Carboxylesterase nucleic acid molecules, proteins and uses thereof

The present invention relates to arthropod esterase proteins; to arthropod esterase nucleic acid molecules, including those that encode such esterase proteins; to antibodies raised against such esterase proteins; and to other compounds that inhibit arthropod esterase 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 hematophagous arthropod infestation.

FIELD OF THE INVENTION 
The present invention relates to arthropod esterase 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 other inhibitors, as well as their use to 
protect an animal from hematophagous arthropod infestation. 
BACKGROUND OF THE INVENTION 
Hematophagous arthropod infestation of animals is a health and economic 
concern because hematophagous arthropods are known to cause and/or 
transmit a variety of diseases. Hematophagous arthropods directly cause a 
variety of diseases, including allergies, and also carry a variety of 
infectious agents including, but not limited to, endoparasites (e.g., 
nematodes, cestodes, trematodes and protozoa), bacteria and viruses. In 
particular, the bites of hematophagous arthropods are a problem for 
animals maintained as pets because the infestation becomes a source of 
annoyance not only for the pet but also for the pet owner who may find his 
or her home generally contaminated with insects. As such, hematophagous 
arthropods are a problem not only when they are on an animal but also when 
they are in the general environment of the animal. 
Bites from hematophagous arthropods are a particular problem because they 
not only can lead to disease transmission but also can cause a 
hypersensitive response in animals which is manifested as disease. For 
example, bites from fleas can cause an allergic disease called flea 
allergic (or allergy) dermatitis (FAD). A hypersensitive response in 
animals typically results in localized tissue inflammation and damage, 
causing substantial discomfort to the animal. 
The medical importance of arthropod infestation has prompted the 
development of reagents capable of controlling arthropod infestation. 
Commonly encountered methods to control arthropod infestation are 
generally focused on use of insecticides. While some of these products are 
efficacious, most, at best, offer protection of a very limited duration. 
Furthermore, many of the methods are often not successful in reducing 
arthropod populations. In particular, insecticides have been used to 
prevent hematophagous arthropod infestation of animals by adding such 
insecticides to shampoos, powders, collars, sprays, foggers and liquid 
bath treatments (i.e., dips). Reduction of hematophagous arthropod 
infestation on the pet has been unsuccessful for one or more of the 
following reasons: (1) failure of owner compliance (frequent 
administration is required); (2) behavioral or physiological intolerance 
of the pet to the pesticide product or means of administration; and (3) 
the emergence of hematophagous arthropod populations resistant to the 
prescribed dose of pesticide. However, hematophagous arthropod populations 
have been found to become resistant to insecticides. 
Prior investigators have described insect carboxylesterase (CE) protein 
biochemistry, for example, Chen et al., Insect Biochem. Molec. Biol., 
24:347-355, 1994; Whyard et al., Biochemical Genetics, 32:924, 1994 and 
Argentine et al., Insect Biochem. Molec Biol, 25:621-630, 1995. Other 
investigators have disclosed certain insect CE amino acid sequences, for 
example, Mouches et al., Proc Natl Acad Sci USA, 87:2574-2578, 1990 and 
Cooke et al., Proc Natl Acad Sci USA, 86:1426-1430, 1989, and nucleic acid 
sequence (Vaughn et al., J. Biol. Chem., 270:17044-17049, 1995). 
Prior investigators have described certain insect juvenile hormone esterase 
(JHE) nucleic acid and amino acid sequences: for example, sequence for 
Heliothis virescens is disclosed by Hanzlik et al., J. Biol. Chem., 
264:12419-12425, 1989; Eldridge et al., App Environ Microbiol, 
58:1583-1591, 1992; Bonning et al., Insect Biochem. Molec. Biol., 
22:453-458, 1992; Bonning et al., Natural and Engineered Pest Management 
Agents, pp. 368-383, 1994 and Harshman et al., Insect Biochem. Molec. 
Biol, 24:671-676, 1994; sequence for Manduca sexta is disclosed by 
Vankatesh et al., J Biol Chem, 265:21727-21732, 1990; sequence for 
Trichoplusia ni is disclosed by Venkataraman et al., Dev. Genet., 
15:391-400, 1994 and Jones et al., Biochem. J., 302:827-835, 1994; and 
sequence for Lymantria dispar is disclosed by Valaitis, Insect Biochem. 
