Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Skinner, S. M.
Right arrow Articles by Dunbar, B. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Skinner, S. M.
Right arrow Articles by Dunbar, B. S.
Agricola
Right arrow Articles by Skinner, S. M.
Right arrow Articles by Dunbar, B. S.
Biology of Reproduction 61, 1373-1380 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Mapping of Dominant B-Cell Epitopes of a Human Zona Pellucida Protein (ZP1)1

Sheri M. Skinnera, Eric S. Schwoebela,b, Sarvamangala V. Prasada, Mary Ogunab, and Bonnie S. Dunbar2,a

a Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030-3411 b Institute of Primate Research, Nairobi, Kenya


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Zona pellucida (ZP) glycoproteins contain numerous antigenic determinants including carbohydrate, protein, and conformational epitopes; and the immunogenicity of these complex glycoproteins varies in different mammalian hosts. Studies have now shown that antibodies from primates immunized with a cDNA-expressed recombinant rabbit ZP protein (the homologue of the human ZP1 [hZP1]) inhibit sperm binding to the ZP without altering ovarian function, unlike immunization with ZP3 and ZP2 protein families. The ZP1 protein or peptides derived from it (recombinant or synthetic) are therefore primary candidates for use in designing safe and reversible human and animal contraceptive vaccines. In order to define peptide epitope(s) that may be critical for eliciting an immune response sufficient to effect immunological contraception without causing any adverse effects on ovarian physiology, studies have been carried out to identify immunodominant B-cell epitopes of the ZP1 protein. The amino acid sequence of the hZP1 was used to design a set of 94 (15-mer) biotinylated peptides having an overlap of 9 amino acids. Using these peptides in a modified enzyme-linked immunoassay, antibodies in sera from rabbits or baboons immunized with native porcine ZP protein were screened for ZP1 peptide recognition.

These studies demonstrate that there are a limited number of peptides recognized by primate antibodies but that the overlapping peptides sharing the sequence GPLTLELQI are recognized by both rabbit and baboon antibodies regardless of the adjuvant system used to induce the immune response. This peptide is 100% conserved in amino acid sequence between the human and pig, although the rabbit protein has two conserved amino acid substitutions (100% similar, 77% identical). Because this peptide is immunogenic as well as antigenic in primates, it could play a major role in the development of human contraceptive vaccines.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The zona pellucida (ZP) is the extracellular glycoprotein matrix formed during mammalian ovarian follicular development. The proteins of this ZP matrix have now been shown to be expressed and secreted by the oocyte as well as the granulosa cells [1–6]. The ZP is involved in several critical stages of the fertilization process, since the capacitated spermatozoa attach and bind to the ZP proteins, inducing the acrosome reaction (see reviews [7–9]). While it has long been noted that the mammalian ZP is composed of three major glycoproteins, the classification of these proteins has been complicated because of extensive species variation in protein posttranslational modification, including glycosylation and sulfation. Because of these modifications, the proteins exhibit extensive heterogeneity. Therefore, the nomenclature, based on electrophoretic mobilities of the ZP proteins of different species, has been difficult. As the cDNAs and genes encoding the ZP proteins have been isolated, it has only in the last decade been possible to distinguish these ZP gene families [9–21]. For clarification of ZP terms used in this study, a summary of nomenclature of these gene families as they relate to the mouse ZP nomenclature is provided in Table 1.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Comparison of the sequence homology of cDNAs of ZP proteins

Although the genes encoding the ZP proteins are evolutionarily conserved, there appear to be major differences in these proteins relative to their immunochemical and functional properties (see reviews by Prasad et al. [22]). In the mouse, ZP3 has been shown to be the primary sperm receptor with a specific class of O-linked oligosaccharides involved in sperm binding [23], while N-linked, but not O-linked, carbohydrates are implicated in sperm binding to porcine ZP [24, 25]. However, the rabbit ZP1 (55 kDa) and pig ZP1 (also referred to as ZP3 alpha), which are homologues of the mouse ZP1, but not the mouse ZP3 family, have been shown to contain sperm receptor activity [16, 26].

Recent studies demonstrate that sperm/ZP interaction in many mammals, including the human, may be more complicated than that proposed for the mouse model. Both protein and carbohydrate moieties appear to be involved in sperm binding in pigs [25–27], and multiple ZP proteins may be involved in this interaction in pigs and rabbits. Regardless of the precise molecular mechanisms of sperm interaction with the ZP, the efficacy of immunization with zona pellucida proteins for contraception will ultimately depend upon the production of sufficient antibody titers to inhibit fertilization at any stage of the fertilization process.

As has been demonstrated, the antigenic repertoire of ZP consisting of carbohydrates, proteins, and their complexes presents a complex immunogenicity profile and results in immune responses of diverse specificity among different mammalian hosts (see reviews by Dunbar and colleagues [9, 13, 22, 28]). To date, most of the studies to identify specific peptide epitopes have used monoclonal antibodies made in mice [14, 29–32]. Since the immunogenicity profile of nonrodent ZP proteins injected into mice is distinct from that in other mammals, and since immunization of mice and rats with porcine ZP does not affect fertility [33,34], it is apparent that it is critical to study the nature of the immune response in primate models if we are to design a safe and effective human contraceptive vaccine.

