Note: Descriptions are shown in the official language in which they were submitted.
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FERTILITY IMPAIRING VACCINE CONTAINING AVIAN ZONA
PELLUCIDA PROTEIN AND METHOD OF USE
This application claims the benefit of U.S. Provisional Applications
Serial No. 60/141,929, filed July 1, 1999, and 60/162,984, filed November 2,
1999.
Background of the Invention
Population control of both domesticated and wild, free-roaming
animals has been a major problem around the world. Overpopulation in dogs and
1 S cats results in millions of euthanasias of unwanted pets yearly in the
United
States alone, and overpopulation of wild animals causes destruction of habitat
and threatens other species within the ecosystem. In humane shelters
population
control of unwanted pets is currently achieved through euthanasia of the
animals. In general, after capture, dogs are held for a period of one week. If
they are not adopted, they are humanely destroyed.
Finding a socially acceptable as well as a safe and effective means
of controlling excess populations of animals has been extremely difficult.
Traditional methods of population control in dogs have been largely
unsuccessful. Surgical spaying is a laborious procedure, requiring the initial
induction of the animal, gas anesthesia during surgery, a surgical pack with
suture materials and post-operative medications. Common surgical
complications include problems associated with the procedure itself, allergic
reactions to anesthetics or post-operative medications, and adverse local or
systemic effects during the recovery period. Examples include ovarian remnant
syndrome, where dogs continue to cycle despite being spayed, uterine
infections,
abdominal hemorrhage, and premature opening of the suture line. A substantial
recovery period is typically needed even after an uncomplicated procedure.
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Surgical spaying is also expensive, and pet owners are often unwilling to
assume
the costs.
The use of a reversible contraceptive is an attractive alternative to
surgical spaying. However, conventional hormonal therapies that have been
used to curb pet overpopulation are inconvenient because they usually require
daily administration of the drug. Furthermore, most protracted hormonal
therapies have undesirable side effects such as uterine infections, mammary
cancer, and diabetes.
A vaccine comprising porcine zona pellucida protein (pZP) and an
adjuvant comprising synthetic trehalose dicorynomycolate has been successfully
used to cause immunocontraception in horses (P. Willis et al., J. Equine Vet.
Sci.
14:364-370 (1994)) and elephants (R. Fayrer-Hosken, Wildlife Soc. Bull.,
25(1):18-21 (1997)). Injection of total heat solubilized pZP into horses
results in
an immunocontraceptive effect that lasts for about 12 months and has been
effective in reducing fertility of free-roaming horses (Kirkpatrick et al.,
J. Reprod. Fert. 94:437-444 (1992)). The pZP vaccine has been used for more
than 8 years in horses with no adverse reactions; nor does the vaccine affect
the
feti of pregnant mares.
A pZP vaccine has been administered to dogs as well, albeit with
somewhat less success. Mahi-Brown et al. have shown that infertility in the
female dog can be achieved by vaccinating with a crude pZP preparation, but
vaccination was accompanied by abnormal estrus cycles and other deleterious
side effects, such as ovarian cyst formation (J. Exp. Zool., 222:89-95 (1982);
Am. J. Reprod. Immunol. Microbiol., 18: 94-103 (1988)).
Porcine zona pellucida protein is obtained from pig ovaries, thus
the amount of vaccine that can be prepared is limited by the supply of pig
ovaries. Dunbar et al. (e.g., EP 599822, U.S. Pat. No. 5,637,300) have
experimented with reproductive control in non-rodent mammals using a
recombinant zona pellucida protein. However, due to limitations imposed by
recombinant DNA technology and available expression systems, the
recombinant protein lacks the glycosylation pattern of the native glycoprotein
and its immunogenicity has not been satisfactorily demonstrated. What is
clearly needed to combat overpopulation of domesticated and wild, free-roaming
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animals is a low cost contraceptive or sterilant that possesses a high degree
of
efficiency, lacks of harmful side effects, is amenable to remote delivery,
requires
a minimal number of administrations, and is easy to produce.
Summar~r of the Invention
The present invention provides a fertility impairing vaccine and a
method for impairing fertility in an animal. One embodiment of the fertility
impairing vaccine, referred to herein as a protein vaccine, comprises a
polypeptide comprising at least one avian zona pellucida protein or an
immunogenic fragment thereof. These proteins are related to the mammalian
zona pellucida proteins. Optionally, the protein vaccine of the invention
further
includes a polypeptide comprising a porcine zona pellucida protein or an
immunogenic fragment thereof.
