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Immunology |
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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contraception, DNA vaccine, follicular, immunize, ZPC
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mammalian zona pellucida (ZP), which surrounds the growing oocytes and ovulated eggs, is a potential immunogen for a contraceptive vaccine [1]. The zona of the mouse is composed of three sulfated glycoproteins (ZPA, ZPB, ZPC) [2]. The ZP induces acrosome reaction on sperm, determines the species specificity of fertilization, and prevents polyspermy in mammals. Bound to ZPC via O-linked oligosaccharide chains initially, the sperm seek ZPB as the secondary receptor for continued binding. Thus, ZPC glycoprotein functions as the primary sperm receptor. Antibodies to ZPC can block sperm-egg binding in vitro and alter the zona surface in such a way that receptor sites are no longer available to spermatozoa. Subsequent attachment and penetration through the zona and fertilization by other sperm are inhibited [3–5].
The efficacy of ZPC as the prototype of contraceptive vaccine antigens is well documented [6, 7]. Monoclonal antibodies directed toward ZPC have been used to inhibit fertilization in vitro and in vivo by passive immunization [8, 9]. A multiepitope ZP vaccine used to actively immunize female mice has been documented [10]. A recombinant ectromelia virus has been constructed to express the entire mouse ZPC glycoprotein for evaluation as a live recombinant immunocontraceptive vaccine for the mouse [11]. However, the major challenge remaining is the assurance of vaccine safety. The problem regarding safety surfaced when female animals injected with ZPC's partial or even entire protein vaccine were found to develop ovarian damage, with loss of oocytes and loss of ovarian function [12].
A desirable ZPC vaccine should induce adequate immune responses against ZPC without serious side effects. Therefore, we have focused our investigation on constructing a safe and effective contraceptive vaccine. We have chosen partial ZPC cDNA sequences that consist of a highly conserved region, and a region highly specific to rabbits was cloned into a eukaryotic expression vector [13]. When this constructed prototype of ZPC DNA vaccine was introduced into animals, a specific high-level antibody against ZPC antigen was observed without any evidence of side effects to the recipients. Furthermore, the prevention of fertility was also evidenced without interfering with normal follicular development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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According to the rabbit ZPC (rZPC) cDNA sequence, we selected the partial sequence of rabbit ZP protein C (amino acid 263–415, which contains both the fragment of a conservative sequence of ZPC and the rabbit species-specific region rZPC') as the target gene. The primers used for PCR cloning were as follows:
Total RNA was isolated from mature female rabbit ovary tissues and purified by using the RNA Gent Total RNA Isolation System (Promega, Madison, WI). The RNA pellets were gently resuspended in 100 µl of nuclease-free water. The quality of total RNA was conformed by the ratio of optical density 260/280. All RNA samples were stored at -20°C until use.
The amplification of the cDNA fragment was performed by reverse transcription-polymerase chain reaction (RT-PCR) according to the manufacturer's instructions and is described briefly as follows. One microliter of total RNA used in the reverse transcription reaction was mixed with 5 µl of 5x avian myeloblastosis virus (AMV)/transcription factor 1 (TFL) reaction buffer, 1 µl of a deoxyribonucleoside triphosphate (dNTP) (25 mmol/L) mixture, 1 µl of downstream primer (50 pmol), 2 µl of total RNA, and nuclease-free water to a final volume of 50 µl. The reaction was allowed to proceed at 42°C for 1 h. The reverse transcriptase was inactivated at 95°C for 2 min. The PCR reaction was set up with an addition of 4 µl of ZPC cDNA, 1 µl of upstream primer, 0.5 µl of downstream primer, 5 µl of AMV/TFL 5x buffer, 5 U/µl TFL DNA polymerase, 1 µl of a dNTP (25 mmol/L) mixture, and nuclease-free water to a final volume of 25 µl. The 30-cycle PCR condition was set as follows: 95°C for 5 min, 95°C for 1 min, 65°C for 1 min, 68°C for 2 min (decreasing 0.5°C every cycle to 50°C), followed by 55°C for 2 min (10 cycles) and 68°C for 10 min [14, 15]. The purified PCR product (15 µl) was detected by electrophoresis in 2.0% agarose gel (Promega).
