U.S. patent application number 09/774377 was filed with the patent office on 2001-09-13 for anti-parasitic helminth macrophage migration inhibitory factor antibodies and uses thereof.
This patent application is currently assigned to Heska Corporation.. Invention is credited to Brandt, Kevin S., Tripp, Cynthia Ann, Wisnewski, Nancy.
Application Number | 20010021517 09/774377 |
Document ID | / |
Family ID | 24230763 |
Filed Date | 2001-09-13 |
United States Patent
Application |
20010021517 |
Kind Code |
A1 |
Tripp, Cynthia Ann ; et
al. |
September 13, 2001 |
Anti-parasitic helminth macrophage migration inhibitory factor
antibodies and uses thereof
Abstract
The present invention relates to parasitic helminth macrophage
migration inhibitory factor (MIF) proteins; to parasitic helminth
MIF nucleic acid molecules, including those that encode such MIF
proteins; to antibodies raised against such MIF proteins; and to
compounds that inhibit parasitic helminth MIF 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: |
Tripp, Cynthia Ann; (Ft.
Collins, CO) ; Brandt, Kevin S.; (Windsor, CO)
; Wisnewski, Nancy; (Ft. Collins, CO) |
Correspondence
Address: |
Angela Dallas-Pedretti
SHERIDAN ROSS P.C.
Suite 1200
1560 Broadway
Denver
CO
80202-5141
US
|
Assignee: |
Heska Corporation.
|
Family ID: |
24230763 |
Appl. No.: |
09/774377 |
Filed: |
January 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09774377 |
Jan 30, 2001 |
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08906408 |
Aug 5, 1997 |
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5808857 |
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08906408 |
Aug 5, 1997 |
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08558735 |
Nov 16, 1995 |
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5681724 |
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Current U.S.
Class: |
435/70.1 ;
424/265.1; 435/184; 435/235.1; 536/23.2 |
Current CPC
Class: |
A61P 33/00 20180101;
A61P 33/02 20180101; H01G 9/055 20130101; Y10S 435/975 20130101;
A61P 33/10 20180101; C07K 2319/00 20130101; C07K 14/52
20130101 |
Class at
Publication: |
435/70.1 ;
435/184; 424/265.1; 536/23.2; 435/235.1 |
International
Class: |
C12P 021/04; C07H
021/04; C12N 009/99; C12N 007/00 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule that hybridizes under
stringent hybridization conditions with a MIF gene selected from
the group consisting of a Dirofilaria immitis MIF gene and an
Onchocerca volvulus MIF gene.
2. The nucleic acid molecule of claim 1, wherein said D. immitis
MIF gene comprises a nucleic acid sequence selected from the group
consisting of SEQ ID NO:17 and SEQ ID NO:19, and wherein said O.
volvulus MIF gene comprises a nucleic acid sequence selected from
the group consisting of SEQ ID NO:6 and SEQ ID NO:9.
3. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule comprises a nucleic acid sequence that encodes a MIF
protein.
4. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule is selected from the group consisting of nematode, cestode
and trematode nucleic acid molecules.
5. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule is selected from the group consisting of filariid,
ascarid, strongyle and trichostrongyle nucleic acid molecules.
6. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule comprises a filariid nematode nucleic acid molecule.
7. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule is selected from the group consisting of D. immitis and O.
volvulus nucleic acid molecules.
8. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule hybridizes under stringent hybridization conditions with a
nucleic acid molecule selected from the group consisting of
nDiMIF(1).sub.532, nDiMIF(2).sub.532, nOvMIF(1).sub.440, and
nOvMIF(2).sub.522.
9. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule comprises a nucleic acid molecule selected from the group
consisting of nDiMIF(1).sub.532 nDiMIF(2).sub.532, nDiMIF.sub.282,
nDiMIF.sub.102, nDiMIF(1).sub.355, nDiMIF(2).sub.333,
nDiMIF(1).sub.345, nDiMIF(2).sub.330, nDiMIF(1).sub.348,
nBvDiMIF(1).sub.348, nRcnDiMIF(1).sub.348, nOvMIF(1).sub.440,
nOvMIF(2).sub.522, nOvMIF(1).sub.345, and nOvMIF(2).sub.342.
10. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule is selected from the group consisting of: a nucleic acid
molecule comprising a nucleic acid sequence, or the complement
thereof, selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and
SEQ ID NO:19; and a nucleic acid molecule comprising an allelic
variant of a nucleic acid molecule comprising a nucleic acid
sequence, or the complement thereof, selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16,
SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.
11. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule encodes a protein comprising an amino acid sequence that
is at least about 55% identical to an amino acid sequence selected
from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8,
and SEQ ID NO:11.
12. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule comprises an oligonucleotide.
13. The nucleic acid molecule of claim 3, wherein said protein,
when administered to an animal elicits an immune response against a
parasitic helminth MIF protein.
14. A recombinant molecule comprising a nucleic acid molecule as
set forth in claim 1 operatively linked to a transcription control
sequence.
15. A recombinant virus comprising a recombinant molecule as set
forth in claim 14.
16. A recombinant cell comprising a nucleic acid molecule as set
forth in claim 1, said cell being capable of expressing said
nucleic acid molecule.
17. An isolated protein comprising a parasitic helminth MIF
protein.
18. The protein of claim 17, wherein said protein is encoded by a
nucleic acid molecule that hybridizes under stringent hybridization
conditions to a gene selected from the group consisting of a
Dirofilaria immitis MIF gene and an Onchocerca volvulus MIF
gene.
19. The protein of claim 18, wherein said D. immitis MIF gene
comprises a nucleic acid sequence selected from the group
consisting of SEQ ID NO:17 and SEQ ID NO:19, and wherein said O.
volvulus MIF gene comprises a nucleic acid sequence selected from
the group consisting of SEQ ID NO:6 and SEQ ID NO:9.
20. The protein of claim 17, wherein said protein, when
administered to an animal, elicits an immune response against a
parasitic helminth MIF protein.
21. The protein of claim 17, wherein said helminth is selected from
the group consisting of nematodes, cestodes and trematodes.
22. The protein of claim 17, wherein said helminth comprises a
nematode selected from the group consisting of filariid, ascarid,
strongyle and trichostrongyle nematodes.
23. The protein of claim 17, wherein said helminth is a filariid
nematode.
24. The protein of claim 17, wherein said helminth is selected from
the group consisting of D. immitis and O. volvulus.
25. The protein of claim 17, wherein said protein is encoded by a
nucleic acid molecule that hybridizes under stringent hybridization
conditions with a nucleic acid molecule selected from the group
consisting of nDiMIF(1).sub.345, nDiMIF(2).sub.330,
nOvMIF(1).sub.345, and nOvMIF(2).sub.342.
26. The protein of claim 17, wherein said protein comprises an
amino acid sequence that is at least about 55% identical to an
amino acid sequence selected from the group consisting of SEQ ID
NO:2, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:11.
27. The protein of claim 17, wherein said protein is selected from
the group consisting of a protein comprising amino acid sequence
SEQ ID NO:2, a protein comprising amino acid sequence SEQ ID NO:5,
a protein comprising amino acid sequence SEQ ID NO:8, a protein
comprising amino acid sequence SEQ ID NO:11, and a protein encoded
by an allelic variant of a nucleic acid molecule encoding a protein
comprising any of said amino acid sequences.
28. An isolated antibody that selectively binds to a protein as set
forth in claim 17.
29. An isolated MIF protein encoded by a nucleic acid molecule that
hybridizes under stringent hybridization conditions to a gene
selected from the group consisting of a Dirofilaria immitis MIF
gene and an Onchocerca volvulus MIF gene.
30. A therapeutic composition to protect an animal from disease
caused by a parasitic helminth, said therapeutic composition
comprising a protective compound selected from the group consisting
of an isolated parasitic helminth MIF protein or a mimetope
thereof, an isolated nucleic acid molecule that hybridizes under
stringent hybridization conditions with a gene selected from the
group consisting of a D. immitis MIF gene and an O. volvulus MIF
gene, an isolated antibody that selectively binds to a parasitic
helminth MIF protein, an inhibitor of MIF protein activity
identified by its ability to inhibit parasitic helminth MIF
activity, and a mixture thereof.
31. The composition of claim 30, wherein said protective compound
is selected from the group consisting of a naked nucleic acid
vaccine, a recombinant virus vaccine and a recombinant cell
vaccine.
32. The composition of claim 30, wherein said composition further
comprises a component selected from the group consisting of an
excipient, an adjuvant, a carrier, and a mixture thereof.
33. The composition of claim 30, wherein said disease is selected
from the group consisting of heartworm and onchocerciasis.
34. A method to protect an animal from parasitic helminth disease,
said method comprising administering to said animal a therapeutic
composition comprising a protective compound selected from the
group consisting of an isolated parasitic helminth MIF protein or a
mimetope thereof, an isolated nucleic acid molecule that hybridizes
under stringent hybridization conditions with a gene selected from
the group consisting of a D. immitis MIF gene and an O. volvulus
MIF gene, an isolated antibody that selectively binds to a
parasitic helminth MIF protein, an inhibitor of MIF protein
activity identified by its ability to inhibit parasitic helminth
MIF activity, and a mixture thereof.
35. The method of claim 34, wherein said composition is selected
from the group consisting of a naked nucleic acid vaccine, a
recombinant virus vaccine and a recombinant cell vaccine.
36. The method of claim 34, wherein said composition further
comprises a component selected from the group consisting of an
excipient, an adjuvant, a carrier, and a mixture thereof.
37. The method of claim 34, wherein said disease is selected from
the group consisting of heartworm and onchocerciasis.
38. A method to produce a parasitic helminth MIF protein, said
method comprising culturing in an effective medium a cell capable
of expressing said protein, said protein being encoded by a nucleic
acid molecule that hybridizes under stringent hybridization
conditions with a gene selected from the group consisting of a D.
immitis MIF gene and an O. volvulus MIF gene.
39. A method to identify a compound capable of inhibiting MIF
activity of a parasitic helminth, said method comprising: (a)
contacting an isolated parasitic helminth MIF protein with a
putative inhibitory compound under conditions in which, in the
absence of said compound, said protein has MIF activity; and (b)
determining if said putative inhibitory compound inhibits said
activity.
40. A test kit to identify a compound capable of inhibiting MIF
activity of a parasitic helminth, said test kit comprising an
isolated parasitic helminth MIF protein having MIF activity and a
means for determining the extent of inhibition of said activity in
the presence of a putative inhibitory compound.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to parasitic helminth
macrophage migration inhibitory factor (MIF) 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, and/or inhibitors, as well as their use to protect
animals from diseases caused by parasitic helminths, such as
heartworm or onchocerciasis.
BACKGROUND OF THE INVENTION
[0002] Parasitic helminth infections in animals, including humans,
are typically treated by chemical drugs, because there are
essentially no efficacious vaccines available. One disadvantage
with chemical drugs is that they must be administered often. For
example, dogs susceptible to heartworm are typically treated
monthly to maintain protective drug levels. Repeated administration
of drugs to treat parasitic helminth infections, 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] It is particularly difficult to develop vaccines against
parasitic helminth infections both because of the complexity of the
parasite's life cycle and because, while administration of
parasites or parasite antigens can lead to the production of a
significant antibody response, the immune response is typically not
sufficient to protect the animal against infection.
[0004] As an example of the complexity of parasitic helminths, the
life cycle of D. immitis, the helminth that causes heartworm,
includes a variety of life forms, each of which presents different
targets, and challenges, for immunization. Adult forms of the
parasite 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. One method of
demonstrating infection in the dog is to detect the circulating
microfilariae.
[0005] If the dog is maintained in an insect-free environment, the
life cycle of the parasite cannot progress. However, when
microfilariae are ingested by the female mosquito during blood
feeding on an infected dog, subsequent development of the
microfilariae into larvae occurs in the mosquito. The microfilariae
go through two larval stages (L1 and L2) and finally become mature
third stage larvae (L3) which can then be transmitted back to the
dog through the bite of the mosquito. It is this L3 stage,
therefore, that accounts for the initial infection. As early as
three days after infection, the L3 molt to the fourth larval (L4)
stage, and subsequently to the fifth stage, or immature adults. The
immature adults migrate to the heart and pulmonary arteries, where
they mature and reproduce, thus producing the microfilariae in the
blood. "Occult" infection with heartworm in dogs is defined as that
wherein no microfilariae can be detected, but the existence of the
adult heartworms can be determined through thoracic
examination.
