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WO1993010225A1 - Vaccin a base de protease anti-dirofilaria immitis - Google Patents

Vaccin a base de protease anti-dirofilaria immitis Download PDF

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Publication number
WO1993010225A1
WO1993010225A1 PCT/US1992/009702 US9209702W WO9310225A1 WO 1993010225 A1 WO1993010225 A1 WO 1993010225A1 US 9209702 W US9209702 W US 9209702W WO 9310225 A1 WO9310225 A1 WO 9310225A1
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WO
WIPO (PCT)
Prior art keywords
protease
amc
lysate
nematode
excretory
Prior art date
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PCT/US1992/009702
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English (en)
Inventor
Robert B. Grieve
Jennifer Richer
Glenn R. Frank
Judy Sakanari
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The Colorado State University Research Foundation
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Publication date
Application filed by The Colorado State University Research Foundation filed Critical The Colorado State University Research Foundation
Priority to JP5509382A priority Critical patent/JPH07501219A/ja
Priority to AU30723/92A priority patent/AU675214B2/en
Priority to EP92924400A priority patent/EP0635058A1/fr
Publication of WO1993010225A1 publication Critical patent/WO1993010225A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/641—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43526—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms
    • G01N2333/4353—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from nematodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96402—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to prevention and treatment 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 protease associated with this organism.
  • the heartworm infection caused by D ⁇ . immitis is a widely distributed problem in dogs in most regions of the world with the exception of Africa.
  • Current treatment is generally chemoprophylactic with agents designed to directly kill the infecting organisms. While this treatment has gained acceptance, because of the inherent toxicity of such treatment, it would be preferable to immunologically protect the host against infection, or to revise the chemoprophylactic regime to include less toxic agents.
  • the present invention is directed to this goal.
  • Other nematode filarial infections are of even greater significance and involve life cycles of the infectious agent similar to those related to heartworm. For example, of more concern are the other filarids which infect humans, and more than 200 million people worldwide are estimated to have such infections.
  • Filarids which infect humans include Brugia malayi. Wuchereria niechereria niethof 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.
  • Heartworm 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 tissue from a mosquito vector. When these larvae are passed into the animal's tissue, their life cycle is continued by molting into a fourth larval stage ( 4) , which then migrates toward the heart and pulmonary arteries where the subsequent stage matures into an adult. The L3 remain at the site of inoculation by the mosquito until molting occurs.
  • Fasciola hepatica also releases a number of proteolytic enzymes (Dalton, J.P., et al., Mol Biochem Parasitol (1989) 15:161) .
  • the adult hookworm Ancylostoma caninum releases a histolytic protease and a protease that acts as an anticlotting agent (Hotez, P.J., et al., J Biol Chem (1985) 2 0:7343) .
  • Toxocara canis larvae secrete proteases which degrade components of extracellular matrix (Robertson, B.D., et al., Experimental Parasitol (1989) 69:30) .
  • a number of filarial nematodes also have been shown to produce proteases that act on extracellular matrix components, including Onchocerca cervipedis. 0. cervicalis. and Bru ia malayi (Lackey, A., et al., Experimental Parasitol (1989) £8.176; Petralanda, I., et al., Mol Biochem Parasitol (1986) 19.:51) .
  • the protease activity includes collagenase in the case of Brugia malayi. 0. cervicalis and Cx. cervipedis.
  • the invention is directed to prevention -and treatment of filarial nematode infection in animal hosts and to purified and isolated forms of the proteases associated with the L3 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 those listed above.
  • the invention provides an approach to the eradication of conditions caused in animals by filarial nematodes, and provides materials useful in these and in in vitro contexts.
  • the invention is directed to a method to protect animal subjects, including humans, against filarial nematode infection, which method comprises administering to the subject an effective amount of a metalloprotease and/or cysteine protease characteristic of transition from the I-3-L4 stage of the relevant filarial nematode effective to i ⁇ tunologically protect the subject against infection.
  • a metalloprotease and/or cysteine protease characteristic of transition from the I-3-L4 stage of the relevant filarial nematode effective to i ⁇ tunologically protect the subject against infection.
  • the characteristic 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.
  • 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.
  • the invention is directed to antibodies immunospecific for filarial L3 or L4 excretory-secretory products or L3 or L4 lysate metalloprotease(s) or to L3 or L4 lysate cysteine protease(s) and to pharmaceutical compositions and vaccines containing them.
