Download Sperm-based contraceptive vaccine for wild rabbit

DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS
Research and Development
CSG 15
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to [email protected]
Project title
Sperm-based contraceptive vaccine for wild rabbit
VC0224
DEFRA project code
Contractor organisation
and location
Professor Harry Moore, Department of Molecular Biology and
Biotechnology, University of Sheffield, Sheffield, S102TN
Total DEFRA project costs
Project start date
£ 255,661
01/04/00
Project end date
31/05/02
Executive summary (maximum 2 sides A4)
World-wide there is considerable interest in the development of contraceptive vaccines for the control
of wild animal pests. Oral vaccination is often the only practical route and an optimal formulation that
stimulates an effective immune response is pivotal to development of this technology. Delivery
systems involving microorganisms (e.g. myxoma virus, vaccinia virus for rabies vaccine) are the most
effective for stimulating a substantial immune response but safety issues and public concerns about
genetically modified organisms have precluded this approach for contraceptive vaccine development
in the U.K. We have pioneered a synthetic vaccine using a peptide antigen (based on sperm-specific
protein) conjugated to a carrier protein (chlora toxin B) and encapsulated in lactide-co-glycolide
microparticles. In order to induce a mucosal response in the gut, antigens or other molecules must
cross the epithelial barrier, the follicle-associated epithelium (FAE) that separates the luminal
intestinal milieu from the lymphoid tissue of Peyer’s patches. The crucial sampling function of the
FAE is performed by specialised M-cells. Thus, the manipulation of the FAE and M-cells represents a
key step in the design of strategies to improve delivery of contraceptive (or other) vaccines, leading to
increased immune responses and enhanced effectiveness.
Previously, we demonstrated that sperm –specific peptide could be used to immunise (by injection)
the laboratory rabbit and generate an immune response that significantly inhibited fertility in vivo.
Specific antibodies generated by the vaccine prevented sperm binding to the egg and fertilisation in
vitro. Moreover during the course of these studies, a novel enhancer of particle uptake by the
intestinal mucosa was discovered and designated Mucosal Antigen Delivery Protein, MADP. The
objectives of the present study were to confirm and verify the reduction in fertility of female laboratory
rabbits and correlate the immune responses both in serum and reproductive tract fluids; to complete
the biochemical and genetic characteristics of Mucosal Antigen Delivery Protein, MADP; to evaluate
CSG 15 (Rev. 6/02)
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Project
title
Sperm-based contraceptive vaccine for wild rabbit
DEFRA
project code
VC0224
any possible toxic effects of multiple applications of vaccine in laboratory rabbits; to investigate the
species-specificity of sperm-based vaccine and in particular the effects of antisera from immunised
rabbits on sperm-egg interactions in vitro in the hare; and to evaluate the development of the vaccine
in fertility trials using group-housed female wild rabbits (in conjunction with Central Science
Laboratory, York).
A further fertility trial with the laboratory rabbit confirmed the efficacy of the vaccine with overall a ~ 3
fold reduction in fertility rate compared with controls. Prolonged immunisation of laboratory rabbits
(n=6) over 18 months caused no ill effects or gross pathological changes to major organs suggesting
that the vaccine was non-toxic to rabbits.
Using a proteomics approach involving protein separation by gel electrophoresis and analysis of
individual protein spots by mass spectrometry (Maldi-tof and qtof) a major component of MADP was
identified as macrophage migratory inhibitory factor, MIF. This cytokine is known to have
pleiomorphic activities related to immune and inflammatory responses. In experiments carried out
with an in vitro cell model involving human epithelial cells (Caco2 cells), MIF increased the number of
microparticles translocated across an epithelial cell monolayer. This finding indicated that MIF is likely
to responsible, in part at least, for the activity of MADP although further studies are required to
confirm this discovery.
Group-housed wild rabbits were successfully established in enclosures (CSL, York) and techniques
developed for sampling blood and vaginal secretions. An initial fertility trial with the contraceptive
vaccine demonstrated no significant reduction in fertility. Since there was some suggestion that
recombinant carrier protein (rCTB) was less effective than native protein a second trial was
undertaken using modified vaccine with peptide conjugated to native CTB. However this vaccine was
equally ineffective in reducing fertility. In both trials a transient immune response (IgG and IgA) was
obtained in serum but was not detected in vaginal flushing samples.