Another embodiment of the fertility impairing vaccine, referred to
herein as a polynucleotide vaccine, comprises a polynucleotide comprising at
least one nucleotide sequence that encodes a polypeptide comprising an avian
zona pellucida protein or an immunogenic fragment thereof. Optionally, the
polynucleotide vaccine of the invention further includes a polynucleotide
comprising a nucleotide sequence that encodes a polypeptide comprising a
porcine zona pellucida protein or an immunogenic fragment thereof.
The fertility impairing vaccine of the invention optionally further
includes an immunological adjuvant or immunostimulant, preferably synthetic
trehalose dicorynomycolate (STDCM). Also optionally, the fertility impairing
vaccine contains an oil, preferably squalene oil.
The method for impairing fertility in the animal comprises
administering to the animal the fertility impairing vaccine of the invention
in a
manner and an amount effective to cause fertility impairment in the animal.
When administered as an immunocontraceptive, the fertility impairing vaccine
causes temporary, reversible infertility in the animal. When administered as
an
immunosterilant, the fertility impairing vaccine causes permanent,
irreversible
infertility in the animal. In dogs and cats, for example, immunosterilization
is
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far preferable to surgical sterilization and hormone regimens as a population
control tool.
The fertility impairing vaccine of the invention can generally be
used for immunological control of fertility and reproduction in any desired
animal population, whether domestic or wild. As an example, the fertility
impairing vaccine can be used to control ferret dog and cat populations by
development of a species-specific oral delivery vehicle. As another example,
the fertility impairing vaccine can be used to control wild horse, deer or
elephant
populations when formulated as a single dose dart or bullet that can be
administered without capture or confinement of the animal.
Detailed Description of the Preferred Embodiments
The fertility impairing vaccine of the invention contains a
polypeptide comprising at least one avian zona pellucida protein (aZP) or an
immunogenic fragment thereof, or a polynucleotide having a nucleotide
sequence encoding a polypeptide comprising an avian zona pellucida protein or
an immunogenic fragment thereof. The avian zona pellucida protein is
preferably, but need not be, substantially pure. The avian zona pellucida
protein
or immunogenic fragment thereof can be a naturally occurring protein, a
chemically or enzymatically synthesized protein, or a recombinant protein. The
avian zona pellucida protein or immunogenic fragment thereof is preferably,
but
need not be, glycosylated. In a glycosylated avian zona pellucida protein
(i.e.,
an avian zona pellucida glycoprotein), the glycosylation pattern is preferably
equivalent to the glycosylation pattern found on a native (i.e., naturally
occurnng) glycoprotein. Naturally occurring avian zona pellucida protein is
preferably obtained from a chicken. Avian zona pellucida protein can be
conveniently obtained from bird ovaries or from an egg cell of the bird and/or
the surrounding extracellular matrix and tissue, particularly the
perivitelline
membrane, for example from an oocyte or unfertilized egg of a bird at any
stage
of development.
In a preferred embodiment of the fertility impairing vaccine, the
avian zona pellucida protein used in the vaccine or encoded by the
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polynucleotide used in the vaccine is a total avian zona pellucida protein. A
total avian zona pellucida protein preparation includes a plurality of
immunoreactive proteins, some of which have been identified in chickens as
gp42 and gp97 (Y. Takeuchi et al., Eur. J. Biochem. 260:736-742 (1999)), and
gp34 and gp95 (M. Waclawek et al., Biol. Reprod. 59:1230-1239 (1998)).
Example I, below, identifies immunoreactive chicken proteins having molecular
weights of 70 kD, 40 kD and 35 kD. The fertility impairing vaccine can,
alternatively, contain fewer than all the avian zona pellucida proteins; for
example it can contain the 40 kD and the 35 kD proteins but not the 70 kD
protein.
Purity of the avian zona pellucida protein or glycoprotein can be
evaluated analytically using a combination or series of two-dimensional
polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) with both silver staining and Coomassie Blue
staining, and Western blot analysis, as described in the following Examples.
