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a Department of Pathology, University of Virginia, Charlottesville, Virginia 22908
b Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mammalian ZP is an extracellular matrix that surrounds growing and mature oocytes. In mice, three sulfated glycoproteins (ZP1, ZP2, ZP3) are found within the ZP [1,2]. In vitro studies indicate that in mice, O-linked oligosaccharide side chains of ZP3 are involved in the primary binding of the sperm to the ZP, while ZP2 contributes to subsequent and persistent ZP binding and functions as a secondary sperm receptor [3, 4]. The ZP proteins have been used extensively in the development of vaccines for the fertility control of animals and humans (reviewed in [5, 6]). The proposed vaccine action is the induction in female subjects of effective, sustained, but reversible levels of ZP-specific antibodies that inhibit sperm-egg binding and/or prevent sperm penetration of the ZP. Indeed, passive immunization of female mice with rat monoclonal antibodies against mouse ZP2 or ZP3 resulted in localization of the antibodies to intra-ovarian oocytes and long-lasting but reversible contraception [7–10]. Active immunization of female mice with ZP3-derived peptides ZP3328–342, containing a B cell epitope recognized by the ZP3-specific contraceptive antibody, also led to reversible, albeit incomplete infertility [11].
However, the ZP3 peptide also induces a T cell response to the ZP3 peptide; and CD4-positive, ZP3-specific T cells adoptively transfer autoimmune ovarian disease to syngeneic recipients [12]. Accordingly, we have mapped the native B cell epitope of the ZP3 peptide, ZP3335–342, and synthesized a modified form of it in tandem with a bovine RNase T cell epitope. The ZP3 chimeric peptide elicited ZP antibody in all mouse strains studied regardless of their histocompatibility (MHC) haplotype. The antibody response correlated with reduction of litter size, and the fertility returned upon reduction of antibody titers [13]. However, the infertility induced by the ZP3 chimeric peptide was incomplete. Therefore, additional B cell epitopes in the functional ZP glycoproteins are being sought in order to develop a more effective multiple-subunit B cell epitope vaccine.
Although the stoichiometry of the three proteins within the ZP matrix remains to be performed, ZP2 is at least as abundant as ZP3. Mice immunized with particulate zonae readily make antibodies to ZP2, and monoclonal antibodies to any of three distinct ZP2 epitopes effectively inhibit fertilization in vitro. One monoclonal antibody, IE-3, was further investigated and shown to cause long-term, reversible contraception after intraperitoneal transfer [9]. In this paper we report the mapping of a B cell epitope in murine ZP2. Active immunization of different inbred mouse strains with a ZP2 peptide containing the B cell epitope elicited antibody to ZP with antifertility effect. Moreover, a chimeric peptide that contained the small ZP2 B cell epitope and a foreign T cell epitope has the capacity to elicit antibody that reacts with native ZP; thus the ZP2 epitope is a potential contraceptive vaccine antigen.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PZP2.1, a 0.9-kilobase pair ZP2 cDNA [14] encoding a polypeptide recognized by the IE-3 monoclonal antibody [7], was randomly cleaved with DNase I in the presence of 1.5 mM MnCl2 [15]. Gel-purified fragments (200–1000 base pairs [bp]) were ligated into lambda ZAP (Stratagene, La Jolla, CA), and the unamplified expression epitope library was screened with IE-3 [11]. Immunoreactive clones were plaque purified, and the sequence of the insert DNA was determined [16].
Random Peptide Display Library Screening
To determine a consensus binding motif for IE-3, the Invitrogen (Carlsbad, CA) FliTrx Random Peptide Display Library was screened with IE-3 according to the manufacturer's instructions. Briefly, after the last panning on the IE-3-coated plate, each randomly selected colony was cultured in 1.5 ml of medium to a nonsaturated density. Expression of the peptide insert was induced by tryptophan, and the cells were harvested 3 h later. For immunoblotting, whole bacterial proteins were solubilized in 100 µl of SDS sample buffer by boiling for 5 min. The proteins, 5 µl per lane, separated on a 12.5% SDS-PAGE minigel (Bio-Rad, Hercules, CA), were stained by Coomassie blue or electroblotted to a membrane (Immobilon-P; Millipore, Bedford, MA). The membrane was blocked with 3% skim milk and sequentially incubated with IE-3 (1:50 000, 1 h at room temperature), biotin-labeled goat anti-rat IgG (1:20 000), and ABC (avidin-biotin-peroxidase) complex (Vector Labs., Burlingame, CA). Enhanced chemilluminescence (ECL kit; Amersham, Arlington Heights, IL) was used to detect the IE-3-reactive protein. The positive clones were amplified, and the sequence of the insert DNA was determined from isolated plasmid DNA. The amino acid sequences of the displayed dodecapeptides were deduced from the sequences of the DNA inserts.
