U.S. patent application number 09/812642 was filed with the patent office on 2002-03-28 for parasitic helminth cuticlin nucleic acid molecules and uses thereof.
Invention is credited to Chandrashekar, Ramaswamy, Morales, Tony H..
Application Number | 20020037294 09/812642 |
Document ID | / |
Family ID | 26776975 |
Filed Date | 2002-03-28 |
United States Patent
Application |
20020037294 |
Kind Code |
A1 |
Chandrashekar, Ramaswamy ;
et al. |
March 28, 2002 |
Parasitic helminth cuticlin nucleic acid molecules and uses
thereof
Abstract
The present invention relates to: parasitic helminth cuticlin
proteins; parasitic helminth cuticlin nucleic acid molecules,
including those that encode such cuticlin proteins; antibodies
raised against such cuticlin proteins; and compounds that inhibit
parasitic helminth cuticlin activity. The present invention also
includes methods to obtain such proteins, nucleic acid molecules,
antibodies, and inhibitory compounds. Also included in the present
invention are therapeutic compositions comprising such proteins,
nucleic acid molecules, antibodies and/or inhibitory compounds as
well as the use of such therapeutic compositions to protect animals
from diseases caused by parasitic helminths.
Inventors: |
Chandrashekar, Ramaswamy;
(Fort Collins, CO) ; Morales, Tony H.; (Fort
Collins, CO) |
Correspondence
Address: |
HESKA CORPORATION
INTELLECTUAL PROPERTY DEPT.
1613 PROSPECT PARKWAY
FORT COLLINS
CO
80525
US
|
Family ID: |
26776975 |
Appl. No.: |
09/812642 |
Filed: |
March 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09812642 |
Mar 20, 2001 |
|
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09323427 |
Jun 1, 1999 |
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Current U.S.
Class: |
424/191.1 ;
435/226; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61P 33/00 20180101;
C07K 14/4354 20130101; A61K 39/00 20130101 |
Class at
Publication: |
424/191.1 ;
435/226; 435/325; 435/69.1; 536/23.2 |
International
Class: |
A61K 039/002; A61K
039/00; C07H 021/04; C12P 021/02; C12N 005/06; C12N 009/64 |
Claims
What is claimed is:
1. An isolated Dirofilaria immitis nucleic acid molecule, wherein
said Dirofilaria immitis nucleic acid molecule hybridizes in a
solution comprising 2X SSC and 0% formamide, at a temperature of
37.degree. C., and washing in 1X SSC and 0% formamide at a
temperature of 64.degree. C., to a nucleic acid sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID
NO:10.
2. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule is selected from the group consisting of SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, and SEQ ID NO:10.
3. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule comprises a nucleic acid sequence that is at least 85%
identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:10, wherein
determination of percent identity between molecules is made by a
DNAsis.TM. computer program, using default parameters.
4. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule encodes a protein comprising an amino acid sequence
selected from the group consisting of SEQ ID NO:4 and SEQ ID
NO:9.
5. A recombinant molecule comprising a nucleic acid molecule as set
forth in claim 1 operatively linked to a transcription control
sequence.
6. A recombinant virus comprising a nucleic acid molecule as set
forth in claim 1.
7. A recombinant cell comprising a nucleic acid molecule as set
forth in claim 1.
8. A method to produce a protein encoded by a nucleic acid molecule
as set forth in claim 1, said method comprising culturing a cell
transformed with a nucleic acid molecule encoding said protein.
9. An isolated nucleic acid molecule selected from the group
consisting of: (a) an isolated nucleic acid molecule comprising a
nucleic acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, and SEQ ID NO:18;
and (b) an isolated Dirofilaria immitis nucleic acid molecule
comprising a homologue of any of said nucleic acid molecules of
(a), or a complement of any of said homologues, wherein said
homologue encodes a protein that elicits an immune response against
a protein selected from the group consisting of SEQ ID NO:4 and SEQ
ID NO:9, and wherein said homologue has at least a 50 contiguous
nucleotide portion identical in sequence to a 50 contiguous
nucleotide portion of a sequence selected from the group consisting
of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10.
10. A recombinant molecule comprising a nucleic acid molecule as
set forth in claim 9 operatively linked to a transcription control
sequence.
11. A recombinant cell comprising a nucleic acid molecule as set
forth in claim 9.
12. An isolated Dirofilaria immitis protein, wherein said
Dirofilaria immitis protein is encoded by a nucleic acid molecule
that hybridizes in a solution comprising 2X SSC and 0% formamide,
at a temperature of 37.degree. C., and washing in 1X SSC and 0%
formamide at a temperature of 64.degree. C., to a nucleic acid
sequence selected from the group consisting of SEQ ID NO:2, SEQ ID
NO:5, SEQ ID NO:7, and SEQ ID NO:10.
13. The protein of claim 12, wherein said protein comprises an
amino acid sequence that is at least about 95% identical to an
amino acid sequence selected from the group consisting of SEQ ID
NO:4 and SEQ ID NO:9, wherein determination of percent identity
between molecules is made by a DNAsis.TM. computer program, using
default parameters.
14. The protein of claim 12, wherein said protein is encoded by a
nucleic acid molecule having a nucleic acid sequence selected from
the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, and
SEQ ID NO:10.
15. The protein of claim 12, wherein said protein comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO:4 and SEQ ID NO:9.
16. An isolated antibody that selectively binds to a protein as set
forth in claim 12.
17. A method to identify a compound capable of inhibiting filariid
cuticlin activity, said method comprising contacting an isolated
Dirofilaria immitis cuticlin protein as set forth in claim 12, with
a putative inhibitory compound under conditions in which, in the
absence of said compound, said protein has cuticlin activity, and
determining if said putative inhibitory compound inhibits said
activity.
18. A therapeutic composition that, when administered to a host
animal, inhibits molting of filariid larvae, said therapeutic
composition comprising: an excipient; and a protective compound
selected from the group consisting of: (a) an isolated Dirofilaria
immitis protein encoded by a nucleic acid molecule that hybridizes
in a solution comprising 2X SSC and 0% formamide, at a temperature
of 37.degree. C., and washing in 1X SSC and 0% formamide at a
temperature of 64.degree. C., to a nucleic acid sequence selected
from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7,
and SEQ ID NO:10; (b) an isolated protein selected from the group
consisting of (i) a protein comprising an amino acid sequence
selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, and
SEQ ID NO:17, and (ii) an isolated Dirofilaria immitis protein
comprising a homologue of a protein of (i), wherein said homologue
comprises at least one epitope that elicits an immune response
against a protein selected from the group consisting of SEQ ID NO:4
and SEQ ID NO:9, and wherein said homologue has at least a 15
contiguous amino acid portion identical in sequence to a 15
contiguous amino acid portion of a sequence selected from the group
consisting of SEQ ID NO:4 and SEQ ID NO:9; (c) an isolated
Dirofilaria immitis nucleic acid molecule that hybridizes in a
solution comprising 2X SSC and 0% formamide, at a temperature of
37.degree. C., and washing in 1X SSC and 0% formamide at a
temperature of 64.degree. C., to a nucleic acid sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID
NO:10; (d) an isolated nucleic acid molecule selected from the
group consisting of (i) an isolated nucleic acid molecule
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16,
and SEQ ID NO:18, and (ii) an isolated Dirofilaria immitis nucleic
acid molecule comprising a homologue of any of said nucleic acid
molecules of (i), or a complement of any of said homologues,
wherein said homologue encodes a protein that elicits an immune
response against a protein selected from the group consisting of
SEQ ID NO:4 and SEQ ID NO:9, and wherein said homologue has at
least a 50 contiguous nucleotide portion identical in sequence to a
50 contiguous nucleotide portion of a sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10; (e) an
isolated antibody that selectively binds to a protein having an
amino acid sequence selected from the group consisting of SEQ ID
NO:4 and SEQ ID NO:9; and (f) an inhibitor of filariid cuticlin
activity identified by its ability to inhibit the activity of a
filariid cuticlin protein having an amino acid sequence selected
from the group consisting of SEQ ID NO:4 and SEQ ID NO:9.
19. The composition of claim 18, wherein said composition further
comprises a component selected from the group consisting of an
adjuvant and a carrier.
20. A method to inhibit molting of filariid larvae in an animal,
said method comprising administering to said animal a composition
comprising a protective compound selected from the group consisting
of:(a) an isolated Dirofilaria immitis protein encoded by a nucleic
acid molecule that hybridizes in a solution comprising 2X SSC and
0% formamide, at a temperature of 37.degree. C., and washing in 1X
SSC and 0% formamide at a temperature of 64.degree. C., to a
nucleic acid sequence selected from the group consisting of SEQ ID
NO:2, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:10; (b) an isolated
protein selected from the group consisting of (i) a protein
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:4, SEQ ID NO:9, and SEQ ID NO: 17, and (ii)
an isolated Dirofilaria immitis protein comprising a homologue of a
protein of (i), wherein said homologue comprises at least one
epitope that elicits an immune response against a protein selected
from the group consisting of SEQ ID NO:4 and SEQ ID NO:9, and
wherein said homologue has at least a 15 contiguous amino acid
portion identical in sequence to a 15 contiguous amino acid portion
of a sequence selected from the group consisting of SEQ ID NO:4 and
SEQ ID NO:9; (c) an isolated Dirofilaria immitis nucleic acid
molecule that hybridizes in a solution comprising 2X SSC and 0%
formamide, at a temperature of 37.degree. C., and washing in 1X SSC
and 0% formamide at a temperature of 64.degree. C., to a nucleic
acid sequence selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, and SEQ ID NO:10; (d) an isolated nucleic acid
molecule selected from the group consisting of (i) an isolated
nucleic acid molecule comprising a nucleic acid sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:16, and SEQ ID NO:18, and (ii) an isolated Dirofilaria
immitis nucleic acid molecule comprising a homologue of any of said
nucleic acid molecules of (i), or a complement of any of said
homologues, wherein said homologue encodes a protein that elicits
an immune response against a protein selected from the group
consisting of SEQ ID NO:4 and SEQ ID NO:9, and wherein said
homologue has at least a 50 contiguous nucleotide portion identical
in sequence to a 50 contiguous nucleotide portion of a sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:10; (e) an isolated antibody that selectively binds to a protein
having an amino acid sequence selected from the group consisting of
SEQ ID NO:4 and SEQ ID NO:9; and (f) an inhibitor of filariid
cuticlin activity identified by its ability to inhibit the activity
of a filariid cuticlin protein having an amino acid sequence
selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:9.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to parasitic helminth cuticlin
nucleic acid molecules, proteins encoded by such nucleic acid
molecules, antibodies raised against such proteins, and inhibitors
of such proteins. The present invention also includes therapeutic
compositions comprising such nucleic acid molecules, proteins,
antibodies, inhibitors, and combinations thereof, as well as the
use of these compositions to protect animals from diseases caused
by parasitic helminths, such as heartworm disease.
BACKGROUND OF THE INVENTION
[0002] Parasitic helminth infections in animals, including humans,
are typically treated by chemical drugs. One disadvantage with
chemical drugs is that they must be administered often. For
example, dogs susceptible to heartworm are typically treated
monthly. Repeated administration of drugs, however, often leads to
the development of resistant helminth strains that no longer
respond to treatment. Furthermore, many of the chemical drugs cause
harmful side effects in the animals being treated, and as larger
doses become required due to the build up of resistance, the side
effects become even greater. Moreover, a number of drugs only treat
symptoms of a parasitic disease but are unable to prevent infection
by the parasitic helminth.
[0003] An alternative method to prevent parasitic helminth
infection includes administering a vaccine against a parasitic
helminth. Although many investigators have tried to develop
vaccines based on specific antigens, it is well understood that the
ability of an antigen to stimulate antibody production does not
necessarily correlate with the ability of the antigen to stimulate
an immune response capable of protecting an animal from infection,
particularly in the case of parasitic helminths. Although a number
of prominent antigens have been identified in several parasitic
helminths, there is yet to be a commercially available vaccine
developed for any parasitic helminth.
[0004] As an example of the complexity of parasitic helminths, the
life cycle of D. immitis, the helminth that causes heartworm
disease, includes a variety of life forms, each of which presents
different targets, and challenges, for immunization. In a mosquito,
D. immitis microfilariae go through two larval stages (L1 and L2)
and become mature third stage larvae (L3), which can then be
transmitted back to the dog when the mosquito takes a blood meal.
In a dog, the L3 molt to the fourth larval stage (L4), and
subsequently to the fifth stage, or immature adults. The immature
adults migrate to the heart and pulmonary arteries, where they
mature to adult heartworms. Adult heartworms are quite large and
preferentially inhabit the heart and pulmonary arteries of an
animal. Sexually mature adults, after mating, produce microfilariae
which traverse capillary beds and circulate in the vascular system
of the dog.
[0005] In particular, heartworm disease is a major problem in dogs,
which typically do not develop immunity, even upon infection (i.e.,
dogs can become reinfected even after being cured by chemotherapy).
In addition, heartworm disease is becoming increasingly widespread
in other companion animals, such as cats and ferrets. D. immitis
has also been reported to infect humans. There remains a need to
identify an efficacious composition that protects animals and
humans against diseases caused by parasitic helminths, such as
heartworm disease. Preferably, such a composition also protects
animals from infection by such helminths.
[0006] The cuticle is an important part of the nematode's
exoskeleton and protects the animal from the environment under a
variety of conditions. In addition, it also mediates the metabolic
interaction of the animal with its environment and, in parasitic
nematodes, the interaction with the host and its immune system. The
nematode cuticle is a complex extracellular structure that is
secreted by an underlying syncytium of hypodermal cells. Recent
studies have demonstrated that the cuticle of parasitic nematodes
is a dynamic structure with important absorptive, secretory, and
enzymatic activities, and not merely an inert protective covering
as was once believed. See, for example, Lustigman, S. 1993,
Parasitology Today, 9:8, 294-297. In addition, immunological
studies have shown the central importance of cuticular antigens as
targets for protective immune responses to parasitic nematodes. In
spite of the wide recognition of the importance of the cuticle in
the nematode physiology and its potential role as a target for
immunoprophylaxis, relatively little is known about the biology of
the cuticle of filarial parasites. Though a number of collagen
genes have been characterized in filarial parasites, very little is
known about the non-collagenous cuticular proteins, including
cuticlin, in filarial parasites. Prior studies in C. elegans have
shown that cuticlin genes are developmentally regulated and that
the message for one of the C. elegans cuticlins, cut-1, is
up-regulated during larval molting. Antibodies raised against a
cuticlin of Ascaris cross-react with the epicuticular structures of
filarial parasites indicating that components of cuticlin are
immunogenic. Since cuticlin proteins are highly conserved among
nematodes, but not among other organisms, they could be an
important target for protective immunity to parasitic
helminths.
SUMMARY OF THE INVENTION
[0007] The present invention is based on the isolation of two D.
immitis nucleic acid molecule isoforms, each encoding a protein
with amino acid sequence similarity to cuticlin cut-1 proteins from
C. elegans and Ascaris lumbricodes.
[0008] The present invention relates to a novel product and process
to protect animals against parasitic helminth infection (e.g., to
prevent and/or treat such an infection). The present invention
provides parasitic helminth cuticlin proteins and mimetopes
thereof; parasitic helminth cuticlin nucleic acid molecules,
including those that encode such proteins; antibodies raised
against such cuticlin proteins (anti-parasitic helminth cuticlin
antibodies); and compounds that inhibit cuticlin activity (i.e,
inhibitory compounds or inhibitors).
[0009] The present invention also includes methods to obtain
parasitic helminth cuticlin proteins, nucleic acid molecules,
antibodies and inhibitory compounds. Also included in the present
invention are therapeutic compositions comprising such proteins,
nucleic acid molecules, antibodies, and inhibitory compounds, as
well as use of such therapeutic compositions to protect animals
from diseases caused by parasitic helminths.
[0010] One embodiment of the present invention is an isolated
nucleic acid molecule that hybridizes under stringent hybridization
conditions with a Dirofilaria immitis (D. immitis) or Brugia malayi
(B. malayi) cuticlin gene. Such nucleic acid molecules are referred
to as cuticlin nucleic acid molecules. A preferred isolated nucleic
acid molecule of this embodiment includes a D. immitis or B. malayi
cuticlin nucleic acid molecule. A D. immitis cuticlin nucleic acid
molecule preferably includes nucleic acid sequence SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, or SEQ ID NO:10, or allelic variants of any of these
sequences. A B. malayi cuticlin nucleic acid molecule preferably
includes nucleic acid sequence SEQ ID NO:16, or SEQ ID NO:18, or
allelic variants of these sequences.
[0011] Another embodiment of the present invention is an isolated
nucleic acid molecule that includes a parasitic helminth cuticlin
nucleic acid molecule. A preferred parasitic helminth cuticlin
nucleic acid molecule of the present invention preferably includes
nucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:16, or SEQ ID NO:18 or allelic variants of any of these
sequences.
[0012] The present invention also relates to recombinant molecules,
recombinant viruses and recombinant cells that include an isolated
cuticlin nucleic acid molecule of the present invention. Also
included are methods to produce such nucleic acid molecules,
recombinant molecules, recombinant viruses and recombinant
cells.
[0013] Another embodiment of the present invention includes a
non-native parasitic helminth cuticlin protein encoded by a nucleic
acid molecule that hybridizes under stringent hybridization
conditions with a parasitic helminth cuticlin gene. A preferred
parasitic helminth protein is capable of eliciting an immune
response when administered to an animal and/or of having parasitic
helminth cuticlin activity. A preferred parasitic helminth cuticlin
protein is encoded by a nucleic acid molecule that hybridizes under
stringent conditions with a nucleic acid molecule including either
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID
NO:18, or allelic variants of any of these sequences.
[0014] Another embodiment of the present invention includes a
parasitic helminth cuticlin protein. A preferred cuticlin protein
includes a D. immitis or B. malayi cuticlin protein. A preferred D.
immitis cuticlin protein comprises amino acid sequence SEQ ID NO:4
or SEQ ID NO:9. A preferred B. malayi cuticlin protein comprises
amino acid sequence SEQ ID NO:17.
[0015] The present invention also relates to: mimetopes of
parasitic helminth cuticlin proteins; isolated antibodies that
selectively bind to parasitic helminth cuticlin proteins or
mimetopes thereof; and inhibitors of parasitic helminth cuticlin
proteins or mimetopes thereof. Also included are methods, including
recombinant methods, to produce proteins, mimetopes, antibodies,
and inhibitors of the present invention.
[0016] Another embodiment of the present invention is a method to
identify a compound capable of inhibiting parasitic helminth
cuticlin activity, comprising the steps of: (a) contacting a
parasitic helminth cuticlin protein with a putative inhibitory
compound under conditions in which, in the absence of the compound,
the protein has cuticlin activity; and (b) determining if the
putative inhibitory compound inhibits the cuticlin activity. Also
included in the present invention is a test kit to identify a
compound capable of inhibiting parasitic helminth cuticlin
activity. Such a test kit includes a parasitic helminth cuticlin
protein having cuticlin activity and a means for determining the
extent of inhibition of the cuticlin activity in the presence of a
putative inhibitory compound.
