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WO1992013560A1 - Reactifs et procedes d'identification de vaccins - Google Patents

Reactifs et procedes d'identification de vaccins Download PDF

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Publication number
WO1992013560A1
WO1992013560A1 PCT/US1992/000848 US9200848W WO9213560A1 WO 1992013560 A1 WO1992013560 A1 WO 1992013560A1 US 9200848 W US9200848 W US 9200848W WO 9213560 A1 WO9213560 A1 WO 9213560A1
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Prior art keywords
infectious agent
reagent
serum
immitis
immunogens
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PCT/US1992/000848
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English (en)
Inventor
Robert B. Grieve
Glenn Frank
Marcia Mika-Grieve
Janice A. Culpepper
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Colorado State University Research Foundation
Paravax, Inc.
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Application filed by Colorado State University Research Foundation, Paravax, Inc. filed Critical Colorado State University Research Foundation
Publication of WO1992013560A1 publication Critical patent/WO1992013560A1/fr

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0003—Invertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • 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

Definitions

  • the invention is directed to reagents and methods to identify and screen candidates for vaccines.
  • the identification and screen depends on the capacity of biological materials used as the screening reagents to destroy or impair the infective agent in an .in vivo incubator. More particularly, the invention concerns the use of cells, serum or fractions thereof obtained from exposed natural hosts wherein a recoverable implant of infectious agent is used to assess the protective effect when these materials are provided passively to the animal incubator.
  • the method is illustratively applied to determine useful active agents for an anti-heartworm vaccine.
  • the general method provided by the invention below to obtain suitable immunogens for use in vaccines is applied specifically to heartworm infection in canines and other mammals, which is caused by the ne atode Dirofilaria im itis. Accordingly, a preliminary discussion of the nature of this infection and the life cycle of the D ⁇ _ immitis parasite may be helpful both in reviewing the background literature and in describing the invention.
  • the adult forms of the parasite are quite large (males are 12-20 cm long and 0.7-0.9 mm wide; females are 25-31 cm long and 1.0-1.3 mm wide) and these preferentially inhabit the heart and pulmonary arteries of the dog.
  • the sexually mature adults, after mating, produce as embryos microfilariae which are only 300 ⁇ m long and 7 ⁇ m wide. These traverse capillary beds and circulate in the vascular system of the dog in concentrations of 10 3-105 microfilariae per ml of blood.
  • One way of demonstrating infection in the dog is to detect the circulating microfilariae.
  • Antibodies which neutralize the infective agent in j_n vitro assays are much more likely to protect against challenge in vivo. Accordingly, the use of "immune" serum simply resulting from “immunization” or from infection by the infectious agent to screen for candidate vaccines does not provide sufficient specificity to identify protective immunogens. On the other hand, serum or other components of blood from immunized animals which is demonstrably protective against infection is assured to contain antibodies, cells, or other factors reactive with an immunogen or infectious agent which will produce responses that protect against challenge.
  • a more useful manner of obtaining blood components and substantiating their protective effects takes advantage of implanted diffusion chambers containing the infectious agents, such as those described by Grieve, R.B., et al. Am J Trop Med Hyg (1988) 39:373- 379, and by Abraham, D., et al. , J Parasit (1988) 74:275- 282.
  • dogs which had been immunized against Dirofilaria immitis infection were supplied diffusion chambers containing infective larvae. The larvae in the chambers could then be evaluated for the effect of the previous immunizations.
  • mice were supplied diffusion chambers containing D ⁇ .
  • immitis L3s labeled with 125I showed that a 35 kd and 6 kd component were shed into the culture medium by developing parasites. They further showed that antibodies from immunized rabbits and infected dogs immunoprecipitated the 35 kd, but not the 6 kd, component.
  • Iodine labeling of surface-associated molecules of L4 gave molecules of apparent molecular weights of 57 kd, 40 kd, 25 kd, 12 kd, and 10 kd; lactoperoxidase-catalyzed labeling showed additional bands of 45 kd, 43 kd, and 3 kd.- However, these were identified using uncharacterized serum sources or without serum identification.
