A Chimeric Sperm Peptide Induces Antibodies and Strain-Specific Reversible Infertility in Mice¹

^a Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 ^b Department of Pharmacology, NV Organon, 5340 BH Oss, The Netherlands

	ABSTRACT

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The development of a contraceptive vaccine based on a gamete-specific antigen requires knowledge of the ability of the antigen to elicit an immune response that inhibits fertilization. A well-defined immune response, as elicited by a synthetic peptide comprising a dominant B-cell epitope coupled to a common promiscuous T-cell epitope, might be preferable. In this study, the immunodominant B-cell epitope of sperm antigen Sp17 has been identified and synthesized as a chimeric peptide with the promiscuous T-cell epitope bovine RNase[94–104] at the N terminal. Immunization of female BALB/c mice with this peptide induced a dose-dependent reduction in fertility. Although antibodies to recombinant and native Sp17 were elicited in these mice, there was no strict correlation between the level of these antibodies and the reduction in fertility. Moreover, the induction of infertility was strain-specific since no effect on fertility could be induced in B6AF1 mice. To understand the mechanism behind this apparent strain-specific infertility induction, a more extended study on both the humoral and the cellular immune response to the chimeric peptide was performed. The antigen-specific T-cell response and the levels of antigen-specific cytokines are the major factors that affect fertility outcome.

	INTRODUCTION

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>The goal of gamete immunocontraception is to define a gamete-specific antigen that can be a target for immunological inhibition of fertilization. To achieve this goal, both defining the target and understanding the presentation of the target immunogen to the immune system are critical for directing the immune response to the native gamete molecule. A number of antigenic targets on spermatozoa with known cDNA sequences have been defined [1, 2]; however, only a few have been successfully tested in fertility trials, and the epitope(s) they presented to the immune system remains poorly understood. Indeed, aside from whole sperm or testis extract preparations, the only molecule shown to elicit one hundred percent male or female infertility is native guinea pig sperm surface hyaluronidase (PH-20) [3]. Earlier studies with anti-acrosomal hyaluronidase were successful in inhibiting fertilization and cumulus dispersion in vitro [4, 5] but proved unsuccessful in in vivo fertility testing in both rabbits and sheep [6, 7]. Other antigens such as native fertilin (PH-30) have been tested in preliminary fertility trials and shown to be successful in males and partially successful in females [8]. Rabbit fertility testing of the recombinant and ß subunits of rabbit fertilin has met with little success [9]. Unfortunately, the hope that vaccinations with recombinant proteins, representing sequences of sperm proteins produced in bacteria or eukaryotic cells, would elicit infertility-inducing antibodies and replace the need for native antigens has yet to be realized.

Synthetic peptides, based on the sequences of sperm proteins, have been used to induce infertility in females with limited but encouraging success. Immunization with peptides from sperm-specific lactate dehydrogenase (LDH-C4) [10], the sperm peptide P10G [11], and peptides from the sperm antigen YWK-II [12] have all achieved varying degrees of infertility. In contrast to immunization with sperm-derived immunogens, the zona pellucida has received considerably more attention and has been the major focus for antigenic targets from female gametes. Recombinant proteins and synthetic peptides constructed from the sequences of zona pellucida proteins have been tested for immunogenicity and their subsequent effect on fertility [13–17]. However, immunizations utilizing the zona pellucida as an immunogen in females may result in the development of ovarian pathology [16, 18] and the subsequent irreversibility of the infertility. Consequently, the zona pellucida as a target for immunological inhibition of fertility requires not only a highly immunogenic molecule whose antibody recognizes the native gamete molecule, but additionally requires the lack of any autoimmune pathologies.

Studies utilizing synthetic, chimeric peptides containing a linear B-cell epitope from a zona pellucida protein and a promiscuous T-cell epitope [14, 18] have demonstrated that the fertility of female mice can be reduced without the induction of ovarian pathology. To test the feasibility of using a chimeric sperm immunogen requires the identification of an appropriate sperm antigen, the identification of its linear B-cell epitopes, and the selection of an appropriate T-cell epitope. Toward this goal, the present study was undertaken with an entirely synthetic, chimeric peptide, constructed from an immunodominant epitope of the sperm protein Sp17 [19] and a known T-cell epitope, eliminating the need for conjugation to a carrier protein. As described by Lou et al. [14] for a mouse zona pellucida peptide, we have used the promiscuous bovine RNase[94–104] peptide, which induces a T-cell response in mice of several different H-2 haplotypes [20], including H-2^d when it is part of a chimeric peptide [14]. The peptide was synthesized such that the RNase[94–104] peptide is on the N-terminal end of the linear B-cell epitope from the sperm protein Sp17. Our results indicate that this synthetic, chimeric peptide can induce infertility in female BALB/c mice but not in B6AF1 mice. To understand the mechanism behind this apparent strain-specific infertility induction, a more extended study on both the humoral and the cellular immune response to the chimeric peptide was performed. These results have important implications for the development of a human gamete immunocontraceptive.

	MATERIALS AND METHODS

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Animals and Chemicals

Female BALB/c and male CD-1 mice were obtained from Charles River Breeding Laboratories (Wilmington, MA), and female B6AF1 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed in the University laboratory animal care facility. All chemicals were purchased from either Fisher Scientific (Raleigh, NC) or Sigma Chemical Company (St. Louis, MO).