Plasmid Construction
Construction of the PCR2.1-rZPC' vector The purified PCR product was ligated into PCR2.1 according to the manufacturer's instructions and successful clones were confirmed by restriction mapping and sequencing. PCR2.1 was purchased from Invitrogen (Carlsbad, CA).
Construction of the pCMV4-rZPC' plasmid To drive expression of the rZPC' cDNA, we chose the mammalian expression vector pCMV4. The 550-base pair (bp) cDNA was removed from PCR2.1-rZPC' by HindIII and XbaI and subcloned into the pCMV4 vector as the pCMV4-rZPC' vaccine.
Expression In Vivo
All experimental BALB/C mice (N = 50) received vaccine i.m. by multispot injection in the leg muscles along with 100 µl of 0.25% bupivacaine-HCl. Twenty-four hours later, pCMV4-rZPC' was inoculated (at 20 µg mouse) in the same fashion, and the mice received the second injection after 2 wk. To examine the level of in vivo expression, total RNA was isolated from muscle of the injection sites Week 10 postinoculation and was analyzed by RT-PCR with rZPC' upstream and downstream primers. This RT-PCR product was conformed by sequencing.
Expression in Cultured HeLa Cells In Vitro
The pCMV4-rZPC' and pCMV4 were transfected into cultured HeLa cells with lipofectamine (Gibco BRL, Rockville, MD). The transfected cells were fixed in 4% paraformalin-PBS for 1 h at room temperature, washed three times in PBS, and directly incubated with the immunized sera (diluted 1:50 with PBS, pH 7.4) at 37°C for 3 h. These immunized sera were obtained from mice (N = 50) 8 wk after being injected with pCMV4-rZPC' or pCMV4. Cells were permeabilized (0.1% Triton x 100 in 0.1% sodium citrate) for 2 min, washed, and incubated with the secondary antibodies (goat anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC), diluted 1:100 with PBS, pH 7.4; Sigma Chemical Company, St. Louis, MO) at 37°C for 1 h. The cells were counterstained with propidium iodine (Sigma Chemical Company) for visualizing nuclei and were analyzed for expression using confocal microscopy (Leica, Solms, Germany).
Immunization
The BALB/C mice and the New Zealand rabbits were purchased from the Institute of Genetics of the Chinese Academy of Sciences and were immunized with DNA constructs as follows.
BALB/C mice (female and male) were divided into two control groups (female, N = 50; male, N = 50) and two experimental groups (female, N = 50; male, N = 50). Each experimental mouse received 20 µg pCMV4-rZPC' vaccine in the leg muscle in the same fashion as described above. All mice were injected with 100 µl 0.25% burpivacaine-HCl 24 h before plasmid inoculation. Control groups were injected in the same way with equal volumes of pCMV4 plasmid using the same injection technique and schedule.
Female rabbits were randomly divided into a control group (N = 50) and an experimental group (N = 50). Each experimental rabbit was injected into the leg muscles with 100 µl of 0.25% burpivacaine-HCl, and 100 µg of pCMV4-rZPC' was injected into the same sites 24 h later. Control groups received pCMV4 plasmid using the same injection technique and schedule.
Sera were obtained from mice and rabbits every 2 wk after all animals received the primary injection of pCMV4-rZPC' or pCMV4 plasmid [16, 17]. All experiments were conducted according to the guidelines of the Chinese Animal Care for Laboratory Animals, and the protocols were approved by the Animal Care and Use Committee at the Institute of Zoology, Chinese Academy of Science.
Immunohistochemistry
Direct and indirect immunohistochemistry were performed to detect the presence of specific serum antibodies binding to the ZP in situ.
