| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a Vertebrate Biocontrol Cooperative Research Centre, CSIRO Wildlife and Ecology, Canberra, ACT, 2601, Australia
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Zona pellucida has been used as an immunocontraceptive antigen in many species [4–7]. In rabbits, heteroimmunization with porcine zona pellucida induces serum antibodies that cross-react with rabbit zonae and cause infertility associated with ovarian degeneration and endocrine dysfunction [8, 9]). In contrast, alloimmunization with rabbit zona pellucida did not induce detectable serum antibodies, although some immunized rabbits were subsequently infertile [8, 10].
The rabbit zona pellucida is composed of three glycoproteins: ZPA (R75), ZPB (R55), and ZPC (R45) [11, 12]. For immunization studies, we chose the ZPB glycoprotein. This protein has sperm receptor activity in rabbits and is the structural homologue of the porcine ZP3
and the human ZPB sperm receptor proteins, but it is distinct from the murine ZP3 (ZPC) sperm-binding protein [13]. On the basis of the cDNA sequence, the rabbit ZPB glycoprotein consists of 540 amino acids with an N-terminal hydrophobic signal sequence and a hydrophobic transmembrane domain close to the C terminus [14]. Mature zona proteins are heavily post-translationally glycosylated and contain both O- and N-linked carbohydrate residues [12]. Immunization of female rabbits with bacterially expressed rabbit ZPB (rc55) did not lead to detectable antibody production [12]. However, when baculovirus-expressed rabbit ZPB (bv55) was used to immunize female rabbits or guinea pigs, antibodies that reacted with ZPB and blocked sperm-egg binding in vitro were elicited [15]. This suggested that post-translational modifications such as glycosylation may be important for immunogenicity of the protein.
To produce rabbit ZPB that was as similar as possible to the native glycoprotein, we expressed the rabbit ZPB cDNA in a rabbit cell line in vitro using a recombinant vaccinia virus T7 expression system [16, 17]. This allowed production of milligram quantities of recombinant rabbit ZPB that was glycosylated and reacted with monoclonal antibodies (mAbs) directed against both carbohydrate and protein epitopes on native ZPB (unpublished results). In addition, the ZPB cDNA was expressed in recombinant myxoma virus for studies on viral delivery. Here we report the results of immunizing male and female rabbits with rabbit ZPB protein delivered either by direct injection of recombinant protein or by a recombinant myxoma virus.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Rabbit ZPB was produced by expression of the 1.7-kilobase (kb) rabbit ZPB (rc55) [14] in the vaccinia virus T7 (VV-T7) expression system [16, 17]. Briefly, the ZPB cDNA was expressed in vaccinia virus ZPB (VV-ZPB) under the control of the T7 promoter. A rabbit kidney cell line (RK 13) was then coinfected with the VV-ZPB and VV-T7 (expressing the T7 polymerase). This allowed production of high levels of the vaccinia virus-expressed ZPB (termed vvZPB), which was predominantly secreted from the infected cells. To purify vvZPB, infected cell culture medium was passed twice over an affinity chromatography column consisting of an mAb, IG7E10, which is directed against the polypeptide component of rabbit ZPB (unpublished results), covalently bound to cyanogen bromide-activated sepharose 4B (Sigma, St. Louis, MO). Purified protein was dialyzed against water. After purification, vvZPB was incubated at 65°C for 60 min to inactivate any residual vaccinia virus. It was then used either in immunization studies or as antigen in ELISA.
Construction of Recombinant Myxoma Virus
The 1.7-kb rabbit ZPB (rc55) cDNA [14] was ligated into the EcoRI site of the myxoma virus expression vector pURTK11 [3], downstream of the vaccinia virus p11 late promoter, generating pURTK11-ZPB. The vector pURTK11 also contains the Escherichia coli gpt (XGPRT: xanthine-guanine phosphoribosyl transferase) gene under the control of the vaccinia virus P7.5 early/late promoter, which allows the selection of the recombinant virus in the presence of mycophenolic acid [18, 19].