[0017] Yet another embodiment of the present invention is a
therapeutic composition that is capable of protecting an animal
from disease caused by a parasitic helminth. Such a therapeutic
composition includes one or more of the following protective
compounds: an isolated parasitic helminth cuticlin protein or a
mimetope thereof; an isolated nucleic acid molecule that hybridizes
under stringent hybridization conditions with a Dirofilaria immitis
cuticlin gene; an isolated antibody that selectively binds to a
parasitic helminth cuticlin protein; or an inhibitor of cuticlin
protein activity identified by its ability to inhibit parasitic
helminth cuticlin activity. A preferred therapeutic composition of
the present invention also includes an excipient, an adjuvant, or a
carrier. Preferred cuticlin nucleic acid molecule therapeutic
compositions of the present invention include genetic vaccines,
recombinant virus vaccines, and recombinant cell vaccines. Also
included in the present invention is a method to protect an animal
from disease caused by a parasitic helminth, comprising the step of
administering to the animal a therapeutic composition of the
present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention provides for isolated parasitic
helminth cuticlin proteins, isolated parasitic helminth cuticlin
nucleic acid molecules, isolated antibodies directed against
parasitic helminth cuticlin proteins, and other inhibitors of
parasitic helminth cuticlin activity. As used herein, the terms
isolated parasitic helminth cuticlin proteins, and isolated
parasitic helminth cuticlin nucleic acid molecules refers to
cuticlin proteins and cuticlin nucleic acid molecules derived from
a parasitic helminths and which can be obtained from their natural
source, or can be produced using, for example, recombinant nucleic
acid technology or chemical synthesis. Also included in the present
invention is the use of these proteins, nucleic acid molecules,
antibodies and other inhibitors as therapeutic compositions to
protect animals from parasitic helminth diseases as well as in
other applications, such as those disclosed below.
[0019] The present invention is based on the isolation of two cDNAs
encoding cuticlin cut-1 like proteins from D. immitis, and the
isolation of a homolog of these cDNAs from B. malayi. Parasitic
helminth cuticlin proteins and nucleic acid molecules of the
present invention have utility because they represent novel targets
for anti-parasite vaccines and drugs. The products and processes of
the present invention are advantageous because they enable the
inhibition of parasite physiological functions that depend on
cuticlin activity.
[0020] One embodiment of the present invention is an isolated
protein comprising a parasitic helminth cuticlin protein. It is to
be noted that the term "a" or "an" entity refers to one or more of
that entity; for example, a protein refers to one or more proteins
or at least one protein. As such, the terms "a" (or "an"), "one or
more" and "at least one" can be used interchangeably herein. It is
also to be noted that the terms "comprising", "including", and
"having" can be used interchangeably. Furthermore, a compound
"selected from the group consisting of" refers to one or more of
the compounds in the list that follows, including mixtures (i.e.,
combinations) of two or more of the compounds. According to the
present invention, an isolated, or biologically pure, protein, is a
protein that has been removed from its natural milieu. The terms
"isolated" and "biologically pure" do not necessarily reflect the
extent to which the protein has been purified. An isolated protein
of the present invention can be obtained from its natural source,
can be produced using recombinant DNA technology or can be produced
by chemical synthesis. When an isolated protein of the present
invention is produced using recombinant DNA technology or produced
by chemical synthesis, the protein is referred to herein as either
an isolated protein or as a non-native protein.
[0021] As used herein, an isolated parasitic helminth cuticlin
protein can be a full-length protein or any homolog of such a
protein. An isolated protein of the present invention, including a
homolog, can be identified in a straight-forward manner by the
protein's ability to elicit an immune response against a parasitic
helminth cuticlin protein or to catalyze the cleavage of asparagine
to aspartic acid and ammonia. Examples of parasitic helminth
cuticlin homologs include parasitic helminth cuticlin proteins in
which amino acids have been deleted (e.g., a truncated version of
the protein, such as a peptide), inserted, inverted, substituted
and/or derivatized (e.g., by glycosylation, phosphorylation,
acetylation, myristoylation, prenylation, palmitoylation,
amidation, or addition of glycerophosphatidyl inositol) so that the
homolog includes at least one epitope capable of eliciting an
immune response against a parasitic helminth cuticlin protein. That
is, when the homolog is administered to an animal as an immunogen,
using techniques known to those skilled in the art, the animal will
produce an immune response against at least one epitope of a
natural parasitic helminth cuticlin protein. As used herein, the
term "epitope" refers to the smallest portion of a protein or other
antigen capable of selectively binding to the antigen binding site
of an antibody or a T-cell receptor. It is well accepted by those
skilled in the art that the minimal size of a protein epitope is
about four amino acids. The ability of a protein to effect an
immune response can be measured using techniques known to those
skilled in the art.
[0022] Parasitic helminth cuticlin protein homologs can be the
result of natural allelic variation or natural mutation. Parasitic
helminth cuticlin protein homologs of the present invention can
also be produced using techniques known in the art including, but
not limited to, direct modifications to the protein or
modifications to the gene encoding the protein using, for example,
classic or recombinant DNA techniques to effect random or targeted
mutagenesis.
[0023] A cuticlin protein of the present invention is encoded by a
parasitic helminth cuticlin nucleic acid molecule. As used herein,
a parasitic helminth cuticlin nucleic acid molecule includes a
nucleic acid sequence related to a natural parasitic helminth
cuticlin gene, and preferably, to a D. immitis or B. malayi
cuticlin gene. As used herein, a parasitic helminth cuticlin gene
includes all regions that control production of the parasitic
helminth cuticlin protein encoded by the gene (such as, but not
limited to, transcription, translation or post-translation control
regions) as well as the coding region itself, and any introns or
non-translated coding regions. As used herein, a gene that
"includes" or "comprises" a nucleic acid sequence may include that
sequence in one contiguous array, or may include that sequence as
fragmented exons. As used herein, the term "coding region" refers
to a continuous linear array of nucleotides that translates into a
protein. A full-length coding region is that coding region which is
translated into a full-length, i.e., a complete, protein as would
be initially translated in its natural milieu, prior to any
post-translational modifications.
[0024] In one embodiment, a parasitic helminth cuticlin gene of the
present invention includes the nucleic acid molecule represented by
the nucleic acid sequence SEQ ID NO:1 (the coding strand), as well
as the complement of SEQ ID NO:1. The production of this molecule
(also referred to herein as nDiCut-1A) is disclosed in the
Examples. The complement of SEQ ID NO:1 (represented herein by SEQ
ID NO:2) refers to the nucleic acid sequence of the strand
complementary to the strand having SEQ ID NO:1, which can easily be
determined by those skilled in the art. Likewise, a nucleic acid
sequence complement of any nucleic acid sequence of the present
invention refers to the nucleic acid sequence of the nucleic acid
strand that is complementary to (i.e., can form a double helix
with) the strand for which the sequence is cited.
[0025] In another embodiment, a parasitic helminth cuticlin gene of
the present invention includes the nucleic acid sequence SEQ ID
NO:6, as well as the complement of SEQ ID NO:6. Nucleic acid
sequence SEQ ID NO:6 represents the nucleic acid sequence of the
coding strand of the nucleic acid molecule denoted herein as
nDiCut-1B, the production of which is disclosed in the Examples.
The complement of SEQ ID NO:6 (represented herein by SEQ ID NO:7)
refers to the nucleic acid sequence of the strand complementary to
the strand having SEQ ID NO:6.
[0026] In another embodiment, a parasitic helminth cuticlin gene of
the present invention includes the nucleic acid sequence SEQ ID
NO:16, as well as the complement of SEQ ID NO:16. Nucleic acid
sequence SEQ ID NO:16 represents the nucleic acid sequence of the
coding strand of the nucleic acid molecule denoted herein as
BmCut-1A, the production of which is disclosed in the Examples. The
complement of SEQ ID NO:16 (represented herein by SEQ ID NO:18)
refers to the nucleic acid sequence of the strand complementary to
the strand having SEQ ID NO:16.
[0027] In another embodiment, a parasitic helminth cuticlin gene
can be an allelic variant that includes a similar, but not
identical, sequence to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ
ID NO:16, or SEQ ID NO:18. For example, an allelic variant of a
parasitic helminth cuticlin gene including SEQ ID NO:1 and SEQ ID
NO:2 is a gene that occurs at essentially the same locus (or loci)
in the genome as the gene including SEQ ID NO:1 and SEQ ID NO:2,
but which, due to natural variations caused by, for example,
mutation or recombination, has a similar but not identical
sequence. Because natural selection typically selects against
alterations that affect function, an allelic variant usually
encodes a protein having a similar activity or function to that of
the protein encoded by the gene to which it is being compared. An
allelic variant of a gene or nucleic acid molecule can also
comprise alterations in the 5' or 3' untranslated regions of the
gene (e.g., in regulatory control regions), or can involve
alternative splicing of a nascent transcript, thereby bringing
alternative exons into juxtaposition. Allelic variants are well
known to those skilled in the art and would be expected to be found
naturally occurring within parasitic helminths because the helminth
genome is diploid, and sexual reproduction will result in the
reassortment of alleles.
[0028] In one embodiment of the present invention, an isolated
cuticlin protein is encoded by a nucleic acid molecule that
hybridizes under stringent hybridization conditions to a gene
encoding a parasitic helminth cuticlin protein (i.e., to a D.
immitis or B. malayi cuticlin gene). The minimal size of a cuticlin
protein of the present invention is a size sufficient to be encoded
by a nucleic acid molecule capable of forming a stable hybrid
(i.e., hybridize under stringent hybridization conditions) with the
complementary sequence of a nucleic acid molecule encoding the
corresponding natural protein. The size of a nucleic acid molecule
encoding such a protein is dependent on the nucleic acid
composition and the percent homology between the parasitic helminth
cuticlin nucleic acid molecule and the complementary nucleic acid
sequence. It can easily be understood that the extent of homology
required to form a stable hybrid under stringent conditions can
vary depending on whether the homologous sequences are interspersed
throughout a given nucleic acid molecule or are clustered (i.e.,
localized) in distinct regions on a given nucleic acid
molecule.
[0029] The minimal size of a nucleic acid molecule capable of
forming a stable hybrid with a gene encoding a parasitic helminth
cuticlin protein is typically-at least about 12 to about 15
nucleotides in length if the nucleic acid molecule is GC-rich and
at least about 15 to about 17 nucleotides in length if it is
AT-rich. The minimal size of a nucleic acid molecule used to encode
a cuticlin protein homolog of the present invention is from about
12 to about 18 nucleotides in length. Thus, the minimal size of a
cuticlin protein homolog of the present invention is from about 4
to about 6 amino acids in length. There is no limit, other than a
practical limit, on the maximal size of a nucleic acid molecule
encoding a parasitic helminth cuticlin protein or protein homolog
because a nucleic acid molecule of the present invention can
include a portion of a gene, an entire gene, or multiple genes. The
preferred size of a protein encoded by a nucleic acid molecule of
the present invention depends on whether a full-length, fusion,
multivalent, or functional portion of such a protein is
desired.
[0030] Stringent hybridization conditions are determined based on
defined physical properties of the gene to which the nucleic acid
molecule is being hybridized, and can be defined mathematically.
Stringent hybridization conditions are those experimental
parameters that allow an individual skilled in the art to identify
significant similarities between heterologous nucleic acid
molecules. These conditions are well known to those skilled in the
art. See, for example, Sambrook, et al., 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et
al., 1984, Anal. Biochem. 138, 267-284, each of which is
incorporated by reference herein in its entirety. As explained in
detail in the cited references, the determination of hybridization
conditions involves the manipulation of a set of variables
including the ionic strength (M, in moles/liter), the hybridization
temperature (.degree. C.), the concentration of nucleic acid helix
destabilizing agents (such as formamide), the average length of the
shortest hybrid duplex (n), and the percent G+C composition of the
fragment to which an unknown nucleic acid molecule is being
hybridized. For nucleic acid molecules of at least about 150
nucleotides, these variables are inserted into a standard
mathematical formula to calculate the melting temperature, or
T.sub.m, of a given nucleic acid molecule. As defined in the
formula below, T.sub.m is the temperature at which two
complementary nucleic acid molecule strands will disassociate,
assuming 100% complementarity between the two strands:
T.sub.m=81.5.degree. C.+16.6 log M+0.41(% G+C)-500/n-0.61(%
formamide).
[0031] For nucleic acid molecules smaller than about 50
nucleotides, hybrid stability is defined by the dissociation
temperature (T.sub.d), which is defined as the temperature at which
50% of the duplexes dissociate. For these smaller molecules, the
stability at a standard ionic strength is defined by the following
equation:
T.sub.d=4(G+C)+2(A+T).
[0032] A temperature of 5.degree. C. below T.sub.d is used to
detect hybridization between perfectly matched molecules.
[0033] Also well known to those skilled in the art is how base pair
mismatch, i.e. differences between two nucleic acid molecules being
compared, including non-complementarity of bases at a given
location, and gaps due to insertion or deletion of one or more
bases at a given location on either of the nucleic acid molecules
being compared, will affect T.sub.m or T.sub.d for nucleic acid
molecules of different sizes. For example, T.sub.m decreases about
1.degree. C. for each 1% of mismatched base pairs for hybrids
greater than about 150 bp, and T.sub.d decreases about 5.degree. C.
for each mismatched base pair for hybrids below about 50 bp.
Conditions for hybrids between about 50 and about 150 base pairs
can be determined empirically and without undue experimentation
using standard laboratory procedures well known to those skilled in
the art. These simple procedures allow one skilled in the art to
set the hybridization conditions (by altering, for example, the
salt concentration, the formamide concentration or the temperature)
so that only nucleic acid hybrids with greater than a specified %
base pair mismatch will hybridize. Stringent hybridization
conditions are commonly understood by those skilled in the art to
be those experimental conditions that will allow less than or equal
to about 30% base pair mismatch (i.e., at least about 70%
identity). Because one skilled in the art can easily determine
whether a given nucleic acid molecule to be tested is less than or
greater than about 50 nucleotides, and can therefore choose the
appropriate formula for determining hybridization conditions, he or
she can determine whether the nucleic acid molecule will hybridize
with a given gene under stringent hybridization conditions and
similarly whether the nucleic acid molecule will hybridize under
conditions designed to allow a desired amount of base pair
mismatch.
[0034] Hybridization reactions are often carried out by attaching
the nucleic acid molecule to be hybridized to a solid support such
as a membrane, and then hybridizing with a labeled nucleic acid
molecule, typically referred to as a probe, suspended in a
hybridization solution. Examples of common hybridization reaction
techniques include, but are not limited to, the well-known Southern
and northern blotting procedures. Typically, the actual
hybridization reaction is done under non-stringent conditions,
i.e., at a lower temperature and/or a higher salt concentration,
and then high stringency is achieved by washing the membrane in a
solution with a higher temperature and/or lower salt concentration
in order to achieve the desired stringency.
[0035] For example, if the skilled artisan wished to identify a
nucleic acid molecule that hybridizes under conditions that would
allow less than or equal to 30% pair mismatch with a flea nucleic
acid molecule of about 150 bp in length or greater, the following
conditions could preferably be used. The average G+C content of D.
immitis DNA is about 35%, as calculated from known flea nucleic
acid sequences. The unknown nucleic acid molecules would be
attached to a support membrane, and the 150 bp probe would be
labeled, e.g. with a radioactive tag. The hybridization reaction
could be carried out in a solution comprising 2X SSC and 0%
formamide, at a temperature of about 37.degree. C. (low stringency
conditions). Solutions of differing concentrations of SSC can be
made by one of skill in the art by diluting a stock solution of 20X
SSC (175.3 gram NaCl and about 88.2 gram sodium citrate in 1 liter
of water, pH 7) to obtain the desired concentration of SSC. The
skilled artisan would calculate the washing conditions required to
allow up to 30% base pair mismatch. For example, in a wash solution
comprising 1X SSC and 0% formamide, the T.sub.m of perfect hybrids
would be about 79.degree. C.:
81.5.degree. C.+16.6 log
(0.15M)+(0.41.times.0.35)-(500/150)-(0.61.times.0- )=79.degree.
C.
[0036] Thus, to achieve hybridization with nucleic acid molecules
having about 30% base pair mismatch, hybridization washes would be
carried out at a temperature of less than or equal to 49.degree. C.
It is thus within the skill of one in the art to calculate
additional hybridization temperatures based on the desired
percentage base pair mismatch, formulae and G/C content disclosed
herein. For example, it is appreciated by one skilled in the art
that as the nucleic acid molecule to be tested for hybridization
against nucleic acid molecules of the present invention having
sequences specified herein becomes longer than 150 nucleotides, the
T.sub.m for a hybridization reaction allowing up to 30% base pair
mismatch will not vary significantly from 49.degree. C.
[0037] Furthermore, it is known in the art that there are
commercially available computer programs for determining the degree
of similarity between two nucleic acid sequences. These computer
programs include various known methods to determine the percentage
identity and the number and length of gaps between hybrid nucleic
acid molecules. It is further known that the various available
sequence analysis programs produce substantially similar results
when the two compared molecules encode amino acid sequences that
have greater than 30% amino acid identity. See Johnson et al., J.
Mol. Biol., vol. 233, pages 716-738, 1993, and Feng et al., J. Mol.
Evol, vol. 21, pages 112-125, 1985, both of which are incorporated
by reference herein in their entirety. Preferred methods to
determine the percent identity among amino acid sequences and also
among nucleic acid sequences include analysis using one or more of
the commercially available computer programs designed to compare
and analyze nucleic acid or amino acid sequences. These computer
programs include, but are in no way limited to, GCG.TM. (available
from Genetics Computer Group, Madison, Wis.), DNAsis.TM. (available
from Hitachi Software, San Bruno, Calif.) and MacVector.TM.
(available from the Eastman Kodak Company, New Haven, Conn.). A
particularly preferred method to determine the percent identity
among amino acid sequences and also among nucleic acid sequences is
to perform the analysis using the DNAsis.TM. computer program,
using default parameters.
[0038] A preferred parasitic helminth cuticlin protein of the
present invention is a compound that when administered to an animal
in an effective manner, is capable of protecting that animal from
disease caused by a parasitic helminth. In accordance with the
present invention, the ability of a cuticlin protein of the present
invention to protect an animal from disease by a parasitic helminth
refers to the ability of that protein to, for example, treat,
ameliorate or prevent disease caused by parasitic helminths. In one
embodiment, a parasitic helminth cuticlin protein of the present
invention can elicit an immune response (including a humoral and/or
cellular immune response) against a parasitic helminth.
[0039] Suitable parasites to target include any parasite that is
essentially incapable of causing disease in an animal administered
a parasitic helminth cuticlin protein of the present invention.
