contraceptive effect lasting for 16-36 weeks was observed in the remaining mice and it was directly related to antibody titre. Ovaries removed from the immunized mice were tested with an anti-mouse IgG horseradish perox&% cbnjugate to determine whether circulating antibodies bound exclusively to the ZP or whether there was any evidence of cross-reactivity. Millar et al. [5] found that antibodies were localized to the ZPs surrounding growing oocytes and that the ovaries looked normal, with no evidence of inflammation or cellular cytotoxicity. They suggested several reasons for the success of their vaccine in producing
Development of vaccinestargetingthe zona pellucida Paterson and Aitken
long-term infertility in the absence of any visible ovarian dysfunction. Firstly, the synthetic ZP3-KU-I immunogen has a well defined sequence, unique to the ZP3 molecule, and antibodies raised against it should not cross-react with any other ovarian antigen. Secondly, the lack of Tcell epitopes within this short amino acid sequence may have prevented a cytotoxic T cell response to the ZP. The identication of B-cell and T-cell epitopes located within the amino acid sequence of the ZP3 polypeptide may be an important step towards production of a vaccine capable of eliciting an effective antibody response without any cytotoxic effects.
Annotated
a l
o
l
Several groups [6,7] have observed the transfer of an oviductal antigen to the surface of the ZP after ovulation. Studies using monoclonal antibodies against the oviductal ZP of the golden hamster have provided evidence that this glycoprotein, when released into the follicular fluid by the non-ciliated secretory cell of the oviduct 161, becomes intimately associated with the matrix of the ovulated ZP. Sakai ef al [7] have identified a similar antigen (ZP-0) that is only present on ovulated eggs. Initial investigations showed that this was a high molecular weight glycoprotein of about 200-240kD. A monoclonal antibody that recognized this antigen was able to form an immunoprecipitate on the surface of the ZP and to block fertilization in a dose-dependent manner. However, this antigen is not necessary for fertilization as follicular oocytes can be fertilizd in vitro without exposure to oviductal fluids. The antigen seems to be associated with early embryonic development and survivzl and its value as a candidate antigen for contraceptive purposes has yet to be fully investigated.
Conclusions Recent publications have emphasized the importance of identifying the epitopes on the surface of the ZP that are uniquely involved in the fertilization process. A potential vaccine should involve antibodies that inhibit fertilization by preventing sperm binding to and penetrating the oocyte without causing ovarian dysfunction. From the literature, it would seem that the use of highly purilied well defined epitopes as imrnunogens is a significant step in this direction. A rwersible contraceptive vaccine must provide a well de&d period in which a woman is protected et pregnancy. With a knowiedge of the precise antigenic dete rminants involved in fertilization, and their mechanism of action, it should be possible to engineer vaccines in which the disturbance of ovarian function is minimized or eliminated.
Of interest Of outstanding interest
with porcine zona pe&ci&. Am J Reprod Immund Micro bid 1988, 1894-103. Report on the immunization of bitches with crude or paaiallypuw porcine Zp to determine the cause of abnormal oestrus cycles.
l
antigens
and recommended
MAHI-BROWN CA, YANAGIMACHI R, NELSONMI, YNWGIMACHIH, PALIJMBO N: Ovarian histopathology of bitches immunized
1.
2.
Oviductal
references
reading
e
SACCO AG, YUREWICZEC, SUEIRAWNLWMG, MATZAT PD: Porcine zona pellucidaz association of the sperm recep
tor activity with the a-glycoproteti component of the Mr=55,OOO family. Bid Re_Dnxl1989,41:52%532. Report on the immunogenicity and sperm receptor activity of liw difof ZP3. Demonstrates the kxalization of the sperm ferent preparations receptor to ZP3a. S, UPAJXWAY SN, ?%IUAlKOOTHANP, BAMEZU 4 JAYARAMAN TALWARGP: Role of adjuvants in inhibitory infhwnce of
3. l
immunization with porcine zona pellucida antigen (ZP3) on ovarian folliculogenesis in bonnet monkeys: a morphological study. Bid R@roci 1989, 41:665-673. Examines the use of FCA and SPLPSas adjuvan@ when immunizingwith ZP3. FCA caused ovarian follicular atrophy whereas no morphological damage was obsetved with SPLPS.Howeve r, the paper does not include any adjuvant controls. 4.