[0006] Heartworm not only is a major problem in dogs, which
typically cannot even develop immunity upon infection (i.e., dogs
can become reinfected even after being cured by chemotherapy), but
is also becoming increasingly widespread in other companion
animals, such as cats and ferrets. Heartworm infections have also
been reported in humans. Other parasitic helminthic infections are
also widespread, and all require better treatment, including a
preventative vaccine program. O. volvulus, for example, causes
onchocerciasis (also known as river blindness) in humans. Up to 50
million people throughout the world are reported to be infected
with O. volvulus, with over a million being blinded due to
infection.
[0007] 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, including in Dirofilaria and Onchocerca, there is yet to
be an effective vaccine developed for any parasitic helminth.
[0008] As such, there remains a need to identify an efficacious
composition that protects animals against diseases caused by
parasitic helminths and that, preferably, also protects animals
from infection by such helminths.
[0009] Macrophage migration inhibitory factors (MIFs), which are
about 13 kilodaltons (kD) in size, have been identified in several
mammalian and avian species; see, for example, Galat et al, 1993,
Fed. Eur. Biochem. Soc. 319, 233-236, Wistow et al, 1993, Proc.
Natl. Acad. Sci. USA 90, 1272-1275, Weiser et al, 1989, Proc. Natl.
Acad. Sci. USA 86, 7522-7526, Bernhagen, et al, 1993, Nature 365,
756-759, Blocki et al, 1993, Protein Science 2, 2095-2102, and
Blocki et al, 1992, Nature 360, 269-270. Although MIF was first
characterized as being able to block macrophage migration, MIF also
appears to effect macrophage-macrophage adherence; induce
macrophage to express interleukin-1-beta, interleukin-6, and tumor
necrosis factor alpha; up-regulate HLA-DR; increase nitric oxide
synthase and nitric oxide concentrations; and activate macrophage
to kill Leishmania donovani tumor cells and inhibit Mycoplasma
avium growth, by a mechanism different from that effected by
interferon-gamma. In addition to its potential role as an
immunoevasive molecule, MIF can act as an immunoadjuvant when given
with bovine serum albumin or HIV gp120 in incomplete Freunds or
liposomes, eliciting antigen induced proliferation comparable to
that of complete Freunds.
[0010] MIF appears to be related to glutathione S-transferase (GST)
since at least some MIFs have GST activity and are able to bind to
glutathione. MIFs, however, are only about half the size of GST
subunits and do not show activity against
1-chloro-2,4-dinitrobenzene, which is the most common substrate
used to detect GST activity. Although GST activity has been
identified in several nematodes, that activity was detected using
1-chloro-2,4-dinitrobenzene, and the enzymes responsible for the
activity were not of the size expected for MIFs. To the inventors'
knowledge MIF homologues have not yet been identified in any
parasitic helminth; efforts to do so have so far proven
unsuccessful.
SUMMARY OF THE INVENTION
[0011] The present invention relates to parasitic helminth
macrophage migration inhibitory factor (MIF) proteins; to parasitic
helminth MIF nucleic acid molecules, including those that encode
such proteins; to antibodies raised against such proteins
(anti-parasitic helminth MIF antibodies); and to compounds that
inhibit parasitic helminth MIF activity (i.e, inhibitory compounds
or inhibitors). 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 use
of such therapeutic compositions to protect animals from diseases
caused by parasitic helminths.
[0012] One embodiment of the present invention is an isolated
nucleic acid molecule that hybridizes under stringent hybridization
conditions with a Dirofilaria immitis macrophage migration
inhibitory factor (MIF) gene (i.e., a D. immitis MIF gene) and/or
with an Onchocerca volvulus MIF gene (i.e., an O. volvulus MIF
gene). A D. immitis MIF gene preferably includes nucleic acid SEQ
ID NO:17 and/or SEQ ID NO:19, and an O. volvulus MIF gene
preferably includes nucleic acid sequence SEQ ID NO:6 and/or SEQ ID
NO:9. A MIF nucleic acid molecule of the present invention can
include a regulatory region of a parasitic helminth MIF gene and/or
can encode a parasitic helminth MIF protein. Particularly preferred
MIF nucleic acid molecules include nucleic acid sequence SEQ ID
NO:1, SEQ ID NO:3) SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19 and/or complements of those SEQ ID NOs, as
well as allelic variants of one or more of those nucleic acid
molecules.
[0013] The present invention also relates to recombinant molecules,
recombinant viruses and recombinant cells that include parasitic
helminth MIF nucleic acid molecules of the present invention. Also
included are methods to produce such nucleic acid molecules,
recombinant molecules, recombinant viruses and recombinant
cells.
[0014] Another embodiment of the present invention includes a
parasitic helminth macrophage migration inhibitory factor (MIF)
protein (i.e., a parasitic helminth MIF protein) or a protein that
includes a parasitic helminth MIF protein. A preferred parasitic
helminth MIF protein, when administered to an animal, is capable of
eliciting an immune response against a natural parasitic helminth
MIF protein. Particularly preferred MIF proteins are proteins that
include amino acid sequence SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8
and/or SEQ ID NO:11, as well as proteins that are encoded by
nucleic acid molecules that are allelic variants of the nucleic
acid molecules that encode proteins having SEQ ID NO:2, SEQ ID
NO:5, SEQ ID NO:8 and/or SEQ ID NO:11.
[0015] The present invention also relates to mimetopes of parasitic
helminth MIF proteins as well as to isolated antibodies that
selectively bind to parasitic helminth MIF proteins or mimetopes
thereof. Also included are methods, including recombinant methods,
to produce proteins, mimetopes and antibodies of the present
invention.
[0016] Another embodiment of the present invention is a method to
identify a compound capable of inhibiting MIF activity of a
parasitic helminth. The method includes the steps of: (a)
contacting an isolated parasitic helminth MIF protein with a
putative inhibitory compound under conditions in which, in the
absence of the compound, the protein has MIF activity; and (b)
determining if the putative inhibitory compound inhibits the MIF
activity. Also included in the present invention is a test kit to
identify a compound capable of inhibiting MIF activity of a
parasitic helminth. Such a test kit includes an isolated parasitic
helminth MIF protein having MIF activity and a means for
determining the extent of inhibition of that 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 MIF protein or a mimetope
thereof, an isolated nucleic acid molecule that hybridizes under
stringent hybridization conditions with a D. immitis MIF gene
and/or an O. volvulus MIF gene, an isolated antibody that
selectively binds to a parasitic helminth MIF protein, and/or an
inhibitor of MIF protein activity identified by its ability to
inhibit parasitic helminth MIF activity. A preferred therapeutic
composition of the present invention also includes an excipient, an
adjuvant and/or a carrier. Preferred MIF nucleic acid molecule
compounds of the present invention include naked nucleic acid
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. The method
includes the step of administering to the animal a therapeutic
composition of the present invention.
[0018] Suitable parasitic helminths to use in the production (e.g.,
recombinant, natural, or synthetic production) of nucleic acid
molecules, proteins, antibodies and inhibitory compounds of the
present invention include nematodes, cestodes and trematodes, with
nematodes (such as filariid, ascarid, strongyle and trichostrongyle
nematodes) being preferred, with filariids being more preferred,
and with D. immitis and O. volvulus being even more preferred.
[0019] Suitable and preferred parasitic helminths from which to
protect animals are as disclosed for use in the production of
nucleic acid molecules, proteins, antibodies and inhibitory
compounds of the present invention. As such, preferred diseases
from which to protect animals include diseases caused by nematodes,
cestodes and/or trematodes, with diseases caused by nematodes being
more preferred targets, and with diseases caused by filariids being
even more preferred targets. Particularly preferred diseases from
which to protect animals include heartworm and onchocerciasis.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention includes the surprising discovery that
parasitic helminths produce a macrophage migration inhibitory
factor that, although reported for mammals and birds, has not been
reported for parasitic helminths. This parasitic helminth protein,
also referred to herein as a parasitic helminth macrophage
migration inhibitory factor protein, or MIF protein, has utility
because it represents a novel target for anti-parasite vaccines and
drugs, particularly since a D. immitis MIF nucleic acid molecule
can encode a protein that binds to immune dog serum; details of
isolation of such a D. immitis MIF nucleic acid molecule are
disclosed in the Examples section. While not being bound by theory,
it is believed that parasitic helminth MIF proteins have a role in
immune evasion, by, for example, detoxifying compounds harmful to
the parasite and/or blocking recruitment of macrophage to the
location of the parasite. For example, the potential GST activity
of MIF could be used enzymatically in minimizing the effect of
electrophilic attack on the extracellular parasite, since GST is
known to catalyze the conjugation of glutathione to electrophilic
compounds, rendering them nontoxic. Furthermore, the inventors have
discovered MIF to be expressed in both D. immitis larvae and O.
volvulus adults, which are both migrating forms of the parasites,
suggesting that MIF plays a role in preventing recruitment of
macrophage and other effector cells to the proximity of the larvae
and adult parasites residing in and migrating through the skin and
tissue. Such parasite activities could otherwise induce an
inflammatory response harmful to the parasite.
[0021] The present invention includes not only parasitic helminth
MIF proteins but also parasitic helminth MIF nucleic acid
molecules, antibodies directed against parasitic helminth MIF
proteins and other inhibitors of MIF proteins. Also included 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.
[0022] One embodiment of the present invention is an isolated
protein comprising a parasitic helminth MIF protein. According to
the present invention, an isolated, or biologically pure, protein,
is a protein that has been removed from its natural milieu. As
such, "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. As used herein, a helminth MIF
protein can be a full-length protein or any homologue of such a
protein. Examples of MIF homologues include MIF 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,
myristylation, prenylation, palmitoylation, amidation and/or
addition of glycerophosphatidyl inositol) such that the homologue
includes at least one epitope capable of eliciting an immune
response against a parasitic helminth MIF protein. That is, when
the homologue is administered to an animal as an immunogen, using
techniques known to those skilled in the art, the animal will
produce a humoral and/or cellular immune response against at least
one epitope of a parasitic helminth MIF protein. The ability of a
protein to effect an immune response, can be measured using
techniques known to those skilled in the art. MIF protein
homologues of the present invention also include MIF proteins that
bind to glutathione (i.e., have glutathione binding activity)
and/or that selectively bind to immune serum. Examples of methods
to measure such activities are disclosed herein, and are known to
those skilled in the art. As used herein, the term "selectively
binds to" immune serum refers to the ability of isolated proteins
and mimetopes thereof to bind to serum collected from animals that
are immune to parasitic helminth infection but essentially not to
bind, according to standard detection techniques, to serum
collected from animals that are not immune to parasitic helminth
infection. Preferably, such isolated proteins and mimetopes are
able to bind to anti-parasitic helminth immune serum with high
affinity. Methods to produce and use immune serum are disclosed,
for example, in Grieve et al., PCT Publication No. WO 94/15593,
published Jul. 21, 1994; this reference (also referred to herein as
WO 94/15593) is incorporated by reference herein in its
entirety.
[0023] Parasitic helminth MIF protein homologues can be the result
of natural allelic variation or natural mutation. MIF protein
homologues 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.
[0024] Isolated proteins of the present invention, including
homologues, can be identified in a straight-forward manner by the
proteins' ability to elicit an immune response against parasitic
helminth MIF proteins, to bind to glutathione and/or to selectively
bind to immune serum.
[0025] Isolated proteins of the present invention have the further
characteristic of being encoded by nucleic acid molecules that
hybridize under stringent hybridization conditions to at least one
of the following genes: (a) a gene encoding a Dirofilaria immitis
MIF protein (i.e., a D. immitis MIF gene); and (b) a gene encoding
an Onchocerca volvulus MIF protein (i.e., an O. volvulus MIF gene.
It is to be noted that the term "a" or "an" entity refers to one or
more of that entity; for example, a gene refers to one or more
genes or at least one gene. 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.
[0026] As used herein, stringent hybridization conditions refer to
standard hybridization conditions under which nucleic acid
molecules, including oligonucleotides, are used to identify
molecules having similar nucleic acid sequences. Stringent
hybridization conditions typically permit isolation of nucleic acid
molecules having at least about 70% nucleic acid sequence identity
with the nucleic acid molecule being used as a probe in the
hybridization reaction. Such standard conditions are disclosed, for
example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Labs Press. The reference Sambrook et
al., ibid., is incorporated by reference herein in its entirety.