Accordingly, a parasite to target includes any parasite that
produces a protein having one or more epitopes that can be targeted
by a humoral or cellular immune response against a parasitic
helminth cuticlin protein of the present invention or that can be
targeted by a compound that otherwise inhibits parasite cuticlin
activity, thereby resulting in the decreased ability of the
parasite to cause disease in an animal. Preferred parasites to
target include parasitic helminths such as nematodes, cestodes, and
trematodes, with nematodes being preferred. Preferred nematodes to
target include filariid, ascarid, capillarid, strongylid,
strongyloides, trichostrongyle, and trichurid nematodes.
Particularly preferred nematodes are those of the genera
Acanthocheilonema, Aelurostrongylus, Ancylostoma, Angiostrongylus,
Ascaris, Brugia, Bunostomum, Capillaria, Chabertia, Cooperia,
Crenosoma, Dictyocaulus, Dioctophyme, Dipetalonema,
Diphyllobothrium, Diplydium, Dirofilaria, Dracunculus, Enterobius,
Filaroides, Haemonchus, Lagochilascaris, Loa, Mansonella,
Muellerius, Nanophyetus, Necator, Nematodirus, Oesophagostomum,
Onchocerca, Opisthorchis, Ostertagia, Parafilaria, Paragonimus,
Parascaris, Physaloptera, Protostrongylus, Setaria, Spirocerca,
Spirometra, Stephanofilaria, Strongyloides, Strongylus, Thelazia,
Toxascaris, Toxocara, Trichinella, Trichostrongylus, Trichuris.
Uncinaria, and Wuchereria. Preferred filariid nematodes include
Dirofilaria, Onchocerca, Acanthocheilonema, Brugia, Dipetalonema,
Loa, Parafilaria, Setaria, Stephanofilaria and Wuchereria filariid
nematodes, with D. immitis being even more preferred.
[0040] The present invention also includes mimetopes of parasitic
helminth cuticlin proteins of the present invention. As used
herein, a mimetope of a parasitic helminth cuticlin protein of the
present invention refers to any compound that is able to mimic the
activity of a parasitic helminth cuticlin protein (e.g., has the
ability to elicit an immune response against a parasitic helminth
cuticlin protein of the present invention or ability to inhibit
parasitic helminth cuticlin activity). The ability to mimic the
activity of a parasitic helminth cuticlin protein is likely to be
the result of a structural similarity between the parasitic
helminth cuticlin protein and the mimetope. It is to be noted,
however, that the mimetope need not have a structure similar to a
parasitic helminth cuticlin protein as long as the mimetope
functionally mimics the protein. A mimetope can be, but is not
limited to: a peptide that has been modified to decrease its
susceptibility to degradation (e.g., as an all-D retro peptide); an
anti-idiotypic or catalytic antibody, or a fragment thereof; a
non-proteinaceous immunogenic portion of an isolated protein (e.g.,
a carbohydrate structure); or a synthetic or natural organic
molecule, including a nucleic acid. Such a mimetope can be designed
using computer-generated structures of proteins of the present
invention. A mimetope can also be obtained by generating random
samples of molecules, such as oligonucleotides, peptides or other
organic molecules, and screening such samples by affinity
chromatography techniques using the corresponding binding
partner.
[0041] In one embodiment, a parasitic helminth cuticlin protein of
the present invention is a fusion protein that includes a parasitic
helminth cuticlin protein-containing domain attached to one or more
fusion segments. Suitable fusion segments for use with the present
invention include, but are not limited to, segments that can:
enhance a protein's stability; act as an immunopotentiator to
enhance an immune response against a parasitic helminth cuticlin
protein; or assist purification of a parasitic helminth cuticlin
protein (e.g., by affinity chromatography). A suitable fusion
segment can be a domain of any size that has the desired function
(e.g., imparts increased stability, imparts increased
immunogenicity to a protein, or simplifies purification of a
protein). Fusion segments can be joined to the amino or carboxyl
termini of a parasitic helminth cuticlin protein-containing domain,
and can be susceptible to cleavage in order to enable
straight-forward recovery of a parasitic helminth cuticlin protein.
A fusion protein is preferably produced by culturing a recombinant
cell transformed with a fusion nucleic acid molecule that encodes a
protein including a fusion segment attached to either the carboxyl
or amino terminal end of a cuticlin protein-containing domain.
Preferred fusion segments include a metal binding domain (e.g., a
poly-histidine segment); an immunoglobulin binding domain (e.g.,
Protein A; Protein G; T cell; B cell; Fc receptor or complement
protein antibody-binding domains); a sugar binding domain (e.g., a
maltose binding domain); and/or a "tag" domain (e.g., at least a
portion of .beta.-galactosidase, a strep tag peptide, a T7-tag
peptide, a FLAG.TM. peptide, or other domain that can be purified
using compounds that bind to the domain, such as monoclonal
antibodies). More preferred fusion segments include metal binding
domains, such as a poly-histidine segment; a maltose binding
domain; a strep tag peptide, such as that available from
Biometra.RTM. in Tampa, Fla.; and an S10 peptide.
[0042] In another embodiment, a parasitic helminth cuticlin protein
of the present invention also includes at least one additional
protein segment that is capable of protecting an animal from one or
more diseases. Such a multivalent protective protein can be
produced by culturing a cell transformed with a nucleic acid
molecule comprising two or more nucleic acid domains joined
together in such a manner that the resulting nucleic acid molecule
is expressed as a multivalent protective compound containing at
least two protective compounds, or portions thereof, capable of
protecting an animal from diseases caused, for example, by at least
one infectious agent.
[0043] Examples of multivalent protective compounds include, but
are not limited to, a parasitic helminth cuticlin protein of the
present invention attached to one or more compounds protective
against one or more other infectious agents, particularly an agent
that infects humans, cats, dogs, ferrets, cattle or horses, such
as, but not limited to: viruses (e.g., adenoviruses, caliciviruses,
coronaviruses, distemper viruses, hepatitis viruses, herpesviruses,
immunodeficiency viruses, infectious peritonitis viruses, leukemia
viruses, oncogenic viruses, panleukopenia viruses, papilloma
viruses, parainfluenza viruses, parvoviruses, rabies viruses, and
reoviruses, as well as other cancer-causing or cancer-related
viruses); bacteria (e.g., Actinomyces, Bacillus, Bacteroides,
Bordetella, Bartonella, Borrelia, Brucella, Campylobacter,
Capnocytophaga, Clostridium, Corynebacterium, Coxiella,
Dermatophilus, Enterococcus, Ehrlichia, Escherichia, Francisella,
Fusobacterium, Haemobartonella, Helicobacter, Klebsiella, L-form
bacteria, Leptospira, Listeria, Mycobacteria, Mycoplasma,
Neorickettsia, Nocardia, Pasteurella, Peptococcus,
Peptostreptococcus, Proteus, Pseudomonas, Rickettsia, Rochalimaea,
Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia;
fungi and fungal-related microorganisms (e.g., Absidia, Acremonium,
Alternaria, Aspergillus, Basidiobolus, Bipolaris, Blastomyces,
Candida, Chlamydia, Coccidioides, Conidiobolus, Cryptococcus,
Curvalaria, Epidermophyton, Exophiala, Geotrichum, Histoplasma,
Madurella, Malassezia, Microsporum, Moniliella, Mortierella, Mucor,
Paecilomyces, Penicillium, Phialemonium, Phialophora, Prototheca,
Pseudallescheria, Pseudomicrodochium, Pythium, Rhinosporidium,
Rhizopus, Scolecobasidium, Sporothrix, Stemphylium, Trichophyton,
Trichosporon, and Xylohypha; and other parasites (e.g., Babesia,
Balantidium, Besnoitia, Cryptosporidium, Eimeria, Encephalitozoon,
Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora, Leishmania,
Microsporidia, Neospora, Nosema, Pentatrichomonas, Plasmodium,
Pneumocystis, Sarcocystis, Schistosoma, Theileria, Toxoplasma, and
Trypanosoma, as well as helminth parasites, such as those disclosed
herein). In one embodiment, a parasitic helminth cuticlin protein
of the present invention is attached to one or more additional
compounds protective against heartworm disease. In another
embodiment, one or more protective compounds, such as those listed
above, can be included in a multivalent vaccine comprising a
parasitic helminth cuticlin protein of the present invention and
one or more other protective molecules as separate compounds.
[0044] In one embodiment, a preferred isolated cuticlin protein of
the present invention is a protein encoded by a nucleic acid
molecule comprising at least a portion of nDiCut-1A, nDiCut-1B, or
nBmCut-1A, or by an allelic variant of any of these nucleic acid
molecules. Also preferred is an isolated cuticlin protein encoded
by a nucleic acid molecule having the nucleic acid sequence SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:16; or by
an allelic variant of a nucleic acid molecule having any of these
sequences.
[0045] Translation of SEQ ID NO:1, the coding strand of nucleic
acid molecule nDiCut-1A, yields an essentially full length
parasitic helminth cuticlin protein of 387 amino acids, referred to
herein as PDiCut-1A, the amino acid sequence of which is
represented by SEQ ID NO:4. The open reading frame spans from
nucleotide 167 through nucleotide 1327 of SEQ ID NO:1 and a
termination (stop) codon spans from nucleotide 1329 through
nucleotide 1331 of SEQ ID NO:1. The coding region encoding
PDiCut-1A, is represented by SEQ ID NO:3 (the coding strand) and
SEQ ID NO:5 (the complementary strand).
[0046] Translation of SEQ ID NO:6, the coding strand of nucleic
acid molecule nDiCut-1B, yields a full length parasitic helminth
cuticlin protein of 271 amino acids, referred to herein as
PDiCut-1B, the amino acid sequence of which is represented by SEQ
ID NO:9, assuming an open reading frame that spans from nucleotide
392 through nucleotide 1203 of SEQ ID NO:6. The coding region
encoding PDiCut-1B is represented by SEQ ID NO:8 (the coding
strand) and SEQ ID NO:10 (the complementary strand). The deduced
amino acid sequence is represented by SEQ ID NO:9.
[0047] Translation of SEQ ID NO:16, the coding strand of nucleic
acid molecule nBmCut-1A, yields a partial length parasitic helminth
cuticlin protein of 245 amino acids, referred to herein as
PBmCut-1A, the amino acid sequence of which is represented by SEQ
ID NO:17. The open reading frame spans from nucleotide 158 through
nucleotide 892 of SEQ ID NO:16.
[0048] One embodiment of the present invention includes a
non-native parasitic helminth cuticlin protein encoded by a nucleic
acid molecule that hybridizes under stringent hybridization
conditions with a parasitic helminth cuticlin gene. A preferred
parasitic helminth cuticlin protein is capable of eliciting an
immune response when administered to an animal and/or of having
parasitic helminth cuticlin activity. A preferred parasitic
helminth cuticlin protein is encoded by a nucleic acid molecule
that hybridizes under stringent conditions with a nucleic acid
molecule including either SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:16, or SEQ ID NO:18, or with allelic variants of any of
these sequences.
[0049] A preferred cuticlin protein includes a protein encoded by a
nucleic acid molecule which is at least about 50 nucleotides and
which hybridizes under conditions which preferably allow about 20%
base pair mismatch, more preferably under conditions which allow
about 15% base pair mismatch, more preferably under conditions
which allow about 10% base pair mismatch, more preferably under
conditions which allow about 5% base pair mismatch, and even more
preferably under conditions which allow about 2% base pair mismatch
with a nucleic acid molecule selected from the group consisting of
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID
NO:18.
[0050] Another preferred cuticlin protein of the present invention
includes a protein encoded by a nucleic acid molecule which is at
least about 150 nucleotides and which hybridizes under conditions
which preferably allow about 30% base pair mismatch, more
preferably under conditions which allow about 25% base pair
mismatch, more preferably under conditions which allow about 20%
base pair mismatch, more preferably under conditions which allow
about 15% base pair mismatch, more preferably under conditions
which allow about 10% base pair mismatch, more preferably under
conditions which allow about 5% base pair mismatch, and even more
preferably under conditions which allow about 2% base pair mismatch
with a nucleic acid molecule selected from the group consisting of
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID
NO:18.
[0051] Another embodiment of the present invention includes a
cuticlin protein encoded by a nucleic acid molecule comprising at
least about 50 nucleotides, wherein said nucleic acid molecule
hybridizes, in a solution comprising 2X SSC and 0% formamide, at a
temperature of 37.degree. C., and washing in 1X SSC and 0%
formamide at a temperature of 64.degree. C., to an isolated nucleic
acid molecule selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:18.
Additional preferred cuticlin proteins include proteins encoded by
oligonucleotides of an isolated nucleic acid molecule comprising at
least about 50 nucleotides, wherein said nucleic acid molecule
hybridizes, in a solution comprising 2X SSC and 0% formamide, at a
temperature of 37.degree. C., and washing in 1X SSC and 0%
formamide at a temperature of 64.degree. C., to an isolated nucleic
acid molecule selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:18, wherein
said oligonucleotide comprises at least about 50 nucleotides.
[0052] Another embodiment of the present invention includes a
cuticlin protein encoded by a nucleic acid molecule comprising at
least about 150 nucleotides, wherein said nucleic acid molecule
hybridizes, in a solution comprising 2X SSC and 0% formamide, at a
temperature of 37.degree. C., and washing in 1X SSC and 0%
formamide at a temperature of 64.degree. C., to an isolated nucleic
acid molecule selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:18.
Additional preferred cuticlin proteins include proteins encoded by
oligonucleotides of an isolated nucleic acid molecule comprising at
least about 150 nucleotides, wherein said nucleic acid molecule
hybridizes, in a solution comprising 2X SSC and 0% formamide, at a
temperature of 37.degree. C., and washing in 1X SSC and 0%
formamide at a temperature of 64.degree. C., to an isolated nucleic
acid molecule selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:18, wherein
said oligonucleotide comprises at least about 50 nucleotides.
[0053] A preferred cuticlin protein of the present invention
comprises a protein that is that is at least about 75%, more
preferably at least about 80%, more preferably at least about 85%,
more preferably at least about 90%, more preferably at least about
95%, and more preferably at least about 98% identical to identical
to PDiCut-1A, PDiCut-1B, or PBmCut-1A. More preferred is a cuticlin
protein comprising PDiCut-1A, PDiCut-1B, or PBmCut-1A, or a protein
encoded by an allelic variant of a nucleic acid molecule encoding a
protein comprising PDiCut-1A, PDiCut-1B, or PBmCut-1A.
[0054] Also preferred is a cuticlin protein comprising an amino
acid sequence that is at least about 75%, more preferably at least
about 80%, more preferably at least about 85%, more preferably at
least about 90%, more preferably at least about 95%, and more
preferably at least about 98% identical to amino acid sequence SEQ
ID NO:4, SEQ ID NO:9, or SEQ ID NO:17. Even more preferred is an
amino acid sequence having the sequence represented by SEQ ID NO:4,
SEQ ID NO:9, or SEQ ID NO:17, or an allelic variant of any of these
an amino acid sequences.
[0055] In one embodiment, a preferred parasitic helminth cuticlin
protein comprises an amino acid sequence of at least about 5 amino
acids, preferably at least about 10 amino acids, more preferably at
least about 15 amino acids, more preferably at least about 20 amino
acids, more preferably at least about 25 amino acids, more
preferably at least about 30 amino acids, more preferably at least
about 35 amino acids, more preferably at least about 50 amino
acids, more preferably at least about 100 amino acids, more
preferably at least about 200 amino acids, more preferably at least
about 250 amino acids, more preferably at least about 275 amino
acids, more preferably at least about 300 amino acids, more
preferably at least about 350 amino acids, more preferably at least
about 375 amino acids, and even more preferably at least about 400
amino acids. In another embodiment, preferred parasitic helminth
cuticlin proteins comprise full-length proteins, i.e., proteins
encoded by full-length coding regions, or post-translationally
modified proteins thereof, such as mature proteins from which
initiating methionine and/or signal sequences or "pro" sequences
have been removed.
[0056] A fragment of a parasitic helminth cuticlin protein of the
present invention preferably comprises at least about 5 amino
acids, more preferably at least about 10 amino acids, more
preferably at least about 15 amino acids, more preferably at least
about 20 amino acids, more preferably at least about 25 amino
acids, more preferably at least about 30 amino acids, more
preferably at least about 35 amino acids, more preferably at least
about 40 amino acids, more preferably at least about 45 amino
acids, more preferably at least about 50 amino acids, more
preferably at least about 55 amino acids, more preferably at least
about 60 amino acids, more preferably at least about 65 amino
acids, more preferably at least about 70 amino acids, more
preferably at least about 75 amino acids, more preferably at least
about 80 amino acids, more preferably at least about 85 amino
acids, more preferably at least about 90 amino acids, more
preferably at least about 95 amino acids, and even more preferably
at least about 100 amino acids in length.
[0057] A particularly preferred parasitic helminth cuticlin protein
of the present invention comprises amino acid sequence SEQ ID NO:4,
including, but not limited to, a cuticlin protein consisting of
amino acid sequence SEQ ID NO:4, a fusion protein or a multivalent
protein; or a protein encoded by an allelic variant of a nucleic
acid molecule encoding a protein having amino acid sequence SEQ ID
NO:4. Also particularly preferred is a parasitic helminth cuticlin
protein of the present invention that comprises amino acid sequence
SEQ ID NO:9, including, but not limited to, a cuticlin protein
consisting of amino acid sequence SEQ ID NO:9, a fusion protein or
a multivalent protein; or a protein encoded by an allelic variant
of a nucleic acid molecule encoding a protein having amino acid
sequence SEQ ID NO:9. Also particularly preferred is a parasitic
helminth cuticlin protein of the present invention that comprises
amino acid sequence SEQ ID NO:17, including, but not limited to, a
cuticlin protein consisting of amino acid sequence SEQ ID NO:17, a
fusion protein or a multivalent protein; or a protein encoded by an
allelic variant of a nucleic acid molecule encoding a protein
having amino acid sequence SEQ ID NO:17.
[0058] Another embodiment of the present invention is an isolated
nucleic acid molecule comprising a parasitic helminth cuticlin
nucleic acid molecule. The identifying characteristics of such a
nucleic acid molecule are heretofore described. A nucleic acid
molecule of the present invention can include an isolated natural
parasitic helminth cuticlin gene or a homolog thereof, the latter
of which is described in more detail below. A nucleic acid molecule
of the present invention can include one or more regulatory
regions, a full-length or a partial coding region, or a combination
thereof. The minimal size of a nucleic acid molecule of the present
invention is a size sufficient to allow the formation of a stable
hybrid (i.e., hybridization under stringent hybridization
conditions) with the complementary sequence of another nucleic acid
molecule. Accordingly, the minimal size of a cuticlin nucleic acid
molecule of the present invention is from about 12 to about 18
nucleotides in length. A preferred cuticlin nucleic acid molecule
includes a parasitic helminth cuticlin nucleic acid molecule.