Peptide Synthesis and Purification
All peptides were synthesized by an automatic peptide synthesizer (Gilson Medical Electronics Inc., Middleton, WI) and purified by HPLC on a C18 reverse-phase column (Waters Associates, Millford, MA). The purity of all peptides exceeded 90%.
Animals and Immunization
C3H, DBA/1, and PL/J female mice were purchased from the Jackson Laboratory (Bar Harbor, ME); A/J, BALB/C.ANCR, C57BL/6 (B6), B6AF1, and SJL/J female mice were purchased from the National Cancer Institute (Frederick, MD). All mice, at 6–9 wk of age, were anesthetized by i.p. injection (0.5 mg/mouse) of tribromoethanol (Aldrich Chemical Co., Milwaukee, MI). Peptide, dissolved in Milli-Q water (Millipore) at 1 mM, was emulsified in an equal volume of complete Freund's adjuvant (CFA). Each mouse received s.c. 0.1 ml of the mixture (containing 50 nM of peptide) either in one footpad and the base of tail or in both footpads. For ovariectomy, each ovary was removed through a small loin incision under general anesthesia. Ovariectomized mice were immunized 1 wk after the surgery. Mouse care and treatments were based on approved protocols in accordance with institutional guidelines.
Ovarian Histopathology and Immunofluorescence (IF) Studies
Ovaries collected at autopsy were fixed in Bouin's fixative and embedded in paraffin, and 5-µm-thick sections were stained with hematoxylin and eosin. Ovarian histopathology was graded with increasing severity from 1 to 4, as described previously [12]. Direct and indirect IF were performed to detect serum antibodies to the native ZP as previously described [17]. Briefly, for indirect IF, normal ovarian frozen sections, fixed in 90% ethanol, were incubated with sera at 2-fold dilutions in PBS containing 3% BSA and then with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-mouse IgM (Cappel, Malvern, PA). Antibody titer was expressed as tube dilutions, and the highest dilution of the serum at which IF was still positive was the serum antibody titer. For competitive IF, sera at proper dilutions (determined by a prior antibody titration) were incubated with various concentrations of peptides at 4°C overnight before performance of indirect IF. For direct IF, frozen sections prepared from ovaries of immunized mice were incubated with FITC-conjugated goat anti-mouse IgG (Cappel), and the intensity of fluorescence was graded with increasing intensity from 1 to 3.
ELISA
Serum antibodies to immunizing peptides were detected by ELISA as described previously [17]. Briefly, 96-well, flat-bottom plates (Corning Glass Works, Corning, NY) were coated with 10 µM peptides and blocked with 3% BSA. Sera at various dilutions in PBS with 3% BSA were added to the wells in duplicate. After incubation, the plates were washed and treated with peroxidase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) diluted at 1:5000 in PBS with 3% BSA. The plates were then washed and treated with O-phenylenediamine and hydrogen peroxide. The reaction was stopped by the addition of sulfuric acid, and the absorbance at 490 nm determined by an ELISA reader (Molecular Devices, Menlo Park, CA). For competitive ELISA, sera at proper dilutions were incubated with various concentrations of peptides at 4°C overnight before addition to the plates.
Fertility Study
Female B6AF1 mice were immunized with ZP2121–140 in CFA, CP2 in CFA, and with CFA alone. Forty-eight days after immunization, they were placed into cohabitation with proven fertile male mice for 30 days at a male:female ratio of 1:1. As soon as mating was confirmed by observation of the presence of a vaginal plug, tail bleedings were obtained from the females and anti-ZP antibody titers were determined by IF. Litter sizes were determined at delivery. Three to four months after delivery, the females were mated again with proven fertile males to observe the return of fertility.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To identify a ZP2 domain recognized by a contraceptive monoclonal antibody [8, 9], an epitope expression library was screened with IE-3, an antibody that reacts with ZP2 polypeptide backbone [14]. The insert DNA sequence of 24 overlapping immunoreactive clones was determined and compared to the known ZP2 sequence (Fig. 1). The 99 bp common to all clones encoded ZP2103–134, corresponding to the major hydrophilic portion of ZP2 (Fig. 1, B and C). This epitope, recognized by the IE-3 monoclonal antibody, represents a candidate antigen for a multivalent contraceptive vaccine.
