Note: Descriptions are shown in the official language in which they were submitted.
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METHOD AND COMPOSITION FOR AFFECTING
REPRODUCTIVE SYSTEMS
This application claims the benefit of U.S. Provisional
Applications Serial No. 60/142,308, filed July 1, 1999, and Serial No.
60/177,903, filed January 25, 2000.
Background of the Invention
Reproductive disorders in birds, rabbits, fish, reptiles and
amphibians are common and can be difficult to treat. Some reproductive
disorders, such as egg binding, are characterized by acute onset and cause a
rapid deterioration in the health and physiological stability of the animal.
Moreover, surgical and pharmacological interventions are often accompanied by
high morbidity and mortality.
In birds, two of the most common clinically recognized
reproductive disorders axe egg binding and dystocia. Egg binding is the
failure
of an egg to pass through the oviduct at a normal rate. Dystocia (egg
retention)
defines a condition in which the developing egg is in the caudal oviduct and
is
either obstructing the cloaca or has caused oviduct tissue to prolapse through
the
oviduct-cloacal opening. Egg movement through the oviduct can stop at various
locations. The most common anatomic areas for problems to occur are the
caudal uterus, vagina and vaginal-cloacal junction. Budgerigars, canaries,
finches, cockatiels and lovebirds most frequently have problems with dystocia.
This is probably because the presentation of a palpable egg for more than a
few
hours in small birds is generally more serious than it is in larger birds.
Dystocias are most critical in passerines and other small birds, many of which
can survive only a few hours without aggressive therapy. Many hens with
dystocia will attempt to lay another egg. Administration of
medroxyprogesterone will temporarily stop ovulation, but there are side
effects
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2
and its use is controversial. Surgical correction, another alternative, is
costly
and dangerous.
Abnormally prolonged presence of an egg in the oviduct causes a
multitude of complications in the hen. The severity of these complications
depends on the species, the bird's previous health, the cause of the egg
binding,
the egg's location in the oviduct, and the time elapsed since egg development
began. An egg lodged in the pelvic canal may compress the pelvic vessels and
the kidneys, causing circulatory disorders and shock. An impacted egg may
cause metabolic disturbances by interfering with normal defecation and
mictruition, inducing ileus and renal dysfunction. Pressure necrosis may occur
to all three layers of the oviduct wall and lead to rupture. Prolapse of the
oviduct, impaction of the oviduct, and bacterial metritis may occur as sequela
to
dystocia or egg binding.
Another common reproductive problem in birds is chronic egg
laying, which occurs when a hen lays eggs beyond the normal clutch size or
produces repeated clutches regardless of the existence of a suitable mate or
breeding season. This problem is particularly common in hand-raised hens that
are imprinted on humans, and many highly domesticated psittacine birds like
cockatiels, lovebirds and budgerigars are notorious chronic egg layers.
Malnutrition and the progressive stress and physiologic demands of egg laying
ultimately compromise the hen. Egg binding is common in hens that chronically
lay eggs. Removing eggs from the hen effectively induces a form of double
clutching and can exacerbate the problem. Medical therapy is directed to
correcting any nutritional imbalances or reproductive tract abnormalities, and
medroxyprogestrone injections can be used to temporarily interrupt the
ovulatory cycle. However, depression, polyuria, weight gain, liver damage,
immunosuppression and occasionally diabetes mellitus (especially in
cockatiels)
can occur with use of medroxyprogesterone. Egg laying may be stopped from
two weeks to several months following therapy and repeat injections are often
necessary, and the long term solution in these cases is a
salpingohysterectomy,
which is costly and dangerous.
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Dystocia is also a common medical problem in reptiles, including
snakes, turtles and lizards. Treatment options include physical manipulation,
hormonal stimulation, percutaneous ovocentesis, and surgery. Common
complications associated with manual palpation include oviductal rupture,
oviductal prolapse, egg rupture, and even death. Furthermore, use of hormones
that cause oviductal contraction on animals that have obstructive dystocia (as
opposed to nonobstructive dystocia) can have detrimental consequences
including egg fracture, oviduct rupture, hemorrhage and death. In lizards,
spaying is often recommended to stop them from ovulating, thus preventing
recurrent dystocias.
Dystocia is also a life-threatening disease in fish. In most cases,
affected fish are either euthanized or the impacted egg mass must be
surgically
removed.
Most rabbit does that are allowed to undergo unrestricted estrus
develop neoplasia of the reproductive tract or mammary tissues that is usually
fatal. The only currently available technique to prevent these fatal
neoplasias is
ovariohysterectomy, preferably performed prior to a doe's first estrus.
The reproductive processes of other egg-producing organisms,
such as amphibians, insects, arachnids, and plant and animal parasites, such
as
nematodes, while not typically targets for therapeutic intervention, offer
opportunities for pest management via prevention of reproduction.
There is, therefore, a need for a safe and effective method for
controlling the reproductive processes in birds, reptiles, fish, rabbits,
amphibians, insects, arachnids, and plant and animal parasites and other egg-
producing organisms. Such a method would be useful not only for population
control, but to treat or prevent the onset of various disorders of the
reproductive
systems in these organisms.
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4
Summary of the Invention
The present invention provides an immunogenic composition and
a method for affecting the reproductive system of an oocyte-producing
organism, preferably a bird, rabbit, fish, reptile, amphibian, insect,
arachnid or
an oocyte-producing parasite. One embodiment of the immunogenic
composition, referred to herein as a protein vaccine, comprises a polypeptide
comprising a zona pellucida protein or an immunogenic fragment thereof.
Another embodiment of the immunogenic composition, referred to herein as a
polynucleotide vaccine, comprises a polynucleotide comprising a nucleic acid
sequence that encodes a polypeptide comprising a zona pellucida protein or an
immunogenic fragment thereof.