Examples of such conditions include, but are not limited to, the
following: oligonucleotide probes of about 18-25 nucleotides in
length with T.sub.m's ranging from about 50.degree. C. to about
65.degree. C., for example, can be hybridized to nucleic acid
molecules typically immobilized on a filter (e.g., nitrocellulose
filter) in a solution containing 5.times.SSPE, 1% Sarkosyl,
5.times.Denhardts and 0.1 mg/ml denatured salmon sperm DNA at
37.degree. C. for about 2 to 12 hours. The filters are then washed
3 times in a wash solution containing 5.times.SSPE, 1% Sarkosyl at
37.degree. C. for 15 minutes each. The filters can be further
washed in a wash solution containing 2.times.SSPE, 1% Sarkosyl at
37.degree. C. for 15 minutes per wash. Randomly primed DNA probes
can be hybridized, for example, to nucleic acid molecules typically
immobilized on a filter (e.g., nitrocellulose filter) in a solution
containing 5.times.SSPE, 1% Sarkosyl, 0.5% Blotto (dried milk in
water), and 0.1 mg/ml denatured salmon sperm DNA at 42.degree. C.
for about 2 to 12 hours. The filters are then washed 2 times in a
wash solution containing 5.times.SSPE, 1% Sarkosyl at 42.degree. C.
for 15 minutes each, followed by 2 washes in a wash solution
containing 2.times.SSPE, 1% Sarkosyl at 42.degree. C. for 15
minutes each.
[0027] As used herein, a D. immitis MIF gene includes all nucleic
acid sequences related to a natural D. immitis MIF gene such as
regulatory regions that control production of the D. immitis MIF
protein encoded by that gene (such as, but not limited to,
transcription, translation or post-translation control regions) as
well as the coding region itself. In one embodiment, a D. immitis
MIF gene of the present invention includes the nucleic acid
sequence SEQ ID NO:17 as well as the complement of SEQ ID NO:17.
Nucleic acid sequence SEQ ID NO:17 represents the deduced sequence
of the coding strand of the apparent coding region of a cDNA
(complementary DNA) nucleic acid molecule denoted herein as
nDiMIF(1).sub.355, the production of which is disclosed in the
Examples. The complement of SEQ ID NO:17 refers to the nucleic acid
sequence of the strand complementary to the strand having SEQ ID
NO:17, 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. It should be noted that since nucleic acid sequencing
technology is not entirely error-free, SEQ ID NO:17 (as well as
other nucleic acid and protein sequences presented herein), at
best, represents an apparent nucleic acid sequence of the nucleic
acid molecule encoding a D. immitis MIF protein of the present
invention.
[0028] In another embodiment, a D. immitis MIF gene can be an
allelic variant that includes a similar but not identical sequence
to SEQ ID NO:17. An allelic variant of a D. immitis MIF gene
including SEQ ID NO:17 is a gene that occurs at essentially the
same locus (or loci) in the genome as the gene including SEQ ID
NO:17, but which, due to natural variations caused by, for example,
mutation or recombination, has a similar but not identical
sequence. Allelic variants typically encode proteins having similar
activity to that of the protein encoded by the gene to which they
are being compared. Allelic variants can also comprise alterations
in the 5' or 3' untranslated regions of the gene (e.g., in
regulatory control regions). Allelic variants are well known to
those skilled in the art and would be expected to be found within a
given parasitic helminth since the genome is diploid and/or among a
group of two or more parasitic helminths. An example of an allelic
variant of the D. immitis MIF gene including SEQ ID NO:17 is a D.
immitis MIF gene including SEQ ID NO:19. Nucleic acid sequence SEQ
ID NO:19 represents the deduced sequence of the coding strand of
the apparent coding region of a cDNA nucleic acid molecule denoted
herein as nDiMIF(2).sub.333, the production of which is disclosed
in the Examples. As such, one embodiment of the present invention
is a D. immitis MIF gene that includes the nucleic acid sequence
SEQ ID NO:19 as well as the complement of SEQ ID NO:19.
[0029] Similarly, an O. volvulus MIF gene includes all nucleic acid
sequences related to a natural O. volvulus MIF gene such as
regulatory regions that control production of the O. volvulus MIF
protein encoded by that gene as well as the coding region itself.
In one embodiment, an O. volvulus MIF gene includes the nucleic
acid sequence SEQ ID NO:6. Nucleic acid sequence SEQ ID NO:6
represents the deduced sequence of the coding strand of the
apparent coding region of a cDNA nucleic acid molecule denoted
herein as nOvMIF(1).sub.440, the production of which is disclosed
in the Examples. In another embodiment, an O. volvulus MIF gene can
be an allelic variant that includes a similar but not identical
sequence to SEQ ID NO:6. An example of such an allelic variant is
an O. volvulus MIF gene including SEQ ID NO:9. Nucleic acid
sequence SEQ ID NO:9 represents the deduced sequence of the coding
strand of the apparent coding region of a cDNA nucleic acid
molecule denoted herein as nOvMIF(2).sub.522, the production of
which is disclosed in the Examples.
[0030] The minimal size of a MIF protein homologue 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. As such, the size of the nucleic acid molecule encoding
such a protein homologue is dependent on nucleic acid composition
and percent homology between the nucleic acid molecule and
complementary sequence. It should also be noted that the extent of
homology required to form a stable hybrid can vary depending on
whether the homologous sequences are interspersed throughout the
nucleic acid molecules or are clustered (i.e., localized) in
distinct regions on the nucleic acid molecules. The minimal size of
such nucleic acid molecules is typically at least about 12 to about
15 nucleotides in length if the nucleic acid molecules are GC-rich
and at least about 15 to about 17 bases in length if they are
AT-rich. As such, the minimal size of a nucleic acid molecule used
to encode a MIF protein homologue of the present invention is from
about 12 to about 18 nucleotides in length.
[0031] There is no limit, other than a practical limit, on the
maximal size of such a nucleic acid molecule in that the nucleic
acid molecule can include a portion of a gene, an entire gene, or
multiple genes, or portions thereof. Similarly, the minimal size of
a MIF protein homologue of the present invention is from about 4 to
about 6 amino acids in length, with preferred sizes depending on
whether a full-length, fusion, multivalent, or functional portions
of such proteins are desired.
[0032] Parasitic helminth MIF proteins of the present invention,
including homologues thereof, preferably are capable of eliciting
an immune response against a parasitic helminth MIF protein and/or
of selectively binding to immune serum. The minimum size of such a
protein is a minimum size sufficient to form an epitope, a size
that typically is at least from about 5 to about 9 amino acids. As
is appreciated by those skilled in the art, an epitope can include
amino acids that naturally are contiguous to each other as well as
amino acids that, due to the tertiary structure of the natural
protein, are in sufficiently close proximity to form an
epitope.
[0033] One embodiment of the present invention includes a parasitic
helminth MIF protein that binds to glutathione and, as such,
includes a glutathione-binding domain. Such a glutathione-binding
domain is believed to be located primarily in the N-terminal
portion of a full-length MIF protein of the present invention.
Methods to detect glutathione binding and to identify glutathione
binding domains are described, for example, in Blocki et al., 1993,
ibid. and references cited therein.
[0034] Suitable parasitic helminths from which to isolate parasitic
helminth MIF proteins of the present invention (including isolation
of the natural protein or production of the protein by recombinant
or synthetic techniques) include nematodes, cestodes, and
trematodes, with nematodes being preferred. Preferred nematodes
from which to isolate MIF proteins include filariid, ascarid,
strongyle and trichostrongyle nematodes. Particularly preferred
nematodes are those of the genera Acanthocheilonema,
Aelurostrongylus, Ancylostoma, Angiostrongylus, Ascaris, Brugia,
Bunostomum, Dictyocaulus, Dioctophyme, Dipetalonema, Dirofilaria,
Dracunculus, Filaroides, Lagochilascaris, Loa, Mansonella,
Muellerius, Necator, Onchocerca, Parafilaria, Parascaris,
Protostrongylus, Setaria, Stephanofilaria, Strongyloides,
Strongylus, Thelazia, Toxascaris, Toxocara, Trichinella, Uncinaria
and Wuchereria. Other particularly preferred nematodes include
parasitic helminths of the genera Capillaria, Chabertia, Cooperia,
Enterobius, Haemonchus, Nematodirus, Oesophagostomum, Ostertagia,
Trichostrongylus and Trichuris. Preferred filariid nematodes
include Dirofilaria, Onchocerca, Acanthocheilonema, Brugia,
Dipetalonema, Loa, Parafilaria, Setaria, Stephanofilaria and
Wuchereria filariid nematodes. Particularly preferred parasitic
helminths are nematodes of the genera Dirofilaria and Onchocerca,
with D. immitis, the parasite that causes heartworm, and O.
volvulus, the parasite that causes onchocerciasis, being even more
preferred.
[0035] A preferred parasitic helminth MIF 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. As such, the parasitic helminth is
essentially incapable of causing disease in an animal that is
immunized with an isolated protein of the present invention. In
accordance with the present invention, the ability of a MIF protein
of the present invention to protect an animal from disease by a
parasitic helminth refers to the ability of that protein to treat,
ameliorate and/or prevent disease, including infection leading to
disease, caused by the parasitic helminth, preferably by eliciting
an immune response against the parasitic helminth. Such an immune
response can include humoral and/or cellular immune responses.
[0036] Suitable parasites to target include any parasite that is
essentially incapable of causing disease in an animal administered
a MIF protein of the present invention. As such, a parasite to
target includes any parasite that produces a protein having one or
more epitopes that can be targeted by a humoral and/or cellular
immune response against a MIF protein of the present invention
and/or that can be targeted by a compound that otherwise inhibits
MIF activity (e.g., a compound that inhibits glutathione binding
and/or GST activity), thereby resulting in the reduced ability of
the parasite to cause disease in an animal. Suitable and preferred
parasites to target include those parasitic helminths disclosed
above as being useful in the production of parasitic helminth
proteins of the present invention.
[0037] It is to be appreciated that the present invention also
includes mimetopes of MIF proteins of the present invention that
can be used in accordance with methods as disclosed for MIF
proteins of the present invention. As used herein, a mimetope of a
MIF protein of the present invention refers to any compound that is
able to mimic the activity of such a MIF protein, often because the
mimetope has a structure that mimics the MIF protein. Mimetopes can
be, but are not limited to: peptides that have been modified to
decrease their susceptibility to degradation; anti-idiotypic and/or
catalytic antibodies, or fragments thereof; non-proteinaceous
immunogenic portions of an isolated protein (e.g., carbohydrate
structures); and synthetic or natural organic molecules, including
nucleic acids. Such mimetopes can be designed using
computer-generated structures of proteins of the present invention.
Mimetopes 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.
[0038] One embodiment of the isolated protein of the present
invention is a fusion protein that includes a parasitic helminth
MIF protein-containing domain attached to a fusion segment.
Inclusion of a fusion segment as part of a MIF protein of the
present invention can enhance the protein's stability during
production, storage and/or use. Depending on the segment's
characteristics, a fusion segment can also act as an
immunopotentiator to enhance the immune response mounted by an
animal immunized with a parasitic helminth MIF protein containing
such a fusion segment. Furthermore, a fusion segment can function
as a tool to simplify purification of a parasitic helminth MIF
protein, such as to enable purification of the resultant fusion
protein using 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, and/or simplifies purification of a protein). It is within
the scope of the present invention to use one or more fusion
segments. Fusion segments can be joined to amino and/or carboxyl
termini of the MIF-containing domain of the protein. Linkages
between fusion segments and MIF-containing domains of fusion
proteins can be susceptible to cleavage in order to enable
straight-forward recovery of the MIF-containing domains of such
proteins. Fusion proteins are preferably produced by culturing a
recombinant cell transformed with a fusion nucleic acid molecule
that encodes a protein including the fusion segment attached to
either the carboxyl and/or amino terminal end of a MIF-containing
domain.
[0039] Preferred fusion segments for use in the present invention
include a metal binding domain, such as a poly-histidine segment
capable of binding to a divalent metal ion; an immunoglobulin
binding domain, such as Protein A, Protein G, T cell, B cell, Fc
receptor or complement protein antibody-binding domains; a sugar
binding domain such as a maltose binding domain from a maltose
binding protein; and/or a "tag" domain (e.g., at least a portion of
.beta.-galactosidase, a strep tag peptide, other domains 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 in Tampa, Fla.; and an S10 peptide. An example of a
particularly preferred fusion protein of the present invention is
PHIS-PDiMIF(1).sub.115, production of which is disclosed
herein.
[0040] Another embodiment of the present invention includes a
parasitic helminth MIF protein that 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.