[0059] In accordance with the present invention, an isolated
nucleic acid molecule is a nucleic acid molecule that has been
removed from its natural milieu (i.e., that has been subject to
human manipulation) and can include DNA, RNA, or derivatives of
either DNA or RNA. As such, "isolated" does not reflect the extent
to which the nucleic acid molecule has been purified. An isolated
parasitic helminth cuticlin nucleic acid molecule of the present
invention can be isolated from its natural source or produced using
recombinant DNA technology (e.g., polymerase chain reaction (PCR)
amplification or cloning) or chemical synthesis. Isolated parasitic
helminth cuticlin nucleic acid molecules can include, for example,
natural allelic variants and nucleic acid molecules modified by
nucleotide insertions, deletions, substitutions, or inversions in a
manner such that the modifications do not substantially interfere
with the nucleic acid molecule's ability to encode a cuticlin
protein of the present invention.
[0060] A parasitic helminth cuticlin nucleic acid molecule homolog
can be produced using a number of methods known to those skilled in
the art. See, for example, Sambrook et al., 1989, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press;
Sambrook et al., ibid., is incorporated by reference herein in its
entirety. For example, a nucleic acid molecule can be modified
using a variety of techniques including, but not limited to,
classic mutagenesis and recombinant DNA techniques such as
site-directed mutagenesis, chemical treatment, restriction enzyme
cleavage, ligation of nucleic acid fragments, PCR amplification,
synthesis of oligonucleotide mixtures and ligation of mixture
groups to "build" a mixture of nucleic acid molecules, and
combinations thereof. A nucleic acid molecule homolog can be
selected by hybridization with a parasitic helminth cuticlin
nucleic acid molecule or by screening the function of a protein
encoded by the nucleic acid molecule (e.g., ability to elicit an
inunune response against at least one epitope of a parasitic
helminth cuticlin protein, or the ability to demonstrate cuticlin
activity).
[0061] An isolated nucleic acid molecule of the present invention
can include a nucleic acid sequence that encodes a parasitic
helminth cuticlin protein of the present invention, examples of
such proteins being disclosed herein. Although the phrase "nucleic
acid molecule" primarily refers to the physical nucleic acid
molecule and the phrase "nucleic acid sequence" primarily refers to
the sequence of nucleotides on the nucleic acid molecule, the two
phrases can be used interchangeably, especially with respect to a
nucleic acid molecule, or a nucleic acid sequence, being capable of
encoding a parasitic helminth cuticlin protein.
[0062] A preferred nucleic acid molecule of the present invention,
when administered to an animal, is capable of protecting that
animal from disease caused by a parasitic helminth. As will be
disclosed in more detail below, such a nucleic acid molecule can
be, or can encode, an antisense RNA, a molecule capable of triple
helix formation, a ribozyme, or other nucleic acid-based drug
compound. In additional embodiments, a nucleic acid molecule of the
present invention can encode a protective protein (e.g., a cuticlin
protein of the present invention), the nucleic acid molecule being
delivered to the animal, for example, by direct injection (i.e, as
a genetic vaccine) or in a vehicle such as a recombinant virus
vaccine or a recombinant cell vaccine.
[0063] One embodiment of the present invention is an isolated
nucleic acid molecule that hybridizes under stringent hybridization
conditions with a parasitic helminth cuticlin gene. Preferred
parasitic helminth cuticlin genes of the present invention are
cuticlin genes from Dirofilaria immitis or B. malayi. Such nucleic
acid molecules are referred to as parasitic helminth cuticlin
nucleic acid molecules. A parasitic helminth cuticlin gene
preferably includes at least one of the following nucleic acid
sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:16, or
SEQ ID NO:18.
[0064] In one embodiment of the present invention, a preferred
parasitic helminth cuticlin nucleic acid molecule includes an
isolated nucleic acid molecule which is at least about 50
nucleotides and which hybridizes under conditions which preferably
allow about 20% base pair mismatch, more preferably under
conditions which allow about 15% base pair mismatch, more
preferably under conditions which allow about 10% base pair
mismatch and even more preferably under conditions which allow
about 5% base pair mismatch with a nucleic acid molecule selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:16, or SEQ ID NO:18.
[0065] Another preferred parasitic helminth cuticlin nucleic acid
molecule of the present invention includes a nucleic acid molecule
which is at least about 150 nucleotides and which hybridizes under
conditions which preferably allow about 30% base pair mismatch,
more preferably under conditions which allow about 25% base pair
mismatch, more preferably under conditions which allow about 20%
base pair mismatch, more preferably under conditions which allow
about 15% base pair mismatch, more preferably under conditions
which allow about 10% base pair mismatch and even more preferably
under conditions which allow about 5% base pair mismatch with a
nucleic acid molecule selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:18.
[0066] Another embodiment of the present invention includes a
nucleic acid molecule comprising at least about 50 nucleotides,
wherein said nucleic acid molecule hybridizes, in a solution
comprising 2X SSC and 0% formamide, at a temperature of 37.degree.
C., and washing in 1X SSC and 0% formamide at a temperature of
64.degree. C., to an isolated nucleic acid molecule selected from
the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ
ID NO:16, or SEQ ID NO:18. Additional preferred nucleic acid
molecules of the present invention include oligonucleotides of an
isolated nucleic acid molecule comprising at least about 50
nucleotides, wherein said nucleic acid molecule hybridizes, in a
solution comprising 2X SSC and 0% formamide, at a temperature of
37.degree. C., and washing in 1X SSC and 0% formamide at a
temperature of 64.degree. C. to an isolated nucleic acid molecule
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:16, or SEQ ID NO:18, wherein said oligonucleotide
comprises at least about 50 nucleotides.
[0067] Another embodiment of the present invention includes a
nucleic acid molecule comprising at least about 150 nucleotides,
wherein said nucleic acid molecule hybridizes, in a solution
comprising 2X SSC and 0% formamide, at a temperature of 37.degree.
C., and washing in 1X SSC and 0% formamide at a temperature of
64.degree. C., to an isolated nucleic acid molecule selected from
the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ
ID NO:16, or SEQ ID NO:18. Additional preferred nucleic acid
molecules of the present invention include oligonucleotides of an
isolated nucleic acid molecule comprising at least about 150
nucleotides, wherein said nucleic acid molecule hybridizes, in a
solution comprising 2X SSC and 0% formamide, at a temperature of
37.degree. C., and washing in 1X SSC and 0% formamide at a
temperature of 64.degree. C., to an isolated nucleic acid molecule
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:16, or SEQ ID NO:18, wherein said oligonucleotide
comprises at least about 50 nucleotides.
[0068] In another embodiment, a parasitic helminth cuticlin nucleic
acid molecule of the present invention includes a nucleic acid
molecule that is at least about 70%, more preferably at least about
75%, more preferably at least about 80%, more preferably at least
about 85%, more preferably at least about 90%, and even more
preferably at least about 95% identical to nucleic acid molecule
nDiCut-1A. nDiCut-1B, or nBmCut-1A, or an allelic variant of any of
these nucleic acid molecules. Also preferred is a parasitic
helminth cuticlin nucleic acid molecule comprising a nucleic acid
sequence that is that is at least about 70%, more preferably at
least about 75%, more preferably at least about 80%, more
preferably at least about 85%, more preferably at least about 90%,
and even more preferably at least about 95% identical to nucleic
acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16,
or SEQ ID NO:18; or an allelic variant of a nucleic acid molecule
having any of these sequences.
[0069] Particularly preferred is a cuticlin nucleic acid molecule
comprising all or part of nucleic acid molecule nDiCut-1A,
nDiCut-1B, or nBmCut-1A, or an allelic variant of any these nucleic
acid molecules. Also particularly preferred is a nucleic acid
molecule that includes at least a portion of nucleic acid sequence
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID
NO:18, or an allelic variant of a nucleic acid molecule having any
of these nucleic acid sequences. Such a nucleic acid molecule can
include nucleotides in addition to those included in the SEQ ID
NOs, such as, but not limited to, nucleotides comprising a
full-length gene, or nucleotides comprising a nucleic acid molecule
encoding a fusion protein or a nucleic acid molecule encoding a
multivalent protective compound.
[0070] The present invention also includes a nucleic acid molecule
encoding a protein having at least a portion of SEQ ID NO:4, or an
allelic variant of a nucleic acid molecule encoding a protein
having at least a portion of SEQ ID NO:4. The present invention
further includes a nucleic acid molecule that has been modified to
accommodate codon usage properties of a cell in which such a
nucleic acid molecule is to be expressed. Also included in the
present invention is a nucleic acid molecule encoding a protein
having at least a portion of SEQ ID NO:9, or an allelic variant of
a nucleic acid molecule encoding a protein having at least a
portion of SEQ ID NO:9. The present invention further includes a
nucleic acid molecule that has been modified to accommodate codon
usage properties of a cell in which such a nucleic acid molecule is
to be expressed. Also included in the present invention is a
nucleic acid molecule encoding a protein having at least a portion
of SEQ ID NO:17, or an allelic variant of a nucleic acid molecule
encoding a protein having at least a portion of SEQ ID NO:17. The
present invention further includes a nucleic acid molecule that has
been modified to accommodate codon usage properties of a cell in
which such a nucleic acid molecule is to be expressed.
[0071] In another embodiment, a preferred parasitic helminth
cuticlin nucleic acid molecule of the present invention comprises a
nucleic acid molecule comprising at least about 15 nucleotides,
more preferably at least about 18 nucleotides, more preferably at
least about 20 nucleotides, more preferably at least about 25
nucleotides, more preferably at least about 30 nucleotides, more
preferably at least about 40 nucleotides, more preferably at least
about 50 nucleotides, more preferably at least about 100
nucleotides, more preferably at least about 150 nucleotides, more
preferably at least about 350 nucleotides, more preferably at least
about 450 nucleotides, more preferably at least about 550
nucleotides, more preferably at least about 650 nucleotides, more
preferably at least about 750 nucleotides, more preferably at least
about 1000 nucleotides, more preferably at least about 1500
nucleotides, more preferably at least about 1750 nucleotides more
preferably at least about 1775 nucleotides, and even more
preferably at least about 2000 nucleotides in length.
[0072] In another embodiment, a preferred parasitic helminth
cuticlin nucleic acid molecule encodes a protein comprising at
least about 5 amino acids, preferably at least about 6 amino acids,
more preferably at least about 10 amino acids, more preferably at
least about 15 amino acids, more preferably at least about 20 amino
acids, more preferably at least about 25 amino acids, more
preferably at least about 30 amino acids, more preferably at least
about 40 amino acids, more preferably at least about 50 amino
acids, more preferably at least about 100 amino acids, more
preferably at least about 150 amino acids, more preferably at least
about 200 amino acids, more preferably at least about 300 amino
acids, more preferably at least about 375 amino acids, and even
more preferably at least about 400 amino acids in length.
[0073] Knowing the nucleic acid sequences of certain parasitic
helminth cuticlin nucleic acid molecules of the present invention
allows one skilled in the art to, for example, (a) make copies of
those nucleic acid molecules, (b) obtain nucleic acid molecules
including at least a portion of such nucleic acid molecules (e.g.,
nucleic acid molecules including full-length genes, full-length
coding regions, regulatory control sequences, truncated coding
regions), and (c) obtain other parasitic helminth cuticlin nucleic
acid molecules. Such nucleic acid molecules can be obtained in a
variety of ways including screening appropriate expression
libraries with antibodies of the present invention; traditional
cloning techniques using oligonucleotide probes of the present
invention to screen appropriate libraries; and PCR amplification of
appropriate libraries or DNA using oligonucleotide primers of the
present invention. Preferred libraries to screen or from which to
amplify nucleic acid molecules include Dirofilaria or B. malayi L3,
L4 or adult cDNA libraries as well as genomic DNA libraries.
Similarly, preferred DNA sources from which to amplify nucleic acid
molecules include Dirofilaria or B. malayi L3, L4 or adult
first-strand cDNA syntheses and genomic DNA. Techniques to clone
and amplify genes are disclosed, for example, in Sambrook et al.,
ibid.
[0074] The present invention also includes a nucleic acid molecule
that is an oligonucleotide capable of hybridizing, under stringent
hybridization conditions, with complementary regions of other,
preferably longer, nucleic acid molecules of the present invention
such as those comprising parasitic helminth cuticlin nucleic acid
molecules; or with complementary regions of other parasitic
helminth cuticlin nucleic acid molecules. An oligonucleotide of the
present invention can be RNA, DNA, or derivatives of either. The
minimum size of such an oligonucleotide is the size required for
formation of a stable hybrid between the oligonucleotide and a
complementary sequence on another nucleic acid molecule. A
preferred oligonucleotide of the present invention has a maximum
size of about 100 nucleotides. The present invention includes
oligonucleotides that can be used as, for example, probes to
identify nucleic acid molecules, primers to produce nucleic acid
molecules, or therapeutic reagents to inhibit parasitic helminth
cuticlin protein production or activity (e.g., as antisense-,
triplex formation-, ribozyme-and/or RNA drug-based reagents). The
present invention also includes the use of such oligonucleotides to
protect animals from disease using one or more of such
technologies. Appropriate oligonucleotide-containing therapeutic
compositions can be administered to an animal using techniques
known to those skilled in the art.
[0075] Another embodiment of the present invention includes a
recombinant vector, which includes at least one isolated nucleic
acid molecule of the present invention inserted into any vector
capable of delivering the nucleic acid molecule into a host cell.
Such a vector contains heterologous nucleic acid sequences, that
is, nucleic acid sequences that are not naturally found adjacent to
nucleic acid molecules of the present invention, and that
preferably are derived from a species other than the species from
which the nucleic acid molecule(s) are derived. The vector can be
either RNA or DNA, either prokaryotic or eukaryotic, and typically
is a virus or a plasmid. Recombinant vectors can be used to clone,
sequence, or otherwise manipulate a parasitic helminth cuticlin
nucleic acid molecule of the present invention.
[0076] One type of recombinant vector, referred to herein as a
recombinant molecule, comprises a nucleic acid molecule of the
present invention operatively linked to an expression vector. The
phrase "operatively linked" refers to insertion of a nucleic acid
molecule into an expression vector in a manner such that the
molecule is able to be expressed when transformed into a host cell.
As used herein, an expression vector is a DNA or RNA vector that is
capable of transforming a host cell and of effecting expression of
a specified nucleic acid molecule. Preferably, the expression
vector is also capable of replicating within the host cell. An
expression vector can be either prokaryotic or eukaryotic, and is
typically a virus or a plasmid. An expression vector of the present
invention includes any vector that functions (i.e., directs gene
expression) in a recombinant cell of the present invention,
including in a bacterial, fungal, parasite, insect, other animal,
or plant cell. A preferred expression vector of the present
invention can direct gene expression in a bacterial, yeast,
helminth or other parasite, insect or mammalian cell, or more
preferably in a cell type disclosed herein.
[0077] In particular, an expression vector of the present invention
contains regulatory sequences such as transcription control
sequences, translation control sequences, origins of replication,
and other regulatory sequences that are compatible with the
recombinant cell and that control the expression of a nucleic acid
molecule of the present invention. In particular, a recombinant
molecule of the present invention includes transcription control
sequences. Transcription control sequences are sequences which
control the initiation, elongation, and termination of
transcription. Particularly important transcription control
sequences are those which control transcription initiation, such as
promoter, enhancer, operator and repressor sequences. A suitable
transcription control sequence includes any transcription control
sequence that can function in at least one of the recombinant cells
of the present invention. A variety of such transcription control
sequences are known to those skilled in the art. Preferred
transcription control sequences include those which function in
bacterial, yeast, helminth or other parasite, insect or mammalian
cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro,
omp/lpp, rmB, bacteriophage lambda (such as lambda P.sub.L and
lambda P.sub.R and fusions that include such promoters),
bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6,
bacteriophage SP01, metallothionein, alpha-mating factor, Pichia
alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis
virus subgenomic promoters), antibiotic resistance gene,
baculovirus, Heliothis zea insect virus, vaccinia virus,
herpesvirus, raccoon poxvirus, other poxvirus adenovirus,
cytomegalovirus (such as immediate early promoters), picomavirus,
simian virus 40, retrovirus, actin, retroviral long terminal
repeat, Rous sarcoma virus, heat shock, phosphate or nitrate
transcription control sequences; as well as other sequences capable
of controlling gene expression in prokaryotic or eukaryotic cells.
Additional suitable transcription control sequences include
tissue-specific promoters and enhancers as well as
lymphokine-inducible promoters (e.g., promoters inducible by
interferons or interleukins). Transcription control sequences of
the present invention can also include naturally occurring
transcription control sequences naturally associated with parasitic
helminths, such as D immitis or B. malayi.
[0078] Suitable and preferred nucleic acid molecules to include in
a recombinant vector of the present invention are as disclosed
herein. Preferred nucleic acid molecules to include in a
recombinant vector, and particularly in a recombinant molecule,
include nDiCut-1A, nDiCut-1B, or nBmCut-1A, the production of which
are described in the Examples section.
[0079] A recombinant molecule of the present invention may also (a)
contain a secretory signal (i.e., a signal segment nucleic acid
sequence) to enable an expressed cuticlin protein of the present
invention to be secreted from the cell that produces the protein or
(b) contain a fusion sequence which leads to the expression of a
nucleic acid molecule of the present invention as a fusion protein.
Examples of suitable signal segments include any signal segment
capable of directing the secretion of a protein of the present
invention. Preferred signal segments include, but are not limited
to, native parasitic helminth signal segments, tissue plasminogen
activator (t-PA), interferon, interleukin,growth hormone,
histocompatibility and viral envelope glycoprotein signal segments.
Suitable fusion segments encoded by fusion segment nucleic acids
are disclosed herein. In addition, a nucleic acid molecule of the
present invention can be joined to a fusion segment that directs
the encoded protein to the proteosome, such as a ubiquitin fusion
segment. A eukaryotic recombinant molecule may also include
intervening and/or untranslated sequences surrounding and/or within
the nucleic acid sequence of the nucleic acid molecule of the
present invention.
[0080] Another embodiment of the present invention includes a
recombinant cell comprising a host cell transformed with one or
more recombinant molecules of the present invention. Transformation
of a nucleic acid molecule into a cell can be accomplished by any
method by which a nucleic acid molecule can be inserted into the
cell. Transformation techniques include, but are not limited to,
transfection, electroporation, microinjection, lipofection,
adsorption, and protoplast fusion. A recombinant cell may remain
unicellular or may grow into a tissue, organ, or a multicellular
organism. Transformed nucleic acid molecules of the present
invention can remain extrachromosomal or can integrate into one or
more sites within a chromosome of the transformed (i.e.,
recombinant) cell in such a manner that their ability to be
expressed is retained. Preferred nucleic acid molecules with which
to transform a cell include cuticlin nucleic acid molecules
disclosed herein. Particularly preferred nucleic acid molecules
with which to transform a cell include nDiCut-1A, nDiCut-1B, and
nBmCut-1A.
