CA2202622A1

CA2202622A1 - Use of protease inhibitors and protease vaccines to protect animals from flea infestation, and flea protease proteins, nucleic acid molecules and uses thereof

Info

Publication number: CA2202622A1
Application number: CA002202622A
Authority: CA
Inventors: Robert B. Grieve; Keith E. Rushlow; Shirley Wu Hunter; Glenn R. Frank; Gary L. Stiegler; Andrew Heath; Miles Yamanaka; Ann Arfsten; Beverly Dale
Original assignee: Individual
Current assignee: Heska Corp
Priority date: 1994-10-18
Filing date: 1995-10-18
Publication date: 1996-04-25
Also published as: EP0787014A1; WO1996011706A1; JPH10507455A; EP0787014A4; NZ296424A; IL115671A0; MX9702776A; AU4103896A; AU705715B2

Abstract

The present invention relates to flea serine protease and aminopeptidase proteins; to flea serine protease and aminopeptidase nucleic acid molecules, including those that encode such proteins; to antibodies raised against such proteins; and to compounds that inhibit flea serine protease and/or aminopeptidase activities. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitors. Also included in 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.

Description

WO 96/11706 PcrluS95/14442 Use of Flea rrul~aseS and Protease Inhibieors to Protect Animals from Flea l,-rtslaLi t Field of the Invention The prèsent invention relates to novel flea protease proteins and their use to reduce fiea infestation of animals. The present invention also relates to the use of anti-flea pPotease antibodies and other _ul~ds that reduce flea protease activity to reduce flea infestation of animals.
~' Backcround of the Invention 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 ~rnn~ i t. concern because fleas are known to cause and/or transmit a variety of ~l;c~:~c,,c. Fleas cause and/or carry infectious agents that cause, for example, flea allergy dermatitis, anemia, murine typhus, plague and td~:W~LIu. 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 in the general environment of the animal.
The medical and vet~ri n:~ry importance of flea infestation has ro ted the development of reagents p mp capable of ~ controlling flea infestation. Commonly wo 96111706 PCT/U595114442 encountered methods to control flea infestation are generally focussed on use of insecticides in formulations such as sprays, shampoos, dusts, dips, or foams, or in pet collars. While some of these products are eff;C~cjollc, 5 most, at best, offer protection of a very limited duration.
Furth~ ~, many of the methods are often not successful in reducing flea populations on the pet for one or more of the following reasons: ( 1) failure of owner compliance (frequent administration is requiredl; (2) behavioral or 10 physiological intolerance of the pet to the pesticide product or means of administration; and (3) the emergence of flea populations resistant to the prescribed dose of pesticide. Additional anti-flea products include nontoxic reagents such as insect growth regulators (IGRs), including 5 methoprene, which mimics flea hl .~-- and affect flea larval development .
An alternative method for controlling flea infestation is the use of flea vaccines to be administered to animals prior to or during flea infestation. However, despite 20 considerable interest in developing anti-flea reagents, no flea vaccine presently exists.

Sun~narv of the Invention The present invention relates to a method to protect a host animal from flea infestation that includes the step ~5 of treating that animal with a composition that includes a _ ' that reduces protease activity of fleas feeding from the treated animal, thereby reducing flea burden on Wo 96/11706 l?CT/US95/14442 the animal and in the environment of the animal. Flea proteases to target can be aminopeptidases, caLI,uxy~Lidases and/or endopeptidases, and can include .
serine proteases, metalloproteases, aspartic acid proteases 5 and cysteine proteases.
Preferred ~;u...~u--ds to include in a composition of the present invention include one or more of the following:
flea protease vaccines, anti-flea pro~ease ant;ho~;Pq and/or protease inhibitors. Also included in the present lO invention are compositions including such ~ _ -c Particularly preferred ~ c are those that reduce the activity of flea ser~ine proteases, with flea serine protease vaccines being even more preferred.
Another embodiment of the present invention is a 15 controlled release formulation that includes one or more compositions of the present invention and use of such a formulation to provide long term protection against flea inf estation .
In a preferred: ;- L, treatment of an animal with 20 a composition of the present invention reduces flea viability by at least about 50 percent within at least about 21 days after fleas begin feeding from a treated animal. In another embodiment, treating an animal with a composition of the present invention reduces flea fecundity 25 by at least about 50 percent within at least about 30 days after fleas begin feeding from a treated animal.
~ he present invention also includes a method to protect a host animal from flea infestation, which includes - , ' . -Wo 96111706 PCrlUS95/14442 treating the animal with a composition comprising a first compound capable of reducing flea protease activity and a second cn~ro~1n~ that reduces flea burden by a method other than by reducing flea protease activity. In this 5 embodiment, the first compound, by reducing proteolytic activity in the flea midgut, can enhance the efficacy of a second compound that also enters the flea through its midgut and otherwise would be susceptible to proteolytic degradation. Also included in the present invention are lO compositions including such first and second ~
Also included in the present invention is a method to reduce flea infestation that includes treating a flea directly or an animal susceptible to flea infestation with a composition comprising a ~_ ~ capable of reducing 15 flea protease activity.
Another aspect of the present invention is a soluble flea midgut preparation having proteolytic activity, wherein at least about 70 percent of that proteolytic activity can be inhibited by 4-2-aminoethyl-20 bPn7~n-~c~11 fonylfluoride hydLu-,1.loride (i.e., wherein about 70 percent of that activity is serine protease activity).
Such a preparation is preferably produced by ta) disrupting a f lea midgut to produce a mixture comprising a liquid portion and a solid portion; and (b) recovering the liquid 25 portion to obtain the preparation. A flea protease can be obtained from such a preparation using any of a variety of purification techniques.
The present invention also includes an isolated Wo 96/11706 pcrNs9sll4442 protein that includes an amino acid sequence encoded by a nucleic acid molecule capable of hybridizing under stringent conditions with a nucleic acid molecule that encodes a midgut protease present in a flea midgut. Such 5 a protein preferably has proteolytic activity and/or the ability to elicit an immune response against a flea midgut protease. Also included in the present invention is an isolated flea protease protein that includes an amino acid sequence encoded by a nucleic acid molecule capable of lO hybridizing under stringent conditions with a nucleic acid molecule that encodes a flea midgut protease having an amino acid sequence including SEQ ID N0: l.
The present invention also includes a nucleic acid molecule capable of hybridizing under stringent conditions 15 with a gene encoding a flea protease present in a flea midgut. Also included are recombinant molecules including one or more nucleic acid molecules of the present invention as well as recombinant cells including one or more nucleic acid molecules of the present invention. Nucleic acid 20 molecules F~nro~;n~ flea protease proteins of the present invention can be used in the production of such proteins.
The present invention also includes isolated antibodies capable of selectively binding to a protease present in a flea midgut. Such antiho~ c have a variety 25 of uses including in passive ;mmlln; 7ation protocols.
Another aspect of the present invention is a method to identify a ~ ' capable of inhibiting the proteolytic activity of a flea protease. Such a method inFludes the Wo 96111706 PCr/USsS/14442 steps of (a) contacting an isolated flea protease protein with a putative inhibitory, _ ~ under conditions in which, in the absence of the _ _ ~, the protease has proteolytic activity; and (b) ~t~rm;n;n~ if=the putative 5 inhibitory ' inhibits the activity of the protease.
Also included in the present invention is a test kit to identify a ~ ' capable of inhibiting proteolytic activity of a flea protease. Such a kit includes an isolated flea protease protein having proteolytic activity 10 and a means for detormin;n~ the extent of inhibition of that activity in the presence of a putative inhibitory ~ul~d.
The present invention also relates to flea serine protease and Am; nl~p~rtidase proteins; to flea serine 15 protease and AminnpPrtidase nucleic acid molecules, including those that encode such proteins to antibodies raised against such proteins: and to, olln~c that inhibit flea serine protease and/or Am;n~lp~rtidase activities. The present invention also includes methods to obtain such 20 proteins, nucleic acid molecules, antibodies, and inhibitors. Also included in 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 25 animal from flea infestation.
One: ;r L of the present invention is an isolated flea serine protease nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea serine Wo 96/11706 PCr/Uss~ 42 protease gene. Particularly preferrea flea serine protease nucleic acid molecules incluae nucleic acid sequences SEQ
ID N0:16, SEQ ID N0:18, SEQ ID N0:20, SEQ ID N0:22, SEQ ID
N0:24, SEQ ID N0:26, SEQ ID N0:28, SEQ ID NO:30, SEQ ID
N0:32, SEQ ID N0:34, SEQ ID N0:36, SEQ ID N0:38, the nucleic acid sequences disclosed in Table 2 ( i . e ., SEQ ID
N0:52, SEQ ID N0:54, SEQ ID N0:56, SEQ ID N0:58, SEQ ID
N0:60, SEQ ID N0:62, SEQ ID N0:64, SEQ ID N0:66, SEQ ID
N0:68, SEQ ID N0:70, SEQ ID N0:72, SEQ ID N0:74, SEQ ID
10 N0:76, SEQ ID N0:78), SEQ ID N0:80, SEQ ID N0:82, SEQ ID
N0:84, SEQ ID N0:86, SEQ ID N0:88, SEQ ID NO:90, SEQ ID
N0:92, SEQ ID N0:94, SEQ ID NO:96, SEQ ID N0:98, SEQ ID
N0:100, SEQ ID N0:102, SEQ ID N0:104, SEQ ID N0:106, SEQ ID
N0:108, SEQ ID N0:110, and/or nucleic acid sequences Pnro~l; nrJ proteins having amino acid sequences SEQ ID N0 :1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID
N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID N0:17, SEQ ID N0:19, SEQ ID N0:21, SEQ ID N0:23, SEQ ID N0:25, SEQ
ID N0:27, SEQ ID N0:29, SEQ ID N0:31, SEQ ID NO:33, SEQ ID
NO:35, SEQ ID N0:37, SEQ ID N0:39, SEQ ID N0:40, SEQ ID
NO:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID
N0:45, SEQ ID N0:46, SEQ ID N0:47, the amino acid 5~qn~n~s disclosed in Table 2 (i.e., SEQ ID N0:53, SEQ ID N0:55, SEQ
ID N0:57, SEQ ID NO:59, SEQ ID N0:61, SEQ ID N0:63, SEQ ID
N0:65, SEQ ID N0:67, SEQ ID N0:69, SEQ ID N0:71, SEQ ID
N0:73, SEQ ID N0:75, SEQ ID N0:77, SEQ ID N0:79), SEQ ID
N0:81, SEQ ID N0:83, SEQ ID N0:85, SEQ ID N0:87, SEQ ID
N0:89, SEQ ID N0:91, SEQ ID N0:93, SEQ ID N0:95, SEQ ID

Wo 96tll706 PCT~S95114442 N0:97, SEQ ID N0:99, SEQ ID N0:101, SEQ ID N0:103, SEQ ID
N0:105, SEQ ID NO:107, SEQ ID N0:109, and/or SEQ ID N0:111, as well as allelic variants of any of those nucleic acid sequences.
~ Another ~ of~ the present invention is an isolated flea aminopeptidase nucleic acid molecule that hy~ridizes under stringent hybridization conditions with a flea aminopeptidase gene. A particularly preferred flea aminopeptidase nucleic acid molecule includes nucleic acid sequence SEQ ID N0:50 or an allelic variant thereof.
Another particularly preferred flea Am;nop~ptidase nucleic acid molecule includes nucleic acid sequence SEQ ID N0:112 or an allelic variant thereof.
The present invention also relates to recombinant molecules, re: ;nAnt viruses and recombinant cells that include flea serine protease and/or Am;nnp.ortidase nucleic acid molecules of the present invention. Also included are methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and r~ ;n~nt cells.
Another: --;r L of the present invention includes an isolated flea serine protease protein, ;n~ A;n~ a protein that includes a f lea serine protease protein . A
preferred flea serine protease protein is capable of eliciting an immune 1c~ ..se against a natural flea 25 protease when administered to an animal and/or of having serine protease activity. Particularly preferred flea serine protease proteins are those encoded by preferred flea serine protease nucleic acid molecules of the present Wo 96/11706 PCr~SsS/l4442 invention .
Yet another ;T ~ of the present invention includes an isolated flea Aminnp~ptidase protein, including a protein that includes a flea AminnpDrtidase protein. A
preferred flea Am;nnpDrtidase protein is capable of eliciting an immune response against a natural flea protease when administered to an animal and/or of having Am; nopDrtidase activity. A particularly preferred flea aminopeptidase protein is a protein that includes SEQ ID
N0: 51 or a protein that is encoded by a nucleic acid molecule that is an allelic variant of a nucleic acid molecule comprising SEQ ID N0:50. Another particularly preferred flea aminopeptidase protein is a protein that includes SEQ ID N0: 113 or a protein that is encoded by a nucleic acid molecule that is an allelic variant of a nucleic acid molecule comprising SEQ ID N0 :112 .
The present invention also relates to m;- op~s of flea serine protease and Am;nopPptidase proteins as well as to isolated antibodies that selectively bind to flea serine protease proteins or m; ' OpDc thereof or to flea aminopeptidase proteins or m;- ' opc~c thereof. Also included are methods, including recombinant methods, to produce proteins, mimetopes and ant; ho~l; DC of the present invention.
Yet another '; ~ of the present invention is a therapeutic composition that is capable of reducing flea infestation. Such a therapeutic composition includes one ~r more of the ~oll~w~ng pro~ecti re ~ ' ~n isola~ed WO g6/11706 PCr/US9~/14442 flea serine protease protein 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 5 selectively binds to a flea serine protease protein; an inhibitor of flea serine protease activity identified by its ability to inhibit flea serine protease activity; an isolated flea Am;nnpprtidase protein or a mimetope thereof;
an isolated flea Am;nor~rtidase nucleic acid molecule that lO hybridizes under stringent hybridization conditions with a flea aminopeptidase gene; an isolated antibody that selectively binds to a flea Am;nnp~rtidase protein; and an inhibitor of flea Am;nnp~ortidase activity identified by its ability to inhibit flea aminopeptidase activity. A
15 preferred therapeutic composition of the present invention also includes an excipient, an adjuvant and/or a carrier.
Also included in the present invention is a method to reduce flea infestation. The method includes the step of administering to the animal a therapeutic composition of

2 0 the present invention .
Another ' '; r - I_ of the present invention is a method to identify a ~ ln-l capable of inhibiting flea serine protease or flea Am1 nnpl~rtidase activity. The method includes the steps of: (a) contacting an isolated 25 flea serine protease protein or a flea Am;nnpl~rtidase protein with a putative inhibitory _ ' under conditions in which, in the absence of the ,_ ', the protein has, respectively, serine protease or WO 9611171~6 PCI~/IJS95/14442 11 ~
~min-~p~ptidase activity; and (b) ~t~rm;n;n~ if the putative inhibitory ., ' inhibits the respective activity. Also included in the present invention is a test kit to identify a ~ capable of inhibiting flea 5 serine protease or flea Am; n~p~ortidase activity. Such a kit includes an isolated flea serine protease protein having serine protease activity or an isolated flea aminopeptidase protein having Flm; nop~rtidase activity and a means for d~t~rm;n;n~ the extent of inhibition of the 10 respective activity in the presence of a putative inhibitory Brief Descri~tion of the Fiqures Fig. 1 depicts a protease substrate gel analysis of the relative proteolytic activity in 1, 2, 5 or 10 midguts 15 from either f ed or unf ed female f leas .
Fig. 2 depicts a protease substrate gel analysis of fed and unfed midgut preparations incubated in the presence or absence of a serine protease inhibitor.
Fig. 3 depicts a protease substrate gel analysis of 20 various fractions obtained in the preparation of a soluble flea midgut preparation incubated in the presence or absence of a serine protease inhibitor.
Fig. 4 depicts a protease ~ub:,~L-~te gel showing midgut protease activity as a function of time after flea blood 25 feeding.
Fig. 5A depicts a ~c eqie stained SDS-PAGE of partially purified (1,3-3~I)-diisopropylfluoro-phosphate Wo 96/11706 PCT/US95/14442 (DFP)-labeled fed flea midgut serine proteases.
Fig. 5B depicts an autoradiogram of the SDS-PAGE gel of Fig. 5A of partially purified DFP-labeled fed flea midgut serine proteases.
Fig. 6 depicts the mean viability of adult (both male and female) fleas fed blood containing certain protease inhibitors .
Fig. 7 depicts the mean fecundity of ~ adult ~female fleas fed blood containing certain protease inhibitors.
Fig. 8 depicts the mean viability of adult (both male and female) fleas fed blood containing certain protease inhibitors .
Fig. 9 depicts the mean fecundity of adult female fleas fed blood containing certain protease inhibitors.
-- Fig. lO depicts ; n~llnti nn over time of DFP-labeled proteases in fleas feeding on a cat.
Fig. ll depicts SDS-PAGE of induction over time of DFP-labeled proteases in fleas feeding on a cat.
Detailed Descri~tion of the Invention The preSQnt invention includes the use of ,. ~l1n-lc that inhibit flea protease activity to protect a host animal from :flea infestation. The inventors have discovered that proteases are significant components of the flea midgut and are good targets for ~immunotherapeutic and/or chemotherapeutic intervention to reduce flea- burden both on the host animal and in the immediate ( i . e ., :~uLL~u~lding) environment of the animal. The inventors have Wo 96/11706 PCrlUS95/14442 shown, for example, that the viability and/or fecundity of fleas cC~n! l~rn;n~ a blood meal is reduced when the blood meal contains ~ ~ullds ~ that reduce flea protease activity, probably because the , ,__llds int~rfere with flea 5 digestion and other functions. C _ .ullds that reduce the amount and/or activity of flea proteases without substantially harming the host animal are included in the present invention. Such, _~-~ullds include flea protease vaccines, anti-flea protease antibodies, flea protease 10 inhibitors, and/or- compounds that ~uLIul~SS protease synthesis; such - '- are discussed in more detail below .
One ' ir 1 of the present invention is a method to protect a host animal from flea infestation by treating the 15 animal with a composition that includes a _ _luulld 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.
20 It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, a __ refers to one or more _,uullds. As such, the terms "a"
(or "an"), "one or more" and "at least one" can be used inter~-h~n~hly herein. Thus, a composition of the present 25 invention can include one or more __ " that target (reduced the activity of ) one or more proteases in the flea .
As used herein, the phrase "to protect an animal from -Wo 96/11706 PcrJuS95/14442 flea infestation" refers to red~ ;nq the potential for flea population ~Yp;~ncir~rl on and around the animal ( i . e., reducing the flea burden). Preferably, the flea population size is decreased, optimally to an extent that the animal 5 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 lO animal in order to feed. At any given time, a certain percentage of a flea population can be on a host animal whereas the L- ;n~.r can be in the environment ~uLL~u-lding the animal (i.e., in the environment of the animal). Such an environment can include not only adult fleas, but also 15 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 20 environment ~uLL~ull~ing the animal.
In accordance with the present invention, a host animal is treated by administering to the animal a _ of the present invention in such a manner that the ~ _ ' itself (e.g., a protease inhibitor, protease synthesis 25 suppressor or anti-flea protease antibody) or a product generated by the animal in response to administration of the o~ln~l (e.g., ~nt;horl;ps produced in response to a flea protease vaccine, or conversion of an inactive WO 96/11706 PC~/US9~i/14442 inhibitor I~L~ dluyl~ to an active protease inhibitor) ultimately enters the flea midgut. An animal is preferably treated in such a way that the ~ ~ ' or product thereof enters the blood stream of the animal.~ Fleas are then 5 exposed to the - ~_ ' when they feed from the animal.
For example, 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. In another embodiment, when a 10 host animal is administered a flea protease vaccine, the treated animal mounts an immune response resulting in the production of antibodies against the protease (anti-flea protease antibodies) which circulate in the animal's blood stream and 2re taken up by fleas upon feeding. ~ Blood taken 15 Up by fleas enters the flea midgut where ~ c of the present invention, or ~L.~lU.:L, thereof, such as anti-flea protease ~ntihQ~ies, flea protease inhibitors, and/or protease synthesis ~u~eSSOrS, interact with, and reduce proteolytic activity in the flea ~midgut. The present 20 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 invention, at least a portion of _ ul~ds of the present invention, or products thereof, in the blood taken 25 Up by the flea are excreted by the flea in feces, which is 511h5~ ntly ingested by flea larvae. It is of note that flea larvae obtain most, if not all, of their nutrition from flea feceo.

Wo 96/11706 Pc~r/uss5ll4442 In accordance with the present invention, reducing proteolytic activity in flea midguts can lead to a number of outcomes that reduce flea burden on treated animals and their ~uLLvullding environments. Such 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 L-~Ludul_Live capacity of male flea6 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., slowing or blocking) molting, and/or otherwise inhibiting maturation to adults).
One ' ~ of the present invention is a composition that includes one or more ~ _ '~ 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 ;n-l;r~ctly (e.g., a flea protease vaccine). Suitable flea proteases to target include flea aminopeptidases, flea caLl,u~y~ idases and/or flea endopeptidases. Preferred flea proteases to target include, but are not limited to, serine proteases, Wo 96/11706 ~ ~P~5114442 metalloproteases, aspartic acid proteases ana/or cysteine proteases. It is ~o be noted that these preferred groups of proteases include ;Im; nnp~ptidases, carboxypeptidases and/or endopeptidases. Preferred flea proteases to target 5 include,~ but are not limited to, proteases that degrade hemoglobin, proteases involved in blood coagulation and/or lytic (anti-coagulation) pathways, proteases involved in the maturation of peptide h~ IF-, proteases that inhibit complement or other host immune response elements (e.g., l0 antibodies) and/or proteases involved in vitellogenesis.
A number of proteases are known to those skilled in the art, including, but not limited to, ~m;nnp~rtidases, such as leucine ~minnp~rtidase and aminopeptidases B and M;
astacin-likemetalloproteases; n~1r~in~; caLl,uxy~:~,tidases, 15 such as caL~.xy~:~Lidases A, P and Y; cath~rq;nc, such as cath~rc;nc B, D, E, G, H, and L, ~ y LL~,sins; cr~l7;pA;nf:;
meprins; papains; pepsins; renins; thermolysins and trypsins. A partic~ ~ly preferred protease to target is a protease having a proteolytic activity that, when 20 targeted with a composition of the present invention, reduces flea burden without ~ubaLdl,Lially har_ing the host animal. Such a protease can be identified using, for example, methods as disclosed herein.
One aspect of the present invention is the discovery 25 that a substantial amount of the proteolytic activity found in flea midguts is serine protease activity. Both in vitro and in vivo studies using a number of protease inhibitors ~ uDaLcll~Liate thls discovery, details of whlch are disclosed Wo 96/11706 ~ uS~5/14442 in the Examples. As such a particularly preferred protease to target is a serine protease. r _ l~c of ~serine proteases, include, but are not limited to, acrosins, bL~ ;nc, cathepsin G, ~ y ~Ly~sins, collagenases, 5 elastases, factor Xa, ficins, kallikreins, papains, plasmins, Staphylococcal V8 proteases, thro_bins and trypsins. In one embodiment, a preferred flea serine protease to target includes a protease having trypsin-like or ~ y LLy~:,in-like activity. It is appreciated by those l0 skilled in the art that 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 ::.LLU~:LULe~ as their referenced counterparts.
Protease inhibitor studies disclosed in the Examples section also indicate that additional preferred proteases to target include ~m;nnpF~rtidases and/or metalloproteases.
Examples of such proteases include exo- and endo-metalloproteases, digestive enzymes, and enzymes involved 20 in peptide hormone maturation. One example of an aminopeptidase that is also a metalloprotease is leucine aminopeptidase .
Suitable _ _ '~ to include in compositions of the present invention include, but are not limited to, a 25 vaccine comprising a flea protease (a ~lea protease vaccine), an antibody that selectively binds to a flea protease (an anti-flea protease antibody), a flea protease inhibitor (a _ _ other than a vaccine or an antibody Wo 96/11706 PCT/US95/144~2 ~ . 19 that inhibits a flea protease), and a mixture of such compounds. As used herein, a mixture thereof refers to a combination of one or more of- the cited entities.
Compositions of the present invention can also include 5 ~ lullds to ~:U~ SS protease synthesis or maturation, such as, but not limited to, protease modulating peptides.
A preferred embodiment of the presént invention is a flea protease vaccine and its use to reduce the flea population on and around an animal. A flea protease 10 vaccine can include one or more proteins capable of eliciting an immune response against a flea protease and can also include other ~_ _ ~ntS Preferred flea protease vaccines include a flea serine protease, a flea metalloprotease, a flea aspartic acid protease and/or a 15 flea cysteine protease, with flea serine protease, flea metalloprotease and/or flea ~m;nl~p~rtidase vaccines being more preferred. Examples of flea protease vaccines include soluble flea midgut preparations of the present invention as well as one or more isolated proteins of the present 20 invention.
One ~ mho~l; L of the present invention is a soluble flea midgut preparation. Such a preparation includes primarily ~ _ naturally present in the lumen of a flea midgut and, ~l~rF~n-l;nJ on the method of preparation, 25 can also include one or more peripheral midgut membrane proteins. Methods to preferentially include, or exclude, me_brane proteins from such a preparation are known to those skilled in the art. The present invention includes Wo 96/11706 PCrlUS9~114442 the discovery that such a preparation has proteolytic activity, of which a suhstantial portion is serine protease activity. Preferably at least - about 70 percent of the proteolytic activity in a soluble flea midgut soluble 5 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-aminoethyl-benzenesulfonylfluoride-hydrochloride (AEBSF). Serine protease activity can also be identified using other known 10 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-diisopropylfluoro-phosphate or leupeptin.
A soluble flea midgut preparation of the present invention includes proteases that range in molecular weight from about 5 kilodaltons (kD) to about 200 kD, as det~rm;n~A by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), with at least a 20 substantial portion of the serine proteases ranging in molecular weight from about 5 kD to about 60 kD, as de~nm; n~ by SDS-PAGE. A ~ LGIlLial portion of protease activity in a soluble flea midgut preparation of the present invention has a pH activity optimum ranging from 25 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 ~ro 96111706 ~ ~CrNsss/l4442 large ~v~o, ~ion 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 . The f; n-l; n~C that proteases in 5 soluble flea midgut preparations of the present invention exhibit a varied pattern of inhibition by protease inhibitors of a given type (e.g., serine protease inhibitors), as well as variances seen in molecular weights and pH optima of the proteases, suggest that there are a lO number of protease isoforms in such preparations.
A soluble flea midgut preparation of the present invention is preferably prepared by a method that includes the steps of (a) disrupting a flea midgut to produce a mixture including a liquid portion and a solid portion and 15 (b) recovering the liquid portion to obtain a soluble flea midgut preparation. Such a method is a simplified version of methods disclosed in U.S. Patent No. 5,356,622, ibid.
It is to be noted that in accordance with the present invention, methods ~ closPd in U.S. Patent No. 5,356,622, 20 ibid. 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 CUIL_ -' a blood meal (i.e., blood-fed fleas). Such midguts are 25 referrea to herein as, respectively, unfed flea midguts and fed flea midguts. Flea midguts can be obtained from either male or female fleas. As (1 LLc~ted in the r 1 ~c:
section, female flea midguts exhibit somewhat more . . . , ~ ~ , Wo 96/11706 PCTrlJS95/14442 proteolytic activity than do male flea midguts.
Fur~h~ fed flea midguts have significantly more proteolytic activity than do unfed flea midguts. While not being bound by theory, it is believed that blood feeding induces in flea midguts the synthesis and/or activation of proteases as well as other factors (e.g., enzymes, other proteins, co-factors, etc. ) important in digesting the blood meal, as well as in neutralizing host molecules potentially damaging to the flea (e.g., complement, immunoglobulins, blood coagulation factors). It is also to be appreciated that unfed flea midguts may contain significant targets not found in fed flea midguts and vice versa. Fur~h ~ although the present application focusses primarily on flea midgut proteases, it is to be ~ noted that the present invention also includes other -ntS of soluble flea midgut preparations of the present invention 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 buf fers being used.
Such methods include any t~rhni qu~ that promotes cell lysis, such as, but are not limited to, chemical disruption t~rhni~l~c (e.q., e~o~ of midguts to a detergent) as well as ---h:lnio:ll disruption t~rhn;~l~c (e-g-, h~ , ; 7~tion, sonication, use of a tissue blender or WO 96/11706 PCI~/~S95/14442 glass beads,- and freeze / thaw t~rhn;qnf~c).
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 5 midguts is separated from the solid portion (e.g., filtration or centrifugation). In a preferred _a; L, 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 10 1 minute to about 15 minutes). The supernatant from such a centrifugation comprises a soluble flea midgut preparation of the present invention.
The present invention also includes an isolated protein that includes an amino acid sequence encoded by a 15 nucleic acid molecule capable of hybridizing under stringent r~nrl;t;r,nc (i.e., that hybridize under stringent hybridization conditions) with a nucleic acid molecule that encodes a protease present in (i.e., can be found in) a flea midgut, such as a midgut from a blood-fed female flea, 20 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 in the lumen of the midgut .
An isolated protein of the present invention, also 25 referred to herein as an isolated protease protein, preferably is capable of eliciting an immune response against a flea midgut protease and/or has proteolytic activity. According to the present invention, an isolated, Wo 96tll706 PCT/US95/14442 or biologically pure, protein, is a protein that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not npl-pc:c~rily reflect the extent to which the protein has been purified. An isolated 5 protease protein can be obtained from its natural source.
Such an isolated protein can also be produced using recombinant DNA technology or ~hPln;C-Al synthesis.
As used herein, an isolated protein of the present invention can be a full-length protein or any homologue of 10 such a protein, such as a protein in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, rh~ srh~rylation~
acetylation, myristylation, prenylation, palmitoylation, 15 amidation and/or addition of glycerophr~srh~tidyl inositol) such that the homologue comprises a protein having an amino acid sequence that is sufficiently similar to a natural flea midgut protease that a nucleic acid ceq-lPn~!p encoding the homologue is capable of hybridizing under stringent 20 conditions to (i.e., with) a nucleic acid sequence encoding the corresponding natural flea midgut protease amino acid sequence. As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules, including oligonucleotides, 25 are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., M~lec~7;9r Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. - -~VO 96/11706 ~ PC'r~llS9~/14442 The minimal size of a protein homologue of the present invention is a size sllff;~ nt to be encoded by a nucleic acid molecule capable of forming a stable hybrid with the compl . ry sequence of a nucleic acid molecule DnrQrl; n~
5 the corrDqp~n~l;n~ natural protein. As such, the size of the nucleic acid molecule Dn~orl;nq such a protein homologue is dependent on nucleic acid composition and percent homology between the nucleic acid molecule and complementary sequence as well as ~ upon hybridization 10 conditions per se (e.g., temperature, salt c~ LLcltion~
and formamide concentration). The minimal size of such nucleic acid molecules is typically at least~ about 12 to about 15 nucIeotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 17 15 bases in length if they are AT-rich. As such, the minimal size of a nucleic acid molecule used to encode a protease protein homologue of the present invention is from about 12 to about 18 nucleotides in length. mere is no limit, other than a practical limit, on the maximal size of such 20 a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes, or portions thereof. Similarly, the minimal size of a protease protein h~ 1OgUD of the present invention is from about 4 to about 6 amino acids in length, 25 with preferred sizes ~c.pDn~;n~ on~ whether a full-length, multivalent (i.e., fusion protein having more than one .
domain each of which has a function), or functional portions of such proteins are desired. Protease protein .

Wo 96111706 PCT~S95/14442 homologues of the present invention preferably have protease activity and/or are capable of eliciting an immune response against a flea midgut protease.
A protease protein homologue of the present invention 5 can be the result of allelic variation of a natural gene ,~n~o~;n~ 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 t~hn; q~ c known in the art including, but not limited to, direct 10 modifications to a gene ~~n~orlin~ a protein using, 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 in a straight-forward manner by the proteins' 15 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 20 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. Also preferred is a flea ;:~m; nr~pl~rtidase or a homologue thereof .
25 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 wo 96/11706 PCT/17S95/14442 from about 5 kD to about 200 kD, as rl~t-~rm;n~ 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 rll~tc~rm;n~l by SDS-PAGE, and 5 homologues of such proteins. Even more preferred are flea serine protease proteins and particularly those having molecular weights of about 26 kD (denoted PfSP26~, about 24 kD (denoted PfSP24), about 19 kD (denoted PfSP19) and about 6 kD (denoted PfSP6), as detPrm;n~ by SDS-PAGE, and 10 homologues of such proteins.

.
One preferred ' ~;r L of the present invention is an isolated flea protease protein that includes an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a 15 flea serine protease gene or with a flea ~sm;nrr~rtidase gene. As used herein, a flea protease gene includes all nucleic acid s,oSrlf~n~ r~q related to a natural flea protease gene such as regulatory regions that control production of a flea protease protein encoded by that gene (such as, but 20 not limited to, transcription, translation or post-translation control regions) as well as the coding region itself.
The inventors have disc~,veLt:d an extensive family of serine proteases, encoded by a family of serine protease 25 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 in the flea genome. As such, the present invention includes flea serine protease genes comprising not only the nucleic acid RPqLPn~ pc disclosed herein te.g., genes including nucleic acid SPq7lf~n~Pq SEQ ID N0:16, SEQ ID
~N0:18, SEQ ID NO:20, SEQ ID N0:22, SEQ ID NO:24, SEQ ID
N0:26, SEQ ID N0:28, SEQ ID N0:30, SEQ ID N0:32, SEQ ID
N0:34, SEQ ID N0:36, SEQ ID N0:38 and/or the nucleic acid sequences disclosed in Table 2, (i.e., SEQ ID N0:52, SEQ ID
NO:54, SEQ ID N0:56, SEQ ID N0:58, SEQ ID N0:60, SEQ ID
lO N0:62, SEQ ID N0:64, SEQ ID N0:66, SEQ ID N0:68, SEQ ID
N0:70, SEQ ID N0:72, SEQ ID NO:74, SEQ ID N0:76, SEQ ID
N0:78), SEQ ID N0:80, SEQ ID N0:82, SEQ ID N0:84, SEQ ID
N0:86, SEQ ID N0:88, SEQ ID N0:90, SEQ ID N0:92, SEQ ID
N0:94, SEQ ID N0:96, SEQ ID :N0:98, SEQ ID N0:100, SEQ ID
N0:102, SEQ ID N0:104, SEQ ID N0:106, SEQ ID N0:108, SEQ ID
N0:110, and/or nucleic acid sPq7lPnrpc Pn~ 7.;n~ proteins having amino acid sPqUpnri c as disclosed herein (e.g., SEQ
ID NO:l, SEQ ID NO:2, SEQ ID N0:3, SEQ ID NO:4, SEQ ID
N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:17, SEQ ID N0:19, SEQ ID N0:21, SEQ ID N0:23, SEQ
ID N0:25, SEQ ID N0:27, SEQ ID N0:29, SEQ ID N0:31, SEQ ID
N0:33, SEQ ID N0:35, SEQ ID N0:37, SEQ ID N0:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID N0:43, SEQ ID
N0:44, SEQ ID NO:45, SEQ ID N0:46, SEQ ID N0:47, the amino acid CPq77Pn~.Pq discrosed ~in Table 2 (i.e., SEQ ID N0:53, SEQ ID N0:55, SEQ ID NO:57, SEQ ID N0:59, SEQ ID N0:61, SEQ
ID N0:63, SEQ ID N0:65, SEQ ID N0:67, SEQ ID N0:69, SEQ ID
NO:71, SEQ ID N0:73, SEQ ID N0:75, SEQ ID N0:77, SEQ ID

WO 96111706 PC~/US95~1444Z

N0:79), SEQ ID N0:81, SEQ ID N0:83, SEQ ID N0:85, SEQ ID
N0:87, SEQ ID NO:89, SEQ ID N0:91, SEQ ID N0:93, SEQ ID
N0:95, SEQ ID N0:97, SEQ ID N0:99, SEQ ID N0:101, SEQ ID
N0:103, SEQ ID N0:105, SEQ ID N0:107, SEQ ID N0:109, and/or 5 SEQ ID N0 : 111), but also allelic variants of any of those nucleic acid s~7Pnr~c. (It should be noted that since nucleic acid sequencing f~rhnnlo~y is not entirely error-free, all S~ Pnr~c ~e~Lels~llLed herein are at best apparent ( i . e., deduced) nucleic acid or amino acid sequences . ) A preferred flea aminopeptidase gene ir,cludes nucleic acid sequence SEQ ID N0:50, which encodes an aminopeptidase protein including SEQ ID N0:51. Another preferred flea Am;nop~rtidase gene ;nrl~ q nucleic acid C~ nr~ SEQ ID
NO:112, which encodes an Aminnp~rtidase protein including 15 SEQ ID N0:113. Additional preferred Am;nop~rtidase genes include allelic variants of SEQ ID N0:50 and of SEQ ID
N0: 112 .
A preferred flea serine protease protein of the present invention is encodea by a nucleic acid molecule 20 that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules:
nfSPl, nfSP2, nfSP3, nfSP4, nfSP5, nfSP6, nfSP7, nfSP8, nfSP9, nfSP10, nfSPll, nfSP12, nfSP13, nfSP14, nfSP15, nfSP16, nfSP17, nfSP18, nfSP19 and nfSP20. As used herein, 25 each of these nucleic acid molecules ~ Lt:s~llL 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 in the Examples).