To date, the primate models that have been used in such studies include baboons (Papio anubis) [35], squirrel monkeys (Saimiri sciureus) [36], marmosets (Callithrix jacchus) [37], and cynomolgous macaques (Macaca fascicularis) [16, 38, 39]. Aitken and colleagues [40] have demonstrated that immunization of marmosets with native porcine ZP3 and recombinant cDNA-expressed ZP3 protein interferes with normal ovarian function as evidenced by histological analysis and fertility. Studies by Dunbar and colleagues, however, have used the cynomolgus monkey model to demonstrate that immunization with recombinant rabbit 55-kDa ZP protein (ZP1 protein family) conjugated to protein A produces a significant humoral immune response when used with the muramyl dipeptide [38, 41]. These monkeys retain normal endocrine function as well as ovarian morphology. However, as importantly, the antibodies developed by these animals inhibited binding of monkey sperm to homologous monkey ZP. Because the ZP1 therefore appears to be an excellent candidate for immunocontraception in the human, the present studies have been designed to define specific peptide epitopes of the hZP1 that are recognized by primate antibodies generated against native ZP proteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
ZP Glycoprotein Immunogen Preparation

Pig ovaries were obtained frozen from slaughterhouses, and large-scale isolation of intact ZP was carried out as previously described by Wood et al. [42]. ZP glycoproteins were solubilized and protein concentration was determined by the method of Lowry et al. [43], and protein purity was evaluated by high-resolution two-dimensional gel electrophoresis [44].

Immunization and Characterization of Baboon Antisera

Eleven sexually mature female baboons were housed in two group cages according to ethics-approved, standardized practices at the Institute of Primate Research, Nairobi, Kenya, and in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction. Animals were fed monkey cubes supplemented with ascorbic acid powder, with fruit and vegetables three times per week. Water was provided ad libitum.

Lyophilized porcine ZP immunogen (heat-solubilized porcine zonae) (HSPZ) was prepared and solubilized in 0.1 M sodium carbonate, pH 9.6, and heated at 60°C for 10 min as previously described [42, 45]. Immunogen was emulsified in Titer Max adjuvant (CytRx Corp., Norcross, GA; 50%) by passage through syringes with 19-gauge needles connected by Tygon tubing (Norton Performance Plastics, Akron, OH). Animals were sedated with ketamine (Fort Dodge Labs, Fort Dodge, IA; 10 mg/kg) before injection with 200 µl containing 150 µg ZP immunogen (primary injection and boosts). Animals were immunized and then boosted at 3-wk and 6-wk intervals.

Blood samples were collected at approximately 3-wk intervals following the booster injections, and were obtained from the femoral vein under ketamine sedation. Serum was isolated by conventional methods and transported at -70°C to the laboratory facility of B.S. Dunbar at Baylor College of Medicine, Houston, TX, for analysis. For the present studies, serum samples taken at 3 wk after the second booster injection were used. The ELISA previously described [46] was used to determine the levels of antibody against immobilized HSPZ. In addition, sera from baboons of a previously published study [35] were used as a positive control for these assays. In that study, electrophoretically purified, denatured porcine ZP proteins were used as the immunogen along with the muramyl dipeptide adjuvant system. The immunogens were prepared using chemical deglycosylation of proteins to remove both O- and N-linked carbohydrates with trifluoromethane sulfonic acid. Deglycosylated proteins were then electrophoretically purified and contained the proteins of the porcine ZP3{alpha} and ZP3ß families that are homologues of the human ZP1 and ZP3 proteins described in Table 1. Nonimmune sera from these animals immunized with adjuvant alone were used as a negative control.

Immunization of Rabbits and Characterization of Rabbit Antibodies

Antisera from infertile rabbits immunized with HSPZ were used as controls in these studies since this antisera has been well characterized and these antibodies inhibit sperm binding to the ZP [16, 42, 47].

Biotinylated Peptide Assays

Preparation of biotinylated peptides Peptides containing 15 amino acid residues, which overlapped by 9 amino acids, were designed to ensure that all potential epitopes would be identified. These biotinylated peptides were synthesized by Chiron Mimotope Peptide Systems (San Diego, CA). Aliquots of these peptides were used once per assay and discarded. This peptide system was found to be preferable to the immobilized peptide Mimotope system also offered by Chiron. For the biotinylated peptide assay, we chose 15-mers with an overlap of 9 amino acids of the hZP1 protein sequence as shown in Figure 1. Additional peptides (peptides #92–96) included rabbit and porcine peptides chosen from the areas of these ZP1 proteins that are homologous with the #25–39 portion of the human ZP1 sequence.



View larger version (109K):
[in this window]
[in a new window]
  		FIG. 1. Summary of peptides used for modified ELISA studies. The amino acid sequence constructs include biotin-SGSG-peptide-NH2 with SGSG as the linker designed by the manufacturer. Peptides #3–91 are derived from the amino acid sequence of the human ZP1 glycoprotein, while peptides #92–95 are derived from the sequence of the rabbit ZP1 protein aligned with sequences #19–51 of hZP1. Peptide #96 is the porcine ZP1 sequence that aligns with hZP1 sequence #25–39. (Note that peptides #1 and #2 are control peptides retained by Chiron, the peptide manufacturer, and are therefore not relevant to this study.)

Initially, each peptide was solubilized in 200 µl of dimethyl sulfoxide according to the manufacturer's instructions and stored long-term at -80°C. Approximately 2 µl is used to make a 1:5000 stock solution diluted in PBS containing 0.1% BSA and 0.1% sodium azide. These stocks are then stored at -20°C and are diluted 1:10 000 for assays.