The method for affecting the reproductive system of an organism
comprises administering to the organism an immunogenic composition of the
invention. The immunogenic composition, whether in the form of a protein
vaccine or a polynucleotide vaccine, can be administered either
prophylactically
to prevent the occurrence, recurrence or onset of a reproductive system
disease,
disorder or condition, or therapeutically to treat a reproductive system
disease,
disorder or condition. When administered as an immunocontraceptive, the
immunogenic composition causes temporary, reversible infertility in the
organism. When administered as an immunosterilant, the immunogenic
composition causes permanent, irreversible infertility in the organism. An
immunogenic composition administered to control reproduction can optionally
further function to treat or prevent one or more other reproductive disorders,
diseases or conditions.
The immunogenic composition of the invention preferably
comprises at least one of a mammalian zona pellucida protein or avian zona
pellucida protein, but alternatively or in addition it can include a zona
pellucida
protein from other animals. A porcine zona pellucida (pZP) protein is a
preferred mammalian zona pellucida protein, and a chicken zona pellucida
protein is a preferred avian zona pellucida protein (aZP). Optionally, the
immunogenic composition of the invention contains an immunological adjuvant.
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The immunogenic composition is administered in a manner and an amount
effective to affect one or more reproductive processes in the organism.
The invention further provides a zona pellucida protein, or a
fragment thereof, conjugated to an immunogenic carrier protein, which can be
used in an immunogenic composition to affect the reproductive system of an
oocyte-producing organism according to the invention. Also provided is a
dually or multiply functional immunogenic composition comprising a zona
pellucida protein, or a fragment thereof, conjugated to at least one
immunogenic
Garner protein, wherein the immunogenic Garner protein is capable of
generating
an independently protective immune response.
Brief Description of the Drawings
Figure 1 shows one version of an oocyte purification apparatus.
Figure 2 shows chicken anti-pZP IgY titers over time for four
pZP-vaccinated chickens and two control chickens.
Figure 3 shows egg production by week after pZP vaccination of
chickens.
Detailed Description
The immunogenic composition of the invention comprises at least
one zona pellucida protein or immunogenic fragment thereof, or at least one
polynucleotide encoding a polypeptide comprising a zona pellucida protein or
an
immunogenic fragment thereof. The zona pellucida protein is preferably, but
need not be, substantially pure. The zona pellucida protein or immunogenic
fragment thereof can be a naturally occurring protein, a chemically or
enzymatically synthesized protein, or a recombinant protein. The zona
pellucida
protein or immunogenic fragment thereof is preferably, but need not be,
glycosylated. In a glycosylated zona pellucida protein (i.e., a zona pellucida
glycoprotein), the glycosylation pattern is preferably equivalent to the
glycosylation pattern found on a native (i.e., naturally occurring)
glycoprotein.
Naturally occurring zona pellucida protein used in this embodiment of the
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6
immunogenic composition of the invention is not limited by the source of the
protein, but is preferably isolated from pigs, birds, or fish. Zona pellucida
proteins have been characterized in many different vertebrates, including, for
example, pigs (e.g., E. Yurewicz et al., Biochim. Biophys. Acta, 1174, 211-214
(1993)), birds (e.g., Y. Takeuchi et al., Eur. J. Biochem., 260, 736-742
(1999);
M. Waclawek et al., Biol. Reprod., 59, 1230-1239 (1998)), fish (e.g., C. Lyons
et
al., J. Biol. Chem., 268, 21351-21358 (1993); K. Murata et al., Dev. Biol.,
127,
9-17 (1995)), mice (e.g., S. Shimizu et al., J. Biol. Chem., 258, 5858-5863
(1983)), rabbits (e.g., V. Lee et al., J. Biol. Chem., 268, 12412-12417
(1993)),
frogs (e.g., J. Yang et al., Dev. Growth Differ., 39, 457-467 (1997)); H. Kubo
et
al., Dev. Growth Differ., 39, 405-417 (1997)), humans (e.g., M. Chamberlin et
al., Proc. Nat'1 Acad. Sci. USA, 87, 6014-6018 (1990)), dogs, cats and
primates.
See J. Harris et al., J. Sect. Map, 4 361-393 (1994), for a review of the ZPC
gene
family in vertebrates; and J. Harris et al., DNA Sea., 4, 361-393 (1994), for
a
review of zona pellucida genes and cDNAs from mammalian species. Zona
pellucida protein can be obtained from an animal's ovaries or from an egg cell
of
an animal and/or the surrounding extracellular matrix and tissue, for example
from an oocyte or unfertilized egg of an animal at any stage of development.
Synthetic or recombinant zona pellucida protein can incorporate all or an
immunogenic portion of the amino acid sequences of a zona pellucida protein
derived from any organism having a zona pellucida.
In a preferred embodiment of the immunogenic composition, the
zona pellucida protein used in the composition or encoded by the
polynucleotide
used in the composition is a porcine zona pellucida protein, preferably a
total
porcine zona pellucida protein, which can be obtained from pig ovaries. A
total
porcine zona pellucida protein preparation includes all three major porcine
zona
pellucida proteins: pZPl, pZP3a and pZP3~i. pZP3a and pZP3~i each have
reported molecular weights of about 55 kD, and pZPl has a reported molecular
weight of about 82 kD. The amino acid sequences of these three proteins are
known (J.D. Harris et al., DNA Sea., 4, 361-393 (1994)). Other reported pZP
proteins are believed to be degradation products of pZP 1. The immunogenic
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composition can, alternatively, contain fewer than all the major porcine zona
pellucida proteins; for example it can contain pZP3 a and pZP3 ~3 but not pZP
1.
