U.S. patent application number 13/948808 was filed with the patent office on 2014-01-30 for zona pellucida binding peptides, expression vectors, compositions, and methods for species-specific immunocontraception of animals.
Invention is credited to Henry J. Baker, Nancy Cox, Stephen Ditchkoff, Tatiana I. Samoylova, Kent Van Kampen.
Application Number | 20140030283 13/948808 |
Document ID | / |
Family ID | 40909248 |
Filed Date | 2014-01-30 |
United States Patent
Application |
20140030283 |
Kind Code |
A1 |
Samoylova; Tatiana I. ; et
al. |
January 30, 2014 |
ZONA PELLUCIDA BINDING PEPTIDES, EXPRESSION VECTORS, COMPOSITIONS,
AND METHODS FOR SPECIES-SPECIFIC IMMUNOCONTRACEPTION OF ANIMALS
Abstract
Disclosed are methods, compositions, zona pellucida binding
peptides and polypeptides, and expression vectors for use in
species-specific immunocontraception of animals. The disclosed
compositions may include immunogenic compositions or vaccines.
Inventors: |
Samoylova; Tatiana I.;
(Auburn, AL) ; Baker; Henry J.; (Auburn, AL)
; Cox; Nancy; (Auburn, AL) ; Ditchkoff;
Stephen; (Auburn, AL) ; Van Kampen; Kent;
(Hoover, AL) |
Family ID: |
40909248 |
Appl. No.: |
13/948808 |
Filed: |
July 23, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13416348 |
Mar 9, 2012 |
8492516 |
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13948808 |
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12419883 |
Apr 7, 2009 |
8158366 |
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13416348 |
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61123275 |
Apr 7, 2008 |
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61130473 |
May 30, 2008 |
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61133201 |
Jun 26, 2008 |
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61150530 |
Feb 6, 2009 |
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Current U.S.
Class: |
424/185.1 ;
435/317.1; 435/320.1; 435/69.1; 506/9; 530/327; 536/23.1 |
Current CPC
Class: |
C07K 7/08 20130101; A61K
2039/53 20130101; Y10S 424/811 20130101; G01N 33/53 20130101; A61K
39/0006 20130101; G01N 33/50 20130101; Y10S 435/853 20130101; A61P
37/04 20180101; A61P 15/00 20180101 |
Class at
Publication: |
424/185.1 ;
506/9; 530/327; 536/23.1; 435/320.1; 435/317.1; 435/69.1 |
International
Class: |
C07K 7/08 20060101
C07K007/08; G01N 33/53 20060101 G01N033/53 |
Claims
1. A method for identifying a peptide that binds specifically to
zona pellucida of oocytes of a target species of animal, the method
comprising (a) isolating oocytes of the target species of animal
and isolating oocytes of one or more non-target species of animals;
(b) contacting the isolated oocytes with a phage library; (c)
selecting phage from the library that bind specifically to the
oocytes of the target species of animal, thereby identifying
peptides that bind to the zona pellucida of oocytes of the target
species of animal.
2. The method of claim 1, wherein the phage library is first
contacted with the oocytes of the one of more non-target species of
animals, and subsequently, members of the phage library that do not
bind to the oocytes of the one of more non-target species of
animals are contacted with the oocytes of the target species of
animal and those members of the phage library that bind to the
oocytes of the target species of animal are selected, thereby
identifying peptides that bind specifically to the zona pellucida
of oocytes of the target species of animal.
3. The method of claim 1, wherein the phage library is first
contacted with the oocytes of the target species of animals, and
subsequently, members of the phage library that bind to the oocytes
of the target species of animals are contacted with the oocytes of
the one or more non-target species of animal and those members of
the phage library that do not bind to the oocytes of the one or
more non-target species of animal are selected, thereby identifying
peptides that bind specifically to the zona pellucida of oocytes of
the target species of animal.
4. The method of claim 1, wherein the target species of animal is a
swine.
5. The method of claim 1, wherein the non-target species of animals
include one or more of felines, canines, and bovines.
6. An isolated polypeptide comprising an amino acid sequence
selected from a group consisting of SEQ ID NO:2 (DANRLPHPANIN), SEQ
ID NO: 18(TLGWTANEAPRR), SEQ ID NO: 19 (LLADTTHHRPWT), SEQ ID
NO:20(SQSPAMYSQTRP), SEQ ID NO:21 (AVTQHLKFKGFN), and SEQ ID
NO:22(ANFNMTHHQGHK).
7. An isolated polypeptide consisting of an amino acid sequence
selected from SEQ ID NO:2 (DANRLPHPANIN), SEQ ID NO: 18
(TLGWTANEAPRR), SEQ ID NO: 19(LLADTTHHRPWT), SEQ ID NO:20
(SQSPAMYSQTRP), SEQ ID NO:21(AVTQHLKFKGFN), and SEQ ID NO:22
(ANFNMTHHQGHK).
8. An isolated polynucleotide encoding the polypeptide of claim
6.
9. A recombinant polynucleotide comprising a promoter sequence
operably linked to the polynucleotide of claim 8.
10. An isolated cell transformed with the recombinant
polynucleotide of claim 9.
11. A method of producing a polypeptide encoded by the
polynucleotide of claim 8, the method comprising: a) culturing a
cell under conditions suitable for expression of the polypeptide,
wherein said cell is transformed with a recombinant polynucleotide,
and said recombinant polynucleotide comprises a promoter sequence
operably linked to the polynucleotide of claim 8; and b) recovering
the polypeptide so expressed.
12. A vector comprising the recombinant polynucleotide of claim
9.
13. The vector of claim 12, where the vector is a viral vector.
14. An antigen comprising the polypeptide of claim 6 conjugated to
one or more carrier proteins.
15. An immunogenic composition comprising; (a) a polypeptide
comprising an amino acid sequence selected from a group consisting
of SEQ ID NO:2 (DANRLPBPANIN), SEQ ID NO: 18 (TLGWTANEAPRR), SEQ ID
NO: 19 (LLADTTHHRPWT), SEQ ID NO:20 (SQSPAMYSQTRP), SEQ ID
NO:21(AVTQHLKFKGFN), and SEQ ID NO:22 (ANFNMTHHQGHK); and (b) a
suitable excipient, carrier, or diluent.
16. The composition of claim 15, further comprising an
adjuvant.
17. The composition of claim 16, comprising two or more
polypeptides, each polypeptide comprising an amino acid sequence
selected from a group consisting of SEQ ID NO:2 (DANRLPHPANIN), SEQ
ID NO:18 (TLGWTANEAPRR), SEQ ID NO:19(LLADTTHHRPWT), SEQ ID NO:20
(SQSPAMYSQTRP), SEQ ID NO:21(AVTQHLKFKGFN), and SEQ ID NO:22
(ANFNMTHHQGHK).
18. An immunogenic composition comprising: (a) the vector of claim
12; and (b) a suitable excipient, carrier, or diluent.
19. The composition of claim 18, further comprising an adjuvant
20. A method for producing antibodies that bind to swine sperm, the
method comprising administering the immunogenic composition of
claim 15 to an animal.
21. The method of claim 20, wherein the animal is a female
swine.
22. A method for producing antibodies that bind to swine sperm, the
method comprising administering the immunogenic composition of
claim 18 to an animal.
23. The method of claim 22, wherein the animal is a female
swine.
24. A method for immunizing a swine against conception, the method
comprising administering the immunogenic composition of claim 15 to
the swine.
25. The method of claim 24, wherein the swine is female.
26. A method for immunizing a swine against conception, the method
comprising administering the immunogenic composition of claim 18 to
the swine.
27. The method of claim 26, wherein the swine is female.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of priority under
35 U.S.C. .sctn.119(e) to U.S. provisional application No.
61/123,275, filed on Apr. 7, 2008; U.S. provisional application No.
61/130,473, filed on May 30, 2008; U.S. provisional application No.