Enzyme immunoassay Streptavidin (Sigma Chemical Co., St. Louis, MO) (#S-4762) was diluted to 5 µg/ml in deionized water and dispensed at 100 µl/well into 96-well microtiter plates (Immulon 2; Dynatech, Dynex Technologies, Chantilly, VA), which were incubated overnight at 32°C to evaporate to complete dryness. Plates were then rinsed four times in wash buffer of pH 7.3, consisting of 0.02 M sodium phosphate containing 0.15 M NaCl and 0.05% Tween 20. The plates were blocked using 200 µl per well of 0.02 M sodium phosphate-buffered saline (PBS) containing 2% nonfat dried milk (pH 8.8) for 2 h at room temperature (25°C), and the rinse was repeated. Blocking and all incubations were carried out using a Minimix shaker (Fisher Scientific, Springfield, NJ). Biotinylated peptides were then dispensed such that the final dilution of each was 1:10 000, made in wash buffer containing 2% nonfat dried milk. Peptides (100 µl per well) were incubated overnight at 4°C on the shaker.

After the plates were rinsed with blocking solution, 100 µl/well of antibody solution was added, and the plates were incubated at 25°C for 2 h with shaking. Appropriate antibody dilutions for this assay were predetermined by titering in an ELISA using antigen related to the biotinylated peptides. Plates were rinsed, and secondary antibody (peroxidase-labeled, affinity-purified goat anti-human IgG [heavy and light chain; #074–1006; Kirkegaard and Perry, Gaithersburg, MD]; 0.25 µg/ml) was added at 100 µl/well. Diluent used for both primary and secondary antibodies was wash buffer containing 2% nonfat dried milk. Secondary antibody was incubated in plates for 1 h at 25°C with shaking. After being rinsed, the plates were loaded with 100 µl/well chromophore/substrate. This consisted of a freshly made solution of 2,2'-azino-di-[3-athyl-benzthiazolinsulfonate] (ABTS; #102–946; Boehringer Mannheim, Indianapolis, IN) in substrate buffer. The substrate buffer was 0.1 M sodium phosphate containing 0.08 M citric acid (pH 4). To 60 ml of this buffer was added 30 mg ABTS, which was dissolved completely before addition of 200 µl of a solution of 4.5 ml filtered H2O (MilliQ filter system; Millipore Corp., Bedford, MA) and 0.5 ml 30% H2O2 just before use. The plates were incubated at room temperature with shaking for 45 min and read in a Titertek Multiskan reader (Flow Laboratories, McLean, VA) at 405 nm.

Controls for these assays included the following: wells lacking streptavidin, streptavidin and primary antibody, or primary antibody; wells in which test antibody was replaced with unrelated (negative control) antibody or with known reactive (positive control) antibody; and wells containing peptides known to react or not with pretitered control (positive or negative) antibodies. Data were processed by subtraction of mean background values (obtained by elimination of primary antibody in the assay), and the signals were expressed as optical density units (O.D.) [48].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of Antibodies Using the Biotinylated Peptide Assay

Antibodies that had been previously characterized by ELISA, immunohistochemistry, and two-dimensional PAGE immunoblotting [11, 35, 47] were used as controls for these studies. These well-characterized antisera were used as positive controls to optimize screening of antibodies recognizing biotinylated hZP1 peptides (Fig. 2). Figure 2A illustrates the results of the identification of peptides recognized by antibodies from control baboons immunized with adjuvant alone (Titer Max adjuvant system) while Figure 2C illustrates the pattern obtained with antisera from baboons immunized with native pig ZP proteins using the Titer Max adjuvant. Figure 2B illustrates peptides recognized by antibodies from rabbits immunized with the same ZP protein (using the Complete Freund's Adjuvant system, GIBCO Labs., Grand Island, NY). While the overall epitope recognition patterns of the serum pools of the two different species were distinct, it was of interest that two peptides (#54 and #55) were recognized by both. These overlapping peptides share the sequence: GPLTLELQI.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 2. Modified ELISA using overlapping 15-mer biotinylated peptides of the hZP1 protein to evaluate specific peptide epitopes recognized by baboon and rabbit antibodies. Complete set of overlapping biotinylated peptides probed with A) antiserum from baboon immunized only with Titer Max adjuvant, B) antisera pooled from infertile rabbits immunized with HSPZ, or C) antisera pooled from baboons immunized with HSPZ. Background signal of control and nonreactive peptides has been subtracted before graphic representation. (Note that peptide #98 is a manufacturer positive internal control for the assay.)

Because of this correlation in antibody recognition of this common peptide epitope by two distinct mammalian species, it was of interest to evaluate the individual responses of nonhuman primates to the ZP1 protein family. Figure 3 shows the peptides recognized by antibodies in sera from four baboons immunized with native ZP proteins using the Titer Max adjuvant (compare with control baboon immunized with adjuvant only in Fig. 2A). These studies demonstrated that these primates had developed antibodies against a limited number of human ZP1 peptides. However, all four of the animals recognized the peptides #54 and #55. While one of these animals (#2032) had a low titer as compared to those of the others, the peptide recognition was still evident. No antibodies against the ZP peptides were observed in the sera of a control adjuvant-injected animal shown in Figure 2A (or in other adjuvant controls, data not shown).



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 3. Modified ELISA using overlapping 15-mer biotinylated peptides of the hZP1 protein to evaluate specific peptide epitopes recognized by antisera of baboons immunized with HSPZ and the Titer Max adjuvant. Complete set of overlapping biotinylated peptides probed with sera collected 3 wk after second booster from individual baboons. Sera dilutions at 1:1000. A) Antiserum from baboon #1994, B) antiserum from baboon #2020, C) antiserum from baboon #2044, D) antiserum from baboon #2032. (Note: Control sera from baboons immunized with Titer Max alone recognized none of the ZP peptides.)