Peptides

The pin block used for mimotope analysis was synthesized by Chiron Mimotopes Peptide Systems (Clayton, Victoria, Australia). The peptide, A9DT (NCAYKTTQANKAEWGAKVDD), was synthesized by Dr. K. Tung (University of Virginia, Charlottesville, VA) and Eurosequence bv (Groningen, The Netherlands). Peptides A9D, derived from rabbit Sp17 (AEWGAKVDD); T (NCAYKTTQANK); G9G, a peptide unrelated to Sp17 (GGGTLPPSG); and G9GT (NCAYKTTQANKGGGTLPPSG) were also synthesized by Eurosequence bv. Peptide hA9DT, derived from human Sp17 (AEWGSKVED), was synthesized at the Salk Institute (under contract N01-HD-0-2906 with the NIH, USPHS) and made available by the Contraceptive Development Branch, Center for Population Research NICHD (Bethesda, MD). Peptide CP1 was a kind gift from Dr. K. Tung.

Recombinant Sp17

Human [21] and mouse [22] Sp17 were cloned into the vector pQE-30 (Qiagen Inc., Chatsworth, CA), and the entire coding region expressed as a six-histidine-tagged recombinant protein as described [19]. Purification of the recombinant human and mouse Sp17 was performed as described by Lea et al. [21].

Immunizations and Sampling

Subcutaneous immunization of BALB/c mice was performed with recombinant human Sp17 emulsified (1:1) in complete Freund's adjuvant (CFA) in a total volume of 50–100 µl. Booster immunizations were given in incomplete Freund's adjuvant (IFA) at 2 and 4 wk, and animals were bled 10 days after the last boost.

Immunizations (s.c.) of BALB/c and B6AF1 mice were performed with the Sp17 peptide A9DT in PBS. The following BALB/c groups were immunized with A9DT: 1) The 10-µg group (n = 14) received a single 10-µg immunization in CFA. In Weeks 4 and 6, these mice received an additional injection of PBS in IFA. 2) The 20-µg group (n = 15) received 10 µg in IFA in Weeks 0 and 2. 3) The 40-µg group (n = 17) received 10 µg in CFA in Week 0, and 10 µg in IFA in Weeks 4, 6, and 8. 4) The 250-µg group (n = 6) received 25 µg in CFA in Week 0, and 25 µg in IFA in Weeks 4 and 6, 50 µg in Weeks 8 and 12, and 75 µg in Week 15. The following B6AF1 groups were immunized with A9DT: 1) The 35-µg group (n = 10) received 10 µg in CFA in Week 0, 10 µg in IFA in Weeks 3 and 5, and 5 µg in ICF in Week 8. 2) The 40-µg group (n = 12) received 10 µg in CFA in Week 0, and 10 µg in IFA in Weeks 4, 6, and 8. 3) The 300-µg group (n = 8) received 75 µg of hA9DT in CFA in Week 0, and 75 µg of hA9DT in IFA in Weeks 3, 5, and 7. Control groups of mice were treated in the same way as the test groups, but instead of A9DT in PBS, PBS only was emulsified with adjuvant and used for the immunizations. From the 40-µg A9DT groups of mice, vaginal washes were taken 3 days before the start of the fertility test (7 days after the last boost). These washes were performed by carefully pipetting 50 µl physiological saline up and down in the vagina. The vaginal samples were filtered by a multiscreen type of membrane filtration system (Millipore, Molsheim, France) and stored at -20°C until further use. In all groups of mice, fertility testing was initiated 7–10 days after the last boost.

To study the T-cell response to the chimeric peptide A9DT and its separate components (A9D and T), BALB/c and B6AF1 mice were immunized (s.c.) with these peptides, which were mixed with dimethyl dioctadecylammonium bromide (Phase Separations, Waddinxveen, The Netherlands) in a total volume of 100 µl. Booster immunizations with the same peptides were given in IFA in Weeks 3 and 6.

Fertility Testing

Male CD-1 mice (Charles River Breeding Laboratories, Wilmington, MA) with proven fertility were housed singly, and groups of 3 immunized females were cycled through their cages daily. The females were checked for plugs twice daily and removed to an individual cage. They were bled via an orbital puncture or from the tail vein (250 µl) when a plug was observed (the blood drawn was referred to as plug serum). If no plug was observed, females remained with the males for a total of 15 days (at least 3 estrous cycles) and were then bled and removed to a separate cage. Second matings were performed in the same way 6–8 wk after the first mating with no additional booster immunizations given (except for the 40-µg group, which received a 1-µg A9DT boost in Week 15).

Mimotope Analysis

Analysis of mouse Sp17 (mSp17) linear B-cell epitopes was performed using a peptide pin block. Antibody reactivity to mSp17 linear B-cell epitopes was determined by ELISA as described in O'Rand and Widgren [23] using both primary (mouse serum) and secondary (horseradish peroxidase [HRP]-conjugated goat anti-mouse IgG, IgA, and IgM; Organon Teknika Corporation, Durham, NC) antisera at a dilution of 1:5000. Baseline reactivity to the peptides was established using secondary antibody alone, and these values were subsequently subtracted from the immune OD450-nm values for each pin. For each decapeptide, a Z score was calculated: z score = (individual peptide pin reactivity) - (mean reactivity for all peptides)/(standard deviation for the antibody used) [24].

ELISA

ELISAs were performed as described in Batova and O'Rand [25], with 100 ng recombinant mSp17 (rmSp17) plated per well and incubated for 2 h (room temperature) in mouse antisera at a 1:1000 dilution (in PBS containing 0.05% Tween 20 and 2% nonfat dried milk). All sera were tested in triplicate, and the mean OD450-nm values were calculated. Immune values were then compared to preimmune values, and the control values were subtracted.