61/133,201, filed on Jun. 26, 2008; and U.S. provisional
application No. 61/150,530, filed on Feb. 6, 2009, the contents of
which are incorporated herein by reference in their entireties.
BACKGROUND
[0002] The present subject matter relates to the fields of peptide
and polypeptide selection, immunology, and targeted contraception
in animals. In particular, the present subject matter relates to
peptides and polypeptides that bind to the zona pellucida (ZP) of
oocytes, methods for selecting such peptides and polypeptides,
vectors that express me selected peptides and polypeptides, and
compositions that comprise such peptides, polypeptides, or vectors
(e.g., compositions for inducing an immune response against
sperm).
[0003] Overpopulation of animals of multiple species including
domestic, feral, and
[0004] wild animals results in various economic, health, and
security problems. For example, feral swine cause significant
physical damage to agricultural crops, soils, vineyards, tree
plantings, turf, rare plant communities, wildlife habitat,
archaeological sites, and vehicles. (See Ditchkoff S S, West B C.
Ecology and management of feral hogs. Human-Wildlife Conflicts
2007; 1 (2): 149-151). Feral swine compete with livestock and
native wildlife for food, and prey on domestic animals and
wildlife. Feral swine carry at least thirty important viral and
bacterial diseases and thirty-seven parasites that affect humans,
pets, livestock, and wildlife (e.g., brucellosis, salmonellosis,
diseases due to pathogenic E. coli strains, rabies, tuberculosis,
and tularemia). Feral swine also could potentially spread
additional human and animal diseases not currently found in the
United States. (See Hutton T, DeLiberto T, Owen S, Morrison B.
Disease risks associated with increasing feral swine numbers and
distribution in the United States. Midwest Association of Fish and
Wildlife Agencies 2006). Control programs for feral swine
eradication such as poisoning, trapping, shooting, etc., are
ineffective, expensive and generally unacceptable to the public.
Currently available contraceptives for animals are not selective
and affect multiple species and, therefore, cannot be permitted for
use in uncontrolled environments such as natural habitats of feral
or wild animals, (See Miller L A, Johns B E, Killian G J.
Immunocontraception of white-tailed deer with GnRH vaccine. Am J
Reprod Immunol 2000; 44(5):266-274; and Killian G, Miller L, Rhyan
J, Doten H. Immunocontraception of Florida feral swine with a
single-dose GnRH vaccine. American Journal of Reproductive
Immunology 2006; 55: 378-384). Thus, there is an urgent need for
immunocontraceptive, species-specific vaccines that can affect the
target species only and be delivered via economically sound oral or
oronasal administration. Additional examples of species, the
overpopulation of which imposes various kinds of economic and
health risks, include coyotes, deer, and raccoons.
SUMMARY
[0005] Disclosed are methods, compositions, zona pellucida
(ZP)-binding peptides., and vectors for expressing the peptides for
use in immunocontraception of animals. The disclosed compositions
may include immunogenic or vaccine compositions that comprise
ZP-binding peptides or vectors that express the ZP-binding
peptides. The disclosed compositions also may include bait
compositions that comprise the immunogenic or vaccine
compositions.
[0006] Preferably, the ZP-binding peptides, the vectors, the
immunogenic or vaccine compositions, and the bait compositions are
species-specific. For example, preferably the peptides and
polypeptides bind specifically to the ZP of oocytes of a target
species of animal and do not bind to the ZP of oocytes of a
non-target species of animal. The peptides and polypeptides may be
expressed via vectors that include viral, bacterial, or other
vectors. Preferably, the vector is species-specific in that the
vector infects or expresses the peptide or polypeptide in a
selected species of animal and does not infect or express the
peptide or polypeptide in a non-selected species of animal. The
compositions disclosed herein may comprise the peptides,
polypeptides, or vectors that express the peptides or polypeptides.
The disclosed compositions may be immunogenic or vaccinogenic and
may be administered to animals for species-specific
immunocontraception via induction of a species-specific anti-sperm
immune response. The compositions may be formulated as bait
compositions that also are species-specific in that they attract a
selected species of animal and do not attract a non-selected
species of animal.
[0007] The disclosed methods include methods for identifying a
peptide or polypeptide that binds specifically to the zona
pellucida of oocytes from a target species of animal. The methods
may include: (a) isolating oocytes from one or more mammals (e.g.,
porcine oocytes, feline oocytes, canine oocytes, or bovine
oocytes); (b) contacting the oocytes with a phage library; (c)
selecting phage that hind specifically to the oocytes of a target
species of animal (e.g. phage that bind specifically to porcine
oocytes as compared to oocytes from other animals (e.g., feline
oocytes, canine oocytes, or bovine oocytes)), thereby identifying
peptides that bind to the ZP of the oocytes of the target animal
species. The methods may include: (a) contacting oocytes of one or
more species of animal with a phage library; (b) separating phage
that do not bind to the oocytes of one or more non-target species
of animal (e.g., feline oocytes, canine oocytes, and bovine
oocytes) from the phage library; and (c) contacting the separated
phage with oocytes of a target species of animal (e.g., porcine
oocytes); and (d) separating phage that bind to the oocytes of the
target species of animal (e.g., porcine oocytes), thereby
identifying peptides that selectively bind to the ZP of the oocytes
of the target animal species (e.g., porcine oocytes).
Alternatively, the methods may include: (a) contacting oocytes of
one or more species of animal with a phage library; (b) separating
phage that bind to the oocytes of the target species of animal
(e.g., porcine oocytes) from the phage library; and (c) contacting
the separated phage with oocytes of one or more non-target species
of animal (e.g., feline oocytes, canine oocytes, or bovine
oocytes); and (d) separating phage that do not bind to the oocytes
of the one or more non-target species of animal thereby identifying
peptides that selectively bind to the ZP of the oocytes of the
target animal species (e.g., porcine oocytes). In the disclosed
methods, the phage library may be contacted with a relatively small
number of oocytes (e.g., less than about 1000 oocytes).
[0008] Also disclosed are peptide or polypeptide identified by the
disclosed methods. In some embodiments, the identified peptides or
polypeptides may include an amino acid sequence selected from a
group consisting of SEQ ID NO:2 (DANRLPHPANIN), SEQ ID NO: 18
(TLGWTANEAPRR), SEQ ID NO: 19 (LLADTTHHRPWT), SEQ ID
NO:20(SQSPAMYSQTRP), SEQ ID NO:21 (AVTQHLKFKGFN), and SEQ ID
NO:22(ANFNMTHHQGHK). Also disclosed are polynucleotides encoding
the identified peptides or polypeptides. The polynucleotide may be
operably linked to a promoter sequence as a recombinant
polynucleotide. The recombinant polynucleotide may be present in a
vector which is utilized to transform an isolated cell. Preferably,
the vector is capable of expressing the encoded peptide or
polypeptide. The encoded peptide or polypeptide may be produced by
a method that includes: a) culturing the transformed cell under
conditions suitable for expression of the polypeptide; and b)
recovering the polypeptide so expressed. Alternatively, the peptide
may be prepared by a synthetic method.
[0009] The identified peptides or polypeptides may be utilized as
antigens. In some embodiments, the identified peptides or
polypeptides may be modified to enhance antigenicity. For example,
the peptides or polypeptides may be conjugated to one or more
carrier proteins (e.g., keyhole-limpet hemocyanin (KLH)).
