WO1997040058A1 - Proteines de protease de puces, molecules d'acides nucleiques et leurs utilisations - Google Patents

Proteines de protease de puces, molecules d'acides nucleiques et leurs utilisations Download PDF

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Publication number
WO1997040058A1
WO1997040058A1 PCT/US1997/006121 US9706121W WO9740058A1 WO 1997040058 A1 WO1997040058 A1 WO 1997040058A1 US 9706121 W US9706121 W US 9706121W WO 9740058 A1 WO9740058 A1 WO 9740058A1
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Prior art keywords
seq
nucleic acid
flea
acid molecule
protease
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PCT/US1997/006121
Other languages
English (en)
Inventor
Robert B. Grieve
Keith E. Rushlow
Shirley Wu Hunter
Glenn R. Frank
Gary L. Steigler
Patrick J. Gaines
Gary Silver
Original Assignee
Heska Corporation
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Publication date
Priority claimed from US08/639,075 external-priority patent/US6150125A/en
Priority claimed from US08/749,699 external-priority patent/US6210920B1/en
Application filed by Heska Corporation filed Critical Heska Corporation
Priority to JP53813497A priority Critical patent/JP2001510324A/ja
Priority to EP97922303A priority patent/EP0900231A1/fr
Priority to AU28015/97A priority patent/AU735717B2/en
Publication of WO1997040058A1 publication Critical patent/WO1997040058A1/fr
Priority to US09/032,215 priority patent/US6204010B1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/6408—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/641—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution

Definitions

  • the present invention relates to novel flea protease proteins and their use to reduce flea infestation of animals.
  • the present invention also relates to the use of anti-flea protease antibodies and other compounds that reduce flea protease activity to reduce flea infestation of animals .
  • Fleas which belong to the insect order Siphonaptera, are obligate ectoparasites for a wide variety of animals, including birds and mammals. Flea infestation of animals is of health and economic concern because fleas are known to cause and/or transmit a variety of diseases. Fleas cause and/or carry infectious agents that cause, for example, flea allergy dermatitis, anemia, murine typhus, plague and tapeworm. In addition, 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 fleas which feed on the pets. As such, fleas are a problem not only when they are on an animal but also when they are m the general environment of the animal.
  • the present invention relates to flea serine protease proteins, to flea ammopeptidase proteins, and to flea cysteine protease proteins; to flea serine protease, ammopeptidase and/or cysteine protease nucleic acid molecules, including those that encode such proteins; to antibodies raised agamst such proteins; and to compounds that inhibit flea serine protease, ammopeptidase and/or cysteine protease activities.
  • the present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitors. Also included m the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies, and/or inhibitors as well as the use of such therapeutic compositions to protect a host animal from flea infestation.
  • One embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene including a serine protease gene comprising a nucleic acid sequence including a nucleic acid molecule including SEQ ID NO: 9, SEQ ID NO-11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID N0:15, SEQ ID NO:17, SEQ ID N0:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID N0:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO: 116, SEQ ID NO-
  • the present invention also includes a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID.
  • a preferred nucleic acid sequence of the present invention includes a nucleic acid molecule comprising a nucleic acid sequence including SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28> SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:45, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 121, SEQ ID NO:131, SEQ ID
  • the present invention also includes an isolated protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO
  • the present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include a nucleic acid molecule of the present invention.
  • nucleic acid molecules recombinant molecules
  • recombinant viruses recombinant cells
  • Yet another embodiment of the present invention is a therapeutic composition that is capable of reducing hematophagous ectoparasite infestation.
  • a therapeutic composition includes an excipient and a protective compound including: an isolated protein or mimetope thereof encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID N0:41, SEQ ID NO:44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:
  • Another embodiment of the present invention is a method to identify a compound capable of inhibiting flea protease activity, the method comprising: (a) contacting an isolated flea protease protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID N0:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO-.157, SEQ ID NO:161, SEQ ID
  • the present mvention also includes an isolated flea protease protem that cleaves an immunoglobulin, when the protem is incubated in the presence of the immunoglobulin m about 100 microliters of about 0.2M Tris-HCl for about 18 hours at about 37°C.
  • a preferred protease protem capable of cleaving immunoglbulm comprises an ammo acid sequence selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO: 96.
  • Another embodiment of the present invention includes a method to identify a compound capable of inhibiting flea immunogloDulm proteinase protem activity, the method comprising: (a) contacting an isolated flea immunoglobulin proteinase protem with a putative inhibitory compound under conditions in which, m the absence of the compound, the protem has immunoglobulin proteinase activity; and (b) determining if the putative inhibitory compound inhibits the activity.
  • Fig. 1 is a scanned image depicting SDS-PAGE of DFP- labeled larval proteases m unfed larvae, fed 1st instar larvae and fed 3rd instar larvae.
  • the present invention includes the use of compounds that inhibit flea protease activity to protect a host animal from flea infestation.
  • proteases are significant components of the flea midgut and are good targets for lmmunotherapeutic and/or chemotherapeutic intervention to reduce flea burden ooth on the host animal and m the immediate (i.e., surrounding) environment of the animal.
  • the inventors have snown, for example, that the viability and/or fecundity of fleas consuming a blood meal is reduced when the blood meal contains compounds that reduce flea protease activity, probably because the compounds interfere with flea digestion and other functions.
  • Such compounds include flea protease vaccines, anti-flea protease antibodies, flea protease inhibitors, and/or compounds that suppress protease synthesis; such compounds are discussed in more detail below.
  • One embodiment of the present invention is a method to protect a host animal from flea infestation by treating the animal with a composition that includes a compound that reduces the protease activity of fleas feeding (includes fleas in the process of feeding as well as fleas having fed) from the treated animal thereby reducing the flea burden on the animal and in the environment of the animal.
  • composition of the present invention can include one or more compounds that target (reduced the activity of) one or more proteases in the flea.
  • the phrase "to protect an animal from flea infestation” refers to reducing the potential for flea population expansion on and around the animal (i.e., reducing the flea burden) .
  • the flea population size is decreased, optimally to an extent that the animal is no longer bothered by fleas.
  • a host animal as used herein, is an animal from which fleas can feed by attaching to and feeding through the skin of the animal. Fleas, and other ectoparasites, can live on a host animal for an extended period of time or can attach temporarily to an animal m order to feed.
  • a certain percentage of a flea 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 fleas, but also flea eggs and/or flea larvae.
  • the environment can be of any size such that fleas in the environment are able to " jump onto and off of a host animal. As such, it is desirable not only to reduce the flea burden on an animal per se, but also to reduce the flea burden in the environment surrounding the animal.
  • a host animal is treated by administering to the animal a compound of the present invention in such a manner that the compound itself (e.g., a protease inhibitor, protease synthesis suppressor or anti-flea protease antibody) or a product generated by the animal in response to administration of the compound (e.g., antibodies produced in response to a flea protease vaccine, or conversion of an inactive inhibitor "prodrug" to an active protease inhibitor) ultimately enters the flea midgut.
  • An animal is preferably treated in such a way that the compound or product thereof enters the blood stream of the animal. Fleas are then exposed ro the compound when they feed from the animal.
  • flea protease inhibitors administered to an animal are administered in such a way that the inhibitors enter the blood stream of the animal, where they can be taken up by feeding fleas.
  • the treated animal mounts an immune response resulting in the production of antibodies agamst the protease (anti-flea protease antibodies) which circulate m the animal's blood stream and are taken up by fleas upon feeding.
  • the present invention also includes the ability to reduce larval flea infestation in that when fleas feed from a host animal that has been administered a therapeutic composition of the present mvention, at least a portion of compounds of the present invention, or products thereof, in the blood taken up by the flea are excreted by the flea m feces, which is subsequently ingested by flea larvae. It is of note that flea larvae obtain most, if not all, of their nutrition from flea feces.
  • reducing proteolytic activity in flea midguts can lead to a number of outcomes that reduce flea burden on treated animals and their surrounding environments.
  • outcomes include, but are not limited to, (a) reducing the viability of fleas that feed from the treated animal, (b) reducing the fecundity of female fleas that feed from the treated animal, (c) reducing the reproductive capacity of male fleas that feed from the treated animal, (d) reducing the viability of eggs laid by female fleas that feed from the treated animal, (e) altering the blood feeding behavior of fleas that feed from the treated animal (e.g., fleas take up less volume per feeding or feed less frequently) , (f) reducing the viability of flea larvae, for example due to the feeding of larvae from feces of fleas that feed from the treated animal and/or (g) altering the development of flea larvae (e.g., by decreasing feeding behavior, inhibiting growth, inhibiting (e.g., slow
  • One embodiment of the present invention is a composition that includes one or more compounds that reduce the activity of one or more flea proteases directly (e.g., an anti-flea protease antibody or a flea protease inhibitor) and/or indirectly (e.g., a flea protease vaccine) .
  • Suitable flea proteases to target include flea ammopeptidases, flea carboxypeptidases and/or flea endopeptidases.
  • Such proteases can include cytosolic and/or membrane-bound forms of a protease.
  • Preferred flea proteases to target include, but are not limited to, serine proteases, metalloproteases, aspartic acid proteases and/or cysteine proteases.
  • proteases include ammopeptidases, carboxypeptidases and/or endopeptidases.
  • Preferred flea proteases to target m include, but are not limited to, proteases that degrade hemoglobin, proteases mvolved m blood coagulation and/or lytic (anti-coagulation) pathways, proteases involved in the maturation of peptide hormones, proteases that inhibit complement or other host immune response elements (e.g., antibodies) and/or proteases mvolved in vitellogenesis.
  • proteases are known to those skilled in the art, including, but not limited to, ammopeptidases, such as leucine ammopeptidase and ammopeptidases B and M; astacin-like metalloproteases; calpams; carboxypeptidases, such as carboxypeptidases A, P and Y; cathepsms, such as cathepsms B, D, E, G, H, and L, chymotrypsms; cruzipams; meprins; papams; pepsins; reruns; thermolysms and trypsms.
  • ammopeptidases such as leucine ammopeptidase and ammopeptidases B and M
  • astacin-like metalloproteases calpams
  • carboxypeptidases such as carboxypeptidases A, P and Y
  • cathepsms such as cathepsms B, D, E, G, H, and L
  • a particularly preferred protease to target is a protease having a proteolytic activity that, when targeted with a composition of the present mvention, reduces flea burden without substantially harming the host animal.
  • a protease can be identified usmg, for example, methods as disclosed herem.
  • One aspect of the present invention is the discovery that a substantial amount of the proteolytic activity found in flea midguts is serine protease activity. Both in vi tro and in vi vo studies usmg a number of protease inhibitors substantiate this discovery, details of which are disclosed m the Examples.
  • a particularly preferred protease to target is a serine protease.
  • serine proteases include, but are not limited to, acrosms, bromelams, cathepsm G, chymotrypsins, coUagenases, elastases, factor Xa, ficms, kallikreins, papains, plasmins, Staphylococcal V8 proteases, thrombms and trypsins.
  • a preferred flea serine protease to target includes a protease having trypsin-like or chymotrypsin-like activity.
  • an enzyme having "like” proteolytic activity has similar activity to the referenced protease, although the exact structure of the preferred substrate cleaved may differ.
  • "Like” proteases usually have similar tertiary structures as their referenced counterparts.
  • Protease inhibitor studies disclosed in the Examples section also indicate that additional preferred proteases to target include aminopeptidases and/or metalloproteases. Examples of such proteases mclude exo- and endo- metalloproteases, digestive enzymes, and enzymes mvolved in peptide hormone maturation.
  • an ammopeptidase that is also a metalloprotease is leucine ammopept-dase .
  • Suitable compounds to include in compositions of the present mvention include, but are not limited to, a vaccine comprising a flea protease (a flea protease vaccine) , an antibody that selectively binds to a flea protease (an anti-flea protease antibody) , a flea protease inhibitor (a compound other than a vaccine or an antibody that inhibits a flea protease) , and a mixture of such compounds.
  • a mixture thereof refers to a combination of one or more of the cited entities.
  • Compositions of the present mvention can also include compounds to suppre-ss protease synthesis or maturation, such as, but not limited to, protease modulating peptides.
  • a preferred embodiment of the present invention is a flea protease vaccine and its use to reduce the flea population on and around an animal.
  • a flea protease vaccine can include one or more proteins capable of eliciting an immune response agamst a flea protease and can also include other components.
  • Preferred flea protease vaccines mclude a flea serine protease, a flea metalloprotease, a flea aspartic acid protease and/or a flea cysteine protease, with flea serine protease, flea metalloprotease and/or flea ammopeptidase vaccines being more preferred.
  • Examples of flea protease vaccines mclude soluble flea midgut preparations of the present invention as well as one or more isolated proteins of the present invention.
  • One embodiment of the present invention is a soluble flea midgut preparation.
  • a preparation mcludes primarily components naturally present m the lumen of a flea midgut and, depending on the method of preparation, can also include one or more peripheral midgut membrane proteins. Methods to preferentially include, or exclude, membrane proteins from such a preparation are known to those skilled m the art.
  • the present mvention mcludes the discovery that such a preparation has proteolytic activity, of which a substantial portion is serine protease activity.
  • At least about 70 percent of the proteolytic activity in a soluble flea midgut soluble preparation is serine protease activity, as can be indicated by the ability to inhibit at least about 70 percent of the proteolytic activity with 4-2-ammoethyl- benzenesulfonylfluo ⁇ de-hydrochloride (AEBSF) .
  • Serine protease activity can also be identified using other known inhibitors or substrates.
  • Other preferred inhibitors that can inhibit at least about 70 percent of the proteolytic activity of a soluble flea midgut preparation of the present invention include soybean trypsin inhibitor, 1,3- dnsopropylfluoro-phosphate or leupeptin.
  • a soluble flea midgut preparation of the present invention includes proteases that range m molecular weight from about 5 kilodaltons (kD or kDa) to about 20 ⁇ KD, as determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), with at least a substantial portion of the serine proteases ranging m molecular weight from about 5 kD to about 60 kD, as determined by SDS-PAGE.
  • proteases that range m molecular weight from about 5 kilodaltons (kD or kDa) to about 20 ⁇ KD, as determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), with at least a substantial portion of the serine proteases ranging m molecular weight from about 5 kD to about 60 kD, as determined by SDS-PAGE.
  • a substantial portion of protease activity in a soluble flea midgut preparation of the present invention has a pH activity optimum ranging from about pH 5 to about pH 10, preferably an activity optimum ranging from about pH 7 to about pH 9, and even more preferably an activity optimum of about pH 8. While not being bound by theory, such a pH optimum suggests that a large proportion of proteases in soluble flea midgut preparations of the present invention are serine proteases. It is also interesting to note that the pH of the flea midgut is also about pH 8.
  • proteases m soluble flea midgut preparations of the present invention exnibit a varied pattern of inhibition by protease inhibitors of a given type (e.g., serine protease inhibitors), as well as variances seen m molecular weights and pH optima of the proteases, suggest that there are a number of protease isoforms in such preparations.
  • protease inhibitors of a given type e.g., serine protease inhibitors
  • a soluble flea midgut preparation of the present mvention is preferably prepared by a method that mcludes tne steps of (a) disrupting a flea midgut to produce a mixture including a liquid portion and a solid portion and (b; recovering the liquid portion to obtain a soluble flea midgut preparation.
  • Such a method is a simplified version of methods disclosed m U.S. Patent No. 5,356,622, ibi d . It is to be noted that in accordance with the present mvention, methods disclosed m U.S. Patent No. 5,356,622, ibi d, can also be used to prepare soluble flea midgut preparations having similar proteolytic activities.
  • Flea midguts can be obtained (e.g., dissected from) from unfed fleas or from fleas that recently consumed a blood meal (i.e., blood-fed fleas) . Such midguts are referred to herein as, respectively, unfed flea midguts and fed flea midguts. Flea midguts can be obtained from either male or female fleas. As demonstrated in the Examples section, female flea midguts exhibit somewhat more proteolytic activity than do male flea midguts. Furthermore, fed flea midguts have significantly more proteolytic activity than do unfed flea midguts.
  • m flea midguts the synthesis and/or activation of proteases as well as other factors (e.g., enzymes, other proteins, co-factors, etc.) important m digesting the blood meal, as well as in neutralizing host molecules potentially damaging to the flea (e.g., complement, immunoglobulins, blood coagulation factors) .
  • unfed flea midguts may contain significant targets not found m fed flea midguts and vice versa.
  • the present application focuses primarily on flea midgut proteases, it is to be noted that the present invention also includes other components of soluble flea midgut preparations of the present mvention that provide suitable targets to reduce flea burden on an animal and in the environment of that animal; see also U.S. Patent No. 5,356,622, ibid.
  • Methods to disrupt flea midguts in order to obtain a soluble flea midgut preparation are known to those skilled in the art and can be selected according to, for example, the volume being processed and the buffers being used.
  • Such methods include any technique that promotes cell lysis, such as, but are not limited to, chemical disruption techniques (e.g., exposure of midguts to a detergent) as well as mechanical disruption techniques (e.g., homogenization, sonication, use of a tissue blender or glass beads, and freeze/thaw techniques) .
  • Methods to recover a soluble flea midgut preparation are also known to those skilled in the art and can include any method by which the liquid portion of disrupted flea midguts is separated from the solid portion (e.g., filtration or centrifugation) .
  • disrupted flea midguts are subjected to centrifugation, preferably at an acceleration ranging from about 10,000 x g to about 15,000 x g for several minutes (e.g., from about 1 min. to about 15 min.) .
  • the supernatant from such a centrifugation comprises a soluble flea midgut preparation of tne present mvention.
  • the present invention also mcludes an isolated protem that includes an ammo acid sequence encoded by a nucleic acid molecule capable of hybridizing under stringent conditions (i.e., that hybridize under stringent nybridization conditions) with a nucleic acid molecule that encodes a protease present (i.e., the nucleic acid molecules hybridize with the nucleic acid strand that is complementary to the coding strand) m (i.e., can be found m) a flea midgut, such as a midgut from a blood-fed female flea, a midgut from a blood-fed male flea, a midgut from an unfed female flea or a midgut from an unfed male flea.
  • a preferred midgut protease is present m the lumen of the midgut.
  • an isolated protem of the present invention also referred to herein as an isolated protease protem, preferably is capable of eliciting an immune response against a flea midgut protease and/or has proteolytic activity.
  • an isolated, or biologically pure, protem is a protem that has been removed from its natural milieu.
  • isolated and biologically pure do not necessarily reflect the extent to which the protem has been purified.
  • An isolated protease protem can be obtained from its natural source. Such an isolated protem can also be produced usmg recombinant DNA technology or chemical synthesis.
  • an isolated protem of the present mvention can be a full-length protem or any homologue of such a protem, such as a protem m which ammo acids have been deleted (e.g., a truncated version of the protem, such as a peptide) , inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl mositol) such that the homologue comprises a protem having an ammo acid sequence that is sufficiently similar to a natural flea midgut protease that a nucleic acid sequence encoding the homologue is capable of hybridizing under stringent conditions to (i.e., with) the complement of a nucleic acid sequence encoding the corresponding natural flea midgut protease ammo acid sequence.
  • 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 Cl oning: A Labora tory Manual , Cold Spring Harbor Labs Press, 1989; Sambrook et al. , ibid. , is incorporated by reference herem m its entirety. Stringent hybridization conditions typically permit isolation of nucleic acid molecules havmg at least about 70 nucleic acid sequence identity v.ith the nucleic acid molecule being used to probe m the hyr>nd-.zat ⁇ or ⁇ reaction.
  • 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 protem.
  • the size of tne nucleic acid molecule encoding such a protem 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 aoout 17 bases m length if they are AT-rich.
  • the minimal size of a nucleic acid molecule used to encode a protease protem homologue of the present invention is from aoout 12 to about 18 nucleotides in length.
  • nc 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.
  • the minimal size of a protease protem homologue of the present invention is from about 4 to about 6 ammo acids in length, with preferred sizes depending on whether a full-length, multivalent (i.e., fusion protem having more than one domain each of which has a function) , or functional portions of such proteins are desired.
  • Protease protem homologues of the present mvention preferably have protease activity and/or are capable of eliciting an immune response agamst a flea midgut protease.
  • a protease protem homologue of the present invention can be the result of allelic variation of a natural gene encoding a flea protease.