Myxoma virus strain Uriarra (UR) was derived from the plaque-purified virus described by Russell and Robbins [20]. This virus was used to infect SIRC cells (a rabbit corneal fibroblast cell line) at a multiplicity of infection (moi) of 0.1 plaque-forming units (pfu) per cell. The infected cell culture was transfected with pURTK11-ZPB using the lipofectACE Reagent (Gibco BRL, Gaithersburg, MD) as recommended by the manufacturer. Recombinant virus was selected in minimal essential medium (MEM; Gibco BRL, Grand Island, NY) containing 5 µg/ml mycophenolic acid, 250 µg/ml xanthine, 15 µg/ml hypoxanthine, 2 µg/ml aminopterin, and 10 µg/ml thymidine at 33°C. The resulting virus UR-ZPB was subjected to three rounds of plaque purification, amplified in RK13 cells, and titered on RK13 cell monolayers by plaque assay. UR for use as control virus infections was prepared as a testis homogenate from an infected rabbit and titered by plaque assay on Vero (green monkey kidney) cell monolayers.
Tissue Culture
RK13, SIRC, and Vero cells were cultured at 37°C in MEM plus 10% newborn bovine serum plus penicillin (200 U/ml) and streptomycin (100 µg/ml) in an atmosphere of 95% air, 5% CO2.
Immunofluorescence Assays
Confluent monolayers of RK13 cells grown on two-well chamber slides (Nunc, Naperville, IL) were infected with UR-ZPB at an moi of 5 pfu/cell and incubated for 24 h at 33°C. The infected cells were fixed to expose either cytoplasmic or cell surface-expressed ZPB for immunofluorescence detection [21] using the murine mAb IG7E10 against rabbit ZPB (unpublished results) as primary antibody.
ELISA
Wells of a 96-well plate were coated with 50 µl of vvZPB (2 µg/ml) in carbonate buffer (pH 9.6) and incubated at 4°C overnight. After being washed with PBS, the wells were coated with blocking buffer (PBS, O.1% Tween 20, 5% w:v skim milk powder) for 2 h at 37°C. Sample dilutions were prepared in blocking buffer as a 2-fold dilution series starting from 1:50, and 50 µl of sample was added to each well. Incubation was for 2 h at 37°C, and detection of antibody binding was performed using an anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad, Richmond, VA) followed by ABTS (2,2'-azino-bis(3 ethylbenzthiazoline-6-sulfonic acid; Sigma), 1 µg/ml plus hydrogen peroxide (0.03% v:v) in acetate buffer (sodium acetate 100 mM, NaH2PO4 50 mM, pH 4). Color development was read at 405 nm after 20-min incubation at room temperature. Titers are the reciprocal of the endpoint dilution. This was defined as the final dilution giving an optical density of at least 0.1 units above the optical density of the 1:50 dilution of the preimmune serum. The preimmune sera normally had an optical density of < 0.1.
Immunoblotting
Confluent monolayers of RK13 cells were infected with UR-ZPB (moi = 5) and incubated at 33°C for 16, 24, 40, 48, 64, and 72 h. At each time point, the culture medium was removed and centrifuged at 2000 x g for 5 min to remove cell debris. Particulate material in the medium was recovered by centrifugation using a TLS-55 swing-out rotor at 55 000 rpm (260 000 x g at r max) in a TL100 ultracentrifuge (Beckman Instruments, Palo Alto, CA), and the pellet containing ZPB was resuspended in a nonreducing SDS-PAGE loading buffer. The infected cell monolayer was washed in PBS (pH 7.2) and lysed using an equivalent volume of double-strength nonreducing SDS-PAGE loading buffer. The cell lysates and media pellets were analyzed by immunoblotting using the mAb IG7E10 mouse anti-rabbit ZPB (unpublished results) as the primary antibody.
Histology and Immunohistochemistry
Ovaries from rabbits immunized with vvZPB, or immunized with UR-ZPB followed by boosting with vvZPB, were fixed in Bouin's solution, paraffin-embedded, and sectioned at 5 µm, and then stained with hematoxylin and eosin. Sections were coded with the rabbit identity and examined without knowledge of the treatment and fertility status of the animal.