In another preferred embodiment of the immunogenic
composition, the zona pellucida protein used in the composition or encoded by
the polynucleotide used in the composition is an avian zona pellucida protein,
preferably a total avian zona pellucida protein, which can be obtained from
the
perivitelline membrane of bird eggs. A total avian zona pellucida protein
preparation includes a plurality 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 VI, below, identifies immunoreactive
chicken proteins having molecular weights of 70 kD, 40 kD and 35 kD. The
immunogenic composition of the invention can, alternatively, contain fewer
than
all the avian zona pellucida proteins; for example it can contain the 40 kD
and
the 3 5 kD proteins but not the 70 kD protein.
In yet another preferred embodiment of the immunogenic
composition, the zona pellucida protein used in the composition or encoded by
the polynucleotide used in the composition is a total fish zona pellucida
protein
obtained from fish oocytes, eggs or liver tissue (C. Lyons et al., J. Biol.
Chem.
268:21351-21358 (1993); K. Murata et al., Dev. Biol. 127, 9-17 (1995)).
It is to be understood that the immunogenic composition of the
invention can contain or encode zona pellucida proteins derived from more than
one source. For example, an immunogenic composition can include both avian
zona pellucida protein, which is readily available and relatively inexpensive
to
isolate, and porcine zona pellucida protein, which is more difficult and
expensive to obtain. The relative amounts of various zona pellucida proteins
used in a protein vaccine of the invention depend on the nature of the animal
being vaccinated and the immunogenic response generated in the animal by the
vaccine. In the polynucleotide vaccine of this aspect of the invention,
nucleic
acid sequences encoding different zona pellucida proteins can be delivered on
the same vector, or on different vectors. It should be further understood that
a
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vector used in the polynucleotide vaccine of the invention can include
multiple
copies of a single nucleic acid sequence encoding a zona pellucida protein.
Purity of a zona pellucida protein or glycoprotein can be
evaluated analytically using a combination or series of two-dimensional sodium
dodecyl sulfate polyacrylamide gels (SDS-polyacrylamide gel electrophoresis,
or SDS-PAGE) with silver staining, 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. For example, a "substantially pure" total zona
pellucida
protein preparation isolated from pig ovaries migrates as two distinct smears
in
the gel electrophoretic experiments (one smaller smear representing pZPl, and
one larger smear representing pZP3a and pZP3~i), and shows 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
zona pellucida protein preparation, there are no detectable contaminating
proteins. The absence of detectable contaminating proteins is determined by
demonstrating that there are no proteins in the preparation that have
electromigration patterns different from those exhibited by the zona pellucida
proteins as determined by two-dimensional SDS-PAGE (silver-stained) or
Western blot analyses of two-dimensional SDS-PAGE gels.
An immunogenic fragment of a 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,
an immune response is evidenced by a detectable anti-ZP antibody level in the
subject using ELISA substantially as described in Example II.
Immunogenicity of the zona pellucida 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-
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ZP fragment antibody titers in serum. Alternatively or additionally,
histochemical analysis of the ovaries of a vaccinated organism can be
performed. Evidence of ovarian pathology indicates that the candidate fragment
is immunogenic. An immunogenic peptide fragment preferably contains more
than seven amino acids, more preferably at least about 10 amino acids, most
preferably at least about 20 amino acids.
The zona pellucida protein used in the immunogenic composition
of the invention is preferably a naturally occurring protein but can also be a
chemically, enzyrnatically, or recombinantly synthesized protein. A
recombinant protein produced in a eukaryotic system is preferred because it is
likely to be at least partially glycosylated (see, e.g., WO 9314786, published
5
August 1993). However, non-glycosylated recombinant or de-glycosylated
wild-type zona pellucida proteins are also envisioned for use in the
immunogenic composition of the invention.
Con~gated vaccine. Another embodiment of the immunogenic
composition of the invention comprises a conjugated immunogenic composition;
that is, a zona pellucida protein, or fragment thereof, conjugated to a
carrier
protein, preferably a carrier protein that is immunogenic in the intended
recipient. Examples of immunogenic Garner proteins include keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA), and ovalbumin. Other
examples of immunogenic Garner proteins include proteins that have be
engineered to include selected epitopes, such as T cell, helper T cell or B
cell
epitopes of the species to be vaccinated or of another species, or viral,
bacterial
or parasitic epitopes.
When the Garner protein is immunogenic, the zona pellucida
protein fragment conjugated thereto can be, but need not be, immunogenic
itself.
Optionally, the zona pellucida protein fragment can take the form of a
"hapten"
that, although able to interact with the products of an immune response,
cannot
itself stimulate a response. Haptens are incomplete immunogens but can be
made fully immunogenic by coupling them to a suitable carrier molecule.
The zona pellucida protein, or fragment thereof, used in the
conjugate can be a naturally occurring protein, a chemically or enzymatically
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synthesized protein, or a recombinant protein. The zona pellucida protein, or
fragment thereof, used in the conjugate is preferably, but need not be,
glycosylated. In a glycosylated zona pellucida protein (i.e., a zona pellucida
glycoprotein), the glycosylation pattern is preferably equivalent to the
5 glycosylation pattern found on a native (i.e., naturally occurnng)
glycoprotein.
The fragment is peptide fragment that preferably contains more than seven
amino acids, more preferably at least about 10 amino acids.
Conjugation methods are well known in the art and can involve
any of the various functional groups on the protein and the carrier (e.g.,
free
10 sulfhydryls, amines, amides, carboxyl groups, and the like) that are
capable of
linkage and/or activation. Optionally, conjugation is achieved using a linker
molecules, for example, a maleimide derivative. An example of a convenient
conjugation system that utilizes maleimide activated carrier proteins is
available
from Pierce Chemical Company (Rockford, IL) under the tradename IMJECT.