[0081] Suitable host cells to transform include any cell that can
be transformed with a nucleic acid molecule of the present
invention. Host cells can be either untransformed cells or cells
that are already transformed with at least one nucleic acid
molecule (e.g., nucleic acid molecules encoding one or more
proteins of the present invention or encoding other proteins useful
in the production of multivalent vaccines). A recombinant cell of
the present invention can be endogenously (i.e., naturally) capable
of producing a parasitic helminth cuticlin protein of the present
invention or can be capable of producing such a protein after being
transformed with at least one nucleic acid molecule of the present
invention. A host cell of the present invention can be any cell
capable of producing at least one protein of the present invention,
and can be a bacterial, fungal (including yeast), parasite
(including helminth, protozoa and ectoparasite), other insect,
other animal or plant cell. Preferred host cells include bacterial,
mycobacterial, yeast, helminth, insect and mammalian cells. More
preferred host cells include Salmonella, Escherichia, Bacillus,
Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia,
BHK (baby hamster kidney) cells, MDCK cells (Madin-Darby Canine
Kidney cells), CRFK cells (Crandell Feline Kidney cells), BSC-1
cells (African monkey kidney cell line used, for example, to
culture poxviruses), COS (e.g., COS-7) cells, and Vero cells.
Particularly preferred host cells are Escherichia coli, including
E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium,
including attenuated strains such as UK-1 .sub.X3987 and SR-11
.sub.X4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK
cells; CRFK cells; BSC-1 cells; COS cells; Vero cells; and
non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).
Additional appropriate mammalian cell hosts include other kidney
cell lines, other fibroblast cell lines (e.g., human, murine or
chicken embryo fibroblast cell lines), myeloma cell lines, Chinese
hamster ovary cells, mouse NIH/3T3 cells, LMTK.sup.31 cells and/or
HeLa cells. In one embodiment, the proteins may be expressed as
heterologous proteins in myeloma cell lines employing
immunoglobulin promoters.
[0082] A recombinant cell of the present invention includes any
cell transformed with at least one of any nucleic acid molecule of
the present invention. Suitable and preferred nucleic acid
molecules as well as suitable and preferred recombinant molecules
with which to transform such a cell are disclosed herein.
[0083] In one embodiment, a recombinant cell of the present
invention can be co-transformed with a recombinant molecule
including a parasitic helminth cuticlin nucleic acid molecule
encoding a protein of the present invention and a nucleic acid
molecule encoding another protective compound, as disclosed herein
(e.g., to produce multivalent vaccines).
[0084] Recombinant DNA technologies can be used to improve
expression of a transformed nucleic acid molecule by manipulating,
for example, the number of copies of the nucleic acid molecule
within a host cell, the efficiency with which that nucleic acid
molecule is transcribed, the efficiency with which the resultant
transcript is translated, and the efficiency of post-translational
modifications. Recombinant techniques useful for increasing the
expression of a nucleic acid molecule of the present invention
include, but are not limited to, operatively linking the nucleic
acid molecule to a high-copy number plasmid, integration of the
nucleic acid molecule into one or more host cell chromosomes,
addition of vector stability sequences to a plasmid, substitution
or modification of transcription control signals (e.g., promoters,
operators, enhancers), substitution or modification of
translational control signals (e.g., ribosome binding sites,
Shine-Dalgarno sequences, or Kozak sequences), modification of a
nucleic acid molecule of the present invention to correspond to the
codon usage of the host cell, deletion of sequences that
destabilize transcripts, and the use of control signals that
temporally separate recombinant cell growth from recombinant enzyme
production during fermentation. The activity of an expressed
recombinant protein of the present invention may be improved by
fragmenting, modifying, or derivatizing a nucleic acid molecule
encoding such a protein.
[0085] Isolated parasitic helminth cuticlin proteins of the present
invention can be produced in a variety of ways, including
production and recovery of natural proteins, production and
recovery of recombinant proteins, and chemical synthesis of the
proteins. In one embodiment, an isolated protein of the present
invention is produced by culturing a cell capable of expressing the
protein under conditions effective to produce the protein, and
recovering the protein. A preferred cell to culture is a
recombinant cell of the present invention. Effective culture
conditions include, but are not limited to, effective media,
bioreactor, temperature, pH and oxygen conditions that permit
protein production. An effective medium refers to any medium in
which a cell is cultured to produce a parasitic helminth cuticlin
protein of the present invention. Such a medium typically comprises
an aqueous base having assimilable carbon, nitrogen and phosphate
sources, and appropriate salts, minerals, metals and other
nutrients, such as vitamins. Cells of the present invention can be
cultured in conventional fermentation bioreactors, shake flasks,
test tubes, microtiter dishes, and petri plates. Culturing can be
carried out at a temperature, pH and oxygen content appropriate for
a given recombinant cell. Such culturing conditions are within the
expertise of one of ordinary skill in the art. Examples of suitable
conditions are included in the Examples section.
[0086] Depending on the vector and host system used for production,
a resultant protein of the present invention may either remain
within the recombinant cell; be secreted into the fermentation
medium; be secreted into a space between two cellular membranes,
such as the periplasmic space in E. coli; or be retained on the
outer surface of a cell or viral membrane.
[0087] The phrase "recovering the protein", as well as similar
phrases, refer to collecting the whole fermentation medium
containing the protein and need not imply additional steps of
separation or purification. Proteins of the present invention can
be purified using a variety of standard protein purification
techniques, such as, but not limited to, affinity chromatography,
ion exchange chromatography, filtration, electrophoresis,
hydrophobic interaction chromatography, gel filtration
chromatography, reverse phase chromatography, concanavalin A
chromatography, chromatofocusing and differential solubilization.
Proteins of the present invention are preferably retrieved in
"substantially pure" form. As used herein, "substantially pure"
refers to a purity that allows for the effective use of the protein
as a therapeutic composition or diagnostic. A therapeutic
composition for animals, for example, should exhibit no substantial
toxicity and preferably should be capable of stimulating the
production of antibodies in a treated animal.
[0088] The present invention also includes isolated (i.e., removed
from their natural milieu) antibodies that selectively bind to a
parasitic helminth cuticlin protein of the present invention or a
mimetope thereof (e.g., anti-parasitic helminth cuticlin
antibodies). As used herein, the term "selectively binds to" a
cuticlin protein refers to the ability of an antibody of the
present invention to preferentially bind to specified proteins and
mimetopes thereof of the present invention. Binding can be measured
using a variety of methods standard in the art including enzyme
immunoassays (e.g., ELISA), immunoblot assays, etc. See, for
example, Sambrook et al., ibid., and Harlow, et al., 1988,
Antibodies, a Laboratory Manual, Cold Spring Harbor Labs Press;
Harlow et al., ibid., is incorporated by reference herein in its
entirety. An anti-parasitic helminth cuticlin antibody preferably
selectively binds to a parasitic helminth cuticlin protein in such
a way as to reduce the activity of that protein.
[0089] Isolated antibodies of the present invention can include
antibodies in serum, or antibodies that have been purified to
varying degrees. Antibodies of the present invention can be
polyclonal or monoclonal, functional equivalents such as antibody
fragments and genetically-engineered antibodies, including single
chain antibodies or chimeric antibodies that can bind to more than
one epitope.
[0090] A preferred method to produce antibodies of the present
invention includes (a) administering to an animal an effective
amount of a protein, peptide or mimetope thereof of the present
invention to produce the antibodies and (b) recovering the
antibodies. In another method, antibodies of the present invention
are produced recombinantly using techniques as heretofore disclosed
to produce cuticlin proteins of the present invention. Antibodies
raised against defined proteins or mimetopes can be advantageous
because such antibodies are not substantially contaminated with
antibodies against other substances that might otherwise cause
interference in a diagnostic assay or side effects if used in a
therapeutic composition.
[0091] Antibodies of the present invention have a variety of
potential uses that are within the scope of the present invention.
For example, such antibodies can be used (a) as therapeutic
compounds to passively immnunize an animal in order to protect the
animal from parasitic helminths susceptible to treatment by such
antibodies, (b) as reagents in assays to detect infection by such
helminths or (c) as tools to screen expression libraries or to
recover desired proteins of the present invention from a mixture of
proteins and other contaminants. Furthermore, antibodies of the
present invention can be used to target cytotoxic agents to
parasitic helminths of the present invention in order to directly
kill such helminths. Targeting can be accomplished by conjugating
(i.e., stably joining) such antibodies to the cytotoxic agents
using techniques known to those skilled in the art. Suitable
cytotoxic agents are known to those skilled in the art.
[0092] One embodiment of the present invention is a therapeutic
composition that, when administered to an animal in an effective
manner, is capable of protecting that animal from disease caused by
a parasitic helminth. A therapeutic composition of the present
invention includes an excipient and at least one of the following
protective compounds: an isolated native parasitic helminth
cuticlin protein; an isolated non-native parasitic helminth
cuticlin protein; a mimetope of a parasitic helminth cuticlin
protein; an isolated parasitic helminth cuticlin nucleic acid
molecule; an isolated antibody that selectively binds to a
parasitic helminth cuticlin protein; or an inhibitor of cuticlin
protein activity identified by its ability to inhibit parasitic
helminth cuticlin activity. As used herein, a protective compound
refers to a compound that, when administered to an animal in an
effective manner, is able to treat, ameliorate, or prevent disease
caused by a parasitic helminth. Preferred helminths to target are
heretofore disclosed. Examples of proteins, nucleic acid molecules,
antibodies and inhibitors of the present invention are disclosed
herein.
[0093] The present invention also includes a therapeutic
composition comprising at least one parasitic helminth
cuticlin-based compound of the present invention in combination
with at least one additional compound protective against one or
more infectious agents. Examples of such compounds and infectious
agents are disclosed herein.
[0094] A therapeutic composition of the present invention can be
administered to any animal susceptible to such therapy, preferably
to mammals, and more preferably to dogs, cats, humans, ferrets,
horses, cattle, sheep and other pets, work animals, economic food
animals, or zoo animals. Preferred animals to protect against
heartworm disease include dogs, cats, humans and ferrets, with dogs
and cats being particularly preferred.
[0095] In one embodiment, a therapeutic composition of the present
invention can be administered to the vector in which the parasitic
helminth develops, such as to a mosquito, in order to prevent the
spread of parasitic helminth to the definitive mammalian host. Such
administration could be orally or by developing transgenic vectors
capable of producing at least one therapeutic composition of the
present invention. In another embodiment, a vector, such as a
mosquito, can ingest therapeutic compositions present in the blood
of a host that has been administered a therapeutic composition of
the present invention.
[0096] A therapeutic composition of the present invention can be
formulated in an excipient that the animal to be treated can
tolerate. Examples of such excipients include water, saline,
Ringer's solution, dextrose solution, Hank's solution, and other
aqueous physiologically balanced salt solutions. Nonaqueous
vehicles, such as fixed oils, sesame oil, ethyl oleate, or
triglycerides may also be used. Other useful formulations include
suspensions containing viscosity enhancing agents, such as sodium
carboxymethylcellulose, sorbitol, or dextran. Excipients can also
contain minor amounts of additives, such as substances that enhance
isotonicity and chemical stability. Examples of buffers include
phosphate buffer, bicarbonate buffer, and Tris buffer, while
examples of preservatives include thimerosal, --or o-cresol,
formalin, and benzyl alcohol. Standard formulations can either be
liquid injectables or solids which can be taken up in a suitable
liquid as a suspension or solution for injection. Thus, in a
non-liquid formulation, the excipient can comprise dextrose, human
serum albumin, preservatives, etc., to which sterile water or
saline can be added prior to administration.
[0097] In one embodiment of the present invention, a therapeutic
composition can include an adjuvant. Adjuvants are agents that are
capable of enhancing the immune response of an animal to a specific
antigen. Suitable adjuvants include, but are not limited to,
cytokines, chemokines, and compounds that induce the production of
cytokines and chemokines (e.g., granulocyte macrophage colony
stimulating factor (GM-CSF), granulocyte colony stimulating factor
(G-CSF), macrophage colony stimulating factor (M-CSF), colony
stimulating factor (CSF), erythropoietin (EPO), interleukin 2
(IL-2), interleukin-3 (IL-3), interleukin 4 (IL-4), interleukin 5
(IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8
(IL-8), interleukin 10 (IL-10), interleukin 12 (IL-1 2), interferon
gamma, interferon gamma inducing factor I (IGIF), transforming
growth factor beta, RANTES (regulated upon activation, normal
T-cell expressed and presumably secreted), macrophage inflammatory
proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmania
elongation initiating factor (LEIF)); bacterial components (e.g.,
endotoxins, in particular superantigens, exotoxins and cell wall
components); aluminum-based salts; calcium-based salts; silica;
polynucleotides; toxoids; serum proteins, viral coat proteins;
block copolymer adjuvants (e.g., Hunter's Titermax.TM. adjuvant
(Vaxcel.TM., Inc. Norcross, Ga.), Ribi adjuvants (Ribi ImmunoChem
Research. Inc., Hamilton, Mont.); and saponins and their
derivatives (e.g., Quil A (Superfos Biosector A/S, Denmark).
Protein adjuvants of the present invention can be delivered in the
form of the protein themselves or of nucleic acid molecules
encoding such proteins using the methods described herein.
[0098] In one embodiment of the present invention, a therapeutic
composition can include a carrier. Carriers include compounds that
increase the half-life of a therapeutic composition in the treated
animal. Suitable carriers include, but are not limited to,
polymeric controlled release vehicles, biodegradable implants,
liposomes, bacteria, viruses, other cells, oils, esters, and
glycols.
[0099] One embodiment of the present invention is a controlled
release formulation that is capable of slowly releasing a
composition of the present invention into an animal. As used
herein, a controlled release formulation comprises a composition of
the present invention in a controlled release vehicle. Suitable
controlled release vehicles include, but are not limited to,
biocompatible polymers, other polymeric matrices, capsules,
microcapsules, microparticles, bolus preparations, osmotic pumps,
diffusion devices, liposomes, lipospheres, and transdermal delivery
systems. Other controlled release formulations of the present
invention include liquids that, upon administration to an animal,
form a solid or a gel in situ. Preferred controlled release
formulations are biodegradable (i.e., bioerodible).
[0100] A preferred controlled release formulation of the present
invention is capable of releasing a composition of the present
invention into the blood of the treated animal at a constant rate
sufficient to attain therapeutic dose levels of the composition to
protect an animal from disease caused by parasitic helminths. The
therapeutic composition is preferably released over a period of
time ranging from about 1 to about 12 months. A controlled release
formulation of the present invention is capable of effecting a
treatment preferably for at least about 1 month, more preferably
for at least about 3 months, even more preferably for at least
about 6 months, even more preferably for at least about 9 months,
and even more preferably for at least about 12 months.
[0101] In order to protect an animal from disease caused by a
parasitic helminth, a therapeutic composition of the present
invention is administered to the animal in an effective manner such
that the composition is capable of protecting that animal from a
disease caused by a parasitic helminth. For example, an isolated
protein or mimetope thereof is administered in an amount and manner
that elicits (i.e., stimulates) an immune response that is
sufficient to protect the animal from the disease. Similarly, an
antibody of the present invention, when administered to an animal
in an effective manner, is administered in an amount so as to be
present in the animal at a titer that is sufficient to protect the
animal from the disease, at least temporarily. An oligonucleotide
nucleic acid molecule of the present invention can also be
administered in an effective manner, thereby reducing expression of
native parasitic helminth cuticlin proteins in order to interfere
with development of the parasitic helminths targeted in accordance
with the present invention.
[0102] Therapeutic compositions of the present invention can be
administered to animals prior to infection in order to prevent
infection (i.e., as a preventative vaccine) or can be administered
to animals after infection in order to treat disease caused by the
parasitic helminth (i.e., as a curative agent or a therapeutic
vaccine).
[0103] Acceptable protocols to administer therapeutic compositions
in an effective manner include individual dose size, number of
doses, frequency of dose administration, and mode of
administration. Determination of such protocols can be accomplished
by those skilled in the art. A suitable single dose is a dose that
is capable of protecting an animal from disease when administered
one or more times over a suitable time period. For example, a
preferred single dose of a protein, mimetope, or antibody
therapeutic composition is from about 1 microgram (.mu.g) to about
10 milligrams (mg) of the therapeutic composition per kilogram body
weight of the animal. Booster vaccinations can be administered from
about 2 weeks to several years after the original administration.
Booster administrations preferably are administered when the immune
response of the animal becomes insufficient to protect the animal
from disease. A preferred administration schedule is one in which
from about 10 .mu.g to about 1 mg of the therapeutic composition
per kg body weight of the animal is administered from about one to
about two times over a time period of from about 2 weeks to about
12 months. Modes of administration can include, but are not limited
to, subcutaneous, intradermal, intravenous, intranasal, oral,
transdermal, and intramuscular routes.
[0104] According to one embodiment, a nucleic acid molecule of the
present invention can be administered to an animal in a fashion to
enable expression of that nucleic acid molecule into a protective
protein or protective RNA (e.g., an antisense RNA, a ribozyme, a
triple helix form, or an RNA drug) in the animal. Nucleic acid
molecules can be delivered to an animal by a variety of methods
including, but not limited to, (a) administering a genetic vaccine
(e.g., a naked DNA or RNA molecule, such as is taught, for example,
in Wolff et al., 1990, Science 247, 1465-1468) or (b) administering
a nucleic acid molecule packaged as a recombinant virus vaccine or
as a recombinant cell vaccine (i.e., the nucleic acid molecule is
delivered by a viral or cellular vehicle).
[0105] A genetic (i.e., naked nucleic acid) vaccine of the present
invention includes a nucleic acid molecule of the present invention
and preferably includes a recombinant molecule of the present
invention that preferably is replication, or otherwise
amplification, competent. A genetic vaccine of the present
invention can comprise one or more nucleic acid molecules of the
present invention in the form of, for example, a dicistronic
recombinant molecule. A preferred genetic vaccine includes at least
a portion of a viral genome (i.e., a viral vector). Preferred viral
vectors include those based on alphaviruses, poxviruses,
adenoviruses, herpesviruses, picornaviruses, and retroviruses, with
those based on alphaviruses (such as Sindbis or Semliki forest
virus), species-specific herpesviruses and poxviruses being
particularly preferred. Any suitable transcription control sequence
can be used, including those disclosed as suitable for protein
production. Particularly preferred transcription control sequences
include cytomegalovirus immediate early (preferably in conjunction
with Intron-A), Rous sarcoma virus long terminal repeat, and
tissue-specific transcription control sequences, as well as
transcription control sequences endogenous to viral vectors if
viral vectors are used. The incorporation of "strong" poly(A)
sequences is also preferred.
[0106] A genetic vaccine of the present invention can be
administered in a variety of ways, with intramuscular,
subcutaneous, intradermal, transdermal, intranasal and oral routes
of administration being preferred. A preferred single dose of a
genetic vaccine ranges from about 1 nanogram (ng) to about 500
.mu.g, depending on the route of administration or method of
delivery, as can be determined by those skilled in the art.
Suitable delivery methods include, for example, by injection, as
drops, aerosolized, or topically. Genetic vaccines of the present
invention can be contained in an aqueous excipient (e.g., phosphate
buffered saline) alone or in a carrier (e.g., lipid-based
vehicles).
[0107] A recombinant virus vaccine of the present invention
includes a recombinant molecule of the present invention that is
packaged in a viral coat and that can be expressed in an animal
after administration. Preferably, the recombinant molecule is
packaging--or replication-deficient or encodes an attenuated virus.