In view of these observations, we tested additional sera from baboons of a previously published study [35] in which electrophoretically purified, denatured porcine ZP proteins were used as the immunogen. These preparations, using chemical deglycosylation of proteins before one-dimensional SDS PAGE purification, contain the proteins of the porcine ZP3{alpha} and ZP3ß families that are homologues of the human ZP1 and ZP3 proteins discussed in this manuscript. The antibodies in the sera of these two baboons recognized more numerous peptides (Fig. 4) than those immunized with the native ZP. However, the peptides #54 and #55 were again recognized.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 4. Modified ELISA using overlapping 15-mer biotinylated peptides of the hZP1 protein to evaluate specific peptide epitopes recognized by antisera of baboons immunized with electrophoretically purified porcine ZP protein (containing both ZP1 and ZP3) using the muramyl dipeptide adjuvant [35]. Complete set of overlapping biotinylated peptides probed with sera collected 3 wk following second booster. Sera dilutions at 1:1000. A) Antiserum from baboon 1 (Zerna); B) antiserum from baboon 2 (Winnie)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Relevance of the Antigenic Repertoire of ZP Proteins to Allogeneic Immunogenicity

The development of a ZP-based contraceptive vaccine has been the subject of many studies for over two decades. As these studies have progressed, they have demonstrated the complex immunogenicity of the three major ZP glycoprotein families. This complexity is clearly a reflection of the antigenic diversity presented to the components of a host's immune system such as major histocompatibility, MHC (human leukocyte antigen [HLA]) complexes. In addition, these studies have shown the variations in amino acid sequence as well as in carbohydrate composition structures of the ZP of different species, complicating the immunogenicity profile further. (See reviews [8, 9, 16, 22, 28].) Major ZP-specific antigenic determinants include not only sequential peptide and conformational determinants, but also carbohydrate moieties, all of which vary among species. In addition to these specific determinants, there are also epitopes shared among species.

It was first observed that when rabbits were immunized with native rabbit ZP (alloimmunization), no antibodies were elicited nor did immunization cause infertility [42]. These and subsequent studies have further demonstrated that rabbits immunized with porcine ZP (heteroimmunization) develop high titers of antibodies that not only react with porcine ZP but also recognize native rabbit ZP [42,45]. These observations corroborate the general rule that mammals, including humans, do not normally generate antibodies to self antigens. However, immunization with a cross-reactive foreign antigen can initiate an immune response to self antigens [49]. Furthermore, mice and rats immunized with porcine ZP do not develop antibodies that result in infertility [33, 34]. These studies clearly demonstrate that, in addition to the immunogenicity of ZP proteins, there is a stringent requirement for appropriate epitopes to engender the functional antibodies capable of blocking effective sperm-ZP binding. Heterogeneity, specificity, and promiscuity among naturally processed antigens have been observed. For the development of a practical contraceptive vaccine effective for humans, it is paramount to identify a promiscuous epitope able to induce antibody responses across heterogeneic HLA types. The present studies, therefore, have focused on identification of such an epitope in the ZP1 protein. Also, to override the diversity of immunogenicity problem in mice as compared to other mammals, we have used antibodies developed in baboons immunized with porcine ZP and different adjuvant systems. These studies have identified a human ZP epitope that is recognized by antibodies in serum pools from rabbits and from baboons as well as in serum from individual baboons.

Effects on Ovarian Function After Immunization with ZP Proteins

Since it was first observed by Dunbar and colleagues that immunization with ZP proteins can result in ovarian dysgenesis as well as infertility [42, 45], it has become necessary to determine whether ZP immunogens could be developed that would inhibit fertilization but not interfere with ovarian function in the human. Because the goal for such a contraceptive vaccine is safety in humans, it has been essential to carry out these studies in primates.

Tung and colleagues have carried out studies using the mouse model ZP3 glycoprotein [50–53]. Although the ZP3 molecule is not homologous to the ZP1 family, which has been shown to be involved in sperm-ZP interaction in the rabbit, pig, and cynomolgous monkey [16, 25, 26, 54], it has served as a valuable model for such studies. These studies have demonstrated that autoimmune oophoritis, as it is mediated by T-cell activity, can in fact be successfully dissected from the B-cell-mediated antibody response to the ZP antigen, which provides contraceptive action. Tung has further shown that T-cell-mediated oophoritis is a temporary phenomenon, giving rise to a refractory condition upon immunological challenge, and is therefore not an appropriate approach to contraceptive vaccine development [14, 52]. It is apparent in these studies, however, that pertussis toxin was used to elicit the observed immune responses. Pertussis toxin has often been used to induce IgE responses [54, 55], and this adjuvant has long been known to induce autoimmune disorders [56–58]. It is not clear that this rodent model is applicable to the primate, including the human, in which such immunization procedures are prohibited.

Studies in primates have also shown that immunization with the ZP proteins can also adversely affect ovarian follicular development [35–40]. However, studies in the cynomolgus monkey [38], demonstrating that the rabbit homologue of the human ZP1 is immunogenic in monkeys and elicits an antibody response without interfering with ovarian function, provide important evidence supporting the use of this ZP protein as a contraceptive immunogen. These studies are also important in that the antibodies produced against this ZP1 immunogen inhibit binding of monkey sperm to the monkey ZP. (Unfortunately, there were no additional sera available from these earlier studies to test using the peptides described in the current studies.)

Identification of Specific ZP Peptides for Immunocontraceptive Development

A number of studies have been carried out to identify ZP peptides that can be used for immunocontraception. These have included ZP3 peptides from the mouse, human, and pig [51, 59–65]. Since the ZP1 protein family in the pig and rabbit has been shown to be important in sperm-zona binding, monoclonal antibodies against pig ZP that inhibit sperm-egg interaction have been used to epitope-map pig ZP1 peptides [48, 60, 62]. Using antisera directed against peptide corresponding to an amino terminal segment of pig ZP1, Yurewicz et al. [66] have demonstrated that the N-terminal region (residues 144–154) is involved in sperm-ZP interaction. The present study, however, is the first to use the biotinylated peptide procedure to identify specific B-cell epitopes of ZP proteins in any species. Other investigators have utilized overlapping peptides in ELISA assays to evaluate B-cell epitopes in pig ZP1 and rabbit sperm antigens [48]. More importantly, however, this procedure is the first to identify human ZP B-cell epitopes that are recognized in primates. Because this peptide is also recognized by rabbit antisera, it is apparent that it may be a species "universal" epitope.