The immunogenic composition of the invention preferably
contains a zona pellucida protein that is heterologous with respect to the
organism to which it is administered. However, conjugation allows effective
administration of an immunogenic composition comprising a homologous zona
pellucida protein, that is, a zona pellucida protein, or fragment thereof,
that is
derived from the species to which the immunogenic composition is intended to
be administered. For example, in a homologous vaccine protocol according to
the invention a conjugate formed by the covalent linkage of avian zona
pellucida
protein to a Garner protein is administered to a bird. The term "derived from"
a
particular species, as used in this context, means that the amino acid
sequence of
the zona pellucida protein, or fragment thereof, is substantially the same as
the
amino acid sequence of the naturally occurring zona pellucida protein obtained
from that species. The zona pellucida protein may, optionally, be
glycosylated;
if glycosylated, it may exhibit a native or a non-native glycosylation
pattern.
Thus, the zona pellucida protein used in a zona pellucida protein-carrier
conjugate prepared for and used in a homologous vaccination can be obtained by
isolating the protein directly from ovarian tissue or an oocyte or egg of that
species, or, alternatively, by chemical or enzymatic synthesis, or using a
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recombinant expression system comprising DNA that encodes zona pellucida
protein, or fragment thereof, of the species to which the immunogenic
composition is intended to be administered. The DNA used in such a
recombinant expression system is conveniently obtained from the intended
species, or, alternatively, can be chemically or enzymatically synthesized.
Preferably, the carrier protein used in the conjugate is a
heterologous protein (i.e., derived from a species that is different from,
more
preferably remote from, the species of the vaccinated organism), as
heterologous
carriers are expected to generate a stronger immune response than homologous
carriers.
It should be understood that the immunogenic composition of the
invention contemplates heterologous as well as homologous zona pellucida
protein-Garner conjugates, together with associated methods comprising
administration of the homologous and heterologous conjugates to organisms of
the same or different species, respectively, as described in more detail
below.
Further, the invention should be broadly understood to include zona pellucida
protein-Garner conjugates themselves, in addition to immunogenic compositions
including said conjugates, and methods of administration and use of said
conjugates and compositions, as further described herein.
In a particularly advantageous embodiment, the invention
provides a dually or multiply functional conjugate comprising a zona pellucida
protein, or fragment thereof, conjugated to at least one immunogenic carrier
protein that is selected so as to elicit a desired, independently protective,
immune response. A dually functional conjugate refers to a zona pellucida
protein, or fragment thereof, conjugated to one independently protective
Garner
protein, and a multiply functional conjugate refers to a zona pellucida
protein, or
fragment thereof, conjugated to two or more independently protective carrier
proteins, or to a single carrier protein or protein construct that is capable
of
eliciting two or more independently protective immune responses. The carrier
protein is advantageously selected to elicit immunological protection against
an
infection or disease state to which the intended subject may be exposed.
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Where a fragment of zona pellucida protein is utilized, it is
preferably, but need not be, an immunogenic fragment, capable of generating an
immune response when administered in an unconjugated form. Because it is
coupled to an immunogenic Garner, a non-immunogenic fragment will
nonetheless elicit an immune response from the organism to which it is
administered.
An example of a dually functional conjugate according to the
invention is a zona pellucida protein, or fragment thereof, conjugated to an
immunogen that stimulates immunity to beak and feather disease virus. This
conjugate can be administered to birds in a single vaccine to simultaneously
cause both immunosterilization (or immunocontraception) and immunity to beak
and feather disease virus. Similarly, zona pellucida protein, or fragment
thereof,
can be conjugated to viruses or viral cell wall components, such as, for
example,
those currently in use as an avian polyomavirus vaccine (e.g., an avian
polyomavirus vaccine from Biomune, Lenexa, KS).
Pol~nucleotide vaccine. The polynucleotide vaccine of the
invention contains one or more polynucleotides encoding one or more
polypeptides comprising a zona pellucida protein or immunogenic fragment
thereof. In a preferred embodiment, the encoded polypeptide also contains at
least one T cell, helper T cell or B cell epitope. The epitope 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 for ZP proteins
(Garza et al., J. Reprod. Immunol., February 1998, pp. 87-101). The use of
epitopes derived from an immunogenic organism, such as a pathogenic parasite,
is preferred. For example, the polynucleotide vaccine can encode a chimeric
peptide comprising a T cell or helper T cell epitope from a parasite and a B
cell
epitope from 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
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use as an immunocontraceptive preferably includes a polypeptide that encodes
at
least one B cell epitope (or a polynucleotide functionally encoding such a
polypeptide).
The polynucleotide vaccine can include DNA, RNA, a modified
nucleic acid, or any combination thereof. The polynucleotide encoding a zona
pellucida protein or immunogenic fragment thereof 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. 8:406-410 (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 polynucleotide vaccine of the
invention is preferably one that functionally encodes a 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 organism. 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 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
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target organism. 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 organism'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
fine-tune the response of the organism's immune system, including both
antibody and cytotoxic T lymphocyte responses, to bring out the specific level
of
response needed to affect the organism's reproductive system.
Alternatively, the vector can be a viral vector, including an
1 S 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)).
Adi,uvanted composition. The immunogenic composition of the
invention, whether it contains a protein (e.g., conjugated or non-conjugated
zona
pellucida protein) or polynucleotide, optionally includes an immunological
adjuvant to enhance the immunological response of the subject to the protein
immunogen. 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 X3(1,4) linked mannan polymer interspersed with O-acetylated
groups, permulum, or 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
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sensitivity to oil-based adjuvants, which cause necrotic granulomas at the
site of
administration. Reptiles are also sensitive to oils. Therefore, a non-oil-
based
adjuvant, such as aluminum hydroxide, is preferred for use in companion birds
and reptiles. An adjuvant comprising synthetic trehalose dicorynemycolate,
5 squalene oil, and a surfactant such as lecithin is preferred for use in
organisms
that are not sensitive to oils. Lecithin typically includes phosphatidyl
choline.
