Flea nucleic acid sequences and uses thereof

The present invention relates to ectoparasite histamine releasing factor (HRF) proteins; to ectoparasite HRF nucleic acid molecules, including those that encode such HRF proteins; to antibodies raised against such HRF proteins; and to compounds that inhibit ectoparasite HRF 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 reduce ectoparasite burden of animals.

FIELD OF THE INVENTION 
The present invention relates to novel ectoparasite histamine releasing 
factor (HRF) nucleic acid molecules, to proteins encoded by such nucleic 
acid molecules, to antibodies raised against such proteins, and to 
inhibitors of such proteins, as well as to the use of such compositions to 
reduce ectoparasite infestation. 
BACKGROUND OF THE INVENTION 
Ectoparasite infestation of animals is of health and economic concern 
because ectoparasites are known to cause and/or transmit a variety of 
diseases. Ectoparasites cause and/or carry infectious agents that cause, 
for example, allergy dermatitis, anemia, murine typhus, plague and 
tapeworm. In addition, ectoparasites, in particular fleas, are a problem 
for animals maintained as pets because the infestation becomes a source of 
annoyance for the pet owner who may find his or her home generally 
contaminated with ectoparasites which feed on the pets. As such, 
ectoparasites are a problem not only when they are on an animal but also 
when they are in the general environment of the animal. 
In addition, ectoparasite bites, such as flea bites, can cause a 
hypersensitive response in animals. For example, hypersensitive responses 
to flea bites is manifested in a disease called flea allergy dermatitis 
(FAD). Hypersensitivity refers to a state of altered reactivity in which 
an animal, having been previously exposed to a compound, exhibits an 
allergic response to the compound upon subsequent exposures. There are 
four major types of hypersensitive responses (described in detail in, for 
example, Janeway et al., in Immunobiology, Garland Publishers, New York, 
N.Y., 1994). FAD can have manifestations of both immediate and 
delayed-type hypersensitivity. Typically, an immediate hypersensitive 
response in an animal susceptible to FAD includes wheal formation at the 
site of a bite. Such wheals can develop into a papule with a crust, 
representative of delayed-type hypersensitivity. 
Foreign compounds that induce symptoms of immediate and/or delayed 
hypersensitivity are herein referred to as allergens. The term "allergen" 
primarily refers to foreign compounds capable of causing an allergic 
response. The term can be used interchangeably with the term "antigen," 
especially with respect to a foreign compound capable of inducing symptoms 
of immediate and/or delayed hypersensitivity. Factors that influence an 
animal's susceptibility to an allergen can include a genetic component 
and/or environmental exposure to an allergen. Animals can be de-sensitized 
to an allergen by repeated injections of the allergen to which an animal 
is hypersensitive. 
The medical and veterinary importance of ectoparasite infestation has 
prompted the development of reagents capable of controlling ectoparasite 
infestation. Commonly encountered methods to control ectoparasite 
infestation are generally focussed on use of insecticides in formulations 
such as sprays, shampoos, dusts, dips, or foams, or in pet collars. 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 ectoparasite populations on the pet 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 ectoparasite populations resistant to the prescribed dose 
of pesticide. Additional anti-ectoparasite products include chemical drugs 
that can, for example, affect the development of ectoparasitic larvae. 
Prior investigators have determined that histamine can be released in 
mammals in response to ectoparasite bites. A variety of biological 
mechanisms can be responsible for the release of histamine in an animal. 
Only mammalian histamine releasing factors, however, have been defined; 
see, for example, Wanstall et al., Toxicon 12:649-655, 1974; Toki et al., 
Biomedical Research 9(1):75-79, 1988; Toki et al., Biomedical Research 
9(1):421-428, 1988; Liao et al., J. Allergy Clin. Immunol. 86:894-901, 
1990; and Matuszek et al., Natural Toxins 2:36-43, 1994. 
Thus, there remains a need to identify an efficacious compound capable of 
reducing ectoparasite burden on animals, desensitizing animals to 
ectoparasite allergens and/or reducing inflammation in an animal. 
SUMMARY OF THE INVENTION 
The present invention relates to a novel product and process for reducing 
ectoparasite infestation, desensitizing animals to ectoparasite allergens 
and/or reducing inflammation in an animal. The present inventors have made 
the surprising discovery that an ectoparasite produces a histamine 
releasing factor-like protein. Such a protein can be a target for vaccines 
and anti-inflammatory reagents useful to prevent or treat, for example, 
allergy dermatitis in an animal. 
The present invention includes ectoparasite histamine releasing factor 
(HRF) proteins; ectoparasite HRF nucleic acid molecules, including those 
that encode such proteins; antibodies raised against such HRF proteins 
(i.e., anti-ectoparasite HRF antibodies); and other compounds that inhibit 
ectoparasite HRF activity (i.e, inhibitory compounds or inhibitors). Also 
included are methods to obtain and use such proteins, nucleic acid 
molecules, antibodies and inhibitory compounds. 
One embodiment of the present invention is an isolated ectoparasite nucleic 
acid molecule that hybridizes under stringent hybridization conditions 
with a flea histamine releasing factor gene (i.e., a flea HRF gene, such a 
nucleic acid molecule is referred to as an ectoparasite HRF nucleic acid 
molecule). A flea HRF gene preferably includes nucleic acid sequences SEQ 
ID NO:1 and SEQ ID NO:3. The present invention also relates to recombinant 
molecules, recombinant viruses and recombinant cells that include one or 
more HRF nucleic acid molecules 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 
ectoparasite histamine releasing factor protein (i.e., an ectoparasite HRF 
protein). A preferred ectoparasite HRF protein comprises amino acid 
sequence SEQ ID NO:2. The present invention also relates to mimetopes of 
ectoparasite HRF proteins as well as to isolated antibodies that 
selectively bind to ectoparasite HRF proteins or mimetopes thereof). Also 
included are methods, including recombinant methods, to produce proteins, 
mimetopes and antibodies of the present invention. 
Yet another embodiment of the present invention is a therapeutic 
composition that is capable of reducing ectoparasite burden of an animal. 
Such a therapeutic composition includes one or more of the following 
compounds: an isolated HRF protein encoded by a nucleic acid molecule that 
hybridizes under stringent hybridization conditions with a flea HRF gene, 
or a mimetope of such protein; an isolated nucleic acid molecule that 
hybridizes under stringent hybridization conditions with a flea HRF gene; 
an isolated antibody that selectively binds to an ectoparasite HRF 
protein; and/or an inhibitor of ectoparasite HRF activity identified by 
its ability to inhibit flea HRF activity (including HRF ligands and 
analogs). 
Another embodiment of the present invention is a therapeutic composition 
that is capable of reducing inflammation in an animal. Such a therapeutic 
composition includes one or more of the following compounds: an isolated 
ectoparasite HRF protein or a mimetope thereof; an isolated ectoparasite 
nucleic acid molecule that hybridizes under stringent hybridization 
conditions with a flea HRF gene; an isolated antibody that selectively 
binds to an ectoparasite HRF protein; a peptide derived from such an 
antibody; and/or an inhibitor of ectoparasite HRF activity identified by 
its ability to inhibit flea HRF activity (including HRF ligands and 
analogs, and mixtures thereof. Also included in the present invention is a 
method to reduce ectoparasite burden in an animal, comprising the step of 
administering to the animal a therapeutic composition of the present 
invention. 
The present invention also includes the use of HRF proteins of the present 
invention to identify or obtain ectoparasite HRF receptors, as well as in 
assays for diagnosing and prescribing treatment of allergic dermatitis. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides ectoparasite histamine releasing factor 
(HRF) proteins and nucleic acid molecules, antibodies directed against 
ectoparasite HRF proteins and other inhibitors of ectoparasite HRF 
activity. Also included in the present invention is the use of these 
proteins, nucleic acid molecules, antibodies and other inhibitors, as well 
as therapeutic compositions, to reduce ectoparasite burden of an animals 
as well as in other applications (e.g., allergic inflammation), such as 
those disclosed below. The invention is particularly advantageous in that 
it provides for unique compounds useful as a target for a vaccine and to 
develop compounds that protect (i.e., treat or prevent) an animal from 
allergy dermatitis. The discovery that ectoparasites express an HRF is 
surprising in that until now only mammalian HRF's have been identified. 
Particularly surprising is that flea HRF proteins of the present invention 
have n-terminal amino acid sequence similar to the human n-terminal amino 
acid sequence of human HRF that has also been identified as a tumor factor 
(MacDonald et al., Science, vol. 269, pp. 688-690, 1995). This discovery 
led to the identification of the novel ectoparasite histamine releasing 
compounds of the present invention. 
One embodiment of the present invention is an isolated protein comprising 
an ectoparasite HRF protein. It is to be noted that the term "a" or "an" 
entity refers to one or more of that entity; for example, a protein refers 
to one or more proteins, or to at least one protein. As such, the terms 
"a" (or "an"), "one or more" and "at least one" can be used 
interchangeably herein. It is also to be noted that the terms 
"comprising", "including", and "having" can be used interchangeably. An 
isolated ectoparasite HRF protein can, for example, be obtained from its 
natural source, be produced using recombinant DNA technology, or be 
synthesized chemically. As used herein, an isolated ectoparasite protein 
can be a full-length ectoparasite HRF protein or any homologue of such a 
protein, such as an ectoparasite HRF protein in which amino acids have 
been deleted (e.g., a truncated version of the protein, such as a 
peptide), inserted, inverted, substituted and/or derivatized (e.g., by 
glycosylation, phosphorylation, acetylation, myristoylation, prenylation, 
palmitation, amidation and/or addition of glycosylphosphatidyl inositol). 