Glycoproteins typically migrate electrophoretically in gels as broad smears
rather than narrow bands, as a result of the variable levels of negative
charge
present in the constituent oligosaccharide chains. A total avian zona
pellucida
protein preparation isolated from bird eggs typically migrates as three
distinct
smears in the gel electrophoretic experiments, and shows the immunological
reactivity in Western blot analysis using a polyclonal antibody raised in
rabbits
to highly purified total porcine zona pellucida protein. In a substantially
pure
avian zona pellucida protein preparation, no contaminating proteins having
electromigration patterns different from those exhibited by the avian zona
pellucida proteins are detectable in the two-dimensional gel, the SDS-PAGE
gel,
or Western blot analyses.
An immunogenic fragment of an avian zona pellucida protein or
glycoprotein is a peptide fragment, preferably a glycosylated peptide
fragment,
that elicits an immune response in a subject to which it is administered. An
immune response includes either or both of a cellular immune response or
production of antibodies, and can include activation of the subject's B cells,
T
cells, helper T cells or other cells of the subject's immune system. For
example,
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an immune response is evidenced by a detectable anti-aZP antibody level in the
subject using ELISA substantially as described in Example III.
Immunogenicity of an avian zona pellucida protein fragment can be
determined, for example, by administering the adjuvanted candidate fragment to
the subject, then observing of the associated immune response by analyzing
anti
aZP fragment antibody titers in serum. An immunogenic peptide fragment
preferably contains more than seven amino acids, more preferably at least
about
amino acids, most preferably at least about 20 amino acids.
In a preferred embodiment of the fertility impairing vaccine, the
10 polypeptide used in the vaccine or encoded by the polynucleotide used in
the
vaccine further includes at least one epitope, such as a T cell, helper T cell
or B
cell epitope. The epitopes can be derived from the species to which the
vaccine
is to be administered, from the species that was the source of the zona
pellucida
protein or immunogenic fragment thereof, or from any other species, including
a
virus, bacterium, or parasite. T cell, helper T cell or B cell epitopes or
epitope
mimics have been identified in zona pellucida proteins (Garza et al., J.
Reprod.
Immunol., February 1998, pp. 87-101). The use of immune cell epitopes
derived from an immunogenic organism, such as a pathogenic parasite, is
preferred. For example, the polynucleotide can encode a chimeric peptide
comprising a T cell or helper T cell epitope from a parasite and a B cell
epitope
from a zona pellucida protein, such as a porcine or avian zona pellucida
protein
(Bagavant et al., Biol. Reprod., March 1997, pp. 764-770). A vaccine for use
as
an immunosterilant preferably contains a polypeptide that contains at least
one T
cell epitope (or a polynucleotide functionally encoding such a polypeptide),
whereas a vaccine for use as an immunocontraceptive preferably includes a
polypeptide that encodes at least one B cell epitope (or a polynucleotide
functionally encoding such a polypeptide).
A polynucleotide encoding the polypeptide comprising the avian
zona pellucida protein or immunogenic fragment thereof can include DNA,
RNA, a modified nucleic acid, or any combination thereof. The polynucleotide
can be supplied as part of a vector or as a "naked" polynucleotide. General
methods for construction, production and administration of polynucleotide
vaccines are known in the art, e.g. F. Vogel et al., Clin. Microbiol. Rev.
8:406-
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410 (1995). Polynucleotides can be generated by means standard in the art,
such
as by recombinant techniques, or by enzymatic or chemical synthesis.
A polynucleotide used in a fertility impairing vaccine of the
invention is preferably one that functionally encodes an avian zona pellucida
protein. A protein is "functionally encoded" if it is capable of being
expressed
from the genetic construct that contains it. For example, the polynucleotide
can
include one or more expression control sequences, such as cis-acting
transcription/translation regulatory sequences, including one or more of the
following: a promoter, response element, an initiator sequence, an enhancer, a
ribosome binding site, an RNA splice site, an intron element, a
polyadenylation
site, and a transcriptional terminator sequence, which are operably linked to
the
coding sequence and are, either alone or in combination, capable of directing
expression in the target animal. An expression control sequence is "operably
linked" to a coding sequence if it is positioned on the construct such that it
does,
or can be used to, control or regulate transcription or translation of that
coding
sequence. Preferred expression control sequences include strong and/or
inducible cis-acting transcription/translation regulatory sequences such as
those
derived from metallothionine genes, actin genes, myosin genes, immunoglobulin
genes, cytomegalovirus (CMV), SV40, Rous sarcoma virus, adenovirus, bovine
papilloma virus, and the like.