  • a natural gene refers to the form of the gene found most often in nature.
  • Protease protein homologues can be produced using techniques known m the art including, but not limited to, direct modifications to a gene encoding a protein usmg, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
  • Isolated protease proteins of the present invention, including homologues can be identified m a straight-forward manner by the proteins' ability to effect proteolytic activity and/or to elicit an immune response against a flea midgut protease. Such techniques are known to those skilled in the art.
  • a preferred protease protein of the present invention is a flea serine protease, a flea metalloprotease, a flea aspartic acid protease, a flea cysteine protease, or a homologue of any of these proteases.
  • a more preferred protease protein is a flea serine protease, a flea metalloprotease or a homologue of either.
  • a flea cysteine protease or a homologue thereof Particularly preferred is a flea serine protease or a homologue thereof.
  • Preferred protease proteins of the present invention are flea protease proteins having molecular weights ranging from about 5 kD to about 200 kD, as determined by SDS-PAGE, and homologues of such proteins. More preferred are flea protease proteins having molecular weights ranging from about 5 kD to about 60 kD, as determined by SDS-PAGE, and homologues of such proteins.
  • flea serine protease proteins particularly those having molecular weights of about 26 kD (denoted PfSP26, now denoted PafSP-26K to distinguish from flea PfSP26 as described in Example 26) , about 24 kD (denoted PfSP24, now denoted PafSP-24K to distinguish from flea PfSP24 as described in Example 27), about 19 kD (denoted PfSP19, now denoted PafSP-19K to distinguish from flea PfSP19 as described m Example 32), about 6 kD (denoted PfSPo, now denoted PafSP-6K to distinguish from flea PfSP6 as described m Example 11), about 31 kD (denoted PfSP28), about 25 kD (denoted PlfSP-25Kl) from 1st instar larvae, about 25 kD (denoted PlfSP-25K3) from 3rd instar larvae, about 28 kD (denoted PlfSP
  • One preferred embodiment of the present invention is an isolated flea protease protem that mcludes an ammo acid sequence encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea serine protease gene, with a flea ammopeptidase gene or with a flea cysteine protease gene.
  • a flea protease gene includes all nucleic acid sequences related to a natural flea protease gene such as regulatory regions that control production of a flea protease protem encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself.
  • the inventors have discovered an extensive family of serine proteases, encoded by a family of serine protease genes. Such a gene family may be due to allelic variants (i.e., genes having similar, but different, sequences at a given locus in a population of fleas) and/or to, the existence of serine protease genes at more than one locus m the flea genome.
  • the present invention includes flea serine protease genes comprising not only the nucleic acid sequences disclosed herein (e.g., genes including nucleic acid sequences SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:127, SEQ ID NO:121,
  • a preferred flea ammopeptidase gene mcludes nucleic acid sequence SEQ ID NO: 110 and/or SEQ ID NO: 112, which encode ammopeptidase proteins having ammo acid sequences including SEQ ID NO: 107, SEQ ID NO: 111 and/or SEQ ID NO: 113. Additional preferred ammopeptidase genes include allelic variants of SEQ ID NO:110 and/or SEQ ID NO: 112.
  • a preferred flea cysteine protease gene includes nucleic acid sequence SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 76 and/or SEQ ID NO: 94, which encode a cysteine protease protem having ammo acid sequences including SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:77, and/or SEQ ID NO: 95.
  • cysteine protease genes include allelic variants of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 76 and/or SEQ ID NO: 94.
  • a preferred flea serine protease protem of the present invention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the followmg nucleic acid molecules: nfSP3, nfSP8, nfSP9, nfSPIO, nfSPll, nfSP19, nfSP20, nfSP21, nfSP23, nfSP25, nfSP26, nfSP27, nfSP29, nfSP30, nfSP31, nfSP34, nfSP36, nfSP37, nfSP38, nfSP39, nfSPl ⁇ , nfSP24, nfSP28, nfSP32, nfSP33 and nfSP40.
  • each of these nucleic acid molecules represent the entire coding region of a flea serine protease gene of the present invention (at least portions of which are also referred to by flea clone numbers, as described m the Examples) .
  • Nucleic acid molecules that contain partial coding regions or other parts of the corresponding gene are denoted by names that include the size of those nucleic acid molecules (e.g., nfSP40 42b ) .
  • Nucleic acid molecules containing apparent full length coding regions for which the size is known also are denoted by names that include the size of tnose nucleic acid molecules (e.g., nfSP40 841 ) .
  • the production, and at least partial nucleic acid sequence, of such nucleic acid molecules is disclosed in the Examples.
  • serine protease proteins are encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfSP18 B34 , nfSP18 77W nfSP18 225 , nfSP24 410 , nfSP24 1089 , nfSP24 774 , nfSP24 711 , nfSP28 P2 ,, nfSP32 qj3 , nfSP32 y33 , nfSP32 924 , nfSP32 699 , nfSP33 42b , nfSP33 71(( , nfSP33 1894 , nfSP33 1?00 , nfSP33 726 , nfSP40 841, nfSP5 806 , nfSPll 307 , nfSP8 sl5 , nfSP8 430, nfSP12 7
  • serine protease proteins include the following amino acid sequences: SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID N0:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID
  • serine protease proteins are encoded by allelic variants of nucleic acid molecules encoding proteins that include the c_.ted ammo acid sequences. Also preferred are flea serine protease proteins including regions that have at least about 50 * :, preferably at least about 75%, and more preferably at least about 90% identity with flea serine protease proteins having ammo acid sequences as cited herem.
  • One embodiment of the present mvention is a flea serine protease that degrades immunoglobulin circulating m a host animal (i.e.-, flea immunoglobulin proteinase or IgGase) .
  • An example of a flea immunoglobulin proteinase is presented in the Examples section.
  • an immunoglobulin proteinase of the present mvention cleaves an immunoglobulin when the protem is incubated m the presence of the immunoglobulin m about 100 microliters of about 0.2M Tris-HCl for about 18 hours at about 37°C.
  • an immunoglobulin proteinase of the present mvention cleaves an immunoglobulin m about 300 microliters of 50 mM Tris-HCl, pH 8.0, for about 1 hour at about 37 C C.
  • Suitable immunoglobulin proteinase proteins of the present invention are capable of cleaving the hinge region of an immunoglobulin heavy chain.
  • the hinge region of an immunoglobulin is the flexible domain that " joins the Fab arms of the immunoglobulin to the Fc portion of the molecule.
  • a more preferred immunoglobulin proteinase protem m cludes a protem havmg a molecular weight ranging from about 25 kD to about 35 kD and more preferably having a molecular weight of about 31 kD, in its mature form.
  • An even more preferred immunoglobulin proteinase protem includes a protem comprising an ammo acid sequence including SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO: 73 and/or SEQ ID NO: 96, which can be encoded by a gene comprising nucleic acid sequence SEQ ID NO: 66.
  • the proteinase activity of an immunoglobulin proteinase of the present invention cleaves an immunoglobulin m such a manner that the immunoglobulin maintains mtact heavy and light chain pairs, either as two Fab fragments or one Ffab') ⁇ fragment.
  • a Fab fragment refers to complete immunoglobulin light chains paired with the variable region and CHI domains of an immunoglobulin heavy chain.
  • a F(ab' ) fragment refers to two Fab fragments that remain linked by a disulfide bond. Both Fab and F(ab') 2 fragments are capable of binding antigen.
  • a preferred immunoglobulin proteinase protem of the present invention is capable of cleaving the hmge region of an immunoglobulin heavy chain at a site comprising an ammo acid sequence including D-C-P-K-C-P-P-P-E-M-L-G-G-P- S-I-F-I-F-P-P-K-P-K-D (SEQ ID NO: 104) , D-C-P-K-C-P-P-P-E-M- L-G-G-P-S-I-F-I-F-P-P-K-D-D-L-L-I-K-R-K-S-E-V (SEQ ID NO: 105) and/or D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K- P-K-D-T-L-S-I-S-R-T-P-
  • a preferred flea ammopeptidase protein of the present invention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfAP and/or nfAP2 (flea ammopeptidase full-length coding regions of a flea ammopeptidase gene of the present mvention) .
  • aminopeptidase proteins are encoded by a nucleic acid rrtolcule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfAP 453 , nfAP 900r nfAP 73 ,, nfAP :580 , nfAP2 38 , and/or nfAP2 537. More preferred is an aminopeptidase protein encoded by a nucleic acid molcule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfAP2 383 and/or nfAP2 537 .
  • aminopeptidase protein that includes amino acid sequence SEQ ID NO: 107, SEQ ID NO: 111 and/or SEQ ID NO: 113, or an ammopeptidase protein encoded by an allelic variant of a nucleic acid molecule that includes SEQ ID NO:llC and/or SEQ ID NO: 112.
  • flea aminopeptidase proteins including regions that have at least about 50%, prefereably at least about 75%, and more preferably at least about 90% identity with flea ammopeptidase proteins having ammo acid sequences as cited herein.
  • a preferred flea cysteine protease protem of the present mvention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfCPl (a flea cysteine protease full-length coding region that includes nfCPl 57 , or nfCPl no (the production of which are described m the Examples) .
  • cysteine protease that includes ammo acid sequence SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO: 95, SEQ ID NO: 77, or a cysteine protease encoded by an allelic variant of a nucleic acid molecule that mcludes SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 76, or SEQ ID NO: 94.
  • flea cysteine protease protem including regions that have at least about 50%, preferably at least about 75%, and more preferably at least about 90% identity with SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO:8, SEQ ID N0:89, SEQ ID NO:92, SEQ ID NO:77, or SEQ ID NO: 95.
  • One embodiment of the present invention is an isolated protem having proteolytic activity that is substantially inhibited by a serine protease inhibitor, an ammopeptidase inhibitor and/or a cysteine protease inhibitor. Such inhibition can be measured by techniques known to those skilled m the art.
  • To be substantially inhibited means, for example, for a serine protease, that at least half of the proteolytic activity of the protease protem is inhibited by a serine protease inhibitor.
  • Preferred serine protease inhibitors include flea serpm proteins, and peptides or analogs thereof.
  • An isolated protem of the present invention can be produced m a variety of ways, including recovering such a protein from a flea midgut and producing such a protein recombinantly.
  • a flea midgut protease can be recovered by methods heretofore disclosed for obtaining a soluble flea midgut preparation.
  • a flea midgut protease protem can be further purified from a disrupted flea midgut by a number of techniques known to those skilled m the art, including, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis (e.g., standard, capillary and flow-through electrophoresis) , hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalm A chromatography, chromatofocusmg and differential solubilization.
  • a flea midgut protease is purified usmg protease inhibitor affinity chromatography, an example of which is disclosed m the Examples section.
  • Another embodiment of the present invention is a method to produce an isolated protem of the present invention using recombinant DNA technology.
  • Such a method includes the steps of (a) culturing a recombinant cell comprising a nucleic acid molecule encoding a protem of the present invention to produce the protem and (b) recovering the protem therefrom. Details on producing recombinant cells and culturing thereof are presented below.
  • the phrase "recovering the protem” refers simply to collecting the whole fermentation medium containing the protem and need not imply additional steps of separation or purification. Proteins of the present invention can be purified usmg a variety of standard protein purification techniques, as heretofore disclosed.
  • Isolated proteins of the present invention are preferably retrieved in "substantially pure” form.
  • substantially pure refers to a purity that allows for the effective use of the protem as a vaccine.
  • a vaccine for animals, for example, should exhibit no substantial toxicity and should be capable of stimulating tne production of antibodies in a vaccinated animal.
  • Another embodiment of the present invention is an solated nucleic acid molecule capable of hybridizing under stringent conditions with a gene encoding a flea protease present in a flea midgut.
  • a nucleic acid molecule is also referred to herem as a flea protease nucleic acid molecule.
  • Particularly preferred is an isolated nucleic acid molecule that hybridizes under stringent conditions with a flea serine protease gene, with a flea aminopeptidase gene or with a flea cysteine protease gene. The characteristics of such genes are disclosed herein.
  • 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) .
  • isolated does not reflect the extent to which the nucleic acid molecule has been purified.
  • An isolated nucleic acid molecule can mclude DNA, RNA, or derivatives of either DNA or RNA.
  • a flea protease gene mcludes all nucleic acid sequences related to a natural flea protease gene such as regulatory regions that control production of a flea protease protem encoded by that gene (such as, but not limited to, transcription, translation or post- translation control regions) as well as the coding region itself.
  • a nucleic acid molecule of the present invention can be an isolated natural flea protease nucleic acid molecule or a homologue thereof.
  • 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 flea protease nucleic acid molecule of the present invention is the minimal size capable of forming a stable hybrid under stringent hybridization conditions with a correspondmg natural gene.
  • Flea protease nucleic acid molecules can also mclude a nucleic acid molecule encoding a hybrid protem, a fusion protem, a multivalent protem or a truncation fragment.
  • An isolated nucleic acid molecule of the present invention can be obtained from its natural source either as an entire (i.e., complete) gene or a portion thereof capable of forming a stable hybrid with that gene.
  • the phrase "at least a portion of" an entity refers to an amount of the entity that is at least sufficient to have the functional aspects of that entity.
  • at least a portion of a nucleic acid sequence is an amount of a nucleic acid sequence capable of forming a stable hybrid with the corresponding gene under stringent hybridization conditions.
  • An isolated nucleic acid molecule of the present invention can also be produced usmg recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • Isolated flea protease nucleic acid molecules mclude natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a flea protease protem of the present invention or to form stable hybrids under stringent conditions with natural nucleic acid molecule isolates.
  • a flea protease 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., ibi d. ) .
  • nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
  • classic mutagenesis techniques and recombinant DNA techniques such as site-directed mutagenesis
  • chemical treatment of a nucleic acid molecule to induce mutations
  • Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid (e.g., the ability of a homologue to elicit an immune response against a flea protease and/or to have proteolytic activity) and/or by hybridization with isolated flea protease nucleic acids under stringent conditions.
  • An isolated flea protease nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one flea protease protem of the present invention, examples of such proteins being disclosed herein.
  • 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 flea protease protem.
  • One embodiment of the present invention is a flea protease nucleic acid molecule of the present mvention that is capable of hybridizing under stringent conditions to a nucleic acid strand that encodes at least a portion of a flea protease or a homologue thereof or to the complement of such a nucleic acid strand.
  • 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.
  • nucleic acid molecules of the present invention for wnich a nucleic acid sequence has been determmed 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.
  • 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.
  • a flea protease nucleic acid molecule that includes a nucleic acid sequence having at least about 65 percent, preferably at least about 75 percent, more preferably at least about 85 percent, and even more preferably at least about 95 percent homology with the corresponding region (s) of the nucleic acid sequence encoding at least a portion of a flea protease protein.
  • a flea protease nucleic acid molecule capable of encoding at least a portion of a flea protease that naturally is present in flea midguts and preferably is included in a soluble flea midgut preparation of the present invention. Examples of nucleic acid molecules of the present invention are disclosed in the Examples section.
  • a preferred flea serine protease nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfSP3, nfSP8, nfSP9, nfSPIO, nfSPll, nfSPl9, nfSP20, nfSP21, nfSP23, nfSP25, nfSP26, nfSP27, nfSP29, nfSP30, nfSP31, nfSP34, nfSP36, nfSP37, nfSP38, nfSP39, nfSPl ⁇ , nfSP24, nfSP28, nfSP32, nfSP33 and/or nfSP40.
  • nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfSP18 534 , nfSP18 775 , nfSP18 225 , nfSP24 410 , nfSP24 10bQ , nfSP24,, 4 , nfSP24 7 ⁇ ; , nfSP28 7n , nfSP28 923 , nfSP32 q1 plausible nfSP32 924 , nfSP32 6qq , nfSP33 426/ nfSP33 77fl , nfSP33 1894 , nfSP33 li00 , nfSP33 72e/ nfSP40 ⁇ 4 plausible nfSP5 8 ⁇ 6 nfSPll 307 nfSP8 M5 nfSP8 436, nfSP12 758 , nfSP26 610 , nfSP27
  • nucleic acid molecules that include nfSP3, nfSP8, nf ⁇ P9, nfSPIO, nfSPll, nfSP19, nfSP20, nfSP21, nfSP23, nfSP25, nfSP26, nfSP27, nfSP29, nfSP30, nfSP31, nfSP34, nfSP36, nfSP37, nfSP38, nfSP39, nfSP18, nfSP24, nfSP28, nfSP32, nfSP33 and/or nfSP40 and even more nfSP18 53, , nfSPl ⁇ T-c, nfSP18 225 , nfSP24 410 , nfSP24 108y , nfSP24 774 , nfSP24 7n , nfSP28 923 , nfSP32 933 , nfSP40
  • nfSP34 390 , nfSP36 197 , nfSP38 341 , nfSP37 2t: , nfSP39,,- nfSP29 cl2 , nfSP30 641 , nfSP31 62b , nfSP32 433 , nfSP15 81! , nfSP19 8C ,, nfSP25 8b4 , nfSP21 5 hail, and/or nfSP40 717 , as well as other specific nucleic acid molecules disclosed in the Examples section.
  • flea serine protease nucleic acid molecules include at least one of the following sequences:SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28-, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO: 116, SEQ ID NO:117, SEQ ID NO:127, SEQ ID NO:121, SEQ ID NO:131, SEQ ID NO: 155, SEQ ID NO: 9
  • a preferred flea ammopeptidase nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with nfAP and/or nfAP2.
  • a more preferred flea ammopeptidase nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with nfAP 4Dl , nfAP 900> nfAP 732 , nfAP l380 , nfAP2 3b and/or nfAP2 53 - More preferred is an ammopeptidase nucleic acid molecule that includes nfAP 453 , nfAP 900> nfAP 732 , nfAP 1580 , nfAP2 38 , and/or nfAP2 ; , 7 .
  • Particularly preferred is a nucleic acid molecule that includes nucleic acid sequence SEQ ID NO: 110 and/or SEQ ID
  • a preferred flea cysteine protease nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with nfCPl 57 ,, or nfCPl no9 (the production of which are described in the Examples) . More preferred is a cysteine protease nucleic acid molecule that includes nfCPlc- or nfCPl UQQ .
  • nucleic acid molecule that mcludes nucleic acid sequence SEQ ID NO:l, SEQ ID N0:3, SEQ ID NO:4, SEQ ID N0:6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93 and/or SEQ ID NO: 94, or allelic variants of such nucleic acid molecules.
  • nucleic acid molecule of a flea protease protem of the present mvention allows one skilled m the art to make copies of that nucleic acid molecule as well as to obtain a nucleic acid molecule including additional portions of flea protease protein-encoding genes (e.g., nucleic acid molecules that include the translation start site and/or transcription and/or translation control regions), and/or flea protease nucleic acid molecule homologues. Knowing a portion of an ammo acid sequence of a flea protease protem of the present invention allows one skilled in the art to clone nucleic acid sequences encoding such a flea protease protem.
  • a desired flea protease nucleic acid molecule can be obtained in a variety of ways including screening appropriate expression libraries with antibodies which bind to flea protease proteins of the present invention; traditional cloning techniques usmg oligonucleotide probes of the present invention to screen appropriate libraries or DNA; and PCR amplification of appropriate libraries, or RNA or DNA using oligonucleotide primers of the present invention (genomic and/or cDNA libraries can be used) .
  • preferred cDNA libraries mclude cDNA libraries made from unfed whole fleas, fed whole fleas, fed flea midguts, unfed flea midguts, and flea salivary glands.
  • the present invention also mcludes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention that encode at least a portion of a flea protease protem.
  • Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either.
  • the minimal size of such oligonucleotides is the size required to form a stable hybrid between a given oligonucleotide and the complementary sequence on another nucleic acid molecule of the present mvention. Minimal size characteristics are disclosed herein.
  • oligonucleotide must also be sufficient for the use of the oligonucleotide m accordance with the present invention.
  • Oligonucleotides of the present invention can be used in a variety of applications including, but not limited to, as probes to identify additional nucleic acid molecules, as primers to amplify or extend nucleic acid molecules or in therapeutic applications to inhibit flea protease production.
  • Such therapeutic applications include the use of such oligonucleotides in, for example, antisense-, triplex formation-, ribozyme- and/or RNA drug-based technologies.
  • the present invention therefore, includes such oligonucleotides and metnods to interfere witn the production of flea protease proteins by use of one or more of such technologies.