Molec. Biol., 22:639-648, 1992. 
Identification of an esterase of the present invention is unexpected, 
however, because even the most similar nucleic acid sequence identified by 
previous investigators could not be used to identify an esterase of the 
present invention. In addition, identification of an esterase protein of 
the present invention is unexpected because a protein fraction from flea 
prepupal larvae that was obtained by monitoring for serine protease 
activity surprisingly also contained esterase proteins of the present 
invention. 
In summary, there remains a need to develop a reagent and a method to 
protect animals or plants from hematophagous arthropod infestation. 
SUMMARY OF THE INVENTION 
The present invention relates to a novel product and process for protection 
of animals or plants from arthropod infestation. According to the present 
invention there are provided arthropod esterase proteins and mimetopes 
thereof; arthropod nucleic acid molecules, including those that encode 
such proteins; antibodies raised against such esterase proteins (i.e., 
anti-arthropod esterase antibodies); and compounds that inhibit arthropod 
esterase activity (i.e, inhibitory compounds or inhibitors). 
The present invention also includes methods to obtain such proteins, 
mimetopes, nucleic acid molecules, antibodies and inhibitory compounds. 
Also included in the present invention are therapeutic compositions 
comprising such proteins, mimetopes, nucleic acid molecules, antibodies, 
and/or inhibitory compounds, as well as use of such therapeutic 
compositions to protect animals from arthropod infestation. 
Identification of an esterase of the present invention is unexpected, 
however, because the most similar nucleic acid sequence identified by 
previous investigators could not be used to identify an esterase of the 
present invention. In addition, identification of an esterase protein of 
the present invention is unexpected because a protein fraction from flea 
prepupal larvae that was obtained by monitoring for serine protease 
activity surprisingly also contained esterase proteins of the present 
invention. 
One embodiment of the present invention is an isolated nucleic acid 
molecule that hybridizes under stringent hybridization conditions with a 
gene comprising a nucleic acid sequence including 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:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ 
ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID 
NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID 
NO:30, SEQ ID NO:32, SEQ I) NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID 
NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID 
NO:59, SEQ ID NO:60 and/or SEQ ID NO:61. 
The present invention also includes a nucleic acid molecule that hybridizes 
under stringent hybridization conditions with a nucleic acid molecule 
encoding a protein comprising at least one of the following amino acid 
sequences: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID 
NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID 
NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID 
NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and/or SEQ ID NO:58; and 
particularly a nucleic acid molecule that hybridizes with a nucleic acid 
sequence that is a complement of a nucleic acid sequence encoding any of 
the amino acid sequences. A preferred nucleic acid molecule of the present 
invention includes a nucleic acid molecule comprising a nucleic acid 
sequence including 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:13, SEQ ID 
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID 
NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID 
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID 
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID 
NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60 and/or SEQ 
ID NO:61, and allelic variants thereof. 
The present invention also includes an isolated carboxylesterase nucleic 
acid molecule comprising a nucleic acid sequence encoding a protein 
comprising an amino acid sequence including SEQ ID NO:5, SEQ ID NO:19, SEQ 
ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID 
NO:44 and/or SEQ ID NO:53. SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ 
ID NO:42, SEQ ID NO:43 and SEQ ID NO:44 represent N-terminal amino acid 
sequences of carboxylesterases isolated from prepupal flea larvae, the 
production of which are described in the Examples of the present 
application. 
The present invention also relates to recombinant molecules, recombinant 
viruses and recombinant cells that include a nucleic acid molecule of the 
present invention. Also included are methods to produce such nucleic acid 
molecules, recombinant molecules, recombinant viruses and recombinant 
cells. 