When an oil adjuvant is used, homogenization of the adjuvant,
such as Freund's adjuvant, with the aqueous zona pellucida protein or
polynucleotide solution can be accomplished using any convenient means
10 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 immunogenic composition comprises a predetermined
amount of zona pellucida protein or polynucleotide and a pharmaceutically
1 S acceptable immunogenic level of adjuvant in an emulsion containing about
10%
oil phase and about 90% aqueous phase.
Non-adjuvanted composition. In another embodiment of the
invention, the immunogenic composition, whether it contains a conjugated or a
non-conjugated zona pellucida protein or a polynucleotide, contains no
adjuvant;
essentially the immunogenic composition of this embodiment is an aqueous zona
pellucida protein or polynucleotide solution that delivers the intended amount
of
zona pellucida protein or polynucleotide to the recipient.
Administration of the immuno~enic composition. The invention
further includes a method for administering the immunogenic composition of the
invention to an oocyte-producing organism such as a bird, a rabbit, a fish, a
reptile, an amphibian, an insect, an arachnid or a parasite. 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.
Fish include but are not limited to koi, goldfish and cichlids. Reptiles
include
but are not limited to lizards, snakes, chelonians (turtles) and crocodilians.
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Amphibians include but are not limited to frogs, toads, and salamanders.
Insects
include but are not limited to roaches, ants, wasps, flies, hornets, mites,
fleas,
and ticks; arachnids include but are not limited to spiders and scorpians.
Parasites include animal and plant parasites, and include, for example,
intestinal
parasites such as nematodes.
Preferably, the zona pellucida included in the immunogenic
composition or encoded by the polynucleotide included in the immunogenic
composition is heterologous with respect to the organism to which it is
administered. For example, a preferred immunogenic composition for
administration to birds contains porcine zona pellucida, which is heterologous
with respect to the bird. Administration of a homologous zona pellucida
protein
or polynucleotide encoding a homologous zona pellucida protein is not excluded
but it is not a preferred method, since the immune response generated by a
homologous protein is typically substantially reduced in comparison to a
heterologous protein. However, if it is conjugated to an immunogenic carrier
protein as described above, a homologous zona pellucida protein is likely to
generate a more significant immune response, and administration of such a
conjugate represents a preferred embodiment of the method of the invention.
The immunogenic composition is administered in a manner and
an amount effective to cause the desired response in the subject. For example,
to
treat an egg-bound bird, reptile or fish, with a protein vaccine of the
invention,
the immunogenic composition is preferably administered in the form of a
plurality of doses (typically about 0.25 mL), each dose containing zona
pellucida
protein, or an immunogenic fragment thereof, in an amount of about 10 ~g to
about 2 mg, more preferably about 50 ~,g to about 400 ~,g. 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
organism, depending on the nature, size and overall health of the organism, as
well as the condition to be treated. An immunostimulant such as STDCM is
optionally present in a per dose amount of about 10 ~,g to about 5 mg,
preferably
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in an amount of about 50 ~g to about 3.5 mg, more preferably in an amount of
about 1 mg to about 3 mg.
When administered to a bird, fish or reptile, the immunogenic
composition preferably contains AIOH as an adjuvant, or contains no adjuvant
at
all.
The immunogenic composition is typically administered by way
of intramuscular injection. However, 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,
transconjunctival administration, including the use of eye drops. For fish,
the
immunogenic composition can be administered by immersing the fish in a
solution containing the zona pellucida protein and the desired adjuvant, if
any.
In addition, liposome-mediated, microsphere-mediated, and microencapsulation
systems are all included as delivery vehicles for the immunogenic composition
of the present invention.
Initial administration of the vaccine, for example by injection, is
preferably followed by two or more administrations, such as booster
injections,
at one to two week intervals, although the boosters can be administered from
about 90 days to several months following the previous vaccination.
Uses of the vaccine. The immunogenic composition of the
invention is used in oocyte-producing organisms such as birds, fish, rabbits,
reptiles, amphibians, insects, arachnids and parasites to affect the
reproductive
systems of these organisms. Administration of the immunogenic compositions
allows for control of reproduction, treatment of reproductive diseases or
disorders, and/or alteration of the behavior of the organism.
Control of reproduction in an organism in accordance with the
invention can take the form of either immunocontraception or
immunosterilization. Immunosterilization means permanent, irreversible
infertility, in contrast to immunocontraception wherein infertility is
temporary or
transient, and reversible. Immunocontraception and immunosterilization are
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both dependent on the immune response of the subject, but immunosterilization
is typically the result of ovarian pathology caused by vaccine administration
and
high titers of anti-ZP antibodies, 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 can lead to a reduced immune response which
results in immunocontraception (i.e., infertility that is temporary and
reversible)
instead of immunosterilization.
Reproductive disorders in birds and reptiles that can be treated or
prevented in accordance with the invention include, but are not limited to,
egg-
binding disease, dystocias, egg-related peritonitis, oophoritis, neoplasias of
the
reproductive tract, prolapsed oviduct and cloaca, salpingitis, metritis,
oviduct
impaction, cloacal problems, cystic hyperplasia, ectopic egg formation, and
chronic egg laying. Reproductive disorders in fish that can be treated in
accordance with the invention include, but are not limited to egg-binding
disease, dystocias, egg-related peritonitis, oophoritis, salpingitis, oviduct
impaction and ectopic egg formation. Reproductive disorders in rabbits that
can
be treated in accordance with the invention include, but are not limited to
dystocia, peritonitis, neoplasias of the reproductive tract, neoplasias of the
mammary glands, metritis and cystic hyperplasia.