[0010] The disclosed compositions may include immunogenic
compositions or
[0011] vaccine compositions. In some embodiments, the compositions
include (a) one or more polypeptides comprising an amino acid
sequence selected from a group consisting of SEQ ID NO:2
(DANRLPHPANIN), SEQ ID NO:18 (TLGWTANEAPRR), SEQ ID NO:
19(LLADTTHHRPWT), SEQ ID NO:20 (SQSPAMYSQTRP), SEQ ID
NO:21(AVTQHLKFKGFN), and SEQ ID NO:22 (ANFNMTHHQGHK); and (b) a
suitable excipient, carrier, or diluent. For example, the
compositions may include two or more polypeptides, where each of
the two or more polypeptides comprise an amino acid sequence
selected from a group consisting of SEQ ID NO:2 (DANRLPHPANIN), SEQ
ID NO: 18(TLGWTANEAPRR), SEQ ID NO: 19 (LLADTTHHRPWT), SEQ ID
NO:20(SQSPAMYSQTRP), SEQ ID NO:21 (AVTQHLKFKGFN), and SEQ ID
NO:22(ANFNMTHHQGHK). The immunogenic compositions or vaccine
compositions may further include an adjuvant as disclosed herein.
The immunogenic compositions or vaccine compositions further may
include an immunostimulatory agent (e.g., an immunostimulatory
oligodeoxynucleotide such as CpG).
[0012] Also disclosed are vectors that express the disclosed
peptides or polypeptides. Suitable vectors include, but are not
limited to, viral vectors and bacterial vectors. The vector may be
species-specific (e.g., a viral vector that specifically infects a
swine). The vectors may be formulated as an immunogenic composition
or a vaccine composition. In some embodiments, the immunogenic
compositions or vaccine compositions comprise one or more vectors
(e.g., species-specific vectors) that express one or more
species-specific peptides or polypeptides (e.g. species-specific
ZP-binding peptides or polypeptides).
[0013] The disclosed compositions also may include bait
compositions for attracting a target species of animal. For
example, the disclosed composition may include species-specific
bait compositions for swine (e.g., feral swine). The bait
compositions may include ZP-binding peptides (e.g., as disclosed
herein), vectors that express the ZP-binding peptides, immunogenic
compositions that include ZP-binding peptides, or vaccine
compositions that include the ZP-binding peptides. The bait
compositions thereof may be specific-specific in one or more
aspects, including but not limited to: (1) the bait compositions
attract a target species of animal (e.g., a swine); (2) the bait
compositions comprise a species-specific vector (e.g., a viral
vector that infects specifically a target species of animal or that
is capable of replicating or expressing an encoding protein only in
a target species of animal); (3) the bait composition comprises
species-specific ZP-binding peptides or polypeptides or comprises
vectors that express species-specific ZP-binding peptides or
polypeptides.
[0014] Also disclosed are methods for using the peptides,
polypeptides, or compositions that contain the peptides or
polypeptides. The disclosed methods may include administering the
disclosed immunogenic compositions or vaccine compositions
(optionally formulated as a bait composition) to an animal in order
to induce an immune response (e.g., an anti-sperm antibody
response, a T-cell response, or both). The disclosed methods may
include methods for producing antibodies that bind to sperm (e.g.,
anti-swine sperm antibodies). As such, the compositions may include
an effective amount of a peptide or polypeptide (e.g., a ZP-binding
peptide) for inducing an immune response against sperm.
Alternatively, the compositions may include a vector that expresses
an effective amount of a peptide or polypeptide (e.g., a ZP-binding
peptide) for inducing an immune response against sperm. In the
methods, the compositions may be administered to an animal of
either sex (i.e., male or female). The methods further may include
isolating the induced antibodies from a sample obtained from the
animal (e.g., from blood or a blood product such as serum or
plasma).
[0015] The disclosed methods also may include methods for
immunizing an animal against conception (i.e., immunocontraceptive
methods). The methods may include administering the disclosed
immunogenic composition or vaccine compositions (optionally
formulated as bait compositions) to an animal such as a female
swine, thereby immunizing the animal against conception. In some
embodiments, the animal is immunized for a temporary period of time
(e.g., for a period of weeks or months).
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1. illustrates a method whereby phage display peptide
libraries are used to
[0017] identify peptides mimicking sperm surface peptides or
proteins that bind to zona pellucida at fertilization.
[0018] FIG. 2. illustrates a strategy for species-specific
overpopulation control for domestic, feral, or wild animals having
three major levels of species-specificity (exemplified for swine in
this figure).
[0019] FIG. 3. illustrates selection of species-specific ZP-binding
peptides using a phage display library. For the peptides to be
species-specific, prior to reaction with oocytes of the target
species, a phage display library is reacted with oocytes of
non-target species that have close homology with respect to ZP
proteins.
[0020] FIG. 4. provides schematics of the experiments described in
Examples 1 and 2.
[0021] FIG. 5. provides a list of porcine ZP-binding peptide
sequences and motifs identified in Experiment 1 by selection from a
PhD-12 phage display library on intact porcine oocytes surrounded
by ZP as described in Example 1.
[0022] FIG. 6. provides a list of porcine-specific ZP-binding
peptide sequences and motifs identified in Experiment 2 by
selection from a PhD-12 phage display library on intact porcine
oocytes surrounded by ZP subsequent to sub-tractive selection on
non-porcine oocytes (i.e., feline oocytes, canine oocytes, and
bovine oocytes).
DETAILED DESCRIPTION
[0023] The disclosed subject matter is further described below.
[0024] Unless otherwise specified or indicated by context, the
terms "a", "an", and "the" mean "one or more."
[0025] As used herein, "about", "approximately;" "substantially,"
and "significantly" will be understood by persons of ordinary skill
in the art and will vary to some extent on the context in which
they are used, if there are uses of the term which are not clear to
persons of ordinary skill in the art given the context in which it
is used, "about" and "approximately" will mean plus or minus
.ltoreq.10% of the particular term and "substantially" and
"significantly" will mean plus or minus >10% of the particular
term.
[0026] As used herein, the terms "include" and "including" have the
same meaning as the terms "comprise" and "comprising."
[0027] The terms "subject" and "patient" may be used
interchangeably herein. A patient or subject may refer to a
non-human patient or subject at risk for conception (e.g., a swine
including a feral swine). The term "swine" as used herein is meant
to include domesticated, wild, and feral swine (e.g., Sus scrofa)
and may be used interchangeably with the term "pig" or
"porcine."
[0028] The term "sample" is used in its broadest, sense. A sample
may comprise a bodily fluid (e.g., blood or a blood product such as
serum or plasma obtained from a subject or patient).
[0029] The disclosed methods may include contacting isolated
oocytes with a phage library. As utilized herein, the term
"contacting" may include placing the isolated oocytes and the phage
library in a reaction vessel and reacting or incubating the
isolated oocytes and phage library under conditions that promote
interaction between the isolated oocytes and the phage library. The
disclosed methods may include separating phage that bind to the
oocytes (or that do not bind to the oocytes) from the phage
library. As utilized herein, the term "separating" may be utilized
interchangeably with the term "isolating" or "removing,"
[0030] One aspect of the present disclosure relates to methods for
isolating peptides and polypeptides that bind to the ZP of oocytes
via phage display. Methods for performing phage display are known
in the art. (See, e.g., U.S. Pat. No. 7,094,868, which discloses
isolating peptides by phage display, the content of which is
incorporated herein by reference in its entirety). Related methods
for phage display and isolation of ZP-binding peptides are
disclosed in U.S. patent application Ser. No. 12/266,944, filed on
Nov. 7, 2008, the content of which is incorporated herein by
reference in its entirety.
[0031] In particular, the methods disclosed herein may be utilized
to isolate peptides and polypeptides that bind selectively to ZP of
oocytes of a target species of animal relative to ZP of oocytes of
a non-target species of animal via phage display. The methods may
include: (a) isolating oocytes from one or more species of animal
(e.g., porcine oocytes, feline oocytes, canine oocytes, or bovine
oocytes); (b) contacting the oocytes with a phage library; (c)
selecting phage that bind to the oocytes (e.g., phage that bind
selectively to oocytes of one species of animal), thereby
identifying peptides that bind to the ZP of the oocytes.