To demonstrate binding of anti-ZPB antibody to zona pellucida, unstained paraffin-embedded sections of these ovaries were probed with horseradish peroxidase-conjugated goat antirabbit Ig (Silenus, Hawthorn, Vic, Australia), and color was developed with H2O2 (0.003%) and diamino benzidine (0.5 g/L). Sections were counterstained with hematoxylin.
Rabbit Inoculations and Infections
All animal experiments were approved by the CSIRO Gungahlin Animal Experimentation Ethics Committee.
Laboratory rabbits (Oryctolagus cuniculus) 6–12 mo old, bred at the Gungahlin animal facility, were housed in individual cages in a temperature-controlled room under physical containment level 2 conditions approved by the Commonwealth of Australia Genetic Manipulation Advisory Committee. For each primary inoculation, 100 µg of vvZPB in water was emulsified in Freund's complete adjuvant (1:1 v:v) and inoculated s.c. into two or four sites using a total inoculum of 100–200 µl per rabbit. Booster injections were prepared similarly except that Freund's incomplete adjuvant was used instead of Freund's complete adjuvant. Myxoma virus or recombinant myxoma virus was diluted in PBS (pH 7.2), and 1000 pfu was inoculated intradermally over the thigh in a volume of 100 µl.
Fertility Trials
Six male rabbits of proven fertility were used to mate both the immunized and the control female rabbits that were infected with myxoma virus. Adjuvant-injected controls were not used, as previous studies (and unpublished data from our own laboratory) have shown no difference in fertility in rabbits after use of Freund's complete adjuvant [8, 9]. For mating, females were placed into the male's cage, and, after mating was observed, the female was left with the male for several hours or overnight. She was then paired with a second male, observed for successful mating, and left again for several hours or overnight. If mating did not occur after 15–30 min or if the female was particularly aggressive towards the male, she was moved to another male until mating did occur. Nesting boxes and nesting material were supplied to each female 4 wk after mating, and these were checked twice daily for nest-building and litters. Numbers of kittens born and numbers alive when the litter was first examined were recorded.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
After immunization with vvZPB, male rabbits had a very rapid antibody response to the protein, which continued to increase after the first boost. Although there was little response to the second boost, antibody titers were maintained for at least 60 days (Fig. 1A). In contrast, primary inoculation of female rabbits with vvZPB initially elicited a poor serum antibody response. Antibodies were detectable at low titers in 2 out of 5 rabbits by Day 10 and 4 of 5 by Day 30 (Fig. 1B). However, only one of the females (rabbit 512) had any substantial antibody response before Day 30, and this was one-quarter of the lowest male response at Day 30. Three of four female rabbits developed high antibody titers by Day 58, following the two booster immunizations (rabbit 310 died of unrelated causes and was not considered further). Antibody titers were maintained for at least 110 days after immunization. The specificity of the antibodies for vvZPB was demonstrated by immunoblotting (data not shown).
|
Each of the four surviving female rabbits was mated at Day 58 (i.e., 14 days after the second boost). After this mating, only rabbit 244, which had the lowest antibody titer, produced a litter (four kittens). Fifty days after this mating, the four rabbits were mated again and then killed by intravenous barbiturate injection 72 h later. The reproductive tracts were removed, and eggs were recovered by separately flushing the fallopian tubes and uterus with PBS. Eggs were found from only two rabbits, and only the four from rabbit 244, which had an antibody titer of 800 and nine corpora lutea, had undergone cleavage. Rabbit 528, which had an antibody titer of 102 400, also had nine corpora lutea, but the four eggs recovered by flushing were all degenerate. Antibody titers (Fig. 1B) had not decreased since the first mating.