  • the invention provides both a general methodology for identification of suitable immunogens for inclusion in vaccines and, specifically, immunogens identified using this method which are useful in controlling heartworm infections in dogs.
  • the general methodology depends on the verification of the protective qualities of serum or cells used to detect or bind candidate antigens by virtue of the ability of the serum or cells to impair or destroy the infectious agent in an in vivo incubator animal wherein an irrelevant host contains diffusion chamber implants of the infectious agent.
  • the invention is directed to a reagent useful to identify immunogens for inclusion in vaccines against infectious agents.
  • the reagent comprises cells, antiseru or fractions thereof which has been demonstrated to be protective against the infective agent by its ability to confer, destroy or impair the infectious agent in an in vivo model wherein an irrelevant host is implanted with a diffusion chamber containing the infectious agent.
  • the method is, of course, limited to infectious agents of sufficient size to be retained by the diffusion chamber.
  • the invention is directed to methods to use these reagents to screen for candidate vaccines in various potential sources of immunogens. These sources of immunogens may be extracts or resolved extracts of the infectious agent, or DNA expression libraries obtained from the infectious agents.
  • the invention is also directed to methods to confer passive immunity on hosts by administration of the reagent.
  • the invention is directed to the application of the reagents and methods of the invention to heartworm infection and, specifically, to a component of the L3 and L4 larval stage of D___ immitis_ said component having a molecular weight of 39 kd. Additional components verified to react exclusively with protective immune serum cells or fractions are also disclosed.
  • the invention permits evaluation of the candidate immunogens by providing a short-term test of their ⁇ protective effect in the target host. Instead of challenging the immunized hosts with infectious agents directly and waiting several months to evaluate the outcome of infection, the host may be bled and the components of the blood or serum tested more directly in the experimental host containing the diffusion chamber. This permits rapid screening of both naturally occurring components of the infectious agent and of synthetic peptides, carbohydrates and glyco- proteins.
  • Figure 1 shows Western blots of D ⁇ . immitis proteins immunoreacted with canine sera derived from immune and nonimmune dogs.
  • Figure 2 shows Western blots of E _ immitis proteins immunoreacted with canine sera at various time points (days) after immunization.
  • Figure 3 shows the results of SDS-PAGE on proteins labeled with S-35 methionine extracted from I _ immitis L4 larvae and reacted with control and immune sera at various time points after immunization.
  • Figure 4 shows the results of proteins analyzed as set forth in Figure 3, but wherein the larval surface proteins are labeled with 1-125.
  • Figure 5 shows the results of proteins analyzed as in Figure 3, but wherein the larval surface proteins are labeled using biotin.
  • Figure 6 shows the results of analysis of proteins present in the excretory/secretory material which characterizes the transition from L3 to L4 and maintenance of L4's for 3-4 days thereafter.
  • the method of the invention takes advantage of a model system which has been used to evaluate immunization protocols using the active response of the model host, but not to ascertain or validate the protective capacity of specific immune reagents generated in the natural target host and passively transferred into the experimental animal system. Thus, the method has not been used to identify suitable reagents which themselves are useful to identify and evaluate vaccine candidates.
  • the active immunization model as applied to heartworm is described in the 1988 papers of Grieve and Abraham cited above. In this model, the infectious agent, third-stage larvae, were first obtained as follows. D___ immitis was maintained in a dog infected with parasites which had been passed once from an infection obtained through the U.S.-Japan Cooperative Medical Sciences Program, National Institutes of Health.