Surprisingly, in the disclosed methods, the phage library may be
contacted with a relatively small number of oocytes (e.g., less
than about 1000 oocytes). It is generally understood in the field
of phage display technology that a significant number of cells
(typically millions) are needed for successful selection of
cell-binding peptides on intact cells. This significant number of
cells is easily achievable for the vast majority of cell types via
propagation in cell culture media. However, oocytes cannot be
obtained through cell culturing and oocytes with surrounding ZP
should be isolated directly from ovaries removed from animals.
Generally, only a small number of oocytes can be isolated from a
single pair of mammalian ovaries (i.e., as a few as 2-3 and on
average, several dozen), depending on the animal species and its
age and condition). As such, it would be expected that many animals
would be required in order to obtain a sufficient number (millions)
of oocytes for phage display selection protocols commonly in use.
In addition to the huge numbers of animals needed for isolating a
sufficient number of oocytes, the oocyte isolation procedure is
very time consuming and generally takes several hours for an
experience technician to isolate even a relatively small number of
oocytes. Thus, it would take thousands of animals and years of work
to isolate the number of oocytes required by currently accepted
protocols. For these reasons, millions of oocytes cannot be readily
available for the use in phage display selection protocols that are
generally utilized in the field. Therefore, herein, a phage display
selection procedure that requires no more than 1000 oocytes
surrounded by ZP was developed.
[0032] The peptides and polypeptides contemplated herein bind
specifically to ZP. Furthermore, the peptides and polypeptides
contemplated herein may be utilized in immunogenic compositions or
vaccines for eliciting antibodies that bind specifically to sperm.
In this regard, the terms "binds specifically" and "bind
specifically" refer to that interaction between the polypeptide (or
peptide) and the ZP; or to that interaction between sperm and an
antibody (or other binding molecule). The interaction is dependent
upon the presence of a particular structure of the protein, e.g.,
the antigenic determinant or epitope present on the polypeptide or
peptide, recognized by the antibody or binding molecule. For
example, if an antibody is specific for epitope "A" the presence of
a polypeptide comprising the epitope A, or the presence of free
unlabeled A, in a reaction containing free labeled A and the
antibody will reduce the amount of labeled A that binds to the
antibody.
[0033] The peptides and polypeptides disclosed herein may be
described via their "amino acid sequence." As used herein, the term
"amino acid sequence" refers to an oligopeptide, peptide,
polypeptide, or protein sequence, or a fragment of any of these,
and to naturally occurring or synthetic molecules. The terms
"peptide" and "polypeptide" may be used interchangeably herein.
Generally, the term "peptide" refers to an amino acid polymer
having a relatively low number of amino acid residues (e.g., no
more than about 50, 40, 30, 20, 15, 12, or 7 amino acid residues).
For the most part, peptides will comprise at least about 7 to about
50 amino acids, preferably at least about 7 to about 30 amino
acids, more preferably about 7 to about 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 amino acids. Generally, the term
"polypeptide" refers to an amino acid polymer having a greater
number of amino acid residues than a peptide. The term "protein"
also may be used herein interchangeably with the term
"polypeptide,"
[0034] The presently disclosed peptides may be synthetic. As used
herein, "synthetic peptide" refers to a peptide which has an amino
acid sequence which is not a native sequence or is not in its
native context and which confers on phage displaying it the ability
to bind or preferentially bind to a particular cell population. By
"not in its native context" is intended that the peptide is
substantially or essentially free of amino acid sequences that
naturally flank the amino acid sequence of the peptide in the
native protein which comprises the amino acid sequence of the
peptide. For example, a synthetic peptide which is not in its
native context may be flanked at either or both ends by no more
than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s)
found in the native protein.
[0035] The peptides and polypeptides disclosed herein may exhibit
at least two-fold, three-fold, four-told, five-fold, six-fold,
seven-fold, ten-fold, twenty-fold, thirty-fold or more increased
binding affinity for ZP of oocytes relative to at least one
category or type of other cell. Peptides and polypeptide that
exhibit such binding characteristics are said to exhibit
preferential binding to ZP. Peptides and polypeptides that do not
exhibit at least a two-fold increased binding affinity for ZP
relative to another category or type of other cell but that bind to
ZP are simply said to bind to ZP.
[0036] Preferably, the peptides and polypeptides disclosed herein
selectively bind to ZP of oocytes of a target species (e.g., ZP of
porcine oocytes) relative to ZP of oocytes of a non-target species
(e.g. feline oocytes, canine oocytes, or bovine oocytes). In this
regard, the peptides and polypeptides may be referred to as
"species-specific." In some embodiments, the species-specific
peptides and polypeptides disclosed herein may exhibit at least
two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold,
ten-fold, twenty-fold, thirty-fold or more increased binding
affinity for ZP of oocytes of a target species relative to ZP of
oocytes of a non-target species.
[0037] As used herein, the term "nucleic acid sequence" refers to a
nucleotide, oligonucleotide, polynucleotide, or any fragment:
thereof. As used herein, the term "polynucleotide" refers to a
nucleotide polymer. A polynucleotide may encode a peptide or
polypeptide as disclosed herein. A polynucleotide may be operably
linked to a heterologous promoter sequence as a recombinant
polynucleotide. "Operably linked" refers to the situation in which
a first nucleic acid sequence is placed in a functional
relationship with a second nucleic acid sequence. For instance, a
promoter is operably linked to a coding sequence if the promoter
affects the transcription or expression of the coding sequence.
Operably linked DNA sequences may be in close proximity or
contiguous and, where necessary to join two protein coding regions,
in the same reading frame. A recombinant polynucleotide comprising
a polynucleotide operably linked to a promoter sequence may be
present in a vector (e.g., a plasmid) which may be utilized to
transform a host cell (e.g., where the vector further includes a
selectable marker).
[0038] The presently disclosed peptides and polypeptide may be
isolated or substantially purified. The terms "isolated" or
"substantially purified" refers to peptides or polypeptides that
are removed from their natural environment and are isolated or
separated, and are at least 60% free, preferably at least 75% free,
and most preferably at least 90% free from other components with
which they are naturally associated.
[0039] Also disclosed are peptide and polypeptides identified by
the phage display method, and preferably include species-specific
ZP-binding peptide and polypeptides. Peptides identified herein
include peptides having the amino acid sequence or motifs of SEQ ID
NOs: 1-26 (preferably SEQ ID NOs:2 or 18-26). Also disclosed are
polypeptides comprising the amino acid sequence or motifs of any of
SEQ ID NOs: 1-26, polynucleotides encodings such polypeptides,
recombinant polynucleotides comprising such polynucleotides,
expression vectors, and methods for expressing the encoded
polypeptide.
[0040] The peptides disclosed herein may be fused or conjugated to
one or more other peptides or non-peptide moieties (e.g., in order
to provide an antigen). For example, a fusion polypeptide as
contemplated herein may include a fusion of any of the peptides or
motifs of SEQ ID NO: 1-26 and one or more other immunogenic
peptides. The peptides disclosed herein may be present in a
polypeptide (e.g., where the polypeptide comprises one or more
copies of the amino acid sequence of the peptide, optionally in
tandem). The disclosed peptides may be modified to enhance
immunogenicity. For example, the peptides disclosed herein may be
conjugated to one or more carrier proteins (e.g., keyhole-limpet
hemocyanin).
[0041] The disclosed methods may include inducing an immune
response against one or more peptides that bind to the ZP (e.g., an
immune response against one or more species-specific peptides that
bind to the ZP). In some embodiments, the methods include inducing
polyclonal antibodies against one or more peptides that bind to the
ZP by administering to an animal an immunogenic composition that
includes one or more of the peptides (and preferably, one or more
specific peptides) or that includes one or more vectors that
express one or more of the peptides. The animal may be a non-human
animal (e.g., a swine). The induced polyclonal antibodies may
include anti-sperm antibodies. The methods disclosed herein also
may include preventing conception by administering to the animal an
immunogenic composition that includes one or more peptides that
bind to the ZP (and preferably, one or more specific peptides that
bind to the ZP) or that includes one or more vectors that express
one or more of the peptides. For example, an animal (e.g., a
non-human animal such as a swine) may be protected against
conception by administering to the animal a composition that
includes one or more peptides that bind to the ZP or that includes
one or more vectors that express one or more peptides that bind to
the ZP.