  • the present invention also includes a recombinant vector, which mcludes a flea protease nucleic acid molecule of the present invention inserted mto any vector capable of delivering the nucleic acid molecule into a host cell.
  • a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to flea protease nucleic acid molecules of the present invention.
  • 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 flea protease nucleic acid molecules of the present invention.
  • recombinant vector herein referred to as a recombinant molecule and described in more detail below, can be used m the expression of nucleic acid molecules of the present invention.
  • Preferred recombinant vectors are capable of replicating in the transformed cell.
  • Preferred nucleic acid molecules to include in recombinant vectors of the present invention are disclosed herein.
  • one embodiment of the present invention is a method to produce a flea protease protem of the present invention by culturing a cell capable of expressing the protem under conditions effective to produce the protem, and recovering the protem.
  • a preferred cell to culture is a recombinant cell that is capable of expressing the flea protease protem, the recombinant cell being produced by transforming a host cell with one or more nucleic acid molecules of the present invention. Transformation of a nucleic acid molecule mto a cell can be accomplished by any method by which a nucleic acid molecule can be inserted mto 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 mto 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 withm a chromosome of the transformed (i.e., recombinant) cell m such a manner that their ability to be expressed is retained.
  • Preferred nucleic acid molecules with which to transform a host cell are disclosed herein.
  • Suitable host cells to transform include any cell that can be transformed and that can express the introduced flea protease protein. Such cells are, therefore, capable of producing flea protease proteins of the present invention after being transformed with at least one 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.
  • Suitable host cells of the present invention can include bacterial, fungal (including yeast), insect, animal and plant cells.
  • Preferred host cells include bacterial, yeast, insect and mammalian cells, with bacterial (e.g., E. coli ) and insect (e.g., Spodoptera) cells being particularly preferred.
  • 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.
  • 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.
  • 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) m recombinant cells of the present invention, including m bacterial, fungal, insect, animal, and/or plant cells.
  • nucleic acid molecules of the present invention can be operatively linked to expression vectors containing regulatory sequences such as promoters, operators, repressors, enhancers, termination 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 mvention.
  • a transcription control sequence includes a sequence which is capable of controlling 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 m at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled m the art.
  • Preferred transcription control sequences include those which function in bacterial, yeast, helminth, insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda ( ⁇ ) (such as ⁇ p L and ⁇ p F and fusions that include such promoters), bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionem, alpha mating factor, Pi chi a alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters) , baculovirus, Heli othi s zea insect virus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus 40, retrovirus actm, retroviral long terminal repeat, Rous sarcoma
  • transcription control sequences include tissue-specific promoters and enhancers as well as lymphokme-mducible promoters (e.g., promoters inducible by interferons or interleukins) .
  • Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with a DNA sequence encoding a flea protease protem.
  • Expression vectors of the present invention may also contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed flea protease protem to be secreted from the cell that produces the protem.
  • Suitable signal segments include a flea protease protem signal segment or any heterologous signal segment capable of directing the secretion of a flea protease protem, including fusion proteins, of the present invention.
  • Preferred signal segments include, but are not limited to, flea protease, tissue plasmmogen activator (t- PA) , interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotem signal segments .
  • Expression vectors of the present invention may also contain fusion sequences which lead to the expression of inserted nucleic acid molecules of the present invention as fusion proteins.
  • Inclusion of a fusion sequence as part of a flea protease nucleic acid molecule of the present invention can enhance the stability during production, storage and/or use of the protem encoded by the nucleic acid molecule.
  • a fusion segment can function as a tool to simplify purification of a flea protease protem, such as to enable purification of the resultant fusion protem usmg affinity chromatography.
  • a suitable fusion segment can be a domain of any size that has the desired function (e.g., increased stability and/or purification tool) .
  • Fusion segments can be joined to ammo and/or carboxyl termini of a flea protease protem.
  • Linkages between fusion segments and flea protease proteins can be constructed to be susceptible to cleavage to enable straight-forward recovery of the flea protease proteins.
  • Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid sequence that encodes a protem including the fusion segment attached to either the carboxyl and/or amino terminal end of a flea protease protein.
  • a recombinant molecule of the present invention is a molecule that can mclude at least one of any nucleic acid molecule heretofore described operatively linked to at least one of any transcription control sequence capable of effectively regulatmg expression of the nucleic acid molecule (s) in the cell to be transformed.
  • a preferred recombinant molecule includes one or more nucleic acid molecules of the present invention, with those that encode one or more flea protease proteins, and particularly one or more flea serine protease, aminopeptidase and/or cysteine protease proteins, being more preferred.
  • a preferred recombinant cell includes one or more nucleic acid molecules of the present invention, with those that encode one or more flea protease proteins, and particularly one or more flea serine protease, aminopeptidase, and/or cysteine protease proteins, being more preferred. It may be appreciated by one skilled in the art that use of recombinant DNA technologies can improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules mto 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, Shme-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 protem production during fermentation.
  • the activity of an expressed recombinant protem of the present mvention may be improved by fragmenting, modifying, or derivatizing the resultant protem.
  • recombinant cells can be used to produce flea protease proteins of the present invention by culturing such cells under conditions effective to produce such a protem, and recovering the protem.
  • Effective conditions to produce a protem include, but are not limited to, appropriate media, bioreactor, temperature, pH and oxygen conditions that permit protem production.
  • An appropriate, or effective, medium refers to any medium in which a cell of the present invention, when cultured, is capable of producing a flea protease protem.
  • Such a medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • the medium may comprise complex nutrients or may be a defined minimal medium.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, which include, but are not limited to, batch, fed-batch, cell recycle, and continuous fermentors. Culturing can also be conducted m shake flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and oxygen content appropriate for the recombinant cell. Such culturing conditions are well withm the expertise of one of ordinary skill in the art.
  • resultant flea protease proteins may either remain withm the recombinant cell; be secreted mto the fermentation medium; be secreted mto a space between two cellular membranes, such as the periplasmic space m E. coli ; or be retained on the outer surface of a cell or viral membrane. Methods to purify such proteins are heretofore disclosed.
  • the present mvention also includes isolated anti-flea protease antibodies and their use to reduce flea infestation on a host animal as well as in the environment of the animal.
  • An anti-flea protease antibody is an antibody capable of selectively binding to a protease present m a flea midgut, including female and male fed midguts as well as female and male unfed midguts.
  • An anti- flea protease antibody preferably binds to the protease m such a way as to reduce the proteolytic activity of that protease.
  • Isolated antibodies are antibodies that have been removed from their natural milieu.
  • isolated does not refer to the state of purity of such antibodies.
  • isolated antibodies can mclude anti-sera containing such antibodies, or antibodies that have been purified to varying degrees.
  • selective binds to refers to the ability of such antibodies to preferentially bind to the protease agamst which the antibody was raised (i.e., to be able to distinguish that protease from unrelated components in a mixture.) . Binding affinities typically range from about 10 ' M to about 10 1 - M " .
  • Binding can be measured using a variety of methods known to those skilled m the art including immunoblot assays, immunoprecipitation assays, radioimmunoassays, enzyme immunoassays (e.g., ELISA) , immunofluorescent antibody assays and immunoelectron microscopy; see, for example, Sambrook et al., lbi a .
  • Antibodies of the present mvention can be either polyclonal or monoclonal antibodies.
  • Antibodies of the present invention mclude functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protem used to obtain the antibodies.
  • Antibodies of the present mvention also include chimeric antibodies that can bind to more than one epitope.
  • Preferred antibodies are raised m response to proteins that are encoded, at least m part, by a flea protease nucleic acid molecule of the present invention.
  • Anti-flea antibodies of the present invention mclude antibodies raised m an animal administered a flea protease vaccine of the present invention that exert their effect when fleas feed from the vaccinated animal's blood containing such antibodies.
  • Anti-flea antibodies of the present invention also include antibodies raised m an animal agamst one or more flea protease proteins, or soluble flea midgut preparations, of the present invention that are then recovered from the animal using techniques known to those skilled m the art.
  • Yet additional antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed for flea protease proteins of the present invention.
  • Antibodies produced agamst defined proteins can be advantageous because such antibodies are not substantially contaminated with antibodies agamst other substances that might otherwise cause interference m a diagnostic assay or side effects if used m a therapeutic composition.
  • Anti-flea protease antibodies of the present mvention have a variety of uses that are within the scope of the present invention. For example, such antibodies can be used m a composition of the present invention to passively immunize an animal m order to protect the animal from flea infestation. Anti-flea antibodies can also be used as tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of proteins and other contaminants. Furthermore, antibodies of the present mvention can be used to target cytotoxic agents to fleas m order to kill fleas. Targeting can be accomplished by conjugating (i.e., stably joining) such antibodies to the cytotoxic agents using techniques known to those skilled m the art.
  • a preferred anti-flea protease antibody of the present invention can selectively bind to, and preferentially reduce the proteolytic activity of, a flea serine protease, a flea metalloprotease, a flea aspartic acid protease and/or a flea cysteine protease.
  • More preferred anti-flea protease antibodies include anti-flea serine protease antibodies, anti-flea metalloprotease antibodies, anti-flea ammopeptidase antibodies, and anti-flea cysteine protease antibodies.
  • anti-flea serine protease antibodies particularly preferred are anti-flea serine protease antibodies, anti-flea ammopeptidase antibodies, and anti-flea cysteine protease antibodies, including those raised agamst flea serine protease proteins, flea ammopeptidase proteins or cysteine protease proteins of the present invention.
  • the present invention also includes the use of protease inhibitors that reduce proteolytic activity of flea proteases to reduce flea infestation of animals and the surrounding environment.
  • protease inhibitors are compounds that interact directly with a protease thereby inhibiting that protease's activity, usually by binding to or otherwise interacting with the protease 's active site.
  • Protease inhibitors are usually relatively small compounds and as such differ from anti- protease antibodies that interact with the active site of a protease.
  • Protease inhibitors can be used directly as compounds m compositions of the present invention to treat animals as long as such compounds are not harmful to the animals being treated.
  • Protease inhibitors can also be used to identify preferred types of flea proteases to target using compositions of the present mvention.
  • the inventors have shown herein the predominance of serine proteases m flea midguts, particularly in soluble flea midgut preparations, usmg protease inhibitors. Such knowledge suggests that effective reduction of flea infestation of an animal can be achieved using serine protease vaccines, anti-flea serine protease antibodies and other inhibitors of serine protease synthesis and activity that can be tolerated by the animal.
  • flea immunoglobulin proteinase activity disclosed herein can be targeted to reduce flea infestation. That other proteases are also present m flea midguts accordmg to the present mvention also suggests targeting such proteases. Methods to use protease inhibitors are known to those skilled in the art; examples of such methods are disclosed herein.
  • a protease inhibitor that can be used m a composition of the present invention to treat an animal is identified by a method including the following steps: (a) identifying candidate (i.e., putative, possible) inhibitor compounds by testing the efficacy of one or more protease inhibitors (l) in vi tro for their ability to inhibit flea protease activity and/or (ii) m a flea feeding assay for their ability to reduce the survival and/or fecundity of fleas by adding the inhibitors to the blood meal of a flea being maintained, for example, m a feeding system, such as that described by Wade et al., 1986, J. Med Entomol .
  • candidate compounds identified usmg the in vi tro assay may work "m the test tube” but may not work in vivo for a number of reasons, including the presence of interfering components m the blood meal that inhibit the activity of such compounds; e.g., although aprotinin can inhibit at least some flea serine proteases in vi tro, aprotinin does not work well m the presence of serum proteins, such as are found m the blood.
  • candidate inhibitor compounds identified by the flea feeding assays can include not only desired compounds but also compounds that reduce the viability and/or fecundity of fleas due to general toxicity (e.g., affecting the mitochondria of fleas) .
  • an inhibitor of a flea protease of the present invention is identified by a method comprising: (a) contacting an isolated flea protease protem comprising an ammo acid sequence including SEQ ID NO:10, SEQ ID N0:13, SEQ ID N0:16, SEQ ID N0:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID N0:41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ
  • a test kit can be used to perform such method.
  • a preferred test kit comprises an isolated flea protease protem comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:
  • protease inhibitors are used m the purification of corresponding proteases by, for example, affinity chromatography, m which, a protease inhibitor is incubated with a mixture containing a desired protease under conditions that the inhibitor forms a complex with the protease. The protease can then be recovered from the complex.
  • the protease inhibitor can be attached to a solid support and/or be labelled with, for example, a radioactive, fluorescent, or enzymatic tag that can be used to detect and/or recover the complex.
  • Suitable protease inhibitors to use in accordance with the present invention include serine protease inhibitors (including immunoglobulin proteinase inhibitors and serpms) , metalloprotease inhibitors, aspartic acid protease inhibitors, cysteine protease inhibitors and ammopeptidase inhibitors.
  • Preferred protease inhibitors include serine protease inhibitors, metalloprotease inhibitors, ammopeptidase inhibitors and cysteine protease inhibitors, particularly those that are broad spectrum inhibitors. More preferred are broad spectrum serine protease inhibitors.
  • protease inhibitors There is a wide variety of protease inhibitors, as is known to one skilled in the art.
  • Examples include, but are not limited to, AEBSF, aprotinin, bestatm, chloromethyl ketones TLCK (N ⁇ -p-tosyl-L-lysme chloromethyl ketone) and TPCK (N-tosyl-L-phenylalanme chloromethyl ketone), chymostatm, cystatm, 3 ' 4-d ⁇ chloro ⁇ socoumarm, E-64 (trans-epoxysuccmyl-L-leucylamido- (4-guan ⁇ dmo) butane) , EDTA (ethylenediammetetraacetic acid) , leupeptin, methyl ketones havmg a variety of leaving groups, oxidized L- leucmethiol, pepstatin, 1, 1O-orthophenanthrolme, phosphoramidon, soybean trypsm/chymotrypsm inhibitor and soybean trypsin inhibitor.
  • Preferred protease inhibitors for use m the present invention include AEBSF, bestatm, E-64 leupeptin, pepstatin, 1, 10-orthophenanthrolme, phosphoramidon, TLCK and TPCK, with AEBSF ⁇ a broad spectrum serine protease inhibitor) , bestatm (an inhibitor of leucine ammopeptidase) and 1, 10-orthophenanthrolme (a broad spectrum metalloprotease inhibitor) being particularly preferred.
  • Another preferred inhibitor of the present invention mcludes an inhibitor of an immunoglobulin proteinase of the present invention.
  • Suitable inhibitors of 6 ⁇ immunoglobulin proteinase activity are compounds that interact directly with an immunoglobulin proteinase protein's active site, thereby inhibiting that immunoglobulin proteinase' s activity, usually by binding to or otherwise interacting with or otherwise modifying the immunoglobulin proteinase' s active site.
  • Immunoglobulin proteinase inhibitors can also interact with other regions of the immunoglobulin proteinase protem to inhibit immunoglobulin proteinase activity, for example, by allosteric interaction.
  • Inhibitors of immunoglobulin protemases are usually relatively small compounds and as such differ from anti-immunoglobulm proteinase antibodies.
  • an immunoglobulin proteinase inhibitor of the present invention is identified by its ability to bind to, or otherwise interact with, a flea immunoglobulin proteinase protem, thereby inhibiting the activity of the flea immunoglobulin proteinase.
  • Preferred immunoglobulin proteinase inhibitors of the present invention include, but are not limited to, flea immunoglobulin proteinase substrate analogs, and other molecules that bind to a flea immunoglobulin proteinase
  • An immunoglobulin proteinase substrate analog refers to a compound that interacts with (e.g., binds to, associates with, modifies) the active site of an immunoglobulin proteinase protem.
  • a preferred immunoglobulin proteinase substrate analog inhibits immunoglobulin proteinase activity.
  • Immunoglobulin proteinase substrate analogs can be of any inorganic or organic composition, and, as such, can be, but are not limited to, peptides, nucleic acids, and peptidomimetic compounds.
  • Immunoglobulin proteinase substrate analogs can be, but need not be, structurally similar to an immunoglobulin proteinase' s natural substrate as _.ong as they can interact with the active site of that proteinase protem. Immunoglobulin proteinase substrate analogs can be designed using computer-generated structures of immunoglobulin proteinase proteins of the present invention or computer structures of immunoglobulin protemases' natural substrates.
  • Substrate analogs can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides, peptidomimetic compounds, or otner inorganic or organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner, (e.g., a flea immunoglobulin proteinase) .
  • molecules such as oligonucleotides, peptides, peptidomimetic compounds, or otner inorganic or organic molecules.
  • affinity chromatography techniques using the corresponding binding partner, (e.g., a flea immunoglobulin proteinase) .
  • a preferred immunoglobulin proteinase substrate analog is a peptidomimetic compound (i.e., a compound that is structurally and/or functionally similar to a natural substrate of an immunoglobulin proteinase of tne present invention, particularly to the region of the substrate that interacts with the proteinase active site, but that inhibits immunoglobulin proteinase activity upon interacting with the immunoglobulin proteinase active site) .
  • Another preferred flea immunoglobulin proteinase inhibitors of the present invention include antibodies that bind specifically to an immunoglobulin proteinase m such a manner that the proteinase activity of the immunoglobulin proteinase is inhibited.
  • Yet another preferred flea immunoglobulin proteinase inhibitor includes an inhibitor from the class of serine proteinase inhibitors. Suitable immunoglobulin proteinase inhibitor include serine proteinase inhibitors disclosed herein.
  • Protease inhibitors can be produced usmg methods known to those skilled m the art.
  • Protem- or peptide- based protease inhibitors such as cystatm or small peptides comprising a protease substrate, can be produced recombinantly and modified as necessary.
  • the present invention also includes the use of proteolytically active flea protease proteins of the present mvention to identify additional protease inhibitors, and preferably protease inhibitor compounds that can be included m a composition of the present invention to be administered to animals.
  • a method to identify a flea protease inhibitor includes the steps of (a) contacting (e.g., combining, mixing) an isolated flea protease protem with a putative (i.e., candidate) inhibitory compound under conditions in which, in the absence of the compound, the protein has proteolytic activity, and (b) determining if the putative inhibitory compound inhibits the proteolytic activity of the protein.
  • Putative inhibitory compounds to screen include organic molecules, antibodies (including functional equivalents thereof) and substrate analogs.
  • protease activity is known to those skilled in the art, as heretofore disclosed.
  • Particularly preferred for use in identifying inhibitors are flea serine protease proteins, flea aminopeptidase proteins and flea cysteine protease proteins of the present invention.
  • the present invention also includes inhibitors isolated by such a method, and/or test kit, and their use to inhibit any flea protease that is susceptible to such an inhibitor.
  • the present invention also includes mimetopes of compounds of the present mvention that can be used in accordance with methods as disclosed for compounds of the present invention.
  • a mimetope of a proteinaceous compound of the present invention e.g., a flea protease protein, an anti- flea protease antibody, a proteinaceous inhibitor of protease activity or synthesis
  • a mimetope of a flea protease protein is a compound that has an activity similar to that of an isolated flea protease protein of the present invention.
  • Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation; anti- idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures) ; and synthetic or natural organic molecules, including nucleic acids. Such mimetopes can be designed using computer- generated structures of proteins of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.
  • the present invention includes therapeutic compositions, also referred to herein as compositions, that include a (i.e., at least one) compound of the present invention.
  • Preferred compounds to include in a composition of the present invention include flea protease vaccines, anti-flea protease antibodies and/or protease inhibitors as disclosed herein.
  • Such a therapeutic composition can protect an animal from flea infestation by reducing flea protease activity, thereby reducing flea burden on the animal and m the environment of the animal.
  • compositions of the present invention include at least one of the followmg compounds: an isolated flea serine protease protem or a mimetope thereof; an isolated flea serine protease nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea serine protease gene; an isolated antibody that selectively binds to a flea serine protease protem; an inhibitor of flea serine protease activity identified by its ability to inhibit flea serine protease activity; an isolated flea ammopeptidase protem or a mimetope thereof; an isolated flea ammopeptidase nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea ammopeptidase gene; an isolated antibody that selectively binds to a flea ammopeptidase protem; an inhibitor of flea ammopeptidase activity identified by its ability to inhibit flea ammopeptidase activity; an isolated flea cyste
  • Another embodiment of the present invention is a therapeutic composition that includes a first compound that reduces flea protease activity and a second compound that reduces flea burden by a method other than by reducing flea protease activity.