The coding and expression control sequences for the avian zona
pellucida protein are preferably constructed in a vector, such as a plasmid of
bacterial origin, a cosmid, episome, or a viral vector, for administration to
a
target animal. A vector useful in the present invention can be circular or
linear,
single-stranded or double stranded. There are numerous plasmids known to
those of ordinary skill in the art useful for the production of polynucleotide
vaccine plasmids. A specific embodiment employs constructs using the plasmid
pcDNA3.1 as the vector (InVitrogen Corporation, Carlsbad, CA). In addition,
the vector construct can contain immunostimulatory sequences (ISS) that
stimulate the animal's immune system. Other possible additions to the
polynucleotide vaccine constructs include nucleotide sequences coding
cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF)
or interleukin-12 (IL-12). The cytokines can be used in various combinations
to
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fme-tune the response of the animal's immune system, including both antibody
and cytotoxic T lymphocyte responses, to bring out the specific level of
response
needed to affect the animal's reproductive system.
Alternatively, the vector can be a viral vector, including an
adenovirus vector, and adenovirus associated vector, or a retroviral vector.
Preferably the viral vector is a nonreplicating retroviral vector such as the
Moloney marine leukemia virus (N2) backbone as described by Irwin et al. (J.
Virology 68:5036-5044 (1994)).
Optionally, a fertility impairing protein vaccine of the invention
further includes a polypeptide comprising a porcine zona pellucida protein or
an
immunogenic fragment thereof. The porcine zona pellucida protein or
immunogenic fragment thereof can be a naturally occurring protein, a
chemically or enzymatically synthesized protein, or a recombinant protein. The
porcine zona pellucida protein or immunogenic fragment thereof is preferably,
but need not be, glycosylated. In a glycosylated porcine zona pellucida
protein
(i.e., a porcine zona pellucida glycoprotein), the glycosylation pattern is
preferably equivalent to the glycosylation pattern found on a native (i.e.,
naturally occurnng) glycoprotein. Immunogenicity of the porcine zona
pellucida fragment is as generally described for avian zona pellucida protein.
Without intending to be bound be any particular theory or mechanism, the
porcine zona pellucida protein or immunogenic fragment thereof can be included
in the fertility impairing vaccine as an adjuvant to enhance the recipient's
immune response, or for its independent and/or synergistic effect in impairing
fertility in the recipient. For example, a fertility impairing protein vaccine
substantially comprising chicken zona pellucida protein, which is readily
available and relatively inexpensive to isolate, can be augmented by the
addition
of a small amount of porcine zona pellucida protein, which is more difficult
and
expensive to obtain. Notwithstanding the above, any desired ratio of avian
zona
pellucida protein to porcine zona pellucida protein can be used in this
embodiment of the vaccine of the invention. The relative amounts of avian zona
pellucida protein and porcine zona pellucida protein used in this embodiment
of
the vaccine depend on the nature of the animal being vaccinated and the
immunogenic response generated in the animal by the vaccine. Preferably the
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ratio of avian zona pellucida to porcine zona pellucida (aZP: pZP) used in
this
embodiment of the vaccine is between 100:1 and 1:100.
Similarly, a polynucleotide vaccine of the invention optionally
includes, in addition to a polynucleotide encoding a avian zona pellucida
protein
or immunogenic fragment thereof, a polynucleotide encoding a porcine zona
pellucida protein or an immunogenic fragment thereof. The porcine zona
pellucida polynucleotide functionally encodes porcine zona pellucida protein
and can take the form of various genetic constructs as generally described for
the
polynucleotide encoding avian zona pellucida protein. Polynucleotides
encoding avian and porcine zona pellucida proteins can constitute part of the
same vector or can be delivered to the recipient on different vectors.
The fertility impairing vaccine of the invention, whether it contains
a polypeptide (e.g., conjugated or non-conjugated avian zona pellucida
protein)
or a polynucleotide encoding a polypeptide, optionally further includes an
immunological adjuvant to enhance the immunological response of the subject
to the immunogenic polypeptide or polynucleotide. Examples of adjuvants
include Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, Freund's
mycotoxin free adjuvant, aluminum hydroxide, EQUIMUNE (a deproteinized
highly purified cell wall extract derived from non-pathogenic Mycobacteria
spp., Acemannan (a long-chain polydispersed (3(1,4) linked mannan polymer
interspersed with O-acetylated groups, permulum, and an adjuvant comprising
an immunostimulant such as synthetic trehalose dicorynomycolate (STDCM)_
The vaccine can also optionally include an oil, such as squalene oil, drakeol,
or
vegetable oil, which can also have an adjuvant effect (see P. Willis et al.,
J.