Another embodiment of the present invention includes an isolated esterase 
protein that is encoded by a nucleic acid molecule that hybridizes under 
stringent hybridization conditions to (a) a nucleic acid molecule that 
includes at least one of the following nucleic acid sequences: SEQ ID 
NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17, 
SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ 
ID NO:35, SEQ ID NO:38, SEQ ID NO:52, SEQ ID NO:59 and SEQ ID NO:61; 
and/or (b) a nucleic acid molecule encoding a protein including at least 
one of the following amino acid sequences: SEQ ID NO:39, SEQ ID NO:40, SEQ 
ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID 
NO:54 and SEQ ID NO:55. One embodiment is a carboxylesterase protein 
encoded by a nucleic acid molecule that hybridizes under stringent 
hybridization conditions to a nucleic acid molecule that encodes a protein 
comprising at least one of the following amino acid sequences: SEQ ID 
NO:5, SEQ ID NO:19, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID 
NO:42, SEQ ID NO:43, SEQ ID NO:44 and/or SEQ ID NO:53. Preferred proteins 
of the present invention are isolated flea proteins including at least one 
of the following amino acid sequences: SEQ ID NO:2, SEQ ID NO:5, SEQ ID 
NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID 
NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID 
NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID 
NO:55 and SEQ ID NO:58; also included are proteins encoded by allelic 
variants of nucleic acid molecules encoding proteins comprising any of the 
above-listed amino acid sequences. 
The present invention also relates to mimetopes of arthropod esterase 
proteins as well as to isolated antibodies that selectively bind to 
arthropod esterase proteins or mimetopes thereof. Also included are 
methods, including recombinant methods, to produce proteins, mimetopes and 
antibodies of the present invention. 
The present invention also includes a formulation of flea carboxylesterase 
proteins, in which the proteins, when submitted to 14% Tris-glycine 
SDS-PAGE, comprise a fractionation profile as depicted in FIG. 3, in which 
the proteins have carboxylesterase activity. 
Also included in the present invention is a formulation of flea 
carboxylesterase proteins, in which the proteins, when submitted to 
IEF-PAGE, comprise a fractionation profile as depicted in FIG. 4, lane 3, 
lane 4, lane 5, lane 6 and/or lane 7, wherein the proteins have 
carboxylesterase activity. 
Another embodiment of the present invention is an isolated flea protein or 
a formulation of flea proteins that hydrolyzes .alpha.-napthyl acetate to 
produce .alpha.-napthol, when the protein is incubated in the presence of 
.alpha.-napthyl acetate contained in 20 mM Tris at pH 8.0 for about 15 
minutes at about 37.degree. C. 
Yet another embodiment of the present invention is an isolated flea protein 
or a formulation of flea proteins that hydrolyzes the methyl ester group 
of juvenile hormone to produce a juvenile hormone acid. 
Another embodiment of the present invention is a method to identify a 
compound capable of inhibiting flea carboxylesterase activity, the method 
comprising: (a) contacting an isolated flea carboxylesterase with a 
putative inhibitory compound under conditions in which, in the absence of 
the compound, the protein has carboxylesterase activity; and (b) 
determining if the putative inhibitory compound inhibits the activity. 
Also included in the present invention is a test kit to identify a 
compound capable of inhibiting flea carboxylesterase activity, the test 
kit comprising an isolated flea carboxylesterase protein having esterase 
activity and a means for determining the extent of inhibition of the 
activity in the presence of a putative inhibitory compound. 
Yet another embodiment of the present invention is a therapeutic 
composition that is capable of reducing hematophagous ectoparasite 
infestation. Such a therapeutic composition includes at least one of the 
following protective compounds: an isolated hematophagous ectoparasite 
carboxylesterase protein or a mimetope thereof, an isolated 
carboxylesterase nucleic acid molecule that hybridizes under stringent 
hybridization conditions with a Ctenocephalides felis carboxylesterase 
gene, an isolated antibody that selectively binds to a hematophagous 
ectoparasite carboxylesterase protein, and an inhibitor of 
carboxylesterase activity identified by its ability to inhibit the 
activity of a flea carboxylesterase. A therapeutic composition of the 
present invention can also include an excipient, an adjuvant and/or a 
carrier. Preferred esterase 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 hematophagous ectoparasite infestation, 
comprising the step of administering to the animal a therapeutic 
composition of the present invention.