[0042] The disclosed compositions may be administered as
immunogenic compositions or vaccines utilizing a selected
"prime-boost vaccination regimen." As used herein, a "prime-boost
vaccination regimen" refers to a regimen in which a subject is
administered a first composition one or more times (e.g., one time
or two or three times with about 2, 3, or 4 weeks between
administrations) and then after a determined period of time after
having administered the first composition (e.g., about 2 weeks,
about 4 weeks, about 2months, about 3 months, about 4 months, about
5 months, about 6 months, or longer), the subject is administered a
second composition. The second composition may also be administered
more than once, with at least 2, 3, or 4 weeks between
administrations. The first and second compositions may be the same
or different.
[0043] Also disclosed are immunogenic compositions and vaccines for
perforating the disclosed methods. An immunogenic composition may
be monovalent or polyvalent. Typically, the immunogenic
compositions include one or more peptides that hind to the ZP
(e.g., species-specific ZP-binding peptides), or the immunogenic
compositions include one or more vectors that express one or more
peptides that bind to the ZP (e.g., species-specific vectors that
express species-specific ZP-binding peptides). The immunogenic
compositions also may include a suitable excipient, carrier, or
diluent.
[0044] Suitable peptides for the immunogenic compositions (or for
expression by vectors of the immunogenic compositions) may include
one or more polypeptides comprising the amino acid sequence of a
peptide as disclosed herein, for example one or more polypeptides
comprising the amino acid sequence or motifs of any of SEQ ID NOs:
1-26. In some embodiments, the immunogenic compositions may include
two or more polypeptides (or two or more vectors that express two
or more polypeptides) where each polypeptide of the two or more
polypeptides comprises the amino acid sequence or motifs of any of
SEQ ID NOs: 1-26. The immunogenic compositions may include an
isolated polypeptide or peptide at a concentration sufficient to
induce an immunogenic response against sperm (e.g., via antibody
induction, a T-cell response, or both), or the immunogenic
compositions may include one or more vectors that express the
polypeptide or peptide at a concentration sufficient to induce an
immunogenic response against sperm (e.g., via antibody induction, a
T-cell response, or both). In some embodiments, the immunogenic
compositions may Include at least about 10 .mu.g of the isolated
polypeptide or peptide (or preferably, at least about 100 .mu.g of
the isolated polypeptide or peptide).
[0045] The "immunogenic compositions" and "vaccines" disclosed
herein are capable of stimulating an immune response in an animal
inoculated with the immunogenic composition or vaccine. An immune
response may include induction of antibodies, induction of a T-cell
response, or both. Herein, the term "prevention" when used in
reference to an immunogenic composition or vaccine may refer to the
partial or complete prevention against conception via an immune
response induced by the immunogenic composition or vaccine.
[0046] An "an immunogenic composition comprising a given peptide or
polypeptide" refers to a composition containing the given peptide
or polypeptide. The composition may comprise a dry formulation or
an aqueous solution. An "immunogenic peptide or polypeptide" is an
antigen which is capable of eliciting an immune response when
introduced into an animal, for example, a swine.
[0047] The methods disclosed herein may include administering an
immunogenic
[0048] composition or a vaccine to an animal. An "animal," as used
herein, may include a non-human animal (e.g., a swine).
[0049] The methods disclosed herein also may include protecting an
animal against conception or preventing an animal from conceiving
by administering to the animal a composition (e.g., a bait
composition) that includes an isolated peptide as disclosed herein
or that includes a vector that expresses the peptide. The
administered composition may include an immunogenic composition or
a vaccine composition. For example, art animal (e.g., a swine) may
be protected against, conception by administering to the animal a
bait composition that includes an isolated polypeptide comprising
an amino acid sequence or motif of any of SEQ ID NOs:1-26 or a
vector that expresses a polypeptide comprising an amino acid
sequence or motif of any of SEQ ID NOs: 1-26. The compositions
disclosed herein may further include a suitable excipient, carrier,
or diluent.
[0050] The presently disclosed peptide or polypeptide may be
expressed by viral vectors or bacterial vectors (e.g., as included
apart of an immunogenic composition, vaccine, or bait composition).
As used herein, a "viral vector" (e.g., an adenovirus, Sendai
virus, or measles virus vector) refers to recombinant viral nucleic
acid that has been engineered to express a heterologous
polypeptide. The recombinant viral nucleic acid typically includes
cis-acting elements for expression of the heterologous polypeptide.
The recombinant viral nucleic acid typically is capable of being
packaged into a helper virus that is capable of infecting a host
cell. For example, the recombinant viral nucleic acid may include
cis-acting elements for packaging. Typically, the viral vector is
not replication competent or is attenuated. An "attenuated
recombinant virus" refers to a virus that has been genetically
altered by modern molecular biological methods (e.g., restriction
endonuclease and ligase treatment, and rendered less virulent than
wild type), typically by deletion of specific genes. For example,
the recombinant viral nucleic acid may lack a gene essential for
the efficient production or essential for the production of
infectious virus. Recombinant attenuated bacteria also may be
utilized as vectors in the pharmaceutical compositions and vaccines
disclosed herein (e.g., recombinant attenuated Shigella,
Salmonella, Listeria, or Yersinia). Recombinant bacterial vaccine
vectors are described in Daudel et al., "Use of attenuated bacteria
as delivery vectors for DNA vaccines," Expert Review of Vaccines,
Volume 6, Number 1, February 2007, pp. 97-110(14); Strata et al.,
"Recent advances with recombinant bacterial vaccine vectors,"
Molec. Med. Today (2000), Volume 6, Issue 2, 1 February 2000, pages
66-71; Clare & Dougan, "Live Recombinant Bacterial Vaccines,"
Novel Vaccination Strategies, Apr. 16, 2004 (Editor Stefan H. E.
Kaufman); Gentschev et al., "Recombinant Attenuated Bacteria for
the Delivery of Subunit Vaccines," Vaccine, Volume 19, Issues
17-19, 21 March 2001, Pages 2621-2628; Garmory et al., "The use of
live attenuated bacteria as a delivery system for heterologous
antigens," J. Drug Target 2003; 11(8-10):471-9; U.S. Pat. No.
6,383,496; and U.S. Pat. No. 6,923,958 (which all are incorporated
by reference herein in their entireties). Preferably, the vector is
species-specific, whereby the vector selectively infects a target
species of animal or the vector selectively expresses an encoded
heterologous peptide in the target species of animal after
infecting the animal. Suitable viral vectors for expressing the
peptides and polypeptides disclosed herein include porcine
adenovirus.
[0051] The immunogenic compositions or vaccines may be formulated
for delivery in any suitable manner. For example, the immunogenic
compositions or vaccines may be formulated for at least one of oral
delivery, intranasal delivery, intramuscular delivery, subdermal
delivery, subcutaneous delivery, intravenous delivery, and
intraperitoneal delivery. The immunogenic compositions or vaccines
can be administered using a variety of methods including intranasal
and/or parenteral (e.g., intramuscular) administration. In some
embodiments of the methods, the immunogenic composition or vaccine
is administered intramuscularly one or more times at suitable
intervals (e.g., at intervals of 2-4 weeks), followed by
administration of the immunogenic composition or vaccine at least
once intramuscularly or intranasally after a suitable time period
(e.g., 2-4 weeks after the last parenteral administration of
vaccine). The immunogenic compositions or vaccines may be
administered to an animal of either sex. In some embodiments, the
animal is female.