Equine Vet. Sci., 14, 364-370 (1994)). It should be noted, however, that
companion birds exhibit a sensitivity to oil-based adjuvants, which cause
necrotic granulomas at the site of administration. An adjuvant comprising
synthetic trehalose dicorynemycolate, squalene oil, and a surfactant such as
lecithin is preferred for use in mammals. Lecithin typically includes
phosphatidyl choline. An adjuvant comprising aluminum hydroxide is preferred
for use in birds.
When an oil adjuvant is used, homogenization of the adjuvant, such
as Freund's adjuvant, with the aqueous zona pellucida protein or
polynucleotide
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solution can be accomplished using any convenient means known in the art,
such that the oil disperses within the aqueous solution to form an oil in
water
emulsion. Oil droplet sizes of about 200 nm or less are particularly preferred
as
they produce a more uniform and stable suspension. A particularly preferred
5 fertility impairing vaccine comprises a predetermined amount of zona
pellucida
protein or polynucleotide and a pharmaceutically acceptable immunogenic level
of adjuvant in an emulsion containing about 10% oil phase and about 90%
aqueous phase.
In another embodiment of the invention, the fertility impairing
10 vaccine, whether it contains a conjugated or a non-conjugated avian zona
pellucida protein or a polynucleotide encoding said polypeptide, contains no
adjuvant; essentially the fertility impairing vaccine of this embodiment is an
aqueous avian zona pellucida protein or polynucleotide solution that delivers
the
intended amount of avian zona pellucida protein or polynucleotide to the
recipient.
In a preferred embodiment of the protein vaccine for use in
mammals, the vaccine comprises oil, preferably a biodegradable oil such as
squalene oil, in an amount of about 2.5% to about 15%, preferably about 8% to
about 12%. In preparing the composition it is advantageous to combine a
concentrated oily adjuvant composition with an aqueous solution of the
immunogen, aZP protein. Typically, the composition is prepared using an
adjuvant concentrate which contains lecithin (about 5% to about 15 % wt/vol,
preferably about 12% wt /vol) and STDCM (preferably about 25 mg/mL to
about 50 mg/mL) in squalene oil. The term % wt/vol means grams per 100 mL
of liquid. The aqueous solution containing the isolated aZP protein is
typically a
phosphate-buffered saline (PBS) solution, and additionally preferably contains
Tween 80 (about 0.2% vol/vol to about 0.8% vol/vol, preferably about 0.4%
vol/vol). See J.A. Rudbach et al., "Ribi Adjuvants: Chemistry, Biology and
Utility in Vaccines for Human and Veterinary Medicine," in The Theory and
Practical Application of Ad'uvants, D.E.S. Stewart-Tull, Ed., John Wiley &
Sons, New York, NY (1995)). Homogenization of the oily adjuvant concentrate
with the aqueous aZP solution can be accomplished using any convenient means
known in the art, such that the oil disperses within the aqueous solution to
form
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an oil in water emulsion. Oil droplet sizes of about 200 nm or less are
particularly preferred as they produce a more uniform and stable suspension. A
particularly preferred vaccine comprises predetermined amounts of aZP and
STDCM in an emulsion containing about 10% squalene oil and about 90%
aqueous phase.
The fertility impairing vaccine is administered in a manner
effective to cause impaired fertility in an animal. An "animal," as that term
is
used herein, is an oocyte-producing organism that is capable of voluntary
mobility. Preferred animals include mammals and birds. More preferably, the
animal is a mammal; most preferably it is a deer, horse, elephant, rat, mouse,
rabbit, ferret, cat or dog. Birds include but are not limited to free-ranging
birds
and domesticated birds, including food birds such as chickens, turkeys, and
waterfowl, and companion birds in the order Psittaciformes, Passeriformes,
Columbiformes, Falconiformes, such as budgerigars, canaries, finches,
cockatiels, lovebirds, pigeons, doves and hawks.