A homologue of an ectoparasite HRF protein is a protein having an amino 
acid sequence that is sufficiently similar to a natural ectoparasite HRF 
protein amino acid sequence that a nucleic acid sequence encoding the 
homologue is capable of hybridizing under stringent conditions to (i.e., 
with) a nucleic acid molecule encoding the natural ectoparasite HRF 
protein (i.e., to the complement of the nucleic acid strand encoding the 
natural ectoparasite HRF protein amino acid sequence). 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 complete double helix with) the strand 
for which the sequence is cited. It is to be noted 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, which may or may not 
be denoted herein. Methods to deduce a complementary sequence are known to 
those skilled in the art. 
As used herein, stringent hybridization conditions refer to standard 
hybridization conditions under which nucleic acid molecules, including 
oligonucleotides, are used to identify similar nucleic acid molecules. 
Such standard conditions are disclosed, for example, in Sambrook et al., 
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 
1989; Sambrook et al., ibid., is incorporated by reference herein in its 
entirety. 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 to probe in the 
hybridization reaction. Formulae to calculate the appropriate 
hybridization and wash conditions to achieve hybridization permitting 30% 
or less mismatch of nucleotides are disclosed, for example, in Meinkoth et 
al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is 
incorporated by reference herein in its entirety. 
The minimal size of a protein homologue of the present invention is a size 
sufficient to be encoded by a nucleic acid molecule capable of forming a 
stable hybrid 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 as well as upon hybridization 
conditions per se (e.g., temperature, salt concentration, and formamide 
concentration). 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 an ectoparasite HRF protein homologue of the 
present invention is from about 12 to about 18 nucleotides in length. 
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 an ectoparasite HRF 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, 
multivalent (i.e., fusion protein having more than one domain each of 
which has a function), or functional portions of such proteins are 
desired. 
Ectoparasite HRF protein homologues can be the result of natural allelic 
variation or natural mutation. HRF 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. 
A homologue of an ectoparasite HRF protein of the present invention also 
includes a homologue that, when the homologue is administered to an animal 
as an immunogen, using techniques known to those skilled in the art, the 
animal will produce an immune response against at least one epitope of a 
natural ectoparasite HRF protein. The ability of a protein to effect an 
immune response, can be measured using techniques known to those skilled 
in the art. HRF protein homologues of the present invention also include 
HRF proteins that selectively bind to antisera that selectively binds to 
flea saliva proteins. Methods to produce and use antiserum are disclosed, 
for example, in PCT Publication No. WO 96/11271, entitled "NOVEL 
ECTOASITE SALIVA PROTEINS AND APATUS TO COLLECT SUCH PROTEINS", 
published Apr. 18, 1996, Application Ser. No. PCT/US95/13,200; which is 
incorporated herein by this reference in its entirety). 
Isolated HRF proteins of the present invention, including full-length 
proteins as well as homologues, can be identified in a straight-forward 
manner by the proteins' ability to elicit an immune response against 
natural ectoparasite HRF proteins, to mediate histamine release and/or to 
selectively bind to antiserum that binds specifically to flea saliva 
proteins. 
Isolated HRF proteins of the present invention have the further 
characteristic of being encoded by nucleic acid molecules that hybridize 
under stringent hybridization conditions to a gene encoding a flea HRF 
protein. As used herein, a flea HRF gene includes all nucleic acid 
sequences related to a flea HRF gene such as regulatory regions that 
control production of the flea HRF 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 flea 
HRF gene of the present invention includes the nucleic acid sequence SEQ 
ID NO:1 as well as the complement of SEQ ID NO:1. Nucleic acid sequence 
SEQ ID NO:1 represents the deduced sequence of the coding strand of a cDNA 
(complementary DNA) nucleic acid molecule denoted herein as nfHRF.sub.693 
(wherein "f" denotes Ctenocephalides felis), the production of which is 
disclosed in the Examples. The complement of SEQ ID NO:1 which refers to 
the nucleic acid sequence of the strand complementary to the strand having 
SEQ ID NO:1 and can easily be determined by those skilled in the art, is 
represented herein as SEQ ID NO:3. It should be noted that since nucleic 
acid sequencing technology is not entirely error-free, SEQ ID NO:1 and 
other nucleic acid and protein sequences presented herein, at best, 
represent apparent sequences of HRF nucleic acid molecules and HRF 
proteins of the present invention. 
In another embodiment, a flea HRF gene can be an allelic variant that 
includes a similar but not identical sequence to SEQ ID NO:1. An allelic 
variant of a flea HRF gene including SEQ ID NO:1, is a gene that occurs at 
essentially the same locus (or loci) in the genome as the gene including 
SEQ ID NO:1, 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. One 
class of allelic variants can encode the same protein but have different 
nucleic acid sequences due to the degeneracy of the genetic code. 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 ectoparasite since the genome is diploid and/or 
among a group of two or more ectoparasites. 
Suitable ectoparasites from which to isolate HRF proteins of the present 
invention (including isolation of the natural protein or production of the 
protein by recombinant or synthetic techniques) include biting gnat, bee, 
wasp, ant, flea, fly, mosquito, tick, mite, lice, spider, ant and true 
bug. Preferred fleas from which to isolate HRF proteins include fleas of 
the genus Ctenocephalides, Ceratophyllus, Diamanus, Echidnophaga, 
Nosopsyllus, Pulex, Tunga, Xenopsylla, Oropsylla or Orchopeus. 
Particularly preferred fleas are those of the species Ctenocephalides 
felis, Ctenocephalides canis, Pulex irritans, Pulex simulans, 
Ceratophyllus pulicidae, Oropsylla (Thrassis) bacchi, Oropsylla (Diamanus) 
montana, Orchopeus howardi, Xenopsylla cheopis. 
The present invention also includes mimetopes of HRF proteins of the 
present invention. As used herein, a mimetope of a HRF protein of the 
present invention refers to any compound that is able to mimic the 
activity of such a HRF protein, often because the mimetope has a structure 
that mimics the HRF 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 or inorganic 
molecules, and screening such samples by affinity chromatography 
techniques using the corresponding binding partner. 
One embodiment of the present invention is a fusion protein that includes 
an ectoparasite HRF protein-containing domain attached to one or more 
fusion segments. Suitable fusion segments for use with the present 
invention include, but are not limited to, segments that can: enhance a 
protein's stability; act as an immunopotentiator to enhance an immune 
response against a HRF protein; and/or assist purification of a HRF 
protein (e.g., by affinity chromatography). A suitable fusion segment can 
be a domain of any size that has the desired function (e.g., imparts 
increased stability, imparts increased immunogenicity to a protein, and/or 
simplifies purification of a protein). Fusion segments can be joined to 
amino and/or carboxyl termini of the HRF-containing domain of the protein 
and can be susceptible to cleavage in order to enable straight-forward 
recovery of a HRF protein. 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 HRF-containing domain. 
Preferred fusion segments include a metal binding domain (e.g., a 
poly-histidine segment); an immunoglobulin binding domain (e.g., Protein 
A; Protein G; T cell; B cell; Fc receptor or complement protein 
antibody-binding domains); a sugar binding domain (e.g., a maltose binding 
domain); and/or a "tag" domain (e.g., at least a portion of 
.beta.-galactosidase, a strep tag peptide, 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. 
In another embodiment, an ectoparasite HRF protein of the present invention 
also includes at least one additional protein segment that is capable of 
protecting an animal from one or more diseases. Such a multivalent 
protective protein can be produced by culturing a cell transformed with a 
nucleic acid molecule comprising two or more nucleic acid domains joined 
together in such a manner that the resulting nucleic acid molecule is 
expressed as a multivalent protective compound containing at least two 
protective compounds, or portions thereof, capable of protecting an animal 
from diseases caused, for example, by at least one infectious agent. 