For 12 BALB/c and 12 B6AF1 mice from the 40-µg A9DT group, serial dilutions of the plug sera were tested in an rmSp17 ELISA, as described above. In this case, HRP-conjugated goat anti-mouse Ig (heavy and light chains), IgG1, IgG2a, and IgA (Southern Biotechnologies Associates, Birmingham, AL) at a 1:5000 dilution were used. Titers in these sera are presented as the ²log dilution of the serum giving an OD450-nm value of 1.5. Titers in the vaginal washes are expressed as the ²log dilution of the vaginal wash giving an OD450 that exceeds twice (for total Ig) or 1.5 times (for IgA) the OD450 values of sera or vaginal washes of controls (mice immunized with PBS and adjuvant), respectively. Spearman correlation coefficients were calculated to determine significance.

Western Blotting

Recombinant mSp17 (1 µg) [22] was separated by 15% SDS-PAGE and blotted according to the method of Towbin et al. [26] using Immobilon P (Millipore) as the transfer membrane.

Fluorescence-Activated Cell Sorting (FACS) Analysis

CD-1 cauda epididymal spermatozoa were capacitated in human tubal fluid medium with HEPES (Anthos Labtec bv, Heerhugowaard, The Netherlands) containing 1.5% (w:v) human serum albumin (HSA; Sigma Chemical Co.) for 2 h at 37°C in a humid atmosphere of 5% CO₂. Cells were then collected, counted, and diluted to 13 x 10⁶ cells per ml in PBS containing 3.5% HSA and 0.01% sodium azide. Fifty microliters of cells (7 x 10⁵) were incubated for 1 h on ice with 50 µl plug serum or control serum (at a 1:1000 dilution). A live/dead staining was performed by addition of 50 µl of a solution of ethidium monoazide bromide (Molecular Probes, Leiden, The Netherlands; final concentration 2 µg/ml) and a 10-min exposure of the cells to an intensive light source. Cells were then washed three times with PBS-0.3% HSA-0.01% sodium azide and were incubated on ice for 45 min with fluorescein isothiocyanate (FITC)-conjugated goat anti mouse Ig (Dako, Glastrup, Denmark; at a 1:100 dilution). Cells were washed twice and fixed in 2% paraformaldehyde dissolved in PBS. FACS analysis was performed on a FACSscan (Beckton Dickinson, Rutherford, NJ). Percentage of cells specifically stained by the immune serum (i.e., cells with a fluorescence intensity exceeding that of cells stained by control serum) was calculated by an FL1 marker setting.

Proliferation Assay

Lymphocyte proliferation assays were performed using mononuclear cells prepared from spleens of immunized mice 4 wk after delivery of pups, i.e., approximately 2 mo after the last immunization (40-µg A9DT group plus control group) or 4 wk after the last immunization with peptide A9DT, A9D, or T. Proliferation was measured by incubating (in triplicate) 2 x 10⁵ cells per well in 200 µl modified Dulbecco's Modified Eagle medium (DMEM)/F12 medium (Gibco BRL, Paisley, UK) supplemented with 10% fetal calf serum (FCS) in flat-bottomed microtiter plates for 3 days in a humidified atmosphere at 37°C and 5% CO₂ with variable amounts of antigen; this was followed by incubation for 16 h with 0.5 µCi [³H]thymidine per well. Viability of each cell population was assessed in 3 wells with 2.5 µg/ml concanavalin A (Sigma Chemical Co.). The cultures were harvested onto glass fiber filters, and the incorporated radioactivity was measured by gas scintillation counting for 5 min in a ß-plate counter. Results are expressed as counts or delta counts (= mean of antigen-specific counts - mean of counts in medium alone).

Interleukin (IL)-4 and Interferon (IFN) Elispot Assay

From the same preparation of spleen cells used for the proliferation assay, cells were used to determine the frequency of antigen-specific IL-4- and IFN-producing cells. Cells were incubated in a multiscreen 96-well filtration plate (Millipore) that had been coated (overnight at 4°C) with 5 µg/ml rat anti-mouse IL-4 (clone 11B11; Pharmingen GmbH, Hamburg, Germany) or rat anti-mouse IFN (clone R4-6A2; Pharmingen GmbH) in PBS and washed with PBS. Incubation with or without antigens took place for 40 h at 37°C and 5% CO₂ with 10⁶ cells per well. All incubations were performed in triplicate. After this incubation, cells were removed, and plates were washed 4 times with PBS containing 0.05% Tween-20 (PBST). Plates were then incubated overnight at 4°C with 2 µg/ml biotinylated rat anti-mouse IL-4 (clone BVD6-24G2; Pharmingen) or rat anti-mouse IFN (clone X MG 1.2; Pharmingen GmbH) in PBST with 1% FCS. Plates were washed with PBST and incubated for 2 h at room temperature with avidin peroxidase solution (2.5 µg/ml; Sigma Chemical Co.). After PBST washing, color development was induced by addition of AEC (3-amino-9-ethylcarbazole; Instruchemie Hilversum bv, The Netherlands) and hydrogen-peroxide (Sigma Chemical Co.). The color reaction was stopped by rinsing the plates with milliQ (Millipore). After air-drying, image analysis was used to count the number of spots per well. Results are expressed as numbers of spots or delta spots (= mean number of antigen-specific spots - mean number of spots of cells in medium alone).

	RESULTS

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Identification of B-Cell Epitopes

To specifically identify the immunodominant linear B-cell epitope(s) on the sperm-specific protein Sp17, female BALB/c mice were immunized with 70 µg of recombinant human Sp17 [21], and their pooled sera were used for mimotope analysis of mouse and rabbit Sp17 decapeptides [23]. As shown in Figure 1, the immunodominant linear B-cell epitope identified from a mimotope analysis of mouse Sp17 decapeptides was AEWGAKV. The chimeric peptide A9DT (NCAYKTTQANKAEWGAKVDD), consisting of the bovine RNase[94–104] peptide (T = NCAYKTTQANK) at the N terminal of the Sp17 peptide (A9D = AEWGAKVDD), was synthesized, and female BALB/c mice were subsequently immunized. Sera from these mice, used in a mimotope analysis of mouse Sp17 decapeptides, identified the immunodominant linear B-cell epitope as AEWGAKV (data not shown).