[0052] The present immunogenic composition and vaccines may be
formulated with a pharmaceutically or veterinarily acceptable
excipient, carrier, or diluent. The forms suitable for injectable
commonly include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. The formulation
should desirably be sterile and fluid to the extent that easy
syringability exists. The dosage form should be stable under the
conditions of manufacture and storage and typically is preserved
against the contaminating action of microorganisms such as bacteria
and fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, liquid polyethylene glycol, and the
like), suitable mixtures thereof and vegetable oils. One possible
carrier is a physiological salt solution. The proper fluidity of
the solution can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of tire required
particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal
(sodium ethyhmercuri-thiosalicylate), deomycin, gentamicin and the
like. In many cases it may be preferable to include isotonic
agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions, if desired, can be
brought about by the use in the compositions of agents delaying
absorption, for example, aluminum monostearate and gelatin.
[0053] The present immunogenic composition or vaccines may include
an adjuvant. The term "adjuvant" refers to a compound or mixture
that is present in an immunogenic composition or vaccine and
enhances the immune response to an antigen present in the
immunogenic composition or vaccine. For example, an adjuvant may
enhance the immune response to a polypeptide present in a vaccine
as contemplated herein, or to an immunogenic fragment or variant
thereof as contemplated herein. An adjuvant can serve as a tissue
depot that slowly releases the antigen and also as a lymphoid
system activator that non-specifically enhances the immune
response. Examples of adjuvants which may be employed include
MPL-TDM adjuvant (monophosphoryl Lipid A/synthetic trehalose
dicorynomycolate, e.g., available from GSK Biologics). Another
suitable adjuvant is the immunostimulatory adjuvant AS021/AS02
(GSK). These immunostimulatory adjuvants are formulated to give a
strong T cell response and include QS-21, a saponin from Quillay
saponaria, the TL-4 ligand, a monophosphoryl lipid A, together in a
lipid or liposomal carrier. Other adjuvants include, but are not
limited to, nonionic block co-polymer adjuvants (e.g., CRL1005),
aluminum phosphates (e.g., AIPO.sub.4), R-848 (a Th1-like
adjuvant), imiquimod, PAM3CYS, poly (I:C), loxoribine, potentially
useful human adjuvants such as BOG (bacille Calmette-Guerin) and
Corynebacterium parvum, CpG oligodeoxynucleotides (ODN), cholera
toxin derived antigens (e.g., CTA1-DD), lipopolysaccharide
adjuvants, complete Freund's adjuvant, incomplete Freund's
adjuvant, saponin, mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil or hydrocarbon emulsions in water (e.g.,
MF59 available from Novartis Vaccines or Montanide ISA 720),
keyhole limpet hemocyanins, and dinitrophenol.
[0054] It is generally advantageous to formulate the present
compositions in dosage unit form for ease of administration and
uniformity of dosage. "Dosage unit form" as used herein refers to
physically discrete units suited as unitary dosages for the animal
subjects to the treated; each unit containing a predetermined
quantity of the active material calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
carrier. The specification for the dosage unit forms are dictated
by and depend on among other factors (a) the unique characteristics
of the active material and the particular therapeutic effect to be
achieved; (b) the limitations inherent in the art of compounding
such active material for the treatment of disease; and (c) the
manner of intended administration of the dosage unit form. In some
embodiments, a dose of the immunogenic composition or vaccine
includes at least about 10 micrograms (preferably 100 micrograms)
of one or more isolated polypeptides or peptides as disclosed
herein.
[0055] Sterile injectable solutions may be prepared by
incorporating the isolated polypeptide or peptide in the desired
amount in an appropriate solvent with various of the other
ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions can be prepared by
incorporating the various active ingredients into a sterile vehicle
which contains the basic dispersion medium and the required other
ingredients horn those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum-drying and the
freeze-drying technique which yield a powder of the active
ingredient (i.e., lyophilized form of the active ingredient) plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0056] It also may be advantageous to add a stabilizer to the
present compositions. Suitable stabilizers include, for example,
glycerol/EDTA, carbohydrates (such as sorbitol, mannitol,
trehalose, starch, sucrose, dextran or glucose), proteins (such as
albumin or casein) and protein degradation products (e.g.,
partially hydrolyzed gelatin). If desired, the formulation may be
buffered by methods known in the art, using reagents such as alkali
metal phosphates, e.g., sodium hydrogen phosphate, sodium
dihydrogen phosphate, potassium hydrogen phosphate and/or potassium
dihydrogen phosphate. Other solvents, such as ethanol or propylene
glycol, can be used to increase solubility of ingredients in the
vaccine formulation and/or the stability of the solution. Further
additives which can be used in the present formulation include
conventional antioxidants and conventional chelating agents, such
as ethylenediamine tetraacetic acid (EDTA).
[0057] The disclosed immunogenic composition, vaccine compositions,
and the peptide or expression vectors included therein may be
formulated as a species-specific bait composition (e.g., as a
contraceptive species-specific bait vaccine). Preferably, the bait
composition attracts a target species of animal, such as feral
swine, and does not attract a non-target species of animal, such as
birds. Feral pig bait compositions are known in the art (see, e.g.,
PIGOUT.RTM. brand feral pig bait (Animal Control Technologies,
Somerton, Victoria AU)) and have been utilized for oral vaccination
of feral pigs (see, e.g., Ballestreros et al., "Evaluation of baits
for oral vaccination of European wild boar piglets," Res. Vet. Sci.
2008Oct. 22 epub; Cowled et al., "Vaccination of feral pigs (Sus
scrofa) using iophenoxic acid as a simulated vaccine," Aust. Vet.
J. 2008 January-February; 86(1-2):50-5; and Kaden et al., "Oral
immunisation of wild boar against classical swine fever; evaluation
of the first field study in Germany," Vet. Micro., April 2000,
73(2-3):239-252, which contents are incorporated herein by
reference in their entireties).
[0058] The disclosed compositions, which may include bait
compositions, may be administered to a target species of animal. As
contemplated herein, "administering.infin. a composition to an
animal may include feeding a bait composition to the animal or
depositing a bait composition in locations where the animal is
likely to come in contact with the bait composition and smell,
touch, taste, or eat the bait composition.
[0059] Also disclosed herein are isolated antisera, antibodies, or
other binding molecules that bind specifically to the peptides
disclosed herein. For example, the antisera, antibodies, or other
binding molecules, may include an isolated antibody that binds
specifically to a polypeptide consisting of an amino acid sequence
or motif of any of SEQ ID NOs:1-26. Preferably, the antisera,
antibodies, or other binding molecules disclosed herein also bind
specifically to sperm (e.g., swine sperm). The isolated antibody or
binding molecule may be of any suitable isotype (e.g., IgG, IgM,
IgE, IgD, IgA, and mixtures thereof). The antibodies may be
polyclonal or monoclonal The term "antibody or other binding
molecule" refers to intact immunoglobulin molecules as well as to
fragments thereof. such as Fab, F(ab').sub.2, and Fv fragments,
which are capable of binding an epitopic determinant. The
antibodies or other binding molecules may be naturally occurring or
synthetic (e.g., scFv). Other binding molecules may include
antibody fragments (e.g., Fab fragments), coupled antibodies, and
coupled antibody fragments. Antibodies or other binding molecules
that bind the presently disclosed peptides and polypeptides can be
induced or elicited using the intact peptide or a polypeptide
comprising the intact peptide as an immunizing antigen. The
polypeptide or oligopeptide used to immunize an animal (e.g., a
swine or other animal) can be derived from the translation of RNA,
or synthesized chemically, and can be conjugated to a carrier
protein if desired. Commonly used carriers that are chemically
coupled to peptides include bovine serum albumin, thyroglobulin,
and keyhole limpet hemocyanin (KLH). The coupled peptide may then
be used to immunize the animal. The antibodies or other specific
binding molecules may be conjugated to a suitable therapeutic agent
(e.g., a toxin) or a label. The antibodies may be modified for use
in therapeutic or diagnostic methods.