Examples of multivalent protective compounds include, but are not limited 
to, a HRF 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., adenoviruses, 
caliciviruses, coronaviruses, distemper viruses, hepatitis viruses, 
herpesviruses, immunodeficiency viruses, infectious peritonitis viruses, 
leukemia viruses, oncogenic viruses, panleukopenia viruses, papilloma 
viruses, parainfluenza viruses, parvoviruses, rabies viruses, and 
reoviruses, as well as other cancer-causing or cancer-related viruses); 
bacteria (e.g., Actinomyces, Bacillus, Bacteroides, Bordetella, 
Bartonella, Borrelia, Brucella, Campylobacter, Capnocytophaga, 
Clostridium, Corynebacterium, Coxiella, Dermatophilus, Enterococcus, 
Ehrlichia, Escherichia, Francisella, Fusobacterium, Haemobartonella, 
Helicobacter, Klebsiella, L-form bacteria, Leptospira, Listeria, 
Mycobacteria, Mycoplasma, Neorickettsia, Nocardia, Pasteurella, 
Peptococcus, Peptostreptococcus, Proteus, Pseudomonas, Rickettsia, 
Rochalimaea, Salmonella, Shigella, Staphylococcus, Streptococcus, and 
Yersinia; fungi and fungal-related microorganisms (e.g., Absidia, 
Acremonium, Alternaria, Aspergillus, Basidiobolus, Bipolaris, Blastomyces, 
Candida, Chlamydia, Coccidioides, Conidiobolus, Cryptococcus, Curvalaria, 
Epidermophyton, Exophiala, Geotrichum, Histoplasma, Madurella, Malassezia, 
Microsporum, Moniliella, Mortierella, Mucor, Paecilomyces, Penicillium, 
Phialemonium, Phialophora, Prototheca, Pseudallescheria, 
Pseudomicrodochium, Pythium, Rhinosporidium, Rhizopus, Scolecobasidium, 
Sporothrix, Stemphylium, Trichophyton, Trichosporon, and Xylohypha; and 
other parasites (e.g., Babesia, Balantidium, Besnoitia, Cryptosporidium, 
Eimeria, Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, 
Isospora, Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas, 
Plasmodium, Pneumocystis, Sarcocystis, Schistosoma, Theileria, Toxoplasma, 
and Trypanosoma, as well as helminth parasites, such as those disclosed 
herein). In such an embodiment, an ectoparasite HRF protein of the present 
invention is attached to one or more additional compounds protective 
against ectoparasites. In another embodiment, one or more protective 
compounds, such as those listed above, can be included in a multivalent 
vaccine comprising an ectoparasite HRF protein of the present invention 
and one or more of the other protective molecules as separate compounds. 
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 molecule nfHRF.sub.693, and particularly with 
nfHRF.sub.501. A further preferred isolated protein is encoded by a 
nucleic acid molecule that hybridizes under stringent hybridization 
conditions with the complement of a nucleic acid molecule having nucleic 
acid sequence SEQ ID NO:1, and particularly SEQ ID NO:4. The complement of 
SEQ ID NO:1 is referred to herein as SEQ ID NO:3; the complement of SEQ ID 
NO:4 is referred to herein as SEQ ID NO:5 
Translation of SEQ ID NO:1 suggests that nucleic acid molecule 
nfHRF.sub.693 encodes a partial ectoparasite HRF protein of about 167 
amino acids, referred to herein as PfHRF.sub.167, represented by SEQ ID 
NO:2, assuming the first codon spans from about nucleotide 1 through about 
nucleotide 3 of SEQ ID NO:1 and a termination (stop) codon spans from 
about nucleotide 502 through about nucleotide 504 of SEQ ID NO:1. The 
coding region encoding PfHRF.sub.167 is represented by nucleic acid 
molecule nfHRF.sub.501, having the nucleic acid sequence represented by 
SEQ ID NO:4 (the coding strand) and SEQ ID NO:5 (the complementary 
strand). The deduced amino acid sequence SEQ ID NO:2 suggests a protein 
having a molecular weight of about 19,307.42 kilodaltons (kD) and an 
estimated pI of about 4.41. 
Comparison of amino acid sequence SEQ ID NO:2 (i.e., the amino acid 
sequence of PfHRF.sub.167) with amino acid sequences reported in GenBank 
indicates that SEQ ID NO:2, showed some homology to HRF proteins of 
eukaryotic origin. The highest scoring match, i.e., about 53% identity, 
was found between SEQ ID NO:2, and mouse p21 or human p23 tumor factors, 
recently also identified to be HRF (MacDonald et al., ibid.). 
Preferred ectoparasite HRF proteins of the present invention include 
proteins comprising amino acid sequences that are at least about 55%, 
preferably at least about 60%, and more preferably at least about 65%, and 
even more preferably at least about 70% identical to amino acid sequence 
SEQ ID NO:2. Particularly preferred are proteins comprising amino acid 
sequences that are at least about 75% and more particularly at least about 
80% identical to amino acid sequence SEQ ID NO:2. More preferred 
ectoparasite HRF 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. Particularly 
preferred HRF proteins of the present invention include SEQ ID NO:2 
(including, but not limited to, SEQ ID NO:2, fusion proteins and 
multivalent proteins) as well as truncated homologues of proteins that 
comprise SEQ ID NO:2. An even more preferred protein includes 
PfHRF.sub.167. 
Another embodiment of the present invention is an isolated nucleic acid 
molecule that hybridizes under stringent hybridization conditions with a 
flea HRF gene. The identifying characteristics of such a gene is 
heretofore described. A nucleic acid molecule of the present invention can 
include an isolated natural ectoparasite HRF 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 a flea HRF gene under 
stringent hybridization conditions. Suitable and preferred ectoparasites 
are disclosed above. 
In accordance with the present invention, an isolated nucleic acid molecule 
is a nucleic acid molecule that has been removed from its natural milieu 
(i.e., that has been subject to human manipulation) and can include DNA, 
RNA, or derivatives of either DNA or RNA. As such, "isolated" does not 
reflect the extent to which the nucleic acid molecule has been purified. 
An isolated ectoparasite HRF nucleic acid molecule of the present 
invention can be isolated from its natural source or produced using 
recombinant DNA technology (e.g., polymerase chain reaction (PCR) 
amplification, cloning) or chemical synthesis. Isolated HRF nucleic acid 
molecules can include, for example, natural allelic variants and nucleic 
acid molecules modified by nucleotide insertions, deletions, 
substitutions, and/or inversions in a manner such that the modifications 
do not substantially interfere with the nucleic acid molecule's ability to 
encode a HRF protein of the present invention or to form stable hybrids 
under stringent conditions with natural gene isolates. An isolated HRF 
nucleic acid molecule can include degeneracies. As used herein, nucleotide 
degeneracies refers to the phenomenon that one amino acid can be encoded 
by different nucleotide codons. Thus, the nucleic acid sequence of a 
nucleic acid molecule that encodes an HRF protein of the present invention 
can vary due to degeneracies. 
An ectoparasite HRF 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, by 
classic mutagenesis and recombinant DNA techniques (e.g., site-directed 
mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of 
nucleic acid fragments and/or PCR amplification), or 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 by hybridization with a flea HRF gene 
or by screening the function of a protein encoded by a nucleic acid 
molecule (e.g., ability to elicit an immune response against at least one 
epitope of a flea HRF protein, ability to selectively bind to antiserum 
that selectively binds to flea saliva proteins, ability to mediate 
histamine release). 
An isolated nucleic acid molecule of the present invention can include a 
nucleic acid sequence that encodes at least one ectoparasite HRF 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 an ectoparasite HRF protein. 
A preferred nucleic acid molecule of the present invention, when 
administered to an animal, is capable of reducing ectoparasite burden of 
an animal. Another preferred nucleic acid molecule of the present 
invention, when administered to an animal, is capable of reducing 
inflammation in an animal, particularly inflammation caused by an 
ectoparasite. 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, for example, 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. 
One embodiment of the present invention is an ectoparasite HRF nucleic acid 
molecule that hybridizes under stringent hybridization conditions with 
nucleic acid molecule nfHRF.sub.693 and preferably with a nucleic acid 
molecule having nucleic acid sequence SEQ ID NO:1 or SEQ ID NO:3. 
Comparison of nucleic acid sequence SEQ ID NO:1 (i.e., the nucleic acid 
sequence of the coding strand of nfHRF.sub.693) with nucleic acid 
sequences reported in GenBank indicates that SEQ ID NO:1, showed some 
homology to HRF proteins of eukaryotic origin. The highest scoring match, 
i.e., about 60% identity, was found between SEQ ID NO:4 and human HRF. 
Preferred ectoparasite HRF nucleic acid molecules include nucleic acid 
molecules having a nucleic acid sequence that is at least about 65%, 
preferably at least about 70%, more preferably at least about 75%, even 
more preferably at least about 80%, and even more preferably at least 
about 85% identical to nucleic acid sequence SEQ ID NO:4 or SEQ ID NO:5. 
Another preferred nucleic acid molecule of the present invention includes 
at least a portion of nucleic acid sequence SEQ ID NO:1 or SEQ ID NO:3, 
that is capable of hybridizing to a flea HRF gene of the present 
invention. A more preferred nucleic acid molecule includes the nucleic 
acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:5, 
as well as allelic variants thereof. 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 nfHRF.sub.693 and nfHRF.sub.501. 
The present invention also includes nucleic acid molecules encoding a 
protein having at least a portion of SEQ ID NO:2, 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. 
Knowing the nucleic acid sequences of certain ectoparasite HRF 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 HRF nucleic acid molecules from other 
ectoparasites. 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 
ectoparasite cDNA libraries as well as genomic DNA libraries. Techniques 
to clone and amplify genes are disclosed, for example, in Sambrook et al., 
ibid. 
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 
ectoparasite HRF genes or other ectoparasite HRF nucleic acid molecules. 