Wo 96/11706 PCTIUS95114442 Nucleic acid molecules that contain partial coding regions or other parts of the ~OLL~ 1J~ ;n~ gene are denoted by names that include the size of those nucleic acid molecules (e.g., nfSP4156) . Nucleic acid molecules containing apparent 5 full length coding regions for which the size is known also are denoted by names that include the size of those nucleic acid molecules (e.g., nfSP467z). The production, and at least partial nucleic acid sequence, of such nucleic acid molecules is disclosed in the Examples.
Particularly preferred 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: nfSP46n, nfSPl1s6~ nfSP2168, nfSP3177, nfSP41s6, nfSP5159, nfSP6168, nfSP7159, nfsp8186~
nfSP9168, nfSPl0120, and nfSPl1162 as well as other specific nucleic acid molecules disclosed in the Examples section, such as, but not limited to, nfSPl779, nfSP2944~ nfSP3177, nfSP4672, nfSP5l57, nfSP5218, nfSP6932, nfSP7gg4, nfSP8zss~
nfSP9266, nfSPl0378, nfSP112s2, nfSPl2144, nfSPl222s~ nfSPl3sso~
nfSPl4213, nfSPl5252, nfSPl6168, nfSPl8s34, nfSPl93s9, and/or nfSP20g41- Even more prefer~red serine protease proteins include the following amino acid Se~l~nr~-q: SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID
N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID N0:17, SEQ ID N0:19, SEQ ID N0:21, SEQ ID N0:23, SEQ ID N0:25, SEQ
ID N0:27, SEQ ID N0:29, SEQ ID N0:31, SEQ ID N0:33, SEQ ID
N0:35, SEQ ID N0:37, SEQ ID N0:39, SEQ ID N0:40, SEQ ID
N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID

wo 96111706 PCTIUS95/14442 No:45, SEQ ID NO:46, SEQ ID N0:47, and/or the amino acid sequences presented in Table 2 (i.e., SEQ ID N0:53, SEQ ID
N0:55, SEQ ID NO:57, SEQ ID N0:59, SEQ ID N0:61, SEQ ID
N0: 63, SEQ ID N0: 65, SEQ ID N0: 67, SEQ ID N0: 69, SEQ ID
N0:71, SEQ ID N0:73, SEQ ID N0:75, SEQ ID N0:77, and/or SEQ
ID N0:79), as well as SEQ ID N0:81, SEQ ID N0:83, SEQ ID
N0:85, SEQ ID N0:87, SEQ ID N0:89, SEQ ID N0:91, SEQ ID
N0:93, SEQ ID N0:95, SEQ ID N0:97, SEQ ID N0:99, SEQ ID
N0:101, SEQ ID N0:103, SEQ ID N0:105, SEQ ID N0:107, SEQ ID
N0:109, and/or SEQ ID N0:111. Additional particularly preferred serine protease proteins are encoded by allelic variants of nucleic acid molecules n~nro~; n~ proteins that include the cited amino acid 5.o~lPnn~c, 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 amino acid ~sequences as cited herein .
A preferred flea Am;nnp~rtidase protein of the present invention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfAP4s3 and/or~nfAP1s8~ the production of which are described in the F~ . Even more preferred is an Am; nnp~ortidase that includes amino acid sequence SEQ ID N0:51 and/or SEQ ID N0:113 or an Am;nnp~rtidase encoded by an allelic variant of a nucleic acid molecule that inn~ c SEQ ID N0:50 and/or SEQ ID
N0:112. Also referred are flea Am;nnr~rtidase proteins P

Wo 96/11706 PCTIUS95/14442 including regions that have at least about 50%, preferably at least about 759~, and more preferably at least about 90%
identity with SEQ ID NO:51 and/or SEQ ID NO:113.
One ~rho~l;r L of the present invention is an isolated 5 protein having proteolytic activity that is substantially inhibited by a serine protease inhibitor. Such inhibition can be measured by techniques known to those skilled in the art.= To be substantially inhibited means that at least half of the proteolytic activity of the protease protein is 10 inhibited by a serine protease inhibitor. Preferably at least about 70 percent, and even more preferably at least about 90 percent of the proteolytic activity of the protease protein is inhibited by a serine protease inhib itor .
An isolated protein of~ the present invention can be produced in a variety of ways, including recovering such a protein from a flea midgut and producing such a protein recombinantly. In one: ': ';r ~, a flea midgut protease can be recovered by methods heretofore disclosed for 20 obtaining a soluble flea midgut preparation. A flea midgut protease protein can be further purified from a disrupted flea midgut by a number of terhn; qn~q known to those skilled in the art, including, but not limited to, affinity chromatography, ion exchange ~ toyl~pl,y, filtration, 25 electrophoresis (e.g., standard, capillary and flow-through electrophoresis), hydLuuhObic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, WO 96/11706 PCT/lTS95/14442 chromatofocusing and differential solubilization. In one embodiment, a flea midgut protease is purified using protease inhibitor affinity chromatography, an example of which is disclosed in the r 1 ~c section.
Another ';- L of the present invention is a method to produce an isolated protein 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 F~nco~in~ a protein of the present invention to produce the protein and (b) recovering the protein therefrom. Details on producing recombinant cells and culturing thereof are presented below. The phrase "recovering the protein" refers simply to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purif ication . Proteins of the present invention can be purified using a variety of standard protein purification techniques, as heretofore disclosed.
Isolated proteins of the present invention are preferably retrieved in ~ul~:.Lcl~lLially pure" form. As used herein, "substantially pure" refers to a purity that allows for the effective use of the protein as a vaccine. A
. .
vaccine for animals, for example, should exhibit no substantial toxicity and should be capable of stimulating the production of ant;ho~ in a vaccinated animal.
Another: ~ ; ~ of the present invention is an isolated nucleic acid molecule capable of hybridizing under stringen= con~itions with a gene l~nl~oll I nq a flea protease = =

Wo 96/11706 Pcr~7S95/14442 present in a flea midgut. Such a nucleic acid molecule is also referred to herein as a flea protease nucleic acid molecule. Particularly preferred is an isolated nucleic acid molecule that hybridizes under stringent conditions 5 with a flea serine protease gene or with a flea aminopeptidase gene. '77he characteristics of such genes are disclosed herein. In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu lO ~i.e., that has been subject to human r-n;L~l7l~7-tion). As such, "isolated" does not reflect the extent to which the nucleic acid molecule has been purif ied . An isolated nucleic acid molecule can include DNA, ~NA, or derivatives of either DNA or 7LWA.
As stated above, a flea protease gene includes all nucleic acid S~l77~n-c,5 related to a natural flea protease gene such as regulatory regions that control production of a flea protease protein encoded by that gene (such as, but not limited to, transcription, translation or post-20 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 25 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 Wo 96/11706 PCr/[Jsss114442 stringent hybridization conditions with a ~.ULL~ i i n~
natural gene. Flea protease nucleic acid molecules can also include a nucleic acid molecule i nro~;n~ a hybrid protein, a fusion protein, a multivalent protein or a 5 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. As used lO herein, 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. For example, at least a portion of a nucleic acid C~ nr.~ ~ as used herein, is an amount of a nucleic acid c.,qll,.nr~ capable of 15 forming a stable hybrid with the corr~cpr~n~l;n~ gene under stringent hybridization conditions.
An isolated nucleic acid molecule of the present invention can also be produced using r~- ; n~nt DNA
technology (e.g., polymerase chain reaction (PCR) 20 amplification, cloning) or rh~m; C:ll synthesis . Isolated flea protease nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid ~ rlll ~c in which nucleotides have been 25 inserted, deleted, substituted, and/or inverted in such a manner that such 'ifir~tions do not :,ub=L~11Lially interfere with the nucleic acid molecule' s ability to encode a flea protease protein of the present invention or ,, ~

Wo 96111706 PCr/USs5/14442 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 5 in the art (see, for example, Sambrook et al., ibid. ) . For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis t~rhn;q~lPc and recombinant DNA
techniques, such as site-directed mutagenesis, rhpTn;t~
10 treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid rL _ ~s, polymerase~chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sPqltpn~:e, synthesis of 15 oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the 20 nucleic acid (e.g., the ability of a homologue to elicit an immune response against a flea protease and/or to have proteolytic actiYity) and/or by hybridization with isolated flea protease nucleic acids under stringent conditions.
An isolated flea protease nucleic acid molecule of the 25 present invention can include a nucleic acid sP~,onl P that encodes at least ohe flea protease protein of the present invention, , lPq of guch proteins being ~liqcloqPcl herein. Although the phrase "nuFleic acid molecule"

wo 96/11706 ~ ~ 4442 primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid s~qu~n~!e~ primarily refers to the sequence Qf nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, ~sr~c;~lly with 5 respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of ~~nco~;n~ an flea protease protein .
One embodiment of the present invention is a flea protease nucleic acid molecule of the present invention 10 that is capable of hybridizing under stringent conditions to a nucleic acid that encodes at least a portion of a flea protease or a homologue thereof. Preferred is a flea protease nucleic acid leclllP that ;nrll~ a nucleic acid sequence having at least about 65 percent, preferably at 15 least about 75 percent, more preferably at least about 85 percent, and even more preferably at least about 95 percent homology with the l_ULL. ~LJ ~ ~l;n~ region(s) of the nucleic acid sequence r~n~o~l;n~ at least a portion of a flea protease protein. Particularly preferred is a flea 20 protease nucleic acid molecule capable of ~n~orl;n~ 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 25 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 wo 96111706 Pcrrusg5rl4442 least one of the following nucleic acid molecules: nfSPl, nfSP2, nfSP3, nfSP4, nfSP5, nfSP6, nfSP7, nfSP8, nfSP9, nfSP10, nfSPll, nfSP12, nfSP13, nfSP14, nfSP15, nfSP16, nfSP17, nfSP18, nfSPl9 and/or nfSP20. More preferred is a 5 nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfSP467z, nfSP1156, nfSP2168' nfSP3177' nfSP41s6, nfSP51s9, nfSP616a, nfSP71sg~ nfSP8186~ nfSP9-68~
nfSP10120, and/or nfSP11162, as Well as other sr~rif;- nucleic 10 acid molecules disclosed in the Examples section. Even more preferred are nucleic acid molecules that include nfSPl, nfSP2, nfSP3, nfSP4, nfSP5, nfSP6, nfSP7, nfSP8, nfSP9, nfSP10, nfSPll, nfSP12, nfSP13, nfSP14, nfSP15, nfSP16, nfSP17, nfSP18, nfSPl9, and/or nfSP20 and eYen more particularly, nfSP467z, nfSPl156~ nfSP2168, nfSP3177, nfSP41s6' nfSP51s9, nfSP6168, nfSP71sg, nfSP81e6, nfSP9168, nfSP101zO, and/or nfSP1116z, as well as other specific nucleic acid molecules disclosed in the r ~1 ~oq section.
Particularly preferred flea serine protease nucleic acid molecules include at least one of the following sequences: SEQ ID N0:16, SEQ ID N0:18, SEQ ID N0:20, SEQ
ID NO:22, SEQ ID N0:24, SEQ ID N0:26, SEQ ID N0:28, SEQ ID
NO:30, SEQ ID N0:32, SEQ ID N0:34, SEQ ID N0:36, SEQ ID
N0:38, and/or nucleic acid S,,qllc,nl-~q disclosed in Table 2 (i.e., SEQ ID N0:52, SEQ ID N0:54, SEQ ID NO:56, SEQ ID
N0:58, SEQ ID N0:60, SEQ ID N0:62, SEQ ID N0:64, SEQ ID
N0:66, SEQ ID N0:68, SEQ ID N0:70, SEQ ID N0:72, SEQ ID
N0:74, SEQ ID N0:76, SEQ ID N0:78), as well as SEQ ID

Wo 96/11706 Pcrlu~95/14442 N0:80, SEQ ID N0:82, SEQ ID N0:84, SEQ ID N0:86, SEQ ID
N0:88, SEQ ID N0:90, SEQ ID N0:92, SEQ ID N0:94, SEQ ID
N0:96, SEQ ID N0:98, SEQ ID N0:100, SEQ ID N0:102, SEQ ID
N0:104, SEQ ID N0:106, SEQ ID N0:108, and/or SEQ ID N0:110.
5 Also preferred are allelic variants of such nucleic acid molecules .
A preferred flea ~m; nnp ~ptidase nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with 10 nfAP4s3 and/or nfAP1s8c More preferred is an ~;nnp-~rtidase nucleic acid molecule that includes nfAP4s3, nfAP930 nfAP73z~
and/or nfAPls80. Particularly preferred is a nucleic acid molecule that includes nucleic acid sequence SEQ ID N0: 50, SEQ ID N0:112, or an allelic variant thereof.
Knowing a nucleic acid molecule of a flea protease protein of the present invention allows one skilled in 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., 20 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 amino acid sequF~n~ of a flea protease protein of the present invention allows one 25 skillea in the art to clone nucleic acid s~ c ~nr~o~; n~
such a flea protease protein. In addition, a desired flea protease nucleic acid --lpl~ can be obtained in a variety of ways i n~ ; n~ screening appropriate expression Wo 96/11706 PCrluss~/l4442 libraries with ant;hs~;~c which bind to flea protease proteins of the present invention; traditional cloning t~rhn;q~ c using ol irJrlnllrlpotide probes of the present invention to screen d~L~Liate libraries or DNA; and PCR
5 amplification of appropriate libraries, or RNA or DNA using oligonucleotide primers of the present invention (genomic and/or cDNA libraries can be used). To isolate flea protease nucleic acid molecules, preferred cDNA libraries include cDNA libraries made from unfed whole fleas, fed 10 whole fleas, fed flea midguts, unfed flea midguts, and flea salivary glands. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., i~id. The Examples section includes examples of the isolation of cDNA
sequences ~nro~;n~ flea protease proteins of the present 15 invention.
The present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the 20 present invention that encode at least a portion of a flea protease protein. Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimal size of such r~ lrleotide5 is the size required to form a stable hybrid between a given oligonucleotide and 25 the compl I_dly sequence on another nucleic acid molecule of the present invention. Minimal size characteristics are disclosed herein. The size of the oligonucleotide must also be sufficient for the use of the ol ;~mllrleotide in wo 96111706 PcrluS9S/1444Z

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 5 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.
10 The present invention, therefore, includes such oligonucleotides and methods to interfere with the production of flea protease proteins by use of one or more of such technologies.
The present invention also ; n~ Af~q a recombinant 15 vector, which includes a flea protease nucleic acid molecule of the present invention inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid c~ n~-e-q that are not 20 naturally founa 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 25 manipulating of flea protease nucleic acid molecules of the present invention. One type of recombinant vector, herein =

referred to as a L~_ ' in~nt molecule and dèscribed in more detail below, can be used in the expression of nucleic acid WO 96/11706 PCI/U59sll4442 molecules of the present invention. Preferred recombinant vectors are capable of replicating in the transformed cell.
Pref erred nucleic acid molecules to include in recombinant vectors of the present invention are disclosed herein.
==As heretofore disclosed, one ' ~ of the present invention is a method to produce a flea Frotease protein of the present invention by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. A
preferred cell to culture is a recombinant cell that is capable of expressing the flea protease protein, the recombinant cell being ~Ludu~;~=d by transforming a host cell with one or more nucleic acid molecules of the present invention. ILa~ oLIIlation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid ~ molecule can be inserted into the cell.
Transformation techniques include, but are not limited to, transfection, elecl-Lu~oLation~ microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachL, - 1 or can integrate into one or more sites within a U11L. ~~ of the transformed (i.e., ro~ ;n~nt) cell in such a manner that their - ability to be tuL~JLe:ssed 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 illLLvdu~d 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 5 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 10 plant cells. Preferred host cells include bacterial, yeast, insect and li;~n cells, with bacterial (e.g., E.
coli ) and insect (e . g., Spodoptera~ cells being particularly preferred.
A recombinant cell is preferably produced by 15 transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively iinked to an expression vector containing one or more transcription control sequences. The phrase operatively linked refers to 20 insertion of a nucleic acid molecule into an eYpression vector in a manner such that the molecule is able to be eYpressed when LL~ r~ ~' into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting 25 ~Yrr~qcion of a crer;f;~-l nucleic acid molecule.
Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotlc, and are typically Wo 96/11706 PClrUss5/14442 viruses or plasmids. Expression vectors of the present invention include any vectors that function ( i . e ., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, insect, animal, 5 and/or~plant cells. As such, nucleic acid molecules of the present invention can be operatively linked to expression vectors containing regulatory S~ nr~-: such as promoters, operators, repressors, ~nh~nr~rs, termination sequences, origins of replication, and other regulatory sequences that 10 are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. As used herein, a transcription control sequence includes a sequence which is capable of controlling the initiation, elongation, and termination of 15 transcription. Par~; r~ r1y important transcription control sequences are those which control transcription initiation, such as promoter, rnh~nr~r, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that 20 can function in at least one of the recombinant cells of the present invention. A variety of such L~ s~iption control sequences are known to those skilled in the art.
Preferred transcription control seouences include those which function in bacterial, yeast, helminth, insect and 25 ~ n cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (1) (such as AP~ and APR and fusions that include such promoters), bacteriophage T7 , T71ac, bacteriophage T3 , CA 02202622 1997-04-14 = ~
Wo 96/11706 PCIIUS95114442 ~ 45 ~
bacteriophage SP6, bacteriophage SPOl, metalloth;~n~;n, alpha mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus ~ J~ ; c promoters), baculovirus, ~{eliothis zea ~insect virus, 5 vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus 40, retrovirus actin, retroviral long t~rm;nAl repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control R~qU~nr.Oq as well as other sequences capable of controlling gene expression in lO prokaryotic or eukaryotic cells. Additional suitable transcription control s~ n~q include tissue-specific promoters and ~nhAnr~R as well as ly _'-k; n~-inducible promoters (e.g., promoters ;n~l~r;hle by interferons or interleukins). Transcription control sequences of the 15 present invention can aIso include naturally occurring transcription control q~qu ~n~ ~q naturally associated with a DNA sequence ~nr~o~; n~ a f lea protease protein .
Expression vectors of the present invention may also contain secretory signals (i.e., signal segment nucleic 20 acid sequences) to enabie an expressed flea protease protein to be secreted from the cell that pL~,du~es the protein. Suitable signal s-- Ls include a flea protease protein signal segment or any heterologous signal segment capable of directing the secretion of a flea protease 25 protein, including fusion proteins, of the present invention. Preferred signal segments include, but are not limited to, flea protease, tissue plAF~;n~ activator (t-PA), interferon, interleukin, growth hormone, Wo 96111706 PCT/US95/14442 histocompatibility and viral envelope glycoprotein signal segments .
Expression vectors of the present invention may also contain fusion s ~ nr~C which lead to the expression of 5 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 protein encoded by the nucleic 10 acid molecule. Furth, l'~:, a fusion segment can function as a tool to simplify purification of a flea protease protein, such as to enable purif ication of the resultant fusion protein using affinity chromatography. A suitable fusion segment can be a domain of any size that has the 15 desired function (e.g., increased stability and/or purification tool). It is within the scope of the present invention to use one or more fusion s~, L~. Fusion segments can be joined to amino and/or carboxyl termini of a flea protease protein. r~ink~g~c between fusion segments 20 and flea protease proteins can be constructed to be susceptible to cleavage to enable straight-forward Le:CUV~l~
of the flea protease proteins. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid sequence that 25 encodes a protein including the fusion segment attached to either the carboxyl and/or amino t~rm;nz~l end of a flea protease protein.
A recombinant molecule of the present invention is a molecule that can include at least one of any nucleic acid molecule heretofore described operatively linked to at least one of any transcription control sequence capable of effectively regulating expression of the nucleic acid 5 m~olecule (s) in the cell to be transformed. A preferred recombinant molecule ; ncl ~ c 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 and/or aminopeptidase proteins, 10 being more preferrea. Similarly, a preferred r~ ;n~nt 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 and/or ;lminnp~rtidase proteins, being more 15 pref erred .
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 r-n;r--7 ~ting, for example, the number of copies of the nucleic acid molecules 20 within a host cell, the ~ff;c~ nf~y 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 t~rhn;q1l~oq useful for increasing the expression 25 of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy num=ber ~ mjrlq~ integration of the nucleic acid molecules into one or more host cell Wo 96~11706 Pcr/u~95114442 ChL - -, addition of vector stability ~Pql-PnrPs to p1 A~ , substitutions or modifications of transcription control signals (e.g., promoters, operators, Pnh;~n~ Prs), substitutions - or modifications of translational control 5 signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of ~sequPn~P~ that destabilize transcripts, and use of control signals that temporally lO separate recombinant cell growth from recombinant protein production during f, - Ldtion. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing the resultant protein.
l~ In accordance with the present invention, recombinant cells can be used to produce flea protease proteins of the present invention by culturing such cells under conditions ef f ective to produce such a protein, and recovering the protein. Effective conditions to produce a protein 20 include, but are not limited to, c-~-~Lu~Liate media, bioreactor, t~ ~LUL~:, pH and oxygen conditions that permit protein produc~ion. An cly~Lv~Liate, or effective, medium refers to any medium in which a cell of the present invention, when cultured, is capable of producing a flea 25 protease protein. Such a medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as d~yLv~Liate salts, minerals, metals and other nutrients, such as vitamins. The medium Wo 96/11706 PCr/Uss5/14442 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 bat~, cell recycle, and continuous fermentors. Culturing can also be conducted in shake flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and oxygen content c~ U~liate for the recombinant cell. Such culturing conditions are well within the expertise of one of ordinary skill in the art.
D~p~n-~;ng on the vector and host system used for production, resultant flea protease proteins may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E.
coli; or be retained on the outer surface of a cell or viral - ~ne. Nethoas to purify such proteins are heretofore disclosed.
The present invention also includes isolated anti-flea protease ant;ho~;eC 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 in a flea midgut, ;nrlllrl;n~ female and male fed midguts as well as female and male unfed midguts. An anti-Elea protease antibody preferably binds to the protease in such a way as to reduce the proteolytic activity of that '' = ~

~o 96/11706 pcrNs9sll4442 protease .
Isolated ant;ho~ q are ant;hQ~i~oq that have been removed from their natural milieu. The term "isolated"
does not refer to the state of purity of such ant;ho~ q.
5 As such, isolated ant;ho~ q can include anti-sera containing such antibodies, or ant; ho~ q that have been purified to varying degrees. As used herein, the term "selectively binds to" refers to the ability of such antibodies to preferentially bind to the protease against lO which the antibody was raised ( i . e ., to be able to distinguish that protease from unrelated Ls in a mixture. ) . Binding affinities typically range from about 103 M-l to about lO12 M-1. Binding can be measured using a variety of methods known to those skilled in the art 15 including immunoblot assays,; cprecipitation assays, raaio; o; csays, enzyme ; --qq~ys (e.g., ELISA), immunof luorescent antibody assays and immunoelectron microscopy see, for example, Samhrook et al., i~id.
Antibodies of the present invention can be either 20 polyclonal or monoclonal ant;ho~ q. Antibodies of the present invention include functional equivalents such as antibody fragments and genetically-~ng;n~Pred ant;ho~; ~q, including single chain ant;horl;Fc, that are capable of selectively binding to at least one of the epitopes of the 25 protein used to obtain the ant;ho~ q. Antibodies of the present invention also include chimeric antibodies that can bind to more than one epitope. Preferred ~nt;hotl; oq are raised in response to proteins that are encoded, at least wo 96111706 PCr~Ss5/14442 in part, by a flea protease nucleic acid molecule of the present invention.
Anti-flea antibodies of the present invention include antibodies raised in an animal administered a flea protease 5 vaccine of the present invention that exert their effect when fleas feed from the vaccinated animal's blood containing such antibodies. Anti-flea antiho~;r~c of the present invention also include ant; ho~l i .,c raised in an animal against one or more flea protease proteins, or 10 soluble flea midgut preparations, of the present invention that are then recovered from the animal using t~ hn; q~ c known to those skilled in the art. Yet additional antibodies of the present invention are produced recombinantly using t~rhn;qllc.q as heretofore disclosed for 15 flea protease proteins of the present invention.
Ant i ho~l i PS produced against def ined proteins can be advantageous because such antibodies are not ~ub~Lal~Lially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay or 20 side effects if used in a therapeutic composition.
Anti-flea protease antihr~ c of the present invention have a variety of uses that are within the scope of the present invention . For example, such ant; horl; PS can be used in a composition of the present invention to passively 25 ; ; 7.e an animal in order to protect the animal from flea infestation. Anti-flea ant;ho~l;es can also be used as tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of Wo 96/11706 PCrlUSs~ll4442 proteins and other contaminants. Furth~ t:, antibodies of the present invention can be used to target cytotoxic agents to fleas in order to kill fleas. Targeting can be 1 ;chec~ by conjugatinq (i.e., stably joining) such 5 = antibodies to the cytotoxic agents using t~hn;qnc-c known to those skilled in 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, 10 a flea metalloprotease, a flea aspartic acid protease and/or a flea cysteine protease. More preferred anti-flea protease antiho~ c include anti-flea serine protease antibodies, anti-flea metalloprotease antibodies, and anti-flea aminopeptidase antibodies. Particularly preferred are 15 anti-flea serine protease ant;ho~ c and anti-flea aminopeptidase ant i ho~ i ~c, including those raised against flea serine protease proteins or flea ~minr~p~rtidase protein6 of the present invention.
The present invention also includes the use of 20 protease inhibitors that reduce proteolytic activity Qf flea proteases to reduce flea infestation of animals and the -iuLLuu~lding environment. As used herein, protease . .
inhibitors are ~ __ that interact directly with a protease thereby inhibiting that protease's activity, 25 usually by binding to or otherwise interacting with the protease's active site. Protease inhibitors are usually relatively small . _ '- and as such differ from anti-protease antibodies that interact with the active site of a protease.
Protease inhibitors can be used directly as _ _ ~ c in compositions of the present invention to treat animals as long as such ~ 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 invention. For example, the inventors have shown herein the ~,~ - ;n~n.e of serine proteases in flea midguts, particularly in soluble flea midgut preparations, using 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 an~;ho~ and other inhibitors of serine protease synthesis and activity that can be tolerated by the animal. ~hat other proteases are also present in flea midguts according to the present invention also auyyl:~L5 targeting such proteases. Methods to use protease inhibitors are known to those skilled in the art; examples of such methods are disclosed herein.
In one ~ , a protease inhibitor that can be used in 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 __ '~ by testing the efficacy of one or more protease inhibitors (i) in vitro for their ability to inhibit flea protease activity andjor (ii) in a flea feeding assay for their ability to reduce the survival and/or fecundity of fleas by adding the inhibltors to the ' =

Wo 96tll706 pcrNs9sll4442 blood meal of a flea being maintained, for example, in a feeding system, such as that described by Wade et al., 1988, J.~ed Entomol. 25, l86-lgO; and (b) testing the ef f icacy of the candidate inhibitor ~ u11ds in animals 5 infested with fleas. Although one does not need both in vitro assay data and flea feeding assay data to ~ t~rlr;n-~which candidate ~ ;1S to administer to animals, evaluation of both sets of data is preferred since data from neither of the assays nr~rr~cc~rily predicts data to be lO obtained from the other assay. For example, candidate -c identified using the in vitro assay may work "in the test tube" but may not work in vivo for a number of reasons, including the presence of interfering r~nr~ntS
in the blood meal that inhibit the activity of such _ ~1.as ; e . g ., although a~rotinin can inhibit at least some flea serine proteases in vitro, aprotinin does not work well in the presence of serum proteins, such as are found in the blood. Furth~ ~:, candidate inhibitor -c identified by the flea feeding assays can include 20 not only desired __ '- but also, _ 'c that reduce the viability and/or fecundity of fleas due to general toxicity (e.g., affecting the mito~ hc nAria of fleas) .
In another ~ -'; L, protease inhibitors are used in the purification of corr~cpon~;n~ proteases by, for 25 example, affinity chromatography, in which, a protease inhibitor is incubated with a mixture containing a desired protease under conditions that the inhibitor f orms a complex with the protease. The protease can then be Wo 96/11706 PcTn~ss5114442 . . .
recovered from the complex. The protease inhibitor can be attached to a solid support and/or be 1 ~h~ol 1 ed with, for example, a radioactive, flu(,Lcscel.~, 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, metalloprotease inhibitors, aspartic acid protease inhibitors, cysteine protease inhibitors, and/or aminopeptidase inhibitors. Preferred protease inhibitors include serine protease inhibitors, metalloprotease inhibitors and i~;nnp~rtidase inhibitors, particularly those that are broad spectrum inhibitors. More preferred are broad spectrum serine 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, beStatin, chloromethyl ketones TLCK (N~-p-tosyl-L-lysine chl-,L yl ketone) and TPCK (N-tosyl-L-phenyl ~l ~n; n~ chloromethyl ketone), chymostatin, cystatin, 3'4-dichloroicocour~rin, E-64 (trans-epoxysuccinyl-L-leucylamido- (4-guanidino) butane), EDTA (ethyl~n~ ~;n~tetraacetic acid), leupeptin, methyl ketones having a variety of leaving groups, n~ d L-l euc ineth iol , pepstatin , 1 ,1 0 - orthophenanthro l i ne , rhnsFhnramidonl soybean trypsin/~l~y LL~ysin inhibitor and soybean trypsin inhibitor. FL~re:LLt:d protease inhibitors for use in =the present invention include AEBSF, bestatin, E-64 leupeptin, pepstatin, 1, lO-orth~ Lhroline, rhosrhAra~idonl TLCK and TPCK, with AEBSF ~a broad spectrum Wo 96111706 PCrlUS95114442 serine protease inhibitor), bestatin (an inhibitor of leucine ~m;ntp~rtidase) and 1,10-orthophenanthroline (a broad spectrum metalloprotease inhibitor) being particularly preferred.
~ Protease inhibitors can be produced using methods known to those skilled in the art. ~=Protein- or peptide-based protease inhibitors, such as cystatin or small peptides comprising a protease ~U~aLL~l~e, can be produced recombinantly and modified as n~r~ccAry.
10The present invention also includes the use of proteolytically active flea protease proteins of the present invention to identify additional protease inhibitors, and preferably protease inhibitor ~ _ that can be included in a composition of the present 15 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 protein with a putative (i.e., candidate) inhibitory ~ ' under conditions in which, in the 20 absence of the __ ', the protein has proteolytic activity, and (b) det~rm;n;n~ if the putative inhibitory ~ulld inhibits the proteolytic activity of the protein.
Putative inhibitory _ ' to screen include organic molecules, ant;ho~ s (including functional equivalents 25 thereof) and :.u~,LL~te analogs. Nethods to determine protease activity are known to those skilled in the art, as heretofore disclosed. Particularly preferred for use in identifying inhibitors are flea serine protease proteins Wo 96111706 PCr/USs5/14442 and flea aminopeptidase proteins of the present invention.
The present invention also ; nf~ DC a test kit to identify a compound capable of inhibiting flea protease activity. Such a test kit includes an isolated flea 5 protease protein having proteolytic activity and a means for detDrm;n;n~ the extent of inhibition of proteolytic activity in the presence of (i.e., effected by) a putative inhibitory The present invention also ; nf~ q inhibitors lO isolated by such a method, and/or test kit, and their use to inhibit any flea protease that is susceptible to such an inhib itor It is to be appreciated that the present invention also includes m;r ~ OpDq of ~ _ 'q of the present 15 invention that can be used in accordance with methods as la,;qrlnqDfl for _ _ of the present invention. As used herein, a mimetope of a proteinaceous ~ .d of the present invention (e.g., a flea protease protein, an anti-flea protease antibody, a protD;nAcDollq inhibitor of 20 protease activity or synthesis) refers to any _ __ ' that is able to mimic the activity of that protD;nAl eollq ~ _ u..d, often because the mi ~ ,~_ has a ~LLU~;LULC: that mimics the protD;nAr~eollq . ~ For example, a mimetope of a flea protease protein is a ~ _ ' that has an 25 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 '; f ~ ~D~ to decrea~e their susceptibility to degradation; anti-Wo 96111706 PCT/US95/14442 idiotypic and/or eatalytic antibodies, or 1L ~s thereof; nu~ in~eeous ; ,_-~ic portions of an isolated protein (e.g., caL~olly~Lte structures); and synthetic or natural organic molecules, including nucleic 5 acids. Such mimetopes can be ~l"c; ~n~cl using computer-generated structures of proteins of the present invention.
~limetopes ean also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity 10 chromatography techniques using the corr~qrnn-l;n~ binding partner .
The present invention includes therapeutic compositions, also referred to herein as compositions, that include a ( i . e., at least one) _ _ _ ' of the present 15 invention. Preferred ~ 'q to include in a composition Gf the present invention include flea protease vaccines, anti-flea protease ant;ho~;eq and/or protease inhibitors as disclosed herein. Sueh a therapeutie eomposition ean proteet an animal from flea infestation by redueing flea 20 protease aetivity, thereby redueing flea burden on the animal and in the environment of the animal.
Partieularly preferred therapeutie eompositions of the present invention inelude at least one of the following emnrolln~q: an isolated flea serine protease protein or a 25 mimetope thereof; an isolated flea serine protease nueleie aeid moleeule that hybridizes under stringent hybridization conditions with a flea serine protease gene; an isolated antibody that selectively binds to a flea serine protease Wo 96111706 PCTIUS95/14442 protein; an inhibitor of ~ flea serine protease activity identified by its ability to inhibit flea serine protease activity; an isolated flea aminopeptidase protein or a mimetope thereof; an isolated flea aminopeptidase nucleic 5 acid molecule that hybridizes under stringent hybridization conditions with a flea Amin-~p~rtidase gene; an isolated antibody that selectively binds to a flea ~;nrpPrtidase protein; and an inhibitor of flea aminopeptidase activity identified by its ability to inhibit flea aminopeptidase 10 activity.
Another ' -'; L of the present invention is a therapeutic composition that includes a f irst ~ that reduces flea protease activity and a second ~ u--d that reduces flea burden by a method other than by reducing flea 15 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 efi~ectively reducinq flea protease activity in the midgut, Pnh~nrps the activity of 20 the second, '. While not being bound by theory, it is believed that a number of anti-flea treatments, particularly those that are protp;n~rpsllq~ are not very effective because they are degraded in the flea midgut.
The present invention permits the effective use of such 25 anti-flea Llt!ai ~s by reducing proteolytic degradation of such treatments by the flea midgut.
Preferred first _ ~ to include in such a composition include flea protease vaccines, anti-flea WO 96/11706 PCr/US95/14442 protease antibodies and/or protease inhibitors as disclosed herein .
Suitable second ~ include any anti-flea agent(s), including, but not limited to, prot~inAo~ us compounds, insecticides and flea collars. Preferred second ~ullds are prot~;nAc~ lS ~ ~- that effect active ; 7~tion (e.g., antigen vaccines), passive; ; ~ation (e.g., antibodies), or that otherwise inhibit a flea activity that when inhibited can reduce flea burden on and around an animal. Examples of second ~_ 'q include a compound that inhibits binding between a flea membrane protein and its ligand (e.g., a ~ ~ that inhibits flea A~Pase activity or a ~ ' that inhibits binding of a peptide or steroid hormone to its receptor), a ~ _ ' that inhibits hormone ( including peptide or steroid hl ~-) synthesis, a '_ ' that inhibits vitellogenesis ( including production of vitellin and transport and maturation thereof into a maj or egg yolk protein), a c ' that inhibits fat body function, a _ ' that inhibits flea muscle action, a ~_ ulld that inhibits the flea nervous system, a ' that inhibits the flea immune system and/or a ~ ,d that inhibits flea f eeding .
Compositions of the present invention can also include other components such as a rhAr--c~lltically acceptable excipient, an adjuvant, and/or a carrier. For example, compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate.

wo 96/11706 PCT/US95114442 Examples of such f~Y- ;r;.~ntS include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically ,~71Anr ed salt solutions.
Nonaqueous vehicles, such as fixea oils, sesame oil, ethyl oIeate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity ~nh;7nr;n~ agents, such as sodium ca~ ,~y ~hylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Exa7~ples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
In one: ; L of the present invention, the composition can also include an; ~ _ Lentiator, such as an adjuvant or a carrier. Adjuvants are typically substances that generally e7 hance the immune response of an animal to a sp~cif;~ antigen. Suitable adjuvants include, but are not li7nited to, Freund's adjuvant; other bacterial cell wall L~; ;7117m;n177~7-based salts; calcium-based salts, silica; polynucleotides; toxoids; serum proteins;
viral coat proteins; other bacterial-derived preparations;

Wo 96/11706 PCTIUS95/14442 gamma interferQn; block copolymer adjuvants, such as Hunter's Titermax aajuvant (VaxcelD', Inc. Norcross, GA);
Ribi adjuvants (available from Ribi ImmunoChem Research, Inc., Hamilton, MT); and saponins and their derivatives, 5 such as Quil A (available from Superfos R30c~ntnr A/S, Denmark). Carriers are typically _ __ ' 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 lO implants, liposomes, bacteria, viruses, oils, esters, and glycols .
One ; L of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the~ present invention into an 15 animal. As used herein a controlled release formulation comprises a composition of ~ the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, hir - ~ tible polymers, other polymeric matrices, capsules, 20 micron~r~lllPS~ microparticles, bolus preparations, osmotic pumps, diffusion devices, 1 ;rnc, -, 1 ;rncrh~res~ and trAnc~ l delivery systems. Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in 25 situ. Preferred controlled release formulations are biodegradable (i.e., bioerodible).
A preferred controlled release formulation of the present invention is capable of releasing a composition of Wo 96/11706 Pcr~Ss5/l4442 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 5 from about l to about 12 months. A controlled release formulation of the present invention is capable of effecting a treatment for preferably at least about l month, more preferakly at least akout 3 months and even more pref erably f or at least about 6 months, even more lO preferably for at least about 9 _months, and even more preferably for at least about 12 months.
In order to protect an animal from flea infestation, a therapeutic composition of the present invention is administered to the animal in an effective manner such that 15 the protease activity of fleas feeding from the blood stream of animals treated with the composition is reduced.
As such, a treated animal is an animal that is competent to reduce the fléa burden by reducing flea protease activity, or by reducing flea protease activity and at least one 20 other flea activity. Preferakly, the protease activity is reduced by at least akout 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 Z5 on the nature of the composition and administration regime.
Animals administered a protease vaccine with at least one booster shot usually become ,~ nt at about the same time as wQuld ke expected for any vaccine treatment. For WO 96/11706 Pc~rluS95114442 example, animals administered a booster dose about 4 to 6 weeks after a primary dose usually become competent within another about 3 to 4 weeks. Animals administered a composition including an anti-flea protease antibody or 5 protease inhibitor become ~ nt as soon as C~ OpL iate serum levels of the ,~ _ a are achieved, usually with one to three days.
In a preferred ~ ;r ~, a composition of the present invention when administered to a host animal is lO able to reduce flea viability by at least about 50 percent within 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 15 :animal is able to reduce flea viability by at least about 65 percent within at least about 14 days after the fleas begin feeding from the treated ani~,al. An even more preferred composition when administered to an animal is able to reduce flea viability by at least about 90 percent 20 within at least about 7 days after the fleas begin feeding from the treated animal.
In another preferred: ~ - , a composition of the present invention when administered to a host animal is able to reduce flea fecundity (i.e., egg laying ability) by 25 at least about 50 percent, more preferably by at least about 70 percent, and even more preferably by at ~ least about 90 percent, within at least about 30 days after the fleas begin feeding from the treated animal. (Note that wo s6rll706 PCl'rUS95/14442 fleas usually do not begin laying eggs until about 7 days after taking a blood meal. ) In ac~oL-ldllce with the present invention, compositions are administered to an animal in a manner such that the 5 animal becomes competent to reduce f lea protease activity in a flea that feeds from the competent; i.e., the animal becomes a treated animal. For example, a flea protease vaccine of the present invention, when administered to an animal in an effective manner, is able to elicit (i.e., 10 stimulate) an immune response that produces an antibody titer in the blood stream of the animal sufficient to reduce flea protease activity. Similarly, an anti-flea protease antibody of the present invention, when administered to an animal in an effective manner, is 15 administered in an amount so as to be present in the animal's blood stream at a titer that is sufficient to reduce flea protease activity. A protease inhibitor __ ' of the present invention, when administered to an animal in an effective manner, is administered in a manner 20 so as to be present in the animal's blood stream at a ;~..Lldtion 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 ani~al, a time Wo 96/11706 PCT~S95/14442 66 ~
period is required for the animal to elicit an immune response before the animal is _ ~~nt to inhibit protease activity of fleas feeding from that animal. MethPds to obtain an immune response in an animal are known to those 5 skilled in the art.
Acceptable protocols to administer compositions in an effective manner include individual dose size, number of doses, frequency of dose ~administration, and mode of administration. Determination of such protocols can be lO 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. For example, a preferred single dose of a protease vaccine or a mimetope 15 thereof ranges from about l microgram (~g, also denoted ug) to about lO milligrams (mg) of the composition per kilogram body weight of the animal. Booster vaccinations can be administered from about 2 weeks to several years after the original administration. Booster vaccinations preferably 20 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 lO ,~g to about l mg of the vaccine per kg body weight of the animal is administered from about one to 25 about two times over a time period of from about 2 weeks to about 12 months . In one ~ ' i L, a booster dose of a composition of the present invention is administered about 4 to 6 weeks after the primary dose, and additional Wo 96/11706 PCT/US95/14442 boosters are administered about once or twice a year.
Modes of administration can include, but are not limited to, oral, nasal, topical, trAns~ ~r~l, rectal, and parenteral routes. Parenteral routes can include, but are 5 not limited to ,, l ,"l ~l~G.:~uSl intradermal, intravenous, and ill~L Cf''~ routes.
In another: _-;r ' ~ a preferred single dose of an anti=flea protease antibody composition or a mimetope thereof ranges from about l ~g to about lO mg of the lO composition per kilogram body weight of the animal. Anti-flea antibodies can be re-administered from about l hour to about biweekly for several weeks following the original administration. Booster ~ ai ~s preferably are administered when the titer of antibodies of the animal 15 becomes insufficient to protect the animal from flea infestation. A preferred administration schedule is one in which from about lO ~g to about l mg of an anti-flea protease antibody composition per kg body weight of the animal is administered about every 2 to every 4 weeks.
20 Suitable modes of administration are as disclosed herein and are known to those skilled in the art.
According to one ~ , a nucleic acid molecule of the present invention can be administered to an animal in a fashion to enable expression of that nucleic acid 25 molecule into a protective protein (e.g., flea protease vaccine, anti-flea protease antibody, or prot~;n~c~ol~c protease inhibitor) or protective RNA (e.g., antisense RNA, ribozyme or RNA drug~ in the animal to be protected from -Wo 96/11706 PCT/US95/14442 disease. Nucleic acid molecules can be delivered to an animal in 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, Science 247, 1465-1468) or (b) pack~ed 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).
A recombinant virus particle vaccine of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule is packaging-deficient. A number of recombinant virus particles can be used, ;n~ l;ng, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses.
When administered to an animal, a recombinant virus particle vaccine of the present invention infects cells within the; i ~d 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 r~ i n~nt virus particle vaccine of the present invention is from about l x 104 to about l x 107 virus plaque forming units (pfu) per kilogram body weight of the animal. Administration protocols are similar to those described herein for protein-based vaccines.

wo 96/11706 PCT/US95/14442 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 L~ ' ;n~nt cells include ~A7 -77i:~, E. coli, 5 Mycobacterium, S. frugiperc7a, baby hamster kidney, myoblast G8, COS, MDCK and CRFK reeombinant cells, with S:~7 -77s~
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 10 at doses ranging from about lo8 to about lo12 bacteria per kilogram body weight.~ Administration protocols are similar to those described herein for protein-based vaccines.
~ecombinant 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, rh;nrh; l l :~c, ferrets, goats, mice, minks, rabbits, raccoons, rats, sheep, squirrels, swine, rh;rk~n~, ostriches, quail and turkeys as well as other furry animals, pets and/or e- ~~1 ; c food animals, being more preferred. Particularly preferred animals to protect are cats and dogs.
~he present invention includes compositions to treat flea infestation by any flea. As such, compositions of the present invention can be derived from any flea species.
Preferred fleas to target include fleas of the following Wo 96/11706 PCr/USs5114442 genera: Ctenocerh 77ir7Fc, Cyopsyllus, Diamanus ~OropsyllaJ, Echidnophaga, Nosopsyllus, ~ulex, Tunga, and Xenopsylla, with those of the species Ctenocerh;~7i~7~s canis, Ctenocephalides felis, Diamanus - n77.c, Echidnophaga 5 gA7 7 in;7C~7,, Nosopsyllus faciatus, Pulex irritans, Pulex simulans, Tunga penetrans and Xenopsylla cheopis being more preferred. Particularly preferred fleas from which to protect animals include fleas of the species Ctenocerh;77i~7~c felis, Cteno~erh;77i~7~c canis, and Pulex o species (e.g., Pulex irritans and Pulex simulans). It is also within the scope of the present invention to administer compositions of the present invention directly to fleas.
The present invention also includes the use of 15 compositions of the present invention 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 Erom any ectoparasite to reduce ectop~rasite 20 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., 25 Ixodes and Am~lyomma) and soft ticks of the family Argasidae (e.g., Ornithodoros, such as 0. parkeri and O.
turicata); flies, such as midges (e.g., ~'777icc~id~C), mosguitos, sand flies, black flies, horse flies, horn wo s6nl706 PCT/US95/14442 flies, deer flies, tsetse flies, stable flies, myiasis-causing flies and biting gnats; ants; spiders, lice; mites;
and true bugs, such as bed bugs and kissing bugs, including those carrying Chagas rl;q~Ace- Even more preferred 5 ectoparasites to target include fleas, mosquitos, midges, sandflies, blackflies, ticks and ~Z2odnius.

Tlle following examples are provided for the purposes of illu6tration and are not intended to limit the scope of the present invention.

Examl~les Examl~le 1 This Example 1' LLates that soluble flea midgut .. ..
preparations contain serine protease activity as well as leucine ~m; n~ p~rtidase activity.
Using a ~ , ; 7ation / sonication protocol as described in U.S. Patent No. 5,356,622, i~id., followed by an about 2 minute centrifugation step at about 10,000 x g, soluble flea midgut preparations were obtained from fed and unfed fleas. Pellets from the centrifugation step were 20 also collected and rF.cllcp~nrl~ for analysis. Also prepared were whole flea lysates. Peptide substrate screening studies using the synthetic ~ L~ iC trypsin substrate BAPNA (Na-benzoyl-DL-arginine-p-nitro~n; ~ ; available from Sigma ~~h~om;c~l Co., St. Louis M0) ~' LL~ted 25 ci~r~if;~nt proteolytic activity in both soluble flea midgut preparations as well as some proteolytic activity in :

Wo 96/11706 PCrlUSs5114442 r~C~crpn~ midgut pellets. Soluble unfed flea midgut preparations exhibited about lO times as much activity as did controls (samples to which no flea midgut fractions were added), whereas soluble fed flea midgut preparations 5 exhibited about 2 0 times as much activity as did controls .
Whole flea ~L~a~ ions exhibited about 2 to 3 times as much activity as did controls.
The ability of soluble fed and unfed flea midgut preparations to cleave BAPNA was almost completely 10 inhibited (i.e., nearly lO09i~ by aprotinin (available from Sigma), whereas PMSF (phenylmethane-7-sulfonyl fluoride;
available from Sigma) inhibited such proteolytic activity by about 50~6. EDTA inhibited proteolytic activity of the preparations by about lOg6, whereas addition of calcium ions 15 stimulated proteolytic activity by about 2596. These results indicate the presence of serine protease activity, and more particularly of trypsin-like activity, in these soluble flea midgut preparations. These results also suggest the presence of serine protease isoforms in the 20 preparations. It is also of interest to note that flea trypsin-like activity appears to be distinctive from that of mosquitos in that mosquito trypsins are not affected by EDTA or calcium ions.
Us=ing a methyl-hemoglobin substrate, the pH optimum of 25 the proteolytic activity in the soluble flea midgut preparations was found to be between pH 7 and pH 9, with a pH of about pH 8 giving the best activity. Such pH optima suggest the presence of serine proteases in soluble flea wo 96111706 PcTfasssfl44~z midgut preparations.
Soluble preparations of both unfed and fed flea midgut soluble preparations also were able to cleave the leucine Am; nop~rtidase specific substrate LPNA (L-leucine-p-5 nitroAn;l i~P; available from Sigma) using standardconditions, indicating the presence of leucine ~m; nop~rtidase (LAP) activity in such preparations.

ExamPle 2 The following example evaluated the number of 10 proteases in flea midguts that could be ;iCCPCCPd by protease substrate gel analysis.
Protease auLaLLclte gels (available from Novex, San Diego, CA, as Novex Zy , 1 c) were 10% polyacrylamide-SDS
gels with 0.1% gelatin. Samples and gels were processed 15 according to Novex instructions. Briefly, samples were diluted in SDS-PAGE sample buffer without reducing agents.
Tris-glycine SDS-PAGE was carried out by standard procedures. After ele~.LLu~llc,L.:sis, gels were incubated in 0.25% Triton X-100 at room temperature for 30 minutes 20 (min), then in developing buffer (50 mN (m; l l ;~ r) Tris-HCl pH 7.0, 5 _N CaC12, 0.02% Brij 35, 0.2 N (molar) NaCl) at room t~, CLLUL~ for 30 min, and then incubated with fresh developing buffer at 37~C, usually overnight. Gels were then stained 30 min in 0.5% ~u~ Cc;e R-250, 40%
25 methanol, 10% acetic acid and destained in 40% methanol, 10% acetic acid.
The followlng flea midguts were ~1; CCPctPd directly .