Antibodies bound in situ to ovarian ZP were analyzed by using direct immunohistochemistry staining. Briefly, for direct immunohistochemistry, frozen sections were prepared from ovaries of pCMV4-rZPC'- or pCMV4-immunized mice (N = 25) and rabbits (N = 10). The sections were blocked with 3%–6% nonfat dry milk for 20 min and then blocked with normal goat serum for 20 min. Sections were washed three times in PBS and directly incubated with the secondary antibodies (goat anti-rabbit/mouse IgG conjugated with horseradish peroxidase [HRP]) in the same buffer at 37°C for 30 min. Slides were washed again in PBS and incubated with avidin-HRP at 37°C for 30 min. The antibody stains were developed in an addition of diaminobenzidine (DAB) and nuclei were stained by hematoxylin.
For indirect immunohistochemistry studies, frozen sections were prepared from ovaries of normal mice (N = 25) and rabbits (N = 10) as above and were incubated with antisera from the animals immunized with either pCMV4-rZPC' or pCMV4 vaccine in the sixth weeks postimmunization because the antisera titer could reach the highest level after 6 wk postimmunization in DNA immunizations in most studies [18]. The sections were blocked with 3%–6% nonfat dry milk for 20 min and then blocked with normal goat serum for 20 min. Sections of ovaries were reacted either with the sera of pCMV4-rZPC'- or pCMV4-immunized mice as the first antibodies overnight at 4°C. The sections were washed three times in PBS and incubated with secondary antibodies (goat anti-rabbit/mouse conjugated with HRP) at 37°C for 30 min. Slides were washed again in PBS and incubated in avidin-HRP 37°C for 30 min. The antibody stains were developed in an addition of diaminobenzidine (DAB) and nuclei were stained by hematoxylin.
Histology Analysis for Morphological Structure
The anesthetized animals were intracardially perfused with 0.9% NaCl solution followed by 4% paraformalin. Histology was performed on representative 8-µm sections of ovaries from mice and rabbits immunized with pCMV4-rZPC' DNA vaccine or with pCMV4 plasmids, which were paraffin embedded and then stained with normal hematoxylin and eosin. Then these sections were used for immunohistochemical staining. We examined 50 mice and 20 rabbits in the histological studies.
Antifertility Affect
To evaluate the safety and efficacy of pCMV4-rZPC' vaccine, each of the immunized female BALB/C mice described above was mated with a normal male at a male:female ratio of 1:1 at 6 wk after the first immunization. Each of immunized male mice described above was mated with a normal female as previously described. Successful mating was confirmed by observation of the presence of a vaginal plug. The newborns from the experimental groups were counted and compared with the controls.
Statistical Analysis
Values were reported as the mean ± SEM. Statistical analyses were done by one-way ANOVA, and when significant effects of treatments were indicated, the Student-Newman-Keuls multirange test was employed among the groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We selected the partial sequence of rabbit ZP protein C (amino acid 263–415, which contains both the conservative and species-specific sequences of rabbit ZP) as the target gene. Using rabbit ovarian total RNA as a sample, the rZPC' cDNA sequence was amplified by our designed primers to generate its cDNA. The resulting PCR product was a 470-bp fragment and was confirmed by digesting with NaeI to release 310 and 160 bp (Fig. 1). This product was subcloned into PCR2.1 cloning vector and confirmed by restriction digestion and sequencing. The rZPC'-containing fragment was removed from PCR2.1-rZPC' and subcloned into a eukaryotic-expressing vector pCMV4 as the pCMV4-rZPC' prototype DNA vaccine.
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To identify the expression of pCMV4-rZPC' in vivo, the presence of rZPC' mRNA from mice muscle inoculated with pCMV4-rZPC' intramuscularly was analyzed. Normal mice muscles were used as the negative control. The total RNA was extracted from muscles of BALB/C mice inoculated with pCMV4-rZPC' vaccine and was subjected to RT-PCR reaction to amplify the rZPC' cDNA fragment with the designed primers. The PCR product was identified for rZPC' by digestion of NaeI and PstI and was analyzed in a 7% agarose gel. Figure 2 shows that expression of rZPC' was not detected in the normal mice, whereas expression of rZPC' in inoculated mice muscle was detected with RT-PCR. The RT-PCR product was further identified by NaeI and PstI digestions and subsequently sequenced to assure its identity. Thus, the constructed pCMV4-rZPC' is proven to be expressed in mice muscle (Fig. 2).