  • 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 useful to assay for the presence or absence of antibodies in the diagnosis of affected individuals and to regulate the growth of cell cultures in vitro, as well as in other therapeutic applications.
  • Figure 1 shows the elution pattern of protease activity from L3/L4 E-S.
  • Figure 2 shows the elution pattern of protease activity from L4 lysate.
  • metalloprotease of L3 and L4 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 L3 and L4 lysates.
  • E-S and lysate preparations from _____ 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 above, and including, specifically, for example, &_,__ malayi. H__ bancrofti, Q,. volvulus. Dipetalonema perstans. ______ streptocerca. Mansonella ozzardi, and Loa loa.
  • the parasites can be cultured in vitro under suitable conditions to provide a source for the E-S preparation or for the L3 or L4 lysates.
  • D. immitis can be cultured as described by Abraham, D., et al., J Parasitol (1987) 71:377-383. Briefly, the mosquito Aedes aegypti Liverpool (black-eyed strain) are infected with 2 ⁇ immitis by feeding on microfilaremic blood obtained from a single experimentally infected dog. Fifteen days after feeding, the mosquitos are anesthetized, surface sterilized and placed on screens in funnels filled with a 1:1 mixture of NCTC-135 and Iscove's modified Dulbecco medium (Sigma) containing
  • the cultures are maintained at a concentration of ten L3 organisms per ml of medium in 5% C0 2 and saturated humidity.
  • the larvae (L3) are cultured at 37° in the foregoing medium, supplemented with 20% fetal calf serum for 1-8 days.
  • the mosquitos are anesthetized and the worms are recovered by dissecting the heads and allowing the worms to emerge 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.
  • L3 ES is collected between 48 and 96 hours of culture on Seru-max free medium.
  • L4 ES is collected between 96 and 144 hours in inden ical 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 10 kd exclusion limit to obtain the fraction of >10 kd M .
  • Larval soluble extracts are prepared from L3 collected on day 2, just after the wash but prior to the molt, and L4 are collected on day 6 in serum-free culture. Pellets of 10,000 worms in PBS are disrupted by ten 10-sec high frequency pulses using a tissue sonicator. Sonicated worms are centrifuged for 5 min at 12,000 x g, and the supernatant collected.
  • 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.
  • metaloprotease 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 larvae or in L3 or L4 lysates of a filarial nematode parasite as ascertained by activity against the synthetic substrate h-phenylalanine-AMC (h-F- AMC, defined below) and which is inhibited by metallo ⁇ protease inhibitors such as 1,10-phenanthroline and EDTA.
  • Metalloprotease activity has been reported in E-S products of third stage larvae of certain species, including B ⁇ . malayi, 0. cervicalis. and Q. 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 (Z-VLR-AMC, defined below) and this activity is inhibited by E64. Again, . 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 methods with use of the appropriate substrate assay to monitor elution fractions as further described below.
  • 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) 8j6:4863.
  • the gene encoding the metalloprotease or cysteine protease is isolated from cDNA prepared from total mRNA of the L3 or L4 stage of the parasite using oligonucleotide primers and the polymerase chain reaction (PCR) and suitable probes.
  • Dj_ immitis genomic or cDNA is used as a source for protease-encoding genes.
  • 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 homologous genes are all metalloproteases, and the cDNAs containing these sequences have been disclosed (Muller, D., et al., Biochem J (1988) 2 ⁇ 1:187-192 and Quantin, B., et al., Biochemistry (1989) 28.:5327-5334) . Primers can be designed based on the conserved regions, including, the active site. PCR amplification is conducted as described by Sakanari et al.
  • primers are designed based on the sequences disclosed in Eakin, A.E. et al., Mol Biochem Parasitol (1990) l :l-8. Otherwise, the retrieval of probes from genomic DNA can be conducted as above.
  • the cDNA library is constructed from messenger RNA isolated from third stage larvae which have been in culture for 48-72 hours.
  • the mRNA is isolated by the single step acid guanidinium thiocyanate/phenol/ chloroform extraction method of Chomczynski, P. and Sacchi, N., Anal Biochem (1987) 1£2:156-159.
  • the RNA is passed over an oligo-dT cellulose column and the poly-A RNA is eluted using standard procedures.
  • cDNA is prepared from the mRNA using standard procedures such as those of Gubler, U. and Hoffman, B.J., Gene (1983) 2J 263.
  • the cDNA is treated by methylation of internal EcoRI sites, and phosphorylated EcoRI linkers are added to the ends of the cDNA and treated again with phosphatase.
  • the treated cDNA contain linkers digested with EcoRI to generate cohesive cloning ends for insertion into ⁇ -gtll arms (Stratagene, San Diego, CA) and packaged using Gigapack (Stratagene) . Standard methods are used to titer and plate the library for screening.
  • 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 expression vectors, or DNA can be synthesized using standard solid phase techniques to obtain any embodiment of the coding sequence to supply all or a portion of the gene.
  • a DNA coding sequence for the protease can be prepared synthetically from overlapping oligonucleotides whose sequence contains codons for the amino acid sequence encoded in the native gene.
  • oligonucleotides are prepared by standard methods and assembled into a complete or partial coding sequence. See, e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; Jay et al. , J Biol Chem (1984) 2 ⁇ 9:6311.
  • a DNA molecule containing the coding sequence for the filarial nematode metalloprotease or cysteine protease can be cloned in any suitable vector and thereby maintained in a composition substantially free of vectors that do not contain the coding sequence for the protease (e.g., other library clones).
  • Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and the host cells which they transform include bacteriophage ⁇ (EL. coli) , pBR322 (EL. coli) , pACYC177 (EL.
  • the coding sequence of the protease gene is placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" 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.
  • the protease is preferably produced by the expression of a coding sequence which does not contain any native signal peptide, or by expression of a coding sequence containing the leader sequence in a eucaryotic system when post-translational processing removes the leader sequence.
  • the protease can also be expressed in the form of a fusion 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 that the protease-encoding sequence is located in the vector with the appropriate control sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the control of the control sequences (i.e., by RNA polymerase which attaches to the DNA molecule at the control sequences) .
  • the control sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequence and an appropriate restriction site downstream from control sequences.
  • the control sequences will be heterologous to the coding sequence. If the host cell is a procaryote, it is also necessary that the coding sequence 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.
  • procaryotic expression vectors are known in the art. See, e.g., U.S. Patent Nos. 4,440,859; 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 expression vectors are known 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 transformed 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 (1988) 253:187-192) .
  • the protease is then isolated from the host cells and purified. If the expression system secretes the protease into growth media, the desired protein can be 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. Antibodv Production
  • Either native 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.
  • a selected mammal e.g., mouse, rabbit, goat, horse, etc.
  • Compositions containing polyclonal antibodies to a variety of antigens in addition to the relevant protease can be made substantially free of antibodies which are not protease antibodies by passing the composition through a column to which the desired protease has been bound. After washing, polyclonal antibodies are eluted from the column. Monoclonal antibodies can also be readily produced by one skilled in the art.
  • Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., Schreier, M., et al., HYBRIDOMA TECHNIQUES (1980); Hammerling et al., MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS (1981); Kennett et al., MONOCLONAL ANTIBODIES (1980) .
  • 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 used.
  • Such antibodies have therapeutic potential if they are able to block the binding of protease to its substrate in vivo.
  • Antibodies which recognize a site on the protease are also useful, for example, in the purification of the desired protease protein from cell lysates or fermentation media, and in its characterization.
  • 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 protease, bound to the immobilized antibodies, is then eluted.
  • the cysteine protease characteristic of the L3 and L4 lysates and the metalloprotease characteristic of these lysates, as well as the L3/L4 E-S can be obtained in isolated and purified form either using the appropriate larval stage of the desired parasitic nematode as starting material, using recombinant production in cell culture and isolating the protease resulting from the cysteine protease or metalloprotease gene expression, or by synthesizing subunits of these proteins using standard peptide synthesis techniques.
  • the nature of the purification method will depend on the origin of the protease or peptide.
  • the lysate or E-S material When isolated from native sources, the lysate or E-S material is subjected to chromatographic techniques, 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 columns, and the like. Optimization of the purification procedure is within the skill of the art, as the fractions eluted from the- columns can be assayed using activity determination with a fluorometric substrate characteristic of the metalloprotease or cysteine protease. For the metalloprotease of D ⁇ . immitis.
  • h-F-AMC is a convenient substrate; for the cysteine protease of this worm, Z-VLR-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 metalloproteases 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 for the E ⁇ . immitis species.
  • 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 forms of the cysteine and metalloproteases of the various filarial nematode species can be used in the production of antibodies (which antibodies, in turn, are useful in immunoassays and separation techniques) , as reagents in immunoassay procedures for the presence or absence of antibodies, and in the regulation of cell culture in vitro by controlling extracellular matrix formation or status.
  • the purified and isolated proteases are useful 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 separations 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 immunoassays is well known in the art.
  • the proteases of the invention are useful as vaccines in immunizing host organisms to protect them against infection by the corresponding filarial nematode.
  • the proteases are administered in standard pharmaceutical formulations systemically, and typically by injection. Injection may be intravenous, intramuscular, peritoneal, or other parenteral. Suitable vehicles for injection include physiological saline, Hank's solution, Ringer's solution and the like, with or without the presence of adjuvants, according to the immunization protocol. Generally, the vaccine is administered at a dosage level sufficient to raise antibody titers to provide effective scavenging of the proteases required for molting from the L3 to the L4 stage in the filarial infective agent. Treatment of Infection with Inhibitors
  • inhibitors of these enzymes are needed for the progression of the parasitic nematode life cycle, administration of inhibitors of these enzymes to infected hosts in suitable dosages inhibits or arrests the course of the infection.
  • the inhibitors are formulated in suitable pharmaceutical compositions such as those described in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA. Administration is preferably by oral formulation, although injection or transdermal or transmucosal routes can also be used.
  • D. immitis were cultured at a concentration of 100 larvae per ml in a 1:1 mixture of NCTC-135 and Iscove's 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 slowly from 1 x 10 4 cpm after 8 hours to about 2 x 10 4 cpm after 56 hours, when L3 molting occurs.
  • a large incremental release of tritium occurs at the time of L3 molting; cpm increase to about 6 x 10 4 cpm after 64 hrs and to over 8 x 10 4 cpm after 72 hrs.
  • the breakdown of matrix mediated by L4 tracked that by L3 until the 56 hour L3 molt event; cpm for L4 continued to increase only slowly after this (to ⁇ 4 x 10 4 cpm after 72 hrs) . In total, after 72 hours the L3 culture degraded 20% of the total ECM, and the L4 culture degraded 13%.
  • controls for nonparasite- derived degradation of ECM constituted either NI alone, mosquito media, or CL_ elegans.
  • Mosquito media were prepared from noninfected mosquito heads processed as if they contained worms.
  • CL. elegans adults and larvae were recovered from NGM agarose plates seeded with EL. coli strain OP50, placed in M9 media at the same concentration as the ____. immitis larvae and incubated at 26°C.
  • Mosquito media were used as a control to assure that mosquito- derived proteases were not responsible for any of the degradation observed.
  • CL_ elegans were used as a control to assure that mosquito- derived proteases were not responsible for any of the degradation observed.
  • Lysates from L3 and L4 were prepared by sonication 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 consisting of amino acids linked to a fluorogenic compound, 7-amido-4-methylcoumarin (AMC) (Bache ) . Some substrates were protected against exopeptidase activity by a benzyloxycarbonyl group, abbreviated 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 spectrofluorometer (Perkin Elmer) with 380 nm excitation wavelength and emission detection at 460 nm.
  • the PBS contained 2 mM DTT except when h-F-AMC was used.
  • reaction mixtures consisted of 10 ⁇ l 5 mM substrate, 10 ⁇ l larval soluble extract or E-S at protein concentration of 1 ⁇ g/ml and 980 ⁇ l of PBS, pH 7.2.
  • the hydrolyzed AMC was measured on a Perkin Elmer LS-2 filter fluorometer with excitation and emission wavelengths as set forth above.
  • the L3 lysate releases about 20 ⁇ mol of AMC after the 3 hr incubation while the L4 lysate releases slightly less than 10 ⁇ mol.
  • Z-FR-AMC and Z-RR-AMC are not effective substrates for 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 ⁇ mol. No AMC was released from the Z-VLR-AMC, Z-FR-AMC or Z-RR-AMC substrates.
  • h-F-AMC was 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 activity; oxidizing conditions inhibit it) (see Table 2) .
  • E64 a potent cysteine protease inhibitor, had essentially no effect on the metalloprotease substrate h-F-AMC; however, E64 was the most effective inhibitor for the cysteine protease substrate, Z-VLR-AMC.
  • DTT The activity of L4 lysates with respect to the various fluorogenic synthetic substrates was also tested in the presence and absence of DTT. DTT seemed to enhance the activity with respect to Z-VLR-AMC, Z-FR-AMC and Z-RR-AMC. DTT is known to enhance the activity of cysteine proteases and to inhibit metalloproteases.
  • the mobile phase used the same buffer, the flow rate was 0.5 ml/min and the detector was set at 220 nm. One minute fractions were collected starting at 12 minutes.
  • FIG. 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 5 mM h-F-AMC in 970 ml PBS, pH 7.2, for 1 hour. Peak enzyme activity was in fraction 10 which corresponded to a molecular weight of approximately 49-58 kd. SDS-PAGE analysis of fraction 10 gave three prominent bands at 58, 30 and 22 kd and three minor bands at 28, 26 and 19 kd under denaturing and reducing conditions.