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Immune Response to the Overlapping Synthetic Peptides of ZP2103–134
Three overlapping peptides, ZP2101–120, ZP2111–131, and ZP2121–140 (Table 1), each emulsified in CFA, were injected into female B6AF1 mice (six per peptide), and their antibody responses were determined at various times after immunization. As shown in Figure 2, mice injected with ZP2101–120 produced a relatively low level of antibody that recognized only ZP2101–120. ZP2111–130 failed to elicit antibody to any of the overlapping peptides, and this is most likely explicable by the absence of a helper T cell epitope in ZP2111–130. In contrast, ZP2121–140 induced a significant amount of antibodies that reacted with itself, and, to a lesser extent, with ZP2111–130. Note that the peptide-binding activity of each antiserum was absorbed quantitatively by the immunizing peptide (Fig. 2, A and C).
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The sera were tested for antibody to ZP by indirect IF on normal mouse ovary sections, and the antibody titer was expressed as the highest reciprocal serum dilutions with positive ZP staining. As shown in Figure 3, only ZP2121–140 induced antibody to native ZP. Thus, a native ZP2 antigenic determinant was tentatively mapped to the ZP2121–140 peptide. Because both peptides ZP2111–130 and ZP2121–140 quantitatively absorbed the ZP antibody activity induced by peptide ZP2121–140 (data not shown), a B cell epitope was likely located within ZP2121–130. Also, because immunization with ZP2121–140 induced IgG class antibody to ZP, the peptide must also possess a T cell epitope that elicited a helper T cell response required for isotype switch of the ZP-specific B cells to produce IgG antibody.
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Both Ovariectomized Female Mice and Normal Female or Male Mice Responded to ZP2121–140
Previously, female mice injected with a T cell peptide of ZP3 had been found to spontaneously produce antibody to ZP3 determinants distant from the immunizing ZP3 T cell peptide [18]. Because the antibody response did not occur in ovariectomized mice, the endogenous ovarian ZP3 antigen must have stimulated the diversified ZP3 antibody response. If this phenomenon of diversified autoantibody response had also occurred in mice injected with ZP2121–140, then the ZP antibody elicited might not be specific for the B cell epitope in the ZP2121–140 peptide. To resolve this potential caveat, the responses of ovariectomized female B6AF1 mice, sham-operated female B6AF1 mice, and normal male B6AF1 mice were compared. Because all three groups of mice produced comparable levels of ZP antibody when injected with ZP2121–140, it was concluded that the ZP antibody in ZP2 peptide-immunized mice was specific to the B epitope within ZP2121–140 (Fig. 4). This conclusion gained further support when the fluorescence ZP antibody activity was completely absorbed with peptide ZP2121–140 (data not shown).
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Mapping the Fine Specificity of the ZP2 B Cell Epitope Using a Peptide Display Library
To precisely map the critical residues of the ZP2 B cell epitope, the IE-3 monoclonal antibody was used to screen a FliTrx Random Peptide Display Library. The library contained random dodecamer peptides expressed within the active-site loop of thioredoxin that was fused to the bacterial flagellin [19]. Four IE-3-reactive clones were plaque purified, and their reactivity to IE-3 was confirmed by Western blot (Fig. 5A). Based on the plasmid DNA sequences and their deduced amino acid sequences, a consensus sequence VXYK (X = variant residue) was identified, which matched the ZP2123–126 sequence VRYK (Fig. 5B). To confirm that ZP2123–126 was indeed a B cell epitope, and to define the relative contribution of each of the conserved residues to the B cell epitope, a panel of ZP2121–127 peptides were synthesized in which the residue valine123, tyrosine125, or lysine126 was substituted by an alanine. The single alanine-substituted peptides were used to displace the binding of antibody to ZP2121–140 on the normal mouse ovarian ZP by indirect IF. As shown in Table 2, while all three consensus residues influenced antibody-binding activity, the quantity of peptide required for competition was found to vary, with tyrosine125 being the most potent and lysine126 the least potent. The relative contributions of the residues were ranked as Y > V > K.