  • the present invention also includes a method to protect an animal from flea infestation by administering to the animal such a composition.
  • the first compound of such a composition by effectively reducing flea protease activity m the midgut, enhances the activity of the second compound. While not being bound by theory, it is believed that a number of anti-flea treatments, particularly those that are proteinaceous, are not very effective because they are degraded m the flea midgut.
  • the present invention permits the effective use of such anti-flea treatments by reducing proteolytic degradation of such treatments by the flea midgut.
  • Preferred first compounds to include m such a composition include flea protease vaccines, anti-flea protease antibodies and/or protease inhibitors as disclosed herem, such compounds that target flea immunoglobulin proteinase activity.
  • a preferred therapeutic composition of the present invention comprises an excipient and a protective compound including: an isolated protem or mimetope thereof encoded by a nucleic acid molecule that hybridizes under stringent nvbridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protem comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ
  • Suitable second compounds mclude any anti-flea agent (s) , including, but not limited to, proteinaceous compounds, insecticides and flea collars.
  • Preferred second compounds are proteinaceous compounds that effect active immunization (e.g., antigen vaccines), passive immunization (e.g., antibodies), or that otherwise inhibit a flea activity that when inhibited can reduce flea burden on and around an animal.
  • second 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 vitellm and transport and maturation thereof mto a major egg yolk protem) , 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 and/or a compound that inhibits flea feeding.
  • 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
  • hormone including peptide or
  • an immunoglobulin proteinase of the present invention can also be used as a second compound in a therapeutic composition of the present invention to promote longevity of antibodies that bind specifically to selected flea proteins.
  • An immunoglobulin proteinase can be administered to an animal tc promote production of antibodies that bind specifically to the immunoglobulin proteinase, thereby inhibiting the activity of the proteinase.
  • An immunoglobulin proteinase can be administered to an animal either together with cr after administration of any desired flea protem to the animal.
  • a preferred immunoglobulin proteinase to include as a second compound in a therapeutic composition includes: an isolated protem or a mimetope thereof encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising ammo acid sequence SEQ ID NO: 67, SEQ ID NO: 68 and/or SEQ ID NO: 69; and/or an isolated nucleic acid molecule that hybridizes under stringent conditions with a gene comprising a nucleic acid sequence including SEQ ID NO: 66 and other nucleic acid sequences encoding an immunoglobulin proteinase of the present mvention disclosed herein.
  • compositions of the present invention can also include other components such as a pharmaceutically acceptable excipient, an adjuvant, and/or a carrier.
  • a pharmaceutically acceptable excipient such as a pharmaceutically acceptable excipient, an adjuvant, and/or a carrier.
  • compositions of the present mvention can be formulated m an excipient that the animal to be treated can tolerate.
  • excipients examples 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 on, 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 mclude 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 m a suitable liquid as a suspension or solution for injection.
  • the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
  • the composition can also include an immunopotentiator, such as an adjuvant or a carrier.
  • adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, Freund's adjuvant; other bacterial cell wall components; alummum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins; viral coat proteins; other bacterial-derived preparations; gamma interferon; block copolymer adjuvants, such as Hunter's Titermax adjuvant (Vaxce!- 1" , Inc.
  • Carriers are typically compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, bacteria, viruses, oils, esters, and glycols.
  • a controlled release formulation that is capable of slowly releasing a composition of the present invention mto an animal.
  • 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 m si tu .
  • 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 reduce protease activity in fleas feeding from the animal over a period of time ranging from about 1 to about 12 months.
  • a controlled release formulation of the present invention is capable of effecting a treatment for preferably at least about 1 month, more preferably at least about 3 months and even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.
  • a therapeutic composition of the present invention is administered to the animal in an effective manner such that the protease activity of fleas feeding from the blood stream of animals treated with the composition is reduced.
  • a treated animal is an animal that is competent to reduce the flea burden by reducing flea protease activity, or by reducing flea protease activity and at least one other flea activity.
  • the protease activity is reduced by at least about 50 percent, more preferably by at least about 70 percent and even more preferably by at least about 90 percent.
  • Methods to administer compositions to the animal in order to render the animal competent depend on the nature of the composition and administration regime.
  • Animals administered a protease vaccine with at least one booster shot usually become competent at about the same time as would be expected for any vaccine treatment. For example, animals administered a booster dose about 4 to 6 weeks after a primary dose usually become competent withm another about 3 to 4 weeks. Animals administered a composition including an anti-flea protease antibody or protease inhibitor become competent as soon as appropriate serum levels of the compound are achieved, usually with one to three days .
  • a composition of the present invention when administered to a host animal is able to reduce flea viability by at least about 50 percent withm at least about 21 days after the fleas begin feeding from the treated animal. (Note that fleas usually live about 40 days to about 50 days on one or more animals.)
  • a more preferred composition when administered to a host animal is able to reduce flea viability by at least about 65 percent withm at least about 14 days after the fleas begin feeding from the treated animal.
  • An even more preferred composition when administered to an animal is able to reduce flea viability by at least about 90 percent withm at least about 7 days after the fleas begin feeding from the treated animal.
  • a composition of the present invention when administered to a host animal is able to reduce flea fecundity (i.e., egg laying abi ⁇ ity) by at least about 50 percent, more preferably by at least about 70 percent, and even more preferably by at least about 90 percent, withm at least about 30 days after the fleas begin feeding from the treated animal. (Note that fleas usually do not begin laying eggs until about 7 days after taking a blood meal.)
  • compositions are administered to an animal in a manner such that the animal becomes competent to reduce flea protease activity in a flea that feeds from the competent; i.e., the animal becomes a treated animal.
  • a flea protease vaccine of the present mvention when administered to an animal m an effective manner, is able to elicit (i.e., stimulate) an immune response that produces an antibody titer in the blood stream of the animal sufficient to reduce flea protease activity.
  • an anti-flea protease antibody of the present invention when administered to an animal in an effective manner, is administered m an amount so as to be present m the animal's blood stream at a titer that is sufficient to reduce flea protease activity.
  • a protease inhibitor compound of the present invention when administered to an animal m an effective manner, is administered m a manner so as to be present m the animal's blood stream at a concentration that is sufficient to reduce flea protease activity.
  • Oligonucleotide nucleic acid molecules of the present invention can also be administered in an effective manner, thereby reducing expression of flea proteases.
  • compositions of the present invention can be administered to animals prior to or during flea infestation. It is to be noted that when vaccines of the present invention are administered to an animal, a time period is required for the animal to elicit an immune response before the animal is competent to inhibit protease activity of fleas feeding from that animal. Methods to obtain an immune response in an animal are known to those skilled in the art. Acceptable protocols to administer compositions m 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 flea infestation when administered one or more times over a suitable time period.
  • a preferred smgle dose of a protease vaccine or a mimetope thereof ranges from about 1 microgram ( ⁇ 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 tc 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 flea infestation.
  • a preferred administration schedule is one in which from about 10 ⁇ 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.
  • 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.
  • a preferred single dose of an anti-flea protease antibody composition or a mimetope thereof ranges from about 1 ⁇ g to about 10 mg of the composition per kilogram body weight of the animal.
  • Anti- flea 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 flea infestation.
  • a preferred administration schedule is one m which from about 10 ⁇ g to about 1 mg of an ant-.-f-.ea protease 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.
  • a nucleic acid molecule of the present invention can be administered to an animal m a fashion to enable expression of that nucleic acid molecule mto a protective protem (e.g., flea protease vaccine, anti-flea protease antibody, or proteinaceous protease inhibitor) or protective RNA (e.g., antisense RNA, ribozyme or RNA drug) in the animal to be protected from disease.
  • a protective protem e.g., flea protease vaccine, anti-flea protease antibody, or proteinaceous protease inhibitor
  • protective RNA e.g., antisense RNA, ribozyme or RNA drug
  • Nucleic acid molecules can be delivered to an animal m a variety of methods including, but not limited to, (a) direct injection (e.g., as "naked” DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Sci ence 247, 1465-1468) or (b) packaged as a recombinant virus particle vaccine or as a recombinant cell vaccine (i.e., delivered to a cell by a vehicle selected from the group consisting of a recombinant virus particle vaccine and a recombinant cell vaccine) .
  • direct injection e.g., as "naked” DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Sci ence 247, 1465-1468
  • packaged as a recombinant virus particle vaccine or as a recombinant cell vaccine i.e., delivered to a cell by a vehicle selected from the group consisting of a recombinant virus particle vaccine
  • a recombinant virus particle vaccine of the present invention includes a recombinant molecule of the present invention that is packaged m a viral coat and that can be expressed in an animal after administration.
  • 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.
  • a recombinant virus particle vaccine of the present invention infects cells within the immunized animal and directs the production of a protective protein or RNA nucleic acid molecule that is capable of protecting the animal from disease caused by a parasite of the present invention.
  • a preferred single dose of a recombinant virus particle vaccine of the present invention is from about 1 x 10 4 to about 1 x 10' virus plaque forming units (pfu) per kilogram body weight of the animal.
  • Administration protocols are similar to those described herein for protein-based vaccines.
  • 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 include Salmonella, E. coli , Mycobacterium, S. frugiperda, baby hamster kidney, myoblast G8, COS, MDCK and CRFK recombinant cells, with Salmonella recombinant cells being more preferred.
  • Such recombinant cells can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about IO 8 to about IO 12 bacteria per kilogram body weight. Administration protocols are similar to those described herein for protem-based vaccines .
  • Recombinant cell vaccines can comprise whole cells or cell lysates.
  • compositions of the present invention can be administered to any animal susceptible to flea 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, bemg more preferred.
  • Particularly preferred animals to protect are cats and dogs.
  • compositions to treat flea infestation by any flea can be derived from any flea species.
  • Preferred fleas to target include fleas of the following genera: Ctenocephalides, Cyopsyll us, Diamanus ( Oropsylla) , Echi dnophaga, Nosopsyll us, Pulex, Tunga, and Xenopsylla, with those of the species Ctenocephalides cams, Ctenocephalides feli s, Diamanus montanus, Echidnophaga gallmacea, Nosopsyll us faciatus, Pulex irri tans, Pul ex simulans, Tunga penetrans and Xenopsylla cheopi s being more preferred.
  • fleas from which to protect animals mclude fleas of the species Ctenocephalides felis, Ctenocephalides cam s, and Pul ex species (e.g., Pulex irri tans and Pulex simulans ) . It is also wit.iin the scope of the present invention to administer compositions of the present mvention directly to fleas.
  • the present invention also includes the use of compositions of the present mvention to reduce infestation by other ectoparasites as well as the use of compositions including protease vaccines, anti-protease antibodies and compounds that inhibit protease synthesis and/or activity derived from any ectoparasite to reduce ectoparasite infestation, particularly controlled release formulations containing such compositions.
  • Preferred ectoparasites to target include arachnids, insects and leeches. More preferred ectoparasites to target include fleas; ticks, including both hard ticks of the family Ixodidae (e.g., Jxodes and Amblyomma) and soft ticks of the family Argasidae (e.g., Orni thodoros, such as 0. parkeri and 0.
  • flies such as midges (e.g., Culicoides) , mosquitos, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis- causmg flies and bitmg gnats; ants; spiders, lice; mites; and true bugs, such as bed bugs and kissing bugs, including those carrying Chagas disease.
  • Even more preferred ectoparasites to target include fleas, mosquitos, midges, sandflies, blackflies, ticks and Rhodm us .
  • the following examples are provided for the purposes cf illustration and are not intended to limit the scope of tne present invention.
  • Example 1 includes a number of molecular biology, microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook et al . , ibi d. , Borovsky, Arch Insect Bi ochem . and Phys . , 7:187-210, 1988, and related references. Examples 1 through 21, and the sequence information provided in the sequence listing therein, of related PCT Publication No. WO 96/11706, published April 25, 1996, are incorporated herein by this reference in their entirety.
  • Example 1 includes a number of molecular biology, microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook et al . , ibi d. , Borovsky, Arch Insect Bi ochem . and Phys . , 7:187-210, 1988, and related references. Examples 1 through 21, and the sequence information provided in the
  • This example describes the determination of internal ammo acid sequence of a flea aminopeptidase.
  • the resulting pellet was resuspended and sonicated m 4 ml buffer comprising 20 mM NaAc, pH 6.0, 0.1% Brij , complete protease inhibitor cocktail (available from Pierce) and 0.25 mM bestatm; the sonicate was centrifuged at about 14,000 rpm for about 20 minutes. Both the pellet and supernatant were recovered. The pellet was re-sonicated and centrifuged as above, and the resulting supernatant was combined with the original supernatant.
  • the pooled supernatant was applied to a polyCAT cation exchange HPLC column and protem was eluted with a NaCl gradient ranging from 0M to IM NaCl in 20 mM NaAc, pH 6.0.
  • Fractions collected from the column were assayed by H-Leu-AMC fluorescence, and active fractions were pooled and applied to a C-1 reverse phase HPLC column (TMS 250, Toso Hass) . Proteins were eluted from the column using an acetonitrile gradient m 0.1% TFA m water, the gradient ranging between 20% and 100% acetonitrile.
  • Proteins contained m fractions from the column were analyzed by SDS-PAGE gel electrophoresis and silver staining. The results of tne gel electrophoresis indicated the presence of an about 95 kDa protem m some of the fractions. This protem correlates with the about 95 kDa protem described in Example 12 of related PCT Publication No. WO 96/11706 which was identified using membrane pellet from flea midgut lysates. To determine internal ammo acid sequence of the 95 kDa protem, those fractions containing the 95 kDa protem were pooled, dried and digested with BNPS-Skatole for about 72 hours at room temperature.
  • the BNPS-Skatole digest was separated by 18% Tris-glycme PAGE gel electrophoresis and blotted onto PVDF membrane. A major band of about 28 kDa was cut out and N-terminally sequenced using techniques as described in Example 7 of related PCT Publication No. WO 96/11706. A partial N-termmal ammo acid sequence of the internal peptide was obtained, namely LATTQFQATHARSAFPCFDEPAM (denoted herem SEQ ID NO:107) .
  • Manduca sexta and rat ammopeptidases having nucleic acid sequence 5' CCC AAA TTT TCC ATW GCN CCN GC 3' (N indicating any nucleotide; represented herein as SEQ ID NO: 108) was used m combination with primer M13 Reverse primer (SEQ ID NO: 87) to PCR amplify a portion of a flea ammopeptidase gene from a bovine blood-fed whole flea cDNA expression library as described m Example 8 of related PCT Publication No. WO 96/11706.
  • the resulting product of the PCR amplification was diluted about 1:50 and used as a template in a second, semi-nested PCR amplification usmg a primer APN3 m combination with degenerate primer APN1C, designed using SEQ ID NO: 107 (described in Example 1), having nucleic acid sequence 5' CAA TTY CAA GCT ACY CAT GC 3' (represented herein as SEQ ID NO: 109) .
  • the resulting PCR product, named nfAP2 383 was approximately 383-bp when visualized on a 1% agarose gel.
  • the PCR product nfAP2 383 was gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques.
  • the nucleotide sequence of nfAP2 38 is denoted SEQ ID NO: 110.
  • Translation of SEQ ID NO: 110 yielded a deduced flea ammopeptidase protem of about 127 ammo acids, denoted herem as PfAP2 ⁇ 27 , having ammo acid sequence SEQ ID NO: 111.
  • nfAP2 38 The PCR product nfAP2 38 , was labelled with "P and used as a probe to screen a bovine blood-fed whole flea phage expression library usmg standard hybridization techniques.
  • a single plaque purified clone was isolated, which included a 2100-nucleot ⁇ de insert, referred to herein as nfAP2 2100 .
  • Partial nucleic acid sequence was obtained using standard techniques from the 5' end of nfAP2 2100 , to yield a flea ammopeptidase nucleic acid molecule named nfAP2. )V . having nucleic acid sequence SEQ ID NO: 112.
  • nucleic acid molecule nfAP2 5 encodes a non-full-length flea ammopeptidase protem of about 178 ammo acids, referred to herein as PfAP ]78 , having ammo acid sequence SEQ ID NO: 113, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID N0:112.
  • SEQ ID NO:113 contains SEQ ID N0:107.
  • Flea ammopeptidase nucleic acid sequence SEQ ID NO: 112 was compared with additional nucleic acid sequences characterized from other organisms.
  • the nucleic acid sequence is about 50% identical to Manduca sexta ammopeptidase N nucleotides between corresponding regions of the two nucleic acid molecules.
  • Example 3 This example describes the cloning and sequencing of a flea cysteine protease nucleic acid molecule.
  • nfCPl s ⁇ A flea cysteine protease nucleic acid molecule, referred to herein as nfCPl s ⁇ was produced by PCR amplification using the following method.
  • Primer Cal3F (designed to obtain a calreticulin gene), having nucleic acid sequence 5' TTG GGA TAC ACT TTG ACT GTT AAC C 3', represented herein as SEQ ID NO: 97 was used in combination with the M13 universal primer, to PCR amplify, using standard techniques, a DNA fragment from a bovine blood-fed whole flea cDNA expression library as described above m
  • the isolated DNA fragment correlated with a cysteine protease nucleic acid sequence.
  • Sequence from this DNA fragment was used to design primer CyslR, having the nucleic acid sequence 5' GTG AGC AAC CAT TAT TTC CAT ATC 3', represented herein as SEQ ID NO: 98, which was used m a second PCR amplification m combination with the M13 reverse primer.
  • a third PCR amplification was performed usmg primer CyslF, having the nucleic acid sequence 5' CTT TCC TCA CAA TAC CAC CAA GGA AGC 3', represented herein as SEQ ID NO: 74, in combination with the M13 universal primer.
  • a fourth PCR amplification was performed using primer Cys2F, having the nucleic acid sequence 5' CTT GTA CGA TTG TCT CAA CAG GC 3', represented herein as SEQ ID NO: 76, m combination with the M13 universal primer.
  • the resulting PCR products were each gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques.
  • a composite nucleic acid sequence representing a flea cysteine protease coding region was deduced, referred to herein as nfCPl 573 , was deduced and is denoted herein as SEQ ID NO:76.
  • SEQ ID NO: 76 suggests that nucleic acid molecule nfCPl 5 -> encodes a non- full-length flea cysteine protease protem of about 191 ammo acids, referred to herem as PfCPl ]91 , having ammo acid sequence SEQ ID NO: 77, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:76.
  • SEQ ID NO: 77 was found to be similar to the ammo acid sequence of P . sa ti vum cysteine protease. The most highly conserved region of contmuous similarity between SEQ ID NO: 77 and P.
  • sa ti vum cysteine protease ammo acid sequences spans from about ammo acid 71 through about ammo acid 165 of SEQ ID NO:77 and from about ammo acid 17 through about ammo acid 168 of the P. sa ti vum cysteine protease, there being about 42% identity between the two regions. Comparison of the nucleic acid sequence encoding ammo acids from about 205 through about 492 of nfCPl, , indicate that those regions are about 54- identical.
  • This example describes the cloning and sequencing of certain flea serine protease nucleic acid molecules. Additional serine protease cDNA nucleic acid molecules have been isolated in a manner similar to that described m Example 8 of related PCT Publication No. WO 96/11706.
  • the actual primers used in PCR amplification of serine protease nucleic acid molecules from a bovme blood-fed flea cDNA expression library included cat-try #2 (SEQ ID NO: 86) in combination with either M13 reverse primer (SEQ ID NO:87, or H57 primer (SEQ ID NO:99) .
  • the resultant PCR products were gel purified and cloned mto the TA VectorTM.
  • Two recombinant TA vector clones were isolated and found to correspond to previously cloned serine protease genes. These newly cloned nucleic acid molecules were subjected to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described in Sambrook et al . , ibi d.
  • a nucleic acid sequence of the flea serine protease nucleic molecule corresponding to flea clone 5 (produced using primers cat try #2 and M13 reverse) , namely nfSP5 «Ct . is represented herem as SEQ ID NO:114.
  • SEQ ID NO: 116 and SEQ ID NO: 117 are both contained withm the sequence of the nucleic acid molecule nfSP5 806 .
  • a Genbank homology search revealed most homology between SEQ ID NO: 114 and a Gall us gall us trypsin gene, there being about 52% identity between correspondmg regions of the two nucleic acid molecules.