View larger version (49K): [in this window] [in a new window]   FIG. 1. Reactivity of mouse Sp17 decapeptides with sera from BALB/c female mice immunized with recombinant human Sp17. The predominant linear B-cell epitope is AEWGAKV.

Infertility of A9DT-Immunized Mice

In a series of fertility experiments, various immunization protocols (varying the amount of immunogen, the type of adjuvant and the immunization schedule) were used to assess the efficacy of the Sp17 peptide, A9DT, at inducing antibody-mediated infertility in BALB/c mice (Table 1). In the first experiments, mice were immunized with either 10 or 20 µg of A9DT (PBS and adjuvant-immunized mice served as the control group). In the 10-µg group there was no reduction in the percentage of mice pregnant (64% compared to 67%) and only a slight reduction in the number of pups per female (5.4 compared 6.3). In the 20-µg group however, the effect was more marked, a reduction in the percentage of mice pregnant from 67% to 47% and a reduction in the number of pups per female to 2.5. When these groups of mice were mated a second time, the percentage of mice pregnant in the 10-µg group was again at control levels and the number of pups per female was still slightly reduced (5.5). In the 20-µg group of mice, the reduction in fertility was sustained, with only 40% of mice becoming pregnant but an increase in the size of the litter (3 pups per female). For mice immunized with 40 µg A9DT, there was a significant decrease in the percentage of pregnancies (29% compared to 72%), with the number of pups per female reduced from 5.9 to 3. Likewise, in the 250-µg group only 33% of females became pregnant, with 1.7 pups per female. Statistical analysis (Student's t-test) of these data shows that the reduction in the number of pups per female in the 20- and 40-µg groups was statistically significant (p < 0.02 and p < 0.05, respectively) when compared to control groups for the first mating. Moreover, for the 20-µg group, this significance was sustained to the second mating (p < 0.04). For the other groups of mice, the induced infertility was reversible, with fertility returning in the second mating (Table 1). There was a dose-response relationship between the percentage of pregnant mice and immunization with A9DT; however, excessive amounts of immunogen (250 µg) did not increase the level of infertility.

We compared the effectiveness of the A9DT peptide at reducing fertility to another peptide known to induce infertility in mice, the zona pellucida peptide CP1 [14]. Using 12 mice immunized with CP1, we found that the effect of A9DT on fertility was comparable to that of CP1 (30 µg CP1: 3.5 pups/female; 20 µg A9DT: 2.5 pups/female) indicating that predicting linear B-cell epitope sequences by mimotope analysis and synthesizing chimeric peptide sequences by linking B- and T-cell epitopes is an effective way of defining immunogens for use as vaccines.

Fertility and Serum Antibody Titer

The relationship between fertility and serum antibody titer is controversial [10–12]; consequently, the data collected in the A9DT experiments were analyzed for a relationship between serum antibody titer and fertility. From all animals, plug sera were tested for mSp17-specific Ig (total) by ELISA and Western blotting. No absolute correlation could be observed between the anti-mSp17 titer, reactivity to mSp17 on a blot, and litter size for each mouse (Fig. 2, A and B). For example, in one group of 15 mice, although there was a tendency toward zero fertility for serum showing strong reactivity to rmSp17 on a Western blot (mice 6C, 7B, 5B, 4C, 3A, 3B: Fig. 2B), there were also some mice that did not conform to this pattern. Mouse 7C had strong anti-rmSp17 reactivity but produced 7 pups; mouse 6A had little anti-rmSp17 reactivity and produced no pups. Possibly, additional factors such as the isotype of serum antibodies or the level of antibodies in the reproductive tract might account for the lack of correlation between serum antibody titer and fertility.

View larger version (89K): [in this window] [in a new window]   FIG. 2. Reactivity of serum to rmSp17 and the litter size of each BALB/c mouse immunized with 20 µg A9DT. A) Western blot showing the reactivity of BALB/c mouse anti-A9DT plug sera from the second mating to rmSp17. Proteins of 26 and 29 kDa were detectable with serum from mice 3A, 3B, 4A, 4B, 4C, 5B, 5C, 6B, 6C, 7B, and 7C. Low levels of protein were detected with sera from mice 3C, 5A, 6A, and 7A; and no proteins were detectable with preimmune serum. The y-axis indicates a protein ladder of molecular weight markers (x 10-3) (Gibco-BRL, Grand Island, NY). B) Litter size of each BALB/c mouse from the second mating.

To investigate whether any correlation could be demonstrated between one of these parameters and litter size, plug sera and vaginal washes from 12 mice of the 40-µg group of BALB/c mice were tested. In addition, to investigate whether the antibodies raised against A9DT would also recognize native mSp17, plug sera were tested on whole CD-1 sperm cells by FACS analysis. All results are presented in Table 2. This analysis did not reveal any significant correlation (p < 0.05) between any one of the parameters studied and the litter size of the animals. A significant correlation (p < 0.02) could be found between anti-rmSp17 Ig titer (both Ig total and IgG1) and the percentage of specific staining of sperm cells by these sera, indicating that the antisera recognize native mSp17.