In a second trial, a further six females were immunized using the protocol already described. Serum antibodies to vvZPB are shown in Figure 1B. The rabbits were mated at Day 58. Rabbits 162 and 167 had antibody titers of 6400 and 100, and had litters of four and five kittens, respectively. The remaining four rabbits, with titers of 12 800 or greater, were infertile. This second group of rabbits was retained and boosted a third time at Day 168. A strong delayed-type hypersensitivity response was observed at the site of the boost in five of the six rabbits 24 and 48 h after inoculation. The six rabbits were mated eleven days later. Only rabbit 167 produced a litter. It was the only rabbit with an antibody titer lower than 12 800 (data not shown) and the only rabbit not to have a delayed-type hypersensitivity response to the boost. The six rabbits were mated again 90 days after this mating and killed 72 h afterwards, and the reproductive tracts were flushed as previously described. Only rabbit 167 produced fertilized eggs (3 of 6 eggs recovered had undergone cleavage). Spermatozoa were observed within the reproductive tracts of all six rabbits, indicating that mating had occurred.
In Vitro Studies of Myxoma Virus Expressing ZPB
A recombinant myxoma virus expressing rabbit ZPB was constructed using the attenuated Uriarra (UR) strain of myxoma virus. This virus was designated UR-ZPB. Expression of ZPB in the cytoplasm and on the plasma membranes of RK13 cells following infection with UR-rZPB was demonstrated by indirect immunofluorescence (Fig. 2). Immunoblotting using cell and supernatant fractions from UR-ZPB-infected RK13 cells indicated that the majority of the virus-expressed ZPB was cell-associated. However, after incubation for longer than 24 h, a substantial proportion of the ZPB was found in the culture medium (data not shown).
|
Immunogenicity of Myxoma Virus Expressing ZPB
The immunogenicity of UR-ZPB was tested by inoculating six female and three male rabbits with 1000 pfu of virus. In contrast to inoculations with vvZPB protein, both male and female rabbits had similar responses, with serum antibodies detected ten days after infection. These antibody titers peaked at Day 15 and then steadily declined to Day 42, when the experiment was terminated (Fig. 3).
|
To examine fertility following immunization with UR-ZPB, 12 female rabbits were immunized with 1000 pfu of the virus, and 12 matched controls were inoculated with 1000 pfu of the parental UR strain of myxoma virus. Each matched pair consisted of litter mates. Inoculations were staggered in groups of six, two or three days apart, to allow the same six males to be used in all the mating trials. The rabbits were monitored daily for clinical signs of myxomatosis, and their rectal temperatures were measured. Over the 30-day monitoring period, the 12 females inoculated with the UR virus had elevated temperatures for 139 rabbit-days (number of animals with a rectal temperature over 40°C x number of days at this temperature). This was associated with mild to severe clinical signs of myxomatosis, although there were no deaths. In contrast, the 12 females infected with UR-ZPB had only 14 rabbit-days with temperatures greater than 40°C, and most animals developed only mild clinical signs of myxomatosis. Thus the recombinant UR-ZPB was attenuated in comparison to the parental virus.
All 12 of the UR-ZPB females were mated at 35 days after infection. However, only one of the controls was considered sufficiently recovered to mate at this time. Seven of the remaining controls were mated at 53 days after infection. A further two controls were not mated in this first phase of the trial because of a slow recovery from the virus infection. These two rabbits had recovered by the second phase of the trial. Finally, two of the controls were removed from the trial because of chronic respiratory disease resulting from the virus infection. After mating, three of the 12 females infected with UR-ZPB did not produce a litter. The antibody titers at mating of these rabbits were 800–1600, and peak antibody titers were 3200–6400. Antibody titers for the fertile rabbits ranged from 400 to 6400 at mating, and peak titers from 1600 to 6400. The mean litter size of the nine fertile rabbits infected with UR-ZPB was 7.0 ± 1.6 (SD). All of the eight mated controls had litters (mean litter size 7.4 ± 1.6). There was no relationship between fertility, peak antibody titer, or antibody titer at mating, and there were no differences between the control and test rabbits either in total number of kittens born or number observed born alive.