Of interest is that the relative reactivity of the antibody binding to the #54 and #55 peptides varies among some individuals. As these two 15-mer peptides have an overlapping sequence of 9 amino acids, it will be of interest to identify the precise antigenic determinant within these overlapping sequences by use of different sera.

These studies also demonstrate that this sequence is highly conserved among the pig, human, bonnet monkey [67], and rabbit, although there are numerous differences from the mouse sequence (Table 2). This may be the reason why immunization of mice or rats [33, 34] with pig ZP proteins does not induce infertility. In contrast, Gupta et al. [30] have identified two regions in the pig ZP1 sequence that are recognized by monoclonal antibodies that inhibit sperm-oocyte interaction. However, the sequence in these regions is not well conserved among the pig, rabbit, and human; and both monoclonal antibodies failed to react with rabbit and human oocytes in immunofluorescence assays.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Comparison of optimally aligned amino acid sequences of dominant peptide epitope of the ZP1 protein family

Of further significance, computer-based sequence comparison of the peptide sequence with other proteins in the protein data bank using the Genetics Computer Group (GCG, Madison, WI) Wisconsin Package show no amino acid similarity or identity to any other human protein yet sequenced other than the ZP protein. This is particularly crucial in view of alarming evidence that some viral proteins and vaccines can induce autoimmune disorders thought to be the result of molecular mimicry of the viral proteins [68–76].

Of interest is that the analysis of this peptide structure (using the program peptide structure of CG) revealed the peptide to be hydrophobic with a helical structure, low surface probability, and hence, low antigenicity index [77]. Given these properties, this peptide would not be predicted to be highly immunogenic. However, these studies demonstrate that the peptide contains a significant antigenic epitope. Therefore, the biotinylated peptide assay is a more definitive method for identifying immunodominant epitopes associated with a protein since the computer-based predictions may miss major epitopes.

The present study has identified a B-cell epitope of human ZP1 that is restricted to the zona, and the specificity of the amino acid sequence of this promiscuous epitope renders it the desired immunogen for developing a ZP-based contraceptive vaccine. Further studies are in progress to dissect the ability of this peptide to induce B- and T-cell responses in animal models including primates. It will be critical to demonstrate that such apparently promiscuous and immunodominant sequences, which show cross-reactivity with rabbit and baboon antibodies, are capable of inducing antibody responses in these animals. Furthermore, it will be critical to establish that such epitopes do not have pathogenic motifs within the sequence. In summary, the identification of this human ZP1 B-cell epitope that is recognized by baboon antibodies has important implications for the continued development of a safe, specific, peptide-based contraceptive vaccine.


   ACKNOWLEDGMENTS
 
The authors wish to thank the support staff of the Institute of Primate Research in Nairobi, Kenya, for their major contributions to this project. We also wish to thank Dr. Balbir Bhogal for his comments on the discussion of these results.


   FOOTNOTES
 
1 This project was supported by funding to B.D. from the Contraception and Research Development Program (CONRAD), The Mellon Foundation, and The National Institutes of Health #HD-17543, and by funding to the Institute of Primate Research, Nairobi, Kenya, by the World Health Foundation, The World Bank, and the National Council for Population and Development. Back

2 Correspondence: Bonnie S. Dunbar, Department of Cell Biology, 112A, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3411. FAX: 713 798 7341; bdunbar{at}bcm.tmc.edu Back

Accepted: June 24, 1999.