A number of recombinant viruses can be used, including, but not
limited to, those based on alphaviruses, poxviruses, adenoviruses,
herpesviruses, picomaviruses, and retroviruses. Preferred
recombinant virus vaccines are those based on alphaviruses (such as
Sindbis virus), raccoon poxviruses, picomaviruses, and
species-specific herpesviruses. Methods to produce and use a
recombinant alphavirus vaccine are disclosed in PCT Publication No.
WO 94/17813, by Xiong et al., published Aug. 18, 1994, which is
incorporated by reference herein in its entirety.
[0108] When administered to an animal, a recombinant virus vaccine
of the present invention infects cells within the immunized animal
and directs the production of a protective protein or RNA nucleic
acid molecule that is capable of protecting the animal from disease
caused by a parasitic helminth as disclosed herein. For example, a
recombinant virus vaccine comprising a parasitic helminth cuticlin
nucleic acid molecule of the present invention is administered
according to a protocol that results in the animal producing a
sufficient immune response to protect itself from heartworm
disease. A preferred single dose of a recombinant virus vaccine of
the present invention is from about 1.times.10.sup.4 to about
1.times.10.sup.8 virus plaque forming units (pfu) per kilogram body
weight of the animal. Administration protocols are similar to those
described herein for protein-based vaccines, with subcutaneous,
intramuscular, intranasal and oral administration routes being
preferred.
[0109] A recombinant cell vaccine of the present invention includes
a recombinant cell of the present invention that expresses at least
one protein of the present invention. Preferred recombinant cells
for this embodiment include Salmonella, E. coli, Listeria,
Mycobacterium, S. frugiperda, yeast (including Saccharomyces
cerevisiae and Pichia pastoris), BHK, BSC-1, myoblast G8, COS
(e.g., COS-7), Vero, MDCK or CRFK recombinant cells. A recombinant
cell vaccine of the present invention can be administered in a
variety of ways but has the advantage that it can be administered
orally, preferably at doses ranging from about 10.sup.8 to about
10.sup.12 cells per kilogram body weight. Administration protocols
are similar to those described herein for protein-based vaccines. A
recombinant cell vaccine can comprise whole cells, cells stripped
of cell walls or cell lysates.
[0110] The efficacy of a therapeutic composition of the present
invention to protect an animal from disease caused by a parasitic
helminth can be tested in a variety of ways including, but not
limited to, detection of protective antibodies (using, for example,
proteins or mimetopes of the present invention), detection of
cellular immunity within the treated animal, or challenge of the
treated animal with the parasitic helminth to determine whether the
treated animal is resistant to disease. Challenge studies can
include implantation of chambers including parasitic helminth
larvae into the treated animal and/or direct administration of
larvae to the treated animal. In one embodiment, therapeutic
compositions can be tested in animal models such as mice. Such
techniques are known to those skilled in the art.
[0111] One preferred embodiment of the present invention is the use
of parasitic helminth cuticlin proteins, nucleic acid molecules,
antibodies or inhibitory compounds of the present invention to
protect an animal from heartworm disease. It is particularly
preferred to prevent L3 that are delivered to the animal by the
mosquito intermediate host from maturing into adult worms.
Accordingly,. a preferred therapeutic composition is one that is
able to inhibit at least one step in the portion of the parasite's
development cycle that includes L3, third molt, L4, fourth molt,
and immature adult prior to entering the circulatory system. In
dogs, this portion of the developmental cycle is about 70 days in
length. A particularly preferred therapeutic composition includes a
parasitic helminth cuticlin-based therapeutic composition of the
present invention, particularly in light of the evidence herein
reported that cuticlin is expressed in both larval and adult stages
of the parasite. Such a composition can include a parasitic
helminth cuticlin nucleic acid molecule, a parasitic helminth
cuticlin protein or a mimetope thereof, anti-parasitic helminth
cuticlin antibodies, or inhibitors of parasitic helminth cuticlin
activity. Such therapeutic compositions are administered to an
animal in a manner effective to protect the animals from heartworm
disease. Additional protection may be obtained by administering
additional protective compounds, including other parasitic helminth
proteins, nucleic acid molecules. antibodies and inhibitory
compounds, as disclosed herein.
[0112] One therapeutic composition of the present invention
includes an inhibitor of parasitic helminth cuticlin activity,
i.e., a compound capable of substantially interfering with the
function of a parasitic helminth cuticlin protein, also referred to
herein as a cuticlin inhibitor. In one embodiment, such an
inhibitor comprises a compound that interacts directly with a
cuticlin protein active site (usually by binding to or modifying
the active site), thereby inhibiting cuticlin activity. According
to this embodiment, a cuticlin inhibitor can also interact with
other regions of a cuticlin protein to inhibit cuticlin activity,
for example, by allosteric interaction. Preferably, a cuticlin
inhibitor of the present invention is identified by its ability to
bind to, or otherwise interact with, a parasitic helminth cuticlin
protein, thereby inhibiting cuticlin activity of that protein. Such
a cuticlin inhibitor is a suitable for inclusion in a therapeutic
composition of the present invention as long as the compound is not
harmful to the host animal being treated.
[0113] A cuticlin inhibitor can be identified using a parasitic
helminth cuticlin protein of the present invention. As such, one
embodiment of the present invention is a method to identify a
compound capable of inhibiting cuticlin activity of a parasitic
helminth susceptible to inhibition by an inhibitor of parasitic
helminth cuticlin activity. Such a method includes the steps of (a)
contacting (e.g., combining, mixing) an isolated parasitic helminth
cuticlin protein, preferably a D. immitis cuticlin protein, with a
putative inhibitory compound under conditions in which, in the
absence of the compound, the protein has cuticlin activity, and (b)
determining if the putative inhibitory compound inhibits the
cuticlin activity. Putative inhibitory compounds to screen include
small organic molecules, antibodies (including mimetopes thereof)
and substrate analogs. Methods to determine cuticlin activity are
known to those skilled in the art; see, for example, Rhee. et al.,
ibid., Lim, et al., ibid., Sauri, et al., ibid., and Kim, et al.,
ibid.
[0114] The present invention also includes a test kit to identify a
compound capable of inhibiting cuticlin activity of a parasitic
helminth. Such a test kit includes an isolated parasitic helminth
cuticlin protein, preferably a D. immitis cuticlin protein, having
cuticlin activity.. and a means for determining the extent of
inhibition of cuticliri activity in the presence of (i.e., effected
by) a putative inhibitory compound. Such compounds are also
screened to identify those that are substantially not toxic in host
animals, e.g., compounds that do not inhibit the activity of
mammalian cuticlin.
[0115] Cuticlin inhibitors isolated by such a method or test kit
can be used to inhibit any parasitic helminth cuticlin protein that
is susceptible to such an inhibitor. A particularly preferred
cuticlin inhibitor of the present invention is capable of
protecting an animal from heartworm disease. A therapeutic
composition comprising a compound that inhibits cuticlin activity
can be administered to an animal in an effective manner to protect
that animal from disease caused by the parasite expressing the
targeted cuticlin enzyme, and preferably to protect that animal
from heartworm disease. Effective amounts and dosing regimens can
be determined using techniques known to those skilled in the
art.
[0116] It is also within the scope of the present invention to use
isolated proteins, mimetopes, nucleic acid molecules and antibodies
of the present invention as diagnostic reagents to detect infection
by parasitic helminths. Such diagnostic reagents can be
supplemented with additional compounds that can detect specific
phases of the parasite's life cycle. Methods to use such diagnostic
reagents to diagnose parasitic helminth infection are well known to
those skilled in the art. Suitable and preferred parasitic
helminths to detect are those to which therapeutic compositions of
the present invention are targeted. Particularly preferred
parasitic helminths to detect using diagnostic reagents of the
present invention are D. immitis or B. malayi.
[0117] A Sequence Listing pursuant to 37 CFR .sctn.1.821 is
submitted herewith on separately numbered pages. A copy in computer
readable form is also submitted herewith. Applicants assert
pursuant to 37 CFR .sctn.1.821(f) that the content of the paper and
computer readable copies of SEQ ID NO:1 through SEQ ID NO:18
submitted herewith are the same.
[0118] The following examples are provided for the purposes of
illustration and are not intended to limit the scope of the present
invention.
EXAMPLES
[0119] It is to be noted that these Examples include a number of
molecular biology, microbiology, immunology and biochemistry
techniques familiar to those skilled in the art. Disclosure of such
techniques can be found, for example, in Sambrook et al., ibid.,
Ausubel, et al., 1993, Current Protocols in Molecular Biology,
Greene/Wiley Interscience, New York, N.Y., and related references.
Ausubel, et al, ibid. is incorporated by reference herein in its
entirety. DNA and protein sequence analyses were carried out using
the PC/GENE.TM. sequence analysis program, version 6.60 (available
from Intelligenetics, Inc., Mountainview, Calif.). The settings for
analysis were as follows. NAlign:(nucleic acid)open gap cost 10;
unit gap cost -10; Palign(protein): open gap cost -2, unit gap cost
-2; CLUSTAL: Protein, K-triple for protein=1, Gap penalty=10,
Window size=10; Nucleic acid, K-triple for nucleic
acids=5;filtering level=2.5; Parameter for final alignment: Open
gap cost=10, Unit gap cost=10; Transitions are WEIGHTED twice as
likely as transversions. It should also be noted that, because
nucleic acid sequencing technology, and in particular the
sequencing of PCR products, is not entirely error-free, the nucleic
acid and deduced protein sequences presented herein represent
apparent nucleic acid sequences of the nucleic acid molecules
encoding parasitic helminth cuticlin proteins of the present
invention.
Example 1
[0120] This example describes the molecular cloning of two related
cuticlin genes from Dirofilaria immitis, referred to herein as
DiCut-1A and DiCut-1B. As is herein described, DiCut-1A and
DiCut-1B are related to the cut-1 cuticlin genes from Ascaris and
C. elegans. Despite this relatedness, initial attempts to isolate
genes encoding cuticlin proteins from D. immitis using degenerate
primers based on C. elegans and Ascaris cut-1 were unsuccessful.
Subsequent to the unsuccessful attempts to isolate cuticlin genes
from D. immitis based on homology to the Ascaris and C. elegans
cut-1 cuticlin genes, D. immitis cDNA sequences with homology to
the Ascaris and C. elegans cut-1 genes were identified from a D.
immitis larval EST DNA sequence, as follows.
[0121] D. immitis larval cDNA was produced and enriched for DNA
representing larval message in relation to adult message using a
CLONTECH PCR-Select.TM. cDNA subtraction kit (available from
CLONETECH, Palo Alto, Calif.) according to the manufacturer's
instructions. These larval cDNAs were subcloned into the
pCRII.TM.vector (available from Invitrogen, San Diego, Calif.)
according to the manufacturer's instructions. The isolated and
subcloned larval cDNAs were sequenced by the Sanger dideoxy chain
termination method, using the PRISM.TM. Ready Dye Terminator Cycle
Sequencing Kit with AmpliTaq.TM. DNA Polymerase, FS (available from
the Perkin-Elmer Corporation, Norwalk, Conn.). PCR extensions were
done in the GeneAmp.TM. PCR System 9600 (available from
Perkin-Elmer). Excess dye terminators were removed from extension
products using the Centriflex.TM. Gel Filtration Cartridge
(available from Advanced Genetics Technologies Corporation,
Gaithersburg, Md.) following the standard protocol. Samples were
resuspended according to ABI protocols and were run on a
Perkin-Elmer ABI PRISM.TM.377 Automated DNA Sequencer.
[0122] The first EST sequence obtained represented a D. immitis
cuticlin nucleic acid molecule 1016 bp long. This sequence
comprises the nucleic acid sequence between base pairs 194 and 1210
of the essentially full-length cDNA sequence of a D. immitis
cuticlin nucleic acid molecule referred to herein as nDiCut-1A (the
coding and complementary strands of which are herein represented by
SEQ ID NO:1 and SEQ ID NO:2, respectively). A second D. immitis
cuticlin EST fragment was obtained which overlapped with the
previous EST fragment at the 3' end, and contributed an additional
569 base pairs of sequence comprising the nucleotide sequence
between base pairs 1211 and 1779 of nDiCut-1A (SEQ ID NO:1). In
order to obtain the 5' end of nDiCut-1A, and to confirm the
sequence of the 3' end of the molecule, sense and antisense primers
specific to the D. immitis cuticlin EST sequence were designed to
hybridize to nDiCut-1A between base pairs 862 and 895 of the final
nDiCut-1A sequence (SEQ ID NO:1). The sense primer specific to the
EST, referred to herein as CUT-3'R (SEQ ID NO:11), consists of the
sequence: 5'G GCT GGC CAA GAA GCT CAC GTA TAC AAA TAT GCG 3'. The
antisense primer referred to herein as CUT-5'R (SEQ ID NO:12),
consists of the sequence: 5' CGC ATA TTT GTA TAC GTG AGC TTC TTG
GCC AGC C 3'. The 5' end of the cuticlin EST nucleic acid molecule
was amplified by standard RT-PCR methods from D. immitis L3-48 hr
first-strand cDNA using the nematode 22-bp splice leader sequence,
referred to herein as SL1 (5' GGTTTAATTA CCCAAGTTTG AG 3'; SEQ ID
NO:13) and the EST-specific antisense primer (SEQ ID NO:12). The
RT-PCR reaction generated an 895 bp product. The composite full
length cDNA sequence of nDiCut-1A comprises a 1779 bp nucleic acid
molecule (the coding and complementary strands of which are herein
represented by SEQ ID NO:1 and SEQ ID NO:2, respectively).
nDiCut-1A encodes a 387 amino acid protein (herein referred to as
PDiCut-1A, represented by SEQ ID NO:4).
[0123] RT-PCR using SL1 (SEQ ID NO:13) and the EST-specific
antisense primer, CUT-5' R (SEQ ID NO:12) was also carried out
using Brugia malayi adult female cDNA as a template. This reaction
resulted in a partial 5' end product of a Brugia cutielin homolog,
referred to herein as nBmcut-1A (the coding strand and reverse
complement of which are herein represented by SEQ ID NO:16 and SEQ
ID NO:18, respectively). nBmCut-1A encodes a 245 amino acid protein
(herein referred to as PBmCut-1A, represented by SEQ ID NO:17).
[0124] In order to confirm the sequence of the 3' nDiCut-1A EST
fragment, 3' RACE PCR was performed using a Marathon.TM. cDNA
Amplification Kit (available from CLONTECH) according to the
manufacturer's instructions. The template for amplification was D.
immitis adult female first-strand cDNA and amplification was
performed using the EST-specific sense primer, CUT-3'R (SEQ ID
NO:11) and an antisense RACE-adapter primer (5'CCA TCC TAA TAC GAC
TCA CTA TAG GGC 3', referred to herein as SEQ ID NO:14). Instead of
obtaining a product of the predicted size of 919 bp (as would be
expected if the amplified product represented nDiCut-1A sequence),
a 643 bp nucleic acid molecule was obtained. This molecule
represented the 3' end of an additional D. immitis cuticlin nucleic
acid molecule referred to herein as nDiCut-1B. The 3' sequence of
nDiCut-1B was very different from the sequence already determined
for the 3' end of nDiCut-1A. The 5' end of nDiCut-1B was amplified
by SL1 RT-PCR using an nDiCut-1B specific antisense primer. This
DiCut-1B primer, referred to as CUTb. consists of the sequence: GGT
TAT ATC AAC CGT GCT AAA ACC GGT ACT GAC GTC CAC CG (herein referred
to as SEQ ID NO:15), and represents the nucleic acid sequence
located between base pairs 981 and 1020 of the essentially
full-length nDiCut-1B cDNA sequence (SEQ ID NO:6). RT-PCR using
CUTb and SL1 as primers generated a 1020 bp nDiCut-1B sequence when
either larval or adult first-strand cDNTA was used as the template.
The composite full length cDNA sequence of nDiCut-1B comprises 1372
bp, herein represented by SEQ ID NO:6 (the coding strand) and SEQ
ID NO:7 (the reverse complement). nDiCut-1B encodes a 271 amino
acids (herein referred to as PDiCut-1B, represented by SEQ ID
NO:9).
[0125] RT-PCR reactions were carried out on total RNA prepared from
0-hr L3, 48-hr L3, 6-day L4, male and female adult worms using
cuticlin-specific primers. The results indicate that gene
expression for both isoforms of cuticlin were up-regulated prior to
the molt, with maximal transcription at 48 hr-L3 and minimal
expression at 0 hr and 6-day L4. There was detectable expression of
both genes in male and female adult worms.
[0126] A homology search of a non-redundant protein database was
performed with SEQ ID NO:4, using the BLASTX program available
through the BLAST.TM. network of the National Center for
Biotechnology Information (NCBI) (National Library of Medicine,
National Institutes of Health, Baltimore, Md.), available on the
World Wide Web. This analysis showed that DiCut-1A had significant
homology to Ascaris cut-1 precursor at the amino acid level (bases
200 through 988 of SEQ ID NO:1 encode an amino acid sequence that
has 91% identity with the Ascaris cut-1 precursor), and nucleotides
434 through 880 of DiCut-1B encode an amino acid sequence that has
81% identity to the same Ascaris homolog.
[0127] Both Dirofilaria cuticlin cDNAs were expressed in a
.lambda.-cro expression vector. DiCut-1A was expressed as a 44.5 kD
histidine fusion protein, and DiCut-1B was expressed as a 31.1 kD
fusion protein. Antibodies raised to larval cuticles in rabbit and
sera from a rabbit immunized with trickle doses of L3 stage larvae
immunoreact with both forms of cuticlin.
[0128] DiCut-1A and DiCut-1B cDNAs were used to probe Southern
blots of genomic DNA from adult D. immitis. Both cDNAs hybridized
to two almost identical genomic fragments suggesting that DiCut-1A
and DiCut-1B are each encoded by a single copy gene. Because
DiCut-1A and DiCut-1B are 75% identical at the nucleotide level, it
is likely that each cDNA may hybridize to the other gene, as was
seen in the present study. Interestingly, the DiCut-1A and DiCut-1B
cDNA probes, were not cut internally by EcoRI, but did hybridize to
a number of fragments in genomic DNA digested with EcoRI. These
results suggest the presence of introns within the cuticlin
genes.
[0129] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention, as set forth in the following claims.