Oligonucleotides of the present invention can be RNA, DNA, or derivatives 
of either. The minimum size of such oligonucleotides is the size required 
for formation of a stable hybrid between an oligonucleotide and a 
complementary sequence on a nucleic acid molecule of the present 
invention. Minimal size characteristics are disclosed herein. The present 
invention includes oligonucleotides that can be used as, for example, 
probes to identify nucleic acid molecules, primers to produce nucleic acid 
molecules or therapeutic reagents to inhibit HRF protein production or 
activity (e.g., as antisense-, triplex formation-, ribozyme- and/or RNA 
drug-based reagents). The present invention also includes the use of such 
oligonucleotides to protect animals from disease using one or more of such 
technologies. Appropriate oligonucleotide-containing therapeutic 
compositions can be administered to an animal using techniques known to 
those skilled in the art. 
One embodiment of the present invention includes a recombinant vector, 
which includes at least one isolated nucleic acid molecule of the present 
invention, inserted into any vector capable of delivering the nucleic acid 
molecule into a host cell. Such a vector contains heterologous nucleic 
acid sequences, that is nucleic acid sequences that are not naturally 
found adjacent to nucleic acid molecules of the present invention and that 
preferably are derived from a species other than the species from which 
the nucleic acid molecule(s) are derived. The vector can be either RNA or 
DNA, either prokaryotic or eukaryotic, and typically is a virus or a 
plasmid. Recombinant vectors can be used in the cloning, sequencing, 
and/or otherwise manipulating of ectoparasite HRF nucleic acid molecules 
of the present invention. 
One type of recombinant vector, referred to herein as a recombinant 
molecule, comprises a nucleic acid molecule of the present invention 
operatively linked to an expression vector. The phrase operatively linked 
refers to insertion of a nucleic acid molecule into an expression vector 
in a manner such that the molecule is able to be expressed when 
transformed into a host cell. As used herein, an expression vector is a 
DNA or RNA vector that is capable of transforming a host cell and of 
effecting expression of a specified nucleic acid molecule. Preferably, the 
expression vector is also capable of replicating within the host cell. 
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 disclosed herein. 
In particular, expression vectors of the present invention contain 
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 (such as lambdap.sub.L and lambdap.sub.R and fusions 
that include such promoters), bacteriophage T7, T7lac, bacteriophage T3, 
bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating 
factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as 
Sindbis virus subgenomic promoters), antibiotic resistance gene, 
baculovirus, Heliothis zea insect virus, herpesvirus, vaccinia virus, 
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 ectoparasites, such as, fleas. 
Suitable and preferred nucleic acid molecules to include in recombinant 
vectors of the present invention are as disclosed herein. Particularly 
preferred nucleic acid molecule to include in recombinant vectors, and 
particularly in recombinant molecules, includes nfHRF.sub.693 and 
nfHRF.sub.501. 
Recombinant molecules of the present invention may also (a) contain 
secretory signals (i.e., signal segment nucleic acid sequences) to enable 
an expressed ectoparasite HRF 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 
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. Suitable 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. 
Another embodiment of the present invention includes a recombinant cell 
comprising a host cell transformed with one or more recombinant molecules 
of the present invention. Transformation of a nucleic acid molecule into a 
cell can be accomplished by any method by which a nucleic acid molecule 
can be inserted into the cell. Transformation techniques include, but are 
not limited to, transfection, electroporation, microinjection, 
lipofection, adsorption, and protoplast fusion. A recombinant cell may 
remain unicellular or may grow into a tissue, organ or a multicellular 
organism. Transformed nucleic acid molecules of the present invention can 
remain extrachromosomal or can integrate into one or more sites within a 
chromosome of the transformed (i.e., recombinant) cell in such a manner 
that their ability to be expressed is retained. Preferred nucleic acid 
molecules with which to transform a cell include ectoparasite HRF nucleic 
acid molecules disclosed herein. Particularly preferred nucleic acid 
molecules with which to transform a cell include nfHRF.sub.501 and 
nfHRF.sub.693. 
Suitable host cells to transform include any cell that can be transformed 
with a nucleic acid molecule of the present invention. Host cells can be 
either untransformed cells or cells that are already transformed with at 
least one nucleic acid molecule (e.g., nucleic acid molecules encoding one 
or more proteins of the present invention and/or other proteins useful in 
the production of multivalent vaccines). Host cells of the present 
invention either can be endogenously (i.e., naturally) capable of 
producing ectoparasite HRF 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), other 
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.x 3987 and 
SR-11 .sub.x 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. 
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. 
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. 
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. 
Recombinant cells of the present invention can also be co-transformed with 
one or more recombinant molecules including ectoparasite HRF 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. 
For example, a multivalent vaccine of the present invention can include 
one or more nucleic acid molecules encoding one or more protective 
compounds in combination with an ectoparasite HRF protein of the present 
invention useful for reducing ectoparasite infestation, desensitizing 
animals to ectoparasite allergens and/or reducing inflammation in an 
animal. 
Recombinant DNA technologies can be used to 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. 
Isolated HRF proteins of the present invention can be produced in a variety 
of ways, including production and recovery of natural proteins, production 
and recovery of recombinant proteins, and chemical synthesis of the 
proteins. In one embodiment, an isolated protein of the present invention 
is produced by culturing a cell capable of expressing the protein under 
conditions effective to produce the protein, and recovering the protein. A 
preferred cell to culture is a recombinant cell of the present invention. 
Effective culture conditions include, but are not limited to, effective 
media, bioreactor, temperature, pH and oxygen conditions that permit 
protein production. An effective, medium refers to any medium in which a 
cell is cultured to produce an ectoparasite HRF protein of the present 
invention. Such medium typically comprises an aqueous medium having 
assimilable carbon, nitrogen and phosphate sources, and appropriate salts, 
minerals, metals and other nutrients, such as vitamins. Cells of the 
present invention can be cultured in conventional fermentation 
bioreactors, shake flasks, test tubes, microtiter dishes, and petri 
plates. Culturing can be.sub.-- carried out at a temperature, pH and 
oxygen content appropriate for a recombinant cell. Such culturing 
conditions are within the expertise of one of ordinary skill in the art. 
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. col; 
or be retained on the outer surface of a cell or viral membrane. 
The phrase "recovering the protein" refers to collecting the whole 
fermentation medium containing the protein and need not imply additional 
steps of separation or purification. Proteins of the present invention can 
be purified using a variety of standard protein purification techniques, 
such as, but not limited to, affinity chromatography, ion exchange 
chromatography, filtration, electrophoresis, hydrophobic interaction 
chromatography, gel filtration chromatography, reverse phase 
chromatography, concanavalin A chromatography, chromatofocusing and 
differential solubilization. Proteins of the present invention are 
preferably retrieved in "substantially pure" form. As used herein, 
"substantially pure" refers to a purity that allows for the effective use 
of the protein as a therapeutic composition or diagnostic. A therapeutic 
composition for animals, for example, should exhibit no substantial 
toxicity and preferably should be capable of stimulating the production of 
antibodies in a treated animal. 
The present invention also includes isolated (i.e., removed from their 
natural milieu) antibodies capable of selectively binding to an 
ectoparasite HRF protein of the present invention or a mimetope thereof 
(e.g., anti-ectoparasite HRF antibodies). 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 standard in the art including enzyme immunoassays (e.g., 
ELISA), immunoblot assays, etc.; see, for example, Sambrook et al., ibid. 
An anti-ectoparasite HRF antibody preferably selectively binds to an 
ectoparasite HRF protein in such a way as to reduce the activity of that 
protein. 
Isolated antibodies of the present invention can include serum containing 
such antibodies, or antibodies that have been purified to varying degrees. 
Antibodies of the present invention can be polyclonal or monoclonal, 
functional equivalents such as antibody fragments and 
genetically-engineered antibodies, including single chain antibodies or 
chimeric antibodies that can bind to more than one epitope. 
A preferred method to produce antibodies of the present invention includes 
(a) administering to an animal an effective amount of a protein, peptide 
or mimetope thereof of the present invention to produce the antibodies and 
(b) recovering the antibodies. In another method, antibodies of the 
present invention are produced recombinantly using techniques as 
heretofore disclosed to produce ectoparasite HRF 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. 
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 infestation by ectoparasites 
susceptible to treatment by such antibodies, (b) as reagents in assays to 
detect the presence, in an animal, of allergens from such ectoparasites 
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. 
One embodiment of the present invention is a formulation that can be used 
to diagnose and/or treat animals susceptible to or having (i.e., suffering 
from) allergic dermatitis. Preferred types of allergic dermatitis to 
diagnose and/or treat using ectoparasite HRF protein, nucleic acid 
molecules, antibodies and inhibitors (the collection of which is referred 
to herein as HRF-related products) of the present invention include flea 
allergy dermatitis, Culicoides allergy dermatitis, mosquito allergy 
dermatitis and food allergies. A preferred type of allergic dermatitis to 
diagnose and/or treat using ectoparasite HRF-related products of the 
present invention is flea allergy dermatitis. As used herein, an animal 
that is susceptible to allergic dermatitis refers to an animal that is 
genetically pre-disposed to developing allergic dermatitis and/or to an 
animal that has been primed with an antigen in such a manner that 
re-exposure to the antigen results in symptoms of allergy that can be 
perceived by, for example, observing the animal or measuring antibody 
production by the animal to the antigen. As such, animals susceptible to 
allergic dermatitis can include animals having sub-clinical allergic 
dermatitis. Sub-clinical allergic dermatitis refers to a condition in 
which allergy symptoms cannot be detected by simply observing an animal 
(i.e., manifestation of the disease can include the presence of 
anti-ectoparasite HRF protein antibodies within an affected animal but no 
dermatitis). For example, sub-clinical allergic dermatitis can be detected 
using in vivo or in vitro assays of the present invention, as described in 
detail below. Reference to animals having allergic dermatitis includes 
animals that do display allergy symptoms that can be detected by simply 
observing an animal and/or by using in vivo or in vitro assays of the 
present invention, as described in detail below. 