In the second phase of this trial, we examined whether a repeat inoculation with UR-ZPB, 140 days after the first immunization, was capable of boosting the antibody response to ZPB. The 12 females previously infected with UR-ZPB and the 10 surviving females previously infected with UR were each inoculated intradermally with 1000 pfu of UR-ZPB and mated 20 days later. As in the earlier part of this experiment, inoculations were staggered to allow the same six males to be used in all matings. Between 9 and 14 days after mating, the rabbits were killed and the reproductive tracts were examined. All 22 rabbits were pregnant, with an average litter size of 7.1 ± 1.5 (SD). There was no increase in antibody titers to ZBP in any of the animals previously immunized with UR-ZPB after this repeat inoculation and no measurable ZPB antibodies in animals previously infected with UR. This indicated that the rabbits were immune to myxoma virus as a consequence of the original infection, and this was supported by the lack of a poxvirus lesion at the site of the repeat inoculation.
Immunizing with UR-ZPB and Boosting with vvZPB Protein
To test whether the ZPB antibodies induced by UR-ZPB could be boosted with ZPB protein, five female and three male rabbits were inoculated with 1000 pfu of UR-ZPB and boosted at Days 30 and 44 with 100 µg of vvZPB in Freund's incomplete adjuvant. The females were then mated on Day 58. The ZPB serum antibody response to infection with UR-ZPB was similar to that obtained previously, with both male and female rabbits having similar titers that peaked at Day 15. There was no response to the first boost with vvZPB; however, the second boost elicited a strong response in both males and females (Fig. 4; compare with unboosted rabbits in Fig. 3). After mating, four of the five females did not produce a litter. The rabbit with the lowest antibody titer (number 518) had a litter of five kittens. Fifty days after the original mating (Day 108), these females were mated again and 18 h later killed, and the reproductive tracts were removed and flushed to recover eggs. Between four and eleven corpora lutea were present on the ovaries, although the ovaries from the infertile rabbits were structurally abnormal. Nine cleaved eggs were recovered from the previously fertile rabbit (number 518), which had an antibody titer of 200 (Fig. 4B). Cleaved eggs were recovered from only one of the other four rabbits (number 521), which had an antibody titer of 12 800; three cleaved eggs, three uncleaved eggs, and one degenerate egg were recovered.
|
Ovarian Pathology
Ovaries from nine rabbits immunized with either vvZPB in Freund's complete adjuvant or UR-ZBP followed by vvZPB in Freund's incomplete adjuvant were examined histologically using coded sections. Seven of nine of these ovaries were from infertile animals, which had antibody titers ranging from 12 800 to 102 400. In general, the infertile animals had a reduced number of primordial follicles, and there were fewer mature follicles compared to the fertile ovaries and to normal ovaries. Ovaries from four of the infertile animals had no developing follicles, and two cases resembled luteinized tissue, whereas ovaries from the other five had some follicular activity although the animals were infertile (Fig. 5). Ovaries from the two fertile animals were active and normal, and these animals had antibody titers of 200 and 800, i.e., less than one-sixteenth that the infertile animals. The histology of the ovaries from the infertile females following a third boost with vvZPB was similar to that of ovaries from the infertile rabbits described above. The ovaries from the 22 animals immunized with either UR-ZPB or UR and then challenged with UR-ZPB were normal in terms of numbers of corpora lutea and overall structure, and they were not examined histologically.
|
Immunohistological studies showed that rabbit immunoglobulin was bound to zonae and granulosa cells from infertile animals (Fig. 6). No follicles from fertile animals stained positively for immunoglobulin.