Strain Specificity of the Fertility of A9DT-Immunized Mice

BALB/c mice are haplotype H-2^d and were reported to be nonresponders to the permissive T-cell epitope RNase[94–104] [20]. However, when used in a chimeric peptide with a zona pellucida peptide, the RNase[94–104]-zona peptide construct generated serum antibodies to zona pellucida in H-2^d mouse strains [14]. Similarly, BALB/c mice also responded to the RNase[94–104]-A9D construct (abbreviated A9DT) as described above.

Since the RNase[94–104]-zona peptide construct also generated antibodies in other mouse strains, including B6AF1 (H-2^bxa; [14]), A9DT was tested in B6AF1 female mice. Immunization with a total of 35 µg (n = 10) or 40 µg (n = 12) of A9DT or 300 µg (n = 8) of hA9DT resulted in a high pregnancy rate with high litter size/female (Table 3) even though serum titers to Sp17 were somewhat higher in B6AF1 mice compared to BALB/c mice (data not shown). Moreover, the reactivity of sera from both BALB/c and B6AF1 mouse strains to mouse spermatozoa in ELISA and FACS analysis was not significantly different (data not shown). It may be concluded that A9DT induces the production of antibodies to Sp17 and spermatozoa in both BALB/c and B6AF1 mouse strains, but the fertility is differentially affected. A difference in the T-cell response to the A9DT peptide may be responsible for this differential effect.

To investigate this possibility, we analyzed the lymphocyte proliferative response and the frequency of A9DT-specific IL-4- and IFN-producing cells isolated approximately 2 mo after the last immunization (4 wk after delivery). From the BALB/c mice, immunized with A9DT, pools of spleen cells were formed from 2–3 mice that did not become pregnant and 2–3 mice that delivered at least 7 pups. Figure 3 shows the lymphocyte proliferative response to two concentrations of A9DT. Figure 4 shows the frequency of IL-4- and INF-producing cells in the same pools of cells incubated with 50 µg/ml A9DT. Similar results were obtained in a second experiment in which pools of spleen cells, derived from the remaining mice of each group, were used. Whereas the groups of control mice (immunized with PBS and adjuvant) showed no or only minor responses to A9DT, the groups of mice immunized with A9DT clearly showed a specific response to A9DT. From these latter groups, the BALB/c mice showed a stronger proliferative response to A9DT than the B6AF1 mice, with those having a litter of at least 7 pups showing the greatest response (Fig. 3). If the IL-4 and INF responses to A9DT by the different groups of mice are compared, the difference between BALB/c and B6AF1 is even more pronounced. B6AF1 mice immunized with A9DT produced a high IFN response, whereas in the BALB/c mice immunized with A9DT, the IL-4 response dominated. Again, this A9DT-specific response seems to be stronger in those mice that become pregnant (Fig. 4).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Proliferative response to A9DT (10 and 50 µg/ml) by spleen cells from 5 groups of mice (cells pooled from two or three animals per group). B6AF1-PBS: B6AF1 mice immunized with PBS and adjuvant only, delivered at least 10 pups; B6AF1-A9DT: B6AF1 mice immunized with a total of 40 µg A9DT, delivered at least 11 pups; BALB/c-PBS: BALB/c mice immunized with PBS and adjuvant only, delivered at least 7 pups; BALB/c-A9DT (> 0): BALB/c mice immunized with a total of 40 µg A9DT, delivered at least 7 pups; BALB/c-A9DT (0); BALB/c mice immunized with a total of 40 µg A9DT delivered no pups.

View larger version (18K): [in this window] [in a new window]   FIG. 4. Frequency of IL-4- and IFN-producing spleen cells from the same 5 groups as indicated in Figure 5, upon stimulation with 50 µg/ml A9DT.

To investigate the difference in T-cell recognition of A9DT and its separate components (A9D and T) between BALB/c and B6AF1 mice, groups of both strains of mice (n = 6) were immunized with A9DT, A9D, or T. Four weeks after the last immunization, spleen cells from each group were pooled and tested in a lymphocyte proliferation assay and an IL-4 and IFN Elispot assay towards A9DT, A9D, T, G9GT (another, unrelated chimeric peptide containing the RNase[94–104] sequence at the N terminal), and G9G. Figure 5 shows the results of the proliferation assays. The only significant proliferative response found in BALB/c mice (Fig. 5A) was the response to A9DT after immunization with A9DT, indicating that the bridge formed by A9D and T in the chimeric peptide contained a T-cell epitope recognized in the context of the H-2^d haplotype (Fig. 5A). In B6AF1 mice (Fig. 5B) however, besides a good proliferative response to A9DT after immunization with A9DT, a significant response to another chimeric peptide containing the RNase[94–104] T-cell epitope, G9GT, could be found. This indicates that B6AF1 mice recognize the RNase[94–104] T-cell epitope in the context of their H-2^bxa haplotype. The lower proliferative response to the separate RNase[94–104] peptide (T) (after immunization with A9DT or T) might be explained by the short length of the peptide. When we look at the cytokine responses in these mice (Fig. 6), the recognition of the RNase T-cell epitope by B6AF1 mice becomes even clearer: B6AF1 mice immunized with A9DT or T responded by a dominant IFN response to A9DT, T, and G9GT (Fig. 6B). However, in BALB/c mice, a cytokine response to these peptides, in this case dominated by an IL-4 response, could be detected too, except for a response to the separate RNase T-cell epitope after immunization with this epitope (Fig. 6A). No T-cell response (neither in BALB/c, nor in B6AF1) was found to A9D. This indicates that A9D itself does not represent a T-cell epitope in the context of H-2^d or H-2^bxa.