One embodiment of the present invention is an in vivo test that is capable 
of detecting whether an animal is hypersensitive to an ectoparasite HRF 
protein of the present invention. An in vivo hypersensitivity test of the 
present invention is particularly useful for identifying animals 
susceptible to or having allergic dermatitis. An in vivo hypersensitivity 
test of the present invention is even more useful for identifying animals 
susceptible to or having FAD. A suitable in vivo hypersensitivity test of 
the present invention can be, but is not limited to, a skin test 
comprising administering (e.g., intradermally injecting or superficial 
scratching) an effective amount of a formulation containing an 
ectoparasite HRF protein, or a mimetope thereof. Methods to conduct skin 
tests of the present invention are known to those of skill in the art. 
Suitable formulations to use in an in vivo skin test include ectoparasite 
HRF protein. A suitable amount of ectoparasite saliva product for use in a 
skin test of the present invention can vary widely depending on the 
allergenicity of the product used in the test and on the site at which the 
product is delivered. Suitable amounts of an ectoparasite HRF protein for 
use in a skin test of the present invention include an amount capable of 
forming reaction, such as a detectable wheal or induration (hardness) 
resulting from an allergic reaction to the product. Preferred amounts of 
an ectoparasite HRF protein in a skin test ranges from about 1 nanogram 
(ng) to about 500 micrograms (.mu.g), more preferably from about 5 ng to 
about 300 .mu.g, and even more preferably from about 10 ng to about 50 
.mu.g of an ectoparasite HRF protein. It is to be appreciated by those of 
skill in the art that such amounts will vary depending upon the 
allergenicity of the protein being administered. 
A skin test of the present invention further comprises administering a 
control solution to an animal. A control solution can include a negative 
control solution and/or a positive control solution. A positive control 
solution of the present invention contains an effective amount of at least 
one compound known to induce a hypersensitive response when administered 
to an animal. A preferred compound for use as positive control solution 
includes, but is not limited to, histamine. A negative control solution of 
the present invention can comprise a solution that is known not to induce 
a hypersensitive response when administered to an animal, such as 
compounds essentially incapable of inducing a hypersensitive response or 
simply a buffer used to prepare the formulation (e.g., saline). An example 
of a preferred negative control solution is phenolated phosphate buffered 
saline (available from Greer Laboratories, Inc., Lenoir, N.C.). 
Hypersensitivity of an animal to a formulation of the present invention can 
be evaluated by measuring reactions (e.g., wheal size, induration or 
hardness; using techniques known to those skilled in the art) resulting 
from administration of one or more experimental sample(s) and control 
sample(s) into an animal and comparing the reactions to the experimental 
sample(s) with reactions resulting from administration of one or more 
control solution. Preferred devices for intradermal injections include 
individual syringes. Preferred devices for scratching include devices that 
permit the administration of a number of samples at one time. The 
hypersensitivity of an animal can be evaluated by determining if the 
reaction resulting from administration of a formulation of the present 
invention is larger than the reaction resulting from administration of a 
negative control, and/or by determining if the reaction resulting from 
administration of the formulation is at least about the same size as the 
reaction resulting from administration of a positive control solution. As 
such, if an experimental sample produces a reaction greater than or equal 
to the size of a wheal produced by administration of a positive control 
sample to an animal, then that animal is hypersensitive to the 
experimental sample. Conversely, if an experimental sample produces a 
reaction similar to the reaction produced by administration of a negative 
control sample to an animal, then that animal is not hypersensitive to the 
experimental sample. 
Preferred wheal sizes for evaluation of the hypersensitivity of an animal 
range from about 16 mm to about 8 mm, more preferably from about 15 mm to 
about 9 mm, and even more preferably from about 14 mm to about 10 mm in 
diameter. 
Preferably, the ability or inability of an animal to exhibit an immediate 
hypersensitive response to a formulation of the present invention is 
determined by measuring wheal sizes from about 2 minutes to about 30 
minutes after administration of a sample, more preferably from about 10 
minutes to about 25 minutes after administration of a sample, and even 
more preferably about 15 minutes after administration of a sample. 
Preferably, the ability or inability of an animal to exhibit a delayed 
hypersensitive response to a formulation of the present invention is 
determined by measuring induration and/or erythema from about 18 hours to 
about 30 hours after administration of a sample, more preferably from 
about 20 hours to about 28 hours after administration of a sample, and 
even more preferably at about 24 hours after administration of a sample. A 
delayed hypersensitivity response can also be measured using other 
techniques such as by determining, using techniques known to those of 
skill in the art, the extent of cell infiltrate at the site of 
administration during the time periods defined directly above. 
In a preferred embodiment, a skin test of the present invention comprises 
intradermally injecting into an animal at a given site an effective amount 
of a formulation that includes an ectoparasite HRF protein, and 
intradermally injecting an effective amount of a control solution into the 
same animal at a different site. It is within the scope of one of skill in 
the art to use devices capable of delivering multiple samples 
simultaneously at a number of sites, preferably enabling concurrent 
evaluation of numerous formulations. 
An ectoparasite HRF protein for use with a skin test of the present 
invention preferably includes an ectoparasite HRF protein encoded by a 
nucleic acid molecule that hybridizes under stringent hybridization 
conditions with a flea HRF gene, more preferably includes an ectoparasite 
HRF protein encoded by nucleic acid SEQ ID NO:1 and/or SEQ ID NO:3 and 
even more preferably includes an ectoparasite HRF protein having the amino 
acid sequence SEQ ID NO:2. 
Animals suitable and preferred to test for hypersensitivity to ectoparasite 
saliva proteins using a skin test of the present invention are disclosed 
herein. Particularly preferred animals to test with a skin test of the 
present invention include dogs, cats and horses, with dogs and cats being 
even more preferred. 
Another embodiment of the present invention is an in vitro immunoabsorbent 
test that is capable of detecting the presence of an antibody capable of 
binding to an ectoparasite HRF protein of the present invention by 
contacting a putative antibody-containing solution with a solution 
containing an ectoparasite HRF protein in such a manner that 
immunocomplexes can form and be detected. Thus, an in vitro 
immunoabsorbent test of the present invention is particularly useful for 
identifying animals susceptible to or having allergic dermatitis by 
demonstrating that an animal has been previously exposed to an 
ectoparasite saliva antigen and, therefore may be hypersensitive to 
further exposure to an ectoparasite HRF protein. 
According to the present invention, an in vitro hypersensitivity test of 
the present invention can be, but is not limited to, an immunoabsorbent 
test comprising: (a) contacting a formulation of the present invention 
with a body fluid from an animal under conditions sufficient for formation 
of an immunocomplex between the formulation and antibodies, if present, in 
the body fluid; and (b) determining the amount of immunocomplex formed, 
wherein formation of the immunocomplex indicates that the animal is 
susceptible to or has allergic dermatitis. The immunoabsorbent test is 
particularly useful for the detection of IgE antibodies in the body fluid, 
thereby indicating immediate hypersensitivity in the animal. Determining 
the amount of immunocomplex formed can include the step of separating 
depending on the mode of detection. Immunoabsorbent assays can be a 
variety of protocols and can be set-up by those of skill in the art. 
A preferred immunoabsorbent test of the present invention comprises a first 
step of coating one or more portions of a solid substrate with a suitable 
amount of an ectoparasite HRF protein of the present invention or a 
mimetope thereof, and of coating one or more other portions of the (or 
another) solid substrate with a suitable amount of positive and/or 
negative control solutions of the present invention. A preferred solid 
substrate of the present invention can include, but is not limited to, an 
ELISA plate, a dipstick, a radioimmunoassay plate, agarose beads, plastic 
beads, immunoblot membranes and paper; a more preferred solid substrate 
includes an ELISA plate, a dipstick or a radioimmunoassay plate, with an 
ELISA plate and a dipstick being even more preferred. As used herein, a 
dipstick refers to any solid material having a surface to which antibodies 
can be bound, such solid material having a stick-like shape capable if 
being inserted into a test tube. Suitable and preferred ectoparasite HRF 
proteins for use with an in vitro hypersensitivity test of the present 
invention are as disclosed for a skin test of the present invention. 
A second step of a preferred in vitro hypersensitivity test of the present 
invention comprises contacting the coated substrate with a body fluid, 
such as serum, plasma or whole blood, from an animal susceptible to 
allergic dermatitis in such a manner as to allow antibodies contained in 
the body fluid that are capable of binding to ectoparasite saliva products 
to bind to such products bound to the substrate to form immunocomplexes. 
Excess body fluid and antibodies are then washed from the substrate. 