Studies on species-specificity of the vaccine could not be undertaken due to difficulties of obtaining
tissue samples from the hare.
The lack of efficacy of the vaccine in group-housed wild rabbits was in contrast to the results in single
house laboratory rabbits and is likely to relate to the relatively weak immune response to oral
vaccination (compared with parenteral route) in association with the repeated matings of wild females
in enclosures. The latter would present high numbers of spermatozoa in the female tract to overcome
the effect of any specific antibodies generated by the vaccine. However, intrinsic genetic differences
in the response of wild and laboratory rabbits to the vaccine cannot be ruled out. Further research is
required before this oral contraceptive vaccine approach can be effective in wild rabbits. In particular
more efficient oral delivery systems are required as this stage is the key to developing a substantial
and long-lasting immune response.
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Sperm-based contraceptive vaccine for wild rabbit
DEFRA
project code
VC0224
Scientific report (maximum 20 sides A4)
Introduction
Fertility control is one option for managing pest species [1]. Many laboratory studies have shown that
parenteral (injection) immunisation with specific reproductive antigens can lead to a reduction in
fertility [2]. World-wide there is considerable interest in the development of contraceptive vaccines for
the control of wild animal pests [3], but developing a safe and effective delivery system has proved
challenging. Oral vaccination is often the only practical route and an optimal formulation that
stimulates an effective immune response is pivotal to development of this technology. In Australia,
(Vertebrate Biocontrol Center, Canberra), the main strategy has been to use a recombinant viral
vector (myxoma virus, cytomegalovirus) expressing a common egg protein (ZPC) as a vaccine.
However, this GMO approach is questionable, since there is limited control after release of virus into
the environment and the egg antigen is not species-specific. In contrast, we have favoured a noninfectious inanimate vector to be delivered by bait and a synthetic antigen based on sperm proteins
that are species-specific.
As well as being practical, an oral approach has important physiological advantages. Maximum
immune responses in the reproductive tract should be achieved by strategies that induce activation,
proliferation and differentiation of immune cells of the common mucosal system which includes the
gastro-intestinal (GI) tract [4]. But immune responses to oral vaccines are often poor unless the GI
tract is stimulated appropriately. In order to induce a mucosal response, antigens or other molecules
must cross the epithelial barrier, the follicle-associated epithelium (FAE) that separates the luminal
intestinal milieu from the lymphoid tissue of Peyer’s patches [5]. The crucial sampling function of the
FAE is performed by specialised M-cells [6]. Thus, the manipulation of the FAE and M-cells
represents an important step in the design of strategies to improve delivery of contraceptive (or other)
vaccines, leading to increased immune responses and enhanced effectiveness. Studies in wild
rabbits will provide essential information concerning the variation in immune responses in outbred
populations and determine the ultimate strategies for antigen design [7].
The correct presentation and delivery of a contraceptive vaccine is imperative if this approach is to be
both effective and practical. Previously, the investigators (VCO205) demonstrated that specific
reproductive antigens (sperm surface proteins, uteroglobin) could be used to immunise (by injection)
the laboratory rabbit and generate an immune response that significantly inhibited fertility. More
recently, research (OC9521, antigen delivery systems for the control of fertility in rabbits) has
focussed on the development of appropriate methods for oral delivery of the contraceptive vaccine.
Our results indicate that significant inhibition of fertility (proportional to antibody response in serum)
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Project
title
Sperm-based contraceptive vaccine for wild rabbit
DEFRA
project code
VC0224
could be obtained in laboratory female rabbits fed a vectored vaccine consisting of microparticleencapsulated peptide antigen conjugated to appropriate carrier protein (i.e. cholera toxin B).
During the course of these studies, a novel enhancer of particle uptake by the intestinal mucosa was
discovered and designated Mucosal Antigen Delivery Protein, MADP [8]. These research findings
may be relevant for oral immunisation procedures in a variety of mammals and are the subject of
patent applications. VC0224 investigated further this oral vaccination approach with further studies in
laboratory rabbits and the initiation of fertility trials in wild rabbits in enclosures. The scientific
objectives were as follows:
1. To confirm and verify the reduction in fertility of female laboratory rabbits administered spermbased contraceptive vaccine with a large-scale fertility trial and a detailed investigation of the
correlation of the immune responses both in serum and reproductive tract fluids.