  • B. A nucleic acid sequence of the flea serine protease nucleic molecule corresponding to flea clone 11 (produced using primers cat try #2 and M13 reverse) , namely nfSPll, 07 , is represented herein as SEQ ID NO: 118.
  • SEQ ID NO: 120 and SEQ ID NO: 121 are withm the sequence of the nucleic acio molecule nfSPll 3C - Translation of SEQ ID NO: 118 suggests tnat nucleic acid molecule nfSPll 30 - encodes a non-full- length flea serine protease protem of about 102 ammo acids, referred to herein as PfSPll 102 , having ammo acid sequence SEQ ID NO: 119, assuming the first codon spans from aoout nucleotide 1 through about nucleotide 3 of SEQ ID NO: 118. C.
  • Translation of SEQ ID NO: 122 suggests that nucleic acid molecule nfSP39 267 encodes a non-full-length flea serine protease protein of about 90 ammo acids, referred to herein as PfSP39 89 , having ammo acid sequence SEQ ID NO: 123, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:122.
  • Example 5 This example describes the cloning and sequencing of certain flea serine protease nucleic acid molecules.
  • Certain flea serine protease cDNA nucleic acid molecules have been isolated in a manner similar to that described m Example 8 of related PCT Publication No. WO 96/11706, using two nucleic acid molecules as probes to screen a bovine blood-fed flea cDNA expression library (produced as described m Example 8 of related PCT Publication No. WO 96/11706), cat-try #1 (SEQ ID NO: 124) and cat-try #2 (SEQ ID NO: 86) .
  • Two clones that hybridized strongly to the probes were isolated and subjected to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described in Sambrook et al . , ibi d.
  • SEQ ID NO: 125 The nucleic acid sequence of a flea serine protease nucleic molecule correlating to flea clone 8, namely nfSP8 436 is represented herein as SEQ ID NO: 125.
  • SEQ ID NO: 127 is within the sequence of the nucleic acid molecule nfSP ⁇ 43b _ Translation of SEQ ID NO: 125 yields a protein of about 145 amino acids, denoted PfSP8 145 , having amino acid sequence SEQ ID NO:126, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 125.
  • a Genbank homology search revealed most homology between SEQ ID NO: 125 and an Anophel es gambiae trypsin precursor gene, there being about 48% identity between corresponding regions of the two nucleic acid molecules .
  • the nucleic acid sequence of a flea serine protease nucleic molecule corresponding to flea clone 12, namely nfSP12 756 is represented herein as SEQ ID NO: 128.
  • SEQ ID NO: 130 and SEQ ID NO: 131 are both contained within the sequence of the nucleic acid molecule nfSP12 758 .
  • Translation cr SEQ ID NO: 128 yields a protem of about 246 ammo acids, denoted PfSP12 4 , having ammo acid sequence SEQ ID NO: 129, assuming an open reading frame m which the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 128 and a stop codon spanning from about nucleotide 739 through about nucleotide 741 of SEQ ID NO: 128.
  • a Genbank homology search revealed most homology between SEQ ID NO: 128 and a rat trypsmogen gene, there being about 57? identity between corresponding regions of the two nucleic acid molecules.
  • Certain flea serine protease cDNA genes have been isolated from a cat blood-fed flea cDNA expression library by screening the library with the cat-try #1 (SEQ ID NO: 124) and cat-try #2 (SEQ ID NO: 86) probes.
  • the cat blood-fed flea library was produced in a similar manner as the bovme blood-fed flea library (described in Example 8 cf related PCT Publication No. WO 96/11706) except the fleas were fed on cat blood.
  • Two clones that hybridized strongly to the probes were isolated and subjected to nucleic acid sequencing using methods described above.
  • the nucleic acid sequence of one of the flea serine protease nucleic molecules, namely nfSP26 Dl0 is represented herein as SEQ ID NO: 132.
  • Translation of SEQ ID NO: 132 yields a non-full-length sequence of about 185 ammo acids, denoted PfSP26 ]8C> , having ammo acid sequence SEQ ID NO: 133, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 132.
  • a Genbank homology search revealed most homology between SEQ ID NO: 133 and a Aedes aegypti trypsin protem sequence, there being about 48% identity between corresponding regions of the two ammo acid sequences.
  • SEQ ID NO: 134 The nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP27 386 is represented herem as SEQ ID NO: 134.
  • Translation of SEQ ID NO: 134 yields a protem of about 128 ammo acids, denoted PfSP27 128 , having amino acid sequence SEQ ID NO: 135, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 134.
  • Example 6 This example describes the cloning and sequencing of certain flea serine protease nucleic acid molecules.
  • Certain serine protease cDNA nucleic acid molecules have been isolated from reverse transcriptase PCR amplification of mRNA isolated from cat blood-fed whole fleas.
  • the mRNA was isolated from fleas gathered over 72 hours after the initiation of feeding on cat blood. As such, the mRNA comprised a mixture of mRNA isolated at different time points over 72 hours.
  • the mRNA was isolated using ground-up fleas, extracting total flea RNA using Tri-Reagent (available from Molecular Research Center, Cincinnati, Ohio) and an Invitrogen Fast Track 1 '"' RNA isolation kit (available from Invitrogen, Inc. San Diego, CA) .
  • cDNA was synthesized using a Stratagene RT-PCR kit (available from Stratagene, Inc, San Diego, CA) .
  • Primers used for first-strand cDNA synthesis included an equal molar mixture of the followmg: 5'dT-2VT3' and 5'dT-2VC3' (as provided m a differential display kit, available from Operon Technologies, Inc. Alameda, CA) .
  • the actual primers used m the PCR amplification of the cDNA described above included cat-try #2 (SEQ ID NO: 86) used m combination with H57 primer (SEQ ID NO: 99) .
  • the resultant PCR products were gel purified and cloned mto the TA VectorTM.
  • Recombinant TA vector clones were isolated and the nucleic acid molecules were subjected to nucleic acid sequencing using analysis as described above.
  • a nucleic acid sequence of one of the flea serine protease nucleic molecules, namely nfSP23 423 is represented herein as SEQ ID NO: 136.
  • nucleic acid molecule nfSP23 42 encodes a non- full-length flea serine protease protem of about 141 ammo acids, referred to herein as PfSP23 141 , having amino acid sequence SEQ ID NO: 137, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 136.
  • a Genbank homology search revealed most homology between SEQ ID NO: 136 and a Homo sapi ens plasmmogen precursor gene, there being about 51% identity between corresponding regions of the two nucleic acid molecules.
  • nucleic acid sequence of a flea serine protease nucleic molecule is represented herein as SEQ ID NO: 78.
  • Translation of SEQ ID NO:78. suggests that nucleic acid molecule nfSP24 41c encodes a non- full-length flea serine protease protein of about 136 ammo acids, referred to herein as PfSP24 136 , having ammo acid sequence SEQ ID NO: 79, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 78.
  • a Genbank homology search revealed most homology between SEQ ID NO:79 and an Anophel es gambiae chymotrypsm protem sequence, there being about 38% identity between corresponding regions of the two amino acid sequences.
  • SEQ ID NO: 82 Another nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP33 426 , is represented herein as SEQ ID NO: 82.
  • Translation of SEQ ID NO: 82 suggests that nucleic acid molecule nfSP33 42e encodes a non- full-length flea serine protease protein of about 142 ammo acids, referred to herein as PfSP33 :42 , having amino acid sequence SEQ ID NO: 83, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 82.
  • SEQ ID NO:138 Another nucleic acid sequence of one of the flea serine protease nucleic molecule, namely nfSP36, q7 , is represented herein as SEQ ID NO:138.
  • SEQ ID NO:138 represents a partial sequence of a PCR amplified nucleic acid molecule nfSP36 S00 .
  • nucleic acid molecule nfSP36 19 - encodes a non- full-length flea serine protease protem of about 65 ammo acids, referred to herem as PfSP36 65 , having ammo acid sequence SEQ ID NO: 139, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 138.
  • a Genbank homology search revealed most homology between SEQ ID NO: 139 and a Drosophila melanogaster easter protem sequence, there being about 42% identity between correspondmg regions of the two ammo acid sequences.
  • SEQ ID NO: 140 Another nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP38 34] , is represented herein as SEQ ID NO: 140.
  • Translation of SEQ ID NO: 140 suggests that nucleic acid molecule nfSP38, 41 encodes a non- full-length flea serine protease protem of about 113 ammo acids, referred to herein as PfSP38 113 , having ammo acid sequence SEQ ID NO: 141, assuming the first codon spans from about nucleotide 3 through about nucleotide 5 of SEQ ID NO: 140.
  • a Genbank homology search revealed most homology between SEQ ID NO: 141 and a rat trypsmogen protem sequence, there being about 30% identity between corresponding regions of the two ammo acid sequences.
  • F. A nucleic acid sequence of one of the flea serine protease nucleic molecules, namely nfSP34 390 , is represented herein as SEQ ID NO: 142.
  • nucleic acid molecule nfSP4 390 encodes a non- full-length flea serine protease protein of about 130 amino acids, referred to herein as PfSP34 130 , having ammo acid sequence SEQ ID NO: 143, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:
  • Genbank homology search revealed most homology between SEQ ID NO: 143 and a Drosophila mel anogaster Delta precursor protein sequence, there being about 33% identity between corresponding regions of the two ammo acid sequences.
  • Example 7 This example describes the cloning and sequencing of a flea serine protease nucleic acid molecule.
  • a serine protease cDNA nucleic acid molecule was isolated in a manner similar to that described in Example 8 of related PCT Publication No. WO 96/11706.
  • the actual primers used in PCR amplification of the serine protease nucleic acid molecule from a cat blood-fed whole flea cDNA expression library included cat-try #2 (SEQ ID NO: 86) in combination with M13 reverse primer (SEQ ID NO:87) .
  • the resulting PCR product was diluted 1:25 and used as a template in a second PCR reaction using the forward vector primer T3 in combination with the reverse primer (derived from the nucleic acid sequence of nfSP33 7 , & , described m Example 6) having the nucleic acid sequence 5' ATT CCT CGT GGT TCA GTC GCT C 3', represented herein as SEQ ID NO: 100.
  • the resultant PCR product was gel purified and cloned mto the TA Vector 1 ⁇ . The clones were subjected to nucleic acid sequencing as described above.
  • a nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP33 77p is represented herein as SEQ ID NO: 84.
  • SEQ ID NO: 84 mcludes a portion of SEQ ID NO: 82
  • Translation of SEQ ID NO: 84 suggests that nucleic acid molecule nfSP33 778 encodes a non-full-length flea serine protease protem of about 259 amino acids, referred to herein as PfSP33 259 , havmg ammo acid sequence SEQ ID NO: 85, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 84.
  • a Genbank homology search revealed most homology between SEQ ID NO: 84 and a Drosophila serine protease stubble gene, there being about 54% identity between nucleotides 23 - 778 of SEQ ID NO: 84 and nucleotides 2324 - 3064 of the Drosophila serine protease stubble gene.
  • This example describes the cloning and sequencing of another flea serine protease nucleic acid molecule.
  • a cDNA clone of a flea serine protease was obtained usmg mRNA isolated from bovme blood-fed whole fleas.
  • the resulting cDNA was used as a template in PCR amplification using the primers cat-try #2 (SEQ ID NO: 86) used m combination with H57 primer (SEQ ID NO: 99) .
  • the resultant PCR products were gel purified and cloned mto the TA VectorTM.
  • TA vector clone was isolated and the flea serine protease nucleic acid molecule and denoted nFS37 50C was subjected to nucleic acid sequencing as described in Sambrook et al . , ibid.
  • nucleic acid sequence of part of the flea serine protease nucleic molecule nFS37 500 is represented herem as SEQ ID NO: 144.
  • Translation of SEQ ID NO: 144 suggests that nucleic acid molecule nfSP37 z61 encodes a non-full-length sequence of a flea serine protease protem of about 87 ammo acids, referred to herein as
  • PfSP37 cr having ammo acid sequence SEQ ID NO: 145, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 144.
  • a Genbank homology search revealed most homology between SEQ ID NO: 145 and a chicken trypsmogen protem sequence, there being about 31% identity between corresponding regions of the two ammo acid sequences.
  • This example describes the cloning and sequencing of certain larval flea serine protease nucleic acid molecules.
  • Certain serine protease cDNA nucleic acid molecules have been isolated from a mixed instar larval cDNA library produced using 1st, 2nd and 3rd instar larvae fed on cat blood, by PCR amplification.
  • the actual primers used in the PCR amplification included either cat-try #2 (SEQ ID NO: 86) m combination with either H57 primer (SEQ ID NO:99)or M13 reverse primer (SEQ ID NO:87) .
  • the resultant PCR products were gel purified and cloned into the TA Vector '1 '' 1 .
  • Three recombinant TA vector clones were isolated containing PCR products using cat-try #2 and M13 reverse as primers and one clone was isolated containing PCR products usmg cat-try #2 and H57 primers. These newly cloned nucleic acid molecules were subjected to nucleic acid sequencing as described above.
  • a nucleic acid sequence of one of the larval flea serine protease nucleic molecules isolated using cat-try #2 and M13 reverse primers, namely nfSP29 61z is represented herein as SEQ ID NO: 146.
  • Translation of SEQ ID NO: 146 suggests that nucleic acid molecule nfSP29 612 encodes a close to full-length flea serine protease protein of about 204 ammo acids, referred to herein as PfSP29 204 , having amino acid sequence SEQ ID NO: 147, assuming an open reading frame m which the first codon spans from about nucleotide 10 through about nucleotide 12 of SEQ IDNO: 146.
  • a Genbank homology search revealed most homology between SEQ ID NO: 146 and a rat trypsinogen gene, there being about 50% identity between corresponding regions of the two nucleic acid molecules.
  • SEQ ID NO: 148 Another nucleic acid sequence of one of the larval flea serine protease nucleic molecules isolated using cat-try #2 and M13 reverse primers, namely nfSP30 641 , is represented herein as SEQ ID NO: 148.
  • Translation of SEQ ID NO: 148 suggests that nucleic acid molecule nfSP30 64: encodes a non-full-length flea serine protease protein of about 213 amino acids, referred to herein as PfSP30 213 , having amino acid sequence SEQ ID NO: 149, assuming the first codon spans from about nucleotide 3 through about nucleotide 5 of SEQ ID NO: 148.
  • a Genbank homology search revealed most homology between SEQ ID NO: 148 and a Anophel es gambiae trypsin gene, there being about 52% identity between corresponding regions of the two nucleic acid molecules.
  • C Another nucleic acid sequence of one of the larval flea serine protease nucleic molecules isolated using cat-try #2 and M13 reverse primers, namely nfSP31 626 , is represented herein as SEQ ID NO: 150.
  • nucleic acid molecule nfSP31 62t encodes a non-full-length flea serine protease protein of about 208 amino acids, referred to herein as PfSP31 208 , having ammo acid sequence SEQ ID NO: 151, a assuming the f_.rst residue spans from about nucleotide 3 through about nucleotide 5 or from a putative start codon spanning from about nucleotide 6 to about nucleotide 8 of SEQ ID NO: 150.
  • a Genbank homology search revealed homology between SEQ ID NO: 150 and an Anophel es gambiae trypsin gene, there being about 52% identity between corresponding regions of the two nucleic acid molecules.
  • Translation of SEQ ID NO: 80 suggests that nucleic acid molecule nfSP32 4 , 3 encodes a non-full-length flea serine protease protem of about 144 ammo acids, referred to herein as PfSP32 144 , having amino acid sequence SEQ ID NO: 81, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 80.
  • a Genbank homology search revealed most homology between SEQ ID NO: 80 and an Anophel es gambiae trypsin gene, there being about 52% identity between corresponding regions of the two nucleic acid molecules.
  • This example describes the cloning and sequencing of another flea serine protease nucleic acid molecule.
  • a bovme blood-fed whole flea cDNA library (prepared as described in Example 8 of related PCT Publication No. WO 96/11706' was immunoscreened with antiserum collected from a rabbit that was immunized with a collection of flea salivary gland products referred to as fspN (as described m PCT Publication No. WO 96.11271, entitled “NOVEL ECTOPARASITE SALIVA PROTEINS AND APPARATUS TO COLLECT SUCH PROTEINS", published April 18, 1996) . Immunoscreenmg was performed as follows.
  • New Zealand White rabbit antiserum developed agamst fspN flea saliva products was used m the immunoscreenmg protocols described in the picoBlueTM Immunoscreenmg Kit instruction manual, available from Stratagene, Inc.
  • the methods for preparation of the cDNA expression libraries for immunoscreenmg i.e., expression of the cDNA clones and procedures for transferring lambda phage plaques to membranes for immunoscreenmg, are described in the ZAP-cDNA Synthesis Kit instruction manual, also available from Stratagene, Inc., La Jolla, California.
  • a nucleotide sequence for a flea serine protease nucleic acid molecule named nfSP15 815 is denoted as SEQ ID NO: 152 and corresponds to SEQ ID NO: 154.
  • Translation of SEQ ID NO: 152 suggests that nucleic acid molecule nfSP15 8)C encodes a close to full-length flea serine protease protem of about 254 ammo acids, referred to herein as PfSP15 2C , 4 , having ammo acid sequence SEQ ID NO: 153, assuming an open reading frame in which the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 152 and a stop codon spanning from about nucleotide 763 through about nucleotide 765 of SEQ ID NO: 152.
  • a Genbank homology search revealed homology between SEQ ID NO: 152 and an Anophel es gambiae trypsin gene,
  • This example describes the cloning and sequencing of additional flea serine protease nucleic acid molecules.
  • flea serine protease cDNA nucleic acid molecules have been isolated in a manner similar to that described in Example 8 of related PCT Publication No. WO 96/11706, using two nucleic acid molecules as probes to screen an unfed flea cDNA expression library, nfSP8 299 (SEQ ID NO: 127) and nfSP19 359 (SEQ ID NO: 155) .
  • a clone that hybridized strongly to the probes was isolated and subjected to nucleic acid sequencing as described above.
  • SEQ ID NO: 156 The nucleic acid sequence of the flea serine protease nucleic molecule, namely nfSP19 855 , is represented herein as SEQ ID NO: 156.
  • SEQ ID NO: 155 is withm the sequence of the nucleic acid molecule nfSP19 855 Translation of SEQ ID NO: 156 yields an apparent full-length protem of about 253 ammo acids, denoted PfSP19 253 , having amino acid sequence SEQ ID NO: 157, assuming the first codon, an apparent start codon, spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 156.
  • a Genbank homology search revealed most homology between SEQ ID NO: 156 and an Aedes aegypti trypsin, there being about 53 identity between corresponding regions of both nucleic acid molecules .
  • SEQ ID NO: 158 The nucleic acid sequence of another flea serine protease nucleic molecule, namely nfSP25 8 €4 , is represented herein as SEQ ID NO: 158.
  • Translation of SEQ ID NO: 158 yields a protem of about 260 ammo acids, denoted PfSP25 260 , having ammo acid sequence SEQ ID NO: 159, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 158 and a stop codon spanning from about nucleotide 782 through about nucleotide 784 of SEQ ID NO: 159.
  • a Genbank homology search revealed most homology between SEQ ID NO: 159 and an Anophel es gambiae chymotrypsm protem sequence, there being about 34% identity between corresponding regions of the two ammo acid sequences.
  • This example describes the cloning and sequencing of another flea serine protease nucleic acid molecule.
  • a flea serine protease cDNA nucleic acid molecule has been isolated m a manner similar to that described m Example 8 of related PCT Publication No. WO 96/11706, usmg nfSPll 25i (SEQ ID NO: 121) as a probe to screen an bovme blood-fed flea cDNA expression library (produced as described m Example 8 of related PCT Publication No. WO 96/11706, .
  • a clone that hybridized strongly to the probe was isolated and subjected to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described m Sambrook et al., ibid.
  • the nucleic acid sequence of the flea serine protease nucleic molecule namely nfSP21 595 , is represented herein as SEQ ID NO: 160.
  • Translation of SEQ ID NO: 160 yields a protem of about 198 ammo acids, denoted PfSP21 ⁇ 98 , having ammo acid sequence SEQ ID NO: 161, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 160 ⁇ nd a putative stop codon spanning from about nucleotide 596 to about nucleotide 598.