A third step of a preferred in vitro hypersensitivity test of the present 
invention comprises contacting the immunocomplexes bound to the substrate 
with a compound capable of binding to the immunocomplexes, such as a 
secondary antibody or other compound that is capable of binding to the 
heavy chain of allergy-related antibodies produced by animals allergic to 
ectoparasites, in such a manner that the compound(s) can bind to the 
immunocomplexes. Preferred binding compounds include, but are not limited 
to, secondary antibodies capable of binding to the heavy chain of IgE 
antibodies and Fc receptors (FcR) that bind to IgE antibodies (i.e., 
epsilon FcR), including single chains of an FcR (e.g., the alpha chain of 
an epsilon FcR), as well as truncated forms with or without transmembrane 
domains. Preferred animals to test are disclosed herein. Compounds capable 
of binding to immunocomplexes are usually tagged with a label which 
enables the amount of compound bound to the antibody from the body fluid 
to be measured. Such labels include, but are not limited to, a radioactive 
label, an enzyme capable of producing a color reaction upon contact with a 
substrate, a fluorescent label, a chemiluminescent label, a chromophoric 
label or a compound capable of being bound by another compound. Preferred 
labels include, but are not limited to, fluorescein, radioisotopes, 
alkaline phosphatases, biotin, avidin, or peroxidases. 
A fourth step of a preferred in vitro hypersensitivity test of the present 
invention comprises measuring the amount of detectable label bound to the 
solid substrate using techniques known to those of skill in the art. It is 
within the scope of the present invention that the amount of antibody from 
the body fluid bound to the substrate can be determined using one or more 
layers of secondary antibodies or other binding compounds. For example, an 
untagged secondary antibody can be bound to a serum antibody and the 
untagged secondary antibody can then be bound by a tagged tertiary 
antibody. 
A hypersensitive animal is identified by comparing the level of 
immunocomplex formation using samples of body fluid with the level of 
immunocomplex formation using control samples. An immunocomplex refers to 
a complex comprising an antibody and its ligand (i.e., antigen). As such, 
immunocomplexes form using positive control samples and do not form using 
negative control samples. As such, if a body fluid sample results in 
immunocomplex formation greater than or equal to immunocomplex formation 
using a positive control sample, then the animal from which the fluid was 
taken is hypersensitive to the ectoparasite saliva product bound to the 
substrate. Conversely, if a body fluid sample results in immunocomplex 
formation similar to immunocomplex formation using a negative control 
sample, then the animal from which the fluid was taken is not 
hypersensitive to the ectoparasite saliva product bound to the substrate. 
A preferred embodiment of an in vitro hypersensitivity test of the present 
invention comprises the steps of: (a) coating one or more portions of an 
ELISA plate with a suitable amount of ectoparasite HRF protein; (b) 
contacting the coated plate with serum, plasma or whole blood from an 
animal susceptible to allergic dermatitis to form immunocomplexes; and (c) 
contacting the immunocomplexes with an antibody that specifically binds to 
IgE or other compounds capable of binding to the immunocomplex, such as an 
epsilon Fc receptor. 
Conversely, another preferred embodiment of an in vitro hypersensitivity 
test of the present invention comprises the steps of: (a) coating one or 
more portions of an ELISA plate with a suitable amount of an antibody that 
specifically binds to IgE or other compounds capable of binding to IgE, 
such as an epsilon Fc receptor; (b) contacting the coated plate with 
serum, plasma or whole blood from an animal susceptible to allergic 
dermatitis to form complexes; and (c) contacting the complexes with a 
suitable amount of ectoparasite HRF protein. 
One embodiment of the present invention is a kit useful for identification 
of an animal susceptible to or having allergic dermatitis. As used herein, 
a suspect animal is an animal to be tested. A kit of the present invention 
comprises a formulation of the present invention and a means for 
determining if an animal is susceptible to or has allergic dermatitis, in 
which the formulation is used to identify animals susceptible to or having 
allergic dermatitis. A means for determining if an animal is susceptible 
to or has allergic dermatitis can include an in vivo or in vitro 
hypersensitivity test of the present invention as described in detail 
above. A kit of the present invention further comprises at least one 
control solution such as those disclosed herein. 
A preferred kit of the present invention comprises the elements useful for 
performing an immunoassay. A kit of the present invention can comprise an 
experimental sample (i.e., a formulation of the present invention) and one 
or more control samples bound to at least one pre-packed dipstick, and the 
necessary means for detecting immunocomplex formation (e.g., labelled 
secondary antibodies or other binding compounds and any necessary 
solutions needed to resolve such labels, as described in detail above) 
between antibodies contained in the bodily fluid of the animal being 
tested and the proteins bound to the dipstick. It is within the scope of 
the invention that the kit can comprise simply a formulation of the 
present invention and that the detecting means can be provided in another 
way. 
An alternative preferred kit of the present invention comprises elements 
useful for performing a skin test. A kit of the present invention can 
comprise at least one pre-packed syringe and needle apparatus containing 
one or more experimental samples and/or one or more control samples. 
It is within the scope of the present invention that two or more different 
in vivo and/or in vitro tests can be used in combination for diagnostic 
purposes. For example, the immediate hypersensitivity of an animal to an 
ectoparasite HRF protein can be tested using an in vitro immunoabsorbent 
test capable of detecting IgE antibodies specific for an ectoparasite HRF 
protein in the animal's bodily fluid. While most animals that display 
delayed hypersensitivity to an ectoparasite HRF protein also display 
immediate hypersensitivity to the protein, a small number of animals that 
display delayed hypersensitivity to an allergen do not display immediate 
hypersensitivity to the protein. In such cases, following negative results 
from the IgE-specific in vitro test, the delayed hypersensitivity of the 
animal to an ectoparasite saliva allergen can be tested using an in vivo 
test of the present invention. 
One embodiment of the present invention is a therapeutic composition that, 
when administered to an animal in an effective manner, is useful for 
immunomodulating the immune response of the animal (i.e., immunomodulating 
the animal) so as to block (i.e., to inhibit, reduce or substantially 
prevent) a hypersensitive response by the animal upon subsequent exposure 
to allergenic components transmitted through bites from ectoparasites. 
Such a therapeutic composition is useful for immunomodulating animals 
known to be hypersensitive to an ectoparasite HRF protein and animals 
susceptible to hypersensitive responses against an ectoparasite HRF 
protein. 
One embodiment of the present invention is a therapeutic composition that 
includes de-sensitizing compounds capable of inhibiting an immune response 
to an ectoparasite saliva product of the present invention. Such 
de-sensitizing compounds include blocking compounds, toleragens and/or 
suppressor compounds. Blocking compounds comprise compounds capable of 
modulating antigen:antibody interactions that can result in inflammatory 
responses, toleragens are compounds capable of immunotolerizing an animal, 
and suppressor compounds are capable of immunosuppressing an animal. A 
de-sensitizing compound of the present invention can be soluble or 
membrane-bound. Membrane-bound de-sensitizing compounds can be associated 
with biomembranes, including cells, liposomes, planar membranes or 
micelles. A soluble de-sensitizing compound of the present invention is 
useful for: (1) inhibiting a Type I hypersensitivity reaction by blocking 
IgE:antigen mediated de-granulation of mast cells; (2) inhibiting a Type 
III hypersensitivity reaction by blocking IgG:antigen complex formation 
leading to complement destruction of cells; and (3) inhibiting a Type IV 
hypersensitivity reaction by blocking T helper cell stimulation of 
cytokine secretion by macrophages. A membrane-bound de-sensitizing 
compound of the present invention is useful for: (1) inhibiting a Type II 
hypersensitivity reaction by blocking IgG:antigen complex formation on the 
surface of cells leading to complement destruction of cells; (2) 
inhibiting a Type II hypersensitivity reaction by blocking IgG regulated 
signal transduction in immune cells; and (3) inhibiting a Type IV 
hypersensitivity reaction by blocking T cytotoxic cell killing of 
antigen-bearing cells. 
A de-sensitizing compound of the present invention can also be covalently 
linked to a ligand molecule capable of targeting the de-sensitizing 
compound to a specific cell involved in a hypersensitive response to 
ectoparasite saliva products. Appropriate ligands with which to link a 
de-sensitizing compound include, for example, at least a portion of an 
immunoglobulin molecule, cytokines, lectins, heterologous allergens, CD8 
molecules, CD4 molecules or major histocompatibility molecules (e.g., MHC 
class I or MHC class II molecules). Preferredportions of immunoglobulin 
molecules to link to a de-sensitizing compound include variable regions 
capable of binding to immune cell specific surface molecules and constant 
regions capable of binding to Fc receptors on immune cells, in particular 
IgE constant regions. Preferred CD8 molecules include at least the 
extracellular functional domains of the .beta. chain of CD8. Preferred CD4 
molecules include at least the extracellular functional domains of CD4. An 
immune cell refers to a cell involved in an immune response, in 
particular, cells having MHC class I or MHC class II molecules. Preferred 
immune cells include antigen presenting cells, T cells and B cells. 