2. To complete the biochemical and genetic characteristics of Mucosal Antigen Delivery Protein,
MADP.
3. To investigate the use of purified MADP, along with improved microparticle technology to further
enhance vaccine efficacy.
4. To evaluate any possible toxic effects of multiple applications of vaccine in laboratory rabbits.
5. To investigate the species-specificity of sperm-based vaccine and in particular the effects of
antisera from immunised rabbits on sperm-egg interactions in vitro in the hare.
6.
To monitor and evaluate the serum and reproductive tract immune responses to contraceptive
vaccine in group-housed female wild rabbits (in conjunction with Central Science Laboratory,
York).
1. To confirm and verify the reduction in fertility of female laboratory rabbits administered spermbased contraceptive vaccine with a large-scale fertility trial and a detailed investigation of the
correlation of immune responses both in serum and reproductive tract fluids.
The fertility trial completed in laboratory rabbits in 2000 indicated that complete vaccine gave a
reduction in pregnancy and fertility rate compared with controls ( CTB and microparticles [CTB-MP],
and microparticles alone [MP]). Overall the results were consistent with the findings in the fertility trial
in 1999 and indicated a significant reduction in fertility rate in laboratory rabbits (treatment 23%;
control 68% fertility rate) after oral vaccination.
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Sperm-based contraceptive vaccine for wild rabbit
Number of
Females
Pregnancy
DEFRA
project code
VC0224
Fertility
rate
rate
Trial 1999
Complete
7
43
20 (15/74)*
CTB-MP
6
83
83 (48/58)
MP
2
100
81 (17/21)
Complete
19
42
24 (50/209)
CTB-MP
6
66
51 (26/51)
MP
3
66
58 (15/26)
Complete
26
42
23 (65/283)
Controls
17
76
68 (106/156)
Trial 2000
Total
(
*(litter size/ corpora lutea)
The immune responses by individual females to vaccine correlated in general with fertility outcome
particularly in relation to local IgA responses in the female genital tract although there were some
exceptions. These results suggested that antibodies generated in the cervix and uterus against
sperm were responsible for a reduction in fertility.
2. To complete the biochemical and genetic characteristics of Mucosal Antigen Delivery Protein,
MADP.
The intestinal mucosal surface is an important delivery point for vaccines and drugs. The antigen
sampling function of the epithelium is performed by specialised M cells, through which antigens are
delivered to the immune system. During studies of the effect of bacterial stimulation on the rabbit
intestinal epithelium carried out under our previous contract, we discovered a protein which enhances
mucosal uptake of particles by increasing M cell number and activity, designated ‘Mucosal Antigen
Delivery Protein’ (MADP) [8]. We believe that this material could be exploited to improve delivery of
the oral contraceptive vaccine.
A proteomics approach, employing 2D gel electrophoresis and mass spectrometry, together with in
vitro cell culture assay, was used to identify MADP. Biological material collected from intestinal loops
after challenge with S. pneumoniae or controls (E. coli or saline solution) was partially purified by
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Project
title
Sperm-based contraceptive vaccine for wild rabbit
DEFRA
project code
VC0224
were directly loaded onto immobilized pH gradient strips for first dimension isoelectric focusing,
followed by SDS polyacrylamide gel electrophoresis for the second dimension to separate proteins
according to their mol. wt. Gels were stained with Sypro Ruby and visualization and imaging analyses
were carried out using Pro-Finder software. This enabled us to identify and select a number of protein
spots that were expressed only in the samples (S. pneumoniae) that showed in vivo MADP activity,
and which are therefore candidates for MADP. Spots were picked using the Pro-Pick robotic system
and subsequently digested with trypsin using an in-gel digestion protocol, followed by peptide
fingerprint analysis by MALDI-Tof and protein sequence determination by Q-Tof mass spectrometry.
Proteins were identified from protein databases on the basis of sequence and size. While the
identification of the differentially expressed proteins is still in progress, results to date have shown
that the MADP extract contains immunoregulatory cytokines, including Macrophage Migration
Inhibitory Factor (MIF). Migration Inhibitory Factor (MIF), MIF [9], and similar multifunctional cytokines
are therefore primary candidates as promoters of conversion of enterocytes into M cells. MIF has
been discovered more that 30 years ago but up to date its production in the gut in response to
bacterial stimuli has not been reported.