[0041] Examples of multivalent protective compounds include, but
are not limited to, a MIF 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, cattle and/or horses, such as, but not limited to: viruses
(e.g., caliciviruses, distemper viruses, hepatitis viruses,
herpesviruses, immunodeficiency viruses, infectious peritonitis
viruses, leukemia viruses, panleukopenia viruses, parvoviruses,
rabies viruses, other cancer-causing or cancer-related viruses);
bacteria (e.g., Leptospira, Bartonella); fungi and fungal-related
microorganisms (e.g., Candida, Cryptococcus, Histoplasma); and
other parasites (e.g., Babesia, Cryptosporidium, Eimeria,
Encephalitozoon, Hepatozoon, Isospora, Leishmania, Microsporidia,
Neospora, Nosema, Plasmodium, Pneumocystis, Toxoplasma, as well as
helminth parasites, such as those disclosed herein). In one
embodiment, a D. immitis MIF protein of the present invention is
attached to one or more additional compounds protective against
heartworm. In another embodiment, an O. volvulus MIF protein of the
present invention is attached to one or more additional compounds
protective against onchocerciasis.
[0042] A preferred isolated protein of the present invention is a
protein encoded by a nucleic acid molecule that hybridizes under
stringent hybridization conditions with nucleic acid molecules
nDiMIF(1).sub.345, nDiMIF(2).sub.330, nOvMIF(1).sub.345, and/or
nOvMIF(2).sub.342. A further preferred isolated protein is encoded
by a nucleic acid molecule that hybridizes under stringent
hybridization conditions with a nucleic acid molecule having
nucleic acid sequence SEQ ID NO:3 (i.e., SEQ ID NO:17 or SEQ
ID:19), SEQ ID NO:6 and/or SEQ ID NO:9.
[0043] Translation of SEQ ID NO:1 suggests that nucleic acid
molecules nDiMIF(1).sub.532 and nDiMIF(2).sub.532 each encodes a
full-length D. immitis MIF protein of about 115 amino acids,
referred to herein as PDiMIF(1).sub.115, and PDiMIF(2).sub.115,
respectively, assuming an open reading frame having an initiation
(start) codon spanning from about nucleotide 8 through about
nucleotide 10 of SEQ ID NO:1 and a termination (stop) codon
spanning from about nucleotide 353 through about nucleotide 355 of
SEQ ID NO:1. Note that PDiMIF(1).sub.115 and PDiMIF(2).sub.115 have
the same amino acid sequence, and as such, are both referred to
herein as PDiMIF(1).sub.115. The open reading frame, excluding the
stop codon, corresponding to nDiMIF(1).sub.532 comprises nucleic
acid molecule nDiMIF(1).sub.345 of the present invention, the
nucleic acid sequence of which is represented herein by SEQ ID
NO:17. The open reading frame, excluding the stop codon,
corresponding to nDiMIF(2).sub.532 comprises nucleic acid molecule
nDiMIF(2).sub.330 of the present invention, the nucleic acid
sequence of which is represented herein by SEQ ID NO:19. SEQ ID
NO:3 represents a composite of SEQ ID NO:17 and SEQ ID NO:19. SEQ
ID NO:19 is truncated at the 5' end compared to SEQ ID NO:17 and
also differs internally in sequence by one nucleotide, as described
in more detail in the Examples section.
[0044] SEQ ID NO:3 and SEQ ID NO:17 encode proteins having the same
amino acid sequence, and as such, each of those proteins is
referred to as PDiMIF(1).sub.115. SEQ ID NO:19, being truncated,
encodes a protein of 110 amino acids referred to herein as
PDiMIF(2).sub.110. The deduced amino acid sequence of
PDiMIF(1).sub.115 is represented herein as SEQ ID NO:2. The deduced
amino acid sequence of PDiMIF(2).sub.110 corresponds to amino acids
6 through 115 of SEQ ID NO:2, since the codons at which the
nucleotide difference between SEQ ID NO:17 and SEQ ID NO:19 occurs
encode the same amino acid. Based on that amino acid sequence,
PDiMIF(1).sub.115 has an estimated molecular weight of about 12.3
kD and an estimated pI of about 8.3. The amino acid sequence of
PDiMIF(1).sub.115 also contains 3 potential N-glycosylation
sites.
[0045] Comparison of amino acid sequence SEQ ID NO:2 (i.e., the
amino acid sequence of PDiMIF(1).sub.115) with MIF amino acid
sequences reported in GenBank indicates that SEQ ID NO:2 is about
52-53% identical to human MIF and about 55% identical to chicken
MIF.
[0046] Translation of SEQ ID NO:4 suggests that nucleic acid
molecule nOvMIF(1).sub.440 encodes a full-length O. volvulus MIF
protein of about 115 amino acids, referred to herein as
POvMIF(1).sub.115, assuming an open reading frame having an
initiation (start) codon spanning from about nucleotide 8 through
about nucleotide 10 of SEQ ID NO:1 and a termination (stop) codon
spanning from about nucleotide 353 through about nucleotide 355 of
SEQ ID NO:4. The open reading frame, excluding the stop codon,
comprises nucleic acid molecule nOvMIF(1).sub.345 of the present
invention, the nucleic acid sequence of which is represented herein
by SEQ ID NO:6. The deduced amino acid sequence of
POvMIF(1).sub.115 is represented herein as SEQ ID NO:5. Based on
that amino acid sequence, POvMIF(1).sub.115 has an estimated
molecular weight of about 12.24 kD and an estimated pI of about
9.21 The amino acid sequence of POvMIF(1).sub.115 also contains 3
potential N-glycosylation sites.
[0047] Translation of SEQ ID NO:7 suggests that nucleic acid
molecule nOvMIF(2).sub.522 encodes a protein of about 114 amino
acids, denoted POvMIF(2).sub.114, assuming a stop codon spanning
from about nucleotide 343 through about nucleotide 345 of SEQ ID
NO:7. The amino acid sequence of POvMIF(2).sub.114 is represented
herein as SEQ ID NO:8. The open reading frame encoding
POvMIF(2).sub.115 is referred to herein as nOvMIF(2).sub.342, the
nucleic acid sequence of which is represented in SEQ ID NO:9.
[0048] SEQ ID NO:4 and SEQ ID NO:7 are allelic variants and are
identical in their coding regions (i.e., SEQ ID NO:6 and SEQ ID
NO:9, respectively), except that (a) SEQ ID NO:7 apparently lacks a
start codon; (b) SEQ ID NO:7 is about 10 nucleotides shorter than
SEQ ID NO:4 at the 5' end; and (c) the region spanning from about
nucleotide 1 through about nucleotide 19 of SEQ ID NO:7 is only
about 47% identical to the region spanning from about nucleotide 10
through about nucleotide 29 of SEQ ID NO:4.
[0049] Comparison of apparent full-length D. immitis and O.
volvulus MIF proteins (i.e., PDiMIF(1).sub.115 and
POvMIF(1).sub.115) indicated that the two MIF proteins were about
88% identical at the amino acid level. Comparison of amino acid
sequence SEQ ID NO:5 (i.e., the amino acid sequence of
POvMIF(1).sub.115) with amino acid sequences reported in GenBank
indicates that SEQ ID NO:5, showed some homology to macrophage
migration inhibition factor proteins of mammalian and avian
origins. The highest scoring match, i.e., 44% identity,, was found
between SEQ ID NO:5 and human and bovine MIFs. SEQ ID NO:5 was
about 43% identical to rat, mouse and chicken MIFs.
[0050] Preferred parasitic helminth MIF proteins of the present
invention include: proteins comprising amino acid sequences that
are at least about 60%, preferably at least about 70%, and more
preferably at least about 80%, and even more preferably at least
about 85% identical to amino acid sequences SEQ ID NO:2, SEQ ID
NO:5 and/or SEQ ID NO:8. Particularly preferred are proteins
comprising amino acid sequences that are at least about 90% and
more particularly at least about 95% identical to amino acid
sequences SEQ ID NO:2, SEQ ID NO:5 and/or SEQ ID NO:8. More
preferred parasitic helminth MIF proteins of the present invention
include: proteins encoded by at least a portion of SEQ ID NO:1 and,
as such, have amino acid sequences that include at least a portion
of SEQ ID NO:2; proteins encoded by at least a portion of SEQ ID
NO:4 and, as such, have amino acid sequences that include at least
a portion of SEQ ID NO:5; and proteins encoded by at least a
portion of SEQ ID NO:7 and, as such, have amino acid sequences that
include at least a portion of SEQ ID NO:8.
[0051] Particularly preferred parasitic helminth proteins of the
present invention are proteins that include SEQ ID NO:2, SEQ ID
NO:5, and/or SEQ ID NO:8 (including, but not limited to the encoded
proteins, full-length proteins, processed proteins, fusion proteins
and multivalent proteins) as well as proteins that are truncated
homologues of proteins that include SEQ ID NO:2, SEQ ID NO:5,
and/or SEQ ID NO:8. Even more preferred proteins include
PDiMIF(1).sub.115, PDiMIF(2).sub.110, PDiMIF.sub.34,
PHIS-PDiMIF(1).sub.115, BvPDiMIF(1).sub.115, POvMIF(1).sub.115 and
POvMIF(2).sub.114. Examples of methods to produce such proteins are
disclosed herein, including in the Examples section.
[0052] Another embodiment of the present invention is an isolated
nucleic acid molecule that hybridizes under stringent hybridization
conditions with a MIF gene selected from the group consisting of a
D. immitis MIF gene and an O. volvulus MIF gene. The identifying
characteristics of such genes are heretofore described. A nucleic
acid molecule of the present invention can include an isolated
natural parasitic helminth MIF gene or a homologue 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, full-length or partial coding regions, or
combinations thereof. The minimal size of a nucleic acid molecule
of the present invention is the minimal size that can form a stable
hybrid with one of the aforementioned genes under stringent
hybridization conditions. Suitable and preferred parasitic
helminths are disclosed above.
[0053] 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). As such, "isolated" does not reflect the
extent to which the nucleic acid molecule has been purified. An
isolated nucleic acid molecule can include DNA, RNA, or derivatives
of either DNA or RNA.
[0054] An isolated parasitic helminth MIF nucleic acid molecule of
the present invention can be obtained from its natural source
either as an entire (i.e., complete) gene or a portion thereof
capable of forming a stable hybrid with that gene. An isolated
parasitic helminth MIF nucleic acid molecule can also be produced
using recombinant DNA technology (e.g., polymerase chain reaction
(PCR) amplification, cloning) or chemical synthesis. Isolated
parasitic helminth MIF nucleic acid molecules include natural
nucleic acid molecules and homologues thereof, including, but not
limited to, natural allelic variants and modified nucleic acid
molecules in which nucleotides have been inserted, deleted,
substituted, and/or inverted in such a manner that such
modifications do not substantially interfere with the nucleic acid
molecule's ability to encode a parasitic helminth MIF protein of
the present invention or to form stable hybrids under stringent
conditions with natural gene isolates.
[0055] A parasitic helminth MIF nucleic acid molecule homologue can
be produced using a number of methods known to those skilled in the
art (see, for example, Sambrook et al., ibid.). For example,
nucleic acid molecules can be modified using a variety of
techniques including, but not limited to, classic mutagenesis
techniques and recombinant DNA techniques, such as site-directed
mutagenesis, chemical treatment of a nucleic acid molecule to
induce mutations, restriction enzyme cleavage of a nucleic acid
fragment, ligation of nucleic acid fragments, PCR amplification
and/or mutagenesis of selected regions of a nucleic acid sequence,
synthesis of oligonucleotide mixtures and ligation of mixture
groups to "build" a mixture of nucleic acid molecules and
combinations thereof. Nucleic acid molecule homologues can be
selected from a mixture of modified nucleic acids by screening for
the function of the protein encoded by the nucleic acid (e.g.,
ability to elicit an immune response against at least one epitope
of a parasitic helminth MIF protein, ability to selectively bind to
immune serum, ability to bind to glutathione) and/or by
hybridization with a D. immitis MIF gene and/or with an O. volvulus
MIF gene.
[0056] An isolated nucleic acid molecule of the present invention
can include a nucleic acid sequence that encodes at least one
parasitic helminth MIF 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 MIF protein. As
heretofore disclosed, parasitic helminth MIF proteins of the
present invention include, but are not limited to, proteins having
full-length parasitic helminth MIF coding regions, proteins having
partial parasitic helminth MIF coding regions, fusion proteins,
multivalent protective proteins and combinations thereof.
[0057] 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 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, the nucleic acid
molecule being delivered to the animal by direct injection (i.e, as
a naked nucleic acid) or in a vehicle such as a recombinant virus
vaccine or a recombinant cell vaccine.