View larger version (39K): [in this window] [in a new window]   FIG. 5. A) Proliferative responses to 10 and 50 µg/ml antigens (A9DT, A9D, T, G9GT, and G9G) or medium (MED) by spleen cells from BALB/c mice immunized with A9DT, A9D, or T (bottom line of x-axis). B) Proliferative responses to 10 and 50 µg/ml antigens (A9DT, A9D, T, G9GT, and G9G) or medium (MED) by spleen cells from B6AF1 mice immunized with A9DT, A9D, or T (bottom line of x-axis).

View larger version (33K): [in this window] [in a new window]   FIG. 6. A) Frequency of IL-4- and IFN-producing spleen cells from BALB/c mice immunized with A9DT, A9D, or T (bottom line of x-axis) upon stimulation with 50 µg/ml antigens (A9DT, A9D, T, or G9GT) or in medium (MED) alone. B) Frequency of IL-4- and IFN-producing spleen cells from B6AF1 mice immunized with A9DT, A9D, or T (bottom line of x-axis) upon stimulation with 50 µg/ml antigens (A9DT, A9D, T, or G9GT) or in medium (MED) alone.

	DISCUSSION

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The goal of this study was to design a chimeric peptide sequence consisting of an Sp17 immunodominant linear B-cell epitope and a promiscuous T-cell epitope that would produce an immune response capable of reducing mouse fertility. Mimotope analysis of mouse anti-Sp17 sera identified the most immunodominant linear B-cell epitope as the 7-mer AEWGAKV. This was synthesized with the bovine RNase T-cell epitope (11-mer NCAYKTTQANK) at the N terminal and was tested in fertility trials for its efficiency at inducing mouse infertility. Immunizing four groups of mice with different amounts of antigen revealed that a single 10-µg immunization was insufficient to significantly affect the fertility of the mice. However, when the immunizing dosage was doubled (two immunizations of 10 µg), there was a marked reduction in the number of mice that became pregnant (a 30% reduction). Moreover, when the amount of immunogen was doubled again (four immunizations of 10 µg), the reduction in the number of pregnancies also doubled (to 43%, a reduction of 60%) reducing the pregnancy rate from 72% to 29% and showing a dose-response relationship between the amount of immunogen and the number of pregnancies. When large excesses of peptide (250 µg) were used, this dose-response relationship was not sustained, suggesting that the antibody response to immunization had reached near maximal levels when the mice were immunized with 40 µg immunogen. In fact, immunizing with excess peptide may be detrimental to the antibody response because induction of antigen tolerance can occur [27].

We have shown that the infertility induced by peptide immunizations was reversible within a 6- to 8-wk time period. Only the 20-µg group of mice had a statistically significant decrease in the number of pups per female for the second mating (Table 1, p < 0.04). The cause of this continued infertility is not clear; possibly this group of mice had a more sustained antibody response (without further boosting).

Controversy surrounds the issue of whether there is any correlation between serum antibody titers and infertility. Fertility testing of LDH-C4 in baboons showed animals with low titers not becoming pregnant and, perhaps more significantly, animals with high titers becoming pregnant [10]. Likewise, we were unable to show any absolute correlation between plug serum Ig (total) titer and reactivity to rmSp17 and fertility status. However, this titer could be an incorrect parameter to be used for such a correlation study. Rather the T-cell response and the resultant isotype of the antibody and/or the level of antibody in the genital tract may be more indicative of the fertility status [28–32]. In order to study all these aspects, a more extensive analysis of the antibody response was performed, including analysis of the anti-Sp17 IgG1 and IgG2a, anti-Sp17 vaginal Ig and IgA, and specificity of the plug sera to spermatozoa. No significant correlation between any of these parameters and the fertility status could be found (Table 2). Nevertheless, in mice it is difficult to detect antibodies present in the genital tract (uterus and oviduct) at the moment of mating, and cyclical changes in the amount and composition of oviductal and vaginal fluid may give rise to changes in the level of immunoglobulin present at the time of fertilization [28, 32].

In contrast to the infertility of A9DT-immunized BALB/c mice, B6AF1 mice immunized with A9DT were completely fertile. The two mouse strains exhibited no detectable difference in A9DT antibody titer or in antibody reactivity to mouse spermatozoa. The difference in fertility status could be a reflection of a difference in the ability of the immunoglobulin to move across the oviductal epithelium, resulting in a greater titer of antibody at the site of fertilization in BALB/c mice, although this seems unlikely. Alternatively, this difference in infertility may be explained by a difference in the cellular immune response to A9DT. Both the T-cell epitope recognized within A9DT and the T helper type of response towards A9DT (Th1 or Th2 [33]) differed between BALB/c and B6AF1.

In several studies it has been demonstrated that factors derived from activated lymphocytes (especially IFN) have a direct inhibiting effect on sperm motility and embryo development [34, 35]. Furthermore, in mice, Th1 type responses have been associated with implantation failure and fetal loss [36]. Therefore, we studied the A9DT-specific proliferative response of spleen cells and the frequency of A9DT-specific IL-4- and IFN-producing cells among spleen cells from the two strains of mice immunized with A9DT. In mice, it is known that during pregnancy and the lactation period the cytotoxic T-cell response is suppressed and the T helper cell response is shifted towards the Th2 type of response, which appears to be regulated by steroid hormones like progesterone (reviewed in [36] and [37]). To prevent interference by this effect, the T-cell responses were tested after the lactation period, at 30-days postpartum. Surprisingly, the T-cell response to A9DT in B6AF1 mice appeared to be dominated by an IFN, i.e., a Th1-like, response, whereas this response in BALB/c mice was clearly shifted towards an IL-4, i.e., a Th2-like, response. A deleterious effect on pregnancy by a Th1 type of response could thus not explain the difference in fertility between BALB/c and B6AF1.