Impairment of fertility in an animal in accordance with the
invention can take the form of either immunocontraception and
immunosterilization. Immunosterilization means permanent, irreversible
infertility, in contrast to immunocontraception wherein infertility is
temporary or
transient, and reversible. Immunocontraception and immunosterilization are
both dependent on the antibody titer level in the serum of the subject, but
immunosterilization is typically the result of ovarian pathology caused by the
immune response of the animal, as evidenced by, for example, total destruction
of the zona pellucida proteins and/or influx of leukocytes into the follicles.
Reducing the number of boosters leads to reduced immune response which
results in immunocontraception (i.e., infertility that is temporary and
reversible)
instead of immunosterilization.
Optionally, the zona pellucida protein can be conjugated to an
immunogenic carrier protein. A conjugated form of an avian zona pellucida
protein is suitable for use in embodiments of the fertility impairing vaccine
containing an avian zona pellucida protein that is homologous with respect to
the
intended recipient; that is, where the avian zona pellucida protein is
isolated
from the same species to which it is to be administered. For example, where a
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fertility impairing vaccine comprising chicken zona pellucida protein is
intended
for administration to chickens, it may be desirable to conjugate the chicken
zona
pellucida protein to an immunogenic carrier to augment the immune response of
the recipient. However, it is to be understood that the invention includes
administration of avian zona pellucida protein from any avian source to any
bird,
whether homologous or heterologous, and whether the avian zona pellucida
protein is conjugated or unconjugated. For example, the invention includes
administration of a fertility impairing vaccine comprising an unconjugated
chicken zona pellucida to psittacine birds.
The fertility impairing vaccine is administered in a manner and an
amount effective to cause the desired infertility in the animal. For example,
to
immunosterilize a dog or a cat using a protein vaccine of the invention, the
vaccine is preferably administered in the form of a plurality of doses
(typically
about 1.0 mL for a dog, 0.5 mL for a cat), each dose containing avian zona
pellucida protein or an immunogenic fragment thereof (or a mixed vaccine
containing both avian and porcine zona pellucida proteins), in an amount of
about 100 ~g to about 2 mg, more preferably about 100 ~,g to about 400 fig. To
treat a bird, for example, a protein vaccine is preferably administered in the
form
of a plurality of doses (typically about 0.25 mL), each dose containing avian
zona pellucida protein or an immunogenic fragment thereof (or a mixed vaccine
containing both avian and porcine zona pellucida proteins), in an amount of
about 10 ~,g to about 2 mg, more preferably about 50 ~g to about 400 fig.
A polynucleotide vaccine is preferably administered in one or more
doses containing the plasmid, viral vector or naked polynucleotide in an
amount
of about 5 ~g to about 100 ~,g. One of skill in the art can readily determine
a
suitable dosage for a particular animal, depending on the nature, size and
overall
health of the animal, as well as the condition to be treated.
For vaccines administered to mammals, an immunostimulant such
as STDCM is typically present in a per dose amount of about 50 ~.g to about 5
mg, preferably in an amount of about 200 ~,g to about 3.5 mg, more preferably
in
an amount of about 400 ~g to about 3.0 mg. For vaccines administered to birds,
an immunostimulant such as STDCM is optionally present in a per dose amount
of about 10 ~.g to about 5 mg, preferably in an amount of about 50 ~,g to
about
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3.5 mg, more preferably in an amount of about 1 mg to about 3 mg. When
administered to a bird, the fertility impairing vaccine preferably contains
AIOH
as an adjuvant, or contains no adjuvant at all.
An initial injection of the vaccine is followed by one or more
booster injections at two to four week intervals, although the boosters can be
administered from about 9 days to about twelve months following the previous
vaccination. Preferably, two booster shots are administered following the
initial
administration. The body's immunological response to the vaccine at this
dosing regimen is expected to render the ovaries permanently inactive as a
result
of, for example, follicle disruption or destruction, as evidenced by
immunocytochemical analysis and histological evaluation of the ovarian tissue
of vaccinated subjects. Sterility is permanent and irreversible.
Immunosterilization of animals in accordance with the present method is not
expected to cause abnormal estrus cycles or other significant undesirable side
1 S effects in the vaccinated subjects.
When the fertility impairing vaccine is administered to a dog or a
cat as described above, but with only one booster instead of two or more
boosters, the vaccine is expected to result in immunocontraception (i.e.,
temporary or transient, reversible infertility) rather than
immunosterilization.
Fertility impairment in dogs in accordance with the present method preferably
is
not expected to cause abnormal estrus cycles or other significant undesirable
side effects in the vaccinated subjects.