[0058] One embodiment of the present invention is a parasitic
helminth MIF nucleic acid molecule that hybridizes under stringent
hybridization conditions with nucleic acid molecule
nDiMIF(1).sub.532, nDiMIF(2).sub.532, nOvMIF(1).sub.440, and/or
nucleic acid molecule nOvMIF(2).sub.522. Such parasitic helminth
nucleic acid molecules can hybridize under stringent hybridization
conditions with a nucleic acid molecule having nucleic acid
sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, and/or a complement of any of
those nucleic acid sequences. The determination of such nucleic
acid sequences is described herein, including in the Examples
section. It is to be noted, as above, that a double-stranded
nucleic acid molecule of the present invention for which a nucleic
acid sequence has been determined for one strand that is
represented by a SEQ ID NO also comprises a complementary strand
having a sequence that is a complement of that SEQ ID NO. As such,
nucleic acid molecules of the present invention, which can be
either double-stranded or single-stranded, include those nucleic
acid molecules that form stable hybrids under stringent
hybridization conditions with either a given SEQ ID NO denoted
herein and/or with the complement of that SEQ ID NO.
[0059] Comparison of nucleic acid molecules containing D. immitis
and O. volvulus MIF coding regions (e.g., nDiMIF(1).sub.345 and
nOvMIF(1).sub.345) indicate that the two MIF coding regions are
about 87% identical at the nucleic acid sequence level. Comparison
of such D. immitis and O. volvulus nucleic acid molecules with
nucleic acid sequences of mammalian and avian MIF genes reported in
GenBank indicates that the coding regions represented in SEQ ID
NO:3 and SEQ ID NO:6 were most similar to those of a chicken
migration inhibitory factor gene, being, respectively, about 52%
and 51% identical, to the chicken gene.
[0060] Preferred parasitic helminth nucleic acid molecules include
nucleic acid molecules having a nucleic acid sequence that is at
least about 55%, preferably at least about 75%, more preferably at
least about 85%, even more preferably at least about 90%, and even
more preferably at least about 95% identical to nucleic acid
sequence SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, and/or a complement
thereof.
[0061] A preferred nucleic acid molecule of the present invention
includes at least a portion of nucleic acid sequence SEQ ID NO:1,
SEQ ID NO:4, SEQ ID NO:7 and/or a complement thereof, that is
capable of hybridizing to a D. immitis MIF gene and/or to a O.
volvulus MIF gene of the present invention. More preferred are
nucleic acid molecules that include a nucleic acid sequence, or the
complement thereof, of nucleic acid sequences SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18
and/or SEQ ID NO:19, as well as nucleic acid molecules that are
allelic variants of nucleic acid molecules that include a nucleic
acid sequence, or the complement thereof, of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18
and/or SEQ ID NO:19. Such nucleic acid molecules can include
nucleotides in addition to those included in the SEQ ID NOs, such
as, but not limited to, a full-length gene, a full-length coding
region, a nucleic acid molecule encoding a fusion protein, or a
nucleic acid molecule encoding a multivalent protective compound.
Particularly preferred nucleic acid molecules include
nDiMIF(1).sub.532, nDiMIF(2).sub.532, nDiMIF.sub.282,
nDiMIF.sub.102, nDiMIF(1).sub.355, nDiMIF(2).sub.333,
nDiMIF(1).sub.345, nDiMIF(2).sub.330, nDiMIF(1).sub.348,
nBvDiMIF(1).sub.348, nRcnDiMIF(1).sub.348, nOvMIF(1).sub.440,
nOvMIF(2).sub.522, nOvMIF(1).sub.345 and/or nOvMIF(2).sub.342.
[0062] The present invention also includes nucleic acid molecules
encoding a protein having at least a portion of SEQ ID NO:2, SEQ ID
NO:5, SEQ ID NO:8 and/or SEQ ID NO:11 including nucleic acid
molecules that have been modified to accommodate codon usage
properties of the cells in which such nucleic acid molecules are to
be expressed.
[0063] Knowing the nucleic acid sequences of certain parasitic
helminth MIF 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 MIF nucleic acid molecules for other parasitic
helminths, particularly since, as described in detail in the
Examples section, knowledge of D. immitis MIF nucleic acid
molecules of the present invention enabled the isolation of O.
volvulus MIF nucleic acid molecules of the present invention. 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 or DNA; 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 molecule include parasitic helminth L3, L4 or adult
libraries as well as genomic DNA libraries. Similarly, preferred
DNA sources to screen or from which to amplify nucleic acid
molecules include parasitic helminth L3, L4 or adult DNA and
genomic DNA. Techniques to clone and amplify genes are disclosed,
for example, in Sambrook et al., ibid.
[0064] The present invention also includes nucleic acid molecules
that are oligonucleotides 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 MIF genes or other
parasitic helminth MIF nucleic acid molecules. Oligonucleotides of
the present invention can be RNA, DNA, or derivatives of either.
The minimal size of such oligonucleotides is the size required to
form a stable hybrid between a given oligonucleotide and the
complementary sequence on another nucleic acid molecule of the
present invention. Minimal size characteristics are disclosed
herein. The size of the oligonucleotide must also be sufficient for
the use of the oligonucleotide in accordance with the present
invention. Oligonucleotides of the present invention can be used in
a variety of applications including, but not limited to, as probes
to identify additional nucleic acid molecules, as primers to
amplify or extend nucleic acid molecules or in therapeutic
applications to inhibit MIF protein production or activity. Such
therapeutic applications include the use of such oligonucleotides
in, for example, antisense-, triplex formation-, ribozyme- and/or
RNA drug-based technologies. The present invention, therefore,
includes such oligonucleotides and methods to protect animals from
disease caused by parasitic helminths by use of 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, either prior to or after
infection by a parasitic helminth such as D. immitis or O. volvulus
in order to protect the animal from disease.
[0065] The present invention also 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 in the cloning,
sequencing, and/or otherwise manipulating of parasitic helminth MIF
nucleic acid molecules of the present invention. One type of
recombinant vector, referred to herein as a recombinant molecule
and described in more detail below, can be used in the expression
of nucleic acid molecules of the present invention. Preferred
recombinant vectors are capable of replicating in the transformed
cell.
[0066] Suitable and preferred nucleic acid molecules to include in
recombinant vectors of the present invention are as disclosed
herein for suitable and preferred parasitic helminth MIF nucleic
acid molecules per se. Particularly preferred nucleic acid
molecules to include in recombinant vectors, and particularly in
recombinant molecules, of the present invention include
nDiMIF(1).sub.532, nDiMIF(2).sub.532, nDiMIF.sub.282,
nDiMIF.sub.102, nDiMIF(1).sub.355, nDiMIF(2).sub.333,
nDiMIF(1).sub.345, nDiMIF(2).sub.330, nDiMIF(1).sub.348,
nBvDiMIF(1).sub.348, nRcnDiMIF(1).sub.348, nOvMIF(1).sub.440,
nOvMIF(2).sub.522, nOvMIF(1).sub.345 and nOvMIF(2).sub.342 .
[0067] Isolated 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 that is capable of expressing the
protein, the recombinant cell being produced by transforming a host
cell with one or more nucleic acid 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. Suitable and preferred nucleic
acid molecules with which to transform a cell are as disclosed
herein for suitable and preferred parasitic helminth MIF nucleic
acid molecules per se. Particularly preferred nucleic acid
molecules to include in recombinant cells of the present invention
include nDiMIF(1).sub.532, nDiMIF(2).sub.532, nDiMIF.sub.282,
nDiMIF.sub.102, nDiMIF(1).sub.355, nDiMIF(2).sub.333,
nDiMIF(1).sub.345, nDiMIF(2).sub.330, nDiMIF(1).sub.348,
nBvDiMIF(1).sub.348, nRcnDiMIF(1).sub.348, nOvMIF(1).sub.440,
nOvMIF(2).sub.522, nOvMIF(1).sub.345 and nOvMIF(2).sub.342.
[0068] 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. Host cells of the present invention either can be
endogenously (i.e., naturally) capable of producing parasitic
helminth MIF proteins of the present invention or can be capable of
producing such proteins after being transformed with at least one
nucleic acid molecule of the present invention. Host cells of the
present invention can be any cell capable of producing at least one
protein of the present invention, and include bacterial, fungal
(including yeast), parasite (including helminth, protozoa and
ectoparasite), insect, other animal and plant cells. 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 (normal dog kidney cell line for canine herpesvirus
cultivation), CRFK cells (normal cat kidney cell line for feline
herpesvirus cultivation), CV-1 cells (African monkey kidney cell
line used, for example, to culture raccoon poxvirus), 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..chi.3987 and SR-11 .sub..chi.4072; Spodoptera
frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells;
CV-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.
[0069] A recombinant cell is preferably produced by transforming a
host cell with one or more recombinant molecules, each comprising
one or more nucleic acid molecules of the present invention
operatively linked to an expression vector containing one or more
transcription control sequences. 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. Expression vectors can be either
prokaryotic or eukaryotic, and are typically viruses or plasmids.
Expression vectors of the present invention include any vectors
that function (i.e., direct gene expression) in recombinant cells
of the present invention, including in bacterial, fungal, parasite,
insect, other animal, and plant cells. Preferred expression vectors
of the present invention can direct gene expression in bacterial,
yeast, helminth or other parasite, insect and mammalian cells and
more preferably in the cell types heretofore disclosed.
[0070] Recombinant molecules of the present invention may also (a)
contain secretory signals (i.e., signal segment nucleic acid
sequences) to enable an expressed parasitic helminth protein of the
present invention to be secreted from the cell that produces the
protein and/or (b) contain fusion sequences which lead to the
expression of nucleic acid molecules of the present invention as
fusion proteins. Examples of suitable signal segments and fusion
segments encoded by fusion segment nucleic acids are disclosed
herein. Eukaryotic recombinant molecules may include intervening
and/or untranslated sequences surrounding and/or within the nucleic
acid sequences of nucleic acid molecules of the present
invention.
[0071] 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, tissue
plasminogen activator (t-PA), interferon, interleukin, growth
hormone, histocompatibility and viral envelope glycoprotein signal
segments.
[0072] Nucleic acid molecules of the present invention can be
operatively linked to expression vectors containing 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 nucleic acid molecules of the present
invention. In particular, recombinant molecules of the present
invention include 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. Suitable transcription control sequences
include 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 and mammalian cells, such as, but not limited to,
tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda
(.lambda.) (such as .lambda.p.sub.L and .lambda.p.sub.R and fusions
that include such promoters), bacteriophage T7, T71ac,
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 intermediate early
promoters, simian virus 40, retrovirus, actin, retroviral long
terminal repeat, Rous sarcoma virus, heat shock, phosphate and
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 a
parasitic helminth, such as a D. immitis or O. volvulus molecule
prior to isolation.
[0073] A recombinant molecule of the present invention is a
molecule that can include at least one of any nucleic acid molecule
heretofore described operatively linked to at least one of any
transcription control sequence capable of effectively regulating
expression of the nucleic acid molecule(s) in the cell to be
transformed, examples of which are disclosed herein. Particularly
preferred recombinant molecules include p.beta.gal-nDiMIF.sub.282,
pHis-nDiMIF(1).sub.348, pVL1393-nDiMIF(1).sub.348, and
pKB3poly-nDiMIF(1).sub.348, pHis-nOvMIF(1).sub.345,
pVL1393-nOvMIF(1).sub.348, and pKB3poly-nOvMIF(1).sub.348. Details
regarding the production of D. immitis MIF nucleic acid
molecule-containing recombinant molecules are disclosed herein. O.
volvulus MIF recombinant molecules are produced in a similar
manner.
[0074] 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 transfer cells are disclosed herein. Particularly
preferred recombinant cells include E.
coli:p.beta.gal-nDiMIF.sub.282, E. coli:pHis-nDiMIF(1).sub.348, S.
frugiperda:pVL1393-nDiMIF(1).sub.348,
BS-C-1:pKB3poly-nDiMIF(1).sub.348, E. coli:pHis-nOvMIF(1).sub.348,
S. frugiperda:pVL1393-nOvMIF(1).sub.348, and
BS-C-1:pKB3poly-nDiMIF(1).sub.348. Details regarding the production
of these recombinant cells are disclosed herein.
[0075] Recombinant cells of the present invention can also be
co-transformed with one or more recombinant molecules including
parasitic helminth MIF nucleic acid molecules encoding one or more
proteins of the present invention and one or more other proteins
useful in the production of multivalent vaccines which can include
one or more protective compounds.