[0060] Also disclosed herein are kits. The kits may include one or
more components for performing the methods disclosed herein. For
example, the kits may include one or more of the immunogenic
compositions or vaccines for immunizing or vaccinating an animal,
where the immunogenic compositions or vaccines optionally are
formulated as species-specific bait compositions. The disclosed
kits may include components for making the immunogenic compositions
or vaccines as disclosed herein, or for formulating bait
compositions comprising the immunogenic compositions or vaccines.
The components of the disclosed kits may be provided in any
suitable form (e.g., liquid form or lyophilized form). Kits further
may include solvents for resuspending or dissolving a lyophilized
protein.
EXAMPLES
[0061] The following Examples are illustrative and are not intended
to limit the scope of the claimed subject matter.
[0062] Summary
[0063] Peptides were Identified that mimic sperm surface
peptides/proteins that bind to zona pellucida (ZP) at fertilization
utilizing phage display selection. (See FIG. 1). ZP is a
glycoproteinaceous protective barrier which surrounds each
mammalian oocyte and is essential for sperm-oocyte interaction and,
therefore, conception. Immunization with ZP-binding peptides
(mimicking sperm antigens) stimulates production of anti-peptide
antibodies which act as anti-sperm antibodies. Anti-sperm
antibodies can reduce fertility by decreasing sperm motility, by
inhibiting the acrosome reaction, or by interfering with
sperm-oocyte binding. (See Chamley L W, Clarke G N. Antisperm
antibodies and conception. Semin Immunopathol 2007; 29(2): 169-184;
Suri A. Contraceptive vaccines targeting sperm. Expert Opin Biol
Ther 2005; 5(3):381-392; and Suri A. Sperm-based contraceptive
vaccines: current status, merits and development. Expert Rev Mol
Med 2005; 7(18): 1-16).
[0064] A strategy was developed for species-specific overpopulation
control of domestic, feral, or wild animals having three major
levels of species-specificity: Level 1: species-specific antigen,
ZP-binding peptide(s) that mimics sperm cell antigens; Level 2:
antigen delivery system, for example, species-specific viral or
bacterial vector; and Level 3: species-preferred bait and feeder.
(See FIG. 2)
[0065] For Level 1, species-specific ZP-binding peptides are
identified using phage display libraries, (See Samoylova T I, Smith
B F. Identification of cell targeting ligands using random
peptide-presenting phage libraries. In: Bird C, Smith B F, editors.
Genetic Library Construction and Screening; Advanced Techniques and
Applications, Heidelberg: Springer-Verlag, 2002: 209-231, the
content of which is incorporated by reference in its entirety).
Phage display libraries are mixtures of billions of genetically
engineered phages which display additional foreign (not of phage
origin) peptides on their surfaces. In general, to identify
target-binding peptides, a peptide library is first reacted with
the target. After that, phage particles not bound to the target are
removed by washing steps, and the target-specific phage (bound to
the target via displayed peptides) are recovered and amplified. To
enrich for the target-specific phage, the whole procedure may be
repeated several times (e.g., three or four times). To identify the
sequences of the peptides responsible for binding to the target,
phage DNAs are isolated, sequenced and translated into peptide
sequences. In the approach developed herein, in order to select for
peptides that are species-specific, prior to reaction with oocytes
of the target species, a phage display library is reacted with
oocytes of non-target species that have close homology with respect
to ZP glycoproteins. In the example shown in FIG. 3, to select for
peptide antigens that are specific for swine, subtractive selection
steps on ZP proteins from species of animals with close homology
were performed prior to selection steps on ZP of porcine oocytes.
These species of animals with close homology included cat, dog and
cow. (See Conner S J, Lefievre L, Hughes D C, Barratt C L. Cracking
the egg: increased complexity in the zona pellucida. Hum Reprod
2005; 20(5): 1148-1152). These subtractive steps remove phage that
bind to ZP on oocytes of non-target species, including those that
are common to the target and non-target species. Subtractive
selection steps on oocytes from non-target species are followed by
selection of species-specific phage binding to ZP on porcine
oocytes.
[0066] For Level 2, oligonucleotide sequences coding for
species-specific ZP-binding peptide antigens are inserted into
species-specific viral vectors, bacterial vectors, or other
vectors. Here, the vector is a delivery mechanism that is used to
transfer specific genetic material (oligonucleotide coding a
peptide antigen) into host cells. As the result, cells that are
transfected with the specific genetic material express the desired
peptide which then stimulates an immune response. In addition,
immune enhancers may be included in the formulation to improve
immunogenicity. Examples of species-specific biological delivery
vectors that could be appropriate delivery systems for domestic,
feral, or wild swine include porcine-specific adenoviruses (see
Hammond J M, Johnson M A. Porcine adenovirus as a delivery system
for swine vaccines and immunotherapeutics. Vet J. 2005 January;
169(1): 17-27) and strains of Salmonella sp. mostly restricted to
infecting swine.
[0067] For Level 3, the vector vaccine is incorporated into
species-preferred bait. The composition of the bait is specifically
formulated for the target species (e.g., feral swine). As an animal
contacts, smells, and eats the bait, it becomes immunized and the
immune response to the vaccine blocks fertilization. Additionally,
the bait can be distributed in feeders designed to exclude species
other than the target species.
Example 1
Identification of Peptides That Bind Pig Oocyte ZP
[0068] Peptides that bind to ZP glycoproteins on intact pig oocytes
were identified using PhD-12 Phage Display Peptide Library
purchased from New England BioLabs. The utilized approach selected
peptides that mimic the sperm antigen at the level of the ZP-sperm
binding. For Experiment 1, three rounds of selection were performed
on pig oocytes. (See FIG. 4.) Pig oocytes and oocytes of non-target
species (i.e., cat, dog, and cow oocytes as utilized in Example 2)
were obtained utilizing a modification of the method disclosed by
Dunbar et al. (Biol. Reprod. 1980; 22: 941-954). In the first
selection round, an aliquot of the primary library was diluted in a
blocking buffer and incubated with 1000 intact pig oocytes
surrounded by ZP. After incubation, phage expressing peptides not
bound to ZP were washed away and the bound phage were recovered by
incubation with a lysis buffer. Two additional selection rounds
were performed similarly. Translation of foreign oligonucleotide
inserts in phage DNA revealed sequences of the peptides that were
responsible for binding to pig oocyte ZP. Peptide sequences from
the phage display selection on intact pig oocytes surrounded by ZP
are shown in FIG. 5. These peptide sequences which bind to pig
oocyte ZP may or may not be species-specific. For example, these
identified peptide sequence may bind to conservative regions of ZP
glycoproteins that are common to multiple species of animals.
Example 2
Identification of Peptides That Bind Specifically to Pig Oocyte
ZP
[0069] Experiment 2 was designed to identify peptides that bind
only to ZP proteins on pig oocytes (pig-specific peptides). (See
FIG. 4.) Prior to reaction with pig oocytes, PhD-12 Phage Display
Peptide Library was reacted with oocytes of non-target species
(cat, dog, and cow oocytes) that have close homology to pig oocytes
with respect to ZP proteins. (See Conner S J, Lefievre L, Hughes D
C, Barratt C L. Cracking the egg; increased complexity in the zona
pellucida. Hum Reprod 2005; 20(5): 1148-1152). For each of these
subtractive selection steps, 2000 oocytes with ZP from each
non-target species were used. Subtractive selection steps were
followed by three rounds of selection on pig oocytes (1000 oocytes
per round), in each round, phage expressing peptides not bound to
ZP of oocytes were washed away and the bound phage were recovered
by incubation with lysis buffer. Translation of foreign
oligonucleotide inserts in phage DNA revealed sequences of the
peptides that were responsible for binding specifically to ZP
proteins on pig oocytes. Peptide sequences from phage display
selection on intact pig oocytes surrounded by ZP are shown in FIG.