DLJNBAR BS, Lo C, POWEUJ, STEVENS VC: Use of a synthetic peptide adjuvant for the immunization of baboons with demuured and deglycosylawd pig xma pellucida &coproteins. Fe&l S&III 1989, 52:3113X Describes the use of muramyI dipeptide as an &ective adjuvant for the immunization of baboons with ZP3 and an 80kD peptide. Antibodies rakwdagainstthesedf%e rminants have a contmceptive effect but may also interfere with normal ovarian function. l
MIUARSE, CHAMOWSM, BAURAw, OLIVERC, ROBEYF, DEW J: Vaccination with a synthetic zona pebcida peptide produces long-term contraception in female mice. Science W89, 246:935-938. This paper describes the kientjfication of a seven-amino-acid sequence from the mouse ZP which is able to prcduce long-lasting contraception. First report of a short synthetic peptide sequence that is capable of inducing long-term infertility. 5. l
e
6.
KANFWK, ST-JACQUES S, BLEW G: Immunocytochemical evidence for the transfer of an oviductal antigen to the zona pellucida of hamster ova after ovulation. Bid Reproci 1989, 40:58%598. These authors use a monoclonal antibody against oviductal zona to demonstrate the secretion of an oviductal antigen which becomes closely associated with the ZP of oocytes during their passage through the oviduct. l
7. l
S&u Y, ARAKIY, Y.U&WITAT, KUWA S, OIKAWAT, HXRO~ M, SENDOF: Inhibition of in vitm fertihaticm by a mOnOdOn& antibody rexting with the zona pellucida of the oviductal egg but not with that of the ovarian egg of be golden
hamste-r. J R@mxi Immund 1988, 14:177-190. This paper demonstmtes that a monoclonal antibody that recognizes a zona antigen @P-O), presentonlyon oviductal oocytes, is able to block fertilization in virra
747
MRC Reproductive
of vaccines targeting the zona pellucida M. Paterson
and R.J. Aitken
Biology Unit, Centre
for Reproductive
Current
Opinion
in immunology
Introduction The idea of a contraceptive vaccine which would provide a safe and reliable form of contraception that would be long lasting and reversible, with a mechanism of action that inhibits fertilization rather than disrupts early development, is an attractive one, particularly given the increasing rate of world population growth. The mammalian zona pellucida (ZP) has provoked a great deal of interest as a potential target for the immunological disruption of fertilization. Studies carried out more than 15 years ago (Shivers et d, Science 1972, 1781211-1213) showed that an antiserum raised against an aqueous extract of hamster ovarian tissue could block the fertilization of hamster ova in vitro, It was rapidly established that the antibodies responsible for this contraceptive effect were directed against antigens on the ZP. This was subsequently coniirmed in a variety of species includlng rabbits (Sacco and Shivers, J Reprod Fertil 1972, 32:415-420), hamsters (Oikawa and Yanigamachi, J Re prod B&Z 1975,45:487+4) and humans (Sacco, Biol Re pod 1977,16:164-173). Since these studies, great efforts have been made to identify and isolate those epitopes on the surface of the ZP that are responsible for this contraceptive elfect. The two most extensively studied systems are those of the mouse and the pig. The porcine ZP, in particular, has been the focus of such research because of its cross-reactivity with human ZP and the ability of anti-porcine ZP antibodies to block sperm-egg interactions in the human. The porcine ZP is highly glycosylated and can be resolved by two-dimensional electrophoresis into four major glyin order of decoproteln families identified as ml-4 creasing molecular mass. The marked microheterogeneity shown by each glycoprotein family can be attributed to the sequential addition of carbohydrate structures to the peptide core. Iectin-binding studies have indicated that the outermost surface of the ZP is rich in carbohydrate residues and that the sperm receptor sites are in this domain (Oikavva et al, Nature 1973, 241~256-259; Oikawa et al, J Rtpod Fertill975, 43~33136). These have been shown to contain carbohydrate and to be lo-
Biology, Edinburgh,
UK
1990, 2:743-747