[0076] It may be appreciated by one skilled in the art that use of
recombinant DNA technologies can improve expression of transformed
nucleic acid molecules by manipulating, for example, the number of
copies of the nucleic acid molecules within a host cell, the
efficiency with which those nucleic acid molecules are transcribed,
the efficiency with which the resultant transcripts are translated,
and the efficiency of post-translational modifications. Recombinant
techniques useful for increasing the expression of nucleic acid
molecules of the present invention include, but are not limited to,
operatively linking nucleic acid molecules to high-copy number
plasmids, integration of the nucleic acid molecules into one or
more host cell chromosomes, addition of vector stability sequences
to plasmids, substitutions or modifications of transcription
control signals (e.g., promoters, operators, enhancers),
substitutions or modifications of translational control signals
(e.g., ribosome binding sites, Shine-Dalgarno sequences),
modification of nucleic acid molecules of the present invention to
correspond to the codon usage of the host cell, deletion of
sequences that destabilize transcripts, and 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 nucleic acid molecules
encoding such a protein.
[0077] In accordance with the present invention, recombinant cells
of the present invention can be used to produce one or more
proteins of the present invention by culturing such cells under
conditions effective to produce such a protein, and recovering the
protein. Effective conditions to produce a protein include, but are
not limited to, appropriate media, bioreactor, temperature, pH and
oxygen conditions that permit protein production. An appropriate,
or effective, medium refers to any medium in which a cell of the
present invention, when cultured, is capable of producing a
parasitic helminth MIF protein of the present invention. Such a
medium is typically an aqueous medium comprising assimilable
carbon, nitrogen and phosphate sources, as well as appropriate
salts, minerals, metals and other nutrients, such as vitamins. The
medium may comprise complex nutrients or may be a defined minimal
medium. Cells of the present invention can be cultured in
conventional fermentation bioreactors, which include, but are not
limited to, batch, fed-batch, cell recycle, and continuous
fermentors. Culturing can also be conducted in shake flasks, test
tubes, microtiter dishes, and petri plates. Culturing is carried
out at a temperature, pH and oxygen content appropriate for the
recombinant cell. Such culturing conditions are well within the
expertise of one of ordinary skill in the art. Examples of suitable
conditions are included in the Examples section.
[0078] Depending on the vector and host system used for production,
resultant proteins 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.
[0079] The phrase "recovering the protein" refers simply 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 should be capable of
stimulating the production of antibodies in a treated animal.
[0080] The present invention also includes isolated antibodies
capable of selectively binding to a parasitic helminth MIF protein
of the present invention or to a mimetope thereof. Such antibodies
are also referred to herein as anti-parasitic helminth MIF
antibodies. Particularly preferred antibodies of this embodiment
include anti-D. immitis MIF antibodies and anti-O. volvulus MIF
antibodies.
[0081] Isolated antibodies are antibodies that have been removed
from their natural milieu. The term "isolated" does not refer to
the state of purity of such antibodies. As such, isolated
antibodies can include anti-sera containing such antibodies, or
antibodies that have been purified to varying degrees.
[0082] As used herein, the term "selectively binds to" refers to
the ability of antibodies 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 known to those skilled in the art including immunoblot
assays, immunoprecipitation assays, radioimmunoassays, enzyme
immunoassays (e.g., ELISA), immunofluorescent antibody assays and
immunoelectron microscopy; see, for example, Sambrook et al., ibid.
An anti-parasitic helminth MIF antibody preferably binds to a
parasitic helminth MIF protein in such a way as to reduce the
activity of that protein.
[0083] Antibodies of the present invention can be either polyclonal
or monoclonal antibodies. Antibodies of the present invention
include functional equivalents such as antibody fragments and
genetically-engineered antibodies, including single chain
antibodies, that are capable of selectively binding to at least one
of the epitopes of the protein or mimetope used to obtain the
antibodies. Antibodies of the present invention also include
chimeric antibodies that can bind to more than one epitope.
Preferred antibodies are raised in response to proteins, or
mimetopes thereof, that are encoded, at least in part, by a nucleic
acid molecule of the present invention.
[0084] A preferred method to produce antibodies of the present
invention includes (a) administering to an animal an effective
amount of a protein 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
parasitic helminth MIF 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.
[0085] 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 immunize 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 and/or (c) as tools to screen expression libraries and/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.
[0086] 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. Therapeutic compositions of the present
invention include at least one of the following protective
compounds: an isolated parasitic helminth MIF protein or a mimetope
thereof, an isolated nucleic acid molecule that hybridizes under
stringent hybridization conditions with a D. immitis MIF gene
and/or an O. volvulus MIF gene, an isolated antibody that
selectively binds to a parasitic helminth MIF protein, an inhibitor
of MIF protein activity identified by its ability to inhibit
parasitic helminth MIF activity, and a mixture thereof (i.e.,
combination) of at least two of the compounds. 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,
and/or prevent disease caused by a parasitic helminth of the
present invention. Preferred helminths to target are heretofore
disclosed. Examples of proteins, nucleic acid molecules, antibodies
and inhibitors of the present invention are disclosed herein.
[0087] The present invention also includes a therapeutic
composition comprising at least one parasitic helminth MIF-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.
[0088] Therapeutic compositions 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, economic food animals and/or
zoo animals. Preferred animals to protect against heartworm include
dogs, cats, humans and ferrets, with dogs and cats being
particularly preferred. Preferred animals to protect against
onchocerciasis include humans, cattle and horses, with humans being
particularly preferred.
[0089] 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 heartworm or to a black fly in order to prevent the
spread of onchocerciasis. 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 or a black fly, can ingest
therapeutic compositions present in the blood of a host that has
been administered a therapeutic composition of the present
invention.
[0090] Therapeutic compositions 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, m- 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.
[0091] In one embodiment of the present invention, the therapeutic
composition can also include an immunopotentiator, such as an
adjuvant or a carrier. Adjuvants are typically substances that
generally enhance the immune response of an animal to a specific
antigen. Suitable adjuvants include, but are not limited to,
Freund's adjuvant; other bacterial cell wall components;
aluminum-based salts; calcium-based salts; silica; polynucleotides;
toxoids; serum proteins; viral coat proteins; other
bacterial-derived preparations; gamma interferon; block copolymer
adjuvants, such as Hunter's Titermax.TM. adjuvant (Vaxcel.TM., Inc.
Norcross, Ga.); Ribi adjuvants (available from Ribi ImmunoChem
Research, Inc., Hamilton, Mont.); and saponins and their
derivatives, such as Quil A (available from Superfos Biosector A/S,
Denmark). Carriers are typically 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 formulations, biodegradable implants, liposomes,
bacteria, viruses, oils, esters, and glycols.
[0092] 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).
[0093] 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 for preferably at least about 1 month, more preferably at
least about 3 months and 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.
[0094] In order to protect an animal from disease caused by a
parasitic helminth of the present invention, 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,
when administered to an animal in an effective manner, is able to
elicit (i.e., stimulate) an immune response, preferably including
both a humoral and cellular 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. Oligonucleotide nucleic acid molecules of the
present invention can also be administered in an effective manner,
thereby reducing expression of parasitic helminth MIF proteins in
order to interfere with development of parasitic helminths targeted
in accordance with the present invention.
[0095] Therapeutic compositions of the present invention can be
administered to animals prior to infection in order to prevent
infection and/or can be administered to animals after infection in
order to treat disease caused by the parasitic helminth. For
example, proteins, mimetopes thereof, and antibodies thereof can be
used as immunotherapeutic agents.
[0096] 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 vaccinations 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.
[0097] 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., antisense RNA, ribozyme or RNA
drug) in the animal to be protected from disease. Nucleic acid
molecules can be delivered to an animal in a variety of methods
including, but not limited to, (a) administering a naked (i.e., not
packaged in a viral coat or cellular membrane) nucleic acid vaccine
(e.g., as naked DNA or RNA molecules, 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).
[0098] A 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. Such a vaccine can comprise any nucleic acid molecule or
recombinant molecule of the present invention. Preferred naked
nucleic acid vaccines include at least a portion of a viral genome
(i.e., a viral vector). Preferred viral vectors include those based
on alphaviruses, poxviruses, adenoviruses, herpesviruses, and
retroviruses, with those based on alphaviruses (such as Sindbis or
Semliki virus), species-specific herpesviruses and species-specific
poxviruses being particularly preferred. Any suitable transcription
control sequence can be used, including those disclosed as suitable
for protein production. Particularly preferred transcription
control sequence include cytomegalovirus intermediate 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 are also preferred.
[0099] Naked nucleic acid vaccines 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
naked nucleic acid vaccine ranges from about 1 nanogram (ng) to
about 100 .mu.g, depending on the route of administration and/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 and/or topically. Suitable excipients
include, for example, physiologically acceptable aqueous solutions
(e.g., phosphate buffered saline as well as others disclosed
above), liposomes (including neutral or cationic liposomes), and
other lipid membrane-based vehicles (e.g., micelles or cellular
membranes).
[0100] 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-deficient and/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, and retroviruses. Preferred recombinant virus
vaccines are those based on alphaviruses (such as Sindbis virus),
raccoon poxviruses, species-specific herpesviruses and
species-specific poxviruses. An example of methods to produce and
use recombinant virus vaccines 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.
[0101] 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 helminths as disclosed herein. For example, a
recombinant virus vaccine comprising a D. immitis MIF 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. 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.7 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.
[0102] A recombinant cell vaccine of the present invention includes
recombinant cells of the present invention that express 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), BHK, CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK
and CRFK recombinant cells. Recombinant cell vaccines of the
present invention can be administered in a variety of ways but have
the advantage that they 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. Recombinant cell
vaccines can comprise whole cells or cell lysates.
[0103] 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. Such techniques are known
to those skilled in the art.
[0104] One preferred embodiment of the present invention is the use
of parasitic helminth MIF proteins, nucleic acid molecules,
antibodies and inhibitory compounds of the present invention, and
particularly D. immitis MIF proteins, nucleic acid molecules,
antibodies and inhibitory compounds of the present invention, to
protect an animal from heartworm. It is particularly preferred to
prevent L3 larvae that are delivered to the animal by the mosquito
intermediate host from maturing into adult worms. As such,
preferred therapeutic compositions are those that are able to
inhibit at least one step in the portion of the parasite's
development cycle that includes L3 larvae, third molt, L4 larvae,
fourth molt, immature adult prior to entering the circulatory
system. In dogs, this portion of the development cycle is about 70
days. Particularly preferred therapeutic compositions include D.
immitis MIF-based therapeutic compositions of the present
invention, particularly since MIF is expressed in L3 and L4. Such
compositions include D. immitis MIF nucleic acid molecules, D.
immitis MIF proteins and mimetopes thereof, anti-D. immitis MIF
antibodies, and inhibitors of D. immitis MIF activity. Such
compositions are administered to animals in a manner effective to
protect the animals from heartworm. Additional protection may be
obtained by administering additional protective compounds,
including other D. immitis proteins, nucleic acid molecules,
antibodies and inhibitory compounds.
[0105] Another preferred embodiment of the present invention is the
use of parasitic helminth MIF proteins, nucleic acid molecules,
antibodies and inhibitory compounds of the present invention, and
particularly O. volvulus MIF proteins, nucleic acid molecules,
antibodies and inhibitory compounds of the present invention, to
protect a human from onchocerciasis. t is particularly preferred to
prevent L3, L4 and adult worms from evading the immune system of
the host. Preferred therapeutic compositions are those that are
able to inhibit at least one step in the portion of the parasite's
development cycle that includes L3 larvae, third molt, L4 larvae,
fourth molt and immature adult prior to entering the subcutaneous
tissues. In humans infected with O. volvulus, this portion of the
development cycle is about 150 days. Additional preferred compounds
inhibit adult worm survival. As such, preferred therapeutic
compositions include O. volvulus MIF-based therapeutic compositions
of the present invention. Such compositions include O. volvulus MIF
nucleic acid molecules, O. volvulus MIF proteins and mimetopes
thereof, anti-O. volvulus MIF antibodies, and inhibitors of O.
volvulus MIF activity. Such compositions are administered to humans
in a manner effective to protect humans from onchocerciasis.
Additional protection may be obtained by administering additional
protective compounds, including other Onchocerca, preferably O.
volvulus, proteins, nucleic acid molecules, antibodies, and
inhibitory compounds.
[0106] One therapeutic composition of the present invention
includes an inhibitor of parasitic helminth MIF activity, i.e., a
compound capable of substantially interfering with the function of
a parasitic helminth MIF susceptible to inhibition by an inhibitor
of parasitic helminth MIF activity.