Received: March 3, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wolgemuth DJ, Celenza J, Bundman DS, Dunbar BS. Formation of the rabbit zona pellucida and its relationship to ovarian follicular development. Dev Biol 1984; 106:1–14.[CrossRef][Medline]
  2. Maresh GA, Timmons T, Dunbar B. Effects of extracellular matrix on the expression of specific ovarian proteins of cultured primary ovarian follicles. Biol Reprod 1990; 43:965–976.[Abstract]
  3. Lee VH, Dunbar BS. Developmental expression of the rabbit 55 kDa zona pellucida protein and messenger RNA in ovarian follicles. Dev Biol 1993; 155:371–382.[CrossRef][Medline]
  4. Kolle S, Sinowatz F, Boie G, Totzauer I, Amselgruber W, Plendl J. Localization of the mRNA encoding the zona protein ZP3{alpha} in the porcine ovary, oocyte and embryo by non radioactive in situ hybridization. Histochem J 1996; 28:441–447.[CrossRef][Medline]
  5. Grootenhuis AJ, Philipsen HLA, de Breet-Grijsbach JTM, van Duin M. Immunocytochemical localization of ZP3 in primordial follicles of rabbit, marmoset, rhesus monkey and human ovaries using antibodies against human ZP3. J Reprod Fertil Suppl 1996; 50:43–54.[Medline]
  6. Totzauer I, Kolle S, Sinowatz F, Plendl J, Amselgruber W, Topfer-Petersen E. Localization of the zona glycoproteins ZPB (ZP3 alpha) and ZPC (ZP3 beta) in the bovine ovary during pre- and postnatal development. Anat Anz 1998; 180:37–43.[Medline]
  7. Wassarman PM. Zona pellucida glycoproteins. Annu Rev Biochem 1988; 57:415–522.[CrossRef][Medline]
  8. Dunbar BS, Prasad SV, Timmons T. Comparative structure and function of the mammalian zonae pellucidae. In: Dunbar BS, O'Rand MG (eds.), A Comparative Overview of Mammalian Fertilization. New York: Plenum Press; 1991: 97–116.
  9. Dunbar BS, Avery S, Lee V, Prasad S, Schwahn D, Schwoebel E, Skinner S, Wilkins B. The mammalian zona pellucida: its biochemistry, immunochemistry, molecular biology and developmental expression. Reprod Fertil Dev 1994; 6:59–76.
  10. Ringuette MJ, Sobieski DA, Chamow SM, Dean J. Molecular analysis of cDNA coding for ZP3, a sperm binding protein of the mouse zona pellucida. Dev Biol 1986; 127:287–295.
  11. Schwoebel E, Prasad S, Timmons T, Cook R, Kimura H, Niu E, Cheung P, Skinner S, Avery S, Wilkins B, Dunbar B. Isolation and characterization of a full length cDNA encoding the 55 kDa rabbit zona pellucida protein. J Biol Chem 1991; 266:7214–7219.[Abstract/Free Full Text]
  12. Lee V, Schwoebel E, Prasad S, Timmons T, Cook R, Dunbar B. Isolation and characterization of a cDNA encoding the rabbit 75-kDa zona pellucida protein. J Biol Chem 1993; 268:12412–12417.[Abstract/Free Full Text]
  13. Skinner SM, Prasad SV, Ndolo T, Dunbar BS. Zona pellucida antigens: targets for contraceptive vaccines. Am J Reprod Immunol 1996; 35:163–174.
  14. Epifano O, Dean J. Biology and structure of the zona pellucida: a target for immunocontraception. Reprod Fertil Dev 1994; 6:319–330.[CrossRef][Medline]
  15. Harris JD, Hibler DW, Fontenot GK, Hsu KT, Yurewicz EC, Sacco AG. Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: the ZPA, ZPB and ZPC gene families. DNA Sequence 1994; 4:361–393.[Medline]
  16. Prasad SV, Wilkins B, Skinner SM, Dunbar BS. Evaluating zona pellucida structure and function using antibodies to 55 kDa ZP protein expressed in baculovirus expression system. Mol Reprod Dev 1996; 43:519–529.[CrossRef][Medline]
  17. Liang LF, Chamowa SM, Dean J. Oocyte-specific expression of mouse ZP2: developmental regulation of the zona pellucida genes. Mol Cell Biol 1990; 10:1507–1515.[Abstract/Free Full Text]
  18. Liang L, Dean J. Conservation of mammalian secondary sperm receptor genes enables the promoter of the human gene to function in mouse oocytes. Dev Biol 1993; 156:399–408.[CrossRef][Medline]
  19. Yurewicz EC, Hibler D, Fontanot GK, Sacco AG, Harris J. Nucleotide sequence of cDNA encoding ZP3{alpha}, a sperm-binding glycoprotein from zona pellucida of pig oocyte. Biochim Biophys Acta 1993; 1174:211–214.[Medline]
  20. Chamberlin M, Dean J. Human homologue of the mouse sperm receptor. Proc Natl Acad Sci USA 1990; 87:6014–6018.[Abstract/Free Full Text]
  21. Thillai-Koothan P, van Duin M, Aitken RJ. Cloning, sequencing and oocyte-specific expression of the marmoset sperm receptor protein, ZP3. Zygote 1993; 1:93–101.[Medline]
  22. Prasad SV, Skinner SM, Dunbar BS. Zona pellucida antigens and the regulation of fertility: an immunocontraceptive approach. In: Coutifaris C, Mastroianni L (eds.), New Horizons in Reproductive Medicine. New York: Parthenon Publishing; 1997: 129–144.
  23. Wassarman PM. Regulation of mammalian fertilization by zona pellucida glycoproteins. J Reprod Fertil 1990; 42:79–87.
  24. Noguchi S, Hatanaka Y, Tobita T, Nakano M. Structural analysis of the N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida. Eur J Biochem 1992; 204:1089–1100.[Medline]
  25. Yonezawa N, Aoki H, Hatanaka Y, Nakano M. Involvement of N-linked carbohydrate chains of pig zona pellucida in sperm-egg binding. Eur J Biochem 1995; 233:35–41.[Medline]
  26. Yurewicz EC, Pack BA, Armant DR, Sacco AG. Porcine zona pellucida ZP3a glycoprotein mediates binding of the biotin-labelled Mr 55,000 family (ZP3) to boar sperm membrane vesicles. Mol Reprod Dev 1993; 36:382–389.[CrossRef][Medline]