calized to the O-linked oligosaccharide side chains of the major ZP glycoprotein, ZP3. Anti-ZP antibodies are thought to exert their contraceptive effect in one of two ways: by direct interaction with the sperm receptor site, or by binding to antigens that are close to the receptor site, masking the sperm receptor by steric hindrance. In heterologous systems, the latter mechanism is thought to be predominant, as the formation of an immunoprecipitate through the crosslinking activity of anti-ZP antibodies is a prerequisite for the inhibition of sperm binding. Univalent antibodies (Fab), which cannot cross-link, have no contraceptive effect (A&ken et al, J Rqtn-od Fertill982,66:327-334). Understanding the precise mechanism by which cross-linking porcine antibodies are able to disrupt the fertilization of human ova in vitro is critical to the development of a contraceptive vaccine. A number of studies of active immunization have been carried out in the past few years in a variety of species, including rabbits (Skinner et d, Enkurinob~ 1984, dogs (hIah&Brown el d, Biol Reprod 115:2418-24321, 1985, 32:761-772) and squirrel monkeys (Sacco et al,
FertiI Sterill983, 39:350-358). In all these studies, antibodies raked against the ZP were found to reduce fertility. However, in many of the immunized animals, hormonal and histological analysis suggested that ovarian function had been disrupted; this could, in part, have contributed to the animals infertility. zany of the recent active immunization studies covered in this review have addressed this problem.
Active immunization Some of the ovarian dysfunction observed in immunization progmmmes may be partly attributed to the use of crude or whole ZP preparations as the immunogens. Mahi-Brown et al. [ll used both crude and partially puriiied porcine ZP to examine the cause of abnormal oestrus cycles in bitches. All the animals became infertile but those bitches that developed high tines of anti-ZP
Abbreviations alum-aluminium hydroxide; EBCD-endo B-galactosidase digestion; FCA-Freund’s complete adjuvant; BlMeyhole limpet haemocyanin; NUFA-non-ulcerative Freund’s adjuvant; SPLPS-sodium phthalylated lipopolysaccharide; ZP-zona pellucida.
@ Current Biology Ltd ISSN0952-7915
743
744
Reproduction
antibodies had abnormal oestrus cycles and their hormonal levels indicated that ovulation had failed. Histopathological examination of their ovaries revealed the presence of follicular cysts lined by a thin layer of granulosa cells; a concomitant loss of oocytes was another major finding. The authors speculated that a layer of anti-ZP antibodies precipitated on the surface of the ZP may have blocked the normal intercellular communication between the oocyte and granulosa cells. Although electron microscopy revealed an electron-dense layer of immunoprecipitate, no changes were seen in the junctional complexes between the granulosa cells and the oocyte surface. However, the ovaries used for this histological examina tion were removed shortly after immunization, and it may be that long-term immunization is required for these effects to manifest themselves. In animals with lower antibody tines, oestradiol and progesterone levels indicated little, if any, impairment of ovarian cyclic& and their infertility was attributed to the presence of anti-ZP antibodies on the surface of the ovulated oocyte. SigniiicantIy, when the ZP immunogen was purified by size exclusion and ion-exchange chromatography, only one of the immunized animals showed any sign of ovarian dysfunction. Experiments with highly purified antigens containing a restricted number of epitopes could lead to an understanding of the underlying mechanisms of these pathological effects. In this context, a large-scale study involving 50 squirrel monkeys was carried out by Sacco et al. (Biol Reprod 1987, 36:481-490) using the highly pur&d porcine glycoprotein ZP3 as immunogen. There seemed to be initial disturbances to ovarian function, as judged by laparoscopic observation and oocyte production data, although these effects were not as severe as those reported by other groups (Gulyas et al, Gamete Res 1983,4:299--307; Skinner et al, 1984; Mahi-Brown et al, 1985). At 6-7 months post-immunization, histological examination revealed a disruption in folliculogenesis but, by 10-15 months, ovarian function was recovering. These initial disturbances are therefore reversible, despite the consistently high titres of anti-ZP antibodies throughout this period. This would suggest that anti-ZP antibodies can cause infertility by disturbing two major reproductive processes: folliculogenesis and sperm-egg interaction. Before a viable vaccine can be produced, it is essential that the antigens responsible for each of these effects are identified and separated.