[0107] An inhibitor of MIF activity can be identified using
parasitic helminth, and preferably D. immitis and/or O. volvulus
MIF proteins of the present invention. One embodiment of the
present invention is a method to identify a compound capable of
inhibiting MIF activity of a parasitic helminth. Such a method
includes the steps of (a) contacting (e.g., combining, mixing) an
isolated parasitic helminth MIF protein with a putative inhibitory
compound under conditions in which, in the absence of the compound,
the protein has MIF activity, and (b) determining if the putative
inhibitory compound inhibits the MIF activity. Putative inhibitory
compounds to screen include organic molecules, antibodies
(including mimetopes thereof) and substrate analogs. Methods to
determine MIF activity are known to those skilled in the art; see,
for example, citations in background section and references
included therein.
[0108] The present invention also includes a test kit to identify a
compound capable of inhibiting MIF activity of a parasitic
helminth. Such a test kit includes an isolated parasitic helminth
MIF protein having MIF activity and a means for determining the
extent of inhibition of MIF 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.
[0109] MIF inhibitors isolated by such a method, and/or test kit,
can be used to inhibit any MIF that is susceptible to such an
inhibitor. Preferred MIF enzymes to inhibit are those produced by
parasitic helminths. A particularly preferred MIF inhibitor of the
present invention is capable of protecting an animal from heartworm
or onchocerciasis. It is also within the scope of the present
invention to use inhibitors of the present invention to target
MIF-related disorders in animals. Therapeutic compositions
comprising MIF inhibitory compounds of the present invention can be
administered to animals in an effective manner to protect animals
from disease caused by the targeted MIF enzymes, and preferably to
protect animals from heartworm and humans from onchocerciasis.
Effective amounts and dosing regimens can be determined using
techniques known to those skilled in the art.
[0110] 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 other 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 Dirofilaria and Onchocerca.
[0111] The following examples are provided for the purposes of
illustration and are not intended to limit the scope of the present
invention.
EXAMPLES
Example 1
[0112] This Example describes the isolation and sequencing of
several D. immitis MIF nucleic acid molecules of the present
invention. It is to be noted that the Examples include a number of
molecular biology, microbiology, immunology and biochemistry
techniques considered to be known to those skilled in the art.
Disclosure of such techniques can be found, for example, in
Sambrook et al., ibid. and related references.
[0113] A. A D. immitis MIF nucleic acid molecule of about 282
nucleotides, denoted nDiMIF.sub.282, was identified by its ability
to encode a protein that selectively bound to at least one
component of immune serum collected from a dog immunized with D.
immitis larvae. Immune serum was produced and used as described in
WO 94/15593, ibid. Specifically, a D. immitis L4 cDNA expression
library was constructed in Uni-ZAp.TM. XR vector (available from
Stratagene Cloning Systems, La Jolla, Calif.), using Stratagene's
ZAP-cDNA Synthesis Kit protocol and fourth stage (L4) larval mRNAs.
Using the protocol described in the Stratagene picoBlue
immunoscreening kit, the L4 larval cDNA expression library was
screened with immune dog sera, prepared as described in WO
94/15593, ibid. Immunoscreening of duplicate plaque lifts of the
cDNA library with the same immune dog serum identified a clone
containing nucleic acid molecule nDiMIF.sub.252.
[0114] The plaque-purified clone including D. immitis nucleic acid
sequence nDiMIF.sub.282 was converted into a double stranded
recombinant molecule, herein denoted as p.beta.gal-nDiMIF.sub.282,
using ExAssist.TM. helper phage and SOLR.TM. E. coli according to
the in vivo excision protocol described in the Stratagene ZAP-cDNA
Synthesis Kit. Double stranded plasmid DNA was prepared using an
alkaline lysis protocol, such as that described in Sambrook et al.,
ibid. The plasmid DNA was digested with EcoRI and XhoI restriction
endonucleases to release a single D. immitis nDiMIF.sub.282 DNA
fragment of about 282 nucleotides in size.
[0115] The plasmid containing D. immitis nDiMIF.sub.282 was
sequenced using the Sanger dideoxy chain termination method, as
described in Sambrook et al., ibid. An about 282 nucleotide
consensus sequence of the entire D. immitis nDiMIF.sub.282 DNA
fragment was determined and is presented as SEQ ID NO:10. The D.
immitis nDiMIF.sub.282 sequence represents a partial cDNA clone
truncated on the amino terminus and spans nucleotides from about
251 through about 532 of SEQ ID NO:1 (the production of which is
described below). The first stop codon within the D. immitis
nDiMIF.sub.282 sequence spans nucleotides from about 103 through
about 105 of SEQ ID NO:10. A putative polyadenylation signal (5'
AATAAA 3') is located in a region spanning from about nucleotide
249 through about 254 of SEQ ID NO:10.
[0116] Translation of SEQ ID NO:10 yields a protein of about 34
amino acids, denoted PDiMIF.sub.34, the amino acid sequence of
which is presented in SEQ ID NO:11. SEQ ID NO:11 corresponds to
about from amino acid 82 through about amino acid 115 of SEQ ID
NO:2 (the production of which is described below). The coding
region of PDiMIF.sub.34 is referred to herein as nDiMIF.sub.102,
the nucleic acid sequence of which is represented in SEQ ID
NO:12.
[0117] B. A D. immitis nDiMIF nucleic acid molecule containing
apparently the entire coding region of nDiMIF was produced using
the following two primers to amplify, by polymerase chain reaction
(PCR), a MIF nucleic acid molecule from a D. immitis L3 cDNA
library: (a) a vector sense primer having the nucleic acid sequence
5' CGCTCTAGAACTAGTGGATC 3', denoted herein as SEQ ID NO:13; and (b)
C3 ant, an antisense primer having nucleic acid sequence 5'
CCAATTATCCGAAAGTAGATCC 3', denoted herein as SEQ ID NO:14, that was
designed from the complement of the region spanning from about
nucleotide 88 through about nucleotide 109 of SEQ ID NO:10
(corresponding to a region spanning from about nucleotide 338
through about nucleotide 359 of SEQ ID NO:1), that region including
the first stop codon detected in the D. immitis nDiMIF.sub.282
sequence. The resultant PCR product of about 355 nucleotides,
denoted D. immitis nDiMIF(1).sub.355 was cloned into the TA cloning
vector (available from Invitrogen, San Diego, Calif.). An antisense
probe having the nucleic acid sequence 5'
CTTCGGAATTTTCAGCTCATCAGCGAGC 3' (denoted herein as SEQ ID NO:15),
representing the complement of nucleotide 6 through about
nucleotide 33 of SEQ ID NO:10, was used to verify the authenticity
of the D. immitis nDiMIF(1).sub.355 PCR product, by hybridization
analysis.
[0118] The nucleic acid sequence of D. immitis nDiMIF(1).sub.355 is
presented in SEQ ID NO:16. Translation of SEQ ID NO:16 yields an
apparent full-length protein of about 115 amino acids, denoted
PDiMIF(1).sub.115, the amino acid sequence of which is presented in
SEQ ID NO:2. The coding region of PDiMIF(1).sub.115 is referred to
herein as nDiMIF(1).sub.345, the nucleic acid sequence of which is
represented in SEQ ID NO:17.
[0119] C. A second, independent PCR clone was amplified from the L3
cDNA library with the vector and C3 ant primers and is denoted
herein as nDiMIF(2).sub.333. The nucleic acid sequence of
nDiMIF(2).sub.333, which is represented herein as SEQ ID NO:18,
differs from that of nDiMIF(1).sub.355 (SEQ ID NO:16) by one base:
SEQ ID NO:18 contains a T at position 37 (i.e., the position
corresponding to position 49 of SEQ ID NO:16) whereas SEQ ID NO:16
contains an A at position 49. As such, nDiMIF(2).sub.333 represents
an allelic variant of nDiMIF(1).sub.355. The deduced amino acid
(arginine) encoded by nDiMIF(2).sub.333 at position 35-37 was the
same as that encoded by the D. immitis nDiMIF(1).sub.355 sequence
at position 47-49. As such, translation of SEQ ID NO:18 yields a
truncated protein of about 110 amino acids, denoted
PDiMIF(2).sub.110, the amino acid sequence of which corresponds to
amino acids 6 through 115 of SEQ ID NO:2. The coding region of
PDiMIF(2).sub.110 is referred to herein as nDiMIF(2).sub.330, the
nucleic acid sequence of which is represented in SEQ ID NO:19.
[0120] To confirm the D. immitis origin of the isolated MIF cDNA
nucleic acid molecules, a Southern blot containing about 10
micrograms of EcoRI restricted Dirofilaria immitis genomic DNA and
Aedes aegypti genomic DNA was hybridized under stringent conditions
with nDiMIF(2).sub.330 DNA radiolabeled by random priming with the
Megaprime DNA Labeling System (available from Amersham Life
Science, Arlington Heights, Ill.). The probe detected two bands of
about 4390 and 1490 nucleotides only in the D. immitis genomic
DNA.
[0121] D. A deduced nucleic acid sequence combining the sequence
information disclosed in Example 1, A-C, is presented in SEQ ID
NO:1. SEQ ID NO:1 was determined by combining the unique and common
nucleotide sequences from the PCR clones D. immitis
nDiMIF(1).sub.355, D. immitis nDiMIF(2).sub.333, and the cDNA clone
D. immitis nDiMIF.sub.282. As such, SEQ ID NO:1 represents the
sequences of two nucleic acid molecules of the present invention,
namely D. immitis nDiMIF(1).sub.532 and D. immitis
nDiMIF(2).sub.532. Nucleotides 1-355 and 23-355, respectively, of
SEQ ID NO:1 were identified from the D. immitis allelic variant
nucleic acid molecules nDiMIF(1).sub.355 and nDiMIF(2).sub.333.
Nucleotides 251 through 532 of SEQ ID NO:1 were identified from D.
immitis nucleic acid molecule nDiMIF.sub.282.
[0122] Translation of the entire 532 nucleotides of SEQ ID NO:1
yields a protein of about 115 amino acids, denoted
PDiMIF(1).sub.115, that has an amino acid sequence as represented
in SEQ ID NO:2, assuming the ATG codon spanning nucleotides from
about 8 through about 10 of SEQ ID NO:1 is the initiation codon,
and that the stop codon is the TAA spanning nucleotides from about
353 through about 355 of SEQ ID NO:1. A putative polyadenylation
signal (5' AATAAA 3') is located in a region spanning nucleotides
from about 499 through about 504 of SEQ ID NO:1. The amino acid
sequence of D. immitis PDiMIF(1).sub.115 (i.e., SEQ ID NO:2)
predicts that PDiMIF(1).sub.115 has an estimated molecular weight
of about 12.3 kD and an estimated pI of about 8.3. There are 3
predicted N-glycosylation sites in the PDiMIF(1).sub.115 deduced
amino acid sequence, which are located in regions spanning amino
acids from about 73 through about 75, from about 103 through about
105 and from about 110 through about 112 of SEQ ID NO:2.
[0123] A homology search of the non-redundant protein sequence
database was performed through the National Center for
Biotechnology Information using the BLAST network. This database
includes +SwissProt+PIR+SPUpdate+G- enPept+GPUpdate. The search was
performed using SEQ ID NO:2 and showed significant homology to
macrophage migration inhibition factor proteins of mammalian and
avian origins, spanning from about amino acid 1 through about amino
acid 115 of SEQ ID NO:2. The highest scoring matches of the
homology search at the amino acid level include: Genbank accession
number M25639: human macrophage migration inhibitory factor, about
53% identical; C47274: chicken macrophage migration inhibitory
factor, about 55% identical; and P80177: human macrophage migration
inhibitory factor, about 52% identical. At the nucleotide level,
the coding regions represented in SEQ ID NO:3 were most similar to
that of chicken macrophage migration inhibitory factor, being about
52% identical.
Example 2
[0124] This Example discloses the production of a recombinant cell
of the present invention.
[0125] Recombinant molecule pHis-nDiMIF(1).sub.348, containing D.
immitis MIF nucleotides from about 8 through about 355 operatively
linked to trc transcription control sequences and to a fusion
sequence encoding a poly-histidine segment comprising 6 histidines
was produced in the following manner. An about 348-nucleotide DNA
fragment containing nucleotides spanning from about 8 through about
355 of SEQ ID NO:16, denoted herein as nDiMIF(1).sub.345, was PCR
amplified from nucleic acid molecule D. immitis nDiMIF(1).sub.355,
produced as described in Example 1, using the primers MIF sen 5'
GGACGGATCCAATGCCATATTTCACGATC 3' (denoted herein as SEQ ID NO:20;
BamHI site in bold) and MIF ant 5' GAGCGAATTCTTATCCGAAAGTAGATCC 3'
(denoted herein as SEQ ID NO:21; EcoRI site in bold). Recombinant
molecule pHis-nDiMIF(1).sub.348 was produced by digesting the
nDiMIF(1).sub.348-containing PCR product with BamHI and EcoRI
restriction endonucleases, gel purifying the resulting fragment and
directionally subcloning it into expression vector pTrcHisB
(available from Invitrogen) that had been cleaved with BamHI and
EcoRI and gel purified.