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Design of an Immunogenic ZP2 Chimeric Peptide
On the basis of the identity of the critical residues of the ZP2121–140 B cell epitope, four chimeric peptides each containing the VRYK motif were designed. ZP2121–127 was synthesized in tandem to bovine RNase94–104 [20] or to the malaria circumsporozoite peptide from Plasmodium falciparum [21]. These foreign peptides were selected because they have been shown to elicit T cell responses in multiple inbred mouse strains regardless of their H-2 haplotype. In each case, the peptide pair was synthesized in both orientations (Table 1). B6AF1 female mice injected with each of the four chimeric peptides were evaluated for serum antibodies to native ovarian ZP by IF. Of the chimeric peptides that contained RNase and ZP2, RNase-ZP2 elicited ZP antibody whereas ZP2-RNase did not (Table 3). In contrast, both chimeric peptides containing the malaria epitope and ZP2 induced ZP antibody, although the titers were consistently below the titer induced by the RNase-ZP2 chimeric peptide. The RNase-ZP2 chimeric peptide was therefore evaluated further.
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Mouse Strains That Responded to ZP2121–140 Versus the Bovine RNase ZP2121–127 Chimeric Peptide
This study determined the antibody responses of inbred mice of different H-2 haplotypes to peptides ZP2121–140 and RNase-ZP2121–127. Contrary to expectation, while all but one of the mouse strains injected with ZP2121–140 produced ZP antibody, the response to the ZP2 chimeric peptide was confined to mice of the H-2k and H-2b haplotypes (Table 4). When the ovaries of B6AF1 mice injected with ZP2121–140 in CFA were examined between Days 14 and 70, no evidence of oophoritis was observed (Fig. 6). Therefore, a preliminary study was conducted to evaluate the effect of ZP2121–140 immunization on female fertility.
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Antifertility Effect of ZP2(121–140) Immunization
Six female B6AF1 mice were immunized either 1) with ZP2121–140 in CFA, 2) with CFA alone, or 3) with the chimeric peptide CP2 in CFA. CP2 contains bovine RNase T cell peptide and a modified ZP3 B cell epitope and was used as a positive control [13]. On Day 48 after immunization, when the ZP antibody levels had reached plateau levels, the mice were individually housed with a proven fertile male mouse. Upon confirmation of mating by the presence of a vaginal plug, the mouse sera were obtained for ZP antibody titers, and the litter sizes were determined at delivery 20 days later. As shown in Figure 7, by Day 48, mice immunized with ZP2121–140 or the ZP3 chimeric peptide had 70% reduction in their litter sizes compared with control females (Fig. 7A), and their sera contained high titers of ZP antibody (Fig. 7B). Four months later, when the ZP antibody titers of mice injected with ZP2121–140 or the ZP3 chimeric peptide had fallen by 3-tube dilutions (Fig. 7B), the mice were again mated. At this point, the litter sizes of ZP2121–140 and the ZP3 chimeric peptide-immunized mice had increased to 50% and 66%, respectively, as compared with those of the control mice (Fig. 7A).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A chimeric peptide contains all of the antigenic elements required to stimulate an epitope-specific antibody response. In the case of the ZP3 chimeric peptide (CP2 in Table 1), the modified B cell epitope of ZP3 devoid of ZP3 T cell epitope is linked to a foreign T cell epitope. The latter stimulates a T cell response to help ZP3-specific B cells to produce ZP3 antibody. Because the helper T cell response is directed to a foreign nonovarian peptide, autoimmune ovarian disease should not occur. Moreover, because the chosen foreign T cell epitope was known to stimulate T cell response in mice of many MHC haplotypes, antibody would be induced in mice regardless of their genetic composition—an important consideration for a vaccine suitable for an outbred population of diverse genetic background.
Although immunization with the CP2 led to significant reduction of fertility, the effect was incomplete [13]. However, because monoclonal antibody to ZP3335–342 completely inhibits fertility in vivo, failure to achieve complete fertility inhibition is likely due to insufficient level or avidity of the ZP3-specific polyclonal antibodies induced by active CP2 immunization. The problem of insufficient immune response, not infrequent in vaccine development, is being addressed by altering the conformation of the B cell epitope, using more potent T cell epitope, and evaluating vaccine formulation including adjuvant selection. In addition, the issue may be resolved by the use of chimeric peptide vaccine containing additional B cell epitopes in gamete macromolecules of fertilization. To identify such an epitope, we have made use of a preexisting monoclonal antibody that recognizes ZP2 [7, 14]. By screening an epitope expression library composed of randomly cleaved ZP2 cDNA fragments, we identified a candidate antigen, the size of which was further refined by immunization with overlapping peptides and screening of a random peptide epitope library. That this epitope was suitable for vaccine development was confirmed by the ability of its cognate antibody to bind to native ZP as detected by IF.