3. To investigate the use of purified MADP, along with improved microparticle technology to further
enhance vaccine efficacy.
In vivo experiments showed that MADP is capable to up-regulate microparticles transport across the
specialized FAE by increasing the number of fully operational M cells. We therefore tested the ability
of MIF to induce similar changes in the function of epithelial cells in an in vitro human model. This
approach was a consequence of the fact that purified rabbit MIF is not commercially available and
that often lymphokines do show high degree of species-specificity. In addition this approach was
more acceptable from ethical point of use regarding the use of experimental animals.
Caco2 epithelial cells [see10] were seeded onto the upper face of 6.5 mm filters and cultured for
about 14 days until they reached confluence. The integrity of the cell monolayer was assessed by
determining the transpepithelial electric resistance. At confluence (electric resistance about 300 ohm.
cm2) cells were treated with various concentration of MIF for about 4 hours or other lymphokines for
control. After 4 hours, fluorescent polystyrene microparticles were added to the culture in the upper
chambers. Samples of culture supernatants from the lower chambers were collected at 15 minutes
intervals for 180 minutes. Samples of culture supernatant were analysed using flow cytometer in
order to assess the number of microparticles translocated across the epithelial monolayer. The
results showed that MIF has the ability to increase translocation of microparticles across Caco2 cells
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Project
title
Sperm-based contraceptive vaccine for wild rabbit
DEFRA
project code
VC0224
(although experiments vary in this effect). It is noteworthy that throughout the experiments exposure
to MIF did not affect the transepithelial electric resistance of the cell monolayer, thus suggesting that
translocation of microparticles was not due to alteration of tight junctions. This was also confirmed by
transmission electron microscopy. To rule out paracellular transport additional tests are in progress
which involve the use of C14-mannitol. Interestingly we have noticed that the differentiation stage of
intestinal epithelial cells does affect the ability of MIF to up-regulate translocation of microparticles.
Consistent with what we have observed and reported during experiments in vivo in rabbits, cell
monolayers that display a lower degree differentiation are more susceptible to the action of MIF
compared with cells that are more differentiated. This lends support to the validity of our in vitro
experimental model. From these experiments it is evident that MIF plays an important role in the
translocation of microparticles across the intestinal epithelium. Experiments are now in progress to
address these events at molecular level by using human microarrays.
4. To evaluate any possible toxic effects of multiple applications of vaccine in laboratory rabbits.
An 18 month study of multiple vaccine administration to 6 laboratory rabbits revealed no pathological
effects of the vaccine. All the animals remained healthy until the end of the study. Gross post mortem
examination revealed no obvious deleterious effect to major organs.
5. To investigate the species-specificity of sperm-based vaccine and in particular the effects of
antiserum from immunised rabbits on sperm-egg interactions in vitro in the hare.
Not undertaken. We have had difficulties in obtaining tissue samples from hares.
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Project
title
DEFRA
project code
Sperm-based contraceptive vaccine for wild rabbit
VC0224
6. To monitor and evaluate the serum and reproductive tract immune responses to contraceptive
vaccine in group-housed female wild rabbits (in conjunction with Central Science Laboratory, York).
The necessary Home office licences were obtained for fertility trials with wild rabbits held in
enclosures at the Central Science Laboratory. Initial investigations on animal handling, administration
of oral vaccine and obtaining fluid samples were successfully completed. The first fertility trial was
undertaken using recombinant CTB carrier protein conjugated to antigen and encapsulated in
microparticles. Females were treated with complete vaccine (13) or control vaccine of microparticlesCTB alone (13). First vaccinations were in March 2001 with the final booster vaccinations in the first
week in April 2001 and animals released into enclosures on the 26 th and 27th of April 2001. This
timing of the vaccination was intended to allow an immune response to develop prior to mating.
There was no obvious change in the behavioural pattern of the rabbits due to their treatments. The
results indicated no significant difference in fertility between treated and control rabbits (figure 1).
There were no significant differences in the date of recorded birth of litters.