In one embodiment, a therapeutic composition of the present invention 
includes an HRF protein of the present invention combined with 
ectoparasite saliva products of the present invention, or mimetopes 
thereof. Preferred therapeutic compositions include formulations 
comprising ectoparasite saliva extracts or at least one ectoparasite 
saliva product (preferably protein) of the present invention or mimetopes 
thereof. 
Suitable therapeutic compositions of the present invention for treating 
flea allergy dermatitis include flea saliva extracts and other 
formulations including at least one flea saliva product, preferably a 
protein, or a mimetope thereof. Preferred therapeutic compositions include 
FS-1, FS-2 and/or FS-3 as well as at least a portion of at least one flea 
saliva product that can be isolated from FS-1, FS-2 and/or FS-3. As such, 
preferred formulations for use as therapeutic compositions include FS-1, 
FS-2, FS-3, and/or at least a portion of one or more of the proteins fspA, 
fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3, fspH, 
fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2, fspN3, 
as well as fspM(A), fspM(B), fspM(C), fspM(D), fspM(E), and fspM(F), 
fspM(G), fspM(H), fspM(I), fspM(J), fspM(K), fspM(L), fspM(M), fspN(B), 
fspN(C), fspN(D), fspN(E), fspN(F), fspN(G), fspN(H), fspN(I), fspN(J), 
fspN(K), fspN(L), fspN(M), fspN(N), fspN(O) and other proteins disclosed 
in U.S. patent application Ser. No. 08/630,822, or homologues thereof. A 
more preferred flea saliva extract for use as a therapeutic compositions 
includes FS-1, FS-2, FS-3,and/or at least a portion of one or more of the 
proteins fspE, fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, 
fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3. A yet more preferred 
flea saliva extract for use as a therapeutic compositions includes FS-1, 
FS-2, and/or at least a portion of one or more of the proteins fspG1, 
fspG2, fspG3, fspH, fspm1, fspM2, fspN1, fspN2 and fspN3. 
One embodiment of the present invention is a therapeutic composition that, 
when administered to an animal in an effective manner, is capable of 
reducing ectoparasite burden of that animal. As used herein, ectoparasite 
burden refers to reducing the potential for ectoparasite population 
expansion on and around an animal (i.e., reducing the ectoparasite 
burden). Preferably, the ectoparasite population size is decreased, 
optimally to an extent that the animal is no longer bothered by 
ectoparasites. A host animal, as used herein, is an animal from which 
ectoparasites can feed by attaching to and feeding through the skin of the 
animal. Ectoparasites can live on a host animal for an extended period of 
time or can attach temporarily to an animal in order to feed. At any given 
time, a certain percentage of an ectoparasite population can be on a host 
animal whereas the remainder can be in the environment surrounding the 
animal (i.e., in the environment of the animal). Such an environment can 
include not only adult ectoparasites, but also ectoparasite eggs and/or 
ectoparasite larvae. 
Therapeutic compositions of the present invention useful for reducing 
ectoparasite burden include at least one of the following compounds: an 
isolated nucleic acid molecule that hybridizes under stringent 
hybridization conditions with a flea HRF gene; an isolated antibody that 
selectively binds to a flea HRF protein; an isolated HRF protein encoded 
by a nucleic acid molecule that hybridizes under stringent hybridization 
conditions with a flea HRF gene, or a mimetope of the protein; and/or an 
inhibitor of ectoparasite HRF activity identified by its ability to 
inhibit flea HRF activity, and a mixture thereof (i.e., combination) of at 
least two of the compounds. Preferred inhibitory compounds to use in a 
composition of the present invention include ligands and/or analogs of HRF 
protein. A suitable ligand includes any molecule capable of binding to an 
HRF protein in such a manner that the activity of the HRF protein is 
inhibited. Preferred ligands include, but are not limited to, antibodies 
and portions of antibodies, such as peptides. A suitable analog of an HRF 
protein includes any molecule (e.g., organic or synthetic compounds, as 
well as proteins) that is capable of binding to an HRF substrate in a 
similar manner as native HRF protein, but itself is incapable of 
stimulating histamine release. An inhibitor of the present invention 
includes non-proteinaceous compounds as well as proteinaceous compounds. 
Suitable ectoparasites to target include any ectoparasite that is 
essentially incapable of causing allergic dermatitis and/or inflammation 
in an animal administered a HRF 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 an humoral and/or cellular 
immune response against a HRF protein of the present invention and/or that 
can be targeted by a compound that otherwise inhibits HRF activity (e.g., 
a compound that inhibits HRF mediated histamine release), thereby 
resulting in the reduced ability of the ectoparasite to infest an animal. 
Preferred ectoparasites to target include ectoparasites disclosed herein 
as being useful in the production of ectoparasite proteins of the present 
invention. Examples of proteins, nucleic acid molecules, antibodies and 
inhibitors of the present invention are disclosed herein. 
Another embodiment of the present invention is a therapeutic composition 
that, when administered to an animal in an effective manner, is capable of 
reducing inflammation in that animal. Inflammation refers to a protective 
response in an animal, generally characterized by the influx of 
inflammatory cells into a specific area (described in detail in Janeway et 
al., ibid.). Therapeutic composition of the of the present invention 
useful for reducing inflammation include at least one of the following 
compounds: an isolated antibody that selectively binds to a flea HRF 
protein, and/or a peptide derived from that antibody, an inhibitor of 
ectoparasite HRF activity identified by its ability to inhibit flea HRF 
activity, and mixtures thereof. A therapeutic composition can also include 
an HRF protein, or portion thereof, that is incapable of inducing 
histamine release. For example, a therapeutic composition can include at 
least a portion of an HRF protein lacking histamine releasing activity 
and/or an antigenic peptide, comprising a portion of an HRF protein, that 
is capable of eliciting an immune response but does not induce histamine 
release when administered to an animal. It is within the scope of the 
present invention that a therapeutic composition can include a nucleic 
acid molecule encoding such protein or portion thereof described 
immediately above. 
The present invention also includes a therapeutic composition comprising at 
least one ectoparasite HRF-based compound of the present invention in 
combination with at least one additional compound that either reduces 
ectoparasite infestation or reduces inflammation in an animal. Examples of 
such additional compounds include any anti-ectoparasite agent(s), 
including, but not limited to, proteinaceous compounds and insecticides. 
Preferred additional compounds are proteinaceous compounds that effect 
active immunization (e.g., antigen vaccines), passive immunization (e.g., 
antibodies), or that otherwise inhibit an ectoparasite activity that when 
inhibited can reduce ectoparasite burden on and around an animal. Specific 
examples of additional compounds include a compound that inhibits binding 
between a flea membrane protein and its ligand (e.g., a compound that 
inhibits flea ATPase activity or a compound that inhibits binding of a 
peptide or steroid hormone to its receptor), a compound that inhibits 
hormone (including peptide or steroid hormones) synthesis, a compound that 
inhibits vitellogenesis (including production of vitellin and transport 
and maturation thereof into a major egg yolk protein), a compound that 
inhibits fat body function, a compound that inhibits flea muscle action, a 
compound that inhibits the flea nervous system, a compound that inhibits 
the flea immune system; a compound that inhibits HRF activity and/or a 
compound that inhibits flea feeding. Examples of additional compounds 
useful for reducing inflammation in an animal include anti-histamines and 
steroids. 
Therapeutic compositions of the present invention can be administered to 
any animal susceptible to ectoparasite infestation, including warm-blooded 
animals. Preferred animals to treat include mammals and birds, with cats, 
dogs, humans, cattle, chinchillas, ferrets, goats, mice, minks, rabbits, 
raccoons, rats, sheep, squirrels, swine, chickens, ostriches, quail and 
turkeys as well as other furry animals, pets and/or economic food animals, 
being more preferred. Particularly preferred animals to protect are cats 
and dogs. 
Therapeutic compositions of the present invention can also be administered 
to any ectoparasite, preferably to fleas, in such a manner that one or 
more components of the compositions are excreted in the feces of an 
ectoparasite. 
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. 
In one embodiment of the present invention, a therapeutic composition can 
include an adjuvant. Adjuvants are agents that are capable of enhancing 
the immune response of an animal to a specific antigen. Suitable adjuvants 
include, but are not limited to, cytokines, chemokines, and compounds that 
induce the production of cytokines and chemokines (e.g., granulocyte 
macrophage colony stimulating factor GM-CSF!, macrophage colony 
stimulating factor M-CSF!, granulocyte colony stimulating factor G-CSF!, 
colony stimulating factor CSF!, erythropoietin EPO!, interleukin-2 
IL-2!, interleukin-3 IL-3!, interleukin-5 IL-5!, interleukin-6 IL-6!, 
interleukin-7 IL-7!, interleukin-8 IL-8!, interleukin-10 IL-10!, 
interleukin-12 IL-12!, gamma interferon IFN-.gamma.!, interferon gamma 
inducing factor IGIF!, transforming growth factor beta, RANTES regulated 
upon activation, normal T cell expressed and presumably secreted!, 
macrophage inflammatory proteins e.g., MIP1.alpha. and MIP1.beta.!, and 
Leishmania elongation initiating factor LeIF!; bacterial components 
(e.g., endotoxins, in particular superantigens, exotoxins and cell wall 
components); aluminum-based salts; calcium-based salts; silica; 
polynucleotides; toxoids; serum proteins, viral coat proteins; block 
copolymer adjuvants (e.g., Hunter's Titermax.TM. adjuvant Vaxcel.TM., 
Inc. Norcross, Ga.!, Ribi adjuvants Ribi ImmunoChem Research, Inc., 
Hamilton, Mont.!; and saponins and their derivatives (e.g., Quil A 
Superfos Biosector A/S, Denmark!. Protein adjuvants of the present 
invention can be delivered in the form of the protein themselves or of 
nucleic acid molecules encoding such proteins using the methods described 
herein. 