To evaluate the response to active immunization with ZP2 B cell epitope ZP2121–140, we compared the antibody response between normal female mice and mice without ovaries (ovariectomized females). The study enabled us to rule out antibody production through the mechanism of T-to-B epitope spreading [17]. With this phenomenon, female mice injected with ZP3 T epitope alone spontaneously produced antibody to diversified B cell epitopes of ZP3 located outside the immunizing ZP3 peptide [18]. The antigens responsible for driving the antibody response originated from the endogenous ovaries, as ovariectomized females did not make the diversified antibody to ZP3. In the present study, by showing the capacity of ovariectomized mice to produce ZP antibody after immunization with ZP2121–140, and by confirming the specificity of the ZP antibody response, it was possible to confirm that ZP2121–140 was indeed a ZP2 B cell epitope. The fact that T-to-B epitope spreading did not occur in response to the ZP2 T cell peptide may also explain why mice injected with the ZP2 peptide did not elicit significant autoimmune ovarian disease, as will be discussed below.
This study has uncovered two interesting and unanticipated findings: 1) the ability of a T cell peptide to induce in mice T cell responses of different MHC haplotypes may be lost when the peptide becomes a component of a chimeric peptide and 2) a ZP peptide that induces both antibody and T cell responses may not induce significant autoimmune ovarian disease when given in CFA.
The bovine RNase T cell peptide is known to induce antibody to ZP in several inbred mouse strains [20]. However, the ZP2 chimeric peptide, which included the RNase T cell peptide, induced antibody only in mice of the H-2k or b haplotype. This discrepancy, not detected in the study on the ZP3 chimeric peptide [13], may be explained by the finding that chimeric peptides rarely elicit a T cell response that recognizes the T cell peptide component of the chimeric peptide. For example, mice injected with the ZP3 or the ZP2 chimeric peptide developed T cell response to the respective chimeric peptide but not to the bovine RNase T cell epitope (unpublished results). That this should occur is due to the emergence of a new and dominant T cell epitope created at the junction between the B cell epitope and the T cell epitope of the chimeric peptide [26, 27]. It is then the new junctional T cell epitopes and not the foreign T cell peptide that will dictate the MHC restriction of the T cell response to the chimeric peptide. In the case of the ZP chimeric peptides, the new T cell epitopes in CP2 evidently remain broadly restricted, thus retaining the capacity to induce T cell and antibody response in multiple H-2 haplotypes ([13], unpublished results). However, this is not the case for the new junctional T cell epitope of the ZP2 chimeric peptide. In practice, the design of a useful chimeric peptide vaccine is therefore not predictable, as demonstrated by the results in Table 3.
Why does the ZP2121–140 peptide induce T cell response detectable by an in vitro assay but fail to induce significant autoimmune ovarian disease? This finding contrasts with those for the ZP3330–342 peptide, which induces both T cell response and autoimmune ovarian disease. The most likely explanation is the differential expression of the respective ZP3 and ZP2 T cell epitopes on the ovarian antigen-presenting cells during the in vivo processing of the ZP3 and ZP2 glycoproteins in the ovaries. If less ovarian ZP2121–140 in relation to ZP3330–342 epitope were processed and presented by the ovarian antigen-presenting cells, only the ZP3 epitope would exist in sufficient quantity to stimulate the T cell-mediated ovarian inflammation. Because the response to T cell peptide presented by antigen-presenting cells is also required for autoantibody induction through T-to-B epitope spreading (reviewed in [28]), the apparent crypticity of the ZP2121–140 T cell epitope might explain why epitope spreading occurred after immunization with the ZP3330–340 but not with the ZP2121–140 peptide.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Kenneth S.K. Tung, Departments of Pathology, University of Virginia, Old Medical School, 4th Floor, Room 4888, Charlottesville, VA 22908. FAX: 804 924 8060; kst7k{at}virginia.edu
Accepted: November 13, 1998.
Received: August 6, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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