Although the fertility impairing vaccine is typically administered
by way of intramuscular injection, other forms of administration are also
contemplated, including subcutaneous or intradermal administration, oral
administration, as by food or water, topical administration, including
transdermal administration, aerosol administration, cloacal or vaginal
administration, intracoelomic administration, intranasal administration, and
transconjunctival administration, including the use of eye drops. In addition,
liposome-mediated, microsphere-mediated, and microencapsulation systems are
all included as delivery vehicles for the fertility impairing vaccine of the
present
invention. The fertility vaccine can be formulated as a single dose vaccine
and
packaged for delivery through a dart or a bullet, such that it can be
administered
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to an animal from a distance. A single shot time-release delivery vehicle is
also
contemplated, wherein the avian zona pellucida protein or immunogenic
fragment thereof (or the polynucleotide encoding the avian zona pellucida or
immunogenic fragment thereof) is supplied in a single dose in two or more
different forms, such as a free protein and a microencapsulated protein, to
allow
administration of the immunogenic agent to the animal over a longer period of
time.
Advantages of the invention are illustrated by the following
examples. However, the particular materials and amounts thereof recited in
these examples, as well as other conditions and details, are to be interpreted
to
apply broadly in the art and should not be construed to unduly restrict or
limit
the invention in any way.
EXAMPLES
Ezample I. Isolation of Avian Zona Pellucida Proteins
Perivitelline membranes were obtained from laid chicken eggs.
Yolks were separated from whites by making a small hole in the egg and
draining the albumin. The yolks were removed from the shell and the chalazae
was removed and discarded. The perivitelline membranes (pvm) were either
manually peeled away or punctured to drain the yolk, and the membranes were
washed in sterile phosphate buffered saline solution. A tissue homogenizer
(Powergam 700D) was used to homogenize the membranes. M. Waclawek et al.
(Biol. Reprod., 59, 1230-1239 (1998)) reported a similar procedure for
isolating
perivitelline membranes from laid eggs that can also be used. Alternatively,
perivitelline membranes can be isolated directly from ovarian follicles by
preparing granulosa cell sheets substantially as described by A. Gilbert et
al, J.
Reprod. Fertil., 50, 179-181 (1977)).
Purity was demonstrated and confirmed using one-dimensional and
two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis
combined with Western blot analysis, silver staining, and, at times, Coomassie
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blue staining, using standard protocols. Proteins having molecular weights of
70
kD, 40 kD and 35 kD reacted with rabbit anti-pZP serum. Yolk and albumin
controls did not react with the rabbit anti-pZP serum. The 70 kD protein was
easily washed away from the perivitelline membrane. The 35 kD protein was
5 approximately twice as abundant as the 40 kD protein and both had strong
reactivity to the anti-pZP serum. One or both of these proteins are likely to
be
homologous to mammalian ZP3 (also known as ZPC), according to published
reports (Y. Takeuchi et al., Eur. J. Biochem., 260, 736-742 (1999); M.
Waclawek et al., Biol. Reprod., 59, 1230-1239 (1998)).
Ezample II. Preparation of Avian Zona Pellucida (aZP) Vaccine
The vaccine is prepared by homogenizing a concentrated oily
adjuvant concentrate with an aqueous antigen solution containing isolated aZP
protein. The oily adjuvant concentrate contains a surfactant, lecithin, and an
immunostimulant, synthetic trehalose dicorynomycolate (STDCM), in squalene
oil. A typical adjuvant concentrate contains about 12% wt/vol (grams/100 mL)
lecithin and about 25-50 mg/mL STDCM in squalene oil. The aqueous antigen
solution contains the aZP protein preparation in saline or phosphate buffered
saline (PBS) and Tween 80. When prepared for use in combination with an
adjuvant concentrate to yield the vaccine composition, the aqueous composition
typically contains 0.4% (vol/vol) Tween 80 and an amount of aZP calculated to
yield a dose of about 100 ~g to about 400 ~g per vaccination. Vaccine doses
for
dogs are about 1 mL in volume.
Homogenizing is accomplished by combining adjuvant concentrate (to a
final concentration of no greater than 10% vol/vol) with aqueous aZP solution
and emulsifying using a Powergam 700D homogenizes at 15,000 rpm for 6
minutes. The resulting emulsion is then homogenized with phosphate buffered
saline (PBS) (containing 0.4% vol/vol Tween 80) at 20,000 for 8-12 minutes.