Sequence CWU 1
1
18 1 1779 DNA Dirofilaria immitis 1 ggtttaatta cccaagtttg
aggtgtctat aacaccgact gcagcaacaa caacaaacaa 60 caaacaacaa
acaacaacaa cagcaataat aaccccatca agtggaggaa gaagacagga 120
agcaatctta gtttttctaa aaatcgaatt tactaaatct tctgaaatga tgattcgtct
180 tattgctttc tgtactacac ttattgcatt gtcttattcg attccggttg
acaatggtgt 240 cgaaggtgag ccagaaattg aatgtggacc aacttcaata
acaatcaatt ttaatacacg 300 taatgcattc gaaggacatg tttatgtgaa
aggtctttat gatcaagaag gttgccgtaa 360 tgatgaaggt ggacgtcaag
ttgccggaat ttcacttcca tttgattcat gcaatgttgc 420 gcgtacacga
tctctgaatc cacgtggtat ttttgtaaca acaactgttg tcatttcgtt 480
tcatccatta tttgttacca aagttgatcg tgcatatcga gtacaatgct tttacatgga
540 agctgataaa acagttagtg cacagattga ggtatctgaa atcacaactg
cttttcaaac 600 tcaaattgtc ccgatgccag tatgccgtta tgaaattttg
gatggtggac caaccggtca 660 accagttcaa tttgctatca ttggtcagcc
agtttatcat aaatggacat gcgattctga 720 aaccgttgat actttctgcg
cggttgtcca ttcctgcttt gtcgatgatg gtaacggtga 780 tactgtggaa
attctaaatg ctgatggatg tgctcttgat aaatatttgc taaataattt 840
ggaatatcca acagatttaa tggctggcca agaagctcac gtatacaaat atgcggatcg
900 atcacagctt ttctatcaat gccagatcag tattaccatt aaagaaccaa
atagcgaatg 960 tgttcgacca caatgttcag aaccacaagg attcggagct
gttaaaacag gtggtgccgc 1020 agcaaaacct gctgcagctg cgcaacttcg
tttactcaag aaaagatctg cagaaccgga 1080 gaatatcatt gatgtacgaa
ctgatatcaa cacccttgaa attagcgatg ataatcaagc 1140 tttgccagtt
gatttacgtc accgtgcact tctgcaacat aatggacaac ctgtaatact 1200
tgctgcagta caaaatggaa tctgcatgtc accatttggc ttctcaatgt ttatgggttt
1260 aagcattgca ttgattgctg ccgtcattat taccatttcg tttaaatttc
gtccaaatca 1320 gaaggcataa aaataatgtt agaatcatcg aagcaataat
aaaactgcca tatatattcg 1380 tttcttctta tcatccttct aataactaat
tttagctaac aaatatatag tatgtaggaa 1440 ataattactg taatacaata
agtgatattt tcatcaaaac ttcttctatc gcttttatag 1500 cttctgaaaa
gcttattcat tattcagtaa tcttttatat gcatactatt gtaaatgttt 1560
catcattagg ccatgaatag tttcgtttgt tattatcatc attatcaact tgtcctattt
1620 tattctaaca gtttatcatt tgtgataata tcacaaatta taccttgtat
tgcccaattt 1680 ttatgggcat catttcctat tctgtaaaca attcacttat
ttgcattatt gcaattaaaa 1740 agtatttcat ttgtgaaaaa aaaaaaaaaa
aaaaaaaaa 1779 2 1779 DNA Dirofilaria immitis 2 tttttttttt
tttttttttt ttttcacaaa tgaaatactt tttaattgca ataatgcaaa 60
taagtgaatt gtttacagaa taggaaatga tgcccataaa aattgggcaa tacaaggtat
120 aatttgtgat attatcacaa atgataaact gttagaataa aataggacaa
gttgataatg 180 atgataataa caaacgaaac tattcatggc ctaatgatga
aacatttaca atagtatgca 240 tataaaagat tactgaataa tgaataagct
tttcagaagc tataaaagcg atagaagaag 300 ttttgatgaa aatatcactt
attgtattac agtaattatt tcctacatac tatatatttg 360 ttagctaaaa
ttagttatta gaaggatgat aagaagaaac gaatatatat ggcagtttta 420
ttattgcttc gatgattcta acattatttt tatgccttct gatttggacg aaatttaaac
480 gaaatggtaa taatgacggc agcaatcaat gcaatgctta aacccataaa
cattgagaag 540 ccaaatggtg acatgcagat tccattttgt actgcagcaa
gtattacagg ttgtccatta 600 tgttgcagaa gtgcacggtg acgtaaatca
actggcaaag cttgattatc atcgctaatt 660 tcaagggtgt tgatatcagt
tcgtacatca atgatattct ccggttctgc agatcttttc 720 ttgagtaaac
gaagttgcgc agctgcagca ggttttgctg cggcaccacc tgttttaaca 780
gctccgaatc cttgtggttc tgaacattgt ggtcgaacac attcgctatt tggttcttta
840 atggtaatac tgatctggca ttgatagaaa agctgtgatc gatccgcata
tttgtatacg 900 tgagcttctt ggccagccat taaatctgtt ggatattcca
aattatttag caaatattta 960 tcaagagcac atccatcagc atttagaatt
tccacagtat caccgttacc atcatcgaca 1020 aagcaggaat ggacaaccgc
gcagaaagta tcaacggttt cagaatcgca tgtccattta 1080 tgataaactg
gctgaccaat gatagcaaat tgaactggtt gaccggttgg tccaccatcc 1140
aaaatttcat aacggcatac tggcatcggg acaatttgag tttgaaaagc agttgtgatt
1200 tcagatacct caatctgtgc actaactgtt ttatcagctt ccatgtaaaa
gcattgtact 1260 cgatatgcac gatcaacttt ggtaacaaat aatggatgaa
acgaaatgac aacagttgtt 1320 gttacaaaaa taccacgtgg attcagagat
cgtgtacgcg caacattgca tgaatcaaat 1380 ggaagtgaaa ttccggcaac
ttgacgtcca ccttcatcat tacggcaacc ttcttgatca 1440 taaagacctt
tcacataaac atgtccttcg aatgcattac gtgtattaaa attgattgtt 1500
attgaagttg gtccacattc aatttctggc tcaccttcga caccattgtc aaccggaatc
1560 gaataagaca atgcaataag tgtagtacag aaagcaataa gacgaatcat
catttcagaa 1620 gatttagtaa attcgatttt tagaaaaact aagattgctt
cctgtcttct tcctccactt 1680 gatggggtta ttattgctgt tgttgttgtt
tgttgtttgt tgtttgttgt tgttgctgca 1740 gtcggtgtta tagacacctc
aaacttgggt aattaaacc 1779 3 1161 DNA Dirofilaria immitis CDS
(1)..(1161) 3 atg atg att cgt ctt att gct ttc tgt act aca ctt att
gca ttg tct 48 Met Met Ile Arg Leu Ile Ala Phe Cys Thr Thr Leu Ile
Ala Leu Ser 1 5 10 15 tat tcg att ccg gtt gac aat ggt gtc gaa ggt
gag cca gaa att gaa 96 Tyr Ser Ile Pro Val Asp Asn Gly Val Glu Gly
Glu Pro Glu Ile Glu 20 25 30 tgt gga cca act tca ata aca atc aat
ttt aat aca cgt aat gca ttc 144 Cys Gly Pro Thr Ser Ile Thr Ile Asn
Phe Asn Thr Arg Asn Ala Phe 35 40 45 gaa gga cat gtt tat gtg aaa
ggt ctt tat gat caa gaa ggt tgc cgt 192 Glu Gly His Val Tyr Val Lys
Gly Leu Tyr Asp Gln Glu Gly Cys Arg 50 55 60 aat gat gaa ggt gga
cgt caa gtt gcc gga att tca ctt cca ttt gat 240 Asn Asp Glu Gly Gly
Arg Gln Val Ala Gly Ile Ser Leu Pro Phe Asp 65 70 75 80 tca tgc aat
gtt gcg cgt aca cga tct ctg aat cca cgt ggt att ttt 288 Ser Cys Asn
Val Ala Arg Thr Arg Ser Leu Asn Pro Arg Gly Ile Phe 85 90 95 gta
aca aca act gtt gtc att tcg ttt cat cca tta ttt gtt acc aaa 336 Val
Thr Thr Thr Val Val Ile Ser Phe His Pro Leu Phe Val Thr Lys 100 105
110 gtt gat cgt gca tat cga gta caa tgc ttt tac atg gaa gct gat aaa
384 Val Asp Arg Ala Tyr Arg Val Gln Cys Phe Tyr Met Glu Ala Asp Lys
115 120 125 aca gtt agt gca cag att gag gta tct gaa atc aca act gct
ttt caa 432 Thr Val Ser Ala Gln Ile Glu Val Ser Glu Ile Thr Thr Ala
Phe Gln 130 135 140 act caa att gtc ccg atg cca gta tgc cgt tat gaa
att ttg gat ggt 480 Thr Gln Ile Val Pro Met Pro Val Cys Arg Tyr Glu
Ile Leu Asp Gly 145 150 155 160 gga cca acc ggt caa cca gtt caa ttt
gct atc att ggt cag cca gtt 528 Gly Pro Thr Gly Gln Pro Val Gln Phe
Ala Ile Ile Gly Gln Pro Val 165 170 175 tat cat aaa tgg aca tgc gat
tct gaa acc gtt gat act ttc tgc gcg 576 Tyr His Lys Trp Thr Cys Asp
Ser Glu Thr Val Asp Thr Phe Cys Ala 180 185 190 gtt gtc cat tcc tgc
ttt gtc gat gat ggt aac ggt gat act gtg gaa 624 Val Val His Ser Cys
Phe Val Asp Asp Gly Asn Gly Asp Thr Val Glu 195 200 205 att cta aat
gct gat gga tgt gct ctt gat aaa tat ttg cta aat aat 672 Ile Leu Asn
Ala Asp Gly Cys Ala Leu Asp Lys Tyr Leu Leu Asn Asn 210 215 220 ttg
gaa tat cca aca gat tta atg gct ggc caa gaa gct cac gta tac 720 Leu
Glu Tyr Pro Thr Asp Leu Met Ala Gly Gln Glu Ala His Val Tyr 225 230
235 240 aaa tat gcg gat cga tca cag ctt ttc tat caa tgc cag atc agt
att 768 Lys Tyr Ala Asp Arg Ser Gln Leu Phe Tyr Gln Cys Gln Ile Ser
Ile 245 250 255 acc att aaa gaa cca aat agc gaa tgt gtt cga cca caa
tgt tca gaa 816 Thr Ile Lys Glu Pro Asn Ser Glu Cys Val Arg Pro Gln
Cys Ser Glu 260 265 270 cca caa gga ttc gga gct gtt aaa aca ggt ggt
gcc gca gca aaa cct 864 Pro Gln Gly Phe Gly Ala Val Lys Thr Gly Gly
Ala Ala Ala Lys Pro 275 280 285 gct gca gct gcg caa ctt cgt tta ctc
aag aaa aga tct gca gaa ccg 912 Ala Ala Ala Ala Gln Leu Arg Leu Leu
Lys Lys Arg Ser Ala Glu Pro 290 295 300 gag aat atc att gat gta cga
act gat atc aac acc ctt gaa att agc 960 Glu Asn Ile Ile Asp Val Arg
Thr Asp Ile Asn Thr Leu Glu Ile Ser 305 310 315 320 gat gat aat caa
gct ttg cca gtt gat tta cgt cac cgt gca ctt ctg 1008 Asp Asp Asn
Gln Ala Leu Pro Val Asp Leu Arg His Arg Ala Leu Leu 325 330 335 caa
cat aat gga caa cct gta ata ctt gct gca gta caa aat gga atc 1056
Gln His Asn Gly Gln Pro Val Ile Leu Ala Ala Val Gln Asn Gly Ile 340
345 350 tgc atg tca cca ttt ggc ttc tca atg ttt atg ggt tta agc att
gca 1104 Cys Met Ser Pro Phe Gly Phe Ser Met Phe Met Gly Leu Ser
Ile Ala 355 360 365 ttg att gct gcc gtc att att acc att tcg ttt aaa
ttt cgt cca aat 1152 Leu Ile Ala Ala Val Ile Ile Thr Ile Ser Phe
Lys Phe Arg Pro Asn 370 375 380 cag aag gca 1161 Gln Lys Ala 385 4
387 PRT Dirofilaria immitis 4 Met Met Ile Arg Leu Ile Ala Phe Cys
Thr Thr Leu Ile Ala Leu Ser 1 5 10 15 Tyr Ser Ile Pro Val Asp Asn
Gly Val Glu Gly Glu Pro Glu Ile Glu 20 25 30 Cys Gly Pro Thr Ser
Ile Thr Ile Asn Phe Asn Thr Arg Asn Ala Phe 35 40 45 Glu Gly His
Val Tyr Val Lys Gly Leu Tyr Asp Gln Glu Gly Cys Arg 50 55 60 Asn
Asp Glu Gly Gly Arg Gln Val Ala Gly Ile Ser Leu Pro Phe Asp 65 70
75 80 Ser Cys Asn Val Ala Arg Thr Arg Ser Leu Asn Pro Arg Gly Ile
Phe 85 90 95 Val Thr Thr Thr Val Val Ile Ser Phe His Pro Leu Phe
Val Thr Lys 100 105 110 Val Asp Arg Ala Tyr Arg Val Gln Cys Phe Tyr
Met Glu Ala Asp Lys 115 120 125 Thr Val Ser Ala Gln Ile Glu Val Ser
Glu Ile Thr Thr Ala Phe Gln 130 135 140 Thr Gln Ile Val Pro Met Pro
Val Cys Arg Tyr Glu Ile Leu Asp Gly 145 150 155 160 Gly Pro Thr Gly
Gln Pro Val Gln Phe Ala Ile Ile Gly Gln Pro Val 165 170 175 Tyr His
Lys Trp Thr Cys Asp Ser Glu Thr Val Asp Thr Phe Cys Ala 180 185 190
Val Val His Ser Cys Phe Val Asp Asp Gly Asn Gly Asp Thr Val Glu 195
200 205 Ile Leu Asn Ala Asp Gly Cys Ala Leu Asp Lys Tyr Leu Leu Asn
Asn 210 215 220 Leu Glu Tyr Pro Thr Asp Leu Met Ala Gly Gln Glu Ala
His Val Tyr 225 230 235 240 Lys Tyr Ala Asp Arg Ser Gln Leu Phe Tyr
Gln Cys Gln Ile Ser Ile 245 250 255 Thr Ile Lys Glu Pro Asn Ser Glu
Cys Val Arg Pro Gln Cys Ser Glu 260 265 270 Pro Gln Gly Phe Gly Ala
Val Lys Thr Gly Gly Ala Ala Ala Lys Pro 275 280 285 Ala Ala Ala Ala
Gln Leu Arg Leu Leu Lys Lys Arg Ser Ala Glu Pro 290 295 300 Glu Asn
Ile Ile Asp Val Arg Thr Asp Ile Asn Thr Leu Glu Ile Ser 305 310 315
320 Asp Asp Asn Gln Ala Leu Pro Val Asp Leu Arg His Arg Ala Leu Leu
325 330 335 Gln His Asn Gly Gln Pro Val Ile Leu Ala Ala Val Gln Asn
Gly Ile 340 345 350 Cys Met Ser Pro Phe Gly Phe Ser Met Phe Met Gly
Leu Ser Ile Ala 355 360 365 Leu Ile Ala Ala Val Ile Ile Thr Ile Ser
Phe Lys Phe Arg Pro Asn 370 375 380 Gln Lys Ala 385 5 1161 DNA
Dirofilaria immitis 5 tgccttctga tttggacgaa atttaaacga aatggtaata
atgacggcag caatcaatgc 60 aatgcttaaa cccataaaca ttgagaagcc
aaatggtgac atgcagattc cattttgtac 120 tgcagcaagt attacaggtt
gtccattatg ttgcagaagt gcacggtgac gtaaatcaac 180 tggcaaagct
tgattatcat cgctaatttc aagggtgttg atatcagttc gtacatcaat 240
gatattctcc ggttctgcag atcttttctt gagtaaacga agttgcgcag ctgcagcagg
300 ttttgctgcg gcaccacctg ttttaacagc tccgaatcct tgtggttctg
aacattgtgg 360 tcgaacacat tcgctatttg gttctttaat ggtaatactg
atctggcatt gatagaaaag 420 ctgtgatcga tccgcatatt tgtatacgtg
agcttcttgg ccagccatta aatctgttgg 480 atattccaaa ttatttagca
aatatttatc aagagcacat ccatcagcat ttagaatttc 540 cacagtatca
ccgttaccat catcgacaaa gcaggaatgg acaaccgcgc agaaagtatc 600
aacggtttca gaatcgcatg tccatttatg ataaactggc tgaccaatga tagcaaattg
660 aactggttga ccggttggtc caccatccaa aatttcataa cggcatactg
gcatcgggac 720 aatttgagtt tgaaaagcag ttgtgatttc agatacctca
atctgtgcac taactgtttt 780 atcagcttcc atgtaaaagc attgtactcg
atatgcacga tcaactttgg taacaaataa 840 tggatgaaac gaaatgacaa
cagttgttgt tacaaaaata ccacgtggat tcagagatcg 900 tgtacgcgca
acattgcatg aatcaaatgg aagtgaaatt ccggcaactt gacgtccacc 960
ttcatcatta cggcaacctt cttgatcata aagacctttc acataaacat gtccttcgaa
1020 tgcattacgt gtattaaaat tgattgttat tgaagttggt ccacattcaa
tttctggctc 1080 accttcgaca ccattgtcaa ccggaatcga ataagacaat
gcaataagtg tagtacagaa 1140 agcaataaga cgaatcatca t 1161 6 1372 DNA
Dirofilaria immitis 6 ggtttaatta cccaagtttg aggcacatgc aattatcatt
attctccttg ttattcctac 60 ttttctactt gacctacgta tcatcgatcc
ctattgacaa tggtgtcgaa ggtgaacctg 120 aaatagaatg tggcgcagct
tcgataacaa tcaatttcaa tactagaaat acatttgaag 180 gacacgtata
tgtaaaagga ctctatgatc aggatgaatg tcgttcagat agtaatggac 240
ggcaggtagc tggaatcgaa ttggcaatgg attcgtgtaa tgttgaacga tcacgatcct
300 taaatcctcg tggtgttttt gtaacaactg tagttgtcat tacatttcat
ccaaaattcg 360 ttacaaaaat agatcgagca tatcgtatac aatgttttta
tatggaaagc tgataagacc 420 gttagtactg gtccttgaag tatctgaaat
gactacagca ttccaaactc aagtggtacc 480 aatgcccgta tgtcgatatg
agattttgga aggtggacca actggtgcac ctgttcgatt 540 tgcaatgatt
ggagatcatg tatatcacaa atggacatgt gattcagaga ctacagatac 600
attctgtgca ttagtacatt catgtgttgt ggatgatgga aaaggtgatg cagtggagat
660 tctgaatgaa gaaggatgtg ctttggacaa atatttactc aataatttgg
aatatattac 720 agatttaatg gctggccaag aagctcatgt ttataaatat
gcagatcgat cagaacttta 780 ctatcaatgc cagattagta taacaattaa
agagccacat agcgaatgtc ctcgaccaca 840 atgcacagag ccacaaggat
ttggtgccat aaaatctgga caaggatttg ctgctgtaaa 900 atctgctgct
gcaccagctc cagaagcttc cttgctttct ccacgattga tcaagaagcg 960