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To identify pCMV4-rZPC' expressed in vitro, pCMV4-rZPC' plasmid was transfected into HeLa cells, whereas pCMV4 plasmid was used as the negative control. The transfected HeLa cells were reacted with antisera from the pCMV4-rZPC'-immunized mice and subsequently reacted with the goat anti-mouse IgG conjugated with FITC as the secondary antibody, whereas antisera from the unimmunized mice were used as another negative control. We have observed that antisera from the animal immunized with pCMV4-rZPC' bound to HeLa cells transfected with pCMV4-rZPC' in cytoplasm, whereas no similar signal was detected from HeLa cells transfected with the control cells (Fig. 3). This suggests that the pCMV4-rZPC' is expressed in vitro.
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Immunohistochemistry
In order to determine with immunohistochemical analysis if the antibodies generated by immunized animals can specifically bind to ZP of ovary in situ, sections of ovaries from rabbits and mice immunized with pCMV4-rZPC' or pCMV4 plasmid were used. We carried out two immunohistochemical analyses.
Direct immunohistochemistry analysis To examine whether the immunized animals developed anti-rZPC antibodies bound to ZP antigen as complexes surrounding ZP, frozen sections from immunized animals and sections from the control plasmid-immunized animals were reacted with goat anti-rabbit/mouse IgG conjugated with HRP. After stain development, we observed that ovarian ZP were surrounded by brown stains in the mice immunized with pCMV4-rZPC' in situ, whereas no staining developed in the animals immunized with the control plasmid (Fig. 4, A and B). It suggests that anti-rZPC antibodies were developed from the mice immunized with pCMV4-rZPC' and were bound to the ZP.
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Indirect immunohistochemistry analysis Alternatively, an indirect immunohistochemistry analysis was performed to examine whether immune sera can bind to ZP in situ generated from these immunized animals. The frozen sections of ovaries from normal animals were reacted with antisera from the pCMV4-rZPC'-immunized mice and subsequently reacted with the goat anti-rabbit/mouse IgG conjugated with HRP as the secondary antibodies. Sera from the unimmunized animals and the control plasmid-immunized animals were used as the negative controls. After stain developed, we observed that antisera from the animals immunized with pCMV4-rZPC' bound to ZP of normal ovaries in situ, whereas no similar stains developed from the sera of animals immunized with the control plasmid or from the normal animals (Fig. 4, C and D). Both direct and indirect immunohistochemical analyses indicated that both mice and rabbits immunized with pCMV4-rZPC' generate specific anti-rZPC antibodies that bind to the ZP of the ovary.
Histologic Analysis for Morphological Structure
Ovaries from experimental rabbit/mice immunized with pCMV4-rZPC' or pCMV4 plasmids were examined using hematoxylin and eosin stain. Histopathologies of ovaries, as coded specimens, were evaluated by an independent observer. All animals, including experimental groups and control groups, had no signs of abnormal development of ovarian follicles at multiple developmental stages. No obvious differences were observed in the ovary structure and follicular development between the experimental groups and control groups. The morphologies of the ovaries from the experimental animals were similar to those of ovaries from the control animals. Our results reveal that rabbits immunized with pCMV4-rZPC' vaccine show no disruption of follicular development compared with the controls. In addition, no ovarian morphological changes were observed by direct immunohistochemical analysis. This demonstrates that immunization with pCMV4-rZPC' vaccine neither causes any detectable adverse side effects on the morphological structure of the ovary nor interferes with normal follicular development by antibodies specifically binding to ZP (Fig. 5).