|
Correlation of Fertility and Antibody Titer
Figure 7 summarizes the fertility data and antibody titers at mating for the entire series of trials. Fourteen rabbits were infertile. These had antibody titers ranging from 800 to 102 400. However, only three of these rabbits had antibody titers less than 12 800, and these three were immunized with UR-ZPB and were fertile at a subsequent mating. There were 13 fertile rabbits that had antibody titers ranging from 100 to 6400. Nine of these animals had been immunized with UR-ZPB. Expressed as log 10 titers, the mean titer ± SD for the infertile animals was 4.13 ± 0.6, and for the fertile animals it was 2.9 ± 0.5. These differences were highly significant (p < 0.001; Student's t-test). Excluding the three rabbits in the UR-ZPB trial that were initially infertile but littered in the second phase of the trial, the lowest antibody titer associated with infertility was 12 800.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
These results contrast with previously reported immunizations of female rabbits using solubilized rabbit zona pellucida in Freund's complete adjuvant [8, 10] or bacterially expressed rabbit ZPB [12]. No serum antibodies were detected in either of these instances. However, when rabbits were immunized with baculovirus-expressed rabbit ZPB, antibodies to rabbit zonae were elicited [15]. No fertility data were reported, but this protein was glycosylated, although differently from native rabbit ZPB. Alloreactivity induced by a baculovirus-expressed protein but not an E. coli-expressed ZPB suggests that post-translational processing, including glycosylation, of ZPB may be important for immunogenicity. Furthermore, deglycoslyated zona proteins induce lower antibody titers than native zona proteins [22–24]. Expression of ZPB in rabbit cells using the vaccinia virus T7 system was intended to produce more authentic post-translational processing than could be obtained with baculovirus expression. Whether glycosylation of this vvZPB is identical to that of native ZPB remains the subject of investigation. Given the failure of solubilized rabbit zonae to induce detectable antibodies [8, 10], it is possible that completely authentic glycosylation may not be optimal for alloreactivity.
Infertility in the immunized rabbits was correlated with high serum antibody to ZPB and the binding of antibody to zona pellucida in developing follicles. Antibody binding to zona pellucida in vitro has been shown to interfere with sperm-binding, fertilization, and subsequent implantation [25, 26], and this provides one mechanism for antibody-induced infertility. But in addition, it seems likely that the follicular degeneration in the ovaries was due to antibody and that this follicular degeneration was a major cause of infertility. Follicular degeneration could also have been caused by an inflammatory or cellular immune response. Although there was no cellular inflammatory response in the ovaries at the time of autopsy, this was more than 3 mo after the original immunization, so an early cell-mediated or inflammatory response cannot be precluded. However, the ovarian histology from these ZPB-immunized rabbits was similar to that of ovaries from rabbits heteroimmunized with porcine zona pellucida [9]. In that study, there was no cellular inflammatory response in ovaries examined between 2 and 21 days after immunization. Similarly, ovaries from dogs studied 2–6 wk after immunization with porcine zona pellucida showed no inflammatory response despite the development of extensive follicular pathology [27]. Taken together, these findings indicate that antibody bound to zonae or to granulosa cells secreting ZPB is sufficient to significantly disrupt follicular development.
It has not been determined how antibody binding to zona might cause follicular degeneration. A simple explanation is that the presence of antibody and complement creates a toxic environment for developing follicles [6, 28]. There is little direct evidence for this, and in one analysis binding of the complement component C3 could not be detected in ovaries from dogs immunized with porcine zonae pellucidae [27]. Alternatively, antibodies bound to ZPB on the developing oocyte and granulosa cells may disrupt junctional complexes between cells and interrupt cell-to-cell communication, causing degeneration of the developing follicle [29]. It has also been suggested that carbohydrate molecules on zona proteins may influence cell-cell interactions in developing follicles [12]. Antibodies binding to these carbohydrate molecules on the zona could disrupt communication between granulosa cells and the oocyte, leading to developmental failure. As already discussed, glycosylation of ZPB appears to be important for immunogenicity. However, it is not clear if antibodies to particular complex carbohydrate epitopes induce ovarian pathology or whether the carbohydrate is structurally important for immunogenicity of the polypeptide chain [30]. The data available suggest that this may vary with antigen and species [5].
The depletion of primordial follicles in the ZPB-immunized rabbits was particularly interesting as this also occurred in primates and other species immunized with zona pellucida proteins and in rabbits heteroimmunized with porcine zona pellucida [6, 9, 28, 31]. The mechanism of depletion of primordial follicles remains to be determined experimentally. It is possible that antibody-induced degeneration of developing follicles could induce development of waves of primordial follicles into zona-secreting primary follicles, which then also degenerate because of antibodies binding to the developing zona [5, 28, 29]. However, it has been reported that ZPB can be detected in oocytes in rabbit primordial follicles [32] and that this protein is accessible to antibody in vivo [28]. If this is the case, then perhaps antibodies binding to ZPB prematurely trigger developmental signal pathways directly in the primordial follicles, thus leading to atresia.