6. Peptide sequences identified in Experiment 2 are pig-specific.
Comparative analysis indicates that peptide sequences shown in
FIGS. 5 and 6 are different (with the exception of the peptide of
SEQ ID NO:2), indicating that peptides common for dog, cat, cow and
swine were removed in pre-selection procedures on dog, cat and cow
oocytes prior to the three rounds of selection on pig oocytes.
Example 3
Bait Compositions that Target Feral Pigs
[0070] Polypeptides comprising or consisting of the peptides of
Example 2 may be formulated as species-specific bait compositions
that target feral pigs. Alternatively, polynucleotides encoding the
peptides identified in Example 2 may be formulated as
species-specific bait compositions that target feral pigs.
Optionally, the polynucleotides may be inserted in species-specific
viral vectors or bacterial vectors for targeting feral pigs, which
subsequently are formulated as species-specific bait compositions
that target feral pigs.
[0071] The bait composition is formulated to attract feral pigs and
is formulated not to attract non-target species of animals (e.g.,
birds). The bait composition may be flavored, colored, or scented,
in order to selectively attract feral pigs while not attracting or
only minimally attracting a non-target species of animal (e.g.,
birds or other non-target species). Ingredients that, may be used
in the bait composition may include, but are not limited to,
cereals or grains (e.g., barley), fish (or fish flavoring), meat
(or meat flavoring), vegetables (e.g., potatoes), fruits (e.g.,
apples), dairy products (e.g., milk or milk powder), and oils
(e.g., vegetable oil or fish oil).
[0072] It will be readily apparent to one skilled in the art that
varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention. The invention illustratively described
herein suitably may be practiced in the absence of any element or
elements, limitation or limitations which is not specifically
disclosed herein. The terms and expressions which have been
employed are used as terms of description and not of limitation,
and there is no intention that in the use of such terms and
expressions of excluding any equivalents of the features shown and
described or portions thereof, but it is recognized that various
modifications are possible within the scope of the invention. Thus,
it should be understood that although the present invention has
been illustrated by specific embodiments and optional features,
modification and/or variation of the concepts herein disclosed may
be resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention. In addition, where features or aspects of the
invention are described in terms of Markush groups or other
grouping of alternatives, those skilled in the art will recognize
that the invention is also thereby described in terms of any
individual member, any subgroup of members of the Markush group or
other group, or the totality of members of the Markush group or
other group. Citations to a number of patent and non-patent
references are made herein. The cited references are incorporated
by reference herein in their entireties.
Sequence CWU 1
1
26112PRTArtificialDodecapeptide sequence obtained from M13 minor
coat protein pIII phage display library 1Asp Ala Asp Asp Gln Thr
His Arg Arg Phe Ser Met 1 5 10 212PRTArtificialDodecapeptide
sequence obtained from M13 minor coat protein pIII phage display
library 2Asp Ala Asn Arg Leu Pro His Pro Ala Asn Ile Asn 1 5 10
312PRTArtificialDodecapeptide sequence obtained from M13 minor coat
protein pIII phage display library 3Asp Leu Asn Gly His Lys Thr Leu
Pro Val Ser Lys 1 5 10 412PRTArtificialDodecapeptide sequence
obtained from M13 minor coat protein pIII phage display library
4Asn Ile Gly Leu Pro His Asp Leu His Lys Arg Leu 1 5 10
512PRTArtificialDodecapeptide sequence obtained from M13 minor coat
protein pIII phage display library 5Gly Leu His Asn Asn Leu His Ala
Thr Thr Pro Glu 1 5 10 612PRTArtificialDodecapeptide sequence
obtained from M13 minor coat protein pIII phage display library
6Gln Ser Ala Ala Trp Tyr Pro Trp Ser Ala Asp His 1 5 10
712PRTArtificialDodecapeptide sequence obtained from M13 minor coat
protein pIII phage display library 7Tyr Thr Val Ser Met Pro Asn Val
Lys Asp Ala Ala 1 5 10 812PRTArtificialDodecapeptide sequence
obtained from M13 minor coat protein pIII phage display library
8Tyr Met Pro Asn Pro Phe Thr Ala Ser Lys Trp Lys 1 5 10
912PRTArtificialDodecapeptide sequence obtained from M13 minor coat
protein pIII phage display library 9Gly Gln Ile Met Pro Leu Pro Thr
Asn Leu Leu Val 1 5 10 1012PRTArtificialDodecapeptide sequence
obtained from M13 minor coat protein pIII phage display library
10Ser Thr Thr Leu Pro Met Gly Ser Asn Ala His Leu 1 5 10
1112PRTArtificialDodecapeptide sequence obtained from M13 minor
coat protein pIII phage display library 11Thr Tyr Leu Lys Ala Asp
Ser Leu Phe Ser Arg Val 1 5 10 1212PRTArtificialConsensus motif of
dodecapeptide sequences obtained from M13 minor coat protein pIII
phage display library 12Thr Thr Leu Xaa Thr Xaa Ser Xaa Xaa His Xaa
Xaa 1 5 10 1312PRTArtificialConsensus motif of dodecapeptide
sequences obtained from M13 minor coat protein pIII phage display
library 13Asp Xaa Asn Xaa Leu Pro His Xaa Xaa Xaa Xaa Xaa 1 5 10
1412PRTArtificialConsensus motif of dodecapeptide sequences
obtained from M13 minor coat protein pIII phage display library
14Xaa Gly Leu Xaa Xaa Xaa Leu His Xaa Thr Xaa Pro 1 5 10
1511PRTArtificialConsensus motif of dodecapeptide sequences
obtained from M13 minor coat protein pIII phage display library
15Xaa Xaa Met Pro Asn Pro Val Xaa Xaa Ala Xaa 1 5 10
1611PRTArtificialConsensus motif of dodecapeptide sequences
obtained from M13 minor coat protein pIII phage display library
16Xaa Gly Ser Xaa Xaa Thr Leu Pro Xaa Ser Xaa 1 5 10
1712PRTArtificialConsensus motif of dodecapeptide sequences
obtained from M13 minor coat protein pIII phage display library
17Gln Xaa Ala Xaa Xaa Xaa Pro Trp Xaa Xaa Xaa Xaa 1 5 10
1812PRTArtificialDodecapeptide sequence obtained from M13 minor
coat protein pIII phage display library 18Thr Leu Gly Trp Thr Ala
Asn Glu Ala Pro Arg Arg 1 5 10 1912PRTArtificialDodecapeptide
sequence obtained from M13 minor coat protein pIII phage display
library 19Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr 1 5 10
2012PRTArtificialDodecapeptide sequence obtained from M13 minor
coat protein pIII phage display library 20Ser Gln Ser Pro Ala Met
Tyr Ser Pro Thr Arg Pro 1 5 10 2112PRTArtificialDodecapeptide
sequence obtained from M13 minor coat protein pIII phage display
library 21Ala Val Thr Gln His Leu Lys Phe Lys Gly Phe Asn 1 5 10
2212PRTArtificialDodecapeptide sequence obtained from M13 minor
coat protein pIII phage display library 22Ala Asn Phe Asn Met Thr
His His Gln Gly His Lys 1 5 10 2312PRTArtificialConsensus motif of
dodecapeptide sequences obtained from M13 minor coat protein pIII
phage display library 23Xaa Xaa Thr Thr His His Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 2412PRTArtificialConsensus motif of dodecapeptide
sequences obtained from M13 minor coat protein pIII phage display
library 24Xaa Xaa Asn Xaa Xaa Leu Xaa Xaa Pro Ala Xaa Xaa 1 5 10
2512PRTArtificialConsensus motif of dodecapeptide sequences
obtained from M13 minor coat protein pIII phage display library
25Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Arg Pro Xaa Xaa 1 5 10
2612PRTArtificialConsensus motif of dodecapeptide sequences
obtained from M13 minor coat protein pIII phage display library
26Xaa Xaa Ser Ser Xaa Xaa Ser Ala Ser Xaa Xaa Xaa 1 5 10
* * * * *