[0126] Recombinant molecule pHis-nDiMIF(1).sub.348 was transformed
into E. coli to form recombinant cell E.
coli:pHis-nDiMIF(1).sub.348 using standard techniques as disclosed
in Sambrook et al., ibid.
Example 3
[0127] This Example discloses the production of a MIF protein of
the present invention in a prokaryotic cell.
[0128] Recombinant cell E. coli:pHis-nDiMIF(1).sub.348, produced as
described in Example 2, was cultured in shake flasks containing an
enriched bacterial growth medium containing 0.1 mg/ml ampicillin
and 0.1% glucose at about 32.degree. C. When the cells reached an
OD.sub.600 of about 0.4, expression of D. immitis nDiMIF(1).sub.348
was induced by addition of about 0.5 mM
isopropyl-.beta.-D-thiogalactoside (IPTG), and the cells were
cultured for about 3 hours at about 32.degree. C. Protein
production was monitored by SDS PAGE of recombinant cell lysates,
followed by Coomassie blue staining, using standard techniques.
Recombinant cell E. coli:pHis-nDiMIF(1).sub.348 produced a fusion
protein, denoted herein as PHIS-PDiMIF(1).sub.115, that migrated
with an apparent molecular weight of about 16 kD.
[0129] Immunoblot analysis of recombinant cell E.
coli:pHis-nDiMIF(1).sub.- 348 lysates indicated that the about 16
kD protein was able to bind to a T7 tag monoclonal antibody
(available from Novagen, Inc., Madison, Wis.) directed against the
fusion portion of the recombinant PHIS-PDiMIF(1).sub.115 fusion
protein.
[0130] The PHIS-PDiMIF(1).sub.115 histidine fusion protein was
separated from E. coli proteins by nickel chelation chromatography
and an imidazole gradient. Immunoblot analysis of the E.
coli:pHis-nDiMIF(1).sub.348 lysate, column eluate and column void
volume indicated that the PHIS-PDiMIF(1).sub.115 16 kD protein
isolated using nickel column chromatography was able to selectively
bind to a T7 tag monoclonal antibody.
Example 4
[0131] This Example describes the production of a MIF protein of
the present invention in a eukaryotic cell.
[0132] Recombinant molecule pVL1393-nDiMIF(1).sub.348, containing a
D. immitis MIF nucleic acid molecule spanning nucleotides from
about 8 through about 355 of SEQ ID NO:16 operatively linked to
baculovirus polyhedron transcription control sequences was produced
in the following manner. In order to subclone a MIF nucleic acid
molecule into baculovirus expression vectors, a MIF nucleic acid
molecule-containing fragment was PCR amplified from D. immitis
nDiMIF(1).sub.355 DNA (produced as in Example 1), using a sense
primer BvMIF sen (5' CGCGGATCCTATAAATATGCCATATT- TCACGATCG 3'
(denoted herein as SEQ ID NO:22; BamHI site in bold) and an
antisense primer BvMIF ant 5' CCGGAATTCTTATCCGAAAGTAGATCC 3'
(denoted herein as SEQ ID NO:23; EcoRI site in bold). The
N-terminal primer (SEQ ID NO:22) was designed from
nDiMIF(1).sub.355 sequence with modifications to enhance expression
in the baculovirus system. The PCR product was digested with BamHI
and EcORI to produce nucleic acid molecule nBvDiMIF(1).sub.348 and
directionally subcloned into the unique BamHI and EcoRI sites of
pVL1393 (available from Invitrogen) baculovirus shuttle plasmid to
produce recombinant molecule pVL1393-nDiMIF(1).sub.348.
[0133] Recombinant molecule pVL1393-nDiMIF(1).sub.348 plasmid DNA
was co-transfected into S. frugiperda Sf9 cells (donated by the
Colorado Bioprocessing Center, Fort Collins, Co.) with wild type
baculovirus DNA (AcMNPV) and insectin cationic liposomes (available
from Invitrogen) to form recombinant cell S.
frugiperda:pVL1393-nDiMIF(1).sub.348. The resulting recombinant
virus, denoted Bv-nDiMIF(1).sub.348, was cultivated for increased
production of recombinant virus and to verify expression of
PDiMIF(1).sub.115. Immunoblot analysis using immune dog 2094-339
antisera demonstrated that total lysates of insect cells
transfected with recombinant baculovirus Bv-nDiMIF(1).sub.348
expressed a protein, encoded by nDiMIF(1).sub.348, namely
BvPDiMIF(1).sub.115, that migrated with an apparent molecular
weight of about 19 kD.
Example 5
[0134] This Example describes the production of a MIF protein of
the present invention in a eukaryotic cell.
[0135] Recombinant molecule pKB3poly-nMIF(1).sub.348, containing a
D. immitis MIF nucleic acid molecule spanning nucleotides from
about 8 through about 355 of SEQ ID NO:16 operatively linked to the
vaccinia virus P.sub.11 late promoter transcription control
sequences was produced in the following manner. The pKB3poly
poxvirus shuttle vector was created by modifying a region of
plasmid pKB3 (P.sub.11-type), pKB3 (P.sub.11-type) plasmid is
described in U.S. Pat. No. 5,348,741, by Esposito et al., issued
Sep. 20, 1994)) such that the initiation codon linked to the
P.sub.11 promoter was mutated and additional unique polylinker
restriction sites were added. The resulting poxvirus vector,
referred to as pKB3poly, requires the insert DNA to provide the ATG
initiation codon when inserted downstream of the P.sub.11 promoter.
The pKB3poly vector is designed such that foreign DNA cloned into
the polylinker region of pKB3poly vector will recombine into the tk
gene of wildtype poxvirus.
[0136] In order to subclone a MIF nucleic acid molecule into
pKB3poly expression vector, MIF nucleic acid molecule-containing
fragments were restricted from D. immitis pVL1393-nMIF(1).sub.348
DNA (produced as in Example 4), by BamHI and EcoRI restriction
endonucleases. The about 348 nucleotide insert DNA (referred to as
Rcn-nDiMIF(1).sub.348) was treated with Klenow enzyme to create
blunt ends resulting in the production of nucleic acid molecule
nRcnDiMIF(1).sub.348, gel purified and subcloned into the pKB3poly
shuttle vector which had been restricted with SmaI restriction
endonuclease, treated with calf intestinal phosphatase and gel
purified to produce recombinant molecule
pKB3poly-nDiMIF(1).sub.348. The proper orientation of the insert
was verified by restriction mapping.
[0137] In order to produce a recombinant raccoon poxvirus capable
of directing the production of PDiMIF(1).sub.115, BS-C-1 African
green monkey kidney cells (obtained from American Type Culture
Collection (ATCC), Rockville, Md.) were infected with wild type
raccoon poxvirus RCN CDC/V71-I-85A) (obtained from Joe Esposito;
Esposito and Knight 1985, Virology 143:230-251) and then
transfected with the pKB3poly-nDiMIF(1).sub.348 vector DNA and
lipofectAMINE (available from Gibco, BRL, Bethesda, Md.) to form
recombinant cell BS-C-1:pKB3poly-nDiMIF(1).sub.348. The resulting
recombinant virus, denoted Rcn-nDiMIF(1).sub.348, was cultivated in
RAT2 rat embryo, thymidine kinase mutant cells (available from
ATCC) in the presence of bromodeoxyuridine to select for TK.sup.-
recombinants.
Example 6
[0138] This Example describes the isolation and sequencing of two
O. volvulus MIF nucleic acid molecules of the present invention.
The O. volvulus MIF nucleic acid molecules were identified using a
D. immitis MIF nucleic acid molecule of the present invention.
[0139] O. volvulus nucleic acid molecules nOvMIF(1).sub.440 and
nOvMIF(2).sub.522 were produced in the following manner. An adult
O. volvulus cDNA library (Touboro, Cameroun, available from ATCC)
was screened with D. immitis MIF nucleic acid molecule
nDiMIF(1).sub.355 using stringent hybridization conditions, which
are known to those skilled in the art (see, for example, Sambrook
et al., ibid.; such conditions typically permit isolation of
nucleic acid molecules having at least about 70% nucleic acid
sequence identity with the nucleic acid probe used in the
hybridization). Several clones that hybridized with
nDiMIF(1).sub.355, two of which were purified and submitted to
nucleic acid sequence analysis. The nucleic acid sequence of an
isolate containing an O. volvulus MIF nucleic acid molecule of
about 440 nucleotides, denoted herein as nOvMIF(1).sub.440, is
represented herein as SEQ ID NO:4. The nucleic acid sequence of the
other isolate, which contained an O. volvulus MIF nucleic acid
molecule of about 522 nucleotides, denoted herein as
nOvMIF(2).sub.522, is represented herein as SEQ ID NO:7.
[0140] Translation of SEQ ID NO:4 yields an apparent full-length
protein of about 115 amino acids, denoted POvMIF(1).sub.115,
assuming a start codon spanning from about nucleotide 8 through
about nucleotide 10, and a stop codon spanning from about
nucleotide 353 through about nucleotide 355 of SEQ ID NO:4. The
amino acid sequence of POvMIF(1).sub.115 is represented herein as
SEQ ID NO:5. The coding region of POvMIF(1).sub.115 is referred to
herein as nOvMIF(1).sub.345, the nucleic acid sequence of which is
represented in SEQ ID NO:6. The amino acid sequence of D. immitis
POvMIF(1).sub.115 (i.e., SEQ ID NO:5) predicts that
POvMIF(1).sub.115 has an estimated molecular weight of about 12.24
kD and an estimated pI of about 9.21. There are 3 predicted
N-glycosylation sites in the POvMIF(1).sub.115 deduced amino acid
sequence, which are located in regions spanning amino acids from
about 14 through about 16, from about 73 through about 75 and from
about 110 through about 112 of SEQ ID NO:5.
[0141] Translation of SEQ ID NO:7 yields a protein of about 114
amino acids, denoted POvMIF(2).sub.114, assuming a stop codon
spanning from about nucleotide 343 through about nucleotide 345 of
SEQ ID NO:7. The amino acid sequence of POvMIF(2).sub.114 is
represented herein as SEQ ID NO:8. The open reading frame encoding
POvMIF(2).sub.115 is referred to herein as nOvMIF(2).sub.342, the
nucleic acid sequence of which is represented in SEQ ID NO:9.
[0142] The two allelic variants SEQ ID NO:4 and SEQ ID NO:7 are
identical in their coding regions (i.e., SEQ ID NO:6 and SEQ ID
NO:9, respectively), except that (a) SEQ ID NO:7 apparently lacks a
start codon; (b) SEQ ID NO:7 is about 10 nucleotides shorter than
SEQ ID NO:4 at the 5' end; and (c) the region spanning from about
nucleotide 1 through about nucleotide 19 of SEQ ID NO:7 is only
about 47% identical to the region spanning from about nucleotide 10
through about nucleotide 29 of SEQ ID NO:4.
[0143] Comparison of nucleic acid molecules containing O. volvulus
and D. immitis MIF coding regions (e.g., nOvMIF(1).sub.345 and
nDiMIF(1).sub.345) indicated that the two MIF coding regions were
about 87% identical at the nucleic acid sequence level. Comparison
of apparent full-length O. volvulus and D. immitis MIF proteins
(i.e., POvMIF(1).sub.115 and PDiMIF(1).sub.115) indicated that the
two MIF proteins were about 88% identical at the amino acid
level.
[0144] A homology search of the non-redundant protein sequence
database, performed as described in Example 1 but using SEQ ID
NO:5, showed significant homology to macrophage migration
inhibition factor proteins of mammalian and avian origins, spanning
from about amino acid 1 through about amino acid 115 of SEQ ID
NO:5. The highest scoring match, i.e., 44% identity, was found
between SEQ ID NO:5 and human and bovine MIFs. SEQ ID NO:5 was
about 43% identical to rat, mouse and chicken MIFs. At the
nucleotide level, the coding region represented in SEQ ID NO:6 was
most similar to that of chicken migration inhibitory factor, being
about 51% identical.
* * * * *