Wo 96/11706 PCr/US95/14442 into sample buffer: 100 midguts from unfed males; 100 midguts from unfed females; 100 midguts from fed males; and 100 midguts from fed females. Samples containing 10 or 20 midguts each were evaluated using protease substrate gel 5 analysis and numerous negative staining bands were observed. The general pattern was the same for female and male midguts, although there appeared to be more activity in gel lanes containing female midguts. There were distinct differences noted between gel lanes containing fed 10 and unfed midguts. There was a definite increase in overall activity in the fed midgut lanes, and, in addition, there were differences in the band patterns.
Fed and unfed female midguts were further evaluated using protease substrate gel analysis and the results are 15 shown in Fig. 1. The protease substrate gel shown in Fig.
1 ~1 L~clLes the relative proteolytic activity in 1, 2, 5 or 10 midguts from either fed or unfed female fleas.
Specifically, lane 1 contains a set of molecular weight markers. Lanes 2 through 5 contain, respectively, 10, 5, 20 2 and 1 unfed midguts. Lanes 6 through 9 contain, respectively, 1, 2, 5 and 10 fed midguts. Lane 10 contains 100 ~g of dried bovine blood.
Proteolytic activity could easily be detected in one fed or one unfQd female midgut, although there was 25 considerably more activity in the fed -midgut. Lane 10 evaluated 100 ~g of dried bovine blood to assess if the increase in activity seen in the fed midgut lane was due to proteases in the blood meal. No activity was seen in the Wo 96111706 PCTIUS95114442 blood lane.

-Exam~le 3 This example evaluated the protease classes present in flea midguts.
Three unfed female midguts and 0.75 fed female midguts were evaluated in duplicate in several protease substrate gels. Each gel was cut in half. Half was ~Loces:,~d as described in Example 2, while the other haif contained protease inhibitors in all incubatiQn buffers. The following inhibitors were evaluated:
(a) the serine protease inhibitor AEBSF (available from Boehringer M:~nnh~;m, Tn~7~n:~rolis~ IN) was used at a final concentration of l mM;
(b) the serine protease inhibitor soybean trypsin inhibitor (available from Sigma) was used at a final concentration of lO0 ~g/ml (milliliter);
(c) the cysteine and serine protease inhibitor leupeptin (available from Sigma) was used at a final concentration of 10 ,ug/ml;
(d) the aminopeptidase inhibitor bestatin (available from Sigma) was used at a final cu.1.:~..LLcl~ion~of 0.25 mM;
(e) the metalloprotease inhibitor EDTA (available from Sigma) was used at a final ~,-.ce--LLation of 2 mM; and (f) the cysteine protease E-6~ (available from Sigma) was 25 used at a final .,.,~ LLc.tion of lO ,~g/ml.
AEBSF, soybean trypsin inhibitor and leupeptin were the only inhibitors to have any effect at the sensitivity Wo 96/11706 PCI~S95/14442 of this assay. It was detPrmin~-1 that= serine proteases were the pr.o~---in~nt, if not only, proteases present in the midgut preparations evaluated. Fig. 2 shows a protease substrate gel with fed (lanes 2 and 4) and unfed (lanes 1 5 and 3) midgut preparations with (lanes 3 and 4) and without (lanes 1 and 2) AEBSF. RPC;~ 1 activity in the inhibitor lanes could have been due to proteolysis that occurred during electrophoresis and prior to saturation of the gel with inhibitor in the incubation buffers.

10 Exam~le 4 This Example evaluates protease activity contained in a soluble fed midgut preparation of the present invention.
Mixed-sex fed flea midguts were processed as described in U.S. Patent No. 5,356,622, i~id. Aliquots of several 15 steps of the procedure were evaluated by loading an equivalent of 0 . 4 midguts per ~ lane of a protease substrate gel as described in Example 2. The results are shown in Fig. 3. Samples were from the low speed supernatant (lanes 2 and 9), sonicated midguts (lanes 3 and 10), high speed 20 supernatant (lanes 4 and 11), combined low and high speed supernatants (FGS) (lanes 5 and 12) and the high ~speed pellet (lanes 6 and 13). Lanes 7 and 8 contained 50 nanograms (ng) of trypsin as a control. Duplicate lanes were evaluated. The gel was cut in half, and lanes 1-7 25 were processed as described in Example 2, and lanes 8-14 were processed with 100 ~Lg/ml soybean trypsin inhibitor in all the incubation buffers.

WO 96/11706 ~ ~ PCT/IJS95114J~2 Protease activity was seen in all preparations, the most being observed in the FGS lane (lane 5). It was also evident that the majority of the activity was inhibited by soybean trypsin inhibitor, a serine protease inhibitor.
ExamPle 5 ~ =
This Example rl LLc-Les the increase in flea midgut protease activity after blood feeding by fleas.
Fleas were fed on a dog for 15 minutes. At timed intervals after feeding, two midguts were dissected dlrectly into sample buffer and proteases evaluated by protease ,ub:.LL.Ite gel analysis as described~ in Example 2.
Fig. 4 depicts a gel showing midgut protease activity at 30 min (lane 1), 1 hr (lane 2), 2 hr (lane 3), 4 hr (lane 4), 6 hr (lane 5), 8 hr (lane 6), 24 hr (lane 7) and 56 hr (lane 8) after blood feeding ended.
Increases in proteolytic activity were first observed 2 hr (lane 3) after feeding, although at 4 hr (lane 4) there was a much greater increase in activity noted. This increase in activity was still noticed 56 hr after feeding (lane 8).
Exam~le 6 This Example evaluates the effect of a number of protease inhibitors on flea viability and fecundity in a flea feeding system as described by Wade et al. ibid.
The following protease inhibitors were tested at the indicated final ~ lCt:llLLCItions in blood meals:

Wo 96/11706 PcrtUS9Stl4442 (a) Am;nnp~rtidase inhibitor bestatin at 1.3 mM and 13 mM;
(b) Aspartic acid protease inhibitor pepstatin A at l tlg/ml and lO t~g/ml;
(c) Cysteine protease inhibitor E-64 at l ,ug/ml and lO
5 ~Lg/ml.
(d) Metalloprotease inhibitor rhncrhnramidon at lO ,~g/ml and lO0 ~g/ml; and (e) the following serine protease inhibitors:
~ AEBSF at 0. 3 mM, 0. 5 mM, 5. 0 mM and 6. 0 mM;
~ Aprotinin at 2 ~Lg/ml and 2 0 ,ug/ml;
~ Leupeptin at 5 ,~g/ml and 50 ~Lg/ml;
~ Soybean trypsin inhibitor at lO ~g/ml and lO0 ~Lg/ml;
~ Soybean trypsin/~ lly LLy~sin inhibitor at lO
~Ig/ml and lO0 ~g/ml;
AEBSF is available from Boehringer MAnnh~;m; all other listed inhibitors are available from Sigma.
Protease inhibitor, '- were tested in groups of

3 to 6 ;n~ ]ll;ng cl~l,L~,l,Liate control groups. Inhibitors 20 were not tested in groups of common inhibition types.
Rather, they were tested in groups based on the diluent needed to dissolve them. (AEBSF, aprotinin, bestatin, leupeptin, rho~rhnramidon, soybean trypsin inhibitor and soybean trypsin/-,1.y ,Lry~sin inhibitor were dissolved in 25 water; E-64 and pepstatin were dissolved in ethanol). This reduced the number of control (diluent only) groups needed within a particular assay. Inhibitor .~ LL~tions were chosen such t_at t_e lower ~ =..L~c-tion used was within Wo g6/11706 PCT/~595/14442 _ 79 the range re~ -n~f~d by the supplier for that inhibitor.
The higher concentration was typically 10 times above the lower ~_~,.,ce..Llcltion and was used to look for aose response.
The general protocol for all of the assays was as 5 follows: Approximately 2000 newly emerged adult fleas were placed in feeding chambers to feed on normal blood for about 24 to 48 hr. The fleas were prefed for two reasons:
The first was to be certain that only fleas that would feed in the feeding system were used in the comparative study.
10 The second was to prime female fleas for egg laying, since female fleas typically do not begin laying maximal numbers of eggs per day until the third day of feeding.
The prefed fleas were placed in "minifeeder" feeding chambers at a ratio of about 80 female fleas to about 20 15 male fleas for a total of about 100 fleas per chamber.
Actual total number of fleas per chamber varied from about 90 to 125 fleas. Previous experiments have not demonstrated any differences in adult survival or fecundity based on such variance in numbers of fleas in a chamber.
20 Three rh: ' :, were prepared for each PYp~r; ~1 and control group. A fresh blood meal containing the a~ t, iate inhibitor in 3 ml total volume was placed on each chamber daily through the 7 day extent of an assay.
On days 3, 5, and 7 of the assay, surviving adult 25 fleas were transferred to clean rh: ' ,. The contents of the original chambers were dissolved in about 4 0 ml of PBS
(phosphate-buffered saline) in a 50 ml Falcon tube. The contents of a given tube were then f iltered through a pre-Wo 96/11706 Pc~r/uss~ll4442 weighed #l Whatman filter=disk inserted into a vacuum filter. The 50 ml tube and the filter funnel were rinsed with distilled water which was then passed through the filter. Once the chamber contents had been filtered, dead adult ~leas were removed from the filter paper and placed in a labelled tube so that they could be counted and sexed.
The filter paper was then placed into a preweighed 12 x 75 polypropylene tube and dried in the SpeedVac for 2 . 5 hr with the heater on. After~ drying the filter paper was weighed. The weight of the filter paper ~and tube was subtracted to obtain the dry weight of the eggs and this value was converted to an estimated number of eggs using the formula y = 41384.361x + 162.37, where x = dry weight of eggs.
On day 7, adult fleas that had survived the study were frozen, counted and sexed. The numbers were added to the number of male and female fleas that had died during the assay to verify the number of male and female fleas in each chamber at the start of the study.
Female, male and total adult flea survival were calculated for all .oYp ~r;~ tal and control groups on days 3, 5, and 7 of each assay. Additionally, -the number of eggs per surviving female was calculated on days 3, 5 and 7. Female fleas found dead on a given collection date were included in the total number of egg-laying females for the days between that date and the previous collection date, providing a conservative estimate of fecundity. Fecundity values were averaged for the three collection dates to Wo 96111706 PCr/USs5ll4442 obtain an average for each group over 7 days.
Results of these studies are presented below in Table l and Fig. 6 through Fig. 9. All survival and fecundity values are presented below as a percent of control value.
~ ~ TABLE 1 Effect of Protease Inhibitors on Flea Viability and Fecundity Compound Conc. Fecundity1 Adult Survival1 DaYs 1--7 Female Male Total lOAEBSF :~ ~
6.0 mU 17.2% 4.1% 0.0 3.4%
5.0 mM 1.4% 6.8% 0.0% 5.6%
0.5 mM 95.0% 103.9% 104.2% 103.6%
0 . 3 mM 82 . 4% 116 . 2% 103 . 0% lll . 9%
15 Aprotinin 20ug/ml 84.2% 100.0% 101.7% 99.9%
2ug/ml 83.2% 103.2% 104.9% 103.3%
~eupeptin 50ug/ml 77.6% 101.5% 111.7% 104.6%
205ug/ml 85.0% 71.0% 61.4% 68.4%
Soybean Trypsin Inhibitor 100 ug/ml 79.1% 76.5% 76.0% 76.3%
10 ug/ml 96 .1% 80 .1% 101. 7% 83 . 9%
Trypsin/~hy LLy~sin Inhibitor 25=100 ug/ml 81.1% 88 . 0% 95. 4% 89 . 9%
10ug/ml 100 . 7% 115.1% 143 . 5% 120 . 7%

10ug/ml 177 . 4% llO . 2% 139 . 0% 114 . 2%
1ug/ml 109.4% 99.9% 102.9% 100.1%
30 10 ug/ml 84.1% 90.2% 91.1% 90.6%
lug/ml 95 . 2% 77 . 3% 80 . 0% 77 . 5%
Phosphoramidon 100 ug/ml 84.9% 70.2% 64.6% 69.7%
10 ug/ml 89 . 0% 98 . 8% 95 . 2% 97 . 8%
35 Pepstatin A
~ lO ug/ml 83.9% 113.6% 133.4% 116.2%
1 ug/ml 67 . 7% 77 . 6% 96 . 6% 80 . 5%
- Bestatin 13.0 mM 23.3% 121.0% 103.4% 117.0%
40 ~ ~1.3 mM 60.4% 119.5% 116.3% 116.8%
All experimental values are expressed as a percent of the n~ co:~trol group.
.

WO 96111706 PCrNS951i4442 The ~m;n~p.ortidase inhibitor bestatin caused a significant (p < 0.05) reduction in fecundity at 13 mM (77%
reduction) and at 1. 3 mM (40% reduction) indicating the presence of an aminopeptidase or other exopeptidase in flea 5 midguts. Bestatin at the con~ .LLc-tions tested, however, had no significant effect on adult viability at either ~ on. ~llLL..tion. These results suggest that aminopeptidases may play a role in ovarian function, or a related process, such as vitellogenesis.
The aspartic acid protease inhibitor pepstatin A
caused a significant reduction (p < 0.05) in fecundity at 1 J~g/ml (32% reduction), but not at 10 ,~g/ml. Pepstatin A
had no significant effect on adult viability at either concentration .
The cysteine protease inhibitor E-64 showed no statistically significant reduction in fecundity in this assay. There was a small, but significant (p < 0.05), reduction in total adult flea survival when E-64 was dissolved in grain alcohol and added to blood at 1 ~g/ml.
20 However, this reduction was not evident in the group that was fed blood containing 10 ILg/ml E-64 in grain alcohol.
The metalloprotease inhibitor phosphoramidon caused a reduction in adult viability of about 30%, which, however was not statistically significant. There was no 25 significant reduction in fecundity.
Results using serine protease inhibitors were particularly interesting and suggest the significance of serine proteases in flea midguts. AEBSF administered at wo 96/11706 PCT/Usg5/14442 83 = ~ ~
concentrations ranging from about 5 mM to about 6 mM
reduced flea fecundity by more than 80%. In addition, adult survival was reduced to near zero (p < 0. 05) .
Aprotinin, however, had no significant effect on 5 either fecundity or viability, likely due to the ability of serum proteins, such as albumin, to interfere with aprotinin's inhibitory activity.
Leupeptin had no effect on fecundity at both concentrations, but reduced adult viability by 30% at 5 10 ,~g/ml. Ilowever, adult viability was not affected by 50 ILg/ml leupeptin and none of the observed reductions were statistically q;gn;f;rqn~.
Soybean trypsin inhibitor caused a small (20%) statistically ins;~n;f;c~nt reduction in fecundity at lO0 15 ~Lg/ml. The lower cullc~llLLdLion had no effect. soybean trypsin inhibitor, on the other hand, is very effective in in vitro studies as disclosed in several of the examples and was used to purify serine proteases as disclosed in Example 7. Soybean trypsin/ully L,~,sin inhibitor had no 20 effect on adult viability or fecundity.

Exam~le 7 ~ =
~ his Example describes the production of a preferred soluble flea midgut ~Le~dLdtion of the present invention and purification of flea serine proteases therefrom. Also 25 included is amino acid sequence analysis of a flea serine protease of the present invention.
The soluble flea midgut preparation was prepared as Wo 96/l1706 PCrrUSs5/l4442 follows. Flea midguts (3,735) from a mix of female and male fed fleas were h~ , ; 7ed in a homogenization buffer comprising 1.5 ml 50 mM Tris-HCl, 0.5 M NaCl, pH 8.5. The ht J~lldte was centrifuged at 14, 000 x g for 10 min. The 5 resultant pellet was processed again in another 1. 5 ml of the homogenization. The two supernatant solutions were combined to form the soluble flea midgut preparation.
The preparation was added to 3 ml of p-~m;nnhPn~m;~linP-sepharose 6 B (affinity matrix for 10 tryp6in-like proteases, available from Sigma) and incubated at 5 ~ C overnight on a rocker. The sepharose beads were drained and washed with 7 . 5 ml of the h~ j i 7ation buffer. The ~snrhPd proteins were eluted with 5 ml 0.1 M
p-aminobPn7Am;~i;nP in the same buffer. This eluate was 15 concentrated and the buffer exchanged to 50 mM Tris-HCl pH
8.5, 0.1 mM CaCl2 by ultrafiltration through a membrane with a 3 kD cutoff, the final volume being 140 ~Ll (microliters).
T.AhPl; ng of proteins was performed by adding 10 lLl of (1,3-3H)-diisopropylfluuLu~l n:,l.hAte (available from New 20 England Nuclear, Beverly, MA, at 6. 0 Ci (Curies) / mmole, 1.0 mCi/ml) to 90 11 of the affinity purified proteins and incubating at 5~C for 18 hours. The reaction was divided in half, each half t_en being separated by C4 reverse phase chromatography according to the following protocol:

Wo 96111706 PCrlUS9~/14442 Buffer A: 0.1% TFA in water Buffer B: 0.085% TFA, 90% Acetonitrile 0. 8 ml/min, 220 nm, 1 min fractions 5. 6% B 15 min ~ =
5 . 6% to 100% B over 60 min .
Ten microliters of each fraction was added to 6cintillation fluid and counted. 7~ost protein-associated counts were found in fractions 44-47. Fig. 5A shows electrophoresis of fractions 40 (lane 2), 44 (lane 3), 46 (lane 4) and 47 10 (lane 5) from one chromatography run through a 14% Tris-glycine polyacrylamide-SDS gel, followed by coomassie staining. This gel was then processed with Entensigy (NEN~
and exposed to film for 18 hours, as shown in Fig. 5B.
Each fraction contained several proteins as shown in Fig.
15 5A, but only 4 bands were labeled, the most ~7LI ;n~nt being 26 kd ~seen in lanes 3, 4 and 5), and denoted herein as PfSP26. A faint band of 24 kd, denoted herein as PfSP24, was also noticed in lane 5. A band of 19 kD, denoted herein as PfSP19, was labeled in lane 4 that was associated 20 with a very faintly staining protein band. Some labeled proteins were seen at the dye front of lanes 4 and 5, indicating a molecular weight less than 6 kd, denoted herein as PfSP6, and could be degradation products.
Fraction 44 (AnAl o~Q7lq to lane 3) from a second C4 25 chromatography separation ~7~r;- L was electrophoresed, blotted onto PVDF, stained with Coomassie R-250 and destained via standard procedures. The 26 kd band, ~ uSL~ 7;n~ to PfSP26 (also referred to herein as PfSP44-=. . ~

Wo 96111706 PCrnJSs5114442 E, indicating the f raction in which the protein eluted and the gel/filter band from which the protein was excised), was excised and subjected to N-t~rn7;nAl amino acid sequencing using terhn;q7l~q known to those skilled in the 5 art. A partial N-t~rn7;nAl amino acid sequence of about 32 amino acids was deducea and is represented herein as SEQ ID
NO: l:
I I G G E V A G E G S A P Y Q V S L R T K E G N .1 F S
G G S I ~, lO It should be noted that since amino acid sequencing technology is not entirely error-free, SEQ ID NO:l represents, at best, an apparent partial N-t~r7~7;nAl amino acid sequence of PfSP26. This caution is particularly relevant in light of the sequencing of this protein having 15 been done at a low p;l -lAr cu...~ Lcltion.
A homology search of the non-redundant protein sequence database was performed through the National Center fo7- Biotorhn~nl o~y Information using the B~AST network.
This database includes +SwissProt + PIR + SPUpdate +
2 0 GenPept + GPUpdate . ole level . Results of the search indicate that the N-7'~rn7;nl7q of PfSP26 shares significant amino sequence homology with a nu7l7ber of serine proteases, including a variety of trypsins, ~ 1.y ~Ly~sins and ~71Aq7n;nq The 32-amino acid N-t~7-n7;n;7l amino acid sequence 25 of PfSP26 shared the highest degree of homology with a hornet u1-y ~Ly~usin II. ~ ~

WO 96111706 PCI~US9~i~14442 Exam~le 8 This example describes the cloning of certain flea protease nucleic acid mQlecules of the present invention.
This example also describes the production of certain 5 recombinant molecules, recombinant cells and flea protease proteins of the present invention.
Several flea serine protease nucleic acid molecules, ranging in size from about 250 to about 500 nucleotides, and representing one or more partial flea serine protease 10 genes, were PCR amplified from a fed flea midgut cDNA
library that was prepared from RNA isolated from fed flea midguts using standard protocols as described in Sambrook et al., ibid. Several pairs of primers were used in PCR
amplification reactions that represented degenerate 15 oligonucleotides flf~qif3nf~d from p~lh~; qhf (l sef~uences of serine protease genes isolated from biting insects (e.g., mos~uitos and black flies) . Each primer pair was flf qiflnf d so that a properly amplified fragment of a flea serine protease gene would include a domain uulL~ ;nf3 to the 20 most conserved domain of trypsin protease genes (thought to be the active site) given that such a domain is contained in flea serine protease gene(s?.
The amplified PCR Ll Ls were of predicted size, ranging from about 250 nucleotides to about 500 25 nucleotides, flf rf nfl i nfl on which primer pairs were used.
PCR fragments that hybridized to a probe flfq;f3nfd from the domain most conserved among all known trypsin genes were gel purified and cloned, for example, into the pCRII

' Wo 96111706 PCT/US95/14442 cloning vector (available from InVitrogen, Corp., San Diego, CA), following manufacturer's instructions. Nucleic acid se~~nr r~e: of the r, . Ls are being det~nm;n~ using standard techniques.
~he amplified PCR L~, - Ls are also being used as probes to identify full-length flea protease genes in unfed and fed flea midgut cDNA libraries and in flea salivary gland cDNA libraries, as well as in flea genomic DNA
libraries, using standard procedures.
Recombinant molecules and recombinant cells including the amplified PCR fragments as well as full-length flea protease genes are being produced using standard procedures. Culturing of such recombYnant cells leads to the production of flea protease proteins of the present invention.
ExamPle 9 This Example describes the testing of a flea protease protein as a flea protease vaccine of the present invention, that is for the ability of such a protein, upon 2 0 administration to an animal, to elicit the production of antibodies that reduce flea protease activity and, as such, reduce flea viability and/or fecundity. ~his Example also ~1 ~rcltes the use of such a flea protease protein as a vaccine on a dog subsequently inf ested with f leas .
2~ A flea protease protein produced as described in Example 7 is administered to rabbits according to a standard i ; 7eltion protocol kno~m to those skilled in the art, including d~ upr iate booster shots. Such a protein is also administered to guinea pigs and to dogs following a similar protocol.
Sera is collected from the treated rabbits and is 5 verified to contain anti-flea protease antibodies. Such sera is then fed to fleas in a feeding system as reported by Wade et al. ibid. Fleas feeding on such a sera show reduced viability compared to fleas feeding on sera collected from rabbits not administered the flea protease 10 protein. Sera from treated guinea pigs and dogs are verified in a similar manner.
Dogs treated with a flea protease protein are then infested with fleas as are dogs not treated with a flea protease protein. Dogs treated with a flea protease ~5 protein show a significant reduction in flea burden compared to untreated dogs.

ExamPle lQ
This Example describes the determination of the partial N-tormin~l amino acid sequence of additional flea 2 0 serine protease proteins of the present invention .
An additiûnal eight flea serine proteases were purified and c~ nconc~e partial N-~orm;n~l amino acid soquonroc were ~lotorrn;no~ as described in Example 7. The results are as follows, the proteins being named by the 25 fraction in which they were eluted and the SDS-PAGE gel band from which they were excised. Each of the proteases bore at least some sequence homology to known proteases, Wo 96/11706 PCTnJss5/14442 the highest percent identity estimated to be no more than about 3 0-4 Og~ .
Flea protease PfSP45-C had a partial N-tPrminRl amino acid sequence of X V G G H D T S I D X H P H Q V T, also 5 represented herein as SEQ ID N0: 2 . PfSP45-C was most similar in amino acid sequence to a fruit fly trypsin epsilon .
Flea protease PfSP46-C had a partial N-tPrm;n~l amino acid sequence of I V G G A D A A P G N A P F Q V S L R D K
lO G, also represented herein as SEQ ID N0:3. PfSP46-C was most similar in amino acid sequence to a collagenolytic 36 kD protease from a Kamchatda crab.
Flea protease PfSP46-A had a partial N-tP~m;n~l amino acid sequence of I V G G Q D A D I A K Y G Y Q A S L Q V
15 F N E H F X G A X I L N N Y, also L~Lese,1~ed herein as SEQ
ID N0:4. PfSP46-A was most similar in amino acid sequence to a hornet ~ y LLy~sin II.
Flea protease PfSP46-B had a partial N-tPrminAl amino acid sequence of I V G G T D V N I E N F G W Q V S L F D R
20 N G H F, also represented herein as SEQ ID N0:5. PfSP46-B
was most similar in amino acid CPql1Pnre to a fruit fly trypsin beta.
Flea protease PfSP48-A had a partial N-tP~n;n~l amino acid sequence of I V G G H D T S I D K H P F Q V S L I D K
25 N, also represented herein as SEQ ID NO: 6. PfSP48-A was most similar in amino acid FPq~lPn~'e to a fruit fly trypsin epsilon.
Flea protease PfSP48-B had a partial N-tPrm;n~l amino Wo 96/11706 PcrluS9S/14442 acid sequence of V V G G L E A A E G S A P Y Q V X L Q W G
N F, also l~Lc~ 1Led herein as SEQ ID NO:7. PfSP48-B was most similar in amino acid sequence to a human Factor 12.
Flea protease PfSP48-D had a partial N-t~rm;n~l amino 5 acid sequence of I V G G E D A E L G E X P T Q, also represented herein as SEQ ID N0:8. PfSP48-D was most similar in amino acid sequence to a bovine Factor 9.
Flea protease PfSP40-B had a partial N-t~rm;n~l amino acid sequence of D E D G K D D S A P G E I, also lO represented herein as SEQ ID N0: 9 . PfSP40-B was most similar in amino acid soqu ~nre to a fruit fly furin-like protease I.

ExamPle 11 This Example describes the isolation of nucleic acid 15 molecules ~nrg~;nq flea serine protease proteins of the present invention.
Several midgut proteinase cDNA genes have been isolated in a manner similar to that described in Example 8, using two degenerate primers, the design of which was 20 based on a highly c~ s,:Lv~d serine proteinase amino acid sequence (C Q/N G D S G G P L, denoted SEQ ID NO:lO) ., located about 195 amino acid residues (based on an average protease size of about 240 residues) from the mature amino t~rm;nl~C in a number of known serine proteases.
25 Complementing primers for use in the PCR amplification reaction were primers corr~crr~n~;n~ to the vectors in which nucleic acid molecules of the present invention had been Wo 96/11706 PCrlUS95/14442 ligated. The actual primers used in PCR amplification oi~
serine protease nucleic acid molecules from whole fed flea cDNA expression libraries (produced as described in Example 8) included the following serine protease specific primers:
5 cat-try #1 having nucleic acid sequence 5' TAA WGG WCC
WCC YGA ATC TCC CTG GCA 3' (Y indicating C or T; W
indicating A or T), represented herein as SEQ ID N0:11; and cat-try #2 having nucleic acid sequence 5' TAA WGG WCC AGA
RTC TCC TTG ACA 3' (R indicating A or G), represented 10 herein as SEQ ID N0: 12 . Vector specific primers included:
M13 Reverse having nucleic acid S~ n~ 5' GGAAACAGCTATGACCATG 3', represented herein as SEQ ID N0:13;
and T3 Primer having nucleic acid sequence 5' ATTAACCCTCACTAaAG 3', represented herein as SEQ ID N0: 14 .
15 The resultant PCR products, obtained using standard PCR
conditions (e.g., Sambrook et al., ibid. ), were about 600 to about 700 nucleotides in length.
The PCR products were hybridized under standard hybridization conditions (e.g., Sambrook et al., ibid . ) 20 with (i.e., to) an internal synthetic oligonucleotide probe named H57, the ~e~ of which Co r ~ uul.~s to a region including a ~U115t:l v.ad histidine residue in known serine proteases . The nucleic acid S ~ nr~ of ~57 is 5' TGG GTW
GTW ACW GCW GCW CAT TG 3', represented herein as SEQ ID
25 N0:15. PCR products which hybridized strongly to the probe were gel purified and cloned into the TA Vector~' (available from InVitrogen, Corp.). Approximately 80 r,~L ' ;n~nt TA
vector clones were isolated. To prevent repetitive -~VO 96111706 PCTIUS95/1444 sequencing of the same serine proteinase clones, a number of the clones were characterized to identify those having unique restriction ~n~ n~ ease patterns using the enzymes ~aeII and lIaeIII. About 11 ~ c apparently containing 5 unique flea serine proteinase nucleic acid molecules of about 600 to about 700 nucleotides in length were isolated using this yLuc~duL~:. These nucleic acid molecules were subj ected to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described in Sambrook 10 et al., i~id.
The complete nucleic acid sequence of one of the flea serine protease nucleic molecules, namely nfSP4672 is represented herein as SEQ ID N0:16. Translation of SEQ ID
N0:16 yields a protein of about 223 amino acids, denoted PfSP4223, having amino acid sequence SEQ ID N0: 17 . Although the entire amino acid sequence of PfSP4223 is not highly conserved to that of known serine proteases, there are several conserved regions of note (as numbered for SEQ ID
N0:17), ;n~ ;n~: (a) the sequence IVGG Sp~nn;n~ from 20 about amino acid 5 through about amino acid 9; (b) the active-site histidine at about amino acid 46 and ~uLLuu-.ding sequences Sp~nn;n~ from about amino acid 41 through about amino acid 47; (c) the ~ul.seLv~d aspartic acid residue at about amino acid 90; (d) the GWG s~qu~nre 25 Sp~nn;ng from about amino acid 124 through about amino acid 126; the conserved cysteines at about amino acid 152 and about amino acid 165; and the conserved sDqllon~-e around the active site serine, Sp~nn;n~ from about amino acid 174 Wo 96tll706 PCrNss5/14442 through about amino acid 182.
Nucleic acid and amino acid sequences of all 11 flea serine protease nucleic acid molecules were detPrm; nPd for the regions corrpcpnn~l; ng to the region in known serine r proteases to span from the conserved GWG SP~lPnre to the conserved ~ L)SG~,~ sequence (denoted SEQ ID NO: 10) . Flea nucleic acid molecule nfSPl156 has the nucleic acid Seq~lpnre represented herein as SEQ ID NO:18, which encodes a protein PfSPls2 having an amino acid sequence represented herein as SEQ ID NO: 19 . Flea nucleic acid molecule nfSP2165 has the nucleic acid sequence represented herein as SEQ ID NO:20, which encodes a protein PfSP256 having an amino acid sequence represented herein as SEQ ID NO:21. Flea nucleic acid molecule nfSP3177 has the nucleic acid sequence represented herein as SEQ ID NO:22, which encodes a protein PfSP359 having an amino acid Cpqn~nre represented herein as SEQ ID NO:23. Flea nucleic acid molecule nfSP4156 has the nucleic acid sequence represented herein as SEQ ID NO: 24, which encodes a protein PfSP4s2 having an amino acid sequence represented herein as SEQ ID NO:25. Flea nucleic acid molecule nfSP51s9 has the nucleic acid sequence represented herein as SEQ ID NO . 2 6, which encodes a protein PfSP553 having an amino acid CPqllPnrP represented herein as SEQ ID NO:27. Flea nucleic acid molecule nfSP6l68 has the nucleic acid sPquPnre represented herein as SEQ ID NO:28, which encodes a protein PfSP656 having an amino acid SPqllPnre Le~LesellLed herein as SEQ ID NO:29. Flea nucleic acid molecule nfSP71s9 has the nucleic acid sequence WO 96111706 PCTflrS9Sfl4442 represented herein as SEQ ID N0:30, which encodes a protein PfSP7s3 having an amino acid C.oTl~nre represented herein as SEQ ID N0: 31. Flea nucleic acid molecule nfSP81l76 has the nucleic acid C~ n~e representea herein as SEQ ID N0: 32, 5 which encodes a protein PfSP862 having an amino acia sequence representea herein as SEQ ID N0: 33 . Flea nucleic acid molecule nfSP9l68 has the nucleic acid sequence representea herein as SEQ ID N0:34, which encoaes a protein PfSP9s6 having an amino acia sequence representea herein as SEQ ID N0:35. Flea nucleic acia molecule nfSPl0120 has the nucleic acia C:~qn~nre representea herein as SEQ ID N0:36, which encodes a protein PfSP1040 having an amino acia sequence representea herein as SEQ ID N0: 37 . Flea nucleic acia molecule nfSP11162 has the nucleic acia sequence 15 representea herein as SEQ ID N0:38, which encoaes a protein PfSP11s4 having an amino acia sequence represented herein as SEQ ID N0:39.
Comparison of the nucleic acid s~ n~ 3 of the flea serine proteases with that of a mosquito (A . aegypti ) tr~fpsin inaicates that SEQ ID N0:18 is about 33% iaentical, SEQ ID N0:20 is about 33% identical, SEQ ID N0:22 is about 24% identical, SEQ ID N0:24 is about 25% identical, SEQ ID
N0:26 is about 32% j~7~nticA~, SEQ ID N0 28 is about 38%
. .
identical, SEQ ID NO:30 is about 33% identical, SEQ ID

N0:32 is about 33% identical, SEQ ID NO:34 is about 40%

iaentical, SEQ ID N0:36 is about 33% ;~7--nt;cAl, and SEQ ID

N0:38 is about 29%; 7~nt;~Al ~ to the CUL ~ 7;n~ region of the mosquito trypsin. Comparison of the nucleic acid .

Wo 96/11706 PCr/uss5/l4442 5~ Pn/ ~c of the flea serine proteases with that of a black fly (S. vittatum) trypsin indicates that SEQ ID N0: 18 is about 34% identical, SEQ ID N0:20 is about 34% identical, SEQ ID N0:22 is about 25% identical, SEQ ID N0:24 is about 28% identical, SEQ ID N0:26 is about 36% identical, SEQ ID
NO:28 is about 45% identical, SEQ ID N0:30 is about 29%
identical, SEQ ID N0:32 is about 36% identical, SEQ ID
N0:34 is about 42% identical, SEQ ID N0:36 is about 34%
identical, and SEQ ID NO:38 is about 30% identical, to the corr~cpQn~;n~ region of the black fly trypsin. It is to be noted that the mosquito and black fly trypsins are about 50% identical in the same regions.
Comparison of the amino acid sequences of the flea serine proteases with that of a mosquito (A. aegypti) trypsin indicates that SEQ ID N0:19 is about Il% identical, SEQ ID N0:21 is about 30% identical, SEQ ID N0:23 is about 19% identical, SEQ ID N0:25 is about 19% identical, SEQ ID
N0:27 is about 28% identical, SEQ ID N0:29 is about 21%
identical, SEQ ID N0:31 is about 14% identical, SEQ ID
N0:33 is about~ 22% identical, SEQ ID N0:35 is about 30%
identical, SEQ ID NO:37 is about 22% identical, and SEQ ID
N0:39 is about 29% identical, to the corr~p~n~;n~ region of the mosquito trypsin. Comparison of the amino acid SPqn~n~~~c of the flea serine proteases with that of a black fly (S. vittatum) trypsin indicates that SEQ ID N0: 19 is about 14% identical, SEQ ID N0:21 is about 28% identical, SEQ ID NO:23 is about 16% identical, SEQ ID N0:25 is about 17% identical, SEQ ID N0:27 is about 35% identical, SEQ ID

CA 02202622 199'7-04-l4 wo 96/11706 Pc~/usgsn444z N0:29 is about 33% identical, SEQ ID NO:31 is about llg6 identical, SEQ ID N0:33 is about 229~ identical, SEQ ID
N0:35 is about 33~ lPnt;cAl, SEQ ID N0:37 is about 2196 identical, and SEQ ID N0:39 is about 25% identical, to the 5 corrPqpnn~l;n~ region of the black fly trypsin. It is to be noted that the mosquito and black fly tr-ypsins are about 50g~ identical in the same regions.
Partial N-t~rm;nAl amino acid sequences were deduced for each of the cloned flea serine protease nucleic acid 10 molecules, four of which were identical to the following amino acid sequences derived from N-t~m;nAl qeqn~nl~in~ of serine proteases as described in Example 10: SEQ ID N0:1, SEQ ID N0:4, SEQ ID N0:6 and SEQ ID N0:7. The r in;ng nucleic acid molecules had the following deduced N-t~rminAl 15 amino acid s~ n--~q: SEQ ID N0:40, namely I V G G E N A
K E K S D V P Y Q V S L R N A E N K H F C G G A I I D D Y
W V L T, which was most similar in amino acid sequence to mite fecal allergen Der pIII; SEQ ID N0:41, namely I V G G
L E A K N G S A P F ~q V S L Q A E D Y F H, which was most 20 similar in amino acid sequence to a ~l.y LLy~sin-like protein; SEQ ID N0:42, namely I I G G E V A G E G S A P Y
Q V S L R T K E G N H F, which was most ~ similar in amino acid sequence to a ~ y ~ly~sin-like protein; SEQ ID N0:43, namely I V G G T A V D I R G F P G R Y Q F K P K P S F L W
25 W F Y, which did not ,ub~ .Lially match any protein in the data base; SEQ ID N0:44, namely I V N G L E A G V G Q F P
I Q V F L D L T N I R D E K S R C G G A L F, which was most similar in amino acid B~ n~ to a trypsin ~Le:~ UL~OL; SEQ

::

Wo 96/11706 PcrluS9Sl14442 ID NO:45, namely I V G G L E A K N G I T P F I G F F A S G
R L F, which was most similar in amino acid sequence to a ~ y LLy~sin-like protease; SEQ ID NO:46, namely I V G G N
D V S X K I F W Q V S I Q S N X Q H F C G, which was most S slmilar in amino acid sequence to a trypsin; and SEQ ID
NO: 47, namely I I G G E D A P E G S A P Y Q V S L R N Q N
L E H F C G G S I, which was most similar in amino acid sequence to a ~11y ~. y~sin-like protein.
Additional amino t~ nRl and carboxyl t~ ;nRl 1~ S,~ n~s of flea serine protease nucleic acid molecules comprising q~qu~nc~q listed ~above as well as additional nucleic acid molecules identif ied using the techniques described herein are presented in Table 2.