When presented in the context of a myxoma virus infection, ZPB was perceived as a foreign antigen by both male and female rabbits. However, the maximum antibody titers obtained were less than 12 800, and only 3 of 12 females were subsequently infertile; this infertility was temporary. The rabbits were solidly immune to reinfection and so did not respond to a subsequent infection with this virus, although they could be boosted with vvZPB in Freund's incomplete adjuvant. In contrast, BALB/c mice immunized with mousepox virus expressing murine ZPC had sustained infertility associated with serum antibodies to ZPC that could be boosted on reexposure to the recombinant virus [33]. The enhanced responses by female rabbits to ZPB presented by myxoma virus may be due to antigen presentation in the context of the inflammatory response to a viral infection [34]. An alternative mechanism is suggested by Jackson et al. [33], in which B-lymphocytes might bind viral proteins complexed with ZBP on their cell surface receptors and present peptides from these viral proteins to helper T cells, bypassing the need for ZPB-specific T cells.
The results reported in this paper demonstrate that alloimmunization with the rabbit ZPB can induce substantial infertility in female laboratory rabbits. A large component of this infertility appears to be due to antibody-induced follicular degeneration. The mechanism of this degeneration remains to be determined, as do critical epitopes on the ZPB protein. While loss of ovarian function may be advantageous in a pest population as it could lead to life-long infertility, this would be highly undesirable for a human contraceptive vaccine, especially if it caused associated endocrine dysfunction. Understanding the mechanism of infertility and composition of critical epitopes is thus very important for further progress in immunocontraception, and the rabbit can provide a model for this. In addition, we have demonstrated that myxoma virus can present ZPB to the rabbit immune system and that presentation in the context of a virus infection overcomes immunological tolerance. The requirement for boosting this response remains to be surmounted, but the success obtained with ZPC presentation by mousepox virus in mice [33] suggests that this may well be possible by manipulating the antigen and the virus presentation.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Accepted: April 9, 1999.
Received: December 29, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. A Duckworth, K. Wilson, X. Cui, F. C Molinia, and P. E Cowan Immunogenicity and contraceptive potential of three infertility-relevant zona pellucida 2 epitopes in the marsupial brushtail possum (Trichosurus vulpecula) Reproduction, January 1, 2007; 133(1): 177 - 186. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. W Cooper and E. Larsen Immunocontraception of mammalian wildlife: ecological and immunogenetic issues. Reproduction, December 1, 2006; 132(6): 821 - 828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.M. Mackenzie, E.A. McLaughlin, H.D. Perkins, N. French, T. Sutherland, R.J. Jackson, B. Inglis, W.J. Muller, B.H. van Leeuwen, A.J. Robinson, et al. Immunocontraceptive Effects on Female Rabbits Infected with Recombinant Myxoma Virus Expressing Rabbit ZP2 or ZP3 Biol Reprod, March 1, 2006; 74(3): 511 - 521. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Clydesdale, J. Pekin, S. Beaton, R. J Jackson, S. Vignarajan, and C. M Hardy Contraception in mice immunized with recombinant zona pellucida subunit 3 proteins correlates with Th2 responses and the levels of interleukin 4 expressed by CD4+ cells Reproduction, December 1, 2004; 128(6): 737 - 745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R.-L. Xiang, F. Zhou, Y. Yang, and J.-P. Peng Construction of the Plasmid pCMV4-rZPC' DNA Vaccine and Analysis of Its Contraceptive Potential1 Biol Reprod, May 1, 2003; 68(5): 1518 - 1524. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Mulryan, M. G. Ryan, K. A. Myers, D. Shaw, W. Wang, S. M. Kingsman, P. L. Stern, and M. W. Carroll Attenuated Recombinant Vaccinia Virus Expressing Oncofetal Antigen (Tumor-associated Antigen) 5T4 Induces Active Therapy of Established Tumors Mol. Cancer Ther., October 1, 2002; 1(12): 1129 - 1137. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. S. Dunbar, S. Prasad, C. Carino, and S. M. Skinner The Ovary as an Immune Target Reproductive Sciences, January 1, 2001; 8(1_suppl): S43 - S48. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