  • Genbank homology search revealed most homology between SEQ ID NO: 161 and Tachypl eus t ⁇ denta tus coagulation factor G protem sequence, there being about 45% identity between corresponding regions of the two ammo acid sequences.
  • This example describes the isolation and characterization of a 31 kD flea serine protease.
  • Guts from about 1500 fleas that had been fed on cat blood for about 24 hours were dissected m Gut Dissection Buffer (50 mM Tris 8.0, 100 mM CaCl 2 ) .
  • the guts were disrupted by freezing and thawing 4 times, followed by sonication.
  • the resulting extracts were clarified by centrifugation for 20 minutes at 14,000 rpm in a microfuge at 4 °C. The supernatant was recovered.
  • the gut supernatant was loaded onto a 3 ml column comprising p-aminobenzamidine cross-linked to Sepharose beads (Sigma) , previously equilibrated in Benzamidine Column Buffer (50 mM Tris 8.0, 100 mM CaCl 2 , 400 mM NaCl) . The supernatant was incubated on the column for about 10 min. Unbound protein was slowly washed off the column using Benzamidine Column Buffer until no protein was detectable by Bradford Assay (Bio Rad) .
  • Proteases bound to the benzamidine column were then eluted using 10 ml Benzamidine Column Buffer supplemented with 10 mM p-aminobenzamidine (brought to pH 8.0 with NaOH) .
  • Proteases in the eluant were concentrated and diafiltered into a volume of about 0.3 ml Gut Dissection Buffer using a Microcon 3 concentrator (Amicon) .
  • the membrane was stained with Coomassie Brilliant Blue. A dominant protem band of about 31 kDa was visualized. The membrane was then used for automated N-terminal sequencing (described in Example 7 of related PCT Publication No. WO 96/11706) .
  • a partial N-terminal amino acid sequence of the flea protease was determined to be IVGGEDVDISTCGWC (denoted SEQ ID NO: 68) .
  • This example describes the isolation and characterization of a 31 kD flea serine protease contained in a formulation having IgGase activity (i.e., ability to proteolyze immunoglobulin G proteins) .
  • Example 13 Cat blood-fed flea gut extracts were prepared and selected on a benzamidine column as described above in Example 13. IgG protease activity was assayed by incubating at 37°C, overnight, the benzamidine eluant with cat immunoglobulin G proteins (IgG) purified on Protein A sepharose. The ability of the flea gut benzamidine eluant to digest cat IgG was detected by resolving the samples by gel electrophoresis through a 14% SDS-PAGE gel and silver staining the gel using standard methods.
  • IgG immunoglobulin G proteins
  • the benzamidine eluant was then purified on a PolyPropylaspartamide hydrophobic interaction chromatography (HIC) column by applying the eluant to the column in buffer containing 0.1 M KP0 4 , pH 6.5 and 2 M (NH 4 ) SO,. Proteases bound to the column were eluted using an ammonium sulfate gradient of 2 M to 0 M in HIC column buffer. Column fractions were tested for IgG protease activity using the method described above.
  • HIC PolyPropylaspartamide hydrophobic interaction chromatography
  • Fractions containing IgG protease activity were pooled and applied to a PolyCat cation exchange column m 20 M sodium acetate, pH 6. The proteins were eluted using a sodium chloride gradient of 0 M to 1 M NaCl in 20 M sodium acetate. Fractions eluted from the column were tested for IgG protease activity and then each fraction was resolved by electrophoresis using SDS-PAGE. Fractions having the highest levels of IgG protease activity included a protem band that migrated at about 31 kDa on the SDS-PAGE gel. Weaker protease activity corresponded to an about 28 kDa band.
  • the 31 kDa protem present on the SDS-PAGE gel was used for N-terminal amino acid sequencing using the blotting method described above.
  • a partial N-terminal ammo acid sequence was determined to be IVGGEDVDIST (C)GWQI (S) FQ (S) ENLHF (C) GG (S) IIAPK (denoted herein as SEQ ID NO: 69) .
  • a comparison of SEQ ID NO: 69 and SEQ ID NO: 68 indicates a single residue difference between the two ammo acid sequences at residue 15 of each sequence (i.e., Q and C, respectively) . Since SEQ ID NO: 69 correlates with IgGase activity, the data suggests that the larval protem containing SEQ ID NO: 68 has IgGase activity.
  • This example describes the cloning and sequencing of a 31 kDa flea serine protease contained in a formulation having IgGase activity.
  • a flea protease nucleic acid molecule was isolated from a cat blood-fed whole flea library (described m Example 6) and a bovme blood-fed whole flea library (described m Example 8 of related PCT Publication No. WO 96/11706) by PCR amplification.
  • the actual primers used m the PCR amplification included FP31A primer designed using the N-termmal ammo acid sequence SEQ ID NO: 68, the primer having the nucleic acid sequence 5' GAA GAT GTW GAT ATT TCW ACA TGT GG 3' (SEQ ID NO: 101) used in combination with the M13 universal primer.
  • the resultant PCR products were gel purified and cloned into the TA Vector 1 *' and subjected to nucleic acid sequencing as described above.
  • a FP31B primer (5' GAA AAT GAA ATC CAC TTA AAC ATT ACG 3'), (represented herein as SEQ ID NO: 102) was designed using the DNA sequence of a DNA fragment from a bovme blood-fed cDNA library.
  • a flea protease cDNA nucleic acid molecule was isolated by PCR amplification of the cat blood-fed whole flea library and the bovine blood-fed whole flea library described above by PCR amplification. PCR amplification was performed using the FP31B primer in combination with M13 reverse primer.
  • PCR products were then diluted 1:25, and used as a template for a second PCR reaction using primer FP31C, having the sequence 5' CTC TTA TTG TAC GAG GGA TGC 3' (denoted herein SEQ ID NO: 103) in combination with T3 primer.
  • primer FP31C having the sequence 5' CTC TTA TTG TAC GAG GGA TGC 3' (denoted herein SEQ ID NO: 103) in combination with T3 primer.
  • the resulting nested PCR product was cloned into TA VectorTM and subjected to DNA sequencing.
  • SEQ ID NO: 66 The nucleic acid sequence of the resulting flea serine protease nucleic molecule, namely nfSP28 923 is represented herein as SEQ ID NO: 66.
  • Translation of SEQ ID NO: 66 yields a protein of about 267 amino acids, denoted PfSP28 267 , having amino acid sequence SEQ ID NO: 67, assuming an open reading frame in which the putative start codon spans from about nucleotide 8 through about nucleotide 10 of SEQ ID NO: 66 or from about nucleotide 11 through about nucleotide 13, and a stop codon spanning from about nucleotide 803 through about nucleotide 805 of SEQ ID NO: 66.
  • SEQ ID NO: 67 contains SEQ ID NO: 68 except Q is substituted for C, and SEQ ID NO: 69.
  • a Genbank homology search revealed most homology between SEQ ID NO: 66 and Bombix mori vitellin- degrading protease gene, there being about 53% identity between corresponding regions of the two nucleic acid sequences .
  • This example describes J H-DFP labelling of larval serine proteases.
  • Fig.l, lane B and fed 3rd instar larvae (Fig.l, lane C) produce serine proteases.
  • fed 1st instar larvae primarily produce a serine protease having a molecular weight of about 25 kD; and fed 3rd mstar larvae produce about serine proteases having molecular weights of about 25 kD, 28 kD and 31 kD.
  • the approximate size of standard molecular weight protem markers are shown in Fig.
  • This example describes the determination of partial N- terminal ammo acid sequences for several larval serine proteases.
  • the supernatant was rocked with the beads overnight at 4°C.
  • the beads were washed m about 45 ml Benzamidine Column Buffer to remove unbound protem.
  • the beads were then mixed 2 hours at 4°C with about 10 ml of Benzamidine Column Buffer containing 100 mM p-ammobenzamidme (pH 8.0 adjusted with NaOH) to elute proteins bound to the beads.
  • the eluted proteins were then collected. The elution process was repeated once more.
  • the eluted protem was concentrated by ultrafiltration with a Centriprep 10 concentrator (available from Amicon) .
  • the concentrate was diluted with Gut Dissection Buffer to a final volume of about 5 ml.
  • Partial N-termmal ammo acid sequence of proteins eluted from the beads was obtained using the method described m Example 11. Two proteins having molecular weights of about 25 kDa and about 26 kDa were identified on the Coomassie Brilliant Blue stained membranes. Partial N- termmal ammo acid sequence obtained for the protem having a molecular weight of about 25 kDa is IVGGVSVNINDYGYQLSLQSNGR, denoted herein as SEQ ID NO: 162. Partial N-termmal ammo acid sequence obtained for the protem having a molecular weight of about 26 kDa is IVGGHDTSIKQHPYQV, denoted herein as SEQ ID NO: 163.
  • Flea serine protease protem PfSPl 21 was m the following manner.
  • Flea serine protease nucleic acid molecule nfSPl 670 produced as described in Example 20 of related PCT Publication No. WO 96/11706, was digested with Xhol restriction endonuclease, gel purified and subcloned mto expression vector ⁇ P R /T 2 ori/S10HIS-RSET-A9 (the production of which is described in Tripp et al,
  • Example 7 The resultant recombinant molecule, referred to herein as pHisCro-nfSPl 670 , was transformed into E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E. coli :pHisCro-nfSP1 670 .
  • the recombinant cell was cultured as described in Example 20 of related PCT
  • PfSPl 2U w as purified by nickel chelation chromatography followed by reverse phase high performance liquid chromatography (HPLC) .
  • Immunoblot analysis of the purified PfSPl 216 indicated that rabbit anti-flea protease antiserum, produced as described in example 14, selectively bound to PfSPl 21b .
  • Flea serine protease protein PfSP2 233 was produced in the following manner.
  • Flea serine protease nucleic acid molecule nfSP2-, 15 produced as described in Example 20 of related PCT Publication No. WO 96/11706, was digested with Xhol restriction endonuclease, gel purified and subcloned mto expression vector ⁇ P [ ⁇ /T * -o.r. ⁇ /S10HIS-RSET-A9 as described in Example 39A.
  • the resultant recombinant molecule referred to herem as pH ⁇ sCro-nfSP2-, t , was transformed mto E.
  • Flea serine protease protein PfSP13 225 was produced m the following manner.
  • Flea serine protease nucleic acid molecule nfSP13 700 produced as described in Example 20 of related PCT Publication No. WO 96/11706, was digested with Xhol restriction endonuclease, gel purified and subcloned into expression vector ⁇ P R /T 2 or ⁇ /S10HIS-RSET-A9 as described in Example 18A.
  • the resultant recombinant molecule referred to herein as pH ⁇ sCro-nfSP13 700 , was transformed mto E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E.
  • PfSP13 22r Flea serine protease protein PfSP13 22r was purified by nickel chelation chromatography followed by reverse phase HPLC. Immunoblot analysis of the purified PfSP13 22b indicated that rabbit anti-flea protease antiserum, produced as described in Example 14 of related PCT Publication No. WO 96/11706, selectively bound to PfSP13 225 .
  • Flea serine protease protein PfSP20 222 was produced in the following manner.
  • An about 669-bp DNA fragment, referred to herein as nfSP20 569 was PCR amplified from flea serine protease clone 20 using the Xhol-site containing primer F27-S (sense) 5' GAG CTC TCG AGA ATC GTA GGA GGA CAC GAT AC 3' (SEQ ID NO: 164) and the EcoRI-site containing primer F20-A (antisense) 5' G GAC GAA TTC TTA AAC ACC AGA CAC TTC CTT G 3' (SEQ ID NO: 165) .
  • the PCR product nfSP20 669 was digested with Xhol and EcoRI restriction endonucleases, gel purified and subcloned into expression vector ⁇ P R /T 2 ori/S10HIS-RSET-A9 as described in Example 18A.
  • the resultant recombinant molecule referred to herein as pHisCro-nfSP20 669 , was transformed into E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E. coli :pHisCro-nfSP20 669 .
  • the recombinant cell was cultured as described in Example 20 of related PCT Publication No. WO 96/11706.
  • This example describes that various flea serine protease nucleic acid molecules described in the foregoing examples can be obtained from multiple sources.
  • Nucleic acid molecules correspondmg to flea clone 4 have been obtained from a bovme blood-fed whole flea library (described in Example 8 of related PCT Publication
  • Nucleic acid molecules corresponding to flea clone 5 have been obtained from a bovme blood-fed whole flea library and a cat blood-fed whole flea library.
  • Nucleic acid molecules corresponding to flea clone 6 have been obtained from a bovme blood-fed whole flea library, a cat blood-fed whole flea library and an unfed whoie flea library.
  • Nucleic acid molecule correspondmg to flea clone 7 have been obtained from a bovme blood-fed whole flea library, and a cat blood-fed whole flea library.
  • Nucleic acid molecules corresponding to flea clone 8 have been obtained from a bovme blood-fed whole flea library and an unfed whole flea library.
  • Nucleic acid molecules corresponding to flea clone 12 have been obtained from a bovme blood-fed whole flea library and a cat blood-fed whole flea library.
  • Nucleic acid molecules corresponding to flea clone 13 have been obtained from a bovine blood-fed whole flea library, a cat blood-fed whole flea library, and an unfed whole flea library.
  • Nucleic acid molecules corresponding to flea clone 20 have been obtained from a bovme blood-fed whole flea library, a cat blood-fed whole flea library, and an unfed whole flea library.
  • Nucleic acid molecules corresponding to flea clone 28 have been obtained from a bovme blood-fed whole flea library and a cat blood-fed whole flea library.
  • This example provides additional nucleic acid and deduced ammo acid sequences of nucleic acid molecules encoding a flea cysteine protease protem of the present which was described m Example 3. This example also provides the production of a cysteine protease protein in E . coli cells.
  • A. Additional Cysteine Protease Nucleic Acid Molecule The PCR products described in Example 3 were submitted to additional nucleic acid sequence analysis in order to obtain the nucleic acid sequence of additional portions of the coding region of the cysteine protease gene.
  • SEQ ID NO: 76 is contained within the sequence of the nucleic acid molecule nfCPl U09.
  • nucleic acid molecule nfCPl 1109 encodes a full- length flea cysteine protease protein of about 327 amino acids, referred to herein as PfCPl 327 , having amino acid sequence SEQ ID NO:2, assuming an open reading frame in which the initiation codon spans from about nucleotide 126 through about nucleotide 128 of SEQ ID NO:l and the termination codon spans from about nucleotide 1107 through about nucleotide 1109 of SEQ ID NO:l.
  • the complement of SEQ ID NO:l is represented herein by SEQ ID NO: 3.
  • the coding region encoding PfCPl 327 is represented by nucleic acid molecule nfCPl 984 , having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 4 and a complementary strand with nucleic acid sequence SEQ ID NO: 6.
  • the proposed mature protem, denoted herein as PfCPl 2 ⁇ contains about 226 ammo acids which is represented herein as SEQ ID NO: 8.
  • the nucleic acid molecule encoding PfCPl is denoted herem as nfCPl 68 , which is represented by SEQ ID NO:7.
  • the ammo acid sequence of PfCPl 3 ⁇ - (i.e., SEQ ID NO: 2) predicts that PfCPl 32 -, has an estimated molecular weight of about 42 kD and an estimated pi of about pi 8.84.
  • SEQ ID NO:l Comparison of nucleic acid sequence SEQ ID NO:l with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:l showed the most homology, i.e., about 55% identity, with the following three genes: a Drosophila cysteine protease gene, a BomJbyx cysteine protease gene and a Sarcophaga cysteine protease gene.
  • SEQ ID NO:2 i.e., the ammo acid sequence of PfCPl 3 _-
  • SEQ ID NO:2 showed the most homology, i.e., about 42- identity, with the following three proteins: a Drosophila cysteine protease protem, a BomJbyx cysteine protease protem and a Sarcophaga cysteine protease protein.
  • nfCPl b60 designed to encode an apparently mature cysteine protease protem
  • nfCPl b60 was PCR-amplifled from a flea mixed instar cDNA library produced usmg unfed 1st instar, bovme blood-fed 1st instar, bovme blood-fed 2 n ⁇ instar and bovme blood-fed 3- d instar flea larvae (this combination of tissues is referred to herein as mixed instar larval tissues for purposes of this example) .
  • Total RNA was extracted from mixed instar tissue using an acid- guanidmium-phenol-chloroform method similar to that described by Chomczynski et al .
  • RNA preparations were used in each RNA preparation.
  • Poly A+ selected RNA was separated from each total RNA preparation by oligo-dT cellulose chromatography using Poly(A) Quick® mRNA isolation kits (available from Stratagene Cloning Systems, La Jolla, CA) , according to the method recommended by the manufacturer.
  • a mixed instar cDNA expression library was constructed m lambda ( ⁇ ) Uni-ZAP XR vector (available from Stratagene Cloning Systems) using Stratagene' s ZAP-cDNA Synthesis Kit® protocol. About 6.34 ⁇ g of mixed instar poly A+ RNA were used to produce the mixed instar library.
  • the resultant mixed instar library was amplified to a titer of about 2.17 x 10 10 pfu/ml with about 97% recombmants.
  • the primers used in the PCR amplification were sense primer CysBS ' having the nucleotide sequence 5' GAT AAG GAT CCG TTA CCA GAT TCT TTC GAC TGG 3' (containing a BamHI-site; denoted SEQ ID NO: 64) and anti-sense primer CysHA having the nucleotide sequence 5' TTA TCA AGC TTC CAT TTA CAT GCC GTA AAA ATC 3' (containing a Hmdlll site; denoted SEQ ID NO: 65) .
  • nfCPl DD was submitted to nucleic acid sequence analysis to obtain a nucleic acid sequence of the coding strand, represented herein as SEQ ID NO: 94.
  • Translation of SEQ ID NO: 94 indicated that nfCPl bqr encodes a protem of about 220 ammo acids, called PfCPl 220 , having SEQ ID NO: 95. It is to be noted that this sequence analysis indicated that the stop codon was actually about 36 base pairs upstream from what had been predicted by SEQ ID NO:l; as such, the protem encoded by nfCPl 6c ⁇ is about 12 ammo acids shorter than would have been predicted by SEQ ID NO:l.
  • the nucleic acid molecule nfCPl 660 contains the coding region for PfCPl 220 .
  • Recombinant cell E. coli:pCro-nfCP1 6D0 was produced in tne following manner.
  • Nucleic acid molecule nfCPl b60 was digested with BamHl and HindiII restriction endonucleases, gel purified, and subcloned mto expression vector IambdaP R /T 2 o ⁇ /S10HIS-RSET-A9 (the production of which is described m Tripp et al, International PCT Publication No. WO 95/24198, published September 14, 1995; see m particular, Example 7), that was digested with BamHl and HindiII and dephosphorylated.
  • the resultant recombinant molecule referred to herem as pCro-nfCPl 6bC
  • pCro-nfCPl 6bC was transformed mto E. coli BL-21 competent cells 'available from Novagen, Madison, WI) to form recombinant cell E. coli:pCro-nfCPl 6b0 .
  • the recombinant cell was cultured as described m Example 20 of related PCT Publication No. WO 95/24198. About 1 ml of culture was collected prior to induction, and about 1 ml of culture was collected about 60 minutes following induction.
  • Example 21 This example provides additional nucleic acid and deduced ammo acid sequences of nucleic acid molecules encoding serine protease proteins of the present invention which are described herein and in the Examples section of related PCT Publication No.WO 96/11706.
  • a DNA probe labeled with 32 P comprising nucleotides from nfAP2 210C (described m Example 23 of related U.S. Patent Application Serial No. 08/639,075, filed April 24, 1996) was used to screen a bovine blood-fed whole flea cDNA library (described in Example 8 of related PCT Publication No.WO 96/11706) using standard hybridization techniques.
  • a clone was isolated having about a 459-nucleot ⁇ de insert, referred to herein as nfSP18 4- Struktur.
  • a nucleic acid sequence of the composite nucleic acid molecule produced using nucleic acid sequence from nfSP16 53 . and nfSP18. tq is referred to herein as nfSPl ⁇ , 7 , having a nucleic acid sequence of the coding strand which is denoted herem as SEQ ID NO: 9.
  • SEQ ID NO: 9 nucleic acid molecule nfSPl ⁇ -,-,- encodes a non-full- length flea serine protease protem of about 228 ammo acids, referred to herein as PfSP18 228 , having ammo acid sequence SEQ ID NO: 10, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 9 and the stop codon spans from about nucleotide 685 through about nucleotide 687 of SEQ ID NO: 9.