In one embodiment of the present invention, a therapeutic composition can 
include a carrier. Carriers include compounds that increase the half-life 
of a therapeutic composition in the treated animal. Suitable carriers 
include, but are not limited to, polymeric controlled release vehicles, 
biodegradable implants, liposomes, bacteria, viruses, oils, other cells, 
esters, and glycols. 
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). 
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 
ectoparasites. The therapeutic composition is preferably released over a 
period of time ranging from about 1 to about 12 months. A controlled 
release formulation of the present invention is capable of effecting a 
treatment preferably for at least about 1 month, more preferably for at 
least about 3 months, even more preferably for at least about 6 months, 
even more preferably for at least about 9 months, and even more preferably 
for at least about 12 months. 
In order to, in an animal, reduce infestation or inflammation caused by an 
ectoparasite 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 reducing infestation or 
inflammation in that animal. Therapeutic compositions of the present 
invention can be administered to animals prior to infestation in order to 
prevent infestation (i.e., as a preventative vaccine) and/or can be 
administered to animals after infestation in order to reduce ectoparasite 
burden or reduce inflammation (i.e., as a therapeutic vaccine). 
Acceptable protocols to administer 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 
ectoparasite infestation when administered one or more times over a 
suitable time period. For example, a preferred single dose of a HRF 
vaccine or a mimetope thereof ranges from about 1 microgram (.mu.g, also 
denoted ug) to about 10 milligrams (mg) of the 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 ectoparasite 
infestation. A preferred administration schedule is one in which from 
about 10 .mu.g to about 1 mg of the vaccine 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. In one embodiment, a 
booster dose of a composition of the present invention is administered 
about 4 to 6 weeks after the primary dose, and additional boosters are 
administered about once or twice a year. Modes of administration can 
include, but are not limited to, oral, nasal, topical, transdermal, 
rectal, and parenteral routes. Parenteral routes can include, but are not 
limited to subcutaneous, intradermal, intravenous, and intramuscular 
routes. 
In another embodiment, a preferred single dose of an anti-HRF antibody 
composition ranges from about 1 .mu.g to about 10 mg of the composition 
per kilogram body weight of the animal. Anti-HRF antibodies can be 
re-administered from about 1 hour to about biweekly for several weeks 
following the original administration. Booster treatments preferably are 
administered when the titer of antibodies of the animal becomes 
insufficient to protect the animal from ectoparasite infestation. A 
preferred administration schedule is one in which from about 10 .mu.g to 
about 1 mg of an anti-HRF antibody composition per kg body weight of the 
animal is administered about every 2 to every 4 weeks. Suitable modes of 
administration are as disclosed herein and are known to those skilled in 
the art. 
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. 
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). 
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. A naked nucleic acid 
reagent of the present invention can comprise one or more nucleic acid 
molecule of the present invention in the form of, for example, a 
dicistronic recombinant molecule. 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. 
Naked nucleic acid vaccine of the present invention can be administered in 
a variety of ways, with intramuscular, subcutaneous, intradermal, 
transdermal, intranasal and oral routes of administration being preferred. 
A preferred single dose of a 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. Naked DNA of the 
present invention can be contained in an aqueous excipient (e.g., 
phosphate buffered saline) alone or a carrier (e.g., lipid-based 
vehicles). 
The present invention also includes a recombinant virus particle 
therapeutic composition. Such a composition 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. A number of recombinant virus 
particles can be used, including, but not limited to, those based on 
alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses. 
Preferred recombinant particle viruses are those based on alphaviruses 
(such as Sindbis virus), herpesviruses and poxviruses. Methods to produce 
and use recombinant virus particle vaccines are disclosed in U.S. patent 
application Ser. No. 08/015/414, filed Feb. 8, 1993, entitled "Recombinant 
Virus Particle Vaccines", U.S. Pat. No. 5,266,313, by Esposito et al., 
issued Nov. 30, 1993 and U.S. patent application Ser. No. 08/602,010, by 
Haanes et al., filed Jan. 15, 1996, entitled "Recombinant Canine 
Herpesvirus", each of the patents and patent application referred to in 
this section is incorporated by reference herein in its entirety. 
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 an ectoparasite as 
disclosed herein. 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. 
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. 
The efficacy of a therapeutic composition of the present invention to 
reduce ectoparasite infestation of an animal and their surrounding 
environments can be tested in a variety of ways including, but not limited 
to, determining (a) reduced the viability of ectoparasites that feed from 
the treated animal, (b) reduced fecundity of female ectoparasites that 
feed from the treated animal, (c) reduced reproductive capacity of male 
ectoparasites that feed from the treated animal, (d) reduced viability of 
eggs laid by female ectoparasites that feed from the treated animal, (e) 
altered blood feeding behavior of ectoparasites that feed from the treated 
animal (e.g., ectoparasites take up less volume per feeding or feed less 
frequently), (f) reduced viability of ectoparasite larvae, for example due 
to the feeding of larvae from feces of fleas that feed from the treated 
animal and/or (g) altered development of ectoparasite larvae (e.g., by 
decreasing feeding behavior, inhibiting growth, inhibiting (e.g., slowing 
or blocking) molting, and/or otherwise inhibiting maturation to adults). 
One preferred embodiment of the present invention is the use of 
ectoparasite HRF proteins, nucleic acid molecules, antibodies and 
inhibitory compounds of the present invention, to protect an animal from 
flea infestation. Therapeutic compositions are administered to animals in 
a manner effective to protect the animals from flea infestation. 
Additional protection may be obtained by administering additional 
protective compounds, including other flea proteins, nucleic acid 
molecules, antibodies and inhibitory compounds. 
One therapeutic composition of the present invention includes an inhibitor 
of ectoparasite HRF activity, i.e., a compound capable of substantially 
interfering with the function of an ectoparasite HRF protein susceptible 
to inhibition by an inhibitor of ectoparasite HRF activity. An inhibitor 
of HRF activity can be identified using ectoparasite HRF proteins of the 
present invention. 
One embodiment of the present invention is a method to identify a compound 
capable of inhibiting HRF activity of an ectoparasite. Such a method 
includes the steps of (a) contacting (e.g., combining, mixing) an isolated 
ectoparasite HRF protein with a putative inhibitory compound under 
conditions in which, in the absence of the compound, the protein has HRF 
activity, and (b) determining if the putative inhibitory compound inhibits 
the HRF activity. Putative inhibitory compounds to screen include small 
organic molecules, antibodies (including mimetopes thereof) and analogs. 
Methods to determine HRF activity are known to those skilled in the art; 
see, for example, citations in background section and references included 
therein. 
The present invention also includes a test kit to identify a compound 
capable of inhibiting HRF activity of an ectoparasite. Such a test kit 
includes an isolated ectoparasite HRF protein having HRF activity and a 
means for determining the extent of inhibition of HRF 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. 
HRF inhibitors isolated by such a method, and/or test kit, can be used to 
inhibit any HRF that is susceptible to such an inhibitor. Preferred HRF 
enzymes to inhibit are those produced by ectoparasites. A particularly 
preferred HRF inhibitor of the present invention is capable of, in an 
animal, reducing inflammation caused by an ectoparasite. It is also within 
the scope of the present invention to use inhibitors of the present 
invention to target HRF-related disorders in animals. Therapeutic 
compositions comprising HRF inhibitory compounds of the present invention 
can be administered to animals in an effective manner to protect animals 
from disease caused by the targeted HRF enzymes. Effective amounts and 
dosing regimens can be determined using techniques known to those skilled 
in the art. 
One embodiment of the present invention is a method to identify a receptor 
capable of binding to an isolated HRF protein of the present invention. 
Such method includes the steps of (a) contacting an isolated ectoparasite 
HRF protein with a putative receptor compound under conditions in which 
the HRF protein binds to an HRF receptor obtained by a method comprising: 
(1) combining a flea HRF protein with a sample having an HRF receptor to 
form an HRF protein:HRF receptor complex; and (2) isolating the HRF 
receptor portion of the HRF protein:HRF receptor complex. Preferably, the 
sample comprises a cell lysate, in which the cell including a mast cell or 
a basophil. In particular, the step of isolating comprises: (1) 
immunoreacting the HRF protein:HRF receptor complex with a ligand capable 
of selectively binding to the HRF protein to form an immune complex; (2) 
recovering the immune complex; and (3) purifying the recovered HRF protein 
from the recovered immune complex. 
Another method to identify an HRF receptor, includes the steps of: (a) 
contacting an isolated flea HRF protein with a putative HRF receptor; and 
(b) determining if the putative HRF receptor binds to the HRF protein. 
Preferably, the putative HRF receptor is a protein isolated from a cell 
including a mast cell or a basophil.