Another difference between BALB/c and B6AF1 mice was found in the recognition of A9DT. Lymphocytes from B6AF1 mice showed a specific proliferative and IFN response to A9DT, T, and G9GT, indicating recognition of the permissive T-cell epitope RNase[94–104] (T) in the context of H-2^bxa. Spleen cells from BALB/c mice, however, showed a specific proliferative response only to the chimeric peptide A9DT, indicating a recognition of the bridge formed by A9D and T in the chimeric peptide. In agreement with Chen et al. [20], in BALB/c (H-2^d) mice we did not find a proliferative response to RNase[94–104] (T) alone. The finding that the IL-4 response was only detectable after priming by A9DT or after priming by T with a restimulation by the chimeric peptides (A9DT or G9GT), might indeed indicate a lower major histocompatability complex (MHC) class II binding affinity of the short peptide (T) than of the chimeric peptides. Consequently, it may be concluded that the difference in fertility between BALB/c and B6AF1 mouse strains immunized with A9DT resides in the different T-cell responses. The recognition of different T-cell epitopes leads to different cytokine responses and subsequently may lead to significant fertility differences.

Within the group of BALB/c mice, those that were pregnant showed a stronger T-cell response to A9DT (Figs. 5 and 6). No shift in the Th1/Th2 balance was found between pregnant and nonpregnant mice (no difference in IL-4/IFN ratio or IgG1/IgG2a ratio) but the absolute number of A9DT-specific IL-4-producing cells was higher in pregnant mice. Even though it may be speculated that in BALB/c mice a low level of IL-4 is detrimental to a successful pregnancy, an understanding of the differences between pregnant and nonpregnant mice within the BALB/c group will require further study. It should be noted that studying T-cell responses at 30 days postpartum may not reflect the type of T helper cell cytokine response that occurred with the initial immunization or that was present at the time of fertilization and implantation.

The design of an effective immunocontraceptive has proven to be a complex problem. In this study, we have designed a synthetic chimeric peptide containing a linear B-cell epitope (from the sperm protein Sp17) and a promiscuous T-cell epitope that can cause a reversible reduction in the fertility rate of BALB/c mice. We have also analyzed the immune response in more detail to determine what components of the cellular and humoral immune response might affect the fertility outcome. Using two different mouse strains, the MHC and the T-cell response clearly are the major factors that affect fertility outcome, regardless of the antibody titer and reactivity to the antigen. Moreover, the difference between fertility and infertility may lie within an early T-cell response and the cytokine profile that is not sustained beyond the first few weeks after initial immunization. By studying these parameters in more detail, we hope to move closer to the goal of designing an effective immunocontraceptive.

	ACKNOWLEDGMENTS

 
The authors would like to thank M. Dooper for her contribution to the development of the Elispot assay.

	FOOTNOTES

 
¹ This work was supported by a National Institutes of Health grant and postdoctoral fellowship through the Center for Recombinant Gamete Vaccinogens, University of Virginia and United States Public Health Service (U54HD29099), Bethesda, MD, and by an Organon Research Agreement.

² Correspondence: M.G. O'Rand, Department of Cell Biology & Anatomy, University of North Carolina at Chapel Hill, CB #7090, 210 Taylor Hall, Chapel Hill, NC 27599-7090. FAX: (919) 966-1856; morand{at}unc.edu

Accepted: April 15, 1998.