location of the sperm receptor In this context, several groups have investigated the role of ZP3, in which the sperm attachment and species specificity functions are thought to reside (Sacco et al, J Reprod Immunol 1984, 6:8!+103). Biochemical investigations have shown that ZP3 is composed of two structumlly and immunologically distinct molecules, ZP3a (deglycosylated molecular weight, 37 kD) and ZP3p (deglycosylated molecular weight, 32 kD; Yurewicz et al, J Biol C?wm 1987, 262:567-571). The two components
ZP3-EBGDol and ZP3-EBGDP have been isolated after partial deglycosylation by endo g-galactosidase digestion (EBGD) [2] and their relative immunogenicity and antigenicity studied. Five preparations of the ZP3 glycoprotein family were investigated, namely ZP3, three enzymically deglycosylated preparations (ZP3-EBGD, ZP3EBGDu and ZP3EBGDp) and a chemically deglycosylated preparation, ZP3-DG. Antisera raised in both mice and rabbits revealed a distinct order of immunogenicity for the different preparations: ZP3 and ZP3-EBGDP > ZP3EBGD and ZP3-EBGM > ZP3-DG. Therefore, when carbohydrate is removed, either enzymically or chemically, ZP3 becomes less immunogenic, suggesting that carbohydrate side chains are important in conferring immunogenic&y. Although all the antisera formed a dense immunoprecipitate on the surface of porcine ZP, antisera to ZP3-EBGDa, ZP3-EBGDB and ZP3DG only formed a discontinuous precipitation layer, possibly because the epitopes recognized by these antisera are less numerous and are spatially distant. Pretreatment of ZPs with antisera to ZP3,ZP3EBGD and ZP3EBGDu inhibited boar sperm binding in a dose-dependent manner but pretreatment with antisera to ZP3-EBGDP and ZP3-DG did not inhibit binding. The authors concluded that sperm receptor activity is associated with ZP3EBGDu. These results contrast with those of Henderson et al. (Gamete Res 1987, 18:251-265), who showed that antisera to DGZP32/36 (similar to ZP3-DG) inhibited the binding of human sperm to salt-stored human ZPs by approximately 90% and that a surface immunoprecipitate was formed (Fig. 1). In addition, antisera against DGZP-32 (equivalent to chemically deglycosylated ZP3-EBGDP) inhibited sperm binding by almost 95%. Whether these discrepancies are due to differences in the method of isolation of the immunogen or are species-related has yet to be ascertained.
Influence of carbohydrate The carbohydrate moieties of porcine ZP3 are clearly important because of their immunogenic&y and involvement in sperm receptor functions. However, a contraceptive vaccine based on carbohydrate residues may be technically d&At to achieve at present because of the problems associated with synthesizing precisely deiined carbohydrate sequences, either chemically or as part of a recombinant glycoprotein molecule. Consequently, several groups have tried to determine whether antigens with contraceptive potential reside within the polypeptide backbone. when porcine ZP is chemically deglycosylated using the reagent trifluoromethane sulphonic acid, it resolves into iive major bands on sodium dodecyl sulphate polyacrylamide gel electrophoresis; these have molecular weights of 66, 52, 36, 32 and 16kD. Antisera against the 32kD peptide are capable of suppressing human sperm-human egg interactions in vittr, (Henderson et al, 1987). Aitken et al. (In CONR4DETZO Znternutionul Work.dh@ on Gamete Interaction eded by N.