~ innRl Flea Serine Protease Sequences A. The apparent N--terminal nucleic acid seguence (SEQ ID NO:52), a3 well as deduced amino acid sequence (SEQ ID NO:53) of nfSP1 is:
TQ GCA CTC GTT GCC TTG TCT GCA GCT ATT CCT QC TCC AAC AGA GTC
S A L V A L S A a I P H S N R V

V G G L E A A E G S A P Y Q V S
TTG CAA GTT GGC AAC TTC QC TTC TGT GGT GGT TCA ATT CTG AAC GAA
L Q V G N F El F C G G S I L N E
TAT TGG GTT TTG ACT GCT GCT CAC TGT TTG GGT TAT GAC TTC GAC GTG
Y w v L T A A Ei C L G Y D F D V
GTA GTT GGA ACA AAC AAA CTT GAT CAA CQ GGT GAA AGA TAC CTC GTA
V V G T N R L D Q P G E R Y L V
GAA QA ACT TTT GTT QC
E Q T F V ~1 ~
30 B. The apparent N-terminal nucleic acid sequence (SEQ ID NO:54), as well as deduced amino acid sequence (SEQ ID NO:55) of nfSP2 is:
TTA GAT GGG CGC ATT GTT GGA GGA QA GAT GCT GAT ATT GCC AAA TAT
L D G R I Y G G Q D A D I A R Y

GGC TAT CAA GCT TQ CTC QA GTA TTT AAC GAA QT TTC TGT GGA GCT
35 G Y Q A S L Q V F N E E~ F C G A

TCA ATA TTG AAT AAT TAT TGG ATT GTC ACA GCA GCT CAT TGC ATA TAT
S I L N N Y W I V T A A EI C ~ I Y
GAT GAA TTC ACG TAT TCA GTT CGA GTC GGC ACC AGT TTC CAA GGA AGA
D E F T Y S V R V G T S F Q G R
CGT GGT TCC GTT CAT CCT GTG GCA CAA ATT ATC AAG CAT CCT GCA TAC
R G S V H P V A Q I I R El P A Y
C. The apparent N-terminal nucleic acid seguence (SEQ ID N0:56), as well as deduced amino acid seguence (SEQ ID N0:57) of nfSP4 is:
AGG GAA CAA AAG CTG GAG CTC CAC CGC GGT GCG CCG GCT CTA GAA CTA

GTG GAT CCC CCG GGT CTG CAG GAA TTG GCA CGA GGA TGT TCT TGG CTG
V D P P G L Q E L A R G C S W L
TGT TTA GTA GCT ATT CTT TGT GCA GTG GCT GCT GGG CCT ACT AAT CGC
C L V A I L C A V A A G P ~T N R
ATT GTT GGA GGA TTG GAG GCG AaA AAT GGA ATC ACC CCA TTC ATC GGT
V G G L E A K N G I T P F I G
TTC TTT GCA AGC GGA AGA CTA TTT CA
F F A S G R L F
..
D. The apparent N-terminal nucleic acid seguence (SEQ ID N0:58), as well as deduced amino acid seouence (SEQ ID NO:59) of nfSP5 i8:
ACG AGG TTT CGC TTA GCA ATT GTA TGT GCT CTC GCT GTC TGC ACA TTC
T R F R L A I V C A L A V C T F>
GGT GCC AGT GTT CCA GAA CCA TGG A~A AGA TTA GAT GGT AGA ATC GTA
G A S V P E P W R R L D G R I V>
2 5 GGA GGA CAC GAT ACC AGC ATC GAT A~A CAC CCT CAT CAA GTA TCT TTA
G G }~ D T S I D R El P E Q V S L>
TTG TAC TCC AGC CAC AAT TGT GGT GGT TCC TTG ATT GCC AaA AAC TGG
L Y S S 11 N C G G S L I A R N W>
GTT TTG ACT GCA GCT CAT TGC ATT GGA GTT AAC AAA TAC AAT GTC CGT
3 0 V L T A A E~ C I G V N K Y N V R>
E. The apparent N-terminal nucleic acid seguence (SEQ }D NO:60), as well as deduced amino acid seouence (SEQ ID N0:61) of nfSP6 is:
CCC TCA CTA AAG GGA ACA AaA GCT GGA GCT CCA CCG CGG TGC GCC GCT
P S L R G T R A G A P P R C A A

L E L V D P P G C R N S A R A F
GGT TGG ATT GAG CGC GTC TCA TCT TAC AAG ATA AAG GAT AGA TTA GAT
G W I E R V S S Y R I R D R L D
GGG CGC ATT GTT GGA GGA CAA GAT GCT GAT ATT GCC AAA TAT GGC TAT
G R I V G G Q D A D I A R Y G Y
CAA GCT TCA CTC CAA GTA CTT AAC GAA CAT TTC TGT GGA GCT
Q A S L Q V L N E El F C G A

WO 96/11706 PCI/USg5114442 F. The apparent N-terminal nucleic acid sequence (SEQ ID N0:62), as well as deduced amino acid sequence tSEQ ID N0:63) of nfSP7 is:
GCG GTG ATT GTG TCA TTT GTT CTG GCT TGT GCA TTT TCT GTA CAG GCT
A V I V S F V L A C A F S V Q A
CTT CCA TCA AGC AGA ATT GTC AAT GGA CTT GAA ~GCA GGA GTT GGA CaA
L P S S R I V X G L E A G V G Q
TTT CCA ATT CAG GTT TTC TTA GAC TTG AQ AAT ATC AGA GAC GAA AaA
F P I Q V F L D L T N I R D E K
TCC AGA TGT GGT GGT GCT TTG TTA TCA GAT TCA TGG GTT TTG ACT GCT
S R C G G A L L S D S W V L T A
GCT CAT TGT TTT GAT GAT TTG AAG TCT ATG GTA GTG TCC GTT GGT GCT
A H C F D D L K S N V V S V G A
CAT GAT GTC AGC AaA TCT GAA GAA CCT CAC AGG CAA ACC AGG AaA CCT
E D V S K S E E P H R Q T R K P
l 5 GAA
E

G. The apparent N-terminal nucleic acLd sequence (SEQ ID NO:64~, as well as deduced amino acid sequence (SEQ ID N0:6~) of nfSP12 is:
GTA CTG ATC GTT TTA GCA GTC ATT GAA TTC GCA TCA GCG TCT TCA ATC
V L I V L A V I E F A S A S S
GGC TGG AGA ATC GTG GGT GGT GAA AAT GCT AaA GAA AaA TCG GTG CCC
G W R I V G G E N A K E K S V P
TAT CAA GTT TCN CTT CGA AAT GCT GAA AAC AaA CAT TTY TGT GGR GGR
Y Q V S L R N A E N ~ H F C G G
H. The apparent N-terminal nucleic acid sequence (SEQ ID NO:66), as well as deduced amino acid ~3equence (SEQ ID NO:67) of nfSP13 is:
TTC GGC TTC AAG CTA AGT CAT TTG GTA AGT AAG TAC TGT GCT TGT GCA
F G F R L S H L V S K Y C A C A
TTA GCA TCG GCA CTG AAG TAC TCC ATC GAT CAT GGT CCT CGT ATC ATC
L A S A L K Y S I D H G P R
GGA GGT GAA GTT GCA GGT GAA GGA TCA GCA CCT TAC CAG GTG TCC TTA
G G E V A G E G S A P Y Q V S L
AGA ACC AAG GAA GGA AAT CAT TTT TGC GGT GGA TCA ATA CTA AAT AAG
R T K E G N E F C G G S I L N K

R W V V T A A H C L E P E I L D
TCG GTA TAC GTC GGA T.CC AAT CAC TTA GAC CGA AaA GGC AGA TAT TAC
S V Y V G S N H L D R K G R Y Y

GAC GTA GAA CGG TAT ATA ATT CAT GAA AaA TAT ATA GGA GAA CTA AAT
D V E R Y I I H E K Y I G E L N
AAT TTT TAT GCT GAC ATC GGT CTA ATA AaA CTT GAT GGA AGA CTT AGA
N F Y A D I G L I K L D G R L R
ATT CAA
Q

WO 96/117~6 PCT/US95/14442 I. The apparent N-terminal nucleic acid sequence (53Q ID N0:68), as well as deducea amino acid sequence ~SEQ ID N0:69) of n~5P14 is:
CGG GCT GQ GGA ATT CGG QC GBG AAG AaA CTG CCA ATA TTA ATC GCC
R A A G I R E E R R L P I L I A
TTG ATC GGA TGC GTT CTT TCT GAA GAA ATA GAG GAT CGC ATT GTC GGC
L I G C V L S E E I E D R I V G
GGA ACG GCA GTT GAT ATA AGA GGT TTT CCC TGG CAG GTA TQ ATT CAA
G T A V D I R G F P W Q V S I Q
ACC GAA AAC CGT CAT TTT TGT GGT GGT TCT ATT ATC GAT AaA AGC TGG
T E N R H F C G G S I I D R S W
ATA TTA ACT GCC GCA QT TGT GTA CNC GAT ATG AAG ATG TCG Aac TGG
L T A A H C V X D M R M S N W
J. The apparent N-termLnal nucleic acid sequence (SEQ ID N0:70), as well as deduced amino acid sequence (SEQ ID N0:71) of nfSPlS i5:
CAC GAG ATT TTA TTA AGC GQ TTA TTT GCA AGT GTA ATT TGC TCC TTT
H E I L L S A L F A S V I C S F
AAC GCG GAA GTA CAA AAT CGA ATC GTT GGT GGC AAT GAT GTA AGT ATT
N A E V Q N R I V G G N D V S
2 0 TQ AaA ATT GGG TGG QA GTA TCT ATT CAA AGT AAT AaA QA CAT TTC
S R I G W Q Y S I Q S N R Q E F
.
TGT GGT GGT TQ ATC ATT GCT AaA GAT GGG TCC
C G G S I I A R D G S
R. The apparent N-terminal nuoleic acid seguence (SEQ ID N0:72), as well as deduced amino acid sequence (SEQ ID N0:73) of nfSP16 is:
ATC ATG GCA AAT TTT AGG CTA TTC ACC TTA CTA GCC TTG GTT TCA GTA
N A N F R L F T L L A L V S V
GQ ACT TCC AaA TAT ATT GAT CQ AGA ATA ATT GGA GGC GAA GAT GCT
A T S R Y I D P R I I G G E D A

P E G S A P Y Q V S L R N Q N L
GAG CAT TTC TGT GGT GGT TCC ATT
E H F C G G S
L. The apparent N-terminal nucleic acid sequence (SEQ ID N0:74), as well as deduced amino acid sequence (SEQ ID N0:75) of nfSPl7 is:
GCA CGA GAT CGC ATT GTT GGA GGA TTG GAG GCG AaA AAT GGA TCA GCC
A R D R I V G G L E A R N G S A
CCA TTC ATG GTT TCT TTG QA GCG GAA GAC TAT TTT CAT TTT TGT GGA
P F M V S L Q A E D Y F E F C G

4 0 TCC TCT ATT CTG AAT GAG AGA TGG GTT CTT ACT GCT GCT QC TGT ATC
S S I L N E R W V L T A A H C
, CAA CQ AAT GTA QC AAG TAC GTT TAC GTC GGT TCG AAC AAC GTA GAA
Q P N V H R Y V Y V G S N N V E

WO 96/11706 PCT/11S9~/14442 M. The apparent C-terminal nucleic acid sequence (SEQ ID N0:76), as well as deduced amino acid sequence (SEQ ID N0:77) of nfSPl2 Ls:
CCA ATC CAC GAT AGC CAA TAT GCA CTT TTG CAG ATA TGG GTC AAG GGT
P I H D S Q Y A L L Q I W V R G>

5 GCA TGT AAG GGT GAT TCC GGT GGC CCC TTA GTC ATC AAT GGA CAA CTT
A C X G D S G G P L V I N G Q L>
CAT GGA ATT GTT TCC TGG GGC ATT CCT TGC GCT GTC GCA AGC CTG ATG
H G I V S W G I P C A V A S L M>
TAT TCA CAA GAG TTT CTC ATT ATG TCG ATT GGA TTA AAT CCA AaA TTG
Y S Q E F L I M S I G L N P R L>
AAT AaA ATT GTT TAG
N R I V *
N. The apparent C-termlnal nucleic acid sequence (SEQ ID N0:78), as well as deduced amino acid sequence (SEQ ID N0:79) of nfSPl3 is (the 15 initial GGPL i9 next to the conserved active-site serine):
GGA GGT CCT TTG GCA ATC AAT GGT GAA CTT GTT GGT GTT ACT TCA TTC
G G P L A I N G E L V G V T S F
ATT ATG GGG ACA TGT GGA GGA GGA CAT CCT GAT GTC TTC GGT CGA GTC
M G T C G G G H P D V F G R V
2 0 CTT GAC TTC AaA CCA TGG ATT GAT TCT CAT ATG GCA AAT GAC GGC GCT
L D F R P W I D S H M A N D G A
AAT TCT TTT ATT TAA
N S F I * .
ExamPle 12 This Example describes the purification of a flea ~m; nnpPrtidase of the present invention.
The starting material for the isolation of a flea ~m;nnpPrtidase was a flea midgut lysate preparation that had been depleted of serine proteases by passage over a bPn7Am;~;nP-Sepharose affinity column. To assay for ;~m;noppptidase activity, the synthetic substrate L-Leucine-Ar~C (Leu-AMC), which releases a fluorescent AMC
leaving group upon proteolytic cleavage, was incubated with the serine protease-depleted flea midgut preparation.
~m;nnpPptidase activity was easily detectable with as little as 1.2 ~g of lysate, both cnnf;rm;n~ the presence of wo 96/11706 PCr~TJS9~ 42 an ;lm; nop~ortidase (as indicated in other Examples herein) and allowing for the detection of Ami nnp ~ptidase activity in fractions collected throughout subsequent fractionation and purification procedures.
Serine protease-depleted flea midgut lysates (samples of about l. 2 ~g and about 12 ,ug) were incubated with Leu-AMC in the presence of the following inhibitors l mM
pefabloc, l mg/ml trypsin/~l.y ~L~sin lnhibitor, l mg/ml trypsin inhibitor, l mM TPCK, 1 ug/ml pepstatin, 10 ~Lg/ml E-64, 10 ~g/ml of leupeptin, 10 mM EDTA, and 86 ug/ml of bestatin. Only bestatin inhibited the ~lea protease that cleaved Leu--AMC, whereas both EDTA and bestatin inhibited the control protease, a leucine aminopeptidase. These results indicated that the flea protease being characterized was an ~m;nop~rtidase~ but~apparently was not a metallo-Am; nop~ortidase like the ~ classic~ leucine aminopeptidases .
A flea ~m;nnp~rtidase was purified using the following protocol. Flea midgut lysates cleared of serine protease activity were fractionated by anion-exchange chromatography. Those fractions containing aminopeptidase activity were pooled and subj ected to cation-exchange chromatography, and the resulting fractions were again assayed for activity with L-Leu-AMC in 96-well plates.
Fractions containing ~m; nnp~rtidase activity were subjected to SDS-PAGE and silver-stained to identify the protein(s) exhibiting that activity. Am;nnp~rtidase activity was found to be associated wlth proteins that ~ =

Wo 96111706 PCrlUS95/14442 migrated at a molecular weight of about 95 kD and about 56 kD when subjected to SDS-PAGE. The 95 kD and 56 kD
proteins may each be ;7m; nc7r~rtidases or they may be subunits of a larger enzyme. A number of known 5 ;7m;nhp~rtidase5 are multi-subunit enzymes comprised of subunits ranging from about 45 kD to about 55 kD and from about 90 kD to about 95 kD.
Additional purification studies have indicated that the majority of ~7m;nnp~rtidase activity was found to be 10 associated with the membrane pellet preparation and could be solubilized with detergent. Aminopeptidase activity in such preparations was also monitored during purif ication using L-J eu-AMC, and appeared to be associated with the 95 kD and 56 kD proteins when active fractions were analyzed 15 by SDS-PAGE and silver staining. The 95 kD and 56 kD
protein were co-purified to greater than 90% purity by cation exchange ~ LOyLap.-ly, affinity chromatography using w-;7~;nnh~Yyl agarose, and C-4 reverse phase chromatography. N-t~rm;n;~l amino acid sequence analysis 20 indicated that both isolated aminopeptidases appeared to be blocked at the amino t~rm; m7c .

. .
Example 13 This Example describes the isolation of a flea .7m;nnpeptidaSe nucleic acid molecule of: the present 25 invention A nucleic acid lec~ oncn~',;n~ a flea ~m;nnpl~rtidase was isolated in the following manner. A DNA fragment was

6 PCr~Ss5/14442 PCR amplified from a whole fed flea cDNA expression library (prepared as described in Example 8 using degenerate primers, the design of which was based on conserved regions of bovine lens leucine aminopeptidase (LAP) . The cr ~r; f; C
5 LAP-based primers used included: deye~ te LAP sense primer A, corr~cp~nA;n~ to bovine lens LAP amino acid sequence from about amino acid Z47 through 257 and having nucleic acid sequence 5' GTW GGW AAA GGW WTW ACW TTY GAT
TCW GGW GG 3', represented herein as SEQ ID N0: 48; and 10 degenerate IAP antisense primer C, ~OLL~ )A;n J to bovine lens LAP amino acid sequence from about amino acid 335 through 329 and having nucleic acid sequence 5' CG WCC TTC
WGC ATC WGT ATT 3', represented herein as SEQ ID N0:49.
Also used were~ vector primers having SEQ ID N0 :13 and SEQ
ID N0 :14, described in Example 11.
In a first ~Yp~; , the LAP primer C having SEQ ID
N0:49 and the M13 reverse vector primer having SEQ ID N0:13 were used to PCR amplify DNA fragments from the expression library. The resultant PCR ~LUdU~ were screened by 2 0 hybridization under standard hybridization conditions with LAP primer A having SEQ ID N0: 48 . A PCR product that hybridized with SEQ ID N0:48 was subjected to nested (actually semi-nested) PCR _ 1 i f; c~tion using LAP primer C and the T3 vector primer having SEQ ID N0: 14 . The 25 resulting PCR product, which was about 900 nucleotides in length (denoted nfAP900) and hybridized under standard (i.e., stringent) hybridization conditions with LAP primer A, was cloned into the TA'Y vector and analyzed by DNA se~l.~nre =

Wo 96111706 PCr/US95/14442 analysis as describea in Example 11.
The nucleic acid SF~ nre of a portion of nfAP900, namely of nfAP453, is represented herein as SEQ ID N0:50.
Translation of SEQ ID N0:50 yields a protein of about 151 5 amino acids, denoted herein as PfAP15~, the amino acid sequence of which is represented herein as SEQ ID NO:51.
Analysis of SEQ ID N0:51 suggests that the sequence includes a leader segment of about 15 amino acids followed by a mature protein that has about 32% identity with the 10 bovine lens LAP. The corresponding bovine and flea nucleotide qrq~T~nc.oc are about 29% identical.

Exam~le 14 This Example describes the production of an anti-flea midgut protease antiserum and its use to inhibit flea 15 protease activity thereby supporting the utility of protease-based vaccines as anti-flea agents.
Anti-flea protease antiserum was produced in the following manner. A rabbit was i ; 7ed 3 times with approximately 40-50 ~g of a flea midgut protease 20 preparation that had been affinity-purified using b.on7~m;~1;nr sepharose as described in Example 7 and then . .
combined with Freund' s complete adjuvant for the first ; 7ation and with incomplete adjuvant for the second and third ;mmlln; 7~tions according to standard procedures.
25 After the second ; ; 7~tion, endpoint titers of around 1:3200 were obtained, while the third; ;7~tion boosted the anti-protease titers to about 1:6400. Western blot WO 96/11706 PCI~/US95/14442 =~ 107 , ~ =
analysis of the; ~activity of the resultant anti-flea protease antiserum against the affinity-purified midgut protease preparation .1 Ll~ted the presence of at least

7-8 reactive protease bands. This was an important 5 observation since there are numerous reports in the literature of difficulties associated with generating high-titered antisera against certain classes of proteases.
To assess the inhibitory activity of the rabbit anti-flea protease antiserum against flea midgut proteases, an in vitro assay which measures trypsin activity as a function of Ahcn~h~n~-e at OD4so using a defined protein :~uba~.ate (i.e., succinylated casein) was established using a commercially available kit (available from Pierce, Rockford, IL) . In prol ;min~ry assays, the proteolytic 15 activity of the affinity-purified flea midgut protease preparation was about 25-3096 of the activity observed with the trypsin control. This lower activity is not unexpected since the flea proteases may require different reaction conditions than the trypsin control for optimal activity.
20 Also, the primary amino acid 5o~loncos detorm;nocl for the flea proteases as described in Examples 7, 10 and 11 are suggestive of highly specialized functions that may require specific ~ub~Llc~t~S for detorm;n;n~ optimal activity.
Incubation of the affinity purified midgut protease 25 preparation with the succinylated casein substrate in the presence of about 500 ng of the rabbit anti-flea protease antibody-containing serum cnl 1 ecto~ after the second ln; ~tion reduced the proteolytic activity of the Wo 96/11706 PcrluS95/14442 protease preparation by about 20%. This result, using a suboptimal assay, ~u~,LLs ~ the feasibility of using immunological methods to inhibit flea midgut prQtease activity .
5 ~ Using a similar ; ~ ~ation protocol, anti-flea protease antiserum has also been generated in cats that has exhibited immunoreactivity, as identified by Western blot analysis, against several proteases in the affinity-purified midgut protease preparation. The cat anti-flea lO protease antiserum also reduced proteolytic activity of an affinity purified midgut protease preparation by about 20%, using the same assay as descr~ibed for analyzing the rabbit antiserum .

ExamPle 15 ~ ~
15 = ~ This Example .1 LLclLes that fiea larvae have protlnm; n~ntly serine-type proteases .
Newly hatched flea larvae were raised in colony rearing dishes and fed on larval rearing media containing dried bovine blood using standard techniques. About 300 to 20 500 larvae were collected at different developmental stages, h~ , ;7ed by sonication in flea gut dissection buffer (50 mM Tris, 100 mM CaCl2, pH 8.0) and centrifuged to pellet cell debris. About 25 larval equivalents were i n c u b a t e d w i t h a b o u t 2 . 5 ~ ~L C i 25 t1,3-3H]-diisopropylfluororhn~rh~te (DFP) overnight at 4'C.
After incubation, about 10 larval equivalents were spotted onto filter paper, precipitated with 10% trichloroacetic -wo 96nl706 PCT/U595~l4442 109 ~ ~
acid, and counted in a liquid scintillation counter Rc~ r;n~ SDS-PAGE was performed on samples comprising about 2.5 larval equivalents, and autoradiography was pt:LL~ -' using standard techniques. In addition, adult flea midgut 5 proteases were extrac~ed and 3H-labeled in the same manner and ~'YAm; n~cl by SDS-PAGE and autoradiography. Analysis of the gel indicated that, based on their ability to be labeled with DFP, larval proteases appear to be pr~lnm;n~ntly serine-type proteases, the production of 10 which appears to be induced by blood feeding as occurs in adults. Blood-fed 3rd instars appeared to have the highest amount of proteolytic activity.

Exam~le 16 ~ :
The Example ~ LL~tes that flea feces has 15 proteolytic activity, that is pr~ rm;n~ntly due to serine proteases .
Flea feces were collected from fleas fed in flea cages placed on a live cat or in a flea feeding system as described in Example 6 in which the fleas were fed bovine 20 blood. Fresh feces were collected every 9-17 hours, resuspended in water at 150 mg feces/ml, and centrifuged to pellet insoluble material. The soluble fractions were then assayed using two terhn;~l~c~ Western blot analysis was pe r~ ~ on~ samples subjected to reducing SDS-PAGE, each 25 lane having about 40 ~g of protein. Blotted proteins were incubated overnight at 4 C with 1:500 rabbit anti-flea protease ~ antise um ~L-Jdu-:~d as described in Example 14.

wo 96111706 PCrlUS95/14442 Goat anti-rabbit ql~c~n~l~ry antibody was used at 1: 2000 to develop the Western blot. Analysis of the Western blot indicated the presence of serine-type proteases in flea feces. Appearance of such proteases migrating at about 25 5 to about 3 0 kD in such a system suggests the presence of full-length serine proteases.
Zymogram analysis was performed by loading approximately 50 ,ug protein into each lane of the electrophoresis gel in non-reducing sample buf fer. After 10 electrophoresis, the ~y ,Lalll gel was soaked in 2.5%
Triton-X-100 to renature the samples and developed at 37 C
in 50 mM Tris, pH 7.6, 200 ~M NaCl, 5 mM CaCl2, 0.02% Brij 35. Coomassie staining the gel revealed clear plaques where active proteases digested the gelatin in the gel matrix.
15 ~ Both of these techniques indicated the presence of serine-type proteases in f lea f eces .

Exam~le 17 This Example cl ~L- t_es that fleas that have fed on antibody-containing blood have ant;horl;~c in their feces, 20 suggesting an ; lo~i~Al method to eradicate flea larvae, which feed from flea feces.
The ability of Ant;ho~ in a blood meal to be taken up by fleas, pass through the midgut and be excreted in the feces was d ~L~Led in the following manner. A
25 commercially available rabbit antibody against ovalbumin was added at near physiological ~ 1LLC~t.iOn (i.e., at about 2 mg/ml) to the blood meal of adult fleas in a flea Wo 96/11706 PCrlUS95/1444Z

feeding system as aescribed in Example 6. Feces were collectea at 24 hr and 48 hr after feeding and rehydrated in phosphate saline buffer. The rehydrated fecal samples were subjected to Western blot analysis and shown to 5 contain rabbit anti-ovalbumin antibodies that were apparently full-length, using a rabbit-sp~r;~ s~ nnrl~ry antibody screen against the Fc region of rabbit antibodies.
Supernatants of flea midguts collected at the same time periods showed residual amounts of rabbit anti-ovalbumin lO antibodies. - --In a second experiment, fleas were fed in a similar manner a blood meal containing cat-specific antiserum generated against keyhole limpet hemocyanin (KL~I) and feces were collected at 24, 48 and 72 hours post-feeding. The 15 sample collected at 24 hours was divided into halves, with one half LehydLclted im~ediately in PBS while the second half was rehydrated 7 days later. Fecal samples collected at 48 and 72 hours were held for 6 and 5 days, respectively, after collection in desiccated form prior to 20 rehydration. Aliquots of the bloodmeal containing the KLEI
antiserum fed to the fleas were also sampled at l and 2 days . All of the recovered ant; horl; ~ were reactive against KLH by Western blot analysis, with a pattern or reactivity indistinguishable from the cat anti-KLEI serum 25 alone.
These studies ~ -LLaLe that antibodies are able to pass through the flea midgut in intact form and are able to maintain their antigen-binding characteristics, thereby Wo 96111706 PC r/US95~14442 supporting the feasibility of an immunologieal method to target larval development, since flea larvae in their normal habitat feed from flea feces.
Exam le 18 P . _ . .
This Example provides additional nueleie aeid and dedueed amino aeid s~rl~n~l~s of nucleic acid moleeules r~nr orlinrJ flea serine protease proteins of the present invention, examples of the isolation of ~whieh were described in Example 11.
A. Flea serine protease clone 1 was fl~t~rm; n~l to comprise flea serine protease nucleic acid molecule nfSPl779, the nueleie aeid sequenee of whieh is denoted herein as SEQ
ID NQ:80. Translation of- SEQ ID N0:80 yielded a predicted serine protease protein referred to herein as PfSPl232, the amino aeid spq~ nre of whieh is denoted herein as SEQ ID
N0:81, SEQ ID N0:80 ineluding an apparent stop codon cp~nn;nrJ about nueleotides 699 through 701. ~he N-t~rm;nllc of the mature form of PfSPl232 apparently occurs at about amino acid 17 of SEQ ID N0:81, and a conserved GWG seq~ nr.,~
spans about amino acids 132 through 134 of SEQ ID .N0:81.
PfSPl232 apparently also include~s SEQ ID N0:7 (a -partial amino t~rm;n~l 5~qn~n~ ~ of a purified serine protease protein, the production of which is rl.ocer; hP~ in Example 10). nfSPl779 also d~aLellLly includes SEQ ID N0:52 and SEQ
ID N0:18 and, as such, PfSPl232 apparently ;nr~lllr~r~c SEQ ID
NO:53 and SEQ ID N0:19. A BLAST homology seareh indieated that SEQ ID NO:81 was most similar in amino aeid s~

wo 96111706 PcT~sgsn4442 to an Anopheles gamoiae ~ lly LLy~in II and to a kallikrein. As is the case for any of these molecules, variations between sequences may be due to a number of factors, such as but not limited to, sequencing errors or allelic variation.
B. Flea serine protease clone 2 was ~ tc~n; n~d to comprise flea serine protease nucleic acid molecule nfSP2944, the nucleic acid sequence of which is denoted herein as SEQ
ID N0:82. Translation of SEQ ID N0:82 yielded a predicted serine protease protein referred to herein as PfSP2255, the amino acid sequence of which is denoted herein as SEQ ID
N0:83, SEQ ID NO:82 including an apparent stop codon spanning about nucleotides 768 through 770. The N-t~rm;n .c of the mature form of PfsP22ss apparently occurs at about amino acid 23 of SEQ ID N0:81, and a L~ seLv~:d GWG qFqu~nrr spans about amino acids 148 through 150 of SEQ ID NO: 83 PfSP2zss apparently also includes SEQ ID N0:5 (a partial amino tF~rm;nA~ q~qn ~nre of a purified serine protease protein, the production of which is described in Example 10). nfSP2944 also apparently includes SEQ ID NO:20, and, as such, PfSP2zss apparently ;nrl~ q SEQ ID N0:21. A BLAST
homology search indicated that SEQ ID N0:83 was most similar in amino acid sequence to a Bom~ix mori vitellin-degrading protease ~Le.:ULi,~L.
~ C. Flea serine protease clone 3 was ~ t~rm; n~d to comprise flea serine protease nucleic acid molecule nfsP3177, the nucleic acid q~ e of which is denoted herein as SEQ
ID NO:22. ~ Translation of SEQ ID N0:22 yielded a predicted wo 96111706 PCr/US9jll44~2 serine protease protein referred to herein as PfSP3s9, the amino acid s~ nre of which is denoted herein as SEQ ID
N0: 23 . Flea serine protease protein PfSP3s9 ; n~r~ c a conserved GWG sequence that spans about amino acids 1 5 through 3 of SEQ ID N0:23. A BLAST homology search indicated that SEQ ID N0: 23 was most similar in amino acid sequence to a rat trypsinogen.
D. Flea serine protease clone 4 was det~ min~l to comprise flea serine protease nucleic acid molecule nfSP4672 lO described in Example 11. Nucleic acid molecule nfSP4672 has nucleic acid sequence SEQ ID N0:16, translation of which yielded a predicted serine protease protein referred to herein as PfSP4223, the amino acid sequence of which is denoted herein as SEQ ID NO:17, SEQ ID N0:16 including an 15 apparent stop codon s~:~nn;n-J about nucleotides 670 through 672. As stated above, the N-tr~l~m;nllc of the mature form of PfSP4z23 apparently occurs at about amino acid 5 of SEQ ID
N0:17, and a cu.lselv~d GWG cr~ nre spans about amino acids 124 through 126 of SEQ ID N0:17. PfSP4223 apparently 20 includes SEQ ID N0:41 and a 5~ nre that is very similar to SEQ ID N0:45. nfSP4672 also apparently includes SEQ ID
N0:24, SEQ ID NO:56 (following nucleotide 141 of ~SEQ ID
NO:56) and SEQ ID N0:74. As such, PfSP4z23 apparently contains SEQ ID N0:25 and SEQ ID N0:75 as well as a 25 sequence that is very similar to SEQ ID N0:57 (following amino acid 47 of SEQ ID N0:57). A BI~ST homology search indicated that SEQ ID N0: 17 was most similar in amino acid sequence to an A. gambiae ~ lly LLy~sin I precursor.

WO 96/11706 PCT/~JS95/14442 - 115 __ E. Flea serine protease clone 5 was det~m;nprl to comprise flea serine protease nucleic acid molecules:
nfSP51s7, the nucleic acid CPq~lPn~e of which is denoted herein as SEQ ID NO: 84; and nfSP5z~8, the nucleic acid 5 sequence of which is denoted herein as SEO ID N0:86.
Translation of SEQ ID N0:84 yielded a predicted serine protease protein referred to herein as PfSP5s2, the amino acid sequence of which is denoted herein as SEQ ID N0:85.
The N-tPrm; nllc of the mature form of the serine protease 10 protein encoded by flea clone 5 apparently occurs at about amino acid 29 of SEQ ID N0:85. SEQ ID N0:85 apparently includes the first 10 amino acids of SEQ ID N0:2 as well as the first 10 amino acids of SEQ ID N0:6. SEQ ID N0:2 and SEQ ID N0: 6 are partial N-tpl-m;nAl qpq~lpncoc of purified 15 serine protease proteins, the production of which are described in Example 10. ~ranslation of SEO ID NO: 86 yielded a predicted 6erine protease protein referred to herein as PfSP572, the amino acid 5pqnpnce of which is denoted herein as SEQ ID N0:87. PfSP572 includes a 20 conserved GWG sequence SpAnn;ng about amino acids 14 through 16 of SEQ ID N0:87. nfSP5218 apparently ;nrllltlPc SEQ
ID N0:26, ana, as such, PfSP572 apparently includes SEQ ID
N0:27. A BI,AST homology gearch indicated that the protein encoded by flea clone 5 was most similar in amino acid 25 sequence to a Drosophila trypsin eta precursor.
F. Flea serine protease clone 6 was detP~n;nPd to comprise flea serine protease nucleic acid molecule nfSP6932, the nucleic acid sPquPn~e of which is denoted herein as SEQ

Wo 96/11706 PCTIUS95/14442 lI6 ID N0:88. Translation of SEQ ID N0:88 yielded a predicted serine protease protein referred to herein as PfSP6zs6, the amino acid SP~7Pn,~e of which is denoted herein as SEQ ID
N0:89, SEQ ID NO:88 including an apparent stop codon ~r~nn;n~ nucleotides 770 through 772. The N-tPrmimlq of the mature form of PfSP62s6 apparently occurs at about amino acid 26 of SEQ ID N0:89, and a conserved GWG sequence spans about amino acids 149 through 151 of SEQ ID N0:89. PfSP6zs6 apparently also includes SEQ ID N0: 4 (a partial amino 10 tP~rn;n;~l SPq~nre of a purified serine protease protein, the production of which is described in Example=~ 10).
nfSP6932 also apparently includes SEQ ID N0:28, SEQ ID N0:54 and SEQ ID N0: 60 (following about nucleotide 111 of SEQ ID
N0:60). As such, PfSP6z56 apparently includes SEQ ID N0:29, SEQ ID N0:55 and SEQ ID N0:61 (following about amino acid 37 of SEQ ID N0: 61) . A BLAST homology search indicated that SEQ ID N0:89 was most similar in amino acid sequence to a B. mori vitellin-degrading protease. ~
G. Flea serine protease clone 7 was flPtP~m; nPd to comprise flea serine protease nucleic acid molecule nfSP7894, the nucleic acid Se~l~onre of which is denoted herein as SEQ
ID N0:90. Translation of SEQ ID N0:90 yielded a predicted serine protease protein referred to herein as PfSP7zss, the amino acid sPquPnre of which is denoted herein as SEQ ID
NO:91, SEQ ID N0:90 including an apparent stop codon Sp~nn;n~ about nucleotides 766 through 768. The N-tPrm;n~

of the mature form of PfSP7zss d~ar~.,l,ly occurs at about amino acid 23 of SEQ ID N0:91, and a conserved GWG (in this -wo 96111706 PCTrlJS95rl4442 case GWA) 5~qn~n~ spans about amino acids 152 through 154 of SEQ ID N0:91. PfSP7zss apparently also includes SEQ ID
N0:44. nfSP7894 also apparently includes SEQ ID NO:30 and SEQ ID N0:62. As such, PfSP7zss apparently includes SEQ ID
N0:31 and SEQ ID N0:63. A BLAST homology search indicated that SEQ ID N0: 91 was most similar in amino acid sequence to a hornet L:lly LL~sin II and to collagenase.
EI. Flea serine protease clone 8 was ~,~t.orm;n~ to comprise flea serine protease nucleic acid molecule nfSP8z99, the nucleic acid sequence of which is denoted herein as SEQ
ID N0:92. Translation of SEQ ID N0:92 yielded a predicted serine protease protein referred to herein as PfSP899, the amino acid sequence of which is denoted herein as SEQ ID
N0:93. PfSP8n includes a conserved GWG .C.~Tll~nre that spans about amino acids 31 through 33 of SEQ ID N0:93. nfSP8z99 also apparently ;nrll~ c SEQ ID N0:32, and, as such, PfSP899 apparently ;n~ c SEQ ID N0:33. A BLAST homology search indicated that SEQ ID N0: 93 was most similar in amino acid c~ e to a Tachypleus tridentatus coaguiation factor B.
I. Flea serine protease clone 9 was det~mn;n~d to comprise flea serine protease nucleic acid molecule nfSP9z66, the nucleic acid sequence of which is denoted herein as SEQ
ID N0:94. Translation of SEQ ID N0:94 yielded a predicted serine protease protein referred to herein as PfSP98a, the amino acid ~ n~e of which is denoted herein as SEQ ID
N0:95. PfSP988 includes a conserved GWG sequence that spans amino acids about 33 through 35 of SEQ ID N0:95. nfSP9z66 also d~J~lLellLly includes SEQ ID N0:34, and, as such, PfSP988 Wo 96tll706 PCrNS95ll4442 apparently includes SEQ ID N0: 35. A ~AST homology search indicated that SEQ ID N0: 95 was most similar in amino acid sequence to an A. gambiae trypsin 2 precursor.
J. Flea serine protease clone 10 was det-~m; n~ to 5 comprise flea serine protease nucleic acid molecule nfSP10378, the nucleic acid sequence of which is denoted herein as SEQ ID N0:96. Translation of SEQ ID NO:96 yielded a predicted serine protease protein referred to herein as PfSP10126, the a~ino acid sequence of which is denoted herein as SEQ ID NO:97. PfSPl0126 includes a conserved GWG sequence that spans about amino acids 63 through 65 of SEQ ID N0:97. nfSPl0378 also apparently includes SEQ ID NO:36, and, as such, PfSPl0126 apparently includes SEQ ID N0:37. A BLAST homology search indicated that SEQ ID N0: 97 was most similar in amino acid s~qu~nre to an A. gambiae trypsin 1 precursor.
K_ Flea serine protease clone 11 was rg~t~rm;n~ to comprise flea- serine protease nucleic acid molecule nfSP112s2, the nucleic acid s~qU~nre of which is denoted herein as SEQ ID N0:98. Translation of SEQ ID N0:98 yielded a predicted serine protease protein referred to herein as PfSPl184, the amino acid sequence of which is denoted herein as SEQ ID N0: 99 . PfSP1184 includes a conserved GWG 5~qnrnce that spans about amino acids 23 through 25 of SEQ ID N0:99. nfSPl12s2 also aRparently includes SEQ ID N0:38, and, as such, PfSPl184 apparently includes SEQ ID N0:39. A BIAST homology search indicated that SEQ ID N0: 99 was most similar in amino acid S~ nre wo 96tll706 PCT/US9Sn4~42 to a Mus musculus ~l~F~1nn~en precursor.
L. Flea serine protease clone 12 was det~rm;n~ to comprise flea serine protease nucleic acid molecules:
nfSPl2144, the nucleic acid qPq~ nre of which is denoted herein as SEQ ID N0:64; and nfSP12zs, the nucleic acid sequence of which is denoted herein as SEQ ID N0: 100.
Translation of SEQ ID N0: 64 yielded a predicted serine protease protein referred to herein as PfSPl2s2, the amino acid sequence of which is denoted herein as SEQ ID N0: 65.
The N-tGrm; nl7q of the mature form of the serine protease protein encoded by flea clone 12 apparently occurs at about amino acid 20 of SEQ ID N0:65. SEQ ID NO:65 apparently includes the first 30 amino acids of SEQ ID N0:40.
Translation of SEQ ID N0 :100 yielded a predicted serine protease protein referred to herein as PfSPl269, the amino acid sequence of which is denoted herein as SEQ ID N0: 101, SEQ ID NO:100 apparently containing a stop codon sp:~nn;n~
from about nucleotides 208 through 210. nfSPl222s apparently includes SEQ ID N0:76, and, as such, PfSPl269 apparently includes SEQ ID N0: 77 . A BLAST homology search indicated that the protein encoded by flea clone 12 was most similar in amino acid sequence to an A. gaml~iae trypsin.
M. Flea serine protease clone 13 was detrrm; n~cl to comprise flea serine protease nucleic acid molecule nfSP13850, the nucleic acid q~l.onre of which is denoted herein as SEQ ID N0: 102 . Translation of SEQ ID N0: 102 yielded a predicted serine protease protein referred to herein as PfSP132s2, the amino acid SF~ onne of which is .

Wo 96/11706 PCrlUSs5/14442 denoted herein as SEQ ID NO:103, SEQ ID N0:102 ;n~ 1n~ an apparent stop eodon spanning about nucleotides 758 through 760. The N-tPrm;n~lc of the mature form of PfSP132sz apparently oeeurs at about amino aeid 28 of SEQ ID N0:103, 5 and a eonserved GWG sequenee spans about amino aeids 137 through 139 of SEQ ID N0 :103 . PfSPl32sz apparently also ineludes SEQ ID N0:1 (a partial amino tprm;n~l sequence of a purified serine protease protein, the production of which is described in Example 10) and SEQ ID NO:42. nfSPl3850 also apparently includes SEQ ID N0:66 and SEQ ID N0:78 and, as such, PfSP132s2 apparently includes SEQ ID N0: 67 and SEQ ID
N0:79. A BLAST homology search indicated that SEQ ID
N0: 103 was most similar in amino acid sequence to an A.
gambiae ~;I.y ~Ly~sin.
N. Flea serine protease clone 14 was detPrm;nP~ to comprise flea serine protease nucleic acid molecule nfSP142~3, the nucleic aeid ~Pq-lPnr~ of whieh is denoted herein as SEQ ID N0:68. Translation of SEQ ID N0:68 yielded a predicted serine protease protein referred to 20 herein as PfSPl471, the amino aeid CPqnPnce of whieh is denoted herein as SEQ ID N0: 69. The N-torm;nllc of the mature form of the serine protease protein eneoded by flea clone 12 apparently oceurs at about amino acid 29 of SEQ ID
N0:69. SEQ ID N0:69 apparently includes the first 13 amino 25 acids of SEQ ID N0: 43 . A BLAST homology search indicated that the protein encoded by SEQ ID N0:69 was most similar in amino aeid SP~lPnre to an A. gambiae trypsin.
o. Flea serine protease clone 15 was ~lptprm; nPd to WO 96~11706 PCI'~US95A4442 comprise flea serine protease nucleic acid molecule nfSP152s2, the nucleic acid sequence of which is denoted herein as SEQ ID NO:104. Translation of SEQ ID N0:104 yielded a predicted serine protease protein referred to 5 herein as PfSPl584, the amino acid seqn~nre of which is denoted herein as SEQ ID N0:105. The N-tor-m;nllq of the mature form of the serine protease protein encoded by flea clone 15 apparently occurs at about amino acid 28 of SEQ ID
NO:105. SEQ ID NO:105 apparently includes SEQ ID N0:46.
nfSP152s2 also apparently includes SEQ ID N0:70, and, as such, PfSPl584 apparently includes SEQ ID N0:71. A BLAST
homology search indicated that the protein encoded by SEQ
ID N0 :105 was most similar in amino acid seqn~n~e to an A.
gambiae trypsin .
~ P. Flea ~serine protease clone 16 was detc~ ; n~ to comprise flea serine protease nucleic acid molecule nfSP16168, the nucleic acid 5c,qn~.nro of which is denoted herein as SEQ ID N0:72. Translation of SEQ ID N0:72 yielded a predicted serine protease protein referred to 20 herein as PfSP16~6, the amino acid qPqnPn~e of which is denoted herein as SEQ ID N0:73. The N-t~;mlq of the mature f orm of the serine protease protein encoded by f lea clone 16 apparently occurs at about amino acid 26 of SEQ ID
N0:73. SEQ ID N0:73 apparently includes SEQ ID N0:47. A
25 BLAST homology search indicated that the protein encoded by SEQ ID N0:73 was most similar in amino acid sequence to an acrosin .

Wo 96111706 PCrlUSs5ll4442 Q. Flea serine protease clone 18 was rlet.~rmin~d to comprise flea serine Rrotease nucleic acid molecule nfSP18s34, the nucleic acid sequence of which is denoted herein as SEQ ID N0: 106. Translation of SEQ ID N0: 106 5 yielded a predicted serine protease protein referred to herein as PfSP18178, the amino acid sequence of which is denoted herein as SEQ ID N0:107. The N-t~rm;nllc of the mature form of PfSP18178 apparently occurs at about amino acid 284 of SEQ ID N0:107, and a conserved GWG sequence spans about amino acids 126 through 128 of SEQ ID N0:107.
A BLAST homology search indicated that SEQ ID N0: 107 was most similar in amino acid sequence to a~ y ~L~sin.
R. Flea serine protease clone 19 was ~ t~rm;n.--1 to comprise flea serine protease nucleic acid molecule nfSPl93s9, the nucleic acid s~ n~-e of which is denoted herein as SEQ ID N0: 108 . Translation of SEQ ID N0: 108 yielded a predicted serine protease protein referred to herein as PfSPl9119, the amino acid q~ nre of which is denoted herein as SEQ ID N0 :109 . A conserved GWG sequence spans about amino acids 69 through 71 of SEQ ID N0:109. A
BLaST homology search indicated that SEQ ID N0:109 was most similar~ in amino acid s~ n~-e to bovine dl~n~F~nAce I.
S. Flea serine protease clone 20 was det~rm;n~o~ to =
comprise flea serine protease nucleic acid molecule nfSP20K1, the nucleic acid sequence of which is denoted herein as SEQ ID N0: llO. Translation of SEQ ID N0: llO
yielded a predicted serine protease protein referred to herein as PfSP20248, the amino acid sPqu~n-e of which is wo 96/11706 Pcrrrrsgsrl4442 denoted herein as SEQ ID N0 :111, SEQ ID N0 :110 including an apparent stop codon crRnnin~ about nucleotides 746 through 748. The N-t~rm;nllc of the mature form of PfSP20248 apparently occurs at about amino acid 27 of SEQ ID N0: 111, 5 and a conserved GWG sequence spans about amino acids 147 through 149 of SEQ ID N0:111. PfSP20248 apparently also includes SEQ ID N0:2 and SEQ ID N0:6 (partial amino tGrm;nRl sequences of purified serine protease proteins, the production of which is described in Example 10).
nfSP20841 also apparently includes SEQ ID N0: 58, and, as such, PfSP20248 apparently ;nrl~ c SEQ ID N0:59. A BLAST
homology search indicated that SEQ ID N0: 111 was most similar in amino acid s~ c~nre to a ~trypsin.