  • the complement of SEQ ID NO: 9 is represented herem by SEQ ID NO: 11.
  • the coding region encoding PfSP18 22 ⁇ is represented by nucleic acid molecule nfSP18 22 volunteer, having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 12 and a complementary strand with nucleic acid sequence SEQ ID NO: 14.
  • the ammo acid sequence of PfSP18 228 (i.e., SEQ ID NO: 10) predicts that PfSP18 228 has an estimated molecular weight of about 25 kD and an estimated pi of about 9.09.
  • Comparison of nucleic acid sequence SEQ ID NO: 9 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 9 showed the most homology, i.e., about 51% identity, between SEQ ID NO: 9 and an Anophel es st ephensi trypsin 1 gene.
  • Comparison of ammo acid sequence SEQ ID NO: 10 i.e., the ammo acid sequence of PfSP18 22r
  • ammo acid sequences reported in GenBank indicates that SEQ ID NO: 10 showed the most homology, i.e., about 59? identity between S ⁇ Q ID NO: 10 and Vespa crabro protein.
  • flea serine protease nucleic molecule clone 24 (described in Example 6 was determined using primers designed from nfSP24 410 to amplify DNA from the bovine biood-fed whole flea cDNA library.
  • Sense primer Flea 24F having the nucleotide sequence 5' GGA CAA ACT GTT CAT TGC AG 3' (denoted SEQ ID NO: 46) was used in combination with the M13 universal primer in a first PCR reaction.
  • Anti-sense primer Flea 24R having the nucleotide sequence 5' CCC TCA TTT GTC GTA ACT CC 3' (denoted SEQ ID NO: 47) was used in combination with the M13 reverse primer in a second PCR reaction.
  • the resulting PCR products were each gel purified and cloned into the TA Vector® System, and subjected to standard DNA sequencing techniques.
  • nfSP24 :089 A composite nucleic acid sequence representing a flea serine protease coding region was deduced, referred to herein as nfSP24 :089 , was deduced and is denoted herein as SEQ ID NO: 15.
  • SEQ ID NO:78 is contained within the sequence of the nucleic acid molecule nfSP24 1089 _ Translation of SEQ ID NO: 15 suggests that nucleic acid molecule nfSP24 1089 encodes a full-length flea serine protease protein of about 258 amino acids, referred to herein as PfSP24 2f)8 , having amino acid sequence SEQ ID NO: 16, assuming an open reading frame in which the initiation codon spans from about nucleotide 33 through about nucleotide 35 of SEQ ID NO: 15 and the termination codon spans from about nucleotide 807 through about nucleotide 809 of SEQ ID NO: 15.
  • SEQ ID NO: 15 The complement of SEQ ID NO: 15 is represented herem by SEQ ID NO: 17.
  • the coding region encoding PfSP24 258 is represented by nucleic acid molecule nfSP24 774 , having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 18 and a complementary strand with nucleic acid sequence SEQ ID NO: 20.
  • the proposed mature protem, denoted herein as PfSP24 237 contains about 237 ammo acids which is represented herein as SEQ ID NO:22.
  • the nucleic acid molecule encoding PfSP24 23 -7 is denoted herein as nfSP24 7n , which is represented by SEQ ID NO: 21.
  • the ammo acid sequence of PfSP24 258 i.e., SEQ ID NO: 16 predicts that PfSP24 258 has an estimated molecular weight of about 28 kD and an estimated pi of about pi 6.70.
  • SEQ ID NO: 15 showed the most homology, i.e., about 51% identity between SEQ ID NO: 15 and an Anopheles stephensi trypsin 1 gene. Comparison of ammo acid sequence SEQ ID NO: 15
  • Anti- sense primer Flea 32R having the nucleotide sequence 5' GCA AAT CAG TTC CAG AAT CCA CTA ACC 3' was used m combination with the M13 reverse primer m a second PCR reaction.
  • the resultmg PCR products were each gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques.
  • SEQ ID NO:23 A composite nucleic acid sequence representing a flea serine protease coding region was deduced, referred to nerem as nfSP32 924 , was deduced and is denoted herein as SEQ ID NO:23.
  • SEQ ID NO: 80 is contained withm the sequence of the nucleic acid molecule nfSP32 924 Translation of SEQ ID NO:23 suggests that nucleic acid molecule nfSP32 9: consult encodes a full-length flea serine protease protem of about 268 ammo acids, referred to herein as PfSP32 268 , havmg ammo acid sequence SEQ ID NO: 24, assuming an open reading frame in which the initiation codon spans from about nucleotide 6 through about nucleotide 8 of SEQ ID NO: 23 and the termination codon spans from about nucleotide 810 through about nucleotide 812 of SEQ ID NO:23.
  • SEQ ID NO:25 The complement of SEQ ID NO:23 is represented herein by SEQ ID NO:25.
  • the coding region encoding PfSP32 268 is represented by nucleic acid molecule nfSP32 b0q , having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 26 and a complementary strand with nucleic acid sequence SEQ ID NO.28.
  • the ammo acid sequence of PfSP32 26p i.e., SEQ ID NO: 24 predicts that PfSP32 268 has an estimated molecular weight of about 28.6 kD and an estimated pi of about pi 7.36.
  • Comparison of nucleic acid sequence SEQ ID NO:23 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 23 showed the most homology, i.e., about 52% identity between SEQ ID NO:23 and a Fusari um oxysporum preprotrypsm gene.
  • Comparison of ammo acid sequence SEQ ID NO: 24 (i.e., the ammo acid sequence of PfSP32 268 ) with ammo acid sequences reported in GenBank indicates that SEQ ID NO:24 showed the most homology, i.e., about 63% identity between SEQ ID NO:24 and a Bombyx mon vitellm -degrading protease precursor protem. D.
  • the remainder of flea serine protease nucleic molecule clone 33 was determined using primers designed from nfSP33-, 78 to amplify DNA from the flea mixed instar larvae cDNA library described above in Example 20.
  • Sense primer Flea 33F having the nucleotide sequence 5' CAG GGC GCT CTG CAG AAC GCA AC 3' (denoted SEQ ID NO: 50) was used in combination with the M13 universal primer m a first PCR reaction.
  • Anti-sense primer Flea 33R having the nucleotide sequence 5' ATT CCT CGT GGT TCA GTC GCT C 3' was used m combination with the M13 reverse primer m a second PCR reaction.
  • the resulting PCR products were each gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques .
  • nfSP33 1894 A composite nucleic acid sequence representing a flea serine protease coding region was deduced, referred to herein as nfSP33 1894 , was deduced and is denoted herein as SEQ ID NO: 29.
  • SEQ ID NO: 84 and SEQ ID NO: 82 are contained withm the sequence of the nucleic acid molecule nfSP33, 894 _ Translation of SEQ ID NO:29 suggests that nucleic acid molecule nfSP33 1894 encodes a full-length flea serine protease protem of about 400 ammo acids, referred to herein as PfSP33 400 , having amino acid sequence SEQ ID NO: 30, assuming an open reading frame m which the initiation codon spans from about nucleotide 335 through about nucleotide 337 of SEQ ID NO:29 and the termination codon spans from about nucleotide 1535 through about nucleotide 1537 of SEQ ID NO:29.
  • SEQ ID NO: 31 The complement of SEQ ID NO:29 is represented herein by SEQ ID NO: 31.
  • the coding region encoding PfSP33 400 is represented by nucleic acid molecule nfSP33 1200 , having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 32 and a complementary strand with nucleic acid sequence SEQ ID NO: 34.
  • the proposed mature protein, denoted herein as PfSP33 4 contains about 242 ammo acids which is represented herein as SEQ ID NO: 36.
  • the nucleic acid molecule encoding PfSP33. 4 is denoted herein as nfSP33 72b , which is represented by SEQ ID NO: 35.
  • the ammo acid sequence of PfSP33 40C i.e., SEQ ID NO:30 predicts that PfSP33 400 has an estimated molecular weight of about 44 kD and an estimated pl of about pi 7.59.
  • Comparison of nucleic acid sequence SEQ ID NO:29 with nucleic acid sequences reported m GenBank indicates that SEQ ID NO: 29 showed the most homology, i.e., about 48% identity between SEQ ID NO:29 and a Drosophil a melanogaster serine protease stubble gene.
  • Comparison of ammo acid sequence SEQ ID NO: 30 (i.e., the amino acid sequence of PfSP33 400 ) with ammo acid sequences reported m GenBank indicates that SEQ ID NO: 30 showed the most homology, i.e., about 63 identity between SEQ ID NO: 30 and a Drosophila melanogaster serine protease stubble protem.
  • Example 22 This example provides nucleic acid and deduced ammo acid sequence of another nucleic acid molecule encoding a serine protease protem of the present invention.
  • a serine protease cDNA nucleic acid molecules has been isolated in a manner similar to that described in Example 8 of related PCT Publication No.No.WO 96/11706.
  • the actual primers used in PCR amplification of the serine protease nucleic acid molecule from a cat blood-fed flea cDNA expression library included cat-try #2 (SEQ ID NO: 86) m combination with H57 primer (SEQ ID NO: 99) .
  • the resultant PCR product was gel purified and cloned into the TA VectorTM.
  • a recombinant TA vector clone was isolated and subjected to nucleic acid sequencing.
  • SEQ ID NO: 37 A composite nucleic acid sequence of a flea serine protease nucleic molecule corresponding to flea clone 40, namely nfSP40 428 was deduced and is denoted herem as SEQ ID NO: 37.
  • Translation of SEQ ID NO: 37 suggests that nucleic acid molecule nfSP40 428 encodes a non-full-length flea serine protease protem of about 142 ammo acids, referred to herein as PfSP40 142 , represented herem by SEQ ID NO: 38.
  • the complement of SEQ ID NO: 37 is represented herem by SEQ ID NO: 39.
  • the remainder of flea serine protease nucleic molecule clone 40 was determined using primers designed from nfSP40 4 _ 8 to amplify DNA from the cat blood-fed whole flea cDNA library.
  • Sense primer Flea 40F having the nucleotide sequence 5' GGC AAG TTT CGT TTC ACA ATA GG 3' (denoted SEQ ID NO: 52) was used in combination with the M13 universal primer in a first PCR reaction.
  • Anti-sense primer Flea 40R having the nucleotide sequence 5' TCC AAC CCT AAC TTT TAA ACC TTC 3' (denoted SEQ ID NO: 53) was used in combination with the M13 reverse primer m a second PCR reaction.
  • nfSP40 841 A composite nucleic acid sequence representing a flea serine protease coding region was deduced, referred to herein as nfSP40 841 , was deduced and is denoted herein as SEQ ID NO: 40.
  • SEQ ID NO: 37 is contained withm the sequence of the nucleic acid molecule nfSP40 841 Translation of SEQ ID NO: 40 suggests that nucleic acid molecule nfSP40 841 encodes a non-full-length flea serine protease protem of about 242 ammo acids, referred to herein as PfSP40 24i , havmg ammo acid sequence SEQ ID NO: 41, assuming an open reading frame in which the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 40 and tne termination codon spans from about nucleotide 728 through about nucleotide 730 of SEQ ID NO: 40.
  • SEQ ID NO:42 The complement of SEQ ID NO: 40 is represented herein by SEQ ID NO:42.
  • the coding region encoding PfSP40 242 is represented by nucleic acid molecule nfSP40 7 ⁇ , having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 43 and a complementary strand with nucleic acid sequence SEQ ID NO: 45.
  • the amino acid sequence of PfSP40 242 i.e., SEQ ID NO:41 predicts that PfSP40 242 has an estimated molecular weight of about 26 kD and an estimated pl of about pi 6.5.
  • SEQ ID NO: 40 Comparison of nucleic acid sequence SEQ ID NO: 40 with nucleic acid sequences reported m GenBank indicates that
  • SEQ ID NO: 40 showed the most homology, i.e., about 57% identity between SEQ ID NO: 40 and a Derma tophagoi des pteronyssmus Der P3 allergen gene.
  • Comparison of ammo acid sequence SEQ ID NO:41 (i.e., the ammo acid sequence of PfSP40_ 42 ) with ammo acid sequences reported m GenBank indicates that SEQ ID NO: 41 showed the most homology, i.e., about 40% identity between SEQ ID NO: 41 and a Bombyx mon vitellm-degradmg protease precursor protem.
  • nfSP24 714 (designed to encode an apparently mature serine protease protem) was PCR amplified from nfSP24 1089 usmg sense primer Flea 24 EF having the nucleotide sequence 5' CAC AGG ATC CAA TAA TTT GTG GTC AAA ATG C 3' (containing a BamHI-site; denoted SEQ ID NO: 54) and anti-sense primer Flea 24 ER havmg the nucleotide sequence 5' AAA AAG AAA GCT TCT TTA ATT TTC TGA CAT TGT CGT G 3' (containing a Hindlll; denoted SEQ ID NO: 55) .
  • nfSP24 714 was digested with BamHl and HindiII restriction endonucleases, gel purified, and subcloned mto expression vector lambdaP R /T-on/S10HIS-RSET-A9, that had been digested with BamHl and HindiII and dephosphorylated.
  • the resultant recombinant molecule referred to herein as pCro-nfSP24 714 , was transformed mto E. coli BL-21 competent cells (available from Novagen, Madison, WI) to form recombinant cell E.
  • the recombinant cell was cultured as described in Example 20 of related PCT Publication No.WO 95/24198.
  • nfSP32 b98 designed to encode an apparently mature serine protease protem
  • sense primer Flea 32 EF having the nucleotide sequence 5' GCG GGA TCC TAT TGT GGG TGG TGA AGC AGT G 3' (containing a BamHI-site; denoted SEQ ID NC:56) and anti-sense primer Flea 32 ER having the nucleotide sequence 5' GAC GGT ACC ATG TAT AAA ATA ATA TTA AAC TCC GG 3' (containing a Kpnl; denoted SEQ ID NO:57) .
  • nfSP32 698 was digested with BamHl and Kpn ⁇ restriction endonucleases, gel purified, and subcloned into expression vector 1 pTrcHisB (available from Ir.Vitrogen Corp., San Diego, CA) , that had been digested with BamHl and Kpnl and dephosphorylated.
  • the resultant recombinant molecule referred to herein as pTrc-nfSP32 b98 , was transformed into E. coli BL-21 competent cells to form recombinant cell E. coli :pTrc-nfSP32 698 .
  • the recombinant cell was cultured and protein production resolved by SDS- PAGE as described above in Section A. Immunoblot analysis of the proteins using a T7 antibody showed expression of an about 38 kD protein in the induced sample but not in the unmduced sample.
  • Flea serine protease protein PfSP33 400 was produced in the following manner.
  • An about 1200 bp nucleic acid molecule, referred to herein as nfSP33 1200 (designed to encode an apparently mature serine protease protein) was PCR amplified from nfSP33 1894 using sense primer Flea 33 EF having the nucleotide sequence 5' CCG GGA TCC TAT GTT AGC GAT CGT CCC GTC AAA C 3' (containing a BamHI-site; denoted SEQ ID NO: 58) and anti-sense primer Flea 33 ER having the nucleotide sequence 5' CCG GAA TTC TTA TCC CAT TAC TTT GTC GAT CC 3' (containing a EcoRI; denoted SEQ ID NO:59) .
  • nfSP33 1200 was digested with BamHl and EcoRI restriction endonucleases, gel purified, and subcloned mto expression vector lambdaP R /T 2 ori/S10HIS-RSET- A9, that had been digested with BamHl and EcoRI and dephosphorylated.
  • the resultant recombinant molecule referred to herein as pCro-nfSP33 1200 , was transformed into £. coli BL-21 competent cells to form recombinant cell E. coli :pCro-nfSP33 1200 .
  • the recombinant cell was cultured using the method described above in Section A. D.
  • Flea serine protease protein PfSP40 242 was produced in the following manner.
  • An about 716 bp nucleic acid molecule, referred to herein as nfSP40 71b (designed to encode an apparently mature serine protease protein) was PCR amplified from nfSP40 841 using sense primer Flea 40 EF having the nucleotide sequence 5' GCG GGA TCC AAT AGT AGG AGG TGA AGA TGT AG 3' (containing a BamHI-site; denoted SEQ ID NO: 60) and anti-sense primer Flea 40 ER having the nucleotide sequence 5' CCG GAA TTC TTC TAA CAA ATT TTA TTT GAT TCC TGC 3' (containing a EcoRI; denoted SEQ ID N0:61) .
  • nfSP40 716 was digested with BamHl and EcoRI restriction endonucleases, gel purified, and subcloned into expression vector lambdaP R /T 2 ori/S10HIS-RSET- A9, that had been digested with BamHl and EcoRI and dephosphorylated.
  • the resultant recombinant molecule referred to herein as pCro-nfSP40 716
  • pCro-nfSP40 716 was transformed into E. coli BL-21 competent cells to form recombinant cell E. coli :pCro-nfSP40 7U - .
  • the recombinant cell was cultured and protein production resolved using the methods described above in Section A. Immunoblot analysis of the proteins using a T7 antibody showed expression of an about 38 kD protein in the induced sample but not in the uninduced samp1e.
  • This Example demonstrates the production of another serine protease protein of the present invention in E. coli cells.
  • nfSP28 71 An about 711 bp nucleic acid molecule, referred to herein as nfSP28 71 , (designed to encode an apparently mature serine protease protein) was PCR amplified from nfSP28 923 using sense primer Flea 28 F having the nucleotide sequence 5' GGA TCC AAT CGT TGG AGG TGA AGA TG 3' (containing a BamHI-site shown in bold; denoted SEQ ID NO: 62) and anti-sense primer Flea 28 R having the nucleotide sequence 5' GAA TTC GAA ATC CAC TTA AAC ATT AGC 3' (containing a EcoRI shown in bold; denoted SEQ ID NO: 63) .
  • nfSP28 7n was digested with BamHl and EcoRI restriction endonucleases, gel purified, and subcloned into expression vector lambdaP R /T 2 ori/S10HIS-RSET-A9, that had been digested with BamHl and Xbal and dephosphorylated.
  • the resultant recombinant molecule referred to herein as pCro-nfSP28 ⁇ , was transformed mto E. coli BL-21 competent cells (available from Novagen, Madison, WI) to form recombinant cell E. coli :pCro-nfSP2 ⁇ n , .
  • the recombinant cell was cultured and protem production resolved using the methods described above m Example 20.
  • Immunoblot analysis of the proteins using a T7 antibody showed expression of an about 36 kD protem in the induced sample but not m the unmduced sample.
  • Immunoblot analysis using a rabbit anti- flea midgut protease polyclonal antibody identified an about 38 kD protem in the induced sample.
  • Example 25 This Example demonstrates the production of another serine protease protem of the present invention m eukaryotic cells.
  • Recombinant molecule pBv-nfSP28 792 containing a flea serine protease nucleic acid molecule spanning nucleotides from about 11 through about 802 of SEQ ID NO: 66, operatively linked to baculovirus polyhedron transcription control sequences were produced m the following manner.
  • nfSP28- A PCR fragment of 792 nucleotides, named nfSP28-.
  • the N-termmal primer was designed from the pol h sequence of baculovirus with modifications to enhance expression in the baculovirus system.
  • Bv-nfSP28-, 9Z the about 792 base pair PCR product
  • BamHl and Xbal subcloned mto BamHl and Xbal digested to produce the recombinant molecule referred to herein as pVL-nfSP28 79t .
  • the resultant recombinant molecule,pVL-nfSP28 792 was verified for proper insert orientation by restriction mapping.
  • the recombinant molecule was co-transfected with a linear Baculogold baculovirus DNA (available from Pharmmgen) mto S. frugiperda Sf9 cells (available from InVitrogen) to form the recombinant cells denoted 5.
  • frugiperda:pVL-nfSP28 -, 92 was cultured m order to produce a flea serine protease protem PfSP28 2b4 . Immunoblots of supematants from cultures of S .
  • frugiperda:pVL-nfSP28-, 92 cells producing the flea serine protease protem PfSP28 2b4 was performed using a cat anti- fSPFlea 26 polyclonal antibody which was produced as follows.
  • Recombinant Flea 28 protein (referred to herein as rSPFlea 28 protein) produced in E. coli described above in Example 24 was used to immunize cats.