Received: February 23, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. O'Rand MG. Antigenic targets on sperm for immunological interception. In: Kurpisz M, Fernandez N (eds.), Immunology of Reproduction. Oxford: BIOS Sci. Pub. Ltd.; 1995: 473–484.
2. Lee AY, Silver LM. Contraceptive vaccine formulations with sperm proteins. In: Bronson R (ed.), Reproductive Immunology. UK: Blackwell Scientific Publ.; 1996: 693–712.
3. Primakoff P, Lathrop W, Woolman L, Cowan A, Myles D. Fully effective contraception in male and female guinea pigs immunized with the sperm protein PH-20. Nature 1988; 335:543–546.[CrossRef][Medline]
4. Metz CB. Effects of antibodies on gametes and fertilization. Biol Reprod 1972; 6:358–383.[Medline]
5. Dunbar BS, Munoz MG, Cordle CT, Metz CB. Inhibition of fertilization in vitro by treatment of rabbit spermatozoa with univalent isoantibodies to rabbit sperm hyaluronidase. J Reprod Fertil 1976; 47:381–384.[Abstract/Free Full Text]
6. Morton DB. Immunoenzymatic studies on acrosin and hyaluronidase in ram spermatozoa. In: Edidin M, Johnson MH (eds.), Immunobiology of Gametes. Cambridge: Cambridge University Press; 1977: 115–155.
7. Metz CB. Immunological inhibition of sperm and egg function: prospects for an infertility vaccine from gametes. In: Contraception: Science, Technology, and Application. Proceedings of a Symposium. National Academy of Sciences; 1979; Washington, DC. pp. 180–220.
8. Ramarao CS, Myles DG, White JM, Primakoff P. Initial evaluation of fertilin as an immunocontraceptive antigen and molecular cloning of the cynomologus monkey fertilin beta subunit. Mol Reprod Dev 1996; 43:70–75.[CrossRef][Medline]
9. Hardy CM, Clarke HG, Nixon B, Grigg JA, Hinds LA, Holland MK. Examination of the immunocontraceptive potential of recombinant rabbit fertilin subunits in rabbit. Biol Reprod 1997; 57:879–886.[Abstract]
10. O'Hern PA, Bambra CS, Isahakia M, Goldberg E. Reversible contraception in female baboons immunized with a synthetic epitope of sperm-specific lactate dehydrogenase. Biol Reprod 1995; 52:331–339.[Abstract]
11. O'Rand MG, Beavers J, Widgren EE, Tung KSK. Inhibition of fertility in female mice by immunization with a B-cell epitope, the synthetic sperm peptide, P10G. J Reprod Immunol 1993; 25:89–102.[CrossRef][Medline]
12. Vanage G, Lu YA, Tam JP, Koide SS. Infertility induced in rats by immunization with synthetic peptide segments of a sperm protein. Biochem Biophys Res Commun 1992; 183:538–543.[CrossRef][Medline]
13. Millar SE, Lader E, Liang LF, Dean J. Oocyte-specific factors bind a conserved upstream sequence required for mouse zona pellucida promoter activity. Mol Cell Biol 1991; 11:6197–6204.[Abstract/Free Full Text]
14. Lou YH, 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]
15. Mahi-Brown CA. Primate response to immunization with a homologous zona pellucida peptide. J Reprod Fertil 1996; 50:165–174.
16. Paterson M, Wilson MR, van Duin M, Aitken RJ. Evaluation of zona pellucida antigens as potential candidates for immunocontraception. In: Gupta SK, Doberska C (eds.), Zona Pellucida Glycoproteins and Immunocontraception. J Reprod Fertil Suppl 1996; 50:175–182.[Medline]
17. VanderVoort CA, Schwoebel ED, Dunbar BS. Immunization of monkeys with recombinant complimentary deoxyribonucleic acid expressed zona pellucida proteins. Fertil Steril 1995; 64:838–847.[Medline]
18. 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]
19. Richardson RT, Yamasaki N, O'Rand MG. Sequence of a rabbit sperm zona pellucida binding protein and localization during the acrosome reaction. Dev Biol 1994; 165:688–701.[CrossRef][Medline]
20. Chen JS, Lorenz RG, Goldberg J, Allen PM. Identification and characterization of a T-cell-inducing epitope of bovine ribonuclease that can be restricted by multiple class II molecules. J Immunol 1991; 147:3672–3678.[Abstract]
21. Lea IA, Richardson RT, Widgren EE, O'Rand MG. Cloning and sequencing of cDNAs encoding the human sperm protein Sp17. Biochim Biophys Acta 1996; 1307:263–266.[Medline]
22. Kong M, Richardson RT, Widgren EE, O'Rand MG. Sequence and localization of the mouse autoantigenic protein, Sp17. Biol Reprod 1995; 53:579–590.[Abstract]
23. O'Rand MG, Widgren EE. Identification of sperm antigen targets for immunocontraception: B-cell epitope analysis of Sp17. Reprod Fertil Dev 1994; 6:289–296.[CrossRef][Medline]
24. Pauls JD, Edworthy SM, Fitzler MJ. Epitope mapping of histone (H5) with systemic lupus erythematosus, procainamide-induced lupus and hydralazine-induced lupus sera. Mol Immunol 1993; 30:709–719.[CrossRef][Medline]
25. Batova I, O'Rand MG. Histone-binding domains in a nuclear autoantigenic sperm protein. Biol Reprod 1996; 54:1238–1244.[Abstract]
26. Towbin H, Staehelin TT, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76:3450–3454.[Abstract/Free Full Text]
27. Burstein HJ, Abbas AK. In vivo role of interleukin 4 in T-cell tolerance induced by aqueous protein antigen. J Exp Med 1993; 177:457–463.[Abstract/Free Full Text]
28. Lea IA, Kurth B, O'Rand MG. Immune response to immunization with sperm antigens in the macaque oviduct. Biol Reprod 1998; 58:794–800.[Abstract/Free Full Text]
29. Ulcova-Gallova Z. Mixed antiglobulin reaction and tray agglutination test for detection of sperm antibodies. Int J Fertil Menopausal Study 1994; 39:185–191.
30. Parr MB, Parr EL. Local immunization for antifertility immunity. In: Johnson PM, Griffin D (eds.), Scientific Basis of Fertility Regulation. Oxford: Oxford University Press; 1993: 441–458.
31. Hjort T. Quantitative determination of IgG and IgA on sperm from infertile patients with and without antisperm antibodies. Am J Reprod Immunol 1996; 36:211–215.
32. Parr MB, Parr EL. Mucosal immunity in the female and male reproductive tracts. In: Ogra PL, Mestecky EL, Strober W, McGhee JR, Bienenstock J (eds.), Handbook of Mucosal Immunity. San Diego: Academic Press Inc.; 1994: 677–689.
33. Mosmann TR, Schumacher JH, Street NF, Budd R, O'Garra A, Fong TA, Bond MW, Moore KW, Sher A, Fiorentino DF. Diversity of cytokine synthesis and function of mouse CD4+ T-cells. Immunol Rev 1991; 123:209–229.[Medline]
34. Hill JA, Haimovici F, Anderson DJ. Products of activated lymphocytes and macrophages inhibit mouse embryo development in vitro. J Immunol 1987; 138:2250–2254.
35. Hill JA, Haimovici F, Politch JA, Anderson DJ. Effects of soluble products of activated lymphocytes and macrophages (lymphokines and monokines) on human sperm motion parameters. Fertil Steril 1987; 47:460–465.[Medline]
36. Raghupathy R. Th1-type immunity is incompatible with successful pregnancy. Immunol Today 1997; 18:478–482.[CrossRef][Medline]
37. Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bi-directional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a T_H2 phenomenon? Immunol Today 1993; 14:353–356.[CrossRef][Medline]