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Antifertility Analysis
To detect the effects of pCMV4-rZPC' vaccine on fertility, the mice were mated to examine their rates of reproduction after immunized. The results of this experiment indicate that animals immunized with pCMV4-rZPC' vaccine have obvious fertility effects. This effect on fertility is apparent with immunized female mice, of which only 40% were able to give birth, but there was little effect on the immunized male mice, of which nearly 100% were fertile (Table 1). This suggests that immune responses caused by the pCMV4-rZPC' vaccine are directly against ZP of female animals as the target and cause no effect on the male animals even though antisera were generated in these male partners.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The recent cloning of the ZPC gene and the characterization of its transcript and protein product have provided sufficient molecular detail of the ZPC to suggest an alternative contraceptive strategy based on active immunization with the ZPC. Hinsch et al. used synthesized peptides (151–165 and 360–369) derived from the conserved sequences of mouse ZPC conjugated with keyhole limpet hemocyanin (KLH) to immunize experimental animals; the antibodies could cross-react with several species of ZPC, including human and monkey [19]. This led us to construct a contraceptive vaccine by choosing epitopic regions of the ZPC.
The purpose of this study was to find a minimum antigenic region that could provide an effective and safe ZPC vaccine. Such epitopic constructs could be sufficient to induce infertility by inhibiting sperm binding with anti-ZPC responses without sacrificing safety. The key factor is to select the immunogenic epitopes from the ZPC sequences. Because ZPC primary structures are highly conserved among many mammalians and anti-rZPC antibodies can also inhibit fertilization by reacting with the oviduct in different mammalian [20], we selected regions from the sequence of rabbit ZPC as the target in the present study; the sequence contains both the conservative sequence of ZPC and a specific region for rabbit. This region from amino acid 263–415, designated as rZPC', was used to construct a eukaryotic expression construct, pCMV4-rZPC', a prototype of the vaccine [13].
From the results of these experiments, when introducing the pCMV4-rZPC' construct into mice, partial ZPC protein can be expressed in vivo [21]. Anti-rZPC antibodies can generate after animals are immunized with this construct. Further evidence indicates that the produced antibodies against ZPC can bind specifically in situ to the ovarian ZP. Although the pCMV4-rZPC' construct is encoded for partial rabbit ZPC protein, observed anti-rZPC antibodies can react with native rabbit ZP protein. Because the O-linked oligosaccharide of ZPC mediates primary sperm binding to the ZP at the time of fertilization, antibodies coating the mature oocytes could prevent fertilization by inhibiting sperm binding to ovulated eggs or penetration of the sperm through the ZPC. Anti-ZPC antibodies are also known to inhibit the shedding of the ZP by the blastocyst before implantation [1]. Antibody to ZPC antigen has been implicated in infertility, and the use of such an antibody has been proposed for contraceptive vaccines either for temporary infertility or for sterilization in animals.
To determine the antifertility effect of pCMV4-rZPC' vaccine in this study, the mice were mated after being immunized. The mating experiments indicate that immunizations of experimental female animals with the pCMV4-rZPC' DNA vaccine significantly affected fertilities, with 60% of female mice giving birth, whereas immunized experimental male mice remained fertile. In contrast, all of the control mice (female and male) had normal birthrates.
Although there are obvious effects of DNA vaccine, the major challenge is the assurance of vaccine safety. It was previously documented that female mice infected with the recombinant ectromelia virus, which expressed the entire mouse ZPC glycoprotein, were observed to produce autoimmune antibodies against ZPC and were infertile for 5–9 mo after the initial infection. For nearly half of the infertile mice, immunity to ZPC was associated with a disruption of ovarian follicular development and the depletion of mature follicles without observable oophoritis [11]. Bagavant [22] used human ZPC (328–341) peptide (hZPCp) as a target antigen to immunize monkeys for a contraceptive vaccine study. Antibodies against hZPCp were generated at high levels in these monkeys and also reacted with native ZPC. However, loss of ovarian function was also observed and a long-term side effect was implicated [23]. We have constructed a rabbit ZPB eukaryotic construct, pCMV4-ZPB, as the ZPB DNA vaccine and used it to immunize rabbits previously. The level of anti-ZPB antibody generated by this construct was barely detectable in these animals. However, undesirable side effects on ovaries were observed. Similar results were also observed by Kerr, who expressed the rabbit ZPB glycoprotein in vitro using a recombinant myxoma virus and used it to immunize rabbits. These immunizations induced sustained infertility in 70% of female rabbits. However, it also induced follicular degeneration and substantial depletion of primordial follicles [24, 25].