The homogenization process results in a vaccine composition that is an oil-in-
water emulsion or possibly a water-in-oil-in-water emulsion. While the
inventors do not intend that the invention be bound by any particular
scientific
theory, it is believed that the STDCM, an amphiphilic glycolipid, partitions
to
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16
the oil/water interfaces in the emulsion, and that the antigen is attracted to
and
associates with the STDCM at these interfaces.
Ezample III. Immunosterilization of Dogs using aZP Vaccine
Vaccinations. Female dogs are vaccinated with 200 ~g of aZP per dose
in a vaccine adjuvanted with synthetic trehalose dicorynomycolate (STDCM,
commercially available from RIBI Immunochem Co., Hamilton, MT) in
squalene oil. The amount of STDCM per dose was about 100 ~,g to about 2.5
mg. A l OX adjuvant concentrate as described in Example II is available from
RIBI Ixnmunochem Co., Hamilton, MT, and vaccines are prepared as described
in Example II. Boosters (containing the same amount of aZP, 200 fig) are
administered at 14-day intervals. Vaccinations are delivered to dogs
infra-muscularly in the longissimus muscle (loin area). Booster injections are
administered on the contra-lateral side.
Antibody titers. Blood is drawn from each dog weekly, and serum
antibody titers are determined using an enzyme linked immunosorbant assay
(ELISA). Adjacent wells of a microwell plate are coated with 2 ~,g aZP, and
incubated for 6 hours. The wells are then blocked with 5% bovine serum
albumin (Sigma Chemical Co., St. Louis, MO) in TBST (Tris-buffered saline +
5% Tween-20) and incubated overnight. Wells are then loaded with the primary
antibody (canine serum) in TBST at a 1:500 and 1:1,000 dilution and incubated
for 4 hours. The wells are then washed and loaded with 50 ~1 of the secondary
antibody (rabbit anti-dog IgG) and incubated for 2 hours. Color change is
observed after the addition ofp-nitrophenyl phosphate for 30 minutes and the
reaction terminated by the addition of 3 M NaOH. The optical density is read
at
a 405-492 nm range on a Spectramax spectrophotometer. The dog's
pre-immune serum serves as the negative control.
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Ezample IV. Administration of Mined Chicken/Porcine ZP Vaccine
to Psittacines
Vaccinations. Chickens are vaccinated with 200 p.g of mixed zona
pellucida protein (1:1, aZP:pZP) per dose (1 mL volumes) in a vaccine
adjuvanted with Freund's complete adjuvant. The birds are vaccinated with
three injections administered at approximately two week intervals. Under
veterinary supervision, vaccinations are delivered to chickens intramuscularly
in
the deep pectoral muscle. Booster injections are administered on the
contra-lateral side.
Antibodv titers. Blood is drawn from each bird at the time of each of the
three injections, and about three weeks following the last injection. Serum
antibody titers (IgG) are determined using an enzyme linked immunosorbant
assay (ELISA). Adjacent wells of a microwell plate are coated with 2 ~,g aZP,
and incubated for 6 hours. The wells are then blocked with 5% bovine serum
albumin (Sigma Chemical Co., St. Louis, MO) in TBST (Tris-buffered saline +
5% Tween-20) and incubated overnight. Wells are then loaded with the primary
antibody (i.e., avian serum) in TBST at a 1:500 and 1:1,000 dilution and
incubated for 4 hours. The wells are then washed and loaded with 50 ~1 of the
secondary antibody (rabbit anti-chicken IgG) and incubated for 2 hours. Color
change is observed after the addition ofp-nitrophenyl phosphate for 30 minutes
and the reaction is terminated by the addition of 3 M NaOH. The optical
density
is read at a 405-492 nm range on a Spectramax spectrophotometer. The
chickens' pre-immune serum serves as the negative control.
Era la~~ Eggs are counted beginning on the day of the first
injection to evaluate reduction in egg production, providing direct evidence
of
fertility impairment.
The complete disclosure of all patents, patent applications, and
publications cited herein are incorporated by reference. The foregoing
detailed
description and examples have been given for clarity of understanding only. No
unnecessary limitations are to be understood therefrom. The invention is not
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18
limited to the exact details shown and described, for variations obvious to
one
skilled in the art will be included within the invention defined by the
claims.