It is further anticipated that the immunogenic composition of the
invention can be used as a growth stimulant in fish, thereby increasing the
rate
of weight gain and the efficiency of food conversion.
Moreover, female birds, reptiles and rabbits can exhibit
undesirable behavior as a result of reproductive activity or reproductive
problems including excess vocalization (birds), aggressiveness, biting,
destruction of the local environment (birds, rabbits), and feather mutilation
(birds). It is anticipated that the immunogenic composition of the invention
can
be used to treat or prevent these behavioral disorders.
It is also anticipated that the immunogenic composition of the
invention can be used as a pest management tool, for example, to inhibit
reproduction of pests by spraying the immunogenic composition onto a pest
population, or by use in baited traps, which can include oral baits or baits
with
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an aerosol. Examples of pest populations that can be controlled thereby
include
ants, particularly fire ants, flies, mosquitoes, ticks, mites and fleas.
Reproduction of fire ants, for example, can be accomplished by baiting with
food that contains one or more zona pellucida proteins or by baiting with a
protozoan engineered to contain a recombinant zona pellucida construct that
can
be expressed in the protozoan or the fire ant or both. Because food digestion
in
ants such as fire ants is facilitated by the internal presence of protozoans,
protozoans can be used as vehicles for administration of the zona pellucida
proteins or immunogenic fragments thereof to the ants.
EXAMPLES
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.
Example I. Isolation of Porcine Zona Pellucida and
Extraction of pZP Proteins
Buffers. Saline buffer (40 L) was made by addition 4 L of the
following solution: 0.9% NaCI, 0.01 M dibasic sodium phosphate, 0.01 M
monobasic sodium phosphate, and 0.002 M sodium citrate dihydrate, pH 7.2, in
triple distilled water, to 36 L of triple distilled water. Tris buffer (3L)
was made
by adding 484 g Tris base, 119 g ethylenediaminetetraacetic acid (EDTA), 47 g
sodium citrate dihydrate and 16 g sodium azide to 3L of triple distilled
water,
then adjusting the pH to 7.9. Tris detergent buffer ( 1 L) was made by
combining
2 mL of NP-40 (Cat. No. N-6507, Sigma Chemical Co., St. Louis, MO) with 998
mL Tris buffer.
Other materials. The oocyte purification apparatus consisted of
three chambers. Each chamber consisted of a stainless steel wire mesh
container
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(Home Depot) suspended inside a buffer container set on an orbital shaker
(shown in Fig. 1 ) or a rotary washing system with an internal agitator. The
pore
size of the wire mesh used to form the wire mesh containers in the first,
second,
and third chambers was 1000 Vim, 500 ~,m, and 150 Vim, respectively. Tubing
5 connecting the chambers allowed fluid transfer from the buffer space
external to
the wire mesh of one chamber to a collection or holding carboy, or,
alternatively,
to the inside of the next succeeding downstream wire mesh container in a
continuous flow process, as shown in Fig. 1. Peristaltic pumps are used to
effect
fluid movement within the tubing between chambers (as shown in Fig. 1 ) or
10 between the chambers and any collection carboys used (not shown in Fig. 1
).
Pig ovaries were obtained from pig slaughterhouses.
Zona pellucida isolation. Porcine ovaries (5-6 lbs.) were twice
ground through a commercial meat grinder (Hobart), and the homogenate was
collected. The homogenate and grinder were rinsed with 4L of saline buffer,
15 and the homogenate solution was placed in the wire mesh container of the
first
chamber of the purification apparatus. The three buffer containers of the
purification apparatus were filled with saline buffer. The shakers were
operated
at an agitation cycler rotation speed of about 20 revolutions per minute
during
the oocyte purification process. Periods of rotary agitation were alternated
with
20 periods of fluid removal from the region surrounding the mesh container.
Filtered oocytes, together with a small amount of tissue, passed through the
1000
~,m mesh and were thus pumped from the buffer space of the first chamber into
a
collection carboy or into the wire mesh container in the second chamber. In
purification procedures making use of a collection carboy, the filtered
oocytes
are subsequently pumped into the wire mesh container in the second chamber.
With rotary agitation and new saline buffer addition, the oocytes were then
passed through the 500 ~,m mesh of the wire mesh container of the second
chamber while the fibrous tissue remained in the mesh container. The oocytes
and saline buffer were then pumped from the buffer space of the second
chamber into a collection carboy or directly into the 150 ~,m wire mesh
container in the third chamber. Rotary agitation was continued in the third
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21
chamber and the solution surrounding the wire mesh (containing the oocytes)
was removed.
The solution containing the oocytes was then passed over a 75
~,m screen (13/4 inches or 21/2 inches in diameter). The oocytes were
collected on
the 75 ~.m screen and were then backwashed into a 100 mL beaker using Tris
buffer. The 100 mL solution was divided into 2 x 50 mL vials and homogenized
at 15,000 rpm for 3 to 5 minutes in a Powergam 700D (Fisher) homogenizer.
The zona fragments were then poured onto a 13/4 inches or 21/z
inches diameter, 0.040mm (40~m) filter screen and washed with Tris detergent
buffer. The zona fragments were removed from the screen by backwashing with
Tris detergent buffer into a small polypropylene beaker, then incubated at
4°C
with constant mechanical stirnng to dissociate any undesired proteins, such as
albumin. The zona material is preferably handled in polypropylene or
siliconized glass beakers to prevent adherence to surfaces which results in
loss
of the material.
After incubation and stirring, the zona fragments were again
poured a 1 3/4 inch diameter, 0.040mm (40~,m) filter screen and washed with
Tris
buffer to remove any protein contaminants. The zona fragments retained on the
screen were collected by spooning or backwashing (using Tris buffer) into a
small polypropylene beaker to a maximal volume of 25 mL. The beaker was
covered and placed in a 75-76°C water bath and incubated for 20 minutes
to
solubilize the zona protein such that the temperature of the zona protein-
containing solution was 73 ~ 1 °C.