Examl~le 19 This Example provides additional nucleic acid and deduced amino acid s~quc~n~c of nucleic acid molecules ~nC~;n~ flea Rm;nnp-~rtidase proteins of the present invention.
The nucleic acid sequence of the rc---; nrl~r of flea 20 aminopeptidase nucleic acid molecule nfAP900 was ~ t~rm;n~d and used to design primers to use in combination with a vector primer (i.e., M13 universal primer) to PCR amplify the 3' torm;nRl ~ _ ~ of the flea Rm;nnp~rtidase coding region from a whole fed flea cDNA expression library using 25 methods as described in Example 13~ The PCR product was subjected to DNA S~l.,nr;n~ analysis, and a composite sequence Le:~L.~ II ;n~ a close to full-length flea Wo 96/11706 PCr/USs~ll4442 :~m; n~p~rtida5e coding region was deduced. The nucleic acid sequence of the composite nucleic acid molecule, referred to herein as nfAP1s80, is denoted herein as SEQ ID N0: 112 .
The primer used to obtain the 3' t~rmin:~l r,. ~ spans from about nucleotide 849 through 877 of SEQ ID N0:112. A
probe sr~nn;n~ from about nucleotide 918 through 938 of SEQ
ID N0:112 was used to verify that the 3' tormin~l fragment was a flea aminopeptidase nucleic acid molecule. The flea aminopeptidase gene-containing sequence of the 3' t~rmin:~l ~fragment, referred to herein as nfAP73z, spans from about nucleotide 849 through 1580 of SEQ ID N0: 112 .
Translation of SEQ ID N0:112 yielded a deduced flea ~m;n~p~rtida5e protein of about 496 amino acids, denoted herein as PfAP496, having amino acid sequence SEQ ID N0:113.
The deduced mature flea Am; nn~rtidase is about 48%
identical to mature bovine leucine ~minnrPrtidase The L~ l;nJ bovine and flea nucleic acid s~ onn~R are about 3 3 % identical .
ExamPle 20 This Example ~ tes the production of certain flea serine protease proteins of the present invention.
A. Flea serine protease protein PfSP1216 was produced in the following manner. An about 670-bp DNA fragment, referred to herein as nfSP1670 and d~Riqn~ to encode an apparently mature serine protease protein, was PCR
,lifi.~d from flea serine protease clone 1 using the XhoI-site containing primers F1 sense 5' Wo 96/11706 PCr/USs5ll4442 GAGCTCTCGAGA~~ TGGAAGC 3' (SEQ ID NO:114) and F1 antisense 5' GGACCTCGAGAATTAGTTATTTTCCATGGTC 3' tSEQ ID
NO:115). The PCR product nfSP167c was digested with XhoI
restriction Pn~nn~ l P~Ce~ gel purified and subcloned into 5 Pxpression vector pTrcHisB (available from InVitrogen) that had been digested with XhQI and ~lprhncrhnnylated. The resultant recombinant molecule, referred to herein as pHis-nfSPl670, was transformed into E. co1i EIB101 competent cells ~available from Gibco BRL, Gaithersburg, MD) to form 10 recombinant cell E. cQli :pHis-nfSP1670. The recombinant cell was cultured in enriched bacterial growt_ medium containing 0.1 mg/ml ampicillin and 0.1% glucose at about 32 C. When the cells reached an OD600 of about 0.4-0.5, expression of nfSP167c was induced by the addition of 0. 5 mM
15 isopropyl-B-D-thiogalactoside (IPTG), and the cells were cultured for about 2 hours at about 32~C. Immunoblot analysis of recombinant cell E. coli :pHis-nfSPl670 lysates using a T7 tag nnlnnAl antibody (available from Novagen, Inc., Madison, WI) directed against the fusiQn portion of 20 the recombinant PEIIS-PfSPl2l6 fusion protein identified a protein of the d~)~LV~I' iate size, namely an about 29 kD
protein .
B. Flea serine protease protein PfSP2233 was produced in the following manner. An about 715-bp DNA fragment, 25 referred to herein as nfSP271s and r9Pc; ~nPd to encode an apparently mature serine protease protein, was PCR
amplified from flea serine protease clone 2 using the XhoI-site containing primers F2 sense 5' ~- -Wo 96111706 PcrluS9S/14442 GAGCTCTCGAGCATCGTCGGCGGCACCAGTG 3' (SEQ ID N0:116) and F2 antisense 5' (~ r~A~T(~TTA~Ar~ V~ 3 (SEQ ID
N0: 117) . The PCR product nfSP271s was digested with XhoI
restriction Pn~lnn~ lea5e, gel purified and subcloned into expression vector pTrcHisB (available from InVitrogen) that had been digested with XhoI and ~l~rhnc~hnrylated. The resultant recombinant molecule, referred to herein as pHis-nfSP2715, was transformed into E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E. coli :pHis-nfSP27ls~ The recombinant cell was cultured as described in Example 20A. ImmunoblQt analysis of recombinant cell E. coli:pEIis-nfSP271s lysates using a T7 tag monoclonal antibody (available from Novagen, Inc. ) directed against the fusion portion of the recombinant PHIS-PfSP2233 fusion protein identified a protein of ~ the c.~ yr iate size, namely an about 35-kD protein.
C. Flea serine protease protein PfSP1322s was 1~ 'P~
in the following manner~ An about 700-bp DNA fragment, referred to herein as nfSPl3700 and ~-c;~ned to encode an apparently mature serine protease protein, was PCR
amplified from flea serine protease clone 13 using the XhoI-site containing primers F13 sense 5' GAGCTCTCGAGTATCATCGGAGGTGAAGTTGC 3' (SEQ ID N0:118) and F13 antisense 5' GGACCTCGAGAATTATGCGCCGTCATTTGC 3' (SEQ ID
N0: 119) . The PCR product nfSPl3700 was digested with XhoI
restriction Pn~nn~rlease, gel purified and subcloned into expression vector pTrcHisB (available from InVitrogen) that had been digested with XhoI and ~prhncrhnrylated~ The Wo 96/11706 PC~/US95114442 resultant recombinant molecule, referred to herein as plIis-nfSP13700, was transformed into E. coli ~BlOl competent cells (available ~rom Gibco BRL) to form recombinant cell E. coli :pEIis-nfSPl3700. The recombinant cell was cultured as described in Example 20A. T -hl ot analysis of recombinant cell E. coli :pHis-nfSPl3700 lysates using a T7 tag monoclonal antibody (available from Novagen, Inc. ) directed against the fusion portion of the recombinant PllIS-PfSP1322s fusion protein identified a protein of the appropriate size, namely an about 33-kD protein.
Exam~le 21 This Example .1 LLc-tes the temporal induction of serine proteases in fleas feeding on cats.
Fleas contained in rh:~mh~rs similar to those used for in vitro feeding experiments were placed on cats and were allowed to feed for various periods of time. Upon removal from the cats, soluble extracts of flea midgut tissues were prepared as described herein. Proteases contained within the extracts were quantitated by l;~h~l ;n~ the extracts with [1,3-3H]DFP using a method similar to that described in Borovs]cy et al, 1988, Arcll. Insect Biochem. Physiol. 7, 187-210. The labeled samples either (a) were precipitated and the radioactivity in the precipitate quantitated or (b) were applied to SDS-PAGE and exposed by autoradiography.
Data generated from counting samples are shown in Fig.
10, which plots [3EI]-DFP in counts per minute (cpm) per lO
flea midguts versus hours of flea feeding on cats. Also Wo 96111706 PCr/USs5/l4442 shown in Fig. 10 for comparison is a plot of the number of eggs laid per female flea per day for chamber-contained fleas feeding on cats for 1, 2, 3 and 4 days, respectively.
These results suggest that DFP-labeled proteases ( i . e ., 5 pr~n~;n~ntly serine proteases) are induced in fleas in response to feeding. Induction is ~uite rapid once feeding begins and, unlike in mosquitos, is sustained over~ time.
The results also suggest a positive correlation between flea protease activity and fecundity.
In order to obtain a profile of the sizes of DFP-labeled proteases temporally induced in fleas during feeding, samples were applied to SDS-PAGE and autoradiographed. Fig. 11 indicates protein molecular weight standards and samples of soluble flea midgut 15 extracts obtained from fleas having fed for various times on cats (i.e., at, respectively, 3, 8, 15, 18, 24, 30, 34, 40, 44, 48, 52, 58, 68, 72, 78 and 88 hours of feeding).
Analysis of the results indicates that primarily proteases migrating with a molecular weight of about 25-35 kD are 20 induced in a flea relatively soon after the flea has begun feeding (i.e., at least within about 3 to about 8 hours).
The amount of such proteases increases over time for about the first 2 days. Over time, several intensely labeled bands of lower molecular weight (primarily in the range of 25 about 12-15 kD) also appear that may be representative of proteases having u,ldelyu,.e degradation.

:i ~ yu~ c~; LISTING
The following Sequence Listing is submitted ursuant P
to 37 CFR 1.821. A copy in computer readable form is also submitted herewith.
Applicants assert ~urauclllL to 37 CFR 1.821(f) that the content of the paper and computer readable copies of SEQ ID NO :1 through SEQ ID NO :119 submitted herewith are the same.
(1) GENERAL INFORMATION:
lû (i) APPLICANTS: Grieve, Robert B.
Rushlow, Keith E.
Hunter, Shirley Wu Frank, Glenn R.
Heath, Andrew W.
~ ~ ~ Yamaka, MilOE Yamanaka Arf sten Ann Dale, Béverly Stiegler, Gary (ii) TITLE OF lNV~~ )N: USE OF PROTEASE lN~ll~l'lUlCS AND
2 û PROTEASE VACCINES TO PROTECT ANIMALS FROM FLEA
INFESTATION, AND FLEA PROTEASE PROTEINS, NUCLEIC
ACID MOLECULES, AND USES THEREOF
(iii) NUMBER OF ~ U~N~ S: 119 (iv) UO~S.:i~UNL)~:;NC~; ADDRESS:
(Aj AnnRRs~RR: Sheridan Ross & McIntosh ~B STREET: 1700 Lincoln Street, Suite 3500 ~ C I CITY: Denver (Dl STATE: Colorado 3 0 ( E ) COUNTRY: USA
(F) ZIP: 80203 (V) ~U._~U'l'~:K RRAnART.R FORN- ~
(A) MEDIUM TYPE: Floppy disk (B) ~:o..~u ~s~: IBM PC compatible (C) OPERATING SYSTEM: PC--DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l.o, Version 1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) ClASSIFICATION:
, .

Wo 96~11706 PCr/USs5/14442 ~viii~ ATTORNEY/AGENT INFORMATION:
(A) NAME: Gary J. Connell (B) REGISTRATION N~r~BER: 32, 020 (C) ~EFERENCE/DOC~ET NUMBER:
~ =(ix) TELECON~qUNICATION INFORMATION:
(A) TELEPHONE: (303) 863--9~00 (B~ TELEFAX: (303 863--0223 . .

wo 96/11706 Pcr/usg5/l4442 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE ~ R~r~RTc~Tcq (A) LENGTE~: 32 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: Linear ( ii ) MOLECULE TYPE: protein (xi) SEQBENCE L~:;sL:Kl~LluN: SEQ ID NO:1:
Ile Ile Gly Gly Glu Val ala Gly Glu Gly Ser Ala Pro Tyr Gln Val Ser Leu Arg Thr Lys Glu Gly Asn Eis Phe Ser Gly Gly Ser Ile Leu (2) INFOR~ATION FOR SEQ ID NO:2:
( i) SEQUENCE r~ RT~ .'.Ll~;b (A) LENGT}~: 17 amino acids (B) TYPE amino acid (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
2 0 Xaa Val Gly Gly is Asp Thr Ser Ile Asp Xaa Hi3 Pro }lis G n Val Thr , (2) INFOR~SATION FOR SEQ ID NO:3:
(i) SEQ~ENCE ~ R1~oT7;~RTcTTl~c (A) LENGTEi: 22 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) ~OLECULE TYPE: protein (xi) SEQUENCE I~ISSl:Kl~LlUN: SEQ ID NO:3:
Ile Val Gly Gly Ala Asp Ala Ala Pro Gly Asn Ala Pro Phe Gln Val Ser Leu Arg Asp Lys Gly _ .

(2) INFORUATION FOP~ SEQ ID NO:4:
(i) SEQUENCE r~l~RP~ I'.LlUD:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) ~IOLECULE TYPE: protein (xi) SEQUENOE LlL~D~l~'lUW: SEQ ID NO:4:
Ile Val Gly Gly Gln Asp Ala Rsp Ile Ala Lys Tyr Gly Tyr Gln Ala 5 l0 15 Ser Leu Gln Val Phe Asn Glu His Phe Xaa Gly Ala Xaa Ile Leu Asn Asn Tyr ~2) INFORUATION FOR SEQ ID NO:5:
15 (i) SEQUENCE rT~R~"rTRTC~T
(A) LENGTH: 25 amino acids ( B ) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) NOLECULE TYPE: protein (xi) SEQUENOE u:;Dunl~LluN: SEQ ID NO:5:
Ile Val Gly Gly Thr Asp Val Asn Ile Glu Asn Phe Gly Trp Gln val Ser Leu Phe Asp Arg Asn Gly His Phe (2) INFORUATION FOR SEQ ID NO:6:
( i) SEQUENOE r~n~Rp", ~."T~ ", ~, (A) LENGTE~: 22 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) ~SOLECULE TYPE: protein (xi) SEQUENOE L~DDL~ 1UN: SEQ ID NO:6:
Ile Val Gly Gly His Asp Thr Ser Ile Asp Lys His Pro Phe Gln Val Ser Leu Ile Asp Lys Asn 12) INFOR~SATION FOR SEQ ID NO:7:
~ i ) SEQUENCE r~7DDD~ O:s:
- (A) LENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear IOLECULE TYPE: protein (xi) SEQUENOE i~E~-.:KlrLluN: SEQ ID NO:7:
Val Val Gly Gly Leu Glu Ala Ala Glu Gly Ser Ala Pro Tyr Gln Val 5 -~ 10 15 Xaa Leu Gln Trp Gly Asn Phe (2) INFORIIATION FOR SEQ ID NO:8:
(i) SEQUENCE ~T7DDD~T~RTcTIcs:
(A) LENGTH: lS amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: protein (xi) SEQUENOE Il~ Kl~lUl~: SEQ ID NO:8:
Ile Val Gly Gly Glu Asp Ala Glu Leu Gly Glu Xaa Pro Thr Gln l 5 l0 lS
(2) INFODQ5ATION FOR SEQ ID NO:9:
(i) SEQUENCE ~'TT~DT~'~DTCTTt'C
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) ~OLECULE TYPE: protein (xi) SEQJENCE D~ ;K1~1~JN: SEQ ID NO:9:
Asp Glu Asp Gly Lys Asp Asp Ser Ala Pro Gly Glu Ile .
.

(2) INFORMATION FOR SEQ ID NO:10:
i ) SEQUENCE rTTpo~rTForcTIcs (A) LENGTH: 9 amino acids ( B ) TYPE: amino acid (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: protein ( ix ) FEATURE:
(A) NAME/l~EY: Xaa = Gln or Asn (B) LOCATION: 2 10 (xi) SEQUENOE Ll~:S~:n~ uN: SEQ ID NO:10:
Cys Xaa Gly Asp Ser Gly Gly Pro :Leu (2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQ~ENCE ~T7P~P~T~RTcTICS
15 ~ (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STOP~Fn'--''9: single (D) TOPOLOGY: linear (ii) 15OLECULE TYPE: DNA (primer) 20 (xi) SEQUENOE l~S~nl~Ll~: SEQ ID NO:ll:
T~rM~ rw CCYGAATCTC CCTGGCA 27 (2) INFOF~MATION FOR SEQ ID NO:12:
(i) SEQUENCE ~TI~rP~ ~L~
(A) LENGTH: 24 base pairs (B) TYPE: nucleLc acid (C) sTPr : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (primer) (xi) SEQUENCE ~ nl~Lluw: SEQ ID NO:12:
30 TPr ~ ~p GARTCTCCTT GACA 24 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENOE O~TPOPrTr~orcTIc5 ..
(A) LENGTH: 19 base pairs ( B ) TYPE nucleic acid (C) .eTl~PNnlPnN~!CC: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (primer) (xi) SEQUENOE u~ nl~Ll~: SEQ ID NO:13:
;~Pr~Pl'r~-:CT ATGACCATG 19 WO 96/11706 ~ = PCT/US95/14442 (2) INFOR~5ATION FOR SEQ ID NO:14:
( i ) SF!QUENCE I~TTPRP~ L l~
A) LENGTH: 17 ba~e pairs B ) TYPE: nucleLc acid , C) .5'rRPNnP'nN~CC: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (primer) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

l0 (2) INFORXATION FOR SEQ ID NO:15:
( i ) SEQUENCE r~7PRP~T~RT~cTT~c (A) LENGTE~: 23 base pairs (B) TYPE: nucleic acid (C) S~RPNnFnN~CC: single ( D ) TOPOLOGY: linear (ii~ I~OLECULE TYPE: DNA (probe) (xi) SEQUENCE L~ :,s:nl~J~ : SEQ ID NO:15:
LW~ r~ TTG 23 =

.~ .
~~ ~

(2) INFORNATION FOR SEQ ID NO:16:
(i) SEQUENCE r~P~R~rTF~TeTTCS
(A) LENGTH: 672 ~ase pairs (B) TYPE: nucleic a~id (C) sT~ n~ee: single (D) TOPOLOGY: linear (ii) ~OLECULE TYPE: cDt~A
( ix ) FEATURE:
( A ) NA!SE / KEY: CDS
(B) LOCATION: 1......... 672 (xi) SEQUENCE D~ 1~l1UI~: SEQ ID NO:16:

Ala Arg Asp Arg Ile Val Gly GIy Leu Glu Ala Lys Asn Gly Ser Ala 15CCA TTC ATG GTT TCT TTG CAA GCG GAA GAC TAT TTT_QT TTT TGT GGA 96 Pro Phe Net Val Ser Leu Gln Ala Glu Asp Tyr Phe }~is Phe Cys Gly Ser Ser Ile Leu Asn Glu Arg Trp Val Leu Thr Ala Ala His Cys Ile Gln Pro Asn Val }~is Lys Tyr Val Tyr Val Gly Ser Asn Asn Val Glu GTA GGC GGA AQ QC TAC GAA ATC GAA A~A GCT TTC TAT CAC GAA GAA 240 25Val Gly Gly Thr His Tyr Glu Ile Glu Lys Ala Phe Tyr }~is Glu Glu Tyr Asp Gly Val Asp Leu Val Asp E~is Asp Val Ile Asp Gln Ser Glu 30ACA AAC ATT GAT TTA ATG AAG TGT CAA CCC ATT A~A TTA CGA AGA AAG 336 Thr Asn Ile Asp Leu Net Lys Cys GIn Pro Ile Lys Leu Arg Arg Lys Pro Leu Val Gly Gly Glu Glu Leu Arg Ala Val Gly Trp Gly Asn Thr 35llS 120 125 AAT TQ GCA GGG GAA AAT TTT CCA TTG AaA CTT CAA GAA TTG TAC GTG 432 Asn Ser Ala Gly Glu Asn Phe Pro Leu Lys Leu Gln Glu Leu Tyr Val 130 135 ' 140 A~A GCT TTG ACT AAT GAG GAG TGC AAA GCT A~A TCA CCA ATT CQ CCA 450 40Lys Ala Leu Thr Asn Glu Glu Cys Lys Ala Lys ser Pro Ile Pro Pro Thr Thr Gln Val Cy3 Thr Leu Leu Glu Lys Asn His Gly Val Cys Ser Gly Asp Ser Gly Gly Pro Leu Leu Leu Asp Gly Glu Gln Val Gly Ile GCC TCA TTT GTT ATC TTC AhA TGC GQ ATG GGG TAC CCT GAC
Ala Ser Phe Val Ile Phe Lys Cys Ala Net Gly Tyr Pro Asp Tyr Phe 624 l9S 200 205 ~t~ . ~ ., . I 1 199~

Thr Arg Leu Ser Leu Tyr Val Asp Trp Ile Glu Gln His Met Asp (2) INFORMATION FOR SEQ ID NO:17:
~ (1) SEQUENCE rl~ r~rF.RT~;T
(A) LENGTH: 223 an?ino acids (B) TYPE: an?ino acid (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: protein (xi1 SEQUENCE DESCRIPTION: SEQ ID NO:17:
Ala Arg Asp Arg Ile Val Gly Gly Leu Glu Ala Lys Asn Gly Ser Ala 5 10 : 15 Pro Phe Met Val Ser Leu Gln Ala Glu Asp Tyr Phe His Phe Cys Gl 20 25 . . _ 30 Ser Ser Ile Leu Asn Glu Arg Trp Val Leu Thr Ala Ala His Cys Ile Gln Pro Asn Val Hia Lys Tyr Val Tyr Val Gly Ser Asn Asn Val Glu Val Gly Gly Thr His Tyr Glu Ile Glu Lys Ala Phe Tyr His Glu Glu 65 . 70 75 80 Tyr Asp Gly Val Asp Leu Val As~L.His Asp_ Val Ile Asp Gln Ser Glu 85 90 . 95 Thr Asn Ile Asp Leu Met Lys Cys Gln Pro Ile Lys Leu Arg Arg Lys Pro Leu VaL Gly Gly Glu Glu Leu Arg Ala Val Gly Trp Gly Asn Thr Asn Ser Ala Gly Glu Asn Phe Pro Leu Lys Leu Glu Glu Leu Tyr Val 130 135 . ~ 1 40 Lys Ala Leu Thr Asn Glu Glu Cys Lys Ala Lys Ser Pro Ile Pro Pro 145 - ' 150 155 ' 160 Thr Thr Gln Val Cys Thr Leu Leu Glu Lys Asn His Gly Val Cys Ser 165. 170 . 175 Gly Asp Ser Gly Gly Pro Leu Leu Leu Asp Gly Glu Gln Val Gly Ile 180 : 185 190 Ala Ser Phe Val Ile Phe Lys Cys Ala Met Gly Tyr Pro Asp Tyr Phe 195 ~: ~ 200~ ' ' ' 205 ~ -Thr Ar~ Leu Ser Leu Tyr Val Asp Trp Ile Glu Gln His Met As 210 215 . 220 ~?,~ ~) SHFET

(2) INFORNAT}ON FOR SEQ ID NO:lS:
(i) SEQUENCE ~ T~r~r~RTqrT~q (A) LENGTH: 156 base pairs (B) TYPE: nucleic acid (C) ql~Rr~MnFnM~:qs: single ( D ) TOPOLOGY: linear ~ii) MOLECULE TYPE: cDNA
( ix ) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: 1.... 156 (xi) SEQUENOE DESCRIPTION: SEQ ID NO:18:

Gly Trp Gly Arg Leu Gly Ala Asn Leu Ann Gly Pro Asn Glu Leu Gln 15GAA CTT ~AC ACT GTC ACA TTA AGC CAC CAG CAA TGT GTA AGA CAA CAA 96 Glu Leu Asn Thr Val Thr Leu Ser E~is Gln Gln Cys Val Arg Gln Gln 20Ile Tyr Pro Val Tyr Asp Ser Gln Leu Cys Thr Phe Val Gly Ser Gly CGA GGC GCC TGC
Arg Gly Ala Cys 156 (2) INFORNATION FOR SEQ ID NO:19: ~
25(i) SEQUENOE ~ R~ ~TqTICS:
(A) LENGT~: 52 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein 30(xi) SEQUENOE D~ n~ : SEQ ID NO:19:
Gly Trp Gly Arg Leu Gly Ala Asn Leu Asn Gly Pro Asn Glu Leu Gln Glu Leu Asn Thr Val Thr Leu Ser EIis Gln Gln Cys Val Arg Gln Gln 35 Ile Tyr Pro Val Tyr Asp Ser Gln Leu Cys Thr Phe Val Gly Ser Gly Arg Gly Ala Cys (2) INFORMATION FOR SEQ ID NO:20:
4 0( i ) SEQUENCE ~ R~
(A) LENGTB: 168 base pairs (B) TYPE: nucleic acid (C) .qTRr : single (D) TOPOLOGY: llnear 45(ii) MOLECULE TYPE: cDNA
-( ix ) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATIOX: 1..168 (xi) SEQUENOE LI~LSUKlr~lUN: SEQ ID NO:20:
5GGA TGG GGC AaA TTA AGT GAA TCA GGA CCC AAG CCA GTA AAT CTA CAA 48 Gly Trp Gly Lys Ley Ser Glu Ser qly Pro Lys Pro Val Asn Leu Gln 5 10 lS
qqA GTA AAA GTG CCT TAT GTG ACC AAG ATA CAT GCT CTG ACA GCT ACG 9 6 10 2 0 2 5 : 3 0 TCT TTG CAG GTA AaA GAT ATC ACC GAA AAC ATG TTG TGT GCC GGA GTT 144 Ser Leu Gln Val Lys Asp Ile Thr Glu Asn ~let Leu Cys Ala Gly Val AGA AGA GGT GGC AAG GAC
15Arg Arg Gly Gly Lys Asp Ser Cys 168 50 55 :~
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE rTT~R7~e~F~rcTIcs (A) LENGTE3: 56 amino acids 20 (B) TYPE: amino acid (D) TOPoLoqY: linear (ii) ~OLECULE TYPE: protein (xi) SEQUENCE L~;~WC1~1UN: SEQ ID NO:21:
25 Gly Trp Gly Lys Leu Ser Glu Ser Gly Pro LyG Pro Val Asn Leu Gln Gly Val Lys Val Pro Tyr Val Thr Lys Ile HLs Aia Leu Thr Ala Thr Ser Leu Gln Val Lys Asp Ile Thr Glu Asn ~5et Leu Cys Ala Gly Val 3 0 Arg Arg Gly Gly Lys Asp Ser Cy9 ~" .
-=::

WO g6111706 PCTIUS95114442 (2) INFOR~5ATION FOR SEQ ID NO:22:
(i) SEQUENCE rTT~o~rT~!o~fiTIcs:
, A) LENGTEI: 177 base palr5 , B) TYPE: nucleic acid . C) ST~ : single ~D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAME/EEy: CDS
(B) LOCATION: 1.... 177 (xi) SEQUENOE DESCRIPTION: SEQ ID NO:22:

Gly Trp Gly Ser Arg Ser Thr Ser Asn Phe Pro Ser Tyr Pro Asn Leu 15~ CAG ACC GTT GAC A~A CCA ATT GTA TCT TAT GCC GAA TGT GAG AAA 96 Leu Gln Thr Val Asp Lys Pro Ile Val Ser Tyr Ala Glu Cys Glu Lys GTA TTG GGA GGT CCT GGA GCC TCA CCA CTT CAC CCC TTG AAC c'rc TGC 144 Val Leu Gly Gly Pro Gly Ala Ser Pro Leu E~is Pro Leu Asn Leu Cys ACT GGA CCC TTG ACC GGT GGA G~A AGC GCT TGT 177 Thr Gly Pro Leu Thr Gly Gly Val Ser Ala Cys (2) INFOR15ATION FOR SEQ ID NO:23:
(i) SEQUENCE r~ o~
(A) LENGTE3: 59 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: protein (xi) SEQUENCE DESC~IPTION: SEQ ID NO:23:
~ly Trp Gly Ser Arg Ser Thr Ser Asn Phe Pro Ser Tyr Pro Asn Leu ~eu Gln Thr Val Asp Lys Pro Ile Val Ser Tyr Ala Glu Cys Glu Ly3 35 Val Leu Gly Gly Pro Gly Ala Ser Pro Leu Bis Pro Leu Asn Leu Cys ~hr Gly Pro Leu Thr Gly Gly Val Ser Ala Cys (2) INFORMATION FOR SEQ ID NO:24:
( i ~ SEQUENCE r~z,R~TF!RTc:TTcq .
(A) LENGTII: 156 base pairs (B) TYPE: nucleic acid (C) .CTR~ Fn~!qc- single (D) TOPOLOGY: linear ( ii ) NOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NA~5E/~Y. CDS
(B) LOCATION. 1. .156 (xi) SEQUENCE L~E5~:K~ JN: SEQ ID NO:24:
GGC TGG GGA AAT ACA AAT TCA GCA GGG GAA AAT TTT CCA TTG AaA CTT 48 Gly Trp Gly Asn Thr Asn Ser Ala Gly Glu Asn Phe Pro Leu Lys Leu 15CAa GAA TTG TAC GTG AaA GCT TTG ACT AAT GAG GAG TGC AaA GCT AaA 96 Gln Glu Leu Tyr Val Lys Ala LeY Thr A~n Glu Glu Cys Lys Ala Lys Ser Pro Ile Pro Pro Thr Thr Gln Val Cys Thr Leu Leu Glu Lys Asn Nis Gly Val Cys (2) INFORNATION FOR SEQ ID NO:25:
2 5 ( i ) SEQ~ENCE ~R7~
(A) LENGTE~: 52 amino acids (B) TYPE: amLno acid (D) TOPOLOGY: linear ( ii ) ItOLECULE TYPE: protein (xi) SEQUENCE Ll~ ;Kl~LL~JN: SEQ ID NO:25:
~ly Trp Gly Asn Thr Asn Ser Ala Gly Glu Asn Phe Pro Leu Lys Leu ~ln Glu Leu Tyr Val Lys Ala Leu Thr Asn Glu Glu Cys Lys Ala Lys 35 Ser~Pro Ile Pro Pro Thr Thr Gln Val Cys Thr Leu Leu Glu Lys Asn ~, 35 , 40 45 ~lis Gly Val Cy~
- '-- ,, WO 961117~6 PCIIIJS95/14442 12) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE rT~x~ TqTIcs:
(A) LENGTH: 159 base pairs (B~ TYPE: nucleic acid : (C) S~R7~ n~cC: single ( D ~ TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAME/gEY: CDS
(B) LOCATION: 1.... 159 (xi) SEQUENCE LJ~ 'llUN: SEQ ID NO:26:

Gly Trp Gly Ser Thr Gly Ser Gly Gly Pro Ile Thr Acn Val Leu Gln Glu Val Glu Val Pro Phe Ile Asp Phe Asn Thr Cy~ Arg Lys Ser Tyr 20 Ser Thr Ser Leu Thr Asp Arg Uet Phe Cys Ala Gly Phe Leu Gly Ile Gly Gly Lys Ala Cys (2) INFORMATION FOR SEQ ID NO:27:
25~ (i) SEQUENCE r~q~T~D~TcTT~c (A) LENGT~: 53 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein 30(xi) SEQUENCE IJ~;~Kl~.~lUN: SEQ ID NO:27:
~ly Trp Gly Ser Thr Gly Ser Gly Gly Pro Ile Thr Asn Val Leu Gln 5 10 ~ 15 ~lu Val Glu Val Pro Phe Ile Asp Phe Asn Thr Cys Arg Ly Ser Tyr 35 Ser Thr Ser Leu Thr Asp Arg Uet Phe Cys Ala Gly Phe Leu Gly Ile Gly Gly Lys Ala Cys (2) INFORMATION FOR SEQ ID NO:28:
(i~ SEQUENCE rT~3~
(A) LENGT~: 168 base pairs (B) TYPE: nucleic acid ( C ~ .CTl~ .C: s ingle (D) TOPOLOGY: linear ( ii ) NOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAME/KEY: CDS
(B) LOQTION: 1..... 168 (xi) SEQUENCE J~ LluN: SEQ ID NO:28:

Gly Trp Gly A6n Leu Gly Glu Asp Glu Asp Asp Pro Glu Gln Leu Gln 15TAT GTA AAG GTA CCT ATT GTT AAC TGG ACT CAG TGC A.3A ACT ATA TAT 96 Tyr Val Lys Val Pro }le Val Asn Trp Thr Gln Cys Lys Thr Ile Tyr Gly Asn Glu Gly Leu Ile }le Thr Gln Asn Met }le Cys Ala Gly Tyr CCT GAT GGC GGT AAG GAC TCT TGC : 168 Pro Asp Gly Gly Lys Asp Ser Cys (2) INFORMAT}ON FOR S3Q ID NO:29:
25( i) SEQUENCE r~7D~3., ~
(A) LENGTE~: 56 amino acids ( B ) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: protein 30(xi) SEQUENCE LI1;S~:K1~L1UI~: SEQ }D NO:29:
~ly Trp Gly Asn Leu Gly Glu Asp Glu Asp Asp Pro Glu Gln Leu Gln 5 10 lS
~yr Val Lys Val Pro Ile Val Asn Trp Thr Gln Cys Lys Thr Ile Tyr 35 Gly Asn Glu Gly Leu Ile Ile Thr Gln Asn Net Ile Cys Ala Gly Tyr 35 g0 45 Pro Asp Giy Gly Lys Asp Ser Cys .
.

= ~

(2) INFOP~25AT}ON FOR SEQ ID NO:30:
(i) SEQUENCE rr~PoD~ ~r. .I r.~
A) LENGTH: 159 base pairs B) TYPE: nucleic acid C) S~Rpl'Trl~n~qq: single D) TOPOLOGY: linear ( ii ) XOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NA~E/REY: CDS
(B) LOCATION: 1. .159 ( xi ) SEQUENCE DESC.~IPTION: SEQ ID NO: 30:

GIy Trp Ala Ser Pro Lys Ile Ser Pro Ala Phe Glu Leu Pro Asp Lys Leu Gln Tyr Thr Thr Leu Glu Val Gln Pro Ser Glu Asp Cys Lys Lys 20 25 ~ 30 Val Trp Ala Pro Tyr Xet Arg Asp Tyr Ile Leu Cys Ala Lys Phe Glu Lys Gln Asn Ile Cys (2) INFOP~5ATION FOP~ SEQ ID NO:31:
25 ( i) SEQUENOE ~~:7PRP.,, .~ D
(A) LENGT~: 53 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) IOLECULE TYPE: protein (xi) SEQUENOE LI~DUK~ Ul~: SEQ ID NO:31:
~ly Trp Ala Ser Pro Lys Ile Ser Pro Ala Phe Glu Leu Pro Asp Lys ~eu Gln Tyr Thr Thr Leu Glu Val Gln Pro Ser Glu Asp Cys Lys Lys 35 Val Tr~ Ala Pro Tyr ~et Arg Asp Tyr Ile Leu Cys Ala Lys Phe Glu Lys Gln Asn Ile Cys ~ CA 02202622 1997-04-14 ~PE~AnJ~S 9~ 19~
~ 145=
(2) INFORIIATION FOR SEQ ID NO:32:
(i) SEQUENCE r~T~R~r~RT!;TIcs (A) LENGTE~: 186 base pairs (B) TYPE: nucleic acid (C) s~NnFnN~R.c: . single (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAME/KEY: CDS
10 (B) LOCATION: 1.. 186 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Gly Trp Gly Lys Ile Asp Tyr Ser Glu Ser Arg Ser Asp Asp Leu Leu 5 ~ 10-' 15 Lys Val Val Leu Lys Ile Ile Asp Asn Arg Gln Cy8 Xaa Pro Leu Tyr Val Asp Gln Ile Asn Arg Arg Arg Leu Arg Asn Gly Ile Val Glu Thr Gln Met Cy8 Ala Gly Glu Leu Asp Gly Gly Lys Asp Thr Cys 50 SS . 60 ~2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE rT~ rT~ TqTICS:
(A) LENGTE~: 62 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein 3 0 ~ix ) FEATURE:
(A) NAI~E/~;EY: Xaa = Val, Ala, Asp, Glu or Gly (B) LOCATION: 29 (xi) SEQUENCE DESCRIPTION: SEQ ID No:33:
Gly Trp Gly Lys Ile Asp Tyr Ser Glu ser Arg Ser Asp Asp Leu Leu 3 5 1 ~ S = 10 = lS
Lys VaL Val Leu Lys :Ble I le Asp Asn Arg Gln Cys Xaa Prc Leu Tyr Val Asp Gln Ile Asn Arg Arg Arg Leu Ary Asn Gly Ile ~~al Glu Thr 35 ~ 40 ~ 45 40 Gln Met Cys Ala Gl.y Glu Leu Asp Gly Gly Lys Asp Thr Cys WO 96/11706 PCltlJS9~114442 (2) INFORMATION POR SEQ ~D No:34:
(i) SEQUENCE rTTP~P~
A) LENGT}~: 16& base pairs B) TYPE: nucleic acid C) S~r~P~ n~Rcc single 'D~ TOPOLOGY: linear (ii) NOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAUE/REY: CDS
(3) LOCATION: 1_.168 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
GGA TGG GGA AGA ACA TCG TTC GGT GGC CAA TTG TCT AaA AAT CTG CGA 48 Gly Trp Gly Arg Thr Ser Phe Gly Gly Gln Leu Ser Lys Asn Leu Arg Gly Val Glu Leu Glu Ile Ile Asp Leu Phe Asp Cys Phe Leu Ser Tyr ATG GAT AAa GTA AAC GTG TCC GAA AGG CAA GTT TGC GCT GGA ATC CCC 144 20 Net Asp Lys Val Asn Val Ser Glu Arg Gln Val Cys Ala Gly Ile Pro GTT GTA GGT GGT AAa GAT TCT TGC 168 Val Val Gly Gly Lys Asp Ser Cys (2) INFORUATION FOR 8EQ ID NO:35:
25(i) SEQUENCE rTT;~ Ir ~.
(A) LENGT~I: 56 amino acids ( B ) TYPE: amino acid (D) TOPOLOGY: linear (ii) NOLECULE TYPE: protein 30(xi) SEQUENCE D!!;~ Lur: SEQ ID NO:35:
~ly Trp Gly Arg Thr Ser Phe Gly Gly Gln Leu Ser Lys Asn Leu Arg ~ly Val Glu Leu Glu Ile Ile Asp Leu Phe Asp Cys Phe Leu Ser Tyr 35 Net Asp Lys Val Asn Val Ser Glu arg Gln Val Cys Ala Gly Ile Pro Val Val Gly Gly Ly5 A5p Ser Cy5 .

(2) INFOR2~ATION FO~ SEQ ID NO:36:
(i) SEQUENCE ~ Tli~UT5TTI~q (A1 LENGTH: l20 base pairs (31 TYPE: nucleic acid (CI .qT~TI : single (D I TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAlE/REY: CDS
0 (q) LOCATION: 1..... 120 (xi) SEQUENCE l~ lr~lUN: SEQ ID NO:36:

Gly Trp Gly Ala Val Tyr Glu Gly Gly Ala Gly Ser Thr Gln Leu Leu 5 ~ l0 15 Tyr Ser Gln Phe Gly Gly Val Ala Pro Ser ~5et Ile Cys Ala Gly Phe 2 0 Asp Gln G y Gly Lys Asp Ala C4q WO 96/11706 PCr/US95/14142 (2~ INFORMATION FOR SEQ ID NO:37:
(i) SEQUENOE ~DDa~r~DTcTIcs:
(A) LENGTU: 40 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear .
(ii) NOLECULE TYPE: protein (xi) SEQUENCE Ll~;s~:Kl~LluN: SEQ ID NO:37:
~ly Trp Gly Ala Val Tyr Glu Gly Gly Ala Gly Ser Thr Gln Leu Leu 10 Tyr Ser Gln Phe Gly Gly Val Ala Pro Ser Met Ile Cys Ala Gly Phe 20 25 . 30 ~sp Gln Gly Gly Lys Asp Ala Cys ~2) INFORMATION FOR SEQ ID NO:38:
15(i) SEQUENCE r'T~pDaf~ RTcTIcs:
A) LENGTE~: 162 base pairs B ~ TYPE: nucleic acid C) c~PNn~nN~cc: single D) TOPOLOGY: linear 2 0( ii ) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAME/REY- CDS
(B) LOCATION. 1. .162 (xi) SBQUENOE L~;Kl~LlUI~: SEQ ID No:38:
25GGT TGG GGa ACT ACA GAG AGT ACT GAA TCA TCA CAC CAC CTG A-aA GAA 48 Gly Trp Gly Thr Thr Glu Ser Thr Glu Ser Ser E~is Eis Leu Lys Glu Val Glu Val Asn Ala Val Ser Asn Ser Glu Cys Gln Arg Pro Asn Glu Asp Leu Ala Thr Ile Ser Ser 5~is Glu Ile Cys Ala Ser Val Pro Gly GGC GGC AaA GAT TCT TGT 162 35Gly Gly Lys Asp Ser Cys (2) INFOR~ATION FOR SEQ ID NO:39:
(i) SEQUENC3 f'T7~ ''Ll~:
(A) LENGT~: 54 amino acids IB) TYPE: amino acid (D) TOPOLOGY: linear (ii) 15OLECULE TYPE: protein (xi) SEQUENCE U~ lUN: SEQ ID NO:39:
Gly Trp Gly Thr Thr Glu Ser Thr Glu Ser Ser His E~i5 Leu Lys Glu 5 l0 15 l0 Val Glu Val Asn Ala Val Ser Asn Ser Glu Cys Gln Arg Pro Asn Glu As Leu Ala Thr Ile Ser Ser Eli5 Glu Ile Cys Ala Ser Val Pro Gl 15 Gly Gly Lys Asp Ser Cys (2) lN~UKl~IlUI~ FOR SEQ ID NO:40:
(i) SEQUENCE rTTpl~D~ ,.."~,, (A) LENGT}~: 40 amino acids (B) TYPE- amino acid lD) TOPOLOGY: linear (ii) ~OLECULE TYPE: protein (xi) SEQUENCE IJlS~l~ lUN: SEQ ID NO:40:
Ile Val Gly Gly Glu Asn Ala Lys Glu Lys Ser Asp Val Pro Tyr Gln 5 l0 15 Val Ser Leu Arg Asn Ala Glu Asn Lys E~is Phe Cys Gly Gly Ala Ile Ile Asp Asp Tyr Trp Val Leu Thr ~, (2) INFORMATION FOR SBQ ID NO:41:
(i) SEQUENCE rT7~R~r~TRTTr~
(A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) ~5OLECULE TYPE: protein (xi) SEQUENOE ~ K1~1UI~: SEQ ID NO:41:
Ile Val Gly GIy Leu Glu Ala Lys Asn Gly Ser Ala Pro Phe Met Val Ser Leu Gln Ala Glu Asp Tyr Phe His (2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENOE rT~P,~Drl'l;~l~TCTTCS:
(A) LENGTH: 26 amino acids (B) TYPE: amino acid (D) TOPOLO&Y: linear ( ii ) 15OLECULE TYPE: protein (xi) SEQUENCE l~S-:K1~ : SEQ ID NO:42:
le Ile Gly Gly Glu Val Ala Gly Glu Gly Ser Ala Pro Tyr Gln Val Ser Leu Arg Thr Lys Glu Gly A3n His Phe (2) INFORMATION FOR SEQ ID NO:43:
( i ) SEQUENCE r~ " ~ -(A) LENGTH: 29 amino acids (B) TYPE: amino acid (D) TOPOLO&Y: linear ( ii ) 15OLECULE TYPE: protein (xi) SEQUENOE DESCRIPTION: SEQ ID NO:43:
3 0 Ile Val Gly Gly Thr Ala Val Asp Ile Arg Gly Phe Pro Gly Arg Tyr Gln Phe Lys Pro Lys Pro Ser Phe Leu Trp Trp Phe Tyr (2) INFORXATION FOR SEQ ID NO:44:
(i) SEQUENOE r;~R~rTRRTqTICS:
(A) LENGTH: 35 amino ac ~ d ( B ) TYPE: aminO acid (D) TOPOLOGY: linear (ii) ~OLECDLE TYPE: protein (xi) SEQ~IENCE L~ Un: SEQ ID NO:44:
Ile Val Asn Gly Leu Glu Ala Gly Val Gly Gln Phe Pro Ile Gln Val Phe Leu Asp Leu Thr Asn Ile Arg Asp Glu Lys Ser Arg Cys Gly Gly Ala Leu Phe (2) INFORUATION FOR SEQ ID NO:45:
15 (i) SEQIJENCE r~T~Rplr~RRT~cTIcs:
(A) LENGTB: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) ~OLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Ile Val Gly Gly Leu Glu Ala Lys Asn Gly Ile Thr Pro Phe Ile Gly Phe Phe Ala Ser Gly Arg Leu Phe 25 (2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE rT7~ TRRT~TTr~ -(A) LENGTB: 26 amino acids ( B ) TYPE: amino acid (D) TOPOLOGY: linear 30 ~ii) ~OLECULE TYPE: protein (xi) SEQUENOE L)~;:7uKl~lun: SEQ ID NO:46:
Ile Val Gly Gly Asn Asp Val Ser Xaa Lys Ile Phe Trp Gln Val Ser 5 . l0 15 Ile Gln Ser Asn Xaa Gln llis Phe Cy8 Gl ,..