Development of vaccines targeting the zona pellucida Paterson and Aitken
juvant used, the difference in immunogenkity between ZP3 and DGZP-32 was immediately apparent. A primary injection of 100 clg glycosylated ZP3 induced an immediate elevated antibody response whereas primary administration of DGZP-32 gave a weak antibody response or no response at all. The poor immunogeniclty of DGZP32 combined with the mild adjuvant induced low antibody titres, and three out of the four marmosets conceived shortly after the introduction of a male. In these animals, the induction of immunity was associated with a reversible disruption of ovarian cyclicity. The fourth animal, which developed the highest antibody titres, continued to cycle normally and remalned infertile throughout the course of the study ( > 12 months). The animals immunized with DGZP-32 emuls&d in NUFA had higher antibody tines; this was directly related to their infertility as three of the four marmosets with the highest titres remained infertile. Sporadic minor disruptions in the normal ovulatory pattern were reported but the pattern quickly returned to normal. Only one out of the five animals immunized with ZP3 showed any sign of ovarian dysfunction, but this group of animals had the highest antibody levels of all, quickly attaming titres of about 1: 20480 (Fig. 2). It is therefore difficult to ascribe these disruptions in ovarian function directly to the presence of anti-ZP antibodies. These disturbances do, however, seem to coincide with the administration of antigen. It has been suggested that the type of adjuvant used in an immunization programme may alkt ovarian folliculogenesis. In a study by Upadhyay et al [3], bonnet monkeys were immunized with ZP3 using either Freund’s complete adjuvant (FCA) or sodium phthalylated lipopolysaccharide (SPLITS) as adjuvants. Whereas immunlzation with SPLPS elicited an antibody response without affecting ovarian follicular development, immunization with FCA resulted in high antibody titres and ovarian follicu lar atrophy. This suggests that FCA is not the adjuvant of choice when immunizing with ZP antigens. However, in the study by Aitken et al. (1989), control animals given NUFA alone continued to cycle normally.
Fig. 1. Cross-reactivity of human and pig zonae pellucidae demonstrated by dark-field and indirect immunofluorescence studies. Salt-stored human ova were incubated for 1 h at 37°C in a poiyclonal antiserum raised against porcine DCZP-32. (a) Examined under dark-field illumination. (b) Incubated for a further Mmin in a second, fluorescein-labelled antibody raised against rabbit IgG and observed using a fluorescence microscope.
Alexander. Alan R I&s, 1989) compared this deglycosylated polypeptide with ZP~, its fully glycosylated coun terpart, using an active immunization protocol in a primate model, the common marmoset. Two different adjuvants were used to enhance the immune response: a mlld adjuvant composed of peanut oil, muramyl dipeptide and aluminuim hydroxide (alum), or, alternatively, one composed of non-ulcerative Freund’s adjuvant (NUFA), bacille Calmette-G&in and alum. Irrespective of the ad-
Dunbar et al. [4] investigated the use of a synthetic peptide adjuvant (muramyl dipeptide) in the immunization of baboons with glycosylated and deglycosylated antigens. Using ZP3 and an SOkD deglycosylated porcine peptide (equivalent to deglycosylated ZPl) as immunogens, they found that the glycosylated molecule elicited a higher immunological response. In two of the live animals that received ZP3, cyclic&y was disrupted; they become amenorrhoic by the eighth month. No visible changes in Q~licity were observed in the group receiving the SOkD peptide. Histopathologkal examination of the ovaries of both groups after nine cycles (or their equivalent), indicated that long-term immunization had altered the development of primary follicles, but these effects were more apparent in the ZP3-immunized group. Future research should, therefore, focus on identifying individual antigenie epitopes that inhibit fertilization but do not alter ovarian function.