  • the rSPFlea 28 protein was diluted to a concentration of about 1 mg/ml in PBS and emulsified in an equal volume of TiterMax research adjuvant (available from CytRx Corp., Norcross, GA) .
  • a series of cats were immunized each with about 50 ⁇ g of rSPFlea 28 protein in adjuvant by subcutaneous injection.
  • a second injection of the same dose of rSPFlea 28 protein in adjuvant was administered 32 days later.
  • Blood samples were obtained prior to immunization (pre-bleed) , 32 days and 47 days after the initial immunization.
  • Sera samples from the pre-immunization and Day 47 bleeds were used for subsequent immunoblot experiments. The latter is referred to as anti-fSPFlea 28 polyclonal antibody. Analysis of the immunoblots identified an about 33 kD protein and an about 36 kD protein.
  • This example describes the production of peptides from the 31 kD flea midgut serine protease and the generation of internal sequence data.
  • Midguts from about 30,000 cat blood-fed fleas were dissected as described m U.S. Patent No. 5,356,622, ibid.
  • m gut dissection buffer 50 mM Tris 8.0, 100 mM CaCl .
  • the guts (m three batches of about 10,000 each) were disrupted by a freeze-thaw cycle, followed by somcation.
  • the resulting extracts were clarified by centrifugation for 20 minutes at 3050 rpm m a swinging bucket centrifuge at 4°C.
  • the supematants were recovered, and adjusted to 400 mM NaCl m preparation for benzamidine column chromatography.
  • gut supematants were loaded into a 5 ml disposable column containing p-ammobenzamidme cross- linked to Sepharose beads (available from Sigma, St. Louis, MO) , previously equilibrated in benzamidine column buffer (50 mM Tris, pH 8.0, 100 mM CaCl 2 , 400 mM NaCl) and incubated with rocking overnight at 4°C. Unbound protem was slowly washed off the column using benzamidine column buffer until no protem was detectable by Bradford Assay (available from Bio-Rad Laboratories, Hercules, CA) .
  • Proteases bound to the benzamidine column were eluted using 4 ml benzamidine column buffer supplemented with 100 mM p-ammobenzamidme (brought to pH 8.0 with NaOH) .
  • Residual bound proteases were washed off with about 21 ml of additional benzamidine column buffer. The recovered proteases were then concentrated to a volume of about 2 ml using a Ultrafree 20 10-kD centrifugal concentrator
  • protease pools from the 3 preparations were combined for a total of about 30,000 gut equivalents m about 6 ml. Protem concentration was measured by Bradford assay and found to be about 0.5 mg/ml. About 150 ⁇ g of the isolated protease pool was resolved by polyacrylamide gel electrophoresis (PAGE) on a preparative-well 14% Tris-glycme gel (available from Novex, San Diego, CA) .
  • PAGE polyacrylamide gel electrophoresis
  • the proteins in the gel were visualized by staining for about 30 minutes in Coomassie brilliant blue stain (0.1% (w/v) Coomassie blue R, 40% (v/v) methanol, 10% (v/v) acetic acid) and oestammg for about 2.5 hours in 50% (v/v) methanol.
  • the band correspondmg to the 31-kD protease was excised with a razor blade.
  • the protem was electroeluted, concentrated, and partially digested for 24 hours with cyanogen bromide (CNBr) (Silver, et al. , 1995, J. Bi ol . Chem.
  • CNBr is known to cleave after methionine (M) residues under the conditions used for this digestion.
  • M methionine
  • the peptides in the sample were resolved by PAGE on an 18% Tris-glycme gel.
  • electrophoresis the separated protease peptides were electroblotted onto a PVDF membrane using a CAPS buffer (10 mM CAPS pH 11, 0.5 mM DTT, 10% (v/v) methanol) .
  • the membrane was stained with Coomassie Brilliant Blue and destamed with 50% (v/v) methanol. Three stained peptide bands were identified having apparent molecular weights of about 14 kD, 21 kD, and 22 kD, respectively. The portions of the membrane containing the 21 kD and 22 kD bands were excised separately. Peptides contained m each membrane segment were subjected to N- termmal ammo acid sequencing using a 473A Protem Sequencer (available from Applied Biosystems, Foster City, CA) using standard techniques.
  • N-termmal ammo acid sequence of the 21-kD peptide was H/R-V/P- G/A/S-Y/G-E/N-D/K-V/R-D/A-D-Y- D-F-D/P-V-A, denoted herein as SEQ ID NO: 70 and the N-terminal amino acid sequence of the 22-kD peptide to be I/Q-V-G-Y/G-E/N/T-D/M/P-V-K/D-I- N/S-M/T/N-F/C herein denoted as SEQ ID NO: 71.
  • the N- texmmal ammo acid sequence of the mtact 31-kD protease is either I-V-G-G- E-D-V-D-I-S-T-C-G-W-C (SEQ ID NO: 59, as disclosed m Example 34 m co-pending U.S. Patent Application Serial No. 08/639,075) , or IVGGEDVDIST(C)GWQI(S)FQ(S)ENLHF(C)GG(S) IIAPK (SEQ ID NO: 69, as disclosed in Example 35 in co-pending U.S. Patent Application Serial No. 08/639,075) .
  • SEQ ID NO: 68 contains a cysteine and SEQ ID NO: 69 contains a glutamine.
  • SEQ ID NO: 70 the sequences of both the 21-kD (SEQ ID NO:70) ano 22-kD (SEQ ID NO:71) peptides, though it is much stronger m the 22-kD peptide, leading to the conclusion that the SEQ ID NO: 71 represents the N-terminus of the 31- kD protease.
  • sequence for the 21-kD (SEQ ID NO:70) peptide is H/R-P-A/S-Y-N- K-R-A-D-Y-D-F-D-V-A, denoted herein as SEQ ID NO:72.
  • This sequence of ammo acids aligns with a stretch of deduced ammo acids from about residue 107 to residue 121 immediately following a methionine residue in SEQ ID NO: 67.
  • This example demonstrates that a 31-kD flea midgut serine protease contained in a formulation is able to proteolyze cat immunoglobulin G, A, and M proteins as well as bovme, dog, human, and rabbit immunoglobulin G proteins.
  • the 31-kD flea midgut serine protease was purified from cat blood-fed fleas as follows. Cat blood-fed flea midgut extracts were prepared and selected on a benzamidine column as described above m Example 26. The benzamidine eluate was then further purified as described in Example 35 of co-pending U.S. Patent Application Serial No. 08/639,075 by PolyCAT A cation exchange chromatography (available from PolyLC, Inc., Columbia, MD) to isolate a protein band which migrated at about 31 kD on a silver stained SDS-PAGE gel.
  • a Sepharose or of purified dog, rabbit, or human IgG
  • This example describes the ability of a 31-kD flea midgut serine protease contained m a formulation to proteolyze cat immunoglobulin G at a specific site.
  • the 31-kD flea midgut serine protease was purified from cat blood-fed flea midgut extracts as described above in Examples 26 and 27.
  • a band of about 33 kD was excised and subjected to N-termmal amino acid sequencing using techniques known to those skilled in the art.
  • a partial N- terminal ammo acid sequence of about 28 ammo acids was determined and is represented herem as SEQ ID NO: 73: X- P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K-D-D-L-L-I-K-R-K.
  • GenBank homology search using SEQ ID NO: 73 revealed most homology to Oryctolagus cani cul us gamma H-cham constant region 2, there being about 71 % identity over the 28 ammo acids.
  • the cleavage site was compared to that of a known protease, papain, as follows.
  • Cat immunoglobulin G 100 mg
  • papain purified from cat blood on Protein A sepharose
  • the reaction mixture was resolved on a 14% Tris-glycine SDS-PAGE gel, blotted onto PVDF membrane, stained with Coomassie R-250 and destained according to standard procedures.
  • a band of about 33 kD was excised and subjected to N-terminal amino acid sequencing using techniques known to those skilled in the art.
  • a partial N-terminal amino acid sequence of about 25 amino acids was deduced and is represented herein as SEQ ID NO: 96: X-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-K-K-D-D-L-L-I .
  • This Example demonstrates the kinetics of cat IgG degrading activity in the midguts of fleas fed on live cats .
  • the fleas' midguts were removed as described in U.S. Patent No. 5,356,622, ibid. , homogenized by freeze-fracture and sonicated in a Tris buffer comprising 50 mM Tris, pH 8.0 and 100 mM CaCl 2 .
  • the extracts were centrifuged at about 14,000 x g for 20 min. and the soluble material recovered.
  • the soluble material was then diluted to a final concentration of about 1.2 midgut equivalents per microliter ( ⁇ l) of Tris buffer.
  • the proteins contained in 1 midgut equivalent of each timepomt were then resolved by SDS-PAGE under reducing conditions, and the proteins visualized by silver staining.
  • the flea chambers were removed and placed in a 28°C, 75% relative humidity growth incubator.
  • Fleas were subjected to dissection at time points of 0, 1, 2, 4, and 8 hr. following removal from the cats.
  • Midguts were homogenized, and the midgut contents were examined by silver stained SDS-PAGE and immunoblot analysis, as described in Section A.
  • the fleas fed for 1 hour had high molecular weight proteins, including the heavy chain and light chain of cat IgG detectable in their midguts at the 0 and 1 hour dissection timepoints, while the flea midguts evaluated at time points of 2 hours or greater had no detectable IgG heavy chain bands.
  • This example describes the ability of a 31-kD flea midgut serine protease contained in a formulation to proteolyze cat immunoglobulin G at a specific site.
  • the 31-kD flea midgut serine protease was purified from cat blood-fed flea midgut extracts as described above in Examples 14 and 15.
  • SEQ ID NO: 104 A partial N-termmal ammo acid sequence of about 25 ammo acids was determined and is represented herem as SEQ ID NO: 104: D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K- D. An additional 10 amino acids were also obtained beyond the last ammo acid of SEQ ID NO: 104.
  • SEQ ID NO: 105 D-C-P-K-C-P-P-P-E-M-L- G-G-P-S-I-F-I-F-P-P-K-P-K-D-D-L-L-I-K-R-K-S-E-V.
  • GenBank homology search using SEQ ID NO: 105 revealed most homology to Homo sapi en immunoglobulin gamma 3 heavy chain constant region 2 exon hinge IGHG3 gene (GenBank Accession No. X99549) , there being about 69% identity over the 35 amino acids.
  • SEQ ID NO: 105 Further alignments of SEQ ID NO: 105 with cat, rabbit, bovine and human IgG amino acid sequences indicated that purified cat blood-fed 31-kD flea midgut serine protease cleaved the cat IgG heavy chain about 6 amino acids just before the predicted C-terminal end of the IgG hinge region.
  • the first 6 amino acids, aspartic acid, cysteine, proline, lysine, cysteine and proline occur within the predicted hinge region while the remaining 29 amino acids of SEQ ID NO: 105, starting with the seventh amino acid proline, occur within the predicted constant heavy chain-2 region.
  • IgG (represented herein by SEQ ID NO: 106) is shown below:
  • SEQ ID NO:105 (continued) -D-D-L-L-I-K-R-K-S-E-V
  • SEQ ID NO:106 (continued) -D-T-L-S-I-S-R-T-P-E-V
  • SEQ ID NO: 105 Discrepancies between SEQ ID NO: 105 and SEQ ID NO: 106 are shown in bold. Applicants believe that the difference between SEQ ID NO: 105 and SEQ ID NO: 106 may be due to sequencing error of the last 10 amino acids of SEQ ID NO: 105.
  • ATGAAGTAAA AACCTTGCGT TGGTTTCCCC GGTCCCAGGA TCAGGAACAG TTGCACTTTA 120
  • GCC AAA AAT AAT AAT GCG GAT TTG ACG ATC ACT TAT TTA CTA TGT ACT 1079 Ala Lys Asn Asn Asn Ala Asp Leu Thr Ile Thr Tyr Leu Leu Cys Thr 305 310 315
  • MOLECULE TYPE protein
  • Asn Asn Asn Asn Ala Asp Leu Thr lie Thr Tyr Leu Leu Cys Thr Thr Phe 305 310 315 320 Lys Ile Asp Phe Tyr Gly Met
  • CAGGAACTTC ACCATGAGTT TGATTGGTCA ATCCCCATCC AGTAATTTTC AATTTTTCAC 420 CTCCGCGTAT AAAATCTTTG TGCAATTTGA TGGGACGAAC ATTTTTGCTA AGTTTTATAG 480
  • Met Asn Arg Trp lie Leu Thr Ala Ala His Cys Leu Thr Asp Gly Tyr 35 40 45
  • MOLECULE TYPE DNA (genomic)
  • CTTATNGACC AGAGGACCAC CAGAGTCACC CATGCATACA CCCTTTTGAG GTGCTTTGAA 180
  • AAA AAA AAA GGA ACC GGA TCT TGT AAG GGT GAT TCT GGT GGT CCA TTA GTC 677 Lys Lys Gly Thr Gly Ser Cys Lys Gly Asp Ser Gly Gly Pro Leu Val 200 205 210 215
  • MOLECULE TYPE protein
  • MOLECULE TYPE DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: ATTTTCTGAC ATTGTCGTGT TAATCCAGTC CAAAAACGAT GTAATTCTTG TGTAAACGTC 60 AGGATAATAA CCACTTCCAC AAGGTTGCAT ACCCCAGGAT ACTAAACCGA CCAATGTATT 120 GTTTCCTTGG ACTAATGGAC CACCAGAATC ACCCTTACAA GATCCGGTTC CTTTTTTAGC 180 CTGTGCACAA ATTTGGCTTT CGAAAGTCTT TTTATAAATT GCGTTTTTGC AATCCTCATA 240
  • AGT CAA AAA TTA CAG GTC ATG ACA GCC AAA TCA CTA ACT TAT GAG GAT 480 Ser Gin Lys Leu Gin Val Met Thr Ala Lys Ser Leu Thr Tyr Glu Asp 145 150 155 160
  • CAG GCT AAA AAA GGA ACC GGA TCT TGT AAG GGT GAT TCT GGT GGT CCA 576 Gin Ala Lys Lys Gly Thr Gly Ser Cys Lys Gly Asp Ser Gly Gly Pro
  • TCT TCT TTA AAT TTG AAT GGA GGT TCT ATT CGA CCG GCT AGG TTA GTG 431 Ser Ser Leu Asn Leu Asn Gly Gly Ser Ile Arg Pro Ala Arg Leu Val 130 135 140

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Abstract

Protéines de sérine protéase de puces et protéines de cystéine protéase de puces, molécules d'acides nucléiques de sérine protéase et de cystéine protéase de puces, y compris celles qui codent lesdites protéines, anticorps contre lesdites protéines et composés qui inhibent l'activité de la sérine protéase et/ou de la cystéine protéase de puces. La présente invention comporte également des procédés permettant d'obtenir lesdites protéines, des molécules d'acides nucléiques, des anticorps et/ou des inhibiteurs, ainsi que l'utilisation desdites compositions thérapeutiques pour protéger un animal hôte de l'infestation par les puces.
PCT/US1997/006121 1995-06-07 1997-04-24 Proteines de protease de puces, molecules d'acides nucleiques et leurs utilisations WO1997040058A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP53813497A JP2001510324A (ja) 1996-04-24 1997-04-24 ノミのプロテアーゼタンパク質、核酸分子、およびそれらの使用
EP97922303A EP0900231A1 (fr) 1996-04-24 1997-04-24 Proteines de protease de puces, molecules d'acides nucleiques et leurs utilisations
AU28015/97A AU735717B2 (en) 1996-04-24 1997-04-24 Flea protease proteins, nucleic acid molecules, and uses thereof
US09/032,215 US6204010B1 (en) 1995-06-07 1998-02-27 Flea protease proteins, nucleic acid molecules, and uses thereof

Applications Claiming Priority (6)

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US08/639,075 US6150125A (en) 1991-12-13 1996-04-24 Flea protease proteins and uses thereof
US08/749,699 US6210920B1 (en) 1991-12-13 1996-11-15 Flea protease proteins, nucleic acid molecules, and uses thereof
US4294597P 1997-04-04 1997-04-04
US08/749,699 1997-04-04
US08/639,075 1997-04-04
US60/042,945 1997-04-04

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000061621A2 (fr) * 1999-04-09 2000-10-19 Heska Corporation Molecules d'acides nucleiques et proteines issues de la tete, de la moelle epiniere, de l'intestin posterieur et du tube de malpighi de puces et utilisations correspondantes
US6204010B1 (en) * 1995-06-07 2001-03-20 Heska Corporation Flea protease proteins, nucleic acid molecules, and uses thereof
WO2001046442A1 (fr) * 1999-12-22 2001-06-28 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, cysteine protease 10, et polynucleotide codant pour ce polypeptide
US6596291B2 (en) 1997-12-05 2003-07-22 Thomas A. Bell Compositions and methods for treating surfaces infected with ectoparasitic insects
EP1710252A2 (fr) * 1999-04-09 2006-10-11 Heska Corporation Molécules d'acides nucléiques et protéines issues de la tête, de la moelle épinière et du tube de Malpighi de puces et les utilisations correspondantes

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WO1993023542A1 (fr) * 1992-05-08 1993-11-25 The Biotechnology And Biological Sciences Research Molecules d'adn recombinees codant des aminopeptidases et leur utilisation dans la preparation de vaccins contre les infections helminthiques
EP0571911A2 (fr) * 1992-05-26 1993-12-01 Becton, Dickinson and Company Sondes pour mycobactéries
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WO1990003433A1 (fr) * 1988-09-26 1990-04-05 Biotechnology Australia Pty. Ltd. Vaccin
US5304482A (en) * 1989-03-06 1994-04-19 The Board Of Regents Of The University Of Texas System Serine protease mutants of the chymotrypsin superfamily resistant to inhibition by their cognate inhibitors
US5288612A (en) * 1991-07-03 1994-02-22 The Scripps Research Institute Assay methods for detecting serum proteases, particularly activated protein C
WO1993023542A1 (fr) * 1992-05-08 1993-11-25 The Biotechnology And Biological Sciences Research Molecules d'adn recombinees codant des aminopeptidases et leur utilisation dans la preparation de vaccins contre les infections helminthiques
EP0571911A2 (fr) * 1992-05-26 1993-12-01 Becton, Dickinson and Company Sondes pour mycobactéries

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FEBS LETTERS, December 1975, Vol. 60, No. 2, ZWILLING R. et al., "The Amino-Terminal Sequence of an Invertebrate Trypsin (Crayfish Astacus Leptodactylus): Homology With Other Serine Proteases", pages 247-249. *
IMMUNOLOGY, 1985, ROITT et al., ST. LOUIS: THE C.V. MOSBY COMPANY, pages 5.4-5.5. *
J. BIOL. CHEM., 1982, Vol. 257, No. 12, CUYPERS T.H. et al., "The Primary Structure of Leucine Aminopeptidase from Bovine Eye Lens", pages 7077-7085. *
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6204010B1 (en) * 1995-06-07 2001-03-20 Heska Corporation Flea protease proteins, nucleic acid molecules, and uses thereof
US6596291B2 (en) 1997-12-05 2003-07-22 Thomas A. Bell Compositions and methods for treating surfaces infected with ectoparasitic insects
WO2000061621A2 (fr) * 1999-04-09 2000-10-19 Heska Corporation Molecules d'acides nucleiques et proteines issues de la tete, de la moelle epiniere, de l'intestin posterieur et du tube de malpighi de puces et utilisations correspondantes
WO2000061621A3 (fr) * 1999-04-09 2001-07-05 Heska Corp Molecules d'acides nucleiques et proteines issues de la tete, de la moelle epiniere, de l'intestin posterieur et du tube de malpighi de puces et utilisations correspondantes
EP1710252A2 (fr) * 1999-04-09 2006-10-11 Heska Corporation Molécules d'acides nucléiques et protéines issues de la tête, de la moelle épinière et du tube de Malpighi de puces et les utilisations correspondantes
EP1710252A3 (fr) * 1999-04-09 2006-12-27 Heska Corporation Molécules d'acides nucléiques et protéines issues de la tête, de la moelle épinière et du tube de Malpighi de puces et les utilisations correspondantes.
WO2001046442A1 (fr) * 1999-12-22 2001-06-28 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, cysteine protease 10, et polynucleotide codant pour ce polypeptide

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IL120704A0 (en) 1998-04-05
JP2001510324A (ja) 2001-07-31

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