(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE rTT~R~rT~RT.CTTrC
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE U~:Sl.:Kl~llUN: SEQ ID NO:47:
Ile Ile Gly Gly Glu Asp Ala Pro Glu Gly Ser Ala Pro Tyr Gln Val S l0 15 lû Ser Leu Arg Asn Gln Asn Leu Glu Hi3 Phe Cys Gly Gly Ser Ile (2) INFORNATION FOR SEQ ID NO:4e:
( i ) SEQUENCE r~R~r~RT.CTICS
A) LENGTH: 32 base pairs , B) TYPE: nucleic acid C ) STR ~ MTlli nM~ C C: ~i ingle ~ D) TOPOLOGY: linear (ii) ~IOLECULE TYPE: DNA (primer) (xi) SEQUENCE ~ri~ ~L~.Ll~JN: SEQ ID NO:48:
2 0 GTWGGWAhAG GWWTWACWTT YGATTCWGGW GG 3 2 (2) INFORNATION FOR SEQ ID NO:49:
( i ) SEQUELENGTH ~ 20 ba~3e pa B, TYPE : nucleic acid C, cTR7~ nMr.c.c: ~3ingle D,~ TOPOLOGY: linear (ii) ~5OLECULE TYPE: DNA (primer) (xi) SEQUENOE IJ~ lr~ N: SEQ ID NO:49:

(2) INFOP~MATION FOR SEQ ID NO:50:
(i) SEQUENOE CU~R~
- (A) LENGT~: 453 base pairs (B) TYPE: nucleic acid (C) ST~Nn~:n~TPS.C single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAME/KEY: CDS
(E) LOCATION: 1. .453 (xi) SEQUENCE ll~L51.:Klr.Ll~ : SEQ ID NO:50:

UiS Glu Phe Cys Ala ser Val Arg Tyr Cys ser Ser Met Ser Asn Lys AaA GGA TTA GTA CTG GGC ATC TAC GAC AAT GAA TTC GAT AaA AaA ATA
Lys Gly Leu Val Leu Gly Ile Tyr Asp Asn Glu Phe Asp Lys Lys Ile 20 25 : 30 2 0 Arg Leu Thr Pro Thr Ala Glu Gln Phe Asn Arg Arg Leu Gln Gly Arg TTA CTA GAT CTA ATT CAT TTG AGT GGA CCC ATT AaA TTG GGC AAG AGC 192 Leu Leu Asp Leu Ile ~lis Leu Ser Gly Pro Ile Lys Leu Gly Lys Ser Arg Ile Phe Trp Asp Leu Asp Glu Phe Gly Ala Val Ala Val Ala Gly Leu Gly Asn Elis Ser Pro Cy5 Glu Leu Leu Glu Glu Leu Asp Val Leu Arg Glu Asn Ala Arg Ile Ala Ala Gly Ala Gly Cys Gln Ala Leu Ala GCC. GAT GGA ATC ACT ACC ATT AGC GTT GAA GTA TGG AGC ACC CGG AGG 384 Ala Asp Gly Ile Thr Thr Ile Ser Val Glu Val Trp Ser Thr Arg Arg llS 120 125 CGG CQ TGC GAA GGT GCA ATA CTA TCG ACG TTC AaA TTC AGG TCA AQ 432 Arg Pro Cys Glu Gly Ala Ile Leu Ser Thr Phe Lys Phe Arg Ser Thr 130 135 .. 140 Glu Val Val Gln Cys Ser Gly (2l INFORISATION FOF~ SEQ ID NO:51:
(i) SEQuEncE rT7~ TF~TcTTI 5 (A) LENGTH: lSl amino acids ( B ) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE ~:S~ JN: SEQ ID NO:Sl:
His Glu Phe Cys Ala Ser Val Arg Tyr Cys Ser Ser ~5et Ser Asn Lys l0 Lys Gly Leu Va Leu Gly Ile Tyr Asp Asn Glu Phe ASp Lys Lys Ile Arg Leu Thr Pro Thr Ala Glu Gln Phe Asn Arg Arg Leu Gln Gly Arg 15 Leu Leu Asp Leu Ile Hil3 Leu Ser Gly Pro ~Ile Lys Leu Gly Lys Ser Arg Ile Phe Trp Asp Leu Asp Glu Phe Gly Ala Val Ala Val Ala Gl ~eu Gly Asn His Ser Pro Cys Glu Leu Leu~Glu Glu Leu A~p Val Leu 20 Arg Glu Asn Ala Arg ~le Ala Ala Gly Ala Gly Cys Gln Ala Leu Ala Ala Asp Gly Ile Thr Thr Ile Ser Val Glu Val Trp Ser Thr Arg Arg llS : 120 125 Arg Pro cy5 Glu Gly Ala Ile Leu Ser Thr Phe Lys Phe Arg Ser Thr Glu Val Val Gln Cys Ser Gl 145 lS0 WO 96/11706 PCI~/US95114442 (2) INFOP~MATION FOK SEQ ID NO:52:
(i) SEQIJENCE ~~P~P~
(A LENGTE~: 258 }~ase pairs (B TYPE: nucleLc acid (C .qTl~iNn~nNFCC: single (D, TOPOLOGY: linear ( ii ) MOLECULE TYPE: cDNA
( ix ) FEATUPE:
(A) NAME/KEY: CDS
(B) LOCATION: 1._258 (xi) SEQUENOE LlE5~:Kl~lUW: SEQ ID NO:52:
TQ GCA CTC GTT GCC TTG TCT GQ GCT ATT CCT CAC TCC AaC AGA GTC 48 Ser Ala Leu Val Ala Leu Ser Ala Ala Ile Pro E~is Ser Asn Arg Val Val Gly Gly Leu Glu- Ala Ala Glu Gly Ser Ala Pro Tyr Gln Val ser Leu Gln Val Gly Asn Phe Eli5 Phe Cys Gly Gly Ser Ile Leu Asn Glu Tyr Trp Val Leu Thr Ala Ala }Iis Cys Leu Gly Tyr Asp Phe Asp val, 25Val Val Gly Thr Asn Lys Leu Asp Gln Pro Gly Glu Arg Tyr Leu Val G1u Gln Thr Phe Val ~lis -12) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENOE t'FT~RP~ RTcTTr5 (A) LENGTH: 86 amino acidn ( B ) TYPE: amino acid (D) TOPOLOGY: linear (ii) ~5OLECULE TYPE: protein (xi) SEQUENCE Sl:5~;~L~~ : SEQ ID NO:53:
~er Ala Leu Val Ala Leu Ser Ala Ala Ile Pro His Ser Asn Arg Val S l0 15 ~0 Val Gly Gly Leu Glu Ala Ala Glu Gly Ser Ala Pro Tyr Gln Val Ser ~eu Gln Val Gly Asn Phe His Phe Cy~ Gly Gly Ser Ile Leu Asn Glu 15 Tyr Trp Val Leu Thr Ala Ala His cy8 Leu Gly Tyr Asp Phe Asp Val Val Val Gly Thr A~n Lys Leu Asp Gln Pro Gly Glu Arg Tyr Leu Val Glu Gln Thr Phe Val His (2) INFOP~ATION FOD~ SEQ ID NO:54:
(i) SEQUENCE rT~D~TFRTcTIcs:
(A) LENGT~: 240 base pairs (B) TYPE: nucleic acLd (C) ql'D~ n~TFcq: single (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: cDNA
( ix ) FEATU.D~E:
(A) NA!IE/XEY: CDS
(B) LOQTION: 1. .240 (xi) SEQUENCE IJ~ lUN: SEQ ID No:54:
TTA GAT GGG CGC ATT GTT GGA GGA CAA GAT GCT GaT A~TT GCC AAA TAT 48 Leu Asp Gly Arg Ile Val Gly Gly Gln Asp Ala Asp Ile Ala Lys Tyr Gly Tyr Gln Ala Ser Leu Gln Val Phe Asn Glu ~is Phe Cys Gly Ala Ser Ile Leu Asn Asn Tyr Trp Ile Val Thr Ala Ala His Cys Ile Tyr Asp Glu Phe Thr Tyr Ser Val Arg Val Gly Thr Ser Phe Gln Gly Arg 25Arg Gly Ser Val Bis Pro Val Ala Gln Ile Ile Lys ~is Pro Ala Tyr 65 ~ ~ 70 ~5 80 2) INFOD~MATION FOP. SEQ ID NO:55:
(i) SEQUENOE ~ D~ lL >
(A) LENGTH: 80 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: protein (xi) SEQUENOE ~:S~KL~I.lUW: SEQ ID NO:55:
Leu Asp Gly Arg Ile Val Gly Gly GIn Asp Ala Asp Ile Ala Lys Tyr Gly Tyr Gln Ala Ser Leu Gln Val Phe A~n Glu E~is Phe Cys Gly Ala ~er Ile Leu Asn Asn Tyr Trp Ile Val Thr Ala Ala l~is Cy5 Ile Tyr 4 0 Asp Glu Phe Thr Tyr Ser Val Arg Val Gly Thr ser Phe Gl:~ Gly Ar Arg Gly Ser Val Eis Pro Val Ala Gln Ile Ile Lys E~is Pro Ala Tyr 65 ~ 70 75 80 .

WO 96/11706 PCI'IUS9S/14442 (2) INFORMATION FOR SEQ ID NO:56:
(i) SEQ~EXCE ry3R3( ~
A I LENGTY.: 218 base pairs B TYPE: nucleic acid C. 51'~7~ : single ,D, TOPOLOGY: linear ( ii ) NOLECULE TYPE: cDNA
( ix ) FEATURE:
( A ) NAME /XEY: CDS
(B) LOCATION: 1....... 216 (xi) SEQUENCE l~ ~L~UIY: SEQ ID NO:56:

Ary Glu Gln Lys Leu Glu Leu Eis Arg Gly Ala Pro Ala Leu Glu Leu Val Asp Pro Pro Gly Leu Gln Glu Leu Ala Arg Gly Cy5 Ser Trp Leu Cys Leu Val Ala Ile Leu Cys Ala Val Ala Ala Gly Pro Thr Asn Arg ATT GTT GGA GGA TTG GAG GCG AaA AAT GGA ATC ACC CCA TTC ATC GGT 192 Ile Val Gly Gly Leu Glu Ala Lys Asn Gly Ile Thr Pro Phe Ile Giy 25 Phe Phe Ala Ser G~:y Arg Leu Phe (2) INFORMATION FOR SEQ ID NO:57:
( i ) SEQUE~CE rY"~rT~i!YT.C~'TCA
(A) LENGTY.: 72 amino acids 3 0 ( B ) TYPE: amino acid (D) TOPOLOGY: linear (ii) l~OLECllLE TYPE: protein (xi) SEQUENCE D~;51.~ 1Y: SEQ ID NO:57:
Arg Glu Gln Lys Leu Glu Leu llis Arg Gly Ala Pro 3l a Leu Glu Leu 3 5 1 5 10 lS
Val Asp Pro Pro Gly Leu Gln Glu Leu Ala Arg Gly Cys Ser Trp Leu Cys Leu Val Ala Ile Leu Cys Ala Val Ala Ala Gly Pro Thr Asn Ar 40 Ile Val Gly Gly Leu Glu Ala Lys Asn Gly Ile Thr Pro Phe Ile Gl Phe Phe Ala ser Gly Arg Leu Phe WO 96111706 ~ PCIIUS95/14442 159 ~~
(2) INFORMATION FOR SEQ ID NO:58:
(i~ SEQUENCE ~TPRPI ,~ D:
A) LENGTE~: 240 ba9e pairs B) TYPE: nucleic acid C) CTP''~T)~!n'~ : single D) TOPOLOGY: linear ( ii ) !5OLECULE TYPE: cDNA
(ix) FEA=:
(A) NAME/KEY- CDS
(B) LOCATION- 1.... 240 (xi) SEQUENCE l~:D~X1~l1UI~: SEQ ID NO:58:

Thr Arg Phe Arg Leu Ala Ile Val Cys Ala Leu Ala Val Cys Thr Phe 15GGT GCC AGT GTT CcA GAA CCA TGG APA AGA TTA GAT GGT AGA ATC GTA
Gly Ala Ser Val Pro Glu Pro Trp Lys Arg Leu Asp Gly Arg Ile Val 20 Gly Gly ~lis Asp Thr Ser Ile Asp Lys E~is Pro His G n Val Ser Leu Leu Tyr Ser Ser His Asn Cys Gly Gly Ser Leu Ile Ala Lys Asn Trp 25Val Leu Thr Ala Ala llis Cys Ile Gly Val Asn Lys Tyr Asn Val Arg (2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENOE f-~7Pl2P, r,.T~ OD:
(A) LENGTE~: 80 amino acids 30 (B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: protein (xi) SEQUENOE I~S~:Kl~L~JN: SEQ ID NO:59:
35Thr Arg Phe Arg Leu Ala Ile Val Cys Ala Leu Ala Val Cys Thr Phe Gly Ala Ser Val Pro Glu Pro Trp Lys Arg Ley Asp Gly Arg Ile Val Gly Gly ~lis Asp Thr Ser Ile Asp Lys E~is Pro ~is Gln Val Ser Leu ~0 Leu Tyr Ser Ser E~is Asn Cys Gly Gly Ser Leu Ile Ala Lys Asn Trp Val Leu Thr Ala Ala Elis Cys Ile Gly Val Asn Lys Tyr Asn Val Arg (2) lNJtU~llUl1 FO.~~ S3Q ID NO:60:
~i~ SEQJENCE ~T~IR~. . _1... ~
A, LENGT}I: 234 'oase pair3 , B TYPE: nucleic acid C, Sl'R~ : 3ingle ,D, TOPOLOGY: linear ( ii ) I~OLECULE TYPE: cDNA
( ix ) FEATbRE:
(A) NAME/~EY: CDS
(B) LûCATION: 1. .234 (xi) SEQUENCE l/~ lUI~: SEQ ID NO:60:
CCC TCA CTA AAG GGA ACA AAA GCT GGA GCT CCA CCG C
Pro Ser Leu Lys Gly Thr Lys Ala Gly Ala Pro Pro Arg Cy3 Ala Ala 48 15CTA GAA CTA GTG GAT CCC CCG GGC TGC AGG Ai~T TCG GCA CGA GCG TTT 96 Leu Glu Leu Val A3p Pro Pro Gly Cys Arg Asn Ser Ala Arg Ala Phe Gly Trp Ile Glu Arg Val Ser Ser Tyr Lys Ile Lys Asp Arg Leu Asp Gly ArsT Il e Val Gly Gly Gln Asp Ala Asp Ile Ala Lys Tyr Gly Tyr CAA GCT TCA CTC CAA GTA CTT AAC

(2) lN~u~.AIlul~ FOR SEQ ID NO:61:
(i) SEQUENCE ~~~
30 ~B) I'YPE amino acid ( D ) TOPOLOGY: linear (ii) ~OLECIJLE TYPE: protein (xi) SEQUENCE 1~5U~lr~lUI~: SEQ ID NO:61:
Pro Ser Leu Lys Gly Thr Lys Ala Gly Ala Pro Pro Arg Cys Ala Ala ~eu Glu Leu Val Asp Pro Pro Gly Cys Arg Asn Ser Ala Arg Ala Phe ~ly Trp Ile Glu Arg Val ser Ser Tyr Lys Ile Lys Asp Arg Leu Asp 4 0 Gly Arg Ile Val Gly Gly Gln Asp Ala Asp Ile Ala Lys Tyr Gly Tyr ~ln Ala Ser Leu Gln Val Leu A3n GlU Pis Phe Cy3 Gly Ala WO 96/11706 ~ PCI/U~;95/14442 (2) INFORNATION FOR SEQ ID NO:62:
( i ) SEQU3NCE OT~
, A) L3NGT~: 291 ~ase pairs , B) TYPE: nuoleLc ac ' d ~C) .ql~Rr~ cq: ~ingle (D) TOPOLOGY: linear (ii) 25OLECUL3 TYP3: cDNA
( ix ) FEA=
(A) NAM3/lOEy: CDS
(B) LOCATION: 1. .291 (xi) SEQU3NOE IJl~ Kl~..lUW: SEQ ID NO:62:

Ala Val Ile Val Ser Phe Val Leu Ala Cys Ala Phe Ser Val Gln Ala Leu Pro Ser Ser Arg Ile Val Asn Gly Leu Glu Ala Gly Val Gly Gln Phe Pro Ile Gln Val Phe Leu Asp Leu Thr Asn Ile Arg Asp Glu Ly~

Ser Arg Cys Gly Gly Ala Leu Leu Ser Asp Ser Trp Val Leu Thr Ala 25Ala }~is Cys Phe Asp Asp Leu Lys Ser ~et Val Val Ser Val Gly Ala 65 ~O 75 80 CAT GAT GTC AGC AaA TCT GAA GAA CCT CAC AGG CAA ACC AGG A~A CCT 288 His Asp Val Ser Lys Ser Glu Glu Pro ~is Arg Gln Thr Arg Lys Pro Glu (2) INFORUATION FOR 53Q ID NO:63:
( i ) 53QU3NOE ~71~R~
(A) L3NGTEI: 97 a~ino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) ISOL3CUL3 TYP3: protein ~
(xi) SEQU3NC3 IJ~ iKlS'.LlUW: 53Q ID NO:63:
Ala Val Ile Val Ser Phe Val Leu Ala Cys Ala Phe Ser Val Gln Ala Leu Pro Ser Ser Arg Ile Val Asn Gly Leu Glu Ala Gly Val Gly Gln 20 25 . 30 ~he Pro Ile Gln Val Phe Leu Asp Leu Thr Asn Ile Arg Asp Glu Lys 45 Ser Arg Cys Gly Gly Ala Leu Leu Ser Asp Ser Trp Val Leu Thr Ala WO 96/11706 PCl'lUS9!i/14442 Ala His Cys Phe Asp Asp Leu Lys Ser !5et Val Val Ser Val Gly Ala His Asp Val Ser Lys Ser Glu Glu Pro His Arg Gln Thr Arg Lys Pro Glu (2) INPORISATION FOR SEQ ID NO:64:
( i ) SEQUENCE rT~Rl~rTTi!RTq'TTl.q (A) LENGTH: 144 base pairs (B) TYPE: nucleic acid (C) .q'l'P~- : single (D) TOPOLOGY: linear (ii) XOLECULE TYPE: cDNA
( ix ) PEATU~IE:
(A) NA!~E/REY: CDS
( B ) LOCAT ION: l . . l 4 4 (xi) SEQUENCE L/~ w: SEQ ~D NO:64:

Val Leu Ile Val Leu Ala Val Ile Glu Phe Ala Ser Ala Ser Ser Ile 2 0l S l0 15 GGC TGG AGA ATC GTG GGT GGT GAA AAT GCT AhA GAA AaA TCG GTG CCC 96 Gly Trp Arg Ile Val Gly Gly Glu Asn Ala Lys Glu Lys Ser Val Pro TAT CAA GTT TCN CTT CGA AAT GCT GAA AAC AaA CAT TTY TGT GGR GG~ 144 25Tyr G~n Val Ser Leu Arg Asn Ala Glu Asn Lys Elis Phe Cys Gly Gly . .

WO 96/11706 PCI/US9~/14442 .

(2) lN~uAI~IluN FOR 53Q ID NO:65:
( i ) SEQUENCE ~'UI~R:I~r~l;:RTql'Trq (A) L3NGTH: 48 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) NOLECULE TYPE: protein (xi) SEQUENCE ~JK5~:Kl~LluN: SEQ ID NO:65:
Val Leu Ile Val Leu Ala Val Ile Glu Phe Ala Ser Ala Ser Ser Ile 5 lO 15 ~0 Gly Trp Arg Ile Val Gly Gly Glu Asn Ala Lys Glu Lys Ser Val Pro Tyr Gln Val Ser Leu Arg Asn Ala Glu Asn Lys His Phe Cys Gly Gly (2) lNJ~ --T/~N FOR SEQ ID NO:66:
(i) SEQUENCE r~u7iR~c~FRTeTIcs (A) LENGTE: 390 base pairs (B) TYPE: nucleic acid (C) STR7' : 13ingle (D) TOPOLOGY: linear 20(ii) !~OLECULE TYPE: cDNA
( ix ) FEATURE
(A) NAUE/3~EY: CDS
(B) LOCATION: 1._390 (xi) SEQUENOE ~ ;5UKlr.LlC./I~: SEQ ID NO:66:

Phe Gly Phe Lys Leu Ser Hi~ Leu Val Ser Lys Tyr Cys Ala Cys Ala .

Leu Ala Ser Ala Leu Lys Tyr Ser Ile Asp His Gly Pro Arg Ile Ile GGA GGT GAA GTT GCA GGT GAA GGA TCA GQ CCT TAC CAG GTG TCC T
Gly Gly Glu Val Ala Gly Glu Gly Ser Ala Pro Tyr Gln Val Ser Leu 144 35Arg Thr Lys Glu Gly Asn Eis Phe cys Gly Gly Ser Ile Leu Asn Lys Arg Trp Val Val Thr Ala Ala His Cys Leu Glu Pro Glu Ile Leu Asp 40TCG GTA TAC GTC GGA TCC AAT QC TTA GAC CGA AaA GGC AGA TAT TAC 288 Ser Val Tyr Val Gly Ser Asn His Leu Asp Arg Lys Gly Arg Tyr Tyr GAC GTA GAA CGG TAT ATA ATT CAT GAA AaA TAT ATA GGA GAA CTA AAT 336 Asp Val Glu Arg Tyr Ile Ile His Glu Lys Tyr Ile Gly Glu Leu Asn 100 105 llO

WO 96/11706 PCT/~JS9S/14442 AAT TTT TAT GCT GAC aTC GGT CTA ATA AAA CTT GAT GGA AGA CTT AGA 384 Asn Phe Tyr Ala Asp Ile Gly Leu Ile Lys Leu Asp G y Arg Leu Arq Ile G

(2) INFORI~ATION FOR SEQ ID NO:67:
( i ) SEQUENCE ~-u~ l .LL >
(A) LENGTH: 130 amlno acLdn (B) TYPE: al[lino acid (D) TOPOLOGY: linear ( ii ) ~OLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
Phe Gly Phe Ly6 Leu Ser ~li6 Leu Val Ser Lys Tyr Cy6 Ala Cy6 Ala 1 5 10 lS
Leu Ala Ser Ala Leu ~y6 Tyr Ser Ile A6p Hi6 Gly Pro Arg Ile Ile 20 25 = 30 ~ly Gly Glu Val Ala Gly Glu Gly Ser Ala Pro Tyr Gln Val ser Leu 35 40 ' 45 20 Arg Thr Lys Glu Gly Asn E;i6 Phe Cys Gly Gly Ser Ile Leu Asn Lys ~rg Trp Val Val Thr Ala Ala Hi6 Cy6 Leu Glu Pro Glu Ile Leu A6 65 70 _ 75 80 2~ Ser Val Tyr Val Gly Ser A6n /}i6 Leu Asp Arg Lys Gly Arg Tyr Tyr ~6p Val Glu Arg Tyr Ile Ile E~is Glu Ly6 Tyr I le Gly Glu Leu Asr A6n Phe Tyr Ala A6p Ile Gly Leu Ile Lys Leu A6p Gly Arg Leu Ar 3 0 Ile Gl l3on ,, wo s6m706 PCI-IUS95n4442 ~ 165 (2) INFORMATION FOR SEQ ID NO:68:
( i ) SEQUENCE r:T~ OD
A) LENGTH: 240 'oase pairs (B) TYPE: nucleic acid ~ C ) .cTTn~ nN~!qq: single ~D) TOPOLOGY: linear ( ii ) NOLECULE TYPE: cDNA
( ix ) FEA=
(A) NAME/KEY: CDS
(B) LOCATION: 1.. 240 ( ix ) FEATURE:
(A) NAME/KEY: Xaa = any amino acid (B) LOCATION: 73 (xi) SEQUENCE Ll~;Ds:nl}~luN: SEQ ID NO:68:
15CGG GCT GCA GGA ATT CGG CAC GAG AAG AaA CTG CCA ATA TTA ATC GCC 48 Arg Ala Ala Gly Ile Arg His Glu Lys Lys Leu Pro Ile Leu Ile Ala 5 10 lS

20 Leu Ile Gly Cys Val Leu Ser Glu Glu Ile Glu Asp Arg Ile Val Gly Gly Thr Ala Val Asp Ile Arg Gly Phe Pro Trp Gln Val Ser Ile Gln ACC GAA AAC CGT CAT TTT TGT GGT GGT TCT ATT ATC GAT AaA AGC TGG 192 25Thr Glu Asn Arg ~lis Phe Cys Gly Gly Ser_Ile Ile Asp Lys Ser Trp Ile Leu Thr Ala Ala Elis Cys Val Xaa Asp Met Lys Met Ser Asn Trp 30 (2~ INFORMATION FOR SEQ ID NO:69:
(i) SEQ~ENCE r~7~R~rT~l~TCTTrC
(A) LENGTH: 80 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 3 5 ( ii ) MOLECUL3 TYPE: protein (xi) SEQUENOE U~D-:ni~lun: SEQ.ID NO:69:
Arg Ala Ala Gly Ile Arg His Glu Lys Lys Leu Pro Ile Leu Ile Ala 5 10 lS
Leu Ile Gly Cys Val Leu Ser Glu Glu Ile Glu Asp Arg Ile Val Gly 20 25 ~ = 30 ~ly Thr Ala Val Asp Ile Arg Gly Phe Pro Trp Gln Val Ser Ile Gln Thr Glu Asn Arg His Phe Cys Gly Gly Ser Ile Ile Agp Lys Ser Trp 45 Ile Leu~Thr Ala Ala His Cys val Xaa Asp Met Lys Met Ser Asn Trp ~, .

(2) INFORXaTION FOR SEQ ID NO:70:
( i) SEQ13ENCE ~'Pil~D~
(A LENGTH: 1~7 l~ase pairs (B TYPE: nucleic acid S (C ~"'D:I, -' single (D TOPOLOGY: linear (ii) I~OLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NA~5E/KEY: CDS
(B~ LOCATION: 1..... 177 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:

His Glu Ile Leu Leu Ser Ala Leu Phe Ala Ser Val Ile Cys Ser Phe Asn Ala Glu Val Gln Asn Arg Ile Val Gly Gly Asn Asp Val Ser Ile 20 25 . 30 TCA AaA ATT GGG TGG CAA GTA TCT ATT CAA AGT AAT AaA CAA CAT TTC 144 Ser Lys Ile Gly Trp Gln Val Ser Ile Gln Ser Asn Lys Gln His Phe 35 = 40 45 TGT GGT GGT TCA ATC ATT GCT Al~A GAT GGG TCC ~ 177 Cys Gly Gly ser Ile Ile Ala Lys Asp Gly Ser (2~ INFOR15ATION FOR SEQ ID NO:71:
(i) SEQUENCE ~ D3~'TPDT.CTTCS:
(A) LENGTH: S9 amino acidn (B~ TYPE: amino acid (D) TOPOLOGY: linear (ii) 150LECULE TYPE: protein (xi) SEQUENCE L~ ~: SEQ ID NO:71:
~is Glu Ile Leu Leu Ser Ala Leu Phe Ala Ser Val Ile Cys Ser Phe 5 10 lS
~sn Ala Glu Val Gln Asn Arg Ile Val Gly Gly Asn Asp Val Ser Ile 35 Ser Lys Ile Gly Trp Gln Val Ser Ile Gln Ser Asn Lys Gln His Phe Cys Gly Gly Ser Ile Ile Ala Lys Asp Gly Ser (2) INFOR~SATION FOR SEQ ID NO:72:
(i) SEQUENCE (~P~Pr~ T.~
(A I LENGT~: 168 ~ase pairs (B TYPE: nucleic acid (C ,~P~n~nN~q 8 single ~D TOPOLOGY: linear 4 5 ( ii ) I~OLECULE TYPE: cDNA

wo 96/11706 Pcr/usg5n4442 ix ) FEATU~RE:
IA) NA15E/KEY: CDS
(B) LOCATION: l. .168 (xi) SEQUENOE DESCRIPTION: SEQ ID N-0:72:

Ile Met Ala Asn Phe Arg Leu Phe Thr Leu Leu Ala Leu Val Ser Val 5 10 lS
GQ ACT TCC AaA TAT ATT GAT CCA AGA ATA ATT GGA GGC GAA GAT GCT 96 Ala Thr Ser Lys Tyr Ile Asp Pro Arg Ile Ile Gly Gly Glu Asp Ala Pro Glu Gly Ser Ala Pro Tyr Gln Val Ser Leu Arg Asn Gln Asn Leu 15 Glu E~is Phe Cy5 Gly Gly Ser Ile (2) INFORI~ATION FOR SEQ ID NO:73:
( i ) SEQUENCE t~7~R~T~RT~TT~q (A) LENGT~I: 56 amino acids 20 (B) TYPE: a~ino acid (D) TOPOLOGY: linear (ii) IIOLECULE TYPE: protein (xi) SEQUENCE J~s~u~lrL'uN: SEQ ID NO:73:
25 Ile ~5et Ala Asn Phe Arg Leu Phe Thr Leu Leu Ala Leu Val Ser Val Ala Thr Ser Lys Tyr Ile Asp Pro Arg Ile Ile Gly Gly Glu Asp Ala Pro Glu Gly Ser Ala Pro Tyr Gln Val SeF Leu Arg Asn Gln Asn Leu 35 = 40 45 3 0 Glu E~i3 Phe Cys Gly Gly Ser Ile .. = .
,,, =

(2) INFOR~ATION FOR SEQ ID NO:74:
( i ) SEQUENCE rUAT~ArrrR~Tcl~Tcs (A) LENGTH: 192 base pairs (B~ TYPE: nucleic acid (C) ~Tl~AUn~:nNT'~S single (D) TOPOLOGY: linear ( ii ) NOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) N-D~ME/~Ey CDS
(B) LOCATION: 1..... 192 (xi) SEQUENCE D~S~:K1~'~1UI~: SEQ ID NO:74:
GCA CGA GAT CGC ATT GTT GGA GGA TTG GAG GCG AP~A AAT GGA TCA GCC 48 Ala Arg Asp Arg Ile Val Gly Gly Leu Glu Ala Lys Asn Gly Ser Ala Pro Phe Met Val Ser Leu Gln Ala Glu A3p Tyr Phe His Phe Cys Gly 20 Ser Ser Ile Leu Asn Glu Arg Trp Val Leu Thr Ala Ala His Cys Ile CAA CCA AAT GTA CAC AAG TAC GTT TAC GTC GGT TCG AAC AAC GTA GAD. 192 Gln Pro Asn Val His Lys Tyr Val Tyr Val Gly Ser Asn Asn Val Glu (2) INFORMATION FOR SEQ ID NO:75:
25 (i) SEQUENCE ~~:;A~D-~T~l~Tc~rTrc-(A) LENGTH: 64 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENOE LI~S~:Kl~L~: SEQ ID NO:75:
~la Arg Asp Arg Ile Val Gly Gly Leu Glu Ala Lys Asn Gly Ser Ala ~ro Phe Met Val Ser Leu Gln Ala Glu Asp Tyr Phe His Phe Cys Gly 35 Ser Ser Ile Leu Asn Glu Arg Trp Val ~eu Thr Ala Ala His Cys Ile ~ln Pro Asn Val His Lys Tyr Val Tyr Val Gl Ser Asn Asn Val Glu WO 96/11706 PCT/~S9511444Z

(2) INFORMAT}OX POR SEQ ID NO:76:
( i ) SEQUENCE rP~rT~T.qTTrc (A) LENGTE: 207 base pairs (B) TYPE: nucleic acid (c) ST~Nn~nN~CC: single ( D ) TOPOLOGY: linear ( ii ) NOLECllLE TYPE: cDNA
( ix ) FEATURE:
(A) NA15E/KEY: CDS
10 (B) LOQTION: l. .204 (xi) SEQUEN~ L)~i~uK~ un: SEQ ID NO:76:

Pro Ile Eis Asp Ser Gln Tyr Ala Leu Leu Gln Ile Trp Val Lys Gly GCA TGT AAG GGT GAT TCC GGT GGC CCC TTA GT

Eis Gly Ile Val Ser Trp Gly Ile Pro Cys Ala Val Ala Ser Leu 16et TAT TCA CAA GAG TTT CTC ATT ATG TCG ATT GGA TTA AAT CCA AaA
Tyr Ser Gln Glu Phe Leu Ile Iset Ser Ile Gly Leu Asn Pro Lys Leu 192 AAT AaA ATT GTT TAG 2 o A~n Lys Ile Val (2) INFORMATION FOR SEQ ID NO:77:
(i) SEQUENCE rP~ lU5 3 0 ( B ) TYPE: a~ino arid (D) TOPOLOGY: linear ( ii ) IIOLECULE TYPE: protein (xi) SEQUENCE Ll~uKl~Llun: SEQ ID NO:77:
Prlo Ile Eis Asp Ser Gln Tyr Ala Leu Leu Gln Ile Trp Val Lys Gly Ala Cys Lys Gly Asp Ser Gly Gly Pro .Leu Val Ile Asn Gl Gln Leu Eis Gly Ile Val Ser Trp Gly Ile Pro Cys Ala Val Ala Ser Leu 2et Tyr Ser Gln Glu Phe Leu Ile l~et Ser Ile Gl Leu Asn y Pro Lys Leu Asn Lys Ile Val (2~ INFO~MATION FOR SEQ ID NO:78:
(i) SEQUENCE rT~D~
A) LENGTH: lS9 base pairs B) TYPE: nucleic acid ' , C) ~l~DNnP'nM~.C~: single ~ D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: cDNA
( ix ) FEATURE:
( A ) NA~5E /}~EY: CDS
(B) LOCATION: 1......... 156 (xi) SEQUENOE ~c.;~:nl~lON: SEQ ID NO:78:
GGA GGT CCT TTG GCA ATC AAT GGT GAD. CTT GTT GGT GTT ACT TCA TTC 48 Gly Gly Pro Leu Ala Ile Asn Gly Glu Leu Val Gly Val Thr Ser Phe 15ATT~ATG GGG ACA TGT GGA GGA GGA CAT CCT GAT GTC TTC GGT CGA GTC 96 Ile ~5et Gly Thr Cys Gly Gly Gly Ei8 Pro Asp Val Phe Gly Arg Val Leu Asp Phe Ly8 Pro Trp Ile Asp ser Eis Met Ala Asn Asp Gly Ala AAT TCT TTT ATT TAD lS9 Asn Ser Phe Ile (2) INFORMATION FOR SEQ ID NO:79:
25 (i) SEQUENCE rT7~Dr'rF!l~T.c'rT~'C
(A) LEMGTE: 52 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein 30 (xi) SEQUENCE ~D~:K~ ~: SEQ ID NO:79:
~ly Gly Pro Leu Ala }le A8n Gly Glu Leu Val Gly Val Thr Ser Phe s 10 15 ~le let Gly Thr Cys Gly Gly Gly Eis Pro Asp Val Phe Gly Arg Val 35 L'eu Acp Phe Lys Pro Trp Ile Asp Ser His Met Ala Asn A8p Gly Ala Asn Ser Phe Ile WO 96/11706 ~ PCT/lJS9S/14442 (2) INFORMATION FOR SEQ ID NO:80:
( i ) SEQUENCE ~7A~A~ " l ~
A, LENGTB: 779 ~ase pairs I B TYPE: nucleic acid C) s~ ~n~=qC: single . D TOPOLOGY: linear ( ii ) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAME/REY: CDS
= (B) LOCATION: 3.. 699 ( ix ) FEA=:
(A) NAME/REY: Xaa = any amino acid (B) LOCATION: 123 (xi) SEQUENCE Ll~:SL~l~lLN: SEQ ID NO:80:

Phe Ser Ala Leu Val Ala Leu Ser Ala Ala Ile Pro E}is Ser Asn Arg Val Val Gly Gly Leu Glu Ala Ala Glu Gly Ser Ala Pro Tyr Gln 20 20 25 ~ 30 Val Ser Leu Gln Val Gly Asn Phe E~is Phe Cy5 Gly Gly Ser Ile Leu 25Asn Glu Tyr Trp Val Leu Thr Ala Ala His Cys Leu Gly Tyr Asp Phe Asp Val Val Val Gly Thr Asn LyG Leu Asp Gln Pro Gly Glu Arg Tyr Leu Val Giu Gln Thr Phe Val ~is Gln Phe Asp Gln Glu Ser Leu Arg 287 Bis Asp Leu Ala Leu Val Ly~ Val Ser Ser Pro Ile Glu Phe Asn Asp 35~ 100 105 110 Tyr Val Gln Pro Ile Pro Leu Gly Glu Thr Tyr Xaa Gly Gly Glu Val lI5 120 125 40Ala Arg Leu Thr Gly Trp Gly Arg Leu Gly Ala Asn Leu Asn Gly Pro 130 ~ 135 140 Asn Glu Leu Gln Glu Leu Asn Thr Val Thr Leu Ser }~is Gln Gln Cys Val Arg Gln Gln Ile Tyr Pro Val Tyr Asp Ser Gln Leu Cys Thr Phe , . .