  27. Yurewicz EC, Pack BA, Sacco AG. Isolation, composition and biological activity of sugar chains of porcine oocyte zona pellucida 55K glycoproteins. Mol Reprod Dev 1991; 33:182–188.
  28. Skinner SM, Timmons T, Schwoebel E, Dunbar BS. Zona pellucida antibodies; fertility and infertility. Immunol Allergy Clin North Am 1989; 10:185–197.
  29. Epifano O, Liang L, Familari M, Moos MC, Dean J. Coordinate expression of the three zona pellucida genes during mouse oogenesis. Development 1995; 121:1947–1956.[Abstract]
  30. Gupta SK, Bagavant H, Chadha K, Gupta M, Yurewicz EC, Sacco AG. Mapping of immunogenic domains on porcine zona pellucida 3{alpha} and ß glycoproteins by murine monoclonal antibodies. Am J Reprod Immunol 1993; 30:95–100.
  31. Gupta SK, Kaul R, Rajalakshmi S, Sahai P, Yurewicz EC, Sacco AG. Immunoreactivity with native zona pellucida of antibodies against a 19 amino acid synthetic peptide corresponding to human ZP3. J Reprod Immunol 1994; 27:241–247.[CrossRef][Medline]
  32. Gupta SK, Yurewicz EC, Afzalpurkar A, Lrao KVS, Gage DA, Wu H, Sacco AG. Localization of epitopes for monoclonal antibodies at the N-terminus of the porcine zona pellucida glycoprotein pZPC. Mol Reprod Dev 1995; 42:220–225.[CrossRef][Medline]
  33. Drell D, Wood D, Bundman D, Dunbar BS. Comparison of the immunological response in rats and rabbits to porcine zona pellucida. Biol Reprod 1984; 30:445–457.[Abstract]
  34. Sacco AG, Subramanian MC, Yurewicz EC. Active immunization of mice with porcine zona pellucida: immune response and effect on fertility. J Exp Zool 1981; 218:405–418.[CrossRef][Medline]
  35. Dunbar BS, Lo YK, Stevens V. Effect of immunization with purified porcine zona pellucida proteins on ovarian function in baboons. Fertil Steril 1989; 52:311–318.[Medline]
  36. Jones GR, Sacco AG, Subramanian MG, Kruger M, Zhang S, Yurewicz EC, Moghissi KS. Histology of ovaries of female rabbits immunized with deglycosylated zona pellucida macromolecules of pigs. J Reprod Fertil 1992; 95:513–525.[Abstract]
  37. Paterson M, Thillai Koothan P, Morris KD, O'Byrne KT, Braude P, Williams A, Aitken RJ. Analysis of the contraceptive potential of antibodies against native and deglycosylated porcine ZP3 in vivo and in vitro. Biol Reprod 1992; 46:523–534.[Abstract]
  38. VandeVoort CA, Schwoebel ED, Dunbar BS. Immunization of monkeys with recombinant cDNA expressed zona pellucida proteins. Fertil Steril 1995; 64:838–847.[Medline]
  39. Gulyas BJ, Gwatkin RBL, Yuan LC. Active immunization of cynomolgus monkeys (Macaca fascicularis) with porcine zonae pellucidae. Gamete Res 1983; 4:299–307.[CrossRef]
  40. Aitken RJ, Paterson M, van Duin M. The potential of the zona pellucida as a target for immunocontraception. Am J Reprod Imunol 1996; 35:175–180.
  41. Schwoebel ED, VandeVoort CA, Lee VH, Lo YK, Dunbar BS. Molecular analysis of the antigenicity and immunogenicity of recombinant zona pellucida antigens in a primate model. Biol Reprod 1992; 47:857–865.[Abstract]
  42. Wood DM, Liu C, Dunbar BS. Effect of alloimmunization and heteroimmunization with zonae pellucidae on fertility in rabbits. Biol Reprod 1981; 25:439–450.[Abstract]
  43. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193:265–275.[Free Full Text]
  44. Dunbar BS, Liu C, Sammons DW. Identification of the three major proteins of porcine and rabbit zonae pellucidae by two-dimensional gel electrophoresis: comparison with follicular fluid, sera and ovarian cell proteins. Biol Reprod 1981; 24:1111–1124.
  45. Skinner SM, Mills T, Kirchick HJ, Dunbar BS. Immunization with zona pellucida proteins results in abnormal ovarian follicular differentiation and inhibition of gonadotropin-induced steroid secretion. Endocrinology 1984; 115:2418–2432.[Abstract]
  46. Drell D, Dunbar BS. Monoclonal antibodies to rabbit and pig zonae pellucidae differentiate species cross-reactive and unique antigenic determinants. Biol Reprod 1984; 230:435–444.
  47. Timmons TM, Maresh GA, Bundman DS, Dunbar BS. Use of specific monoclonal and polyclonal antibodies to define distinct antigens of porcine zona pellucida. Biol Reprod 1987; 36:1275–1287.[Abstract]
  48. O'Rand MG, Widgren EE. Identification of sperm antigen targets for immunocontraception: B-cell epitope analysis of SP17. Reprod Fertil Dev 1994; 6:17–24.
  49. Mamula MJ, Lin RH, Janeway CA Jr, Hardin JA. Breaking T cell tolerance with foreign and self co-immunogens. A study of autoimmune B and T cell epitopes of cytochrome c. J Immunol 1992; 149:789–795.[Abstract]
  50. Rhim SH, Millar SE, Robey F, Luo A-M, Lou Y-H, Yule T, Allen P, Dean J, Tung KSK. Autoimmune disease of the ovary induced by a ZP3 peptide from the mouse zona pellucida. J Clin Invest 1992; 89:28–35.
  51. Lou Y, Tung KSK. T cell peptide of a self-protein elicits autoantibody to the protein antigen. Implications for specificity and pathogenetic role of antibody in autoimmunity. J Immunol 1993; 151:5790–5799.[Abstract]
  52. Lou Y, Ang J, Thai H, McElveen F, Tung KSK. A zona pellucida 3 peptide vaccine induces antibodies and reversible infertility without ovarian pathology. J Immunol 1995; 155:2715–2720.[Abstract]