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Abstract

La transition entre les étapes larvaires L3 et L4 des nématodes parasites filariens tels que Dirofilaria immitis se produit après l'introduction dans l'animal hôte, et elle est induite par au moins la métalloprotéase et/ou la cystéine spécifique à l'étape larvaire L3 ou L4. L'invention concerne également des procédés de prévention et de traitement d'infections filariennes constituées par des vaccins composés des déterminants immunogènes des protéases L3 ou L4 caractéristiques, et consistant à administrer des inhibiteurs de ces protéases.
PCT/US1992/009702 1991-11-12 1992-11-12 Vaccin a base de protease anti-dirofilaria immitis WO1993010225A1 (fr)

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JP5509382A JPH07501219A (ja) 1991-11-12 1992-11-12 心糸状虫に対するプロテアーゼワクチン
AU30723/92A AU675214B2 (en) 1991-11-12 1992-11-12 Protease vaccine against heartworm
EP92924400A EP0635058A1 (fr) 1991-11-12 1992-11-12 Vaccin a base de protease anti-dirofilaria immitis

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US79220991A 1991-11-12 1991-11-12
US07/792,209 1991-11-12

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WO1994009142A1 (fr) * 1992-10-21 1994-04-28 Mallinckrodt Veterinary, Inc. Vaccin contenant une protease de thiol
WO1995023229A1 (fr) * 1994-02-28 1995-08-31 The University Of Leeds Plantes transgeniques permettant de lutter contre des parasites infestant les animaux
EP0630258A4 (fr) * 1992-05-14 1995-09-27 Univ Colorado State Res Found Vaccin destine a proteger des hotes sensibles contre des parasites non adaptes.
WO1995032988A1 (fr) * 1994-05-26 1995-12-07 Heska Corporation Nouveaux genes et proteines de proteases de parasites
WO1997019107A1 (fr) * 1995-11-22 1997-05-29 Bayer Aktiengesellschaft Identification d'antigenes a partir de nematodes post-infectieux pour le developpement de nouveaux anthelmintiques et vaccins
US5686080A (en) * 1991-02-12 1997-11-11 Heska Corporation Parasitic helminth p4 proteins
AU726774B2 (en) * 1994-05-26 2000-11-23 Heska Corporation Novel parasite protease genes and proteins
US6159477A (en) * 1996-06-27 2000-12-12 Merial Canine herpesvirus based recombinant live vaccine, in particular against canine distemper, rabies or the parainfluenza 2 virus
WO2000063350A3 (fr) * 1999-04-21 2001-02-08 Univ Georgia Res Found Cysteine protease et inhibiteurs destines a la prevention et au traitement des neurocysterocoses
EP0846165A4 (fr) * 1995-06-07 2001-07-18 Univ Colorado State Res Found Nouvelles proteines et molecules d'acide nucleique de protease de cysteine de nematodes filariides et leur utilisation
US6265198B1 (en) 1994-05-26 2001-07-24 Heska Corporation Parasite astacin metalloendopeptidase proteins
US6281345B1 (en) 1994-05-26 2001-08-28 Heska Corporation Parasite astacin metalloendopeptidase nucleic acid molecules and uses thereof
WO2017176950A2 (fr) 2016-04-07 2017-10-12 Merial, Inc. Vaccin contre le ver du cœur, procédés et utilisations associés
EP2667192B1 (fr) * 2004-01-22 2020-10-07 Promega Corporation Dosage multiplexe luminogène et non luminogène

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US4842999A (en) * 1986-08-11 1989-06-27 Adi Diagnostics Inc. Canine heartworm vaccine and diagnostic test
AU6256990A (en) * 1989-09-18 1991-03-21 Synergen, Inc. Anticoagulant and antihelminthic proteins and methods for the production and use of the same
CA2103788A1 (fr) * 1991-02-12 1992-08-13 Robert B. Grieve Reactifs et methodes d'identification de vaccins

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Journal of Biological Chemistry, Vol. 260, No. 12, issued 25 June 1985, HOTEZ et al., "Isolation and Characterization of a Proteolytic Enzyme from the Adult Hookworm Ancylostoma caninum", pages 7343-7348, see entire document. *
Journal of Helminthology, Vol. 60, issued 1986, MAKI et al., "Demonstration of Carboxyl and Thiol Protease Activities in Adult Schistosoma mansoni, Dirofilaria immitis, Angiostrongylus cantonensis, and Ascaris suum", pages 31-37, see entire document. *
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See also references of EP0635058A4 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5686080A (en) * 1991-02-12 1997-11-11 Heska Corporation Parasitic helminth p4 proteins
EP0630258A4 (fr) * 1992-05-14 1995-09-27 Univ Colorado State Res Found Vaccin destine a proteger des hotes sensibles contre des parasites non adaptes.
US5492695A (en) * 1992-05-14 1996-02-20 Colorado State University Research Foundation Vaccinating cats against Dirofilaria immitis with an L4 homogenate
WO1994009142A1 (fr) * 1992-10-21 1994-04-28 Mallinckrodt Veterinary, Inc. Vaccin contenant une protease de thiol
WO1995023229A1 (fr) * 1994-02-28 1995-08-31 The University Of Leeds Plantes transgeniques permettant de lutter contre des parasites infestant les animaux
US5863775A (en) * 1994-02-28 1999-01-26 The University Of Leeds Control of parasites
US6265198B1 (en) 1994-05-26 2001-07-24 Heska Corporation Parasite astacin metalloendopeptidase proteins
US6281345B1 (en) 1994-05-26 2001-08-28 Heska Corporation Parasite astacin metalloendopeptidase nucleic acid molecules and uses thereof
US5750391A (en) * 1994-05-26 1998-05-12 Heska Corporation Filariid nematode cysteine protease proteins
US5691186A (en) * 1994-05-26 1997-11-25 Heska Corporation Filariid cysteine protease genes
AU702915B2 (en) * 1994-05-26 1999-03-11 Heska Corporation Novel parasite protease genes and proteins
AU726774B2 (en) * 1994-05-26 2000-11-23 Heska Corporation Novel parasite protease genes and proteins
WO1995032988A1 (fr) * 1994-05-26 1995-12-07 Heska Corporation Nouveaux genes et proteines de proteases de parasites
EP0846165A4 (fr) * 1995-06-07 2001-07-18 Univ Colorado State Res Found Nouvelles proteines et molecules d'acide nucleique de protease de cysteine de nematodes filariides et leur utilisation
WO1997019107A1 (fr) * 1995-11-22 1997-05-29 Bayer Aktiengesellschaft Identification d'antigenes a partir de nematodes post-infectieux pour le developpement de nouveaux anthelmintiques et vaccins
US6159477A (en) * 1996-06-27 2000-12-12 Merial Canine herpesvirus based recombinant live vaccine, in particular against canine distemper, rabies or the parainfluenza 2 virus
WO2000063350A3 (fr) * 1999-04-21 2001-02-08 Univ Georgia Res Found Cysteine protease et inhibiteurs destines a la prevention et au traitement des neurocysterocoses
EP2667192B1 (fr) * 2004-01-22 2020-10-07 Promega Corporation Dosage multiplexe luminogène et non luminogène
WO2017176950A2 (fr) 2016-04-07 2017-10-12 Merial, Inc. Vaccin contre le ver du cœur, procédés et utilisations associés
WO2017176950A3 (fr) * 2016-04-07 2017-12-21 Merial, Inc. Vaccin contre le ver du cœur, procédés et utilisations associés
EP3439690A2 (fr) * 2016-04-07 2019-02-13 Merial, Inc. Vaccin contre le ver du c ur, procédés et utilisations associés
US10485857B2 (en) 2016-04-07 2019-11-26 Boehringer Ingelheim Animal Health USA Inc. Heartworm vaccine, methods and uses thereof

Also Published As

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EP0635058A4 (fr) 1994-12-07
CA2123420A1 (fr) 1993-05-27
AU675214B2 (en) 1997-01-30
AU3072392A (en) 1993-06-15
EP0635058A1 (fr) 1995-01-25
JPH07501219A (ja) 1995-02-09

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