Either partial ZPC protein or entire ZPC glycoprotein vaccine could interfere with normal follicular development and induce endocrine dysfunction. The antibodies bound to the developing ZP of growing oocytes may lead to malfunction of folliculogenesis mediated by killing of the oocyte via antibody-dependent cell-mediated cytotoxicity of complement lysis [26, 27] or cytotoxic T cells. Alternatively, antibody attachment to the developing ZP could interfere with normal follicular development, which relies on close communication between the oocyte and its surrounding granulosa cells. Loss of ovarian function may be advantageous as a pest population control because it could lead to life-long infertility; this would be highly undesirable, though, for a human contraceptive vaccine, which may limit its usefulness as a contraceptive agent [2].
In our study, development of morphologically normal follicles of all stages was also found to be present in the groups immunized with the pCMV4-rZPC' vector. As with the control group, the presence of multiple stages of ovarian follicles correlates with the measurements, indicating that the mice and rabbits have normal ovulatory cycles. There were not any notable ovarian abnormality of these immunized animals under the direct immunohistochemistry analysis. Therefore, immunization of mice and rabbits using the pCMV4-rZPC' construct does not appear to alter ovarian development. All the immunized animals elicited a good antibody response against rZPC', continued to have normal ovulatory cycles, and showed no signs of disturbance in cyclicity. At the same time, we did measure the profile of estrogen levels with radioimmunoassays from mice immunized with pCMV4-rZPC' or pCMV4 at different time points. We have not found significant differences among the groups.
Although immunization with the pCMV4-rZPC' led to a significant reduction of fertility, the effect was far from satisfactory. The reasons for this may be the limited epitopic sequences in the pCMV4-rZPC', which generate a weak immune responses in these animals. Different genetic backgrounds may also be a contributing factor. As for safety concerns, immunization of mice by the pCMV4-rZPC' construct may have reduced cytotoxic T cell responses to the zona, which in turn avoided abnormal ovarian histopathology and dysfunction of the reproduction system. However, to increase the efficacy of this type of vaccine, more immunogenic sequences may be included and the balance of TH1- versus TH2-type immune responses will also be considered in our next study. Heterologous DNA vaccine may be feasible in the development of a contraceptive vaccine that elicits effective, reversible immune responses and at the same time to avoids ovarian cytotoxicity.
In this study, we have demonstrated the feasibility of using epitopic regions of rabbit ZPC (rZPC') as an immunocontraceptive vaccine for the first time. The results of immunizations in this study achieved a similar level to that of Dr. Hinsch's using peptide vaccine [19]. Though the study presented here is preliminary, it suggests a potentially important method for controlling population in consideration of its cost effectiveness, safety, and ease of production.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 86 10 62529248; pengjp{at}panda.ioz.ac.cn
Received: 29 May 2002.
First decision: 21 June 2002.
Accepted: 13 November 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  J.-J. Chang, J.-P. Peng, Y. Yang, J.-L. Wang, and L. Xu Study on the antifertility effects of the plasmid DNA vaccine expressing partial brLDH-C4' Reproduction, January 1, 2006; 131(1): 183 - 192. [Abstract] [Full Text] [PDF]
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 R. K. Naz, S. K. Gupta, J. C. Gupta, H. K. Vyas, and a. G.P. Talwar Recent advances in contraceptive vaccine development: a mini-review Hum. Reprod., December 1, 2005; 20(12): 3271 - 3283. [Abstract] [Full Text] [PDF]
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