After solubilization, the mixture was centrifuged at 21,000 rpm
for 25 minutes or until a pellet was observed at the base of the tube. The
supernatant was collected, and protein concentration was estimated. The
supernatant was aliquoted (3mg/vial), lyophilized, and stored under NZ gas in
a
desiccator at 4°C. Typically about 1.5 mg to 1.9 mg of highly purified
pZP
protein per pound of ovaries can be produced, amounting to about 10 mg on a
daily basis. Previous techniques produced only about 200 - 300 ~g quantities
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22
over a two day period. It is anticipated that this harvesting technique of the
present invention can be increased to produce even greater amounts.
Purity was demonstrated and confirmed using two-dimensional
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
combined with Western blot analysis, silver staining, and, at times, Coomassie
blue staining, using standard protocols. The preparation was tested for viral
and
bacterial contaminants at the Diagnostic Laboratory at the College of
Veterinary
Medicine at the University of Georgia.
Example II. Preparation of a ZP Vaccine
A pZP vaccine was prepared by homogenizing Freud's complete
adjuvant with an aqueous antigen solution containing isolated pZP protein. The
aqueous antigen solution contained the pZP protein preparation in saline or
phosphate buffered saline (PBS) and Tween 80. When prepared for use in the
vaccine composition, the aqueous composition typically contained 0.4%
(vol/vol) Tween 80 and an amount of pZP calculated to yield the desired pZP
dose. Vaccine volumes for the chickens were about 1 mL; the dose to be used
for smaller birds is about 0.25 mL.
An avian ZP vaccine, for use in organisms other than chickens,
can be prepared using the same procedure, but substituting avian ZP for
porcine
ZP.
Example III. Vaccination of Chickens with pZP Vaccine
Vaccinations. Four experimental SF leghorn chickens were
vaccinated with 200 ~g of pZP per dose (1 mL volumes) in a vaccine adjuvanted
with Freund's complete adjuvant. The chickens were vaccinated with three
injections administered at approximately two week intervals. Three
unvaccinated control chickens were included in the trial. Under veterinary
supervision, vaccinations were delivered to chickens intramuscularly in the
deep
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pectoral muscle. Booster injections were administered on the contra-lateral
side.
No pain or adverse reactions were observed at the injection sites.
Antibod, t~. Blood was drawn from each chicken at the time
of each of the three injections, and about three weeks following the last
S injection. Serum antibody titers (IgG) were determined using an enzyme
linked
immunosorbant assay (ELISA). Adjacent wells of a microwell plate were
coated with 2 ~.g pZP, and incubated for 6 hours. The wells were 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 were 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 were then washed and
loaded with 50 ~,1 of the secondary antibody (rabbit anti-chicken IgG) and
incubated for 2 hours. Color change was observed after the addition of
p-nitrophenyl phosphate for 30 minutes and the reaction terminated by the
addition of 3 M NaOH. The optical density was read at a 405-492 nm range on
a Spectramax spectrophotometer. The chickens' pre-immune serum served as
the negative controls.
The ELISA trials (Fig. 2) revealed that there was a similar
antibody profile in all four experimental chickens characterized by a
significant
rise in antibody titers between the initial injection and the first booster.
Antibody levels remained high after the first and second boosters. These data
clearly show that there is a significant immune response to the adjuvanted pZP
vaccine. The rapidity of the effect suggests an IgM-mediated response in the
chickens.
Eau la,~g. Eggs were counted beginning on the day of the first
injection. After 13 weeks, the three control chickens had laid, as a group, 81
eggs, and the four experimental chickens had laid, as a group, only 4 (Fig.
3).
An immediate reduction in egg production was observed in the experimental
animals.
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Ezample IV. Treatment of Egg-binding Disease in Chicken
A pZP vaccine ( 1 mL volume) containing 200 ~g of pZP
adjuvanted with Freund's complete adjuvant was administered intramuscularly
as described in Example III to an hysterectomized chicken that had behavioral
and physical abnormalities (broodiness, cloacal prolapse) consistent with
ovulation. Within 24 hours of administration, cloacal prolapse was no longer
evident and straining was eliminated.
Example V. Conjugated pZP Vaccine
Materials and Methods. A pZP-KLH conjugate was made using
IMJECT~ Maleimide Activated Keyhold Limpet Hemocyanin kit (Pierce
Chemical Company, Rockford IL) using the method and materials provided with
the kit substantially without modification. The instructions supplied with the
IMJECT kit are hereby incorporated by reference in their entirety. Briefly,
porcine zona pellucida protein (2 mg pZP) was reduced in 2-3 mL buffer (0.1
NH4HC03, pH 8) by the addition of dithiothreitol (DTT, 1.5 mg/mL), then
dialyzed against distilled water containing 10 mM EDTA (pH 7) to remove
excess DTT. The reduced protein was lyophilized, then redissolved using
between 200 ~L to 500 ~,L conjugation buffer supplied in the Pierce kit to a
concentration of between about 4 ~g/~L to about 10 ~,g/~L pZP, respectively.
KLH, activated according to the instructions of the kit (200 ~1), was added to
reduced pZP by mixing the two solutions, then the mixture was sparged with
nitrogen gas for 2 minutes and left at room temperature for 2 hours to
complete
the conjugation. The conjugate was then dialyzed against Purification Buffer
Prod. # 77159 available from Pierce Chemical Co. (Rockford, IL) (i.e., 0.083 M
sodium phosphate, 0.9 M NaCI, pH 7.2, with stabilizers).