  53. Tung SK, Ang J, Lou Y. ZP3 peptide vaccine that induces antibody and reversible infertility without autoimmune oophoritis. Am J Reprod Immunol 1996; 35:181–183.
  54. Noguchi S, Hatanaka Y, Tobita T, Nakano M. Structural analysis of the N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida. Eur J Biochem 1992; 204:1089–1100.
  55. Jarrett EE, Hall E, Karlsson T, Bennich H. Adjuvants in the induction and enhancement of rat IgE responses. Clin Exp Immunol 1980; 39:183–189.[Medline]
  56. Cronkhite RI. Lymphocyte proliferation induced by pertussis toxin utilizes a pathway parallel to transforming growth factor-beta-sensitive growth. Int Arch Allergy Immunol 1993; 102:141–143.[Medline]
  57. Ryan M, McCarthy L, Rappuoli R, Mahon BP, Mills KH. Pertussis toxin potentiates Th1 and Th2 responses to co-injected antigen: adjuvant action is associated with enhanced regulatory cytokine production and expression of the co-stimulatory molecules B7–1, B7–2 and CD28. Int Immunol 1998; 10:651–652.[Abstract/Free Full Text]
  58. Sudweeks JD, Todd JA, Blankenhorn EP, Wardell BB, Woodward SR, Meeker ND, Estes SS, Teuscher C. Locus controlling Bordetella pertussis-induced histamine sensitization (Bphs), an autoimmune disease-susceptibility gene, maps distal to T-cell receptor beta-chain gene on mouse chromosome 6. Proc Natl Acad Sci USA 1993; 90:3700–3704.[Abstract/Free Full Text]
  59. Millar SE, Chamow SM, Baur AW, Oliver C, Robey F, Dean J. Vaccination with a synthetic zona peptide produces long-term contraception in female mice. Science 1989; 246:935–938.[Abstract/Free Full Text]
  60. Gupta SK, Bagavant H, Chadha K, Gupta M, Yurewicz EC, Sacco AG. Mapping of immunogenic domains on porcine zona pellucida 3 {alpha} and ß glycoproteins by murine monoclonal antibodies. Am J Reprod Immunol 1993; 30:95–100.
  61. Gupta SK, Kaul R, Rajalakshmi S, Sahai P, Yurewicz EC, Sacco AG. Immunoreactivity with native zona pellucida of antibodies against a 19 amino acid synthetic peptide corresponding to human ZP3. J Reprod Immunol 1994; 27:241–247.
  62. Bagavant H, Yurewicz EC, Sacco AG, Talwar GP, Gupta SK. Delineation of epitopes on porcine zona pellucida relevant for binding of sperm to oocyte using monoclonal antibodies. Am J Reprod Immunol 1993; 23:265–279.
  63. Mahi-Brown CA, Moran F. Response of cynomolgous macaques to immunization against a synthetic peptide from the human zona pellucida. J Med Primatol 1995; 24:258–270.[Medline]
  64. Afzalpurkar A, Gupta SK. Identification of epitopes of monoclonal antibodies to porcine zona pellucida 3ß glycoprotein, a homologue of the mouse/human sperm receptor. Am J Reprod Immunol 1997; 38:26–32.
  65. Bagavant H, Fusi FM, Baisch J, Kurth B, David CS, Tung KSK. Immunogenicity and contraceptive potential of a human zona pellucida 3 peptide vaccine. Biol Reprod 1997; 56:764–770.[Abstract]
  66. Yurewicz EC, Zhang S, Sacco AG. Generation and characterization of site-directed antisera against an amino-terminal segment of a 55 kDa sperm adhesive glycoprotein from zona pellucida of pig oocytes. J Reprod Fertil 1993; 98:147–152.[Abstract]
  67. Gupta SK, Sharma M, Behera AK, Bisht R, Kaul R. Sequence of complementary deoxyribonucleic acid encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP1 and its high-level expression in Escherichia coli. Biol Reprod 1997; 57:532–538.[Abstract]
  68. Oldstone MB. Molecular mimicry and immune-mediated diseases. FASEB J 1998; 12:1255–1265.[Abstract/Free Full Text]
  69. Dickman S. Possible cause found for Lyme arthritis. Science 1998; 281:631–632.[Free Full Text]
  70. Gross DM, Forsthuber T, Tary-Lehmann M, Etling C, Ito K, Nagy ZA, Field JA, Steere AC, Huber BT. Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis. Science 1998; 281:703–706.[Abstract/Free Full Text]
  71. Pope JE, Stevens A, Howson W, Bell DA. The development of rheumatoid arthritis after recombinant hepatitis B vaccination. J Rheumatol 1998; 25:1687–1693.[Medline]
  72. Grotto I, Mandel Y, Ephrost M, Ashkenazi I, Shemer J. Major adverse reactions to yeast-derived hepatitis B vaccines—a review. Vaccine 1998; 16:329–334.[CrossRef][Medline]
  73. Gout O, Theodorou I, Liblau R, Lyon-Caen O. Central nervous system demyelination after recombinant hepatitis B vaccination: report of 25 cases. Neurology 1997; 48(suppl):A424.
  74. Kakar A, Sethi PK. Guillain Barre syndrome associated with hepatitis B vaccination. Indian J Pediatr 1997; 64:710–712.[Medline]
  75. Guiserix J. Systemic lupus erythematosus following hepatitis B vaccine. Nephron 1996; 74:441.[Medline]
  76. Brezin AP, Massin-Korobelnik P, Boudin M, Gaudric A, LeHoang P. Acute posterior multifocal placoid pigment epitheliopathy after hepatitis B vaccine. Arch Ophthalmol 1995; 113:297–300.[Abstract]
  77. Jameson BA, Wolf H. The antigenic index: a novel algorithm for predicting antigenic determinants. Comput Appl Biosci 1988; 4:181–186.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Skinner, S. M.
Right arrow Articles by Dunbar, B. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Skinner, S. M.
Right arrow Articles by Dunbar, B. S.
Agricola
Right arrow Articles by Skinner, S. M.
Right arrow Articles by Dunbar, B. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS