CA2123420A1

CA2123420A1 - Protease vaccine against heartworm

Info

Publication number: CA2123420A1
Application number: CA002123420A
Authority: CA
Inventors: Robert B. Grieve; Jennifer Richer; Glenn R. Frank; Judy Sakanari
Original assignee: Individual
Current assignee: Colorado State University Research Foundation
Priority date: 1991-11-12
Filing date: 1992-11-12
Publication date: 1993-05-27
Also published as: JPH07501219A; EP0635058A4; EP0635058A1; AU3072392A; WO1993010225A1; AU675214B2

Abstract

The transition between the L3 and L4 larval stages of the nematode filarial parasites such as Dirofilaria immitis occurs after introduction into the animal host and is mediated by at least one metalloprotease and/or cysteine protease unique to the L3 or L4 larval stage. Methods to prevent and treat filarial infection are provided by vaccines comprised of the immunogenic determinants of the characteristic L3 or L4 proteases and by administration of inhibitors of these proteases.

Description

2:123~2{) W~93/10225 PCT/US92/09702 PROTEASE VACCINE AGAINST HEARTWORM

Technical FieId The invention relates to prevention andtreatment of nematode-caused filarial disease in animal hosts, such as heartworm which occurs most commonly in dogs. Heartworm infection is caused by the nematode Dirofilaria immitis, and the treatment and prevention method of the invention can be applied specifically to this disease by employing the characteristic metallo-protease and/or cysteine protea~e associated with this organism.
:
Backqround Art The heartworm i~fection caused by D. immitis is a widely distributed problem in dogs in most regions ofthe world with the exception of Africa. Current treatment is generally chemoprophylactic with ~gents designed to directly kill the infecting organisms. While this treatment has gained accepta~ce, because of the ; 25 inherent toxici~ty of such treatment, it would be preferable to immunologically protect the host against i~fection, or to revise the chemoprophylactic regime to i~clude less toxic agents. The present in~ention is directed to this goal.
Other nematode filarial infections are of even greater significance and in~-olve life cycles of the infectious agent similar to those related to heartworm.
For example, o~ more concern are the other filarids which infect humans, and more than 200 million people worldwide are estimated to have such infections. Filarid~ which W093/l0225 2 1 2 3 ~ 2 0 -2- PCT/US92/09702t infect humans include Brugia malayi, Wuchereria bancrof~i, and Onchocerca volvulus. These are serious infections which can cause blindness and elephantiasis in humans. At present, there is no effective vaccine available against filarial nematode infection.
As the life cycles of the infectious agents are similar in all of these diseases, heartworm infection can be used as an illustration. This life cycle can be described as follows:
Heartworm infection, specifically in dogs, generally occurs through passage of the third-stage larvae (L3) of the nematode D. immitis into the subcutaneous ti~sue from a mosquito vector. When these larvae are passed into the animal~s tis~ue, their life cycle is continued by molting into a fourth larval stage (L4), which then migrates toward the heart and pulmonary arteries where the subsequent stage matures into an adult. The ~3 remain at the site of inoculation by the mosquito until molting occurs. ~4 emigrate through cutaneous tissue and muscle ~nd do not molt to fifth stage for 50-70 days af~er infection (Grieve, R., et al., Epidem Rev (1983) 5:220-246). Adult D immitis, which are on the order of 12-20 cm (males) and 25-31 cm (females) in length, produce, in this location, motile vermiform embryos called microfilariae, which are o~ly 0.3 mm long and which traverse capillary beds and circulate in the ~ascular system. The microfilariae are ingested by mosquitoes and continue their life cycle through L3 in the mosquito vector.
The transition from L3 to h4 i5 thus the initial step in the infection cycle in the animal ho~t.
This transition involves a molting process, which has been studied by Abraham, D., et al., Experimental Parasitol (1990) 70:314-322, incorporated herein by reference. In this study, the morphology of the larvae wO 93/10225 2 ~ ~ 3 ~ 2 0 PCT/US92/09702 during the molt was monitored, and the roles of temperature and of albumin, which appeared to be essential for this process, were evaluated. The L3 stage contains a cuticle and epicuticle which are abandoned, and the body wall of the larva is encased in the L4 stage cuticle and epicuticle. During this molting process, an excretory-secretory (E-S) product which, among other components, contains enzymes presumably employed in the molting proceqs is formed.
The excretory-secretory products of various tissue invasive helminths have been studied. Proteases have been found in a number of them. Both Schistosoma mansoni and Schistosomatium douthitti produce elastases capable of degrading skin (~cKerrow, J.H., et al., Experimental Parasitol (1982) ~3:249; McKerrow, J.H., et al., J ~iol Chem (1985) 231:47; Amiri, P., et al., Mol Biochem Parasitol (1988) 28:113). Fasciola hepa~ica also release~ a number of proteolytic enzymes (Dalton, J.P., et al., ol Biochem Parasitol (19~9) 35:161). The adult hookworm ~ncylos~oma caninum releases a histolytic protease and a protease that acts as an anticlotting agent (Hotez, P.J., et al., J Biol Chem (1985) 260:7343).
Toxocara anis larvae secrete proteases which degrade cQmponents of extracellular matrix (Robertson, B.D., et al., Bxperimenal ~E~_itol ~1989) ~:30).
A number of filarial nematodes also have been hown to produce proteases that act on ex~racellular matrix co~ponents, including Onchocerca cervipedis, O.
cer~icalis, and Bruqia malayi (Lackey, A., et al., Experimental Parasitol (1989) 68:176; Petralanda, I., et al., ~Ql Biochem Parasitol (1986) 19:51). The protease activity includes collagenase in the case of Bruqia malayi, O. cervicalis and O. cervipedis. A collagenase and a leucine aminopeptidase have been found in the molting process of Haemonchus contortus by Rogers, W.P., W093/10225 ~ 1~ 3 4 ~ O PCT/US92/09702 J Parasitol (1982) 12:495, and Gamble, H.R., et al., Mol Biochem PaxasitQl (1989) 33:49.
With respect to D. immii~is, protease activity has been detected i~ soluble extracts of D. immitis adults by Maki, J., et al., J Helminthol ~1986) 60:31-37, and in extracts of microfilariae by Tomashiro, W.K., et al., J Parasitol ~1987) 73:149-154. However, the soluble extracts of D. immitis L3 and L4 and the excretory-secretory ~E-S) products of these larval stages have not previously been studied.
It is also known that collagens form the major structural components of nematode cuticle by virtue of studies conducted on Caenorhabditis eleqans, B. malayi, and B. ~ahangi.
1~
Disclosure of the Invention The invention i8 directed to prevention and treatment of filarial nematode infection in animal hosts and to purified and isolated forms of the proteases ~0 associated with the ~3 and L4 larval stages of the parasites that cause these infections. One of these nematodes is Dirofilaria immitis, which causes heartworm ; ~ in dogs. Other diseases of importance are caused by nematodes such as tho~e listed above. The invention provides an approach to the eradication of co~ditions caused in animals by filarial nematodes, and provides materials useful in these and in in vitro contexts.
Accordingly, i~ one aspect, the invention is directed to a method to protect animal subjects, including humans, against-filarial nematode infection, which method comprises administering to the 8ub; ect an effective amount of a metalloprotease and/or cysteine protease characteristic of transition from the L3-L4 stage of the relevant filarial nematode effective to immunologically protect the subject against infection.

5 ~ d 3 ~ ~ ~ PCT/US92/09702 The characteristlc metalloprotease(s) may be found in the L3 or L4 excretory-secretory material or in L3 or L4 lysates. The cysteine protease is found in L3 and L4 lysates.
In another aspect, the invention is directed to the treatment of nematode filarial infection in animal subjects, including humans, which method comprises administering to that subject an effective amount of a metalloprotease inhibitor and/or cysteine protease inhibitor.
In other aspects, the invention is directed to antibodies immunospecific for filarial L3 or L4 excretory-secretory products or L3 or ~4 lysate metalloprotease(s) or to B3~or L4 lysa~e cysteine protease(s) and to pharmaceutical compositions and vaccines containing them.
In still another aspect, the invention is directed to the L3/L4-associated metalloproteases and cysteine proteases of filarial parasites in isolated and purified form. These purified proteases are additionally u~eful to assay for the presence or absence of a~ti~odies i~ the diagnosis of affected individuals and to regulate the growth of cell cultures in ~itro, as well as in other therapeutic applications.
Brief D~ri ~ion of the Drawings Figure 1 shows the elution pattern of protease activity from L3/L4 E-S.
Figure~2 shows the elution pattern of protease acti~ity from L4 lysate.

WO93/1022~ 212 3 ~ 2 0 -6- PCT/US92/09702 Modes of Carrying Out the Invention As used herein, "metalloprotease" of ~3 and ~4 excretory-secretory preparation (L3 and L4 E-S) or of L3 or L4 lysates refers to metalloprotease enzymes characteristic of the excretory-secretory products obtained during the molting of the L3 larval stage into L4 for filarial infective nematodes; or of whole worm lysates of the L3 or L4 larval stage. At least one "cysteine protease" is also found in ~3 and L4 lysates.
While the E-S and lysate preparations from D. immitis are exemplified below, similar E-S or lysate preparations can be obtained from various other filarial parasites such as those set forth in the Background section ~bove, and including, specificall~y, for example, B. malayi, W. bancrofti, O. volvulus, Dipetalonema per~tans, D streptocerca, Mansonella ozzardi, and Loa loa.

Preparation of~ Larval Cultures The parasites can be cultured ln vitro under suitable conditions~to provide a source for the E-S
preparation or for the L3 or L4 lysates. For example, D.
immitis can be cultured as described by Abraham, D., et al., J Parasitol (1987) 73:377-383. Briefly, the mos~uito Aedes :eqY~ti Liverpool (black-eyed strain) are 25~;~ infected with D. immitis by feeding on microfilaremic blood obtaine~d f~rom a~single experimentally infected dog.
Fifteen days after feeding, the mosquitos are anestheeized, surface sterilized and placed on screens in funnels filled~with a l:l mixture of NCTC-135 and Iscove' 8 modifisd~Dulbecco medium (Sigma) containing 2.5 ~g/ml amphotericin-B; lO0 ~g/ml gentamicin; 50 ~g/ml sulfadiazone; and lO ~g/ml trimethoprim. The larvae are collected from~funnels 90 minutes postincubation.
The cultures are maintained at a concentration of ten L3 organisms per ml of medium in 5% CO2 and .

2 ~ '1 2 0 saturated humidity. The larvae (L3~ are cultured at 37 in the foregoing medium, supplemented with 20~ fetal calf serum for l-~ days.
Alternatively, and preferably, lO days after feeding, the mosquitos are anesthetized and the worms are recovered by dissecting the heads and allowing the worms to emexge into medium with 20~ Seru-max ~Sigma) to induce molting. After 48 hr, the worms are recovered, washed 5 times in medium which does not contain Seru-max, and recultured therein.
: :
Preparation of L3 and L4 E-S
L3 ES is collected between 4~ and 96 hours of culture on Seru-max free medium. L4 ES is collected between 96 and 144 hours in indentical culture conditions. Medium containing ES is collected and filtered through a 0.45 ~m filter. The ES is concentrated and the buffer is exchanged into pH 7.2 PBS
using ultrafiltration and lO kd exclusion limit to obtain the fraction of ~lO kd MW.

Pre~aration of L3 and L4 Lysates Larval~soluble extracts are prepared from L3 collected on day 2, ~ust after the wash but prior to the lt, and L4 are~collected on day 6 in serum-free culture. Pellets of lO,000 worms in P~S are disrupted by ten lO-sec high frequency pulses using a tissue sonicator. Sonicated worms are centrifuged for 5 min at : .
12,000 x g, and the supernatant collected~
Determination of E-S and Lysate Components Protein concentration for both E-S and whole worm soluble extracts may be estimated using a Micro BCA
kit (Pierce Chemical Co., Rockford, IL). All samples are maintained at -20 C prior to further analysis.

2123 ~120 -8-By "metalloprotease" of the L3 and L4 E-S
preparation or of L3 or L4 lysates is meant a protease enzyme which is found in the excretory-secretory product of third or fourth stage lar~ae or in L3 or L4 lysates of a filarial nematode parasite as ascertained by acti~ity against the synthetic substrate h-phenylalanine-AMC (h-F-AMC, defined below) and which is inhibited by metallo-protease inhibitors such as 1,10-phenan~hroline and EDTA.
Metalloprotea~e activity has been reported in E-S
products of third stage larvae of certain species, including B. malayi, O. cervicalis, and O. cervipedis as set forth above. The activity is also present in L3 and L4 lysates.
The invention also relates to "cysteine protease(s)" from L3 or L4 lysates, which lysates may be prepared as described above. The cysteine proteases of the invention are characterized by ability to hydrolyze Z-valine-leucine-arginine-AMC tZ-VLR-AMC, defined below) and this acti~ity is inhibited by E64. Again, D. immitis is used for illustration below, but other filarial nematodes may be used.
Both of these enzymes may be obtained in purified and isolated form using chromatographic me~hods with use of the appropriate substrate as~ay to monitor elution fractions as further described below.
~ ecause of the practical difficulties in obtaining sufficient quantities of the metalloprotease of L3 and L4 E-S preparations or the metalloprotease or cysteine protease from ~3 or L4 lysates to provide material for vaccines, alternati~e methods of production are preferred when large quantities are desired.
Specifically, for the full-length metalloprotease or cysteine protease, recombinant production is the most practical approach; for immunogenic subunits which are capable of eliciting antibodies that neutralize the metalloprotease or cysteine protease activity, ordinary solid phase peptide synthesis may be preferred. However, eYen in ~his instance, it may be desirable to utilize recombinant production to obtain tandem repeats of the immunogenic subunit. Production of tandem repeats may enhance the immunogenicity of the material. In addition, the subunit vaccines may be recombinantly produced as fusion proteins to an immunogenicity-conferring sequence.

Recombinant Production The recombinant sequences necessary for production of the relevant metalloprotease or cysteine protease are obtained in a process analogous to that described by Sakanari, J.A., et al., Proc Natl_Acad Sci (1989) 86:4863. In this process, the gene encoding the metalloprotease or cysteine protease is isolated from cDNA prepared from total mRNA o~ the L3 or L4 stage of the parasite using oligonucleotide primers and the polymerase chain reaction (PCR) and suitabie probes.
D. immitis genomic or cDNA is used as a ~ource ;for protease-encoding genes. For isolation of the metàlloprotease gene, primers are designed based on consensus sequences in the bacterial metalloprotease thermolysin, and~members of the human metalloprotease family which include stromelysin, stromelysin II, and Pump-I. These highly ho logous gene~ are all metalloproteases, and the cDNAs containing these equences ha~e been disclosed (Muller, D., et al., Biochem J ~I988) 253:187-192 and Quantin, B., et al., ioch~mist~y (1989) 28:5327-5334). Primers can be designed based on the conserYed regions, including the active site. PCR amplification is conducted a~ described by Sakanari et al. ~supra), and reaction products are loaded on agarose/NuSieve (FMC). An additional probe designed based on the above-mentioned published sequences WO93/10225 2 1 ~ ~ 1 2 0 - lo - PCT/US92/09702 is used to identify the relevant amplified gene products on Southern blots. The fragments are cut out of the gel, extracted with ~glass milk" (Geneclean) and liga~ed into Bluescript ~Stratagene) to obtain sequences of both coding and anticoding strands, using the dideoxy method of Sanger with Sequenase (USB) and the SK and KS primers for sequencing in both directions. The gene fragments obtained are then u~ed as probes to screen a cDNA
~ibrary. They are labeled with 32p using standard random priming methods.
For preparation of analogous probes for the cysteine protease genes, primers are de~igned based on the sequences disclosed in Eakin, A.E. et al., ~1 Biochem Parasitol (1~90) 39:1-8. Otherwise, the retrie~al of probes from genomic DNA can be conducted as abo~e.
The cDNA library is construc~ed from messenger RNA i~olated from third stage larvae which have been in culture for 4~-72 hours. The mRNA is isolated by the sing}e step acid guanidinium thiocyanate/phenol/
chloroform extraction method of Chomczynski, P. and Sacchi, N., Anal Biochem (1987) 162:156-159. The RNA is passed over an oligo-dT cellulose col~mn and the poly-A
RNA is eluted using ~andard procedures. cDNA is prepared from the mRNA using sta~dard procedures such as tho~e of Gubler, U. and Hoffman, B.J., Gene ~1983) ~:263. The cDNA is treated by methylation of internal EcoRI sites, a~d phosphorylated EcoRI linker~ are added to the ends of the cDNA and trea~ed again with pho~phatase. The treated cDNA contain linkers diges~ed with EcoRI to generate cohesive cloning ends for in~ertion in~o A-gtll arms (Stratagene, San Diego, CA) and packaged using Gigapack (Stratagene). Standard methods are used to titer and plate the library for screening.

- ~1.?3'l2~

The library can be screened either using the probes obtained as described above, heterologous probes, or the expression products can be screened using antibodies prepared against the proteases obtained from the E-S product or lysates. Selected clones are plaque purified, and the isolated coding sequences are used to produce the recombinant protease.
The cloned DNA can be used directly in expres~ion vectors, or DNA can be synthesiæed using standard 901 id phase techniques to obtain any embodiment o~ the coding sequence to supply all or a portion of the gene.
For example, a DNA coding sequence ~or the protease can be prepared synthetically from overlapping oligonucleotides who~e -Qequence contains coduns for the amino acid sequence encoded in the native gene. Such oligonucleotides are prepared by standard methods and as~embled into a cGmplete or partial coding sequence.
See, e.g., Edge, Nature (1981) 2~2:756; Nambair et al., Science (1984) 223:1299; Jay et al., J Biol Chem (1984) 259:6311.
Thus, a DNA molecule containing the coding ~e~uence for the filarial nematode metalloprotease or cysteine protease can be cloned in any suitable vector and thereby maintainéd in a compo~ition substantially free of vectors that do not contain the coding ~equence for the protease (e.g., other library clones). Numerous clo~ing vectors are known to those of skill in the art, and the Yelection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vec~ors for clo~ing and the host cells which they transform include bacteriophage ~ (E. coli), pBR322 (E. coli), pA~YC177 (E. coli), pKT230 (gram-negative bacteria), pBG1106 (gram-negative bacteria), pLAPR1 (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), 2~23~12~ -12-pHVl4 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ6l (Streptomyces), pUC6 (Streptomyces), actinophage ~C31 (Streptomyces), YIp5 (yeast), YCpl9 (yeast), and bovine papilloma virus (mdmmalian cells).
For expression, the coding sequence of the protea~e gene is placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein a~ ~Icontrol~l sequences) so that the protease-encoding sequence is transcribed into RNA in the host cell transformed by the vector. The coding sequence may or may not contain a signal peptide or leader sequence. In bacteria, the protease is preferably produced by the expression of a coding sequence which does not conSain any ~ative signal peptide, or by expression of a coding sequence containing the leader sequence in a eucaryotic system when post-translational processing removes thP
leader sequence. The protease can also be expressed in the form of a fu~ion protein, wherein a heterologous amino acid sequence is expressed at the N- or C-terminus.
See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437.
The recombinant vector is constructed so ~hat the protease-encoding sequence is located in ~he vec~or with the appropriate control sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding ~equence i9 transcribed under the control of the control sequences (i.e., by RNA polymera~e which attaches to the DNA
molecule at the control sequences). The control ~equences may be ligated to the coding sequence prior to in~ertion into a vector, such as the cloning ve~tors described above. Alternatively, the coding ~equence can be cloned directly into an expreYsion ~ector which already contains the control sequence and an appropriate restriction site downstream from control sequences. For WO93/10225 ~12 3 4 2 ~ PCT/US92/09702 expression of the protease-encoding sequence in other than nematodes, the control sequences will be heterologous to the coding sequence. If the host cell is a procaryote, it is also necessary that the coding ~e~uence be free of introns; e.g., cDNA. If the selected host cell is a nematode cell, the control sequences can be heterologous or homologous to the protease-encoding sequence, and the coding sequence can be genomic DNA
containing introns or cDNA. Either genomic or cDNA
coding sequences may be also expressed in yeast.
A number of procaryotic expression vectors are known in the art. See, e.g., U.S. Patent No~. 4,440,B59;
4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437;
4,418,149; 4,411,994; 4,366,246; 4,342,832. Preferred expression vectors, however, are those for use in eucaryotic systems. Yeast expre~sion vectors are ~nown in the art. See, e.g., U.S. Patent Nos. 4,446,235;
4,443,539; 4,430,428. See also European Patent Specifications 103,409; 100,561; 96,491. The recombinant protease can be produced by growing host cells tra~sformed by the expression vector described above under conditions whereby the protease is produced. Human collagenase cDNA has been cloned and expressed in active form in eucaryotic cells (Muller, D., et al., Biochem J
(I988) 2$3:187-192). The protease is then isolated from the host cells and purified. If the expre~sion system - secretes the protease into growth media, the desired protein can ~e purified directly from cell-free media.
If the protease is not secreted, it is isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art; purifications similar to those exemplified below can be used.

WO93/1~22S PCT/US92/097~2 2 1 2 3 Ll 2 ~ - 14-Antibody Production Either nati~e or recombinant proteases of the invention can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, the purified protease is used to immunize a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) and serum from the immunized animal later collected and treated according to known procedures. Compositions containing polyclonal antibodies to a variety of antigens in addition to the relevant protease can be made substantially free of anti~odies which are not protease antibodies by passing the composition through a column to which the desired protease has been bound. After washing, polyclonal antibodi~es are eluted from the column. Monoclonal antibodies can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal, antibody-producing cell lines can also be created by techniques other than fusion, ~uch as dire~t transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., Schreier, M., et al., HYBRIDOMA
TRCHNIQUES (1980); Hammerling et al., MONOCLONAL
ANTIBODI~S AND T-CE~L HYBRIDOMAS (l98l~; Kennett et al., MONOCLONAL ANTIBODIES (1980).
-By employing the metalloprotease or cysteine protease (native or synthetic) as an antigen in the immNnization of the source of the B-cells immortalized for the production of monoclonal antibodie~, a panel of monoclonal antibodies recognizing epitopes at different sites on the protease can be obtained. Antibodies which recognize an epitope in the active site binding region of the protease can be readily identified in competition assays between antibodies and enzyme substrate.
Artificial substrates such as Z-VLR-AMC ~cysteine .

protease) or h-F-AMC (metalloprotease) can also be u~ed.
Such an~ibodies have therapeutic potential if they are able to block the binding of protease to its substrate n vivo. Antibodieg which recognize a site on the protea~e S are also useful, for example, in the purification of the desired protease protein from cell lysates or ~ermentation media, and in its characterization. In general, as is known in the art, the protease antibody is fixed (immobilized) to a solid support, such as a column or latex beads, contacted with a solution containing the protease, and separated from the solution. The protea~e, bound to the immobilized antibodies, is then eluted.

Isolation and Purification of the L3 and L4 Proteases ~s stated above, the cysteine protease characteristic of the L3 and L4 lysates and the metalloprotea~e characteristic of these lysates, a~ well as the L3/L4 E-S, can be obtained in isolated and purified form either using the appropria~e larval stage of the desired parasitic nematode as starting material, using recombinant production in cell culture and isola~ing the protease resulting from the cysteine protease or metalloprotease gene expression, or by ~ynthesiziny 8ubunit8 of these proteins using standard peptide syntheQis techniques. The na~ure of the purification method will depend on the origin of the protease or peptide.
When isolated from natiYe sources, the lyqate or E-S materiaI i9 ~ubjected to chromatographic technigues, typically chromatography using affinity chromatography (e.g., affinity chromatography using antibodies prepared with respect to the protease as affinity ligands), ion-exchange chromatography, sizing columns, reverse-phase colu~ns, and the like.
Optimization of the purification procedure is within the WO93/10225 2 1 2 3 ~ 2 0 - 16- PCT/US92/09702 skill of the art, as the fractions eluted from the columns can be assayed using activity determination with a fluorometric substra~e characteristic of the metalloprotease or cysteine protease. For the metalloprotease of ~ immitis, h-F-AMC is a convenient ~ubstrate; for the cysteine protease of thiY worm, Z-V~R-AMC is appropriately used. The specificity and nature of the protease can be verified by supplementing the assay with various inhibitors known to characterize metalloproteases or cysteine proteases. Modified forms of these substrates may be appropriate for the ~etalloproteases or cysteine proteases of other species of filarial nematodes; the appropriate substrate can be ascertained by the conduct of preliminary assays on the crude extracts, as exemplified herein ~or the D. immitis species .
If the protease is produced recombinantly, similar techniques can~be used, although the starting material generally contains the protease in a more highly concentrated form. ~Further modification of the purification procedure is appropriate for isolation of the peptides prepared by solid-phase synthesis, since the nature of the contaminants is different. Generally, dialysis or other~size-separation methods are appropriate.
The purified and isolated ~orms of the cy3teine and metalloproteases of the various filarial n~matode species can be used in the production of antibodies (which antibodies, in turn, are useful in immunoassays and separation techniques), as reagents in ~mmunoas~ay procedures for the presence or absence of antibodies, and ; in the regulation of cell culture in vitEo by controlling extracellular matrix formation or status.
::

' WO93/10225 ~ 3 '~ 2 0 PCT/US92/09702 U~e of Purified and Isolated Proteases in Diaqno~is The purified and isolated proteases are u~eful in diagnostic immunoassays for the presence or absence of antibodies with respect to filarial nematode species.
These assays can be used to assess the disease state of a host organism or to assay titers in immunization protocols. The assays are conducted in standard immunological format, including RIA, ELISA, and fluorescence-labeled assays. The assays can be conducted in either a direct or a competitive format and rely on ~eparations by virtue of binding to solid support or by virtue of precipitation of immunological complexes. A
large number of protocols suitable for the conduct of immunoa~says i9 well known in the art.
U~e of Purified and Isolated Proteases in Vacçines ;~ The ?roteases of the invention are useful as vaccines in immunizin~ host organisms to protect them against i~fection by the corresponding filarial nematode.
The proteases are administered in standard pharmaceutical formulations ystemically, and typically by injection.
Injection may be~intravenous, intramuscular, peritoneal, or other parenteral. Suitable vehicles for in~ection include physiological saline, Hank's solution, Ringer's solution and the like, with or without the pre3ence of adjuvants, according to the immunization protocol.
Generally, the vaccine is administered at a dosage level sufficient to raise~antibody titers to pro~ide effective cavenging of the proteases required for molting from the ~3 ~o the ~4 stage in the filarial infecti~e agent.
A

~ 35 WO93/10225 2 1 2 3 ~ 2 0 -18- PCT/US92/09702 Treatment of ~nfection with Inhibitors As the proteases of the invention are needed for the progression of the parasitic nematode life cycle, administration of inhibitors of these enzymes to infected hosts in suita~le do ages inhibits or arrests the course of the infection. The inhibitors are formulated in ~uitable pharmaceutical compositions such as those described in Remin~ton's Pharmaceutical Scien~es, latest edition, Mack Publishing Co., Easton, PA. Administration 10 i9 preferably by oral formulation, although injection or transdermal or transmucosal routes can also be used.
The following examples are intended to illustrate, ~ut not to limit the invention.

Example 1 Demon~tration of Protease_Rctivity in D. Immitis D. immitis were cultured at a concentration of lO0 larvae per ml in a 1:1 mixture of NCTC-135 and IYcove' 8 modified Dulbecco medium (Sigma) containing antibiotics (NI) on a model of extracellular matrix (ECM) secreted by rat vascular smooth muscle cells and labelled with tritiated proline. Every 8 hrs a 50 ~l sample was collected and the amount of tritium released from the matrix was counted on a scintillation counter. The counts per minute of tritium released from the ECM for the L3 stage increased 810wly from 1 x 104 cpm after 8 hours to about 2 x 104 cpm after 56 hours, when L3 molting occurs.~ A large incremental release of tritium (indicating degradation of the matrix) occurs at the time of L3 molting; cpm increase to about 6 x 104 cpm after 64 hrs and to over 8 x 104 cpm after 72 hrs. The breakdown of matrix mediated by L4 tracked that by ~3 until the 56 hour L3 molt event; cpm for L4 continued to increase only slowly after this (to ~4 x 104 cpm after 72 hrs). In ~093/10225 2 ~ 2 3 d 2 J~ PCT/US92/09702 total, a~ter 72 hours the L3 culture d~graded 20% of the total ECM, and the L4 culture degraded 13%.
ThP components of the ECM which were degraded were evaluated by sequential enzyme digests of the remaining ECM as described previously (McKerrow, J.H., et al., Lab Invest (1933) 49:195-200.
The results are shown in Table 1. Collagen is shown to be the major component of the ECM degraded by both L3 and ~4 lysates. However, L3 lysates degrade nearly twice as much collagen as lysate from ~4 Table 1 Percent Deqradation of ECM Constituent Proteins (100 larvae lysate/ml) ~3 4 ; Glycoproteins 22 20 Elastin 10 8 Collagen 61 38 In these experiments, controls for nonparasite-derived degradation of ECM constituted either NI alone, mosquito media, or C. ele~ans. Mosquito media were prepared from noninfected mosquito heads processed as if they contained worms. C. elegans adults and larvae were recovered from NGM agarose plates seeded with E. cQli strain OP50, placed in M9 media at the same concentration as the ~ immitis larvae and inc~bated at 26C. Mosquito media were u~ed as a control to aScure that mosquito-; 30 derived proteases were not responsible for any of the degradation observed. C. elegans, a free-living nematode, was used as a comparison to tissue-invasive nematodes and as a control to insure motility alone did not cau~e release of label.

W093/10225 2 l 2. ~ 4 2 ~ PCT/US92/0975~

Lysates from L3 and L4 were prepared by ~onication of the larvae in PBS on ice using 10 x 10 sec high frequency pulses. Lysates containing 10 ~g protein per reaction were tested against artificial substrates con8isting of amino acids linked to a fluorogenic ; compound, 7-amido-4-methylcoumarin (AMC) (Bachem). Some substrates were protected against exopeptidase acti~ity by a benzyloxycarbonyl group, abbre~iated Z; the substrates that are not protected are indicated by a preceding "h". These substrates are Z-Val-Leu-Arg-AMC, h-Phe-AMC, Z-Phe-Arg-AMC, and Z-Arg-Arg-AMC (abbreviated ; Z-VLR-AMC, h-F-AMC, Z-FR-AMC, and Z-RR-AMC respectively).
The lysate was incubated with each substrate for 3 hrs, and the amount of AMC hydrolyzed was measured fluorimetrically. Cleavage of AMC was measured using an LS-2 ~pectrofluorometer (Perkin Elmer) with 380 nm excitation wavelength and e~ission detection at 460 nm.
Initial substrate screening reactions consisted of 10 ~1 ~ubstrate (at 5 mM in DMS0), 980 ~1 PBS, pH 7.2, and 10 ~1 combined L3 and L4 sol~ble extracts. A number of additional sub~trates, found to be uncleaved, were also tested: Z-GPLGP-AMC, Z-GPR-AMC, Z-ARR-AMC, Z-AR-AMC, Z-R-AMC and h-L-AMC. However,;further work was conducted with the four substrates listed above. Two mM dithio-threitol (DTT)~was found to enhance clea~age of Z-VLR-AMC
wofold, but to~inhibit h-F-AMC hydrolysis as shown in Tàble 2. Thus, Z-VLR~AMC i8 shown to be a substrate for this cysteine protease, h-F-AMC is a substrate for the metalloprotease.

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WO93/1022~ 2 ~ 2 ~ ~ 2 ~ PCT~US9~/09702 Table 2 Effect of 2 mM DTT on Hydrolysis of R-AMC and h-F-AMC by L3 Soluble Extract Substr~e nMoles AMC released/hr +DTT -DTT

h-Phe-AMC 99.0 (16.7) 326.4 (25.0) z-Val-Leu-Arg-AMC 54.6 (1.7) 32.6 (2.6) Data represent the means of duplicate samples with ranges indicated in parentheses.
.

Thereafter, in the determinations, the P~S
contained 2 mM DTT except when h-F-AMC was u~ed.
Further, reaction mixtures consisted of 10 ~1 5 ~ ~ mM substrate, 10 ~1 larval soluble extract or E-S at ;~ protein concentration of 1 ~g/ml and 980 ~1 of PBS, pH
7.2. After i~cubation of the lysate or E-S with 3ubstrate at 37C~for a specified length of time, the hydrolyzed AMC was measured on a Perkin Elmer LS-2 filter fluorometer with excitation and emission wavelength~ as set forth abo~e. For h-F-AMC, the L3 lysate relea~es about~20 ~mol of AMC after the 3 hr incubation while the ~4 lysate releaYes slightly less than 10 ~mol. Z-FR-AMC
a~d Z-RR-AMC are not effective substrates ~or either lysate; approximately 5 ~mol of AMC are released from Z-VLR-AMC by either extract.
The excretory-secretory materials were also tested for activity on these substrates. Using 2 ~g of protein per reaction, the L3 E-S composition released about 9 ~mol AMC from h-F-AMC per reaction mixture after 3 hr whereas the L4 E-S composition released only about 2 W O 93/10225 2 1 2 ~ 4 2 0 -22- PC~r/VS92/09702 ~mol. No AMC was released from the Z-VLR-AMC, Z-FR-AMC
or Z-RR-AMC substrates.
The effect of inhibitors was also tested with respect to the synthetic substrates Z-VLR-AMC and h-F-AMC
using the lysates of L3 and L4 prepared as aboYe.
h-F-AMC wa~ shown to be a substrate for this metalloprotease; Z-VLR-AMC was shown to be a substrate for this cysteine protease (DTT enhances cysteine protease acti~ity; oxidizing conditions inhibit it) (see Table 2). Ten ~l (10 ~g total protein~ of L3 or L4 lysate was mixed with 10 ~l 50 ~M synthetic peptide substrate, 960 ~l PBS buffer, pH 7.2 containing 2 mM DTT
when Z-VLR-AMC was used and without DTT when h-F-AMC was u~ed. Twenty ~l of the test~inhibitors were added to the reaction mixtures, the final concentrations of the inhibitors were as follows: PMSF, 2 mM; l,10-phenanthro-line, 10 mM; NEM, 2 mM; E-64, 10 mM; Bestatin, 1 mM;
Cystatin, 4 ~M; and EDTA, 2 mM. Inhibition was calculated as the percent activity remaining as compared to control in absence of the inhibitor. The results are ; shown in Table 3 ~L3) and Table 4 (L4~.

Table 3 L3 hYsate % Control Acti~ity Remaininq ~ Substrate Z-VLR-AMC h-F-AMC

NEM : 54 22 1,10-Phe . 31 8 Bestatin 62 100 Cystatin 60 100 WO93/1022~2 1 2 3 ~1 2 0 PCT/US92/09702 Table 4 L4 h~sate ~ Control Activity Remaininq Substrate Z-VLR-AMC h-F-AMC

E64 15 7~
1,10-Phe 24 5 Bestatin 80 88 Cystatin 60 97 E64, a potent cysteine protease inhibitor, had e~sentially no effect on th~ metalloprotease substrate h-F-AMC; however, E64 was the most effective inhibitor for the cy~teine protease substrate, Z-V~R-AMC.
The activity of L4 lysates with respect to the various fluorogenic synthetic substrates was also te~ted in the presence and ab~ence of DTT. DTT seemed to enhance the activity with respect to Z-VLR-AMC, Z-FR-AMC
;:
and Z-RR-AMC. DTT i~ known to enhance the activity of cysteine proteases and to inhibit metalloprotea~es.
The effect of the same inhibitor~ was tested essentially as de~cribed above with respect to the hydroly~is of h-F-AMC by L3 E-S. The reactions contain 10 ~1 ~3 ~-S, i.e., 10 ~g total protein, 10 ~1 of the h-F-AMC to gi~e 50 ~M final concen~ration, 960 ~1 PBS pH
7 ~ 2 and 20 ~ hibitor to give final concentrations as for the ly~ate~ above. The inhibition pattern, as shown in Table 5, is imilar to that shown by the lysates.

: :
~ 35 W093i10225 2 ~ 2 ~ 4 2 n -24- PCT/US92/09702 Table 5 E-S from L3, ~ of Con~rol ~cti~ity Remaininq Substrate h-F-AMC

l,lO-Phe 5 Bestatin 80 ln Cystatin 98 Taken together, these data show that the ly ates and ~-S preparations~contain metalloprotease, but only the ly~ates contain ~ignificant amounts of cysteine protea~e activity.

Exam~le 2 Pre~aration of_Pr~teases from D. Immitis The L3 or L4 ly~ates or the h3/h4 E-S prepared as described above were ~ubjected to size exclusion chromatography.
The ~3/L4 E-S of 8,000 larvae, collected from ~:: 48 to 144 hours, was concentrated to 7S ~l in 0.05 M
Tris/HCl, pH 6.8, O.lS M NaCl, and injec~ed for size exclusio~ chromatography into a TSK 3,000 SW 7.5 x 300 mm column with a 7.5 x 75 mm guard column (Beckman, : : Fullerton, C~) attached to Beckman Model 338 HP~C. The mobile pha~e used the ~ame buffer, the flow rate was 0.5 ml/min a~d ~he detector was set at 220 nm. O~e mi~ute fractio~ were collected starting at 12 minutes. The : column was calibrated using gel fil~ration molecular weight markers ~NW-GF-200) (Sigma).
:~ The L3/L4 E-S which had been metabolically labeled with S35 methionine and cysteine was collected .

21~3'~2~
~WO93/10225 -25- PCT/US92/09702 and chromatographed under the conditions described above and the fractions were subjected to reducing SDS-PAGE
using standard techniques. Figure 1 shows the chromatogram obtained when h-F-AMC was used as a substrate to assay activity of the fractions--20 ~l of each fraction was incubated with S mM h-F-AMC in 970 ml PBS, pH 7.2, for 1 hour. Peak enzyme acti~ity was in fraction 10 which correspQnded to a molecular weight of approximately 49-58 kd. SDS-PAGE analysis of fraction 10 gave three prominent bands at 5~, 30 and 22 kd and three minor bands at 28, 26 and 19 kd under denaturing and .
reducing conditions.
Similar separations were run using lysates prepared from 6,000 L4 worms collected after 144 hours.
The fractions were assayed using both Z-VLR-AMC and h-F-AMC as sub~trates. The results are ~hown in Figure 2. The h-F-AMC~acti~ity (metalloprotease) eluted at a ,~
position corresponding to 49-54 kd; the Z-VLR-AMC
acti~ity eluted at a position corresponding to 31-34 kd.
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Claims

1. A veterinary or pharmaceutical composition for immunization of an animal host against filarial nematode infection, which composition comprises an amount of at least one protease in purified and isolated form effective to immunize said animal host and which protease is obtainable by isolation from L3 or L4 lysate or from L3/L4 excretory-secretory material of said filarial nematode, or an immunogenic subunit thereof.

2. A veterinary or pharmaceutical composition useful in treating or ameliorating the symptoms of filarial nematode infection in an animal host, which composition comprises an effective amount of an inhibitor of at least one protease which protease is obtainable by isolation from L3 or L4 lysate or from L3/L4 excretory-secretory material of said filarial nematode.

3. The composition of claim 1 or 2 wherein said protease is a metalloprotease or a cysteine protease.

4. The composition of claim 1 or 2 wherein the nematode is D. immitis filarial nematode.

5. A method to immunize an animal host, susceptible to infection by a filarial nematode, against said infection, which method comprises:
administering to a host in need of such immunization an amount effective to immunize said host of at least one protease in isolated and purified form, which protease is obtainable by isolation from L3 or L4 lysate or from L3/L4 excretory-secretory material of said filarial nematode, or an immunogenic subunit thereof.

6. A method to treat or ameliorate filarial nematode infection in an animal host, which method comprises administering to said host an effective amount of an inhibitor of at least one protease which protease is obtainable by isolation from L3 or L4 lysate or from L3/L4 excretory-secretory material of said filarial nematode.

7. The method of claim 5 or 6 wherein said protease is a metalloprotease or a cysteine protease.

8. The method of claim 5 or 6 wherein the nematode is D. immitis filarial nematode.

9. Antibodies specifically immunoreactive with at least one protease which is obtainable by isolation from L3 or L4 lysate or from L3/L4 excretory-secretory material of a D. immitis filarial nematode, wherein said protease is a cysteine protease or a metalloprotease.

10. A protease obtainable by isolation from the L3 or L4 lysate or from L3/L4 excretory-secretory material of a D. immitis infective filarial nematode in purified and isolated form, wherein said protease is a metalloprotease or a cysteine protease.

11. A method to purify a protease from an L3 or L4 lysate or from L3/L4 excretory-secretory material of a filarial nematode, which method comprises subjecting said lysate or excretory-secretory material to a column chromatographic procedure and assaying fractions eluted from said column for proteolytic activity on a synthetic substrate characteristic of said protease.

12. The method of claim 11 wherein said protease is a cysteine protease and the substrate is Z-VLR-AMC, or wherein said protease is a metalloprotease and the substrate is h-F-AMC.

13. A peptide which consists essentially of an immunogenic subunit of the purified protease of claim 10.

14. A DNA in purified and isolated form that encodes the protease of claim 10, or the complement thereof.

15. An expression system capable, when transformed into a recombinant host cell, of expressing a DNA encoding the protease of claim 10.

16. Recombinant host cells transformed with the expression system of claim 15.

17. A method to prepare a protease enzyme which method comprises culturing the cells of claim 15 under conditions that effect the expression of said coding sequence and recovering the protease from the cell culture.