Per dose, the pZP-KLH conjugate (about 0.5-2 mg) was
combined with adjuvant supplied by RIBI Immunochem Co. (Hamilton, MT)
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comprising 2.5 mg STDCM, in about 1-4 mL drakeol oil. Homologous
vaccination was effected by administering the vaccine to three pigs two to
four
times at 2-3 week intervals. Serum titers were evaluated, and ovarian
histopathology was observed.
5 Results. The resulting conjugate is, presumably, in the form of
pZP covalently linked, via a maleimide linker, at the free sulfhydryl (-SH) of
one or more of the constituent cysteines to the free amine (-NHZ) of a KLH
lysine or N-terminus. KL,H is commonly used as a carrier protein to impart
immunogenicity to covalently coupled haptens. In the current experiment, KLH
10 was coupled to porcine zona pellucida protein (which is not a small-
molecule
"hapten") in an effort to induce an immune response in a homologous
vaccination (i.e., vaccination of a pig). Typically, it is difficult to
generate an
immune response in a homologous vaccination protocol because the body does
not treat the potential immunogen as "foreign" or "nonself' material.
15 The KLH-pZP conjugate was found to generate a strong immune
response in the vaccinated pigs. Severe damage to the ovaries was observed,
evidenced by massive infiltration of lymphocytes, neutrophils, and macrophages
into the large follicles, consistent with irreversible sterilization.
Interestingly,
serum titers were lower than expected for this degree of ovarian pathology.
Ezample VI. 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
(Powergen 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
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preparing granulosa cell sheets substantially as described by A. Gilbert et
al, J.
Re~rod. Fertil., 50, 179-181 (1977)).
Purity was demonstrated and confirmed using one-dimensional
and two-dimensional SDS-PAGE combined with Western blot analysis, silver
staining, and, at times, Coomassie 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 rabbit
anti-
pZP serum. The 70 kD protein was easily washed away from the perivitelline
membrane. The 35 kD protein was 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 may 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)).
1 S Ezample VII. Ovarian Response in Fire Ants to Rabbit
Anti-pZP Antibodies
Ovaries of fire ants were sectioned and stained with hematoxylin and
eosin. This confirmed the architecture of the ovaries.
Further, the fire ant ovaries were immunostained with rabbit anti-pZP
serum. Specifically, sections of tissues were heated in a 60°C oven for
20
minutes followed by two washes with xylene in order to remove all paraffin.
The sections were then rehydrated through an ethanol series followed by
incubation in a 9:1 methanol/H202 solution for 30 minutes to remove
endogenous peroxidase. Nonspecific binding sites on the sections were blocked
by incubating sections in 5% bovine serum albumin (BSA) dissolved in Tris
buffered saline (TBS) containing 0.05% Tween 20 (TBST) overnight. Rabbits
that had been vaccinated with highly purified pZP provided the source of the
anti-pZP IgG. The serum was diluted 1:1,000 with TBST and 200 ~.l were
applied to the tissue sections and incubated for one hour in a wet chamber.
Negative control sections were treated with a similarly diluted normal rabbit
serum. Sections were washed twice with TBS after incubation with primary
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antibody followed by incubation with 200 ~1 of goat anti-rabbit IgG biotin
conjugate at a 1:1,000 dilution for 30 minutes in a wet chamber. After
incubation, the sections were washed twice with TBS and then treated with
Extravidin peroxidase (1:100 in TBST, Sigma Chemical, St. Louis, MO) for 30
S minutes in a wet chamber. Tissue sections were washed twice with TBS and
treated with diaminobenzidine (DAB) for 2 minutes. Color reaction was stopped
with running distilled water followed by counterstaining with Mayer's
hematoxylin. Sections were then dehydrated through an ethanol series and
permanently mounted. Observations were made with a Zeiss Axioscope
microscope at 40 and 100 x magnification.
The immunocytochemistry showed that the anti-pZP reacted with the
ovarian tissue of the fire ant queens, indicating that the fire ant ovaries
contained
a pZP-like target and suggesting that the ants would be affected by the zona
pellucida vaccine.
Example VIII. Vaccination of Chickens with Mixed Avian ZP/pZP Vaccine
Vaccinations. Chickens are vaccinated with 200 ~g of mixed
zona pellucida protein ( 1: l, avian ZP:porcine ZP) per dose ( 1 mL volumes)
in a
vaccine adjuvanted with Freund's complete adjuvant. Avian ZP is isolated as in
Example VI. 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.
Antibod. 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
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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.
E~~ la~~ Eggs are counted beginning on the day of the first
injection to evaluate reduction in egg production, providing direct evidence
of
effect on the chicken's reproductive system.
Ezample IX. Preparation of Protein Vaccine for Use in Companion Birds,
Rabbits, Reptiles and Fish
Ad~uvanted vaccine. A vaccine is prepared by mixing aluminum
hydroxide (AIOH) with an aqueous antigen solution containing one or more
isolated ZP protein e.g., avian ZP and/or pZP. The aqueous antigen solution
contains the ZP protein preparation in saline or phosphate buffered saline
(PBS)
and Tween 80. When prepared for use in the vaccine composition, the aqueous
composition typically contains 0.4% (vol/vol) Tween 80 and an amount of ZP
calculated to yield a dose of about 10 ~,g to about 2 mg per vaccination.
Non-adiuvanted vaccine. A vaccine is prepared using an aqueous
antigen solution containing isolated ZP protein(s), without an adjuvant. The
aqueous antigen solution contains the ZP protein preparation in saline or
phosphate buffered saline (PBS) and Tween 80. When prepared for use in the
vaccine composition, the aqueous composition typically contains 0.4% (vol/vol)
Tween 80 and an amount of ZP protein calculated to yield a ZP dose of about 50
~g to about 2 mg per vaccination.
Vaccine volumes for the adjuvanted and the non-adjuvanted
vaccines depend on the size of the animal but are typically about 0.25 mL.
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29
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
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.