Treatment
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Females
Females
Pregnancy
Mean Litter
vaccinated
completing trial
Rate (%)
size
Full vaccine
13
12
83
4.2
control
13
10
90
4.0
8
Project
title
DEFRA
project code
Sperm-based contraceptive vaccine for wild rabbit
VC0224
Figure 1 Fertility trial with contraceptive vaccine on wild rabbits (group-housed enclosures), March –
July 2001. Results after first breeding season (June).
Serum and vaginal secretion samples were obtained from treated and controls females during the
trial. There were indications of elevated serum IgG immune responses in 6 treated animals; however
vaginal secretions gave very low or undetectable response to IgG or IgA.
A second fertility trial was undertaken in 2002. There was a possibility that the failure of the vaccine
to inhibit fertility might have been due to the use of recombinant carrier protein. Recombinant CTB
protein can be produced to a consistent quality and is therefore preferable for vaccine production,
however adjuvant activity has been reported to be reduced compared to that of purified native CTB.
The latter was therefore used for the 2002 trial which was otherwise similar in design to that in 2001.
The results indicated no significant difference in fertility between treated and control rabbits (figure 2).
Treatment
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Females
Females
Pregnancy
Mean Litter
vaccinated
completing trial
Rate (%)
size
Full vaccine
11
11
100
3.7
control
10
10
100
3.3
9
Project
title
Sperm-based contraceptive vaccine for wild rabbit
DEFRA
project code
VC0224
Figure 2 Fertility trail with contraceptive vaccine (native CTB carrier) on wild rabbits (group-housed
enclosures). March –July 2002.
Elevated serum IgG immune responses were detected in 7 treated and 2 control females with the
magnitude of responses similar to those of females in 2001. Immune responses in vaginal secretions
were again very low. Clearly the contraceptive vaccine was ineffective in reducing fertility in wild
rabbits. This failure was in contrast to the success obtained in laboratory rabbits. The reasons for the
difference in efficacy of vaccine in laboratory and wild rabbits are unclear although several factors
need to be taken into account.
(1) Differences in immune response of laboratory and wild rabbits to vaccine: Vaginal IgG and IgA
responses in wild rabbits were very low or non-detectable while significant responses were detected
in laboratory animals. These results suggest that the mucosal response of wild rabbits to the vaccine
is poor. However caution needs to be exercised with this interpretation as the vaginal/cervical sample
from wild rabbits was more difficult to obtain and therefore low values could represent an artefact of
the sampling technique. Genetic differences between the inbred New Zealand white laboratory rabbit
and the wild European rabbit with respect to immune mechanisms are likely although there is little
data on this topic; however the response of wild and laboratory rabbits to strains of myxoma virus
differs markedly [11].
(2) Controlled mating of laboratory rabbits: Laboratory rabbits were given limited access to males
and allowed to mate only once. Wild rabbits in enclosures will mate repeatedly over the behavioural
oestrus period. Thus the number of sperm deposited in the female tract prior to fertilisation is likely to
be considerably higher in wild rabbits than lab rabbits and may overwhelm specific antibodies present
in the tract.
(3) Differences in the sperm antigen: The vaccine is based on a sperm peptide derived from
laboratory rabbit. Subtle differences in the sperm-egg recognition processes in the laboratory and
wild rabbit could mean that the vaccine fails to inhibit fertilisation despite raising an immune response
in wild rabbits.
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Project
title
Sperm-based contraceptive vaccine for wild rabbit
DEFRA
project code
VC0224
Conclusions
Currently our data indicate that the immune response to oral vaccination of wild rabbits (and in a
similar programme, wild grey squirrel) is insufficient to inhibit fertility. In contrast, under conditions
with laboratory rabbit and squirrel where access to breeding was limited or more controlled, a
significant reduction in fertility has been achieved. The relatively weak immune response to oral
vaccine means that blocking antibodies present in the tract can be effectively ‘swamped’ by repeated
sperm ejaculates over the mating period. Previous studies have indicated that parenteral (injection)
administration of vaccine is highly effective suggesting that the antigen itself will provoke antibodies
that specifically block fertility. The problem lies mainly with the inherent difficulty of mounting an
effective and long-lasting immune response by an oral route with synthetic (non-organism) vaccine.
The major rate-limiting factor seems to be presentation and uptake of microparticle in the follicle
associated epithelium.