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Fig.2. (a)Antibody titre and plasma progesterone values for a marmoset immunized with the 32 kD deglycosylated porcine zona peptide.
Note the temporary impairment of ovarian cyclicity following the primary, first and second booster injections. fb) Antibody titres and progesterone levels in a marmoset immunized with ZP3 in an adjuvant composed of non-ulcerative Freund’s adjuvant, bacille CalmetteGuerin and aluminium hydroxide Note the rapid rise in antibody titres and the normal patterns of ovarian cyclicity. Titres have no units as such and are taken to be the highest dilution of antisera at which an antibody/antigen reaction still occurs.
Syntheticantigens Of 4 the mammal& ZPs studied so far, that of the mouse is the best characterized. Using information gained from the cloning of the mouse ZP3 gene (Ringuette et al, Dev Bid 1988, 127:287-2951, hdlar et al. [5] have developed an alternative approach to contraceptive vaccination. Using a monoclonal antibody to ZP3 that blocked fertilization in the mouse, they were able to locate the epitopes responsible for this contraceptive effect A 1.Okilobase complementary DNA encoding these epitopes was cut into random fragments and cloned into the expression vector kgtl 1. This library was then screened using the monoclonal antibody and eight positive clones were obtained. A nucleic acid sequence common to all the positive clones was identified. This encoded a seven-amino-acid peptide (residues 336-342) recognized by the antibody. A 16-amino-acid peptide representing the sequence 328-343 was synthesized and conjugated to keyhole limpet haemocyanin (KLH) via the
N-terminal cysteine residue. Active immunization of mi&e with this conjugate elicited varying antibody titres, separating the mice into two groups, high and low responders. When mating trials were carried out, the control animals (given KLH with adjuvant) gave birth to litters within 3r/2weeks of the introduction of a male. Three animals, found to be low responders, also gave birth within the first month. A contraceptive effect lasting for 16-36 weeks was observed in the remaining mice and it was directly related to antibody titre. Ovaries removed from the immunized mice were tested with an anti-mouse IgG horseradish perox&% cbnjugate to determine whether circulating antibodies bound exclusively to the ZP or whether there was any evidence of cross-reactivity. Millar et al. [5] found that antibodies were localized to the ZPs surrounding growing oocytes and that the ovaries looked normal, with no evidence of inflammation or cellular cytotoxicity. They suggested several reasons for the success of their vaccine in producing
Development of vaccinestargetingthe zona pellucida Paterson and Aitken
long-term infertility in the absence of any visible ovarian dysfunction. Firstly, the synthetic ZP3-KU-I immunogen has a well defined sequence, unique to the ZP3 molecule, and antibodies raised against it should not cross-react with any other ovarian antigen. Secondly, the lack of Tcell epitopes within this short amino acid sequence may have prevented a cytotoxic T cell response to the ZP. The identication of B-cell and T-cell epitopes located within the amino acid sequence of the ZP3 polypeptide may be an important step towards production of a vaccine capable of eliciting an effective antibody response without any cytotoxic effects.
Annotated
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Several groups [6,7] have observed the transfer of an oviductal antigen to the surface of the ZP after ovulation. Studies using monoclonal antibodies against the oviductal ZP of the golden hamster have provided evidence that this glycoprotein, when released into the follicular fluid by the non-ciliated secretory cell of the oviduct 161, becomes intimately associated with the matrix of the ovulated ZP. Sakai ef al [7] have identified a similar antigen (ZP-0) that is only present on ovulated eggs. Initial investigations showed that this was a high molecular weight glycoprotein of about 200-240kD. A monoclonal antibody that recognized this antigen was able to form an immunoprecipitate on the surface of the ZP and to block fertilization in a dose-dependent manner. However, this antigen is not necessary for fertilization as follicular oocytes can be fertilizd in vitro without exposure to oviductal fluids. The antigen seems to be associated with early embryonic development and survivzl and its value as a candidate antigen for contraceptive purposes has yet to be fully investigated.