Wo 96111706 PCT/[1~9~/14442 GTT GGC AGT GGA CGA GGC GCC TGC AAC GGT GAC TCT GGT GGT CCA TTG 5~5 Val Gly Ser Gly Arg Gly Ala Cys Asn Gly Asp Ser Gly Gly Pro Leu 180 185 ~ 190 Val Val Asn Gly Glu Leu ~is Gly Val Val Ser~ Trp Gly le Pro Cys GCC GTT GGA TTA CCC GAT GTC TTC ACA AGA GTT TCA CAC TAC GCT GAC
Ala Val Gly Leu Pro Asp Val Phe Thr Arg Val Ser Eis Tyr Ala Asp 671 TGG ATT AGA GAG ACC ATG GAA AAT AAC T AATTTTTAAT ~e.r~TATT~T 719 Trp Ile Arg Glu Thr 2~et Glu Asn Asn TGTATTGTCT ~.'T~ T0~ TTAATA~AAA CGTGATAGAT T~ 7U~ 779 (2) INFORMATION FOR SEQ ID NO:81:
(i) SEQUENCE ('TT;~R~'TrRT~TICS
(A) LENGT~: 232 amino acids (B~ TYPE: amino acid (D) TOPOLOGY: l~near ( ii ) ~OLECULE TYPE: protein (xi) SEQUENCE L~ : SEQ ID NO:81:
Phe Ser Ala Leu Val Ala Leu Ser Ala Ala Ile Pro ~li8 ser Asn Ar Val Val Gly Gly Leu Glu Ala Ala Glu Gly Ser Ala Pro Tyr Gln Val Ser Leu Gln Val Gly Asn Phe llis Phe Cys Gly Gly Ser Ile Leu Asn Glu Tyr Trp Val Leu Thr Ala Ala E~io Cys Leu Gly Tyr Asp Phe Asp 3 0 Val Val Val Gly Thr Asn Lys Leu Asp Gln Pro Gly Glu Arg Tyr Leu Val Glu Gln Thr Phe Val E~is Gln Phe Asp Gln Glu S
85 90 er Leu Arg }IL8 Asp Leu Ala Leu Val Lys Val 8er ser Pro Ile Glu Phe Asn Asp Tyr Val Gln Pro }le Pro Leu Gly Glu Thr Tyr Xaa Gly Gly Glu Val Ala Arg Leu Thr Gly Trp Gly Arg Leu Gly Ala Asn Leu Asn Gly Pro Asn Glu Leu Gln Glu Leu Asn Thr Val Thr Leu Ser E~is Gln Gln Cys Val ~rg Gln Gln Ile Tyr Pro Val Tyr Asp Ser Gln Leu Cys Thr Phe Val ~ly Ser Gly Arg Gly Ala Cys Asn Gly Asp Ser Gly Gly Pro Leu Val WO 96/11706 PCI~/U59S/14442 Val A3n Gly Glu Leu Elis Gly Val Val Ser Trp Gly Ile Pro cys Ala Val Gly Leu Pro Asp Val Phe Thr Arg Val Ser ~15 Tyr Ala Asp Trp 5 Ile Arg Glu Thr 15et Glu Asn Asn (2) INFORMATION FOR SEQ ID NO.82:
( i ~ SEQUENCE OT7~
A) LENGTl~: 944 base pairs ~B) TYPE: nucleic acid , C) S~ : single ~D) TOPOLOGY: linear (ii) ~5OLECULE TYPE: cDNA
( ix ) FEAT~RE:
(A) NAME/E~EY: CDS
(B) LOCATION: 3. .768 (xi) SEQUENCE Ll,l:;:X.:K1~.L uN: SEQ ID NO:82:

Glu Val Trp Ile EIis Arg cys Phe Pro Cys Gly Gly ser Leu Gly GAA GAT TCG GTC GTC GAC CGC ATC GTC GGC GGC ACC AGT GTT AaA ATT 95 Glu Asp Ser Val Val Asp Arg Ile Val Gly Gly Thr Ser Val Lys Ile Glu Asn Phe Gly Trp Gln Val Ser Leu Phe Asp Arg Lys Gly his Phe Cys Gly Gly Ser Ile Ile Ser Asp Glu Trp Val Leu Thr Ala Ala His Cys Val Tyr Asp Tyr Phe Ser Pro Lys Gln Tyr Gly Val Arg Val Gly AGC AGT TTA CGC AAC AaA GGT GGA GTC CTT CAC AGA ATT TCC AGG GTA 287 Ser Ser Leu Arg Asn Lys Gly Gly Val Leu Elis Arg Ile Ser Arg Val ~is Ile l~is Pro Asp Tyr Asp Thr Val Ser Tyr Asp Asn Asp Val Ala CTC CTG AaA GTT GAA ACC AaA TTT AaA CTA AAC GGC AGG AGC GTT CGC 383 4 0 Leu Leu Lys Val Glu Thr Lys Phe Lys Leu Asn Gly Arg Ser Val Arg AaA GTT AaA TTG GTT GAC GAA GAT CAC GAG GTT GAT GAT GGT GCC CGG 431 Lys Val Lys Leu Val Asp Glu Asp Elis Glu Val Asp Asp Gly Ala Arg CTC ACC GTC ACT GGA TGG GGC AaA TTA AGT GAA TCA GGA CCC AAG CCA 479 Leu Thr Val Thr Gly Trp Gly Lys Leu Ser Glu Ser Gly Pro Lys Pro wo 96rll706 PCT/US95114442 GTA AAT CTA CAA vGA GTA A~A GTG CCT TAT GTG GAC CAA GAT ACA TGC 527 Val Asn Leu Gln Gly Val Lys Val Pro Tyr Val Asp Gln Asp Thr Cys Ser Asp Ser Tyr Val Phe Ala Gly Lys Aop ~le Thr Glu Asn Net Leu Cy5 Ala Gly Val Arg Arg Gly Gly Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Asp Glu Asn Lys Asn Leu Val Gly Val Val Ser Trp Gly Asn Gly Cys Ala Arg Pro Asn Net Pro Gly Val Tyr Ala Lys GTT GCT GCT TCT AGC ATT AGA GAG TTC ATT CGC APA AaA ACT GGT CTT T 768 Val Ala Ala ser Ser le Arg Glu Phe Ile Arg Lys Lys Thr Gly Leu AATTTCCTTA TATGAACAAA ~rG~TCr~rr~ ~D~P~rPT~r.~r TTAGATTTTA r.~P~P'r~P~ 828 2 0 IUL, ~V . V~ TTCATGCAAA TA~ TATTTATTTA TTTACTTTAT TCAAACGAAT 888 GTATAAAGTG AATTAACAAT AaAAATGTTA GTGTTGCCAA ~P''''''7'~7''' AP,AAAA 944 (2) INFOPlATION FOFc SEQ ID NO:83:
( i ) SEQUENC3 rR~ " ~ ~, ~ ~ (A) LENGTE 255 amino arids (D) TOPOLOGY: linear (ii) NOLECULE TYPE: protein (xi) SEQUENCE ~ L~l~ILUr1: SEQ ID NO:83:
Glu Val Trp Ile _is Arg Cys Phe Pro cys Gly Gly Ser Leu Gly Glu Asp Ser Val Val Asp Arg Ile Val Gly Gly Thr ser Val Lys Ile Glu Asn Phe Gly Trp Gln Val Ser Leu Phe A3p Arg Lys Gly _i9 Phe Cys Gly Gly Ser Ile Ile Ser Asp Glu Trp Val Leu Thr Ala Ala Eiis Cys Val Tyr Asp Tyr Phe Ser Pro Lys,Gln Tyr Gly Val Arg Val Gly Ser Ser Leu Arg Asn Lys Gly Gly Val Leu Elis Arg Ile Ser Arg Val _is ~le Ris Pro Asp Tyr Asp Thr Val Ser Tyr Asp Asn Asp Val Ala 1eu 100 105 llO
~eu Lys Val Glu Thr Lys Phe Lys Leu Asn Gly Ary Ser Val Arg Lys WO 96tll706 PCI~S95114442 Val Lys Leu Val Asp Glu Asp Els Glu Val Asp Asp Gly Ala Arg Leu Thr Val Thr Gly Trp Gly Lys Leu Ser Glu Ser Gly Pro Lys Pro Val 5 Asn Leu Gln Gly Val Lys Val Pro Tyr Val Asp Gln Asp Thr cy5 ser Asp Ser Tyr Val Phe Ala Gly Lys Asp Ile Thr Glu Asn Met Leu Cys Ala Gly Val Arg Arg Gly Gly Lys Asp Ser Cys Gln Gly Asp Ser Gl Gly Pro Leu Val Asp Glu Asn Ly3 Asn Leu Val Gly Val Val Ser Tr Gly Asn Gly Cys Ala Arg Pro Asn Met Pro Gly Val Tyr Ala Lys Val 15 Ala Ala Ser Ser Ile Ary Glu Phe Ile Arg Lys Lys Thr Gly Leu (2) INFORMATION FOR SEQ ID NO:84:
( i ) SEQ~lE ~OE rT~
. A ;ENGTE: 157 base pairs I B ~YPE: nucleic acid C ~P : single ,D, TOPOLOGY: linear ( ii ) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NA~/KEY: CDS
(B) LOCATION: 1. .157 (xi) SEQUENOE IJ~ KLt~lUW: SEQ ID NO:84:

Gly Ile Ary Eis Glu Tyr Leu Cy5 Pro Arg Ser Leu Eis Pro Trp Arg Ser Val Pro Asp Phe Trp Asn Arg Leu Asp Gly Arg Ile Val Gly Gly Eis Asp Thr Ser Ile Asp Asn Ile Leu Me~ Gln Val Ser Leu Scr Leu - CAC A~A CCA CAA T 157 Elis Lys Pro Gln (2) INFORXATIOX FOR SEQ ID NO:85:
(i) SEQUENCE ~ t'TTi!l~TSTTOC
(A) LENGTH: 52 ami~o acidD
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) 26OLECULE TYPE: protein (xi) SEQUENCE IJ~ LlUN: SEQ ID NO:85:
~ly Ile Arg E~is Glu Tyr Leu Cys Pro Arg Ser Leu His Pro Trp Ar 10 Ser Val Pro ASp Phe Trp Asn Arg Leu Anp Gly Arg Ile Val Gly Gl 20 ~ 25 30 ~~is Asp Thr Ser Ile Asp Asn Ile Leu 25et Gln Val Ser Leu Ser Leu ~is Lys Pro Gln ~2) lN~u~IluN FOR SEQ ID NO:86:
( i ) SEQUENCE t'T~
A, LENGT}~: 218 base pairs ~ B TYPE: nuc le ic ac id ,C, ST~ : single I D' TOPOLOGY: linear (ii) r5OLECULE TYPE: cDNA
( ix ) FEaTURE:
(A) NANEr~EY: CDS
(B) LOCATION: 3. .218 (xi) SEQUENCE Dl:.~UK1~..1UD/: SEQ ID NO:86:

Asp Val Glu Leu Thr Pro Gly Thr !5et Cys Thr Val Thr Gly Trp 3 û GGA TCA ACT GGA TCT GGT GGT CCA ATT ACA AAT
Gly Ser Thr Gly Ser Gly Gly Pro Ile Thr Asn Val Leu Gln Glu Val GAA GTT CCA TTT ATC GAC TTC AAC ACC TGC CGA AaA TCC TAC TCA ACC 4 Glu Val Pro Phe Ile Asp Phe A~n Thr Cys Arg Lys Ser Tyr Ser Thr 1 3 Ser Leu Thr Asp Arg }5et Phe Cys Ala Gly Phe Leu Gly Ile Gly Gly AAG GAC GCT TGC CAA GGC GAC TCC GGA
Lys Asp Ala Cys Gln Gly Asp Ser Gly 218 WO 96/11706 ~ PCT/US95/14442 l77 (2) INFOR16ATION FOR SEQ ID NO:87:
(i) SEQUENCE r'~7~DP~ L~
B) TYPE amino acid ~ (D) TOPOLCGY: linear ( ii ) MOLECULE TYPE: protein (xi) SEQUENCE L~ K~ JW: SEQ ID No:87:
~sp Val Glu Leu Thr Pro Gly Thr ~5et Cys Thr Val Thr Gly Trp Gly ~0 Ser Thr Gly Ser Gly Gly Pro Ile Thr Asn Val Leu Gln Glu Val Glu 20 25 ~ 30 ~al Pro Phe Ile Asp Phe Asn Thr Cys Arg Lys Ser Tyr Ser Thr Ser 15 Leu Thr Asp Arg Met Phe Cys Ala Gly Phe Leu Gly Ile Gly Gly Lys Asp Ala Cy3 Gln Gly Asp Ser Gly (2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENOE l'~T7DDZ~
20 ,AI LENGTH: 932 base pairs ~B, TYPE: nucleic acid C, cTDr : single D l TOPOLOGY: linear ( ii ) MOLECULE TYPE: cDNA
2 5( ix ) FEATUKE:
(A) NAl~E/KEY: CDS
(B) LOQTION: 2..770 ( ix ) FEA=:
(A) NAME/KEY: Xaa = any amino acid 30 (B) LOCATION: 92 (ix) FEA=:
(A) NAI~E/KEY: Xaa = any amino acid (B) LOCATION: lll (xi) SEQUENOE ~ ~: SEQ ID NO:88:

Phe Ile Phe Val Leu Val Cys Val Gly Leu Ser Ala Val Ser Ser Tyr Lys Ile 1ys Asp Gly Leu Asp Gly Arg Ile Val Gly Gly Gln Asp GCT GAT ATT GCC A~A TAT GGC TAT QA GCT TCA CTC QA GTA TTT AAC 142 Ala Asp Ile Ala Lys Tyr Gly Tyr Gln Ala Ser Leu Gln Val Phe Asn 45Glu Elis Phe Cys Gly Ala Ser Ile Leu Asn Asn Tyr Trp Ile Val Thr Ala Ala His Cy5 Ile Tyr Asp Glu Phe Thr Tyr Ser Val Arg Val Gly ACC AGT TTC CAA GGA AGA CGT GGT TCC GTT QT CCT GKG GC~ CAA ATT 286 Thr Ser Phe Gln Gly Arg Arg Gly Ser Val His Pro Xaa Ala Gln Ile ATC AAG CAT CCT GCA TAC GGT AAT GTA ACT GAC ATC GAT ATG GAA ItGC 334 Ile Lys Hi3 Pro Ala Tyr Gly Asn Val Thr Asp Ile Asp Met Glu Xaa Ala Leu Ile Lys Val Arg Arg Pro Phe Arg Leu Asn Asn Arg Thr Val AGA ACA GTC AaA CTT ACT GAT GTT GGA AaA GAC ATG CCA TQ GGA GAA 430 15 Arg Thr Val Lys Leu Thr Asp Val Gly Lys Asp Met Pro Ser Gly Glu Leu A a Thr Val Thr Gly Trp Gly Asn Leu Gly Glu Asp Glu Asp Asp Pro Glu Gln Leu Gln Tyr Val Lys Val Pro Ile Val Asn Trp Thr Gln 160 _ 165 170 175 TGC AaA ACT ATA TAT GGA AAT GAA GGA CTA ATA ATT ACC CAA AAT ATG 574 Cys Lys Thr Ile Tyr Gly Asn Glu Gly Leu Ile Ile Thr Gln Asn 2set Ile Cys Ala Gly Tyr Pro Glu Gly G13 Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Asn Ser Lys Gly Val Leu His Gly Ile Val Ser 2Tr2p Gly Ile Gly Cy~ Ala Arg Pro Glu Ile Pro Gly Val Tyr Thr CGA GTG GCT TCA AaA CCA ATA AGA GAA TTT ATC AF~A ATG CAC ACT GGA 766 Arg Val Ala Ser Ly~ Pro Ile Arg Glu Phe Ile Lys Met His Thr Gly ATA T A~CF~r-TTTTF~ ACTTATAATA TTAr~''TAT TTTTTGATAT TCCTTAATTT 820 Ile CAATGATATA r.TP~ ATGTTTTACA AaAT~TTGAT ACTCAACTAA CAAATTAaAC 880 CATATTACTA cTcAaATAaA TaTrF~rTaF~T AATCAaAAAA F~AAF~F~F~A~F~F~ AA 932 _ (2) INFORNATION FOR SEQ ID NO.89:
i~ SEQUENCE r~r'rF!l?T.q'rTt'C
(A) LENGTE~: 256 amino acids (B) TYPE- amino acid (D) TOPOLOGY: linear (ii) NOLECULE TYPE: protein (xi) SEQUENCE Li~:5~ : SEQ ID NO:89:
~he Ile Phe Val Leu Val Cy5 Val Gly Leu Ser Ala Val Ser Ser Tyr 10 Lys Ile Ly3 Asp Gly Leu Asp Gly Arg Ile Val Gly Gly G n Asp Ala Asp Ile Ala Lys Tyr Gly Tyr Gln Ala 8er Leu Gln Val Phe Asn G

E~is Phe Cys Gly Ala Ser Ile Leu Asn Asn Tyr Trp Ile Val Thr Ala Ala His Cys Ile Tyr Asp Glu Phe Thr Tyr Ser Val Arg Val Gly Thr Ser Phe Gln Gly Arg Arg Gly Ser Val Eis Pro Xaa Ala Gln Ile Ile Lys His Pro Ala Tyr Gly Asn Val Thr Asp Ile Asp Net Glu Xaa Ala Leu Ile Lys Val Arg Arg Pro Phe Arg Leu Asn Asn Arg Thr Val Arg Thr Val Lys Leu Thr Asp Val Gly Lys Asp l~et Pro Ser Gly Glu Leu Ala Thr Val Thr Gly Trp G1y Asn Leu Gly Glu Asp Glu Asp Asp Pro ~lu Gln Leu Gln Tyr Val Lya Val Pro Ile Val Asn Trp Thr Gln Cys Lys Thr Ile Tyr Gly Asn Glu Gly Leu Ile Ile Thr Gln Asn Net Ile Cys Ala Gly Tyr Pro Glu Gly Gly Lys Asp 8er Cys Gln Gly Asp Ser l9S 200 205 Gly Gly Pro Leu Val Asn Ser Lys Gly Val Leu 3~is Gly Ile Val Ser . 210 215 , 220 Trp Gly Ile Gly Cys Ala Arg Pro Glu Ile Pro Gly Val Tyr Thr Arg 225 . ~ 230 235 240 Val Ala Ser Lys Pro Ile Arg Glu Phe Ile Lys ~5et ~lis Thr Gly Ile ~S 250 255 WO 96/11706 PCT/lJ~i95/14442 (2) INFOR~5ATION FO}~ SEQ ID NO:90:
( i ) SEQUENOE r~
A LENGT~: 894 base pairG
. B TYPE: nucleic acid . C .cT~Mn~nM~qc, single . D TOPOLOGY: linear ( ii ) NOLECULE TYPE: cDNA
( ix ) FEATUP~E:
( A ) NANE /REY CDS
10 (B) LOCATION; 1. .766 ( ix ) FEaTuRE:
(A) NA!SE/REY: Xaa = any amino acid (B) LOCATION: 187 (xi) SEQUENCE DESCP~IPTION: SEQ ID NO:90:
CCG GCG GTG ATT GTG TCA TTT GTT CTG GCT TGT GCA TTT TCT GTA CAG~ro Ala Val Ile Val Ser Phe Val Leu Ala Cya Ala Phe Ser Val Gln 48 ~CT CTT CCA TQ AGC AGA ATT GTC AAT GGA CTT GAA GCA GGA GTT GGA
20 Ala Leu Pro Ser Ser Arg Ile Val Asn Gly Leu Glu Ala Gly Val Gly 96 CAA TTT CCA ATT CAG GTT TTC TTA GAC TTG ACA AAT ATC AGA GAC GAA~ln Phe Pro Ile Gln Val Phe Leu Asp Leu Thr Asn Ile Arg Asp Glu 144 ~AA TCC AGA TGT GGT GGT GCT TTG TTA TCA GAT TCA TGG GTT TTG ACT~5 Lys Ser Arg Cys Gly Gly Ala Leu Leu Ser Asp Ser Trp Val Leu Thr 192 GCT GCT CAT TGT TTT GAT GAT T$G aAG TCT ATG GTA GTG TCC GTT GGT
Ala Ala His Cy5 Phe Asp Asp Leu Lys Ser !~et Val Val Ser Val Gly 240 3 0 GCT CAT GAT GTC AGC AaA TCT GAA GAA CCT CAC AGG CAA ACC AGG AAA~la ~is Asp Val Ser Lys Ser Glu Glu Pro }~is Arg Gln Thr Arg Lys 288 ~CT GAA AGG TAC TTC CAG CAT GAA AAA TAC GAC AGG GCA AAT CTT GCA~ro Glu Arg Tyr Phe Gln Elis Glu Lys Tyr Asp Arg Ala Asn Leu Ala 336 ~AT GAT CTT GGT TTG TTG AaA TTG GAC AaA CCA GTG GAA TTG AAT GAT
Tyr Asp Leu Gly Leu Leu Lys Leu Asp Lys Pro Val Glu Leu Asn Asp 384 115 120 .. 125 TTC GTG AAA CTC ACA AAA TTG AAC AaA GAC AaA ACT GAA ACT TTT GTC
4 0 Phe Val Lys Leu Thr Lys Leu Asn Lys Asp Lys Thr Glu Thr Phe Val 432 GGC AAA ACT GCA ACT GTT AGT GGA TGG GCA TCT CCA AAG ATT TCC CCT
G4y Lys Thr Ala Thr VaO Ser G1y Trp Ala Ser Pro Lys Ile Ser Pro 480 Ala Phe Glu Leu Pro Asp Lys Leu Gln Tyr Thr Thr Leu Glu Val Gln 52 WO 96/1 1~0C PCT~lJS9~/14442 CCA AGT GAA GAC TGC AAA A~A GTA TGG GCC CNT TAC ATG CGC GAC TAC 576 Pro Ser Glu As0p Cys Lys Lyn Val Trp Ala Xaa Tyr let Arg Asp Tyr ATC CTT T&T GCC AAA TTT GAA AAA CAA AAC ATT TGC ACT GGT GAC AGT 6Z4 Ile Leu Cy3 Ala Lys Phe Glu Lys Gln Asn Ile Cys Thr Gly Asp Ser G&C GGT CCA TTG ACC ATT GAT G&T GTC CAA GTT GGT GTG GTG AGT TTT 672 Gly Gly Pro Leu Thr Ile Asp Gly Val Gln Val Gly Val Val Ser Phe Gly Ser Val Pro Cys Ala Arg Gly Asn Pro Ser Gly Phe Thr Asn Val GCT CAT TTT GTG GAT TGG ATT CAA GAA CAT ACT GGA TTG GAA TTG T ~66 Ala ~is Phe Val Asp Trp Ile Gln Glu E~is Thr Gly Leu Glu Leu T~ Zi CTCAAACTAA ~ A;~A~ ATTTAATTGC ACTGAAAAAT TTTTCAAGAA 826 AAGTTTGGAT CGTTTTGTAA TTGAATGACA ~T~A~Grr~T AAATTAGAaA P~ 886 (2) ~NFO~MATION FOR SEQ ID NO:91:
2 0 ( i ) SEQUENCE rT~R~r~E~T~TTrc (A) LENGTP.: 255 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: 1 inear (ii) l~OLECIJLE TYPE: protein (xi) SEQUENOE L~ lCJlN: SEQ ID NO:91:
Pro Ala Val Ile Val Ser Phe Val Leu Ala Cys Ala Phe Ser Val Gln Ala Leu Pro Ser Ser Arg Ile Val Asn Gly Leu Glu Ala G13 Val Gly 3 0 Gln Phe Pro Ile Gln Val Phe Leu Asp Leu Thr Asn Ile Arg Asp Glu Lys ser Arg Cys Gly Gly Ala Leu Leu Ser Asp Ser Trp Val Leu Thr Ala Ala ~is Cys Phe Asp Asp Leu Lys Ser Met Val Val Ser Val Gl ~la E~is Asp Val Ser Lys Ser Glu &lu Pro Eis Arg Gln Thr Arg Lys ~ro Glu Arg Tyr Phe Gln Pis Glu Lys Tyr Asp Arg Ala Asn Leu Ala lO0 105 110 4 0 Tyr Asp Leu Gly Leu Leu Lys Leu Asp Lys Pro Val Glu Leu Asn As Phe Val Lys Leu Thr Lys Leu Asn Lys Asp Lys Thr Glu Thr Phe Val 45 G4y Lys Thr Ala Thr V5a0 Ser Gly Trp Ala 15e5r Pro Lys Ile Ser Plr50o rirll ~ 9~62 Ala Phe Glu Leu Pro Asp Lys Leu Gln Tyr Thr Thr Leu Glu Vai Gln Pro Ser Glu Asp Cys Lys Lys Val Trp Ala Xaa Tyr ~Set Arg Asp Tyr ~ Ile Leu Cys Ala Lys Phe Glu Ly3 Gln Asn Ile Cy9 Thr Gly Asp Ser l9S 20D 205 Gly Gly Pro Leu Thr Ile Asp Gly Val Gln Val Gly Val Val Ser Phe Gly Ser Val Pro Cys Ala Arg Gly Asn Pro Ser Gly Phe Thr Asn Val 10 225 ~ 230 235 240 Ala His Phe Val Asp Trp Ile Gln Glu BiB Thr Gly Leu Glu Leu (2~ INFORIIATION FOR SEQ ID NO:92:
(i) SEQUENCE c~TA~ r~cTIcs:
(A I LENGTB: 299 base pairs (B, TYPE: nucleic acid (C s~ Nn~n~:R.C: single (D, TOPOLOGY: linear (ii) IIOLECULE TYPE: cDNA
2 0 ( ix ) FEATURE:
(A) NA!IE/KEY: CDS
( B) LOCATION: 3 . . 299 ( ix ) FEATURE:
(A~ NAIIE/XEY: Xaa = any amino acid (B) LOCATION: S9 .
(xi) SEQUENOE D13SCRIPTION: SEQ ID NO:92:

Phe Gly Thr Arg Val Ser Leu Ser Asn 8er Ile Arg Pro Ser Cys Leu Trp Ala A3n Asp Glu Phe Asp Thr Asp Ser Ser Ile Ala Thr Gly TGG GGA AAG ATA GAC TAT GCT GAG AGC AGA AGT~ GAT GAC CTA CTG AAA 143 Trp Gly Ly8 Ile Asp Tyr Ala Glu Ser Arg Ser A6p Asp Leu Leu Lys Val Val Leu Lys Ile Ile Asp Asn Arg Gln Cys Xaa Pro Leu Tyr Val Asp Gln Ile Asn Arg Arg Arg Leu Arg Asn Gly Ile Val Asp Thr Gln ATG TGT GCA GGA G.~A TTG GAT GGT GGC AAA GAC ACT TGC CAG GGA GAT 2 87 ~et Cys Ala Gly Glu Leu Asp Gly Gly Lys Asp Thr Cys Gln Gly Asp TCA GGT GGT CCT : . 299 Ser Gly Gly Pro 5MA~1~962 (2) INFOR15ATION FOR SEQ ID NO:93:
(i~ SEQUENCE CHARACTERISTICS:
~ (A) LENGTH: 99 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:
~he Gly Thr Arg Val Ser Leu Ser Asn Ser Ile Arg Pro Ser Cys Leu 10 Trp Ala Asn Asp Glu Phe Asp Thr Asp Ser Ser Ile Ala Thr Gly Trp ~ly Lys Ile Asp Tyr Ala Glu Ser Arg Ser Asp Asp Leu Leu Lys Val ~al Leu Lys Ile Ile A3p Asn Arg Gln Cys Xaa Pro Leu Tyr Val Asp 50 SS : 60 Gln Ile Asn Arg Arg Arg Leu Arg Asn Gly Ile Val Asp Thr Gln ~let Cys Ala Gly Glu Leu Aup Gly Gly Lys Asp Thr Cys Gln Gly Asp Ser .
2 0 Gly Gly Pro (2) INFOR~ATION FOR SEQ ID No:9i:
(i) SEQUENCE ~'W3R3<'TFRr.CTrCS:
(A) LENGTH: 266 oase pairs (B) TYPE: nuoleic acid (C) sTR7~NnFnN~cc: single (D) TOPOLOGY: linear (ii) ~SOLECULE TYPE: cDNA
( ix ) FEATURE:
~ (A) NA2~E/KEY: CDS

(8) LOCATION: 1.. 266 (xi) SEQUENCE uE5~ ~LlOI~: SEQ ID NO:94:
CTT GCA TAT CCG CTC AAG TTT AGC GAA GAC ATC ~3. CCG ATC ATG ATG 45 Leu Ala Tyr Pro Leu Lys Phe Ser Glu Asp Ile Gln Pro Ile llet ~let 5 = ~ ~ 10 15 Ala Glu Lys Asp Tyr Glu Pro Pro Ala Gly Thr Lys Ala Tyr Val Ser Gly Trp Gly Arg Thr Ser Phe Gly Gly Gln Leu Ser Lys Asn Leu Arg Gly Val Glu Leu Glu Ile Ile Asp Leu Phe Asp Cys Phe Leu Ser Tyr ' SS = . 60 CA 02202622 1997-04-14 - =

Met Asp Lys Val Asn Val Ser Glu Arg Gln Val Cys Ala Gly Ile Pro Val Val Gly Gly Lys Asp Ser cy5 (2) lelr J~uqhIl~Jw FOR SEQ ID NO:gS:
( i ) SEQUENOE rPp~
(A) LENGTE: 88 amino acids l 0 ( B ) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: protein (xi) SEQUENCE L~rSL~ : SEQ ID NO:95:
Leu Ala Tyr Pro Leu Lys Phe Ser Glu Asp Ile Gln Pro Ile Met Met l 5 l0 lS
Ala Glu Lys Asp Tyr Glu Pro Pro Ala Gly Thr Lys Ala Tyr Val ser 20 25 ~ 30 Gly Trp Gly Arg Thr Ser Phe Gly Gly Gln Leu Ser Lys Asn Leu Ar 20 Gly Val Glu Leu Glu Ile Ile Asp Leu Phe Asp Cys Phe Leu Ser Tyr Met Asp Lys Val Asn Val Ser Glu Arg Gln Val Cys Ala Gly Ile Pro Val Val Gly Gly Lys A3p Ser Cys BS
(2) INFOPMATION FOR SEQ ID NO:96:
(i) SEQUENCE ~7~PDr~ Tq~rTc~q A) LE~GTE: 378 base pairs B) TYPE: nucleic acid (c) .C~P7'~!nN~!C.C: single (D) TOPOLOGY: linear ~ii) MOLECDLE TYPE: cDNA
( ix ) FEATURE:
~A) NAME/KEY: CDS
35 (B) LOCATION: 1.. 376 ,.
( ix ) FEATURE:
(A) NAME/KEY: Xaa = any amino acid ( B ) LOCATION:
( ix ) FEATUFE
(A) NAME/KEY: Xaa = any amino acid (B) LOCATION: 90 (xi) SEQUENOE IJ~D~:n.Lr~lul~: SEQ ID NO:96:

Xaa Gly Thr Ser Eis Arg Val Ala Gln Ile Val Val Eis Pro Gln Tyr l 5 l0 lS

WO 96/11706 _ PCI/US95114442 AAC GGC AAC ACC AAC ATC AAC GAT GTT GCT GTT CTC CGT GTT CAA
Asn Gly Asn Thr Asn Ile Asn Asp Val Ala Val Leu Arg Val Gln Asp 96 5Lys Phe Val Leu Asn Gly Arg Ser Val Arg Pro Val Asp Uet Ile Ala ser Gly Val Asp Thr Pro Ala Gly Ala Pro Leu Tyr Val Thr Gly Trp Gly Ala Val Tyr Glu Gly Gly Ala Gly Ser Thr Gln Leu Leu Gly val GGT GTA CCC ATG CGT AGC CAC APA AAC ACW TGT AAC AGC AaA TAC TCC 288 15Gly Val Pro Uet Arg Ser Hls Lys Acn Xaa Cys Asn Ser Lys Tyr Ser CAA TTT GGC GGT GTT GCT CCT AGC ATG ATC TGC GCT GGA TTT
Gln Phe Gly Gly Val Ala Pro Ser Uet Ile Cyc Ala Gly Phe Asp Gln 336 20Gly Gly Lys Asp Ala Cyc Gln Gly Acp Ser Gly Gly Pro (2) IN~OP~5ATION FOP. SEQ ID No:97:
(i) SEQIJ3NCE ~'~P~PI ...,.~" ~1'~
(A) LENGTE: 125 amino acids 25 (B) TYI?E: amino acid (D) TOPOLOGY: linear ~ (ii) UOL3CULE TYPE: protein (xi) SEQU3NOE N~UK11~1UN: SEQ ID NO:97:
3 0 Xala Gly Thr Ser Eis5 Arg Val Ala Gln Ile Val Val Eis Pro Gln Tyr Asn Gly AGn Thr Asn Ile Asn Asp Val Ala Val Leu Arg Val Gln As ~ys Phe Val Leu Asn Gly Arg Ser Val Arg Pro Val Asp Uet Ile Ala 3 5 Ser Gly Val Asp Thr Pro Ala Gly Ala Pro Leu Tyr Val Thr Gly Trp Gly Ala Val Tyr Glu Gly Gly Ala Gly Ser Thr Gln Leu Leu Gly Val Gly Val Pro Uet Arg Ser Eis Lys Asn Xaa Cys Asn Ser Lys Tyr Ser ~ln Phe Gly Gly Val Ala Pro Ser Uet Ile Cys Ala Gly Phe Asp Gln ~ly Gly Lys Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro llS 120 125 WO 96/11706 ~ PCTIUS9~/14442 (2) INFORNATION FOR SEQ ID NO:98:
(i) SEQUENCE r~ ~TqTIcs:
(A) LENGTE~: 252 base pairs (B) TYPE: nucleic acid S ~ (C) CT1~7 : single (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAME/KEY~ CDS
(B) LOCATION; 1. .252 (xi) SEQUENCE ~:SUK1r~1Url: SEQ ID NO:98:

Arg Ala Pro Cys Cys Pro Thr Ser Arg Glu Arg Gly Asn Arg Asn Arg Val Thr Leu Thr Val Thr Gly Trp Gly Thr Thr Glu Ser Thr Glu Ser Ser Eis llis Leu Lys Glu Val Glu Val Asn Ala Val Ser Asn Ser Glu Cys Gln Arg Pro A3n Glu Asp Leu Ala Thr Ile Ser Ser ~is Glu Ile 25Cys Ala Ser Val Pro Gly Gly Gly Lys Asp Ser Cys Gln Gly Asp SerO

Gly Gly Pro Leu 30(2~ lNrUK~~ FOR SEQ ID NO:99:
(i) SEQ~ENCE ~ T~~
~A) LENGTh~: 84 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 35(ii) NOLECULE TYPE: protein (xi) SEQUENCE L)~i~-,rl~. ~ur~: SEQ ID NO:99:
Arg Ala Pro Cys Cys Pro Thr Ser Arg Glu Arg Gly Asn Arg Asn Arg Val Thr Leu Thr Val Thr Gly Trp Gly Thr Thr Glu Ser Thr Glu Ser ~er Pis E~is Leu Lys Glu Val Glu Val Asn Ala Val Ser Asn Ser Glu Cys Gln Arg Pro Asn Glu A5p Leu Ala Thr Ilë Ser Ser E~is Glu Ile ~5 Cys Ala Ser Val Pro Gly Gly Gly Lys Asp Ser Cys Gln Gly Asp Ser 65 : = 70 75 80 Gly Gly Pro Leu , (2) INFORMATION FOR SEQ ID NO:lO0:
(i) SEQUENCE rT7DPD~ LC. ~
(A) LENGTH: 225 base pairs (B) TYPE: nucleic acid ( C ) .CTP D ~5 ing l e ( D ) TOPOLOGY: linear (ii) NOLECULE TYPE: cDNA
( ix ) FEATUFE:
10 (A) NA~IE/KEY: CDS
(B) LOCATION: 1. .208 (xi) SEQUENCE L~ CiN: SEQ ID NO:100:

Prc Ile His Asp Ser Gln Tyr Ala Leu Leu Gln Ile Trp Val Lys Gly Ala Cys Lys Gly Asp Ser Gly Gly Prc Leu Val Ile Asn Gly Gln Leu 20 2~ 30 20Hss Gly Ile Val Ser Trp Gly Ile Pro Cys Ala Val Ala Ser Leu Met TAT TCA CAA GAG TTT CTC ATT ATG TCG ATT GGA TTA AAT CCA A~A
Tyr Ser Gln Glu Phe Leu Ile Met ser Ile Gly Leu Asn Pro Lys Leu 192 ~ CCA AAT AaA ATT GTT T ~/~DrT~r~rD~ AD~aaaaA 225 Pro Asn Ly~ Ile Val (2) INFORMATION FOR SEQ ID NO:101:
(i) SEQUENCE ~~TDl~D~ 1C;i (A) LENGTh: 69 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) !5OLECULE TYPE: protein (xi) SEQIJENCE Ll~;s~;Kl~ : SEQ ID NO:101:
35 Pro Ile His AGp Ser Gln Tyr Ala Leu Leu Gln Ile Trp Val Lys Gl 5 ..10 15 Ala Cys Ly3 Gly Asp Ser Gly G1y Pro Leu Val Ile Asn Gl Gln Leu His Gly Ile Val Ser Trp Gly Ile Pro Cys Ala Val Ala Ser Leu Met Tyr Sér Gln Glu Phe Leu Ile het Ser Ile Gly Leu Asn Pro Lys Leu Pro Asn Lys Ile Val (2) INFOR~5ATION FOR SEQ ID NO:102:
(i) SEQUENCE ~ P~P~TF!~TqTTrq A) LENGT~I: 850 base pairs B) TYPE: nucleic acid C) STl~P~ nN~Cq: single ~D) TOPOLOGY: linear (ii) lOLEC~LE TYPE: cDNA
( ix ) FEATURE:
IA) NAl~E/XEY CDS
lo (B) LOCATION: 2. .758 (xi) SEQUENCE IJ~ 01: SEQ ID NO:102:
C CTG GTG A,PA CTT TTC TTT GTA ATG TAC TGT GCT TGT GCA TTA GCA 46 Leu Val Lys Leu Phe Phe Val Net Tyr Cys Ala Cys Ala Leu Ala Ser Ala Leu Lys Tyr Ser Ile Asp ~is Gly Pro Arg Ile Ile Gly Gly 20 25 ~ 30 Glu Val Ala Gly Glu Gly Ser Ala Pro Tyr Gln Val Ser Leu Arg Thr Lys Glu Gly Asn Eis Phe Cys Gly Gly Ser Ile Leu Asn Lys Arg Trp 25Val Val Thr Ala Ala ~lis Cys Leu Glu Pro Glu Ile Leu Asp Ser Val .
TAC GTC GGA TCC AAT QC TTA GAC CGA A,PA GGC AGA TAT TAC GAC GTA 286 Tyr Val Gly Ser Asn E~is Leu Asp Arg Lys Gly Arg Tyr Tyr Asp Val Glu Arg Tyr Ile Ile llis Glu Lys Tyr Ile Gly Glu Leu Asn Asn Phe TAT GCT GAC ATC GGT CTA ATA AP,A CTT GAT GAA GAC TTA GAA TTC AAT 382 Tyr Ala Asp Ile Gly Leu Ile Lys Leu Asp Glu Asp Leu Glu Phe Asn GAC A,PA GTC AAG CQ ATA A,PA ATT CAT GAA AAC AQ ATT
Asp Lys Val Lys Pro Ile Lys Ile l~is Glu Asn Thr }le Gln Gly Gly 130 135 ~ 140 4 0Glu Gly Leu Arg Ala Thr Gly Trp Gly Arg Leu Gly Ala Gly Arg Pro Ile Pro Asn Lys Leu Gln Glu Leu Gln Thr Phe Ala Leu Ser Asp Lys ~5GAT TGT AQ GTA APA ACT GGT CTT GTA CQ AAG TQ QA CTT TGT GTT 574 Asp Cys Thr Val Lys Thr Gly Leu Val Pro Lys Ser Gln Leu Cys Val .

WO 96/11706 PCII~JS95114442 TTC CGT GQ TCG GAA AaA GGA GTT TGC TTT GGT GAT TCG GGA GGT CCT 622 Phe Arg Ala Ser Glu Lys Gly Val C3s Phe Gly Asp Ser G y Gly Pro TTG GCA ATC AAT GGT GAP. CTT GTT GGT GTT ACT TCA TTC ATT ATG GGA 670 5Leu Ala Ile Asn Gly Glu Leu Val Gly Val Thr Ser Phe Ile let Gly Thr Cys Gly Gly Gly His Pro Asp Val Phe Gly Arg Val Leu Asp Phe Lys Pro Trp Ile Asp Ser His ~et Ala Asn Asp Gly Ala TTTAPTAPTG ATTGAATGTA PPDTTPTP~ CAPATTGTAP. ATTGCATADA TGATATAPAT 828 GCAGGAPATT rr ~ P ~ P 7~ D r 7~ AA 8 5 0 (2) INFOR~5ATION FOR SEQ I~ NO:103:
( i ) SEQUENCE ~'~PT DrTPT TCTICS:
(A) LENGT~l: 252 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 2 0 ( ii ) 2~OLECULE TYPE: protein (xi) SEQUENCE L~ Url: SEQ ID NO:103:
Leu Val Lys Leu Phe Phe Val ~et Tyr Cys Ala Cys Ala Leu Ala Ser Ala Leu Lys Tyr Ser Ile Asp Eis Gly Pro Arg Ile Ile Gly Gly Glu Val Ala Gly Glu Gly Ser Ala Pro Tyr Gln Val Ser Leu Arg Thr Lys Glu Gly Asn ~is Phe Cys Gly Gly Ser Ile Leu Asn Lys Arg Trp Val ~0 Val Thr Ala Ala His Cy~ Leu Glu Pro Glu Ile Leu Asp Ser Val Tyr Val Gly Ser Asn His Leu Asp Arg Lys Gly Arg Tyr Tyr Asp Val Glu Arg Tyr Ile Ile His Glu Lys Tyr Ile Gly Glu Leu Asn Asn Phe Tyr = 100 105 ,. - 110 Ala Asp Ile Gly Leu Ile Lys Leu A5p Glu Asp Leu Glu Phe Asn As llS 120 125 Lys Val Lys Pro Ile Lys Ile His Glu Asn Thr Ile Gln Gly Gly Glu 4 0 Gly Leu Arg Ala Thr Gly Trp Gly Arg Leu Gly Ala Gly Arg Pro Ile 145 150 lSS 160 Pro Asn Lys Leu Grn Glu Leu Gln Thr Phe Ala Leu Ser Asp Lys Asp Cys Thr Val Lys Thr Gly Leu Val Pro Lys Ser Gln Leu Cys Val Phe WO 96/11?06 PCT/US95/14442 Arg Ala Ser Glu Lya Gly Val Cys Phe Gly Asp Ser Gly Gly Pro Leu Ala Ile Asn Gly Glu Leu Val Gly Val Thr Ser Phe Ile ~et Gly Thr Cys Gly Gly Gly ~li8 Pro Asp Val Phe Gly Arg Val Leu Asp Phe Lys ~ro Trp Ile Asp Ser His !le~ Ala Asn Asp Gly Ala ~2) INFO,D~ATION FOR SEQ ID NO:104:
(i) SEQIJENCE rTTpDprTF~T~5~rTl~c (A LENGT~: 252 base pairs (B TYPE: nucleic acid (C .c~rDP~nN~c: single (D, TOPOLOGY: linear 15~ (ii) 2OLECULE TYPE: cDNA
( ix ) FEATUDE:
(A) NA~SE/KEY: CDS
(B) LOCATION: 1..251 ( ix ) FEATUD~E:
20 (A) NA?SE/KEY: Xaa = any aminc acid (B) LOQTION: 2 (xi) SEQUENCE l~ Kll:'.LlIJI~: SEQ ID NO:104:

Ala Xaa Ile Arg Elis Glu Asn Leu Leu Ser Ala Leu Phe Ala Ser Val Ile Cys Ser Phe Asn Ala Glu Val Gln Asn Arg Ile Val Gly Gly Asn 30Asp Val Ser Ile Ser Lys Ile Gly Trp Gln Val Ser Ile Gln Ser Asn 35 40 ~ 45 AAC CAA CAT TTC TGT GGT GGT TCA ATC ATT GCT A~A GAT TGG GTA CTG 192 Asn Gln Elis Phe Cys Gly Gly Ser Ile Ile Ala Lys Asp Trp Val Leu 35ACT TCT TCT CAA TGC GTC GTG GAC AaA CAA AGT CCA CCG AAG GAT TTA 240 Thr Ser Ser Gln Cys Val Val Asp Lys Gln Ser Pro Pro Lys Asp Leu .. 75 80 Thr Val Arg ,: . , , (2) INFOR!5ATION FOR SEQ ID NO:105:
(i~ SEQUENCE rT7~r~rT~l2TcTICS
(A) LENGTEI: 83 amino acids (B) TYPE a ~ o 'd mln ac ( ii ) MOLECULE TYPE: protein (xi) SEQUENCE L~;S~:Kl~lO~: SEQ ID NO:105:
~la Xaa Ile Arg ~is Glu Asn Leu Leu Ser Ala Leu Phe Ala Ser Val 10 Ile Cys Ser Phe Asn Ala Glu Val Gln Asn Arg Ile Val Gly Gly Asn ~sp Val Ser Ile Ser Lys Ile Gly Trp Gln Val Ser Ile Gln Ser Asn ~sn Gln Lis Phe Cys Gly Gly Ser Ile Ile Ala Lys Asp Trp Val Leu ~hr Ser Ser Gln Cys Val Val Asp Lys Gln Ser Pro Pro Lys Asp Leu ~hr Val Arg ~2) INFORMATION FOR SEQ ID NO:106:
20 (i) SEQ~ENCE C~ V~r~Fl~Tcl~Tcs (A) LENGT~: 534 base pairs (B) TYPE: nucleic acid (C) S~r~r : single (D) TOPOLOGY: linear 25 (ii) MOLECULE TYPE: CDNA
( ix ) FEATURE:
( A ) NA~5E /};EY: CDS
(B) LOCATION: 1. .534 (xi) SEQUENCE Ll~;S~;Kl~lurl: SEQ ID NO:106: _ 3 0TCA AAT CGG ATT GTT AAT GÇA GTT AAT GCC AAA AAC GGT TCT GCT CCA
Ser Asn Arg Ile Val Asn Gly Val Asn Ala Lys Asn Gly Ser Ala Pro 48 Tyr Met Ala Ser Leu Arg Asp Val Me~ Çlu Thr Ile Ser Val Glu }~is CGA TAT TÇG ATG AAC CGC TGG ATT CTT ACT GCT GCC CAT TGC CTT ACT 144 Arg Tyr Trp Met Asn Arg Trp Ile Leu Thr Ala Ala Eis Cys Leu Thr 4 0Asp Gly Tyr Leu Asp Thr Val Tyr Val Gly Ser Asn Hi8 Leu Ser Gly A3p Gly Glu Tyr Tyr Asn Val Glu Glu Gln Val Ile llis Asp Lys Tyr TTT GGT CAA ACA ACC GGC TTC AAA AAT GA$ ATT GCT CTC GTC AAA GTT 288 Phe Gly Gln Thr Thr Gly Phe Lys Asn Asp Ile Ala Leu Val Lys Val TCT AGT GCT ATA AaA CTT AGC AaA AAT GTT CGT CCC ATC AaA TTG CAC 336 Ser Ser Ala Ile Lys Leu Ser Lys Asn Val Arg Pro Ile Lys Leu Ei3 AAA GAT TTT ATA CGC GGA GGT GAA AAA TTG AaA ATT ACT GGA TGG GGA 384 Lys Asp Phe Ile Arg Gly Gly Glu Lys Leu Lys Ile Thr Gly Trp Gly Leu Thr Asn Gln Thr ~is Gly Glu Val Pro Asp Ala Leu Gln Glu Leu Gln Val Glu Ala Leu Ser Asn Ser Lys Cys Lys Ala Ile Thr Gly Val CAT CTT CCT GCT CAT CTC TGC ACC TTC AGA GCT CCT CaA AAG GGT GTA 528 His Leu Pro Ala E~is Leu Cys Thr Phe Arg Ala Pro Gln Lys Gly Val TGC CAG
2 0 Cys Gln 534 ~2) INFORMATION FOi~ SEQ ID NO:107:
( i ) SEQUE~CE I~TPT'~rl'R~TC~Tl'C
(A) LENGTE~: 178 amino aoids (B) TOPOLOGY linear ( ii ) IOLEC~LE TYPE: protein (xi) SEQI;ENCE Llo~ J~: SEQ ID NO:107:
~er Acn Arg Ile Val Asn Gly Val Asn Ala Lys Asn Gly Ser Ala Pro 30 Tyr ~5et Ala Ser Leu Arg Asp Val ~et Glu Thr Ile Ser Val Glu His ~rg Tyr Trp ~Set Asn Arg Trp Ile Leu Thr Ala Ala ~i8 Cys Leu Thr ~sp Gly Tyr Leu Asp Thr Val Tyr Val Gly Ser Asn }~is Leu Ser Gly Asp Gly Glu Tyr Tyr Asn Val Glu Glu Gln Val Ile E~is Asp Lys Tyr ~he Gly Gln Thr Thr Gly Phe Lys A~n Asp Ile Ala Leu Val Lys Val ~0 Ser Ser Ala Ile Lys Leu Ser Lys Asn Val Arg Pro Ile Lys Leu E~i3 Lys Asp Phe Ile Arg Gly Gly Glu Lys Leu Lys Ile Thr Gly~Trp Gly Leu Thr Asn Gln Thr Elis Gly Glu Val Pro Asp Ala Leu Gln Glu Leu Gln Val Glu Ala Leu Ser Asn Ser Lys Cys Ly~ Ala Ile Thr Gly Val 145 150 . 155 160 ~Iis Leu Pro Ala His Leu Cys Thr Phe Arg Ala Pro Gln Lys Gly Val Cys Gln (2) INFOR~SATION FOR SEQ ID NO:108:
( i ) SEOUENCE rN~
(A) LENGTH: 359 oase pairs (B) TYPE: nucleic acid (C) ST7~NnEnNr<:c: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NA15E/REY: CDS
(B) LOCATION: 1..359 (xl) SEQUENCE DESCRIPTION: SEO ID NO:108:
GGG TTC GAA TTT GTG GAT CGA AaA GGC AGA TAT TAC GAT GTA GAA AGA 48 20 Gly Phe Glu Phe Val Asp Arg Lys Gly Arg Tyr Tyr Asp Val Glu Arg Phe Val Met Ei3 His Asn Tyr Thr Gly Lys Ile Val Ala Asn Val Ala GAT ATA GGT CTA ATA AaA CTA GCA GAA GAT ATA AaA TTC AGT GAC AAG 144 25Asp Ile Gly Leu Ile Lys Leu Ala Glu Asp Ile Lys Phe Ser Asp Lys GTA CaA CCT GTA AaA ATT QT CAA ACT QA ATC ~AG GGC GGA GAG ATT 192 Val Gln Pro Val Lys Ile His Gln Thr Gln Ile Lys Gly Gly Glu Ile 3 0TGC AaA GCT ACT GGA TGG GGC AGG TTG GGT GCT GAT QG CCT GTA CCA 240 cy3 Lys Ala Thr Gly Trp Gly Arg Leu Gly Ala Asp Gln Pro Val Pro AAT AaA TTA QA CAA TTG GAG AQ ATT GCT ATT AGT GAT GAG AaA TGT 288 Asn Lys Leu Gln Gln Leu Glu Thr Ile Ala Ile Ser Asp Glu 1ys Cy~
35 85 ~ 90 9S