atcaattaat tctgataata cggtggacgt cagtaccggt tttagcacgg ttgatataac
1020 cgaagagaat ccgaacttct cagcaaatcg tttatcatca tcaacgagcc
gtgaacaatt 1080 caatggtatc ttctgtattg catcaaatga tattttactt
atcattttgt tcggtgctat 1140 gttagctatt gcttgcatat tttttaccgc
ttttcttgtt cattccaata atcattctaa 1200 atcatagttc tattcgatct
tatcaataat tcttaccggt ttcgagattt tagaagagag 1260 agagagagaa
agagagaaag agagggaaag agagaaagag agagaaagag agagagagag 1320
agaagaaaaa agtactcgga tatttcaaaa aaaaaaaaaa aaaaaaaaaa aa 1372 7
1372 DNA Dirofilaria immitis 7 tttttttttt tttttttttt ttttttgaaa
tatccgagta cttttttctt ctctctctct 60 ctctctttct ctctctttct
ctctttccct ctctttctct ctttctctct ctctctcttc 120 taaaatctcg
aaaccggtaa gaattattga taagatcgaa tagaactatg atttagaatg 180
attattggaa tgaacaagaa aagcggtaaa aaatatgcaa gcaatagcta acatagcacc
240 gaacaaaatg ataagtaaaa tatcatttga tgcaatacag aagataccat
tgaattgttc 300 acggctcgtt gatgatgata aacgatttgc tgagaagttc
ggattctctt cggttatatc 360 aaccgtgcta aaaccggtac tgacgtccac
cgtattatca gaattaattg atcgcttctt 420 gatcaatcgt ggagaaagca
aggaagcttc tggagctggt gcagcagcag attttacagc 480 agcaaatcct
tgtccagatt ttatggcacc aaatccttgt ggctctgtgc attgtggtcg 540
aggacattcg ctatgtggct ctttaattgt tatactaatc tggcattgat agtaaagttc
600 tgatcgatct gcatatttat aaacatgagc ttcttggcca gccattaaat
ctgtaatata 660 ttccaaatta ttgagtaaat atttgtccaa agcacatcct
tcttcattca gaatctccac 720 tgcatcacct tttccatcat ccacaacaca
tgaatgtact aatgcacaga atgtatctgt 780 agtctctgaa tcacatgtcc
atttgtgata tacatgatct ccaatcattg caaatcgaac 840 aggtgcacca
gttggtccac cttccaaaat ctcatatcga catacgggca ttggtaccac 900
ttgagtttgg aatgctgtag tcatttcaga tacttcaagg accagtacta acggtcttat
960 cagctttcca tataaaaaca ttgtatacga tatgctcgat ctatttttgt
aacgaatttt 1020 ggatgaaatg taatgacaac tacagttgtt acaaaaacac
cacgaggatt taaggatcgt 1080 gatcgttcaa cattacacga atccattgcc
aattcgattc cagctacctg ccgtccatta 1140 ctatctgaac gacattcatc
ctgatcatag agtcctttta catatacgtg tccttcaaat 1200 gtatttctag
tattgaaatt gattgttatc gaagctgcgc cacattctat ttcaggttca 1260
ccttcgacac cattgtcaat agggatcgat gatacgtagg tcaagtagaa aagtaggaat
1320 aacaaggaga ataatgataa ttgcatgtgc ctcaaacttg ggtaattaaa cc 1372
8 813 DNA Dirofilaria immitis CDS (1)..(813) 8 atg ttt tta tat gga
aag ctg ata aga ccg tta gta ctg gtc ctt gaa 48 Met Phe Leu Tyr Gly
Lys Leu Ile Arg Pro Leu Val Leu Val Leu Glu 1 5 10 15 gta tct gaa
atg act aca gca ttc caa act caa gtg gta cca atg ccc 96 Val Ser Glu
Met Thr Thr Ala Phe Gln Thr Gln Val Val Pro Met Pro 20 25 30 gta
tgt cga tat gag att ttg gaa ggt gga cca act ggt gca cct gtt 144 Val
Cys Arg Tyr Glu Ile Leu Glu Gly Gly Pro Thr Gly Ala Pro Val 35 40
45 cga ttt gca atg att gga gat cat gta tat cac aaa tgg aca tgt gat
192 Arg Phe Ala Met Ile Gly Asp His Val Tyr His Lys Trp Thr Cys Asp
50 55 60 tca gag act aca gat aca ttc tgt gca tta gta cat tca tgt
gtt gtg 240 Ser Glu Thr Thr Asp Thr Phe Cys Ala Leu Val His Ser Cys
Val Val 65 70 75 80 gat gat gga aaa ggt gat gca gtg gag att ctg aat
gaa gaa gga tgt 288 Asp Asp Gly Lys Gly Asp Ala Val Glu Ile Leu Asn
Glu Glu Gly Cys 85 90 95 gct ttg gac aaa tat tta ctc aat aat ttg
gaa tat att aca gat tta
336 Ala Leu Asp Lys Tyr Leu Leu Asn Asn Leu Glu Tyr Ile Thr Asp Leu
100 105 110 atg gct ggc caa gaa gct cat gtt tat aaa tat gca gat cga
tca gaa 384 Met Ala Gly Gln Glu Ala His Val Tyr Lys Tyr Ala Asp Arg
Ser Glu 115 120 125 ctt tac tat caa tgc cag att agt ata aca att aaa
gag cca cat agc 432 Leu Tyr Tyr Gln Cys Gln Ile Ser Ile Thr Ile Lys
Glu Pro His Ser 130 135 140 gaa tgt cct cga cca caa tgc aca gag cca
caa gga ttt ggt gcc ata 480 Glu Cys Pro Arg Pro Gln Cys Thr Glu Pro
Gln Gly Phe Gly Ala Ile 145 150 155 160 aaa tct gga caa gga ttt gct
gct gta aaa tct gct gct gca cca gct 528 Lys Ser Gly Gln Gly Phe Ala
Ala Val Lys Ser Ala Ala Ala Pro Ala 165 170 175 cca gaa gct tcc ttg
ctt tct cca cga ttg atc aag aag cga tca att 576 Pro Glu Ala Ser Leu
Leu Ser Pro Arg Leu Ile Lys Lys Arg Ser Ile 180 185 190 aat tct gat
aat acg gtg gac gtc agt acc ggt ttt agc acg gtt gat 624 Asn Ser Asp
Asn Thr Val Asp Val Ser Thr Gly Phe Ser Thr Val Asp 195 200 205 ata
acc gaa gag aat ccg aac ttc tca gca aat cgt tta tca tca tca 672 Ile
Thr Glu Glu Asn Pro Asn Phe Ser Ala Asn Arg Leu Ser Ser Ser 210 215
220 acg agc cgt gaa caa ttc aat ggt atc ttc tgt att gca tca aat gat
720 Thr Ser Arg Glu Gln Phe Asn Gly Ile Phe Cys Ile Ala Ser Asn Asp
225 230 235 240 att tta ctt atc att ttg ttc ggt gct atg tta gct att
gct tgc ata 768 Ile Leu Leu Ile Ile Leu Phe Gly Ala Met Leu Ala Ile
Ala Cys Ile 245 250 255 ttt ttt acc gct ttt ctt gtt cat tcc aat aat
cat tct aaa tca 813 Phe Phe Thr Ala Phe Leu Val His Ser Asn Asn His
Ser Lys Ser 260 265 270 9 271 PRT Dirofilaria immitis 9 Met Phe Leu
Tyr Gly Lys Leu Ile Arg Pro Leu Val Leu Val Leu Glu 1 5 10 15 Val
Ser Glu Met Thr Thr Ala Phe Gln Thr Gln Val Val Pro Met Pro 20 25
30 Val Cys Arg Tyr Glu Ile Leu Glu Gly Gly Pro Thr Gly Ala Pro Val
35 40 45 Arg Phe Ala Met Ile Gly Asp His Val Tyr His Lys Trp Thr
Cys Asp 50 55 60 Ser Glu Thr Thr Asp Thr Phe Cys Ala Leu Val His
Ser Cys Val Val 65 70 75 80 Asp Asp Gly Lys Gly Asp Ala Val Glu Ile
Leu Asn Glu Glu Gly Cys 85 90 95 Ala Leu Asp Lys Tyr Leu Leu Asn
Asn Leu Glu Tyr Ile Thr Asp Leu 100 105 110 Met Ala Gly Gln Glu Ala
His Val Tyr Lys Tyr Ala Asp Arg Ser Glu 115 120 125 Leu Tyr Tyr Gln
Cys Gln Ile Ser Ile Thr Ile Lys Glu Pro His Ser 130 135 140 Glu Cys
Pro Arg Pro Gln Cys Thr Glu Pro Gln Gly Phe Gly Ala Ile 145 150 155
160 Lys Ser Gly Gln Gly Phe Ala Ala Val Lys Ser Ala Ala Ala Pro Ala
165 170 175 Pro Glu Ala Ser Leu Leu Ser Pro Arg Leu Ile Lys Lys Arg
Ser Ile 180 185 190 Asn Ser Asp Asn Thr Val Asp Val Ser Thr Gly Phe
Ser Thr Val Asp 195 200 205 Ile Thr Glu Glu Asn Pro Asn Phe Ser Ala
Asn Arg Leu Ser Ser Ser 210 215 220 Thr Ser Arg Glu Gln Phe Asn Gly
Ile Phe Cys Ile Ala Ser Asn Asp 225 230 235 240 Ile Leu Leu Ile Ile
Leu Phe Gly Ala Met Leu Ala Ile Ala Cys Ile 245 250 255 Phe Phe Thr
Ala Phe Leu Val His Ser Asn Asn His Ser Lys Ser 260 265 270 10 813
DNA Dirofilaria immitis 10 tgatttagaa tgattattgg aatgaacaag
aaaagcggta aaaaatatgc aagcaatagc 60 taacatagca ccgaacaaaa
tgataagtaa aatatcattt gatgcaatac agaagatacc 120 attgaattgt
tcacggctcg ttgatgatga taaacgattt gctgagaagt tcggattctc 180
ttcggttata tcaaccgtgc taaaaccggt actgacgtcc accgtattat cagaattaat
240 tgatcgcttc ttgatcaatc gtggagaaag caaggaagct tctggagctg
gtgcagcagc 300 agattttaca gcagcaaatc cttgtccaga ttttatggca
ccaaatcctt gtggctctgt 360 gcattgtggt cgaggacatt cgctatgtgg
ctctttaatt gttatactaa tctggcattg 420 atagtaaagt tctgatcgat
ctgcatattt ataaacatga gcttcttggc cagccattaa 480 atctgtaata
tattccaaat tattgagtaa atatttgtcc aaagcacatc cttcttcatt 540
cagaatctcc actgcatcac cttttccatc atccacaaca catgaatgta ctaatgcaca
600 gaatgtatct gtagtctctg aatcacatgt ccatttgtga tatacatgat
ctccaatcat 660 tgcaaatcga acaggtgcac cagttggtcc accttccaaa
atctcatatc gacatacggg 720 cattggtacc acttgagttt ggaatgctgt
agtcatttca gatacttcaa ggaccagtac 780 taacggtctt atcagctttc
catataaaaa cat 813 11 34 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Primer 11 ggctggccaa gaagctcacg
tatacaaata tgcg 34 12 34 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Primer 12 cgcatatttg tatacgtgag
cttcttggcc agcc 34 13 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Primer 13 ggtttaatta cccaagtttg ag 22
14 27 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 14 ccatcctaat acgactcact atagggc 27 15 41 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Primer 15 ggttatatca accgtgctaa aaccggtact gacgtccacc g 41 16 892
DNA Brugia malayi CDS (158)..(892) 16 ggtttaatta cccaagtttg
agatcattaa aattgatcat caataattca ataatttgtt 60 gcaatttcaa
attaatcatt ttgctaattc tattattcca actattttca tcactaatca 120
ctgagaagaa atcaggaaga aagaagcaaa aagttaa atg ttg cat atg caa att
175 Met Leu His Met Gln Ile 1 5 tgc tca ttt ttg tca tat atg ata ata
gca agt att aat gct att cca 223 Cys Ser Phe Leu Ser Tyr Met Ile Ile
Ala Ser Ile Asn Ala Ile Pro 10 15 20 att gat aat ggt gtc gaa agt
gaa cct gaa att gaa tgt ggt cca aca 271 Ile Asp Asn Gly Val Glu Ser
Glu Pro Glu Ile Glu Cys Gly Pro Thr 25 30 35 tca atc act gtt aat
ttt aat act cga aat cct ttt gaa gga cat gta 319 Ser Ile Thr Val Asn
Phe Asn Thr Arg Asn Pro Phe Glu Gly His Val 40 45 50 tat gct aaa
gga tta tac agt aat caa gat tgt cgt agt gat gaa ggt 367 Tyr Ala Lys
Gly Leu Tyr Ser Asn Gln Asp Cys Arg Ser Asp Glu Gly 55 60 65 70 gga
cgt cag gta gcc gga ata tca tta ccg ttt gat tca tgt aat gtc 415 Gly
Arg Gln Val Ala Gly Ile Ser Leu Pro Phe Asp Ser Cys Asn Val 75 80
85 gca cgt aca cgt tcg tta aat cca cgt gga ata ttt gtc aca gct gtt
463 Ala Arg Thr Arg Ser Leu Asn Pro Arg Gly Ile Phe Val Thr Ala Val
90 95 100 gtg gta att acg ttt cat cca cag ttt atc aca aaa gtt gat
cga aca 511 Val Val Ile Thr Phe His Pro Gln Phe Ile Thr Lys Val Asp
Arg Thr 105 110 115 tat cga ttg caa tgc ttt tac atg gaa gct gat aag
act gtt agc aca 559 Tyr Arg Leu Gln Cys Phe Tyr Met Glu Ala Asp Lys
Thr Val Ser Thr 120 125 130 caa att gaa gtt tcc gaa atg aca acc gta
ttt gct aca caa ttg gta 607 Gln Ile Glu Val Ser Glu Met Thr Thr Val
Phe Ala Thr Gln Leu Val 135 140 145 150 cca atg cct gtg tgt aga tat
gag att ctg gat ggt ggt cca acc gga 655 Pro Met Pro Val Cys Arg Tyr
Glu Ile Leu Asp Gly Gly Pro Thr Gly 155 160 165 caa cct gtc cag tat
gct aat att gga caa ccg gtt tat cat aaa tgg 703 Gln Pro Val Gln Tyr
Ala Asn Ile Gly Gln Pro Val Tyr His Lys Trp 170 175 180 aca tgt gat
tct gaa aca gtt gat acc ttc tgt gct ttg gta cat tcc 751 Thr Cys Asp
Ser Glu Thr Val Asp Thr Phe Cys Ala Leu Val His Ser 185 190 195 tgt
ttt gtt gat gat ggc aat ggt gac agt att aat tta att aat gaa 799 Cys
Phe Val Asp Asp Gly Asn Gly Asp Ser Ile Asn Leu Ile Asn Glu 200 205
210 gaa gga tgt gca tta gat cga tat ctt cta aat aat ttg gaa tat cca
847 Glu Gly Cys Ala Leu Asp Arg Tyr Leu Leu Asn Asn Leu Glu Tyr Pro
215 220 225 230 act gat cta atg gct ggc caa gaa gct cac gta tac aaa
tat gcg 892 Thr Asp Leu Met Ala Gly Gln Glu Ala His Val Tyr Lys Tyr
Ala 235 240 245 17 245 PRT Brugia malayi 17 Met Leu His Met Gln Ile
Cys Ser Phe Leu Ser Tyr Met Ile Ile Ala 1 5 10 15 Ser Ile Asn Ala
Ile Pro Ile Asp Asn Gly Val Glu Ser Glu Pro Glu 20 25 30 Ile Glu
Cys Gly Pro Thr Ser Ile Thr Val Asn Phe Asn Thr Arg Asn 35 40 45
Pro Phe Glu Gly His Val Tyr Ala Lys Gly Leu Tyr Ser Asn Gln Asp 50
55 60 Cys Arg Ser Asp Glu Gly Gly Arg Gln Val Ala Gly Ile Ser Leu
Pro 65 70 75 80 Phe Asp Ser Cys Asn Val Ala Arg Thr Arg Ser Leu Asn
Pro Arg Gly 85 90 95 Ile Phe Val Thr Ala Val Val Val Ile Thr Phe
His Pro Gln Phe Ile 100 105 110 Thr Lys Val Asp Arg Thr Tyr Arg Leu
Gln Cys Phe Tyr Met Glu Ala 115 120 125 Asp Lys Thr Val Ser Thr Gln
Ile Glu Val Ser Glu Met Thr Thr Val 130 135 140 Phe Ala Thr Gln Leu
Val Pro Met Pro Val Cys Arg Tyr Glu Ile Leu 145 150 155 160 Asp Gly
Gly Pro Thr Gly Gln Pro Val Gln Tyr Ala Asn Ile Gly Gln 165 170 175
Pro Val Tyr His Lys Trp Thr Cys Asp Ser Glu Thr Val Asp Thr Phe 180
185 190 Cys Ala Leu Val His Ser Cys Phe Val Asp Asp Gly Asn Gly Asp
Ser 195 200 205 Ile Asn Leu Ile Asn Glu Glu Gly Cys Ala Leu Asp Arg
Tyr Leu Leu 210 215 220 Asn Asn Leu Glu Tyr Pro Thr Asp Leu Met Ala
Gly Gln Glu Ala His 225 230 235 240 Val Tyr Lys Tyr Ala 245 18 892
DNA Brugia malayi 18 cgcatatttg tatacgtgag cttcttggcc agccattaga
tcagttggat attccaaatt 60 atttagaaga tatcgatcta atgcacatcc
ttcttcatta attaaattaa tactgtcacc 120 attgccatca tcaacaaaac
aggaatgtac caaagcacag aaggtatcaa ctgtttcaga 180 atcacatgtc
catttatgat aaaccggttg tccaatatta gcatactgga caggttgtcc 240
ggttggacca ccatccagaa tctcatatct acacacaggc attggtacca attgtgtagc
300 aaatacggtt gtcatttcgg aaacttcaat ttgtgtgcta acagtcttat
cagcttccat 360 gtaaaagcat tgcaatcgat atgttcgatc aacttttgtg
ataaactgtg gatgaaacgt 420 aattaccaca acagctgtga caaatattcc
acgtggattt aacgaacgtg tacgtgcgac 480 attacatgaa tcaaacggta
atgatattcc ggctacctga cgtccacctt catcactacg 540 acaatcttga
ttactgtata atcctttagc atatacatgt ccttcaaaag gatttcgagt 600
attaaaatta acagtgattg atgttggacc acattcaatt tcaggttcac tttcgacacc
660 attatcaatt ggaatagcat taatacttgc tattatcata tatgacaaaa
atgagcaaat 720 ttgcatatgc aacatttaac tttttgcttc tttcttcctg
atttcttctc agtgattagt 780 gatgaaaata gttggaataa tagaattagc
aaaatgatta atttgaaatt gcaacaaatt 840 attgaattat tgatgatcaa
ttttaatgat ctcaaacttg ggtaattaaa cc 892
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