Conclusions Recent publications have emphasized the importance of identifying the epitopes on the surface of the ZP that are uniquely involved in the fertilization process. A potential vaccine should involve antibodies that inhibit fertilization by preventing sperm binding to and penetrating the oocyte without causing ovarian dysfunction. From the literature, it would seem that the use of highly purilied well defined epitopes as imrnunogens is a significant step in this direction. A rwersible contraceptive vaccine must provide a well de&d period in which a woman is protected et pregnancy. With a knowiedge of the precise antigenic dete rminants involved in fertilization, and their mechanism of action, it should be possible to engineer vaccines in which the disturbance of ovarian function is minimized or eliminated.
Of interest Of outstanding interest
with porcine zona pe&ci&. Am J Reprod Immund Micro bid 1988, 1894-103. Report on the immunization of bitches with crude or paaiallypuw porcine Zp to determine the cause of abnormal oestrus cycles.
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MAHI-BROWN CA, YANAGIMACHI R, NELSONMI, YNWGIMACHIH, PALIJMBO N: Ovarian histopathology of bitches immunized
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2.
Oviductal
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SACCO AG, YUREWICZEC, SUEIRAWNLWMG, MATZAT PD: Porcine zona pellucidaz association of the sperm recep
tor activity with the a-glycoproteti component of the Mr=55,OOO family. Bid Re_Dnxl1989,41:52%532. Report on the immunogenicity and sperm receptor activity of liw difof ZP3. Demonstrates the kxalization of the sperm ferent preparations receptor to ZP3a. S, UPAJXWAY SN, ?%IUAlKOOTHANP, BAMEZU 4 JAYARAMAN TALWARGP: Role of adjuvants in inhibitory infhwnce of
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immunization with porcine zona pellucida antigen (ZP3) on ovarian folliculogenesis in bonnet monkeys: a morphological study. Bid R@roci 1989, 41:665-673. Examines the use of FCA and SPLPSas adjuvan@ when immunizingwith ZP3. FCA caused ovarian follicular atrophy whereas no morphological damage was obsetved with SPLPS.Howeve r, the paper does not include any adjuvant controls. 4.
DLJNBAR BS, Lo C, POWEUJ, STEVENS VC: Use of a synthetic peptide adjuvant for the immunization of baboons with demuured and deglycosylawd pig xma pellucida &coproteins. Fe&l S&III 1989, 52:3113X Describes the use of muramyI dipeptide as an &ective adjuvant for the immunization of baboons with ZP3 and an 80kD peptide. Antibodies rakwdagainstthesedf%e rminants have a contmceptive effect but may also interfere with normal ovarian function. l
MIUARSE, CHAMOWSM, BAURAw, OLIVERC, ROBEYF, DEW J: Vaccination with a synthetic zona pebcida peptide produces long-term contraception in female mice. Science W89, 246:935-938. This paper describes the kientjfication of a seven-amino-acid sequence from the mouse ZP which is able to prcduce long-lasting contraception. First report of a short synthetic peptide sequence that is capable of inducing long-term infertility. 5. l
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KANFWK, ST-JACQUES S, BLEW G: Immunocytochemical evidence for the transfer of an oviductal antigen to the zona pellucida of hamster ova after ovulation. Bid Reproci 1989, 40:58%598. These authors use a monoclonal antibody against oviductal zona to demonstrate the secretion of an oviductal antigen which becomes closely associated with the ZP of oocytes during their passage through the oviduct. l
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S&u Y, ARAKIY, Y.U&WITAT, KUWA S, OIKAWAT, HXRO~ M, SENDOF: Inhibition of in vitm fertihaticm by a mOnOdOn& antibody rexting with the zona pellucida of the oviductal egg but not with that of the ovarian egg of be golden
hamste-r. J R@mxi Immund 1988, 14:177-190. This paper demonstmtes that a monoclonal antibody that recognizes a zona antigen @P-O), presentonlyon oviductal oocytes, is able to block fertilization in virra
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