Tyr Ala Asp Thr Gly Phe Leu Glu Pro Thr Ser Gln Ile Cys Val Phe AGT GCA TTT GGA AaA GGA GTT GT _ 3s9 40 Ser Ala Phe Gly Lys Gly Val (2) l~r ~ orl FOR SEQ ID NO:109:
( i ) SEQUENcE rT~
(A) LENGTH: 119 amino acids 4 5 ( B ) TYPE: amino acid (D) TOPOLOGY: linear wo 96/11706 PCT~USg5/14442 l94 (ii) ~OLECIlLE TYPE: protein ~xi) SEQUENCE ~ ;b~l~ W: SEQ ID XO:109:
~ly Phe Glu Phe Val Asp Arg Lys Gly Arg Tyr Tyr Asp Val Glu Arg 5 Phe Val ~et His His Asn Tyr Thr Gly Lys Ile Val Ala Asn Val Ala ~sp Ile Gly Leu Ile Lys Leu Ala Glu Asp Ile Lys Phe Ser Asp Lys ~al Gln Pro Val Lys Ile E~is Gln Thr Gln Ile Lys Gly Gly Glu Ile Cys Lys Ala Thr Gly Trp Gly Arg Leu Gly Ala Asp Gln Pro Val Pro 65 70 7s 80 Asn Lys Leu Gln Gln Leu Glu Thr Ile Ala Ile Ser Asp Glu Lys Cys Tyr Ala Asp Thr Gly Phe Leu Glu Pro Thr Ser Gln Ile Cys Val Phe Ser Ala Phe Gly Lys Gly Val (2) INFORMATION FOR SEQ ID NO:110:
20 (i) SEQ~ENCE rF17'~D~
(A LENGTE/: S41 }:)ase pairs B ! TYPE: nuoleic acid C! ! : single D TOPOLOGY: linear 25 (ii) /50LEC~LE TYPE: cDNA
( ix ) FEATURE:
~A) NAME/REY: CDS
tB) LOQTION: 2 . . 746 ( ix ~ FEATURE:
30 (A) NAME/REY: Xaa = any amino acid (B) LOQTION: 102 ( ix ) FEATURE:
(A) NAME/REY: Xaa = any amino acid (B) LOQTION: 127 3 5 : =: ( ix ) FEATURE .
(A) NAME/REY: Xaa = any ar~ino acid ( B ) LOQTION: 131 (xi) SEQUENOE Ul~ JW: SEQ ID NO:110:

40 Leu Ala Ile Val Cys Ala Leu Ala Val Cys Thr Phe Gly Ala Ser Val Pro Glu Ser Trp Lys Arg Leu Asp Ser Arg Ile Val Gly Gly Bis WO 96/11706 PCI/US9~/14442 GAT ACC AGC ATC GAT AAA CAC CCT CAT CAA GTA TCT I~TA TTG TAC
Asp Thr Ser Ile Asp Lys ~is Pro Elis Gln Val Ser Leu Leu Tyr Ser 142 35 : 40 45 AGC CAC AAT TGT GGT GGT TCC TTG ATT GCC AaA AAC TGG TGG GTT T
Ser E~is Asn Cys Gly Gly Ser Leu Ile Ala Lys Asn Trp Trp Val Leu 190 ACT GCA GCT CAT TGC ATT GGA GTT AAC AaA TAC AAT GTC CGT GTA GGA 238 Thr Ala Ala ~is Cys Ile Gly Val Asn Lys Tyr Asn Val Arg Val Gly AGT TCC ATC GTA AAC AGC GGT GGT ATC TTG CAT AaA GTT AAA AAC CAT 286 Ser Ser Ile Val Asn Ser Gly Gly Ile Leu His Lys Val Lys Asn ~gi5 TAC AGA CAT CCA AaA TAC AMC GCA GCT GCT ATT GAC TTT GAT TAC G
Tyr Arg Elis Pro Lys Tyr Xaa Ala Ala Ala Ile Asp Phe Asp Tyr Al : 100 105 110 CTC TTA GAA CTC GAA ACT CCT GTT CAA CTC ACA AAT GAT GTG TCC AT~l 382 Leu Leu Glu Leu Glu Thr Pro Val Gln Leu Thr Asn Asp Val Ser Xaa ATA AAA TTG GTS GAT GAA GGA GTA GAT CTT AAA CCT GGT ACC TTG T
Ile Lys Leu Xaa Asp Glu Gly Val Asp Leu Lys Pro Gly Thr Leu Leu Thr Val Thr Gly Trp Gly ser Thr Gly Asn Gly Pro Ser Thr Asn Val Leu Gln Glu Val Gln Val Pro ~is Val Asp Gln Thr Thr Cys Ser Lys 3 0 Ser Tyr Pro Gly Ser Leu Thr Asp Arg Met Phe Cys Ala Gly Tyr Leu Gly Gln Gly Gly Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Val Val Val Asn Gly Val Gln Elis Gly Ile Val Ser Trp Gly Arg Gly Cys GCA CTT CCT GAT TAT CCT GGA GTT TAC TCT AaA ATC TCT ACC GCT CGC 715 Ala Leu Pro Asp Tyr Pro Gly Val Tyr Ser Lys Ile Ser Thr Ala Arg AGC TGG ATC AAG GAA GTG TCT GGT GTT T AATTTATTCT TGAaATCTCT 766 Ser Trp Ile Lys Glu Val Ser Gly Val ATTTTGTATT ~ T~rTl~r~a TTGTAaATAT ~AT~-'TT~r ~T~ P. 826 7~7\P,~7\~7\Di~- AAAAA 841 CA 02202622 i997-04-l4 --WO 96/11706 ~ PCTIUS95/14442 (2) INFOR~ATION POR SEQ ID NO:111:
( i ) SEQIJENCE r~
(A) L3NGTH: 24S amino acids (B) TYPE: a~ino acid . (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: protein (xi) SEQUENOE Llc.a~Kl~ ~ : SEQ ID NO:111:
~eu Ala Ile Val Cy8 Ala Leu Ala Val Cys Thr Phe Gly Ala Ser Va Pro Glu Ser Trp Lys Arg Leu Asp Ser Arg Ile Val Gly Gly His As Thr~ Ser Ile Asp Lys His Pro His Gln Val Ser Leu Leu Tyr Ser Ser His Asn Cys Gly Gly Ser Leu I1e Ala Lys As~n Trp Trp Val Leu Thr Ala Ala His Cys Ile Gly Val Asn Lys Tyr Asn Val Arg Val Gly Ser Ser Ile Val Asn Ser Gly Gly Ile Leu His Lys Val Lys Asn His Tyr Arg His Pro L 5 Tyr Xaa Ala Ala Ala Ile Asp Phe Asp Tyr Ala Leu Leu Glu Leu Glu Thr Pro Val Gln Leu Thr Asn Asp Val Ser Xaa Ile Lys Leu Xaa Asp Glu Gly Val Asp Leu Lys Pro Gly Thr Leu Leu Thr . 130 135 . 1~0 Val Thr Gly Trp Gly Ser Thr Gly Asn Gly Pro Ser Thr Asn Val Leu 145 -- ~ 150 155 160 ~ln Glu Val Gln Val Pro His Val Asp Gln Thr Thr cys Ser Lys Ser 3 0 Tyr Pro Gly Ser Leu Thr Asp Arg Met Phe Cys Ala Gly Tyr Leu G

Gln Gly Gly Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Val Val Val Asn Gly Val Gln His Gly Ile Val Ser Trp Gly Arg Gly Cys Ala Leu Pro Asp Tyr Pro Gly Val Tyr Ser Lys Ile Ser Thr Ala Arg Ser Trp Ile Lys Glu Val Ser Gly Val ~2) INFORMATION FOR SEQ ID NO:112:
i ) SEQUENCE rT~ L~:a:
~A) LENGTH: 1580 }~a8e pa-~B) TYPE: nucleic acid ~C) CTP~ : si:lgle ~D) TOPOLOGY: linear ~ii) NOLECULE TYPE: cDNA
ix ) FEATURE:
~ A ) NAllE /I~EY . CDS
10 ~B) LOCATION- 3. .1491 ~xi) SEQUENOE I~S-:K~ : SEQ ID NO:112:

Hi~ Glu Phe Cy8 Ala Ser Val Arg Tyr Cys Ser Ser Met Ser Asn 15~ 5 10 lS
AAG AaA GGA TTA GTA CTG GGC ATC TAC GAC AAT GAA TTC GAT AaA AaA 95 Lys Lys Gly Leu Val Leu Gly Ile Tyr Asp Asn Glu Phe Asp Lys Lys 20Ile Arg Leu Thr Pro Thr Ala Glu Gln Phe As~ Arg Arg Leu Gln Gly CGT TTA CTA GAT CTA ATT CAT TTG AGT GGA CCC ATT AaA TTG GGC AAG 191 Arg Leu Leu Asp Leu Ile His Leu Ser Gly Pro Ile Lys Leu Gly Lys 50 55 60 ~

Ser Arg Ile Phe Trp Asp Leu Asp Glu Phe Gly Ala Val Ala Val Ala Gly Leu Gly Asn His Ser Pro Cys Glu Leu Leu Glu Glu Leu Asp Val 3080 85 90 9s Leu Arg Glu Asn Ala Arg Ile Ala Ala Gly Ala Gly Cys Gln Ala Leu 35Ala Ala Asp Gly Ile Thr Thr Ile Ser Val Glu Val Trp Ser Thr Arg llS 120 125 AGG CGG CCA TGC GAA GGT GCA ATA CTA TCG ACG TTC AaA TTC AGG TCA 431 Arg Arg Pro Cys Glu Gly Ala Ile Leu Ser Thr Phe Lys Phe Arg Ser 130 135 .. 140 40ACA GAA GAA GAG TCC AAG TGT AAG CCG ATA CCT ACC ATA ACC CCT TAC 47g Thr Glu Glu Glu Ser Lys Cys Lys Pro Ile Pro Thr Ile Thr Pro Tyr TGC CTT CAA GAT AaA GAT GCT CCA TTA TGG GAA CTT GGC CAA GTA TCA 527 Cys Leu Gln Asp Lys Asp Ala Pro Leu Trp Glu Leu Gly Gln Val Ser GCA GCA GCT CAA AAC TGG GCT CGT ACA TTG ATG GAT ACA CCA GCA AaT 575 Ala Ala Ala Gln Asn Trp Ala Arg Thr Leu ~Set Asp Thr Pro Ala Asn WO 96111706 PCT/lrS95/14442 CAA ATG ACA CCA TTT TTG TTC GCC GAA GCC GCC A~A GAA AAT TTA GTG 623 Gln Met Thr Pro Phe Leu Phe Ala Glu Ala Ala Lys Glu Asn Leu Val CCA TTA GGA GTG AaA GTT GAA GCT AGA GAT CGG A~A TGG GCG GTA AGC 671 Pro Leu Gly Val Lys Val Glu Ala Arg Asp Arg Lys Trp Ala Val Ser - ATG AaA ATG GGA TCC TTC TTG TCT GTC GCT CGT GGC TCC AAT GAA CCA 719 Net Lys Met Gly Ser Phe Leu Ser Val Ala Arg Gly Ser Asn Glu Pro CCA GTT TTT CTT GAA ATT TCT TAT TGT GGT GGT CCA Aaa GAT GAG GCA 767 Pro Val Phe Leu Glu Ile Ser Tyr Cys Gly Gly Pro Lys Asp Glu Ala Pro Phe Ala Leu Val Gly Lys Gly Val Thr Phe Asp Thr Gly Gly Ile AGC ATC A~A CCG AGT GCA TCC ATG GAC GAA ATG CGT GGA GAT ATG GGA 863 Ser Ile Lys Pro Ser Ala Ser Net Asp Glu Met Arg Gly Asp Net Gly GGA GCT GCT TGC GTT GTT TCT ACA TTG GCA cAa TTG A~A GCA CCA GTC 911 Gly Ala Ala Cys Val Val Ser Thr Leu Ala Gln Leu Lys Ala Pro Val AAC GTC GTC GGT CTT ATC CCC TTA ACC GAG AAT ATG CCA GGT GGT A~A 9 5 9 Asn Val Val Gly Leu rle Pro Leu Thr Glu Asn ~et Pro Gly Gly Lys GCA ACA AAA CCT GGT GAC GTC GTT GTT GCG ATG AAT GGG AaA TCG ATT 1007 Ala Thr Lys Pro Gly Asp Val Val Val Ala Net Asn Gly Lys Ser Ile Cys Val Asp Asn Thr Asp Ala Glu Gly Arg Leu Ile Leu Ala Asp Ala Leu Cys Tyr Ser Ala Elis Phe Lys Pro Lys Trp Val Leu Asp Ile Ala 35 Thr Leu Thr Gly Ala Met Arg Val Ala Leu Gly Asp Cys Ala Thr Gly GTA TTT TCT TCA TGC GAT AAT CTC TGG AAC ACA CTG CAC GAa GCT GGT 1199 Val Phe Ser Ser Cys Asp Asn Leu Trp Asn Thr Leu E~is Glu Ala Gly Arg Val Thr Gly Asp Arg Met Trp Arg Phe Pro Leu Phe Lys His Tyr Ala Asn Arg Val Thr Glu Tyr Ser Gly Tyr Asp Val Asn Asn Ile Gly ~5 420 425 430 AAG GGC A~A GGG GGA GGC AGT TGC CTA GCA GCT GCT TTC CTT AAT CAG 1343 Lys Gly Lys Gly Gly Gly Ser Cys Leu Ala Ala Ala Phe Leu Asn Gln 50 Phe Arg Pro Glu Asp Val Pro Trp Net Ilis Leu Asp Ile Ala Gly Val 15et Ser A~p Cys Ser Asp Gln Ser Tyr Leu Pro Lys Gly ~Set Thr Gly 5 Arg Pro Thr Arg Thr Leu Val Gln Phe Ile Gln Ser Gln Lys Arg ~lis TCT T GAATGQTTT r~ pprpT~ ~rr~Tr~rrTpp QCCTTTACA TGCGCACCTC 1541 Ser TGrTTPCrrP AACCACTTTT GTPrPPPPrr: ATATTTTTA 1580 (2) INFOR15ATION FOR SEQ ID NO:113: -(i) SEQUENCE r~lP~PrT~.CT~r~
(A) LENGTH: 496 amino acids ( B ) TYPE - amino ao d (D) TOPOLOGY: linear (ii) IIOLECULE TYPE: protein (xi) SEQUENCE L~o~:~lr~ : SEQ ID NO:113:
~~iC Glu Phe Cy5 Ala Ser Val Arg Tyr Cys Ser Ser llet Ser Asn Lys ~0 Lys Gly Leu Val Leu Gly Ile Tyr Asp A~2n Glu Phe Asp Lys Lys Ile Arg Leu Thr Pro Thr Ala Glu Gln Phe Asn Arg Arg Leu Gln Gly Arg 25 Leu Leu Asp Leu Ile E~is Leu ser Gly Pro Ile Lys Leu Gly Lys ser Arg Ile Phe Trp Asp Leu Asp Glu Phe Gly Ala Yal Ala Val Ala Gly ~eu Gly Asn His Ser Pro Cys Glu Leu Leu Glu Glu Leu Asp Val Leu 85 , 90 95 ~0 Arg Glu Asn Ala Arg Ile Ala Ala Gly Ala Gly Cys Gln Ala Leu Ala Ala Asp Gly Ile Thr Thr Ile 8er Val Glu Val Trp Ser Thr Arg Arg Arg Pro Cys Glu Gly Ala Ile Leu Ser Thr Phe Lys Phe Arg Ser Thr 130 135 .. 140 Glu Glu Glu Ser Lys Cy5 Lys Pro Ile Pro Thr Ile Thr Pro Tyr Cyc ~eu Gln Acp Lys Asp Ala Pro Leu Trp Glu Leu Gly Gln Val Ser Ala ~ 0 Ala Ala Gln Asn Trp Ala Arg Thr Leu ~Set Asp Thr Pro Al;~ Asn Gln 180 185 l9O
~let Thr Pro Phe Leu Phe Ala Glu Ala Ala Lys Glu Asn Leu Val Pro Leu Gly Val Lys Val Glu Ala Arg Asp Arg Lys Trp Ala Val Ser Met 210 215 2.20 Lys Met Gly Ser Phe Leu Ser Val Ala Arg Gly Ser Asn Glu Pro Pro ~ Val Phe Leu Glu Ile Ser Tyr Cy3 Gly Gly Pro Lys Asp Glu Ala Pro ~he Ala Leu Val Gly Lys Gly Val Thr Phe Asp Thr Gly Gly Ile Ser Ala Ala Cy5 Val Val Ser Thr Leu Ala Gln Leu Lys Ala Pro Val Asn Val Val Gly Leu Ile Pro Leu Thr Glu Asn Met Pro Gly Gly Lys Ala Thr Lys Pro Gly A8p Val Val Val Ala Met Asn Gly Lys Ser Ile Cys Val Asp Asn Thr Asp Ala Glu Gly Arg Leu Ile Leu Ala Asp Ala Leu Cys Tyr Ser Ala His Phe Lys Pro Lys Trp Val Leu Asp Ile Ala Thr Leu Thr Gly Ala Met Arg Val Ala Leu Gly Asp Cys Ala Thr Gly Val Phe Ser Ser Cys Asp Asn Leu Tr ' 8 Glu Ala Gly Arg 3S5 - 390 p A8n Thr 3e95U HL 400 Val Thr Gly Asp Arg Met Trp Arg Phe Pro Leu Phe Lys His Tyr Ala Asn Arg Val Thr Glu Tyr Ser Gly Tyr Asp Val Asn Asn Ile Gly Lys 420 425 ~ 430 Gly Lys Gly Gly Gly Ser Cys Leu Ala Ala Ala Phe Leu Asn Gln Phe Arg Pro Glu Asp Val Pro Trp Met His Leu Asp Ile Ala Gly Val Met Ser Asp Cys Ser Asp Gln Ser Tyr Leu Pro Lys Gly Met Thr Gly Arg ~5 Pro Thr Arg Thr Leu Val Gln Phe Ile Cln Ser Gln Lys Arg His Ser ..~
~ .

(2) INFORMATION FOR SEQ ID NO:114:
(i~ SEQUENCE ru~v~r~wqTcTTrc (A LENGTH: 32 base pairs (B TYPE: nucleic acid (C .qTl~ qWnNWCq: single ( D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (primer) (xi) SEQUENCE ~ unL~luN': SEQ ID NO:114:

10 (2) INFORMATION POR SEQ ID NO:115:
( i ) SEQUENCE rui~q~r~F~TcTTrc (A) LENGTH: 31 ba~e pairs (B) TYPE: nucleic acid (C) q~:~NnFnMWCS single 15 : (D) TOPOLOGY: linear (ii) !5OLECULE TYPE: DNA (primer) (xi) SEQUENCE L)~ unl~lluN: SEQ ID NO:115:

(2) INFOR~ATION FOR SEQ ID NO:116-2 0 ( i ) SEQUENOE ru~
A LENGTH: 31 base pairs B TYPE: nucleic acid c 5~rq~ wnNwqq: single D TOPOLOGY: linear 25 (ii) l~OLECULE TYPE: DNA (primer) (xi) SEQUENOE ~ unl~luN: SEQ ID NO:116:

(2) INFORMATION FOR SEQ ID NO:117.
( i ) SEQUENCE rT~v~
(A) LENGTH: 31 base paira (B) TYPE: nucleic acid (C) c~7~ : single (D) TOPOLOGY: linear .. =
(ii) MOLECULE TYPE: DNA (primer~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:
GGACGAATTC l~m~ r~ G 31 WO 96111706 PCI~/US9S/14442 ~2) INFORMATION FOR SEQ ID NO:118:
(i) SEQUENCE I~UPl:lpr~TP~RT.STT~5 , A) LENGTH: 32 base pairs ~B) TYPE: n.~cleic acid ~ C) STV~ : single , D) TOPOLOGY: linear ~ii) NOLECULE TYPE: DNA (primer) (xi) SEQUENCE Ll~;b-;nlr~ n: SEQ ID NO:118:

10 (2) INFORNATION FOR SEQ ID NO:ll9:
(i) S~QUEN~ ~pvp~ "~
A) LENGT~: 30 ~ase pairs B I TYPE: n-lcleic acid I C) ST~P~ n'~sc: single I~D I TOPOLOGY: linear (ii~ NOLECULE TYPE: DNA (primer) (xi) SEQUENCE L~ nl~lUN: SEQ ID NO:ll9:
GGACCTCGAG AATTATGCGC CGTCATTTGC _ ~ - 30 While various ': ~; r ~ntS of the present invention 2 0 have been described in detail, it is apparent that modifications and adaptations of those: ' ~ q will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, 25 as set forth in the followlng ~lai~s.

Claims

What is claimed is:

1. A method to protect a host animal from flea infestation comprising treating said animal with a composition comprising a compound that reduces protease activity of fleas feeding from said treated animal, thereby reducing flea burden on said animal and in the environment of said animal.

2. A method to protect a host animal from flea infestation, said method comprising treating said animal with a composition comprising a first compound capable of reducing flea protease activity and a second compound that reduces flea burden by a method other than by reducing flea protease activity, wherein said step of treating reduces flea burden on said animal and in the environment of said animal.

3. A method to reduce flea infestation comprising treating an animal with a composition comprising a compound capable of reducing flea protease activity, said animal being selected from the group consisting of fleas and animals susceptible to flea infestation, said step of treating thereby reducing flea burden on said animal and in the environment of said animal.

4. A method to treat flea infestation comprising administering to an animal a controlled release formulation comprising a composition capable of reducing flea protease activity, thereby reducing flea burden on said animal and in the environment of said animal.

5. An isolated protein that comprises an amino acid sequence encoded by a nucleic acid molecule capable of hybridizing under stringent conditions with a nucleic acid molecule that encodes a midgut protease present in a flea midgut selected from the group consisting of a midgut from a blood-fed female flea, a midgut from a blood-fed male flea, a midgut from an unfed female flea, a midgut from an unfed male flea and a mixture thereof.

6. An isolated antibody capable of selectively binding to a protease present in a flea midgut selected from the group consisting of a midgut from a blood-fed female flea, a midgut from a blood-fed male flea, a midgut from an unfed female flea, a midgut from an unfed male flea and a mixture thereof.

7. An isolated nucleic acid molecule capable of hybridizing under stringent conditions with a gene encoding a flea protease present in a flea midgut.

8. A soluble flea midgut preparation having protease activity, wherein at least about 70 percent of said protease activity can be inhibited by 4-2-aminoethyl-benzenesulfonylfluoride-hydrocloridde.

9. A composition comprising a compound selected from the group consisting of a flea protease vaccine, an anti-flea protease antibody, a protease inhibitor and a mixture thereof, said composition, when administered to an animal, being able to reduce flea burden on said animal and in the environment of said animal.

10. A composition comprising a first compound capable of reducing flea protease activity and a second compound that reduces flea burden by a method other than by reducing flea protease activity.

11. A controlled release formulation imparting long-term control of flea infestation comprising a compound that reduces protease activity of fleas feeding from an animal, said compound being formulated in a controlled release vehicle.

12. A method to produce a soluble flea midgut preparation comprising flea proteases, said method comprising:
(a) disrupting a flea midgut to produce a mixture comprising a liquid portion and a solid portion: and (b) recovering said liquid portion to obtain said preparation.

13. A method to produce a flea protease comprising:
(a) disrupting a flea midgut to produce a mixture comprising a liquid portion and a solid portion; and (b) purifying said protease from said liquid portion.

14. A method to produce a flea protease protein comprising:
(a) culturing in an effective medium a recombinant cell capable of expressing a said protein to produce said protein; and (b) recovering said protein.

15. A method to identify a compound capable of inhibiting the proteolytic activity of a flea protease, said method comprising:

(a) contacting an isolated flea protease protein with a putative inhibitory compound under conditions in which, in the absence of said compound, said protease has proteolytic activity; and (b) determining if said putative inhibitory compound inhibits said activity.

16. A test kit to identify a compound capable of inhibiting proteolytic activity of a flea protease, said test kit comprising an isolated flea protease protein having proteolytic activity and a means for determining the extent of inhibition of said activity in the presence of a putative inhibitory compound.

17. An isolated flea nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene selected from the group consisting of a flea serine protease gene and a flea aminopeptidase gene.

18. An isolated flea protein that comprises an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene selected from the group consisting of a flea serine protease gene and a flea aminopeptidase gene.

19. A therapeutic composition that, when administered to an animal, reduces flea infestation, said therapeutic composition comprising a protective compound selected from the group consisting of: an isolated flea serine protease nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea serine protease gene;
an isolated flea serine protease protein or a mimetope thereof; an isolated antibody that selectively binds to a flea serine protease protein; an inhibitor of flea serine protease activity identified by its ability to inhibit flea serine protease activity; an isolated flea aminopeptidase nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea aminopeptidase gene;
an isolated flea aminopeptidase protein or a mimetope thereof; an isolated antibody that selectively binds to a flea aminopeptidase protein; an inhibitor of flea aminopeptidase activity identified by its ability to inhibit flea aminopeptidase activity; and a mixture thereof.

20. A method to reduce flea infestation comprising treating an animal with a therapeutic composition comprising a protective compound selected from the group consisting of: an isolated flea serine protease nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea serine protease gene; an isolated flea serine protease protein or a mimetope thereof; an isolated antibody that selectively binds to a flea serine protease protein; an inhibitor of flea serine protease activity identified by its ability to inhibit flea serine protease activity; an isolated flea aminopeptidase nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea aminopeptidase gene; an isolated flea aminopeptidase protein or a mimetope thereof; an isolated antibody that selectively binds to a flea aminopeptidase protein; an inhibitor of flea aminopeptidase activity identified by its ability to inhibit flea aminopeptidase activity; and a mixture thereof.

21. A method to produce a protein selected from the group consisting of a flea serine protease protein and a flea aminopeptidase protein, said method comprising culturing a cell capable of expressing said protein, said protein being encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene selected from the group consisting of a flea serine protease gene and a flea aminopeptidase gene.

22. The invention of Claim 1-16, wherein said protease is selected from the group consisting of an aminopeptidase, a carboxypeptidase, an endopeptidase, and a mixture thereof.

23. The invention of Claim 1-16, wherein said protease is selected from the group consisting of a serine protease, a metalloprotease, an aspartic acid protease and a cysteine protease.

24. The invention of Claim 1-16, wherein said protease is selected from the group consisting of a serine protease, a metalloprotease, and a mixture thereof.

25. The invention of Claim 1-16, wherein said protease comprises a serine protease.

26. The invention of Claim 1-16, wherein said protease comprises an aminopeptidase.

27. The invention of Claim 1-16, wherein said protease is selected from the group consisting of a trypsin-like protease, a chymotrypsin-like protease, an aminopeptidase, and a mixture thereof.

28. The invention of Claim 1-16, wherein said protease comprises a midgut protease present in the lumen of said midgut.

29. The invention of Claim 1-4 or 10-11, wherein said composition or formulation comprises a compound selected from the group consisting of a vaccine comprising a flea protease, an anti-flea protease antibody, a flea protease inhibitor, and a mixture thereof.

30. The invention of Claim 1-4 or 9-11, wherein said composition or formulation comprises a flea protease vaccine selected from the group consisting of a flea serine protease, a flea metalloprotease, a flea aspartic acid protease, a flea cysteine protease, and a mixture thereof.

31. The invention of Claim 1-4 or 9-11, wherein said composition or formulation comprises a flea protease vaccine selected from the group consisting of a flea serine protease vaccine, a flea metalloprotease vaccine, and a mixture thereof.

32. The invention of Claim 1-4 or 9-11, wherein said composition or formulation comprises an aminopeptidase vaccine.

33. The invention of Claim 1-4 or 9-11, wherein said composition or formulation comprises an anti-flea protease antibody that selectively binds to a flea protease selected from the group consisting of a flea serine protease, a flea metalloprotease, a flea aspartic acid protease, a flea cysteine protease and a mixture thereof.

34. The invention of Claim 1-4 or 9-11, wherein said composition or formulation comprises an anti-flea protease antibody that selectively binds to a flea protease selected from the group consisting of a flea serine protease and a flea metalloprotease.

35. The invention of Claim 1-4 or 9-11, wherein said composition or formulation comprises an anti-flea protease antibody that selectively binds to a flea aminopeptidase.

36. The invention of Claim 1-4 or 9-11, wherein said composition or formulation comprises a flea protease inhibitor selected from the group consisting of a serine protease inhibitor, a metalloprotease inhibitor, an aspartic acid protease inhibitor, a cysteine protease inhibitor, an aminopeptidase inhibitor and a mixture thereof.

37. The invention of Claim 1-4 or 9-11, wherein said composition or formulation comprises a flea protease inhibitor selected from the group consisting of a serine protease inhibitor, a metalloprotease inhibitor, an aminopeptidase inhibitor, and a mixture thereof.

38. The invention of Claim 1-4 or 9-11, wherein said composition or formulation comprises a flea serine protease inhibitor.

39. The invention of Claim 1-4 or 9-11, wherein said composition or formulation further comprises a component selected from the group consisting of a pharmaceutically acceptable excipient, an adjuvant, a carrier, and a mixture thereof.

40. The invention of Claim 1-4 or 9-11, wherein said composition or formulation reduces flea burden by a method selected from the group consisting of reducing the viability of fleas that feed from said treated animal, reducing the fecundity of female fleas that feed from said treated animal, reducing the reproductive capacity of male fleas that feed from said treated animal, reducing the viability of eggs laid by female fleas that feed from said treated animal, altering the blood feeding behavior of fleas that feed from said treated animal, reducing the viability of flea larvae, altering the development of flea larvae, and a mixture thereof.

41. The invention of Claim 1-4 or 9-11, wherein said composition or formulation reduces flea viability by at least about 50 percent within at least about 21 days after said fleas begin feeding from said treated animal.

42. The invention of Claim 1-4 or 9-11, wherein said composition or formulation reduces flea fecundity by at least about 50 percent within at least about 30 days after said fleas begin feeding from said treated animal.

43. The invention of Claim 1-4 or 9-11, wherein said composition or formulation is administered by at least one route selected from the group consisting of oral, nasal, topical, transdermal, rectal, and parenteral routes.

44. The invention of Claim 1-4 or 9-11, wherein said composition or formulation is administered to an animal selected from the group consisting of mammals and birds.

45. The invention of Claim 1-4 or 9-11, wherein said composition or formulation is administered to an animal selected from the group consisting of cats and dogs.

46. The invention of Claim 1-16, wherein said fleas are of a genus selected from the group consisting of Ctenocephalides, Cyopsyllus, Diamanus, Echidnophaga, Nosopsyllus, Pulex, Tunga, and Xenopsylla.

47. The invention of Claim 1-16, wherein said fleas are of a species selected from the group consisting of Ctenocephalides felis, Ctenocephalides canis, Pulex irritans and Pulex simulans.

48. The invention of Claim 1-3 or 9-10, wherein said composition comprises a controlled release formulation.

49. The invention of Claim 1-4 or 9-11, wherein said composition or formulation comprises a biocompatible polymer.

50. The invention of Claim 1-4 or 9-11, wherein said composition or formulation comprises a liquid that upon administration to said animal forms a solid or a gel in situ.

51. The invention of Claim 1-4 or 9-11, wherein said composition or formulation is biodegradable.

52. The invention of Claim 1-4 or 9-11, wherein said composition or formulation is capable of effecting treatment for at least about 1 month after administration of said composition or formulation to said animal.

53. The invention of Claim 2 or 10, wherein said first compound, by reducing flea proteolytic activity, enhances the efficacy of said second compound.

54. The invention of Claim 2 or 10, wherein said first compound is selected from the group consisting of a flea protease vaccine, an anti-flea protease antibody, a protease inhibitor and a mixture thereof.

55. The invention of Claim 2 or 10, wherein said second compound is selected from the group consisting of a compound that effects active immunization and a compound that effects passive immunization.

56. The invention of Claim 2 or 10, wherein said second compound a is selected from the group consisting of a compound that inhibits binding between a flea membrane protein and its ligand, a compound that inhibits hormone synthesis, a compound that inhibits vitellogenesis, a compound that inhibits fat body function, a compound that inhibits flea muscle action, a compound that inhibits the flea nervous system, a compound that inhibits the flea immune system, a compound that inhibits flea feeding, and a mixture thereof.

57. The invention of Claim 5, wherein said protein, when administered to an animal, is capable of eliciting an immune response against a flea midgut protease.

58. The invention of Claim 5, wherein said protein has protease activity.

59. The invention of Claim 1-16, wherein said protease is selected from a group of midgut proteases having molecular weights ranging from about 5 kD to about 200 kD, as determined by SDS-PAGE.

60. The invention of Claim 1-16, wherein said protease comprises a midgut protease selected from the group consisting of a serine protease having a molecular weight of about 26 kD, a serine protease having a molecular weight of about 24 kD, a serine protease having a molecular weight of about 19 kD, and a serine protease having a molecular weight of about 6 kD, said molecular weight being determined by SDS-PAGE.

61. The invention of Claim 1-16, wherein said protease comprises a serine protease having a molecular weight of about 26 kD as determined by SDS-PAGE.

62. The invention of Claim 1-16, wherein said protease comprises a midgut protease having a pH activity optimum ranging from about pH 5 to about pH 10.

63. The invention of Claim 1-16, wherein said protease comprises a midgut protease having a proteolytic activity that is inhibited by at least about 50 percent by a serine protease inhibitor.

64. The invention of Claim 1-5, 8-11 or 15-16, wherein said compound of Claim 1, 3, 9 or 11, said first compound of Claim 2 or 10, said composition of Claim 4, said protein of Claim 5, 15 or 16, or said preparation of Claim 8 comprises a material recovered by a method comprising:
(a) disrupting a flea midgut to produce a mixture comprising a liquid portion and a solid portion; and (b) recovering said liquid portion to obtain a soluble flea midgut preparation comprising said material.

65. The invention of Claim 64, wherein said material is further purified.

66. The invention of Claim 1-5, 8-11, or 15-16, wherein a protein of said invention is produced by a method comprising culturing a recombinant cell comprising a nucleic acid molecule encoding said protein to produce said protein and recovering said protein therefrom.

67. The invention of Claim 6, wherein said antibody comprises a passive immunotherapy composition that when administered to a host animal reduces flea burden on said animal and in the environment of said animal.

68. A recombinant molecule comprising an isolated nucleic acid molecule as set forth in Claim 7 operatively linked to a transcription control sequence.

69. A recombinant cell comprising a cell having at least one nucleic acid molecule as set forth in Claim 7, said cell being capable of expressing said nucleic acid molecule.

70. The invention of Claim 17-21, wherein said serine protease gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ
ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:36, and SEQ ID NO:38, SEQ ID NO:52, SEQ ID NO:54, SEQ ID
NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID

NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID
NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID
NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID
NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID
NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID
NO:104, SEQ ID NO:106, SEQ ID NO:108, and SEQ ID NO:110, and wherein said aminopeptidase gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID
NO:50 and SEQ ID NO:112.

71. The invention of Claim 17-21, wherein said serine protease gene comprises a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO 2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ
ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID
NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID
NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID
NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID
NO:47, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID
NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID
NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID
NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID
NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID
NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID
NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID
NO:107, SEQ ID NO:109, and SEQ ID NO:111, and wherein said aminopeptidase gene comprises a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:51 and SEQ ID NO:113.

72. The invention of Claim 17, 19, or 20, wherein said nucleic acid molecule comprises a nucleic acid sequence that encodes a protein selected from the group consisting of a serine protease protein or an aminopeptidase protein.

73. The invention of Claim 17-21, wherein said nucleic acid molecule hybridizes under stringent hybridization conditions with a nucleic acid molecule selected from the group consisting of nfSP1, nfSP2, nfSP3, nfSP4, nfSP5, nfSP6, nfSP7, nfSP8, nfSP9, nfSP10, nfSP11, nfSP12, nfSP13, nfSP14, nfSP15, nfSP16, nfSP17, nfSP18, nfSP19, nfSP20 and nfAP1580.

74. The invention of Claim 17-21, wherein said nucleic acid molecule hybridizes under stringent hybridization conditions with a nucleic acid molecule selected from the group consisting of nfSP1779, nfSP2 944, nfSP3 177, nfSP4 672, nfSP5 157, nfSP5 218, nfSP6 932, nfSP7 894, nfSP8 299, nfSP9 266, nfSP10 378, nfSP11 252, nfSP12 144, nfSP12 225, nfSP13 850, nfSP14 213, nfSP15 252, nfSP16 168, nfSP18 534, nfSP19 359, nfSP20 841, and nfAP1580

75. The invention of Claim 17-21, wherein said nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of nfSP1 779, nfSP2 944, nfSP3 177, nfSP4 672, nfSP5 157, nfSP5 218, nfSP6 932, nfSP7 894, nfSP8 299, nfSP9 266, nfSP10 378, nfSP11 252, nfSP12 144, nfSP12 225, nfSP13 850, nfSP14 213, nfSP15 252, nfSP16 168, nfSP18 534, nfSP19 359, nfSP20 841, and nfAP 1580.

76. The invention of Claim 17-21, wherein said nucleic acid molecule comprises a nucleic acid sequence that encodes a protein having an amino acid sequence and allelic variants of any of said nucleic acid molecules.

77. The invention of Claim 17-21, wherein said nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting comprises a nucleic acid sequence selected from the group consisting of SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID
NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID

and an allelic variant of any of said nucleic acid molecules.

78. The invention of Claim 17, 19, or 20, wherein said nucleic acid molecule comprises an oligonucleotide.

79. The invention of Claim 72, wherein said protein, when administered to an animal is capable of eliciting an immune response against a flea protein selected from the group consisting of a flea serine protease protein and a flea aminopeptidase protein.

80. The invention of Claim 72, wherein said protein has proteolytic activity.

81. The invention of Claim 17, wherein said nucleic acid molecule, when administered to an animal is capable of reducing flea infestation.

82. A recombinant molecule comprising a nucleic acid molecule as get forth in Claim 17 operatively linked to a transcription control sequence.

83. A recombinant virus comprising a recombinant molecule as set forth in Claim 82.

84. A recombinant cell comprising a nucleic acid molecule as set forth in Claim 17, said cell being capable of expressing said nucleic acid molecule.

85. The invention of Claim 18-21, wherein said protein, when administered to an animal is capable of eliciting an immune response against a flea protein selected from the group consisting of a flea serine protease protein and a flea aminopeptidase protein.

86. The invention of Claim 18-21, wherein said protein has proteolytic activity.

87. The invention of Claim 18-21, wherein said protein comprises an amino acid sequence selected from the group consisting of and a protein encoded by an allelic variant of a nucleic acid molecule encoding a protein comprising any of said amino acid sequences.

88. The invention of Claim 18-20, wherein said protein is produced by a process comprising culturing a recombinant cell transformed with a nucleic acid molecule encoding said protein to produce said protein.

89. The invention of Claim 18 or 21, wherein said protein or a mimetope thereof, when administered to an animal is capable of reducing flea infestation.

90. The invention of Claim 18 or 21, wherein said protein or a mimetope thereof, when administered to a host animal is capable of reducing flea burden on said animal and in the environment of said animal.

91. The invention of Claim 18 or 21, wherein said protein is used to identify an inhibitor selected from the group consisting of an inhibitor of flea serine protease activity and an inhibitor of flea aminopeptidase activity.

92. The invention of Claim 91, wherein said inhibitor, when administered to an animal, is capable of reducing flea infestation.

93. An isolated antibody that selectively binds to a protein as set forth in Claim 18 or 21.

94. The invention of Claim 19 or 20, wherein said composition further comprises a component selected from the group consisting of an excipient, an adjuvant, a carrier, and a mixture thereof.

95. The invention of Claim 19 or 20, wherein said composition comprises a controlled release formulation.

96. The invention of Claim 19 or 20, wherein said composition further comprises a compound that reduces flea burden by a method other than by reducing flea protease activity.

97. The invention of Claim 19 or 20, wherein said animal is selected from the group consisting of adult fleas, flea larvae and animals susceptible to flea infestation.

98. The invention of Claim 19 or 20, wherein larval flea infestation is reduced by flea larvae ingesting adult flea feces comprising said therapeutic composition.

99. The invention of Claim 19 or 20, wherein larval flea infestation is reduced by flea larvae ingesting adult flea feces, said feces comprising anti-flea protease antibodies elicited in a host animal in response to administration of one or more of said isolated flea protease proteins, said adult flea having fed from said host animal after said administration, said protease being selected from the group consisting of a serine protease and an aminopeptidase,

100. The invention of Claim 19 or 20, wherein said animal is selected from the group consisting of mammals and birds.

101. The invention of Claim 19 or 20, wherein said animal is selected from the group consisting of cats and dogs.

102. The invention of Claim 17-21, wherein said fleas are of a genus selected from the group consisting of Ctenocephalides, Cyopsyllus, Diamanus, Echidnophaga, Nosopsyllus, Pulex, Tunga, and Xenopsylla.

103. The invention of Claim 17-21, wherein said fleas are of a species selected from the group consisting of Ctenocephalides felis, Ctenocephalides canis, Pulex irritans and Pulex simulans.