An oral delivery route is clearly the most practical means of targeting small wild animal for population
control and the development of an effective oral vaccine would have many important applications for
disease control. But while empirical reformulation of the microparticle vaccine and testing in fertility
trials may produce incremental improvements and some efficacy, this strategy is expensive in terms
of fertility trials and success is uncertain at present. With the discovery of MIF as a component of
MADP we have made a clear advance in identifying a specific factor that enhances uptake and
processing of antigen within the gut however further studies are required in the laboratory to confirm
our findings and elucidate the cellular and molecular mechanism before it can be used effectively in a
vaccine. Recently human intestinal epithelial cells were shown to be a major source of MIF
supporting the contention that this molecular has a major role in regulating the mucosal immune
response [12]. A problem with the research area currently is that our knowledge generally of
mucosal immunity remains very sketchy.
Glycolide -co-lactide microparticles remain the most effective of the synthetic delivery systems for
vaccines. In vitro assays with epithelial cell lines may allow us to screen various formulations more
rapidly than in vivo experiments and provide a route to test other factors to modify the response of
cells (e.g. RNA interference techniques) [13].
However the immune response obtained by microparticles is at least an order of magnitude lower
than by viral infection. The highly effective rabies vaccine used in continental Europe involves a noninfectious recombinant vaccinia virus with limited replication [14]. Another possibility for future
CSG 15 (1/00)
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Project
title
Sperm-based contraceptive vaccine for wild rabbit
DEFRA
project code
VC0224
research would be to explore the option of a viral delivery system in the laboratory rabbits to assess
its effectiveness.
References
1. Tuyttens, FAM and Mcdonald DW (1998) Fertility control: An option for non-lethal control of
wild carnivores. Animal Welfare, 7: 339.
2. Frayne J and Hall L (1999) The potential use of sperm antigens as targets for
inununocontraception: Past present and future. J.Reprod.Immunol:43,1-33
3. Tyndale-Biscoe, C.H. (1994) Virus-vectored immunocontraception of feral mammals.
Reprod.Fertil.Dev. 6, 281-7.
4. Alexander, N.J. and Bialy, G. (1994) Contraceptive vaccine development. Reprod.Fertil.Dev.
6,273-80.
5. Borghesi C, Rogoli M, Bertifli E, and Nicoletti C (1996) Modifications of the follicle associated
epithelium by short-term exposure to a non-intestinal bacterium. J.Pathol: 180, 326
6. Jepson MA, Simons NL et al. (1993) selective binding and transcytosis of latex nu'crospheres
by rabbit M cells. Cell Tissue Res. 271:399
7. Madara JL (1997) The chameleon within: improving antigen delivery. Science, 277:910
8. F. Lodi, A. Man, M. J. Taussig and C. Nicoletti. Macrophage Migration Inhibitory Factor (MIF)
up-regulates transcellular transport of microparticles across human intestinal epithelial cells.
(ms. in preparation)
9. Lue H. et al. (2002) Macrophage migration inhibitory factor (MIF): mechanisms of action and
role in disease.
10. Nicoletti C. (2000) Unsolved mysteries of intestinal M cells. Gut, 47, 735-739.
11. Best et al (2000) Coevolution of host and virus: cellular localization of virus in myxoma virus
infection of resistant and susceptible European rabbits. Virology;277,:76-91.
12. . Eckmann M. et al. (2002) Ubiquitous production of macrophage migration inhibitory factor by
human gastric and intestinal epithelium. Gastroenterology, 122, 667-80.
13. Hannon G. (2002) RNA interference. Nature, 418, 144-251.
14. Mackowiak M. et al (1999) Vaccination of wildlife against rabies: successful use of a vectored
vaccine obtained by recombinant technology. Adv. Vet. Med.;41:571-83
Papers in preparation
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Project
title
Sperm-based contraceptive vaccine for wild rabbit
DEFRA
project code
VC0224
F. Lodi, A. Man, M. J. Taussig and C. Nicoletti. Macrophage Migration Inhibitory Factor (MIF) upregulates transcellular transport of microparticles across human intestinal epithelial cells. (ms. in
preparation)
Moore, H., Wong, C., Jenkins, N. Taussig, M. Nicolleti, C. Oral immunocontraception in the rabbit
(Ms in preparation).
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