  • Aedes aegypti mosquitos Liverpool (blackeye strain) were infected with the parasite by feeding on microfilaremic blood using the artificial feeding apparatus described by Rutledge, L.L., et al., Mosq News (1964) 2 ⁇ 407-419. Fifteen days after infection, the mosquitoes were cold-anesthetized and surface sterilized by immersion in 95% ethanol followed by a 3 min wash in 1% benzalkonium chloride in 0.01 M phosphate-buffered saline, pH 7.2. The mosquitoes were washed three times in PBS and incubated over a 60 mesh screen inside a funnel filled with medium (the medium was described in Abraham, D. et al. , J Parasitol (1987) 73.:377-383) and the larvae were collected 90 min after incubation.
  • the recovered L3 larvae are then placed in diffusion chambers and implanted subcutaneously.
  • chambers are composed of two 14 mm Lucite rings sealed with 5.0 ⁇ m hydrophilic Durapore membranes (Millipore, Bedford, MA) .
  • the larvae are inserted through a hole in one of the Lucite rings which is subsequently sealed with nylon thread.
  • the dogs are anaesthetized and a subcutaneous pocket is formed in the dorsal skin of the neck; the chamber is implanted in the pocket and the wound closed with suture.
  • mice For implantation in mice, similar chambers are used and implanted into a subcutaneous pocket formed laterally to the lumbar spine. The chambers can be removed at the desired time for evaluation of the contained larvae.
  • a variety of chamber designs can be used, and the porosity of the diffusion membrane chosen according to the nature of the infectious agent and to desired limitation on the nature of the inward diffusion.
  • the diffusion chambers are allowed to remain in vivo in an irrelevant host which has not been actively immunized and a portion of serum or other component believed to be capable of conferring protective immunity is administered to the irrelevant host. A portion is retained for use in the invention method to screen immunogens if destruction or impairment of infectious agent is shown.
  • a portion is retained for use in the invention method to screen immunogens if destruction or impairment of infectious agent is shown.
  • about 0.5 ml of serum for example, is administered by placing the serum in the pocket along with the diffusion chamber.
  • mice similar amounts but fewer chambers are used.
  • the chamber containing the infectious agent is allowed to remain in place for a time sufficient to evaluate the protective effects of the serum or other component. This time is determined by the nature of the infection and the protective capacity of the test sample.
  • the chamber is removed and the infectious agents are retrieved.
  • the protective capacity of the passively transferred components from the natural target host is evaluated by any deleterious effects seen in the infectious agents. These effects may include, but are not limited to, such parameters as killing, stunting, alterations in normal morphology, alterations in measurable metabolism, failure to mature in in vitro culture, and failure to infect conventional target hosts.
  • a fraction of the sample which has been retained during the evaluation is then used to screen candidate immunogens.
  • Any technique which results in the complexation of the validated component with the candidate immunogen can be used.
  • an extract of the infectious agent is subjected to resolution using a variety of chromatographic techniques, including size separation using gel permeation chromatography, electrophoresis on polyacrylamide gels, ion exchange chromatography, affinity chromatography, and the like.
  • the whole extract or resolved extract is then tested for reactive effect with the protective component. If serum is the protective component, a complex will be formed. If the component is a cell subfraction with receptor for antigen, the antigen will be bound. The complex is recovered, and the immunogen recovered from the complex.
  • the protective serum or cells can be used as an affinity ligand in chromatographic techniques to isolate immunoreactive components.
  • the extract can first be resolved and the appropriate fractions identified by complexation with the protective cells or serum.
  • the protective cells or serum can be used as a screening reagent for a cDNA library prepared from the infectious stage or later stage of the infective agent which is constructed in expression vectors.
  • a commonly used and convenient such library is the ⁇ gtll library described by Young, R.A. , and Davis, R.W. , Proc Natl Acad Sci (USA) (1983) 80:1194- 1198.
  • the expression library is plated and screened with the protective cells or serum to identify colonies which produce immunoreactive components. The positive colonies are then purified, and the cDNA inserts in the expression vectors recovered and sequenced to identify the encoded proteins.
  • the cDNA inserts identified as expressing immunogens using the reagent of the invention can then be ligated into alternative conventional expression systems for production of the proteins useful as vaccines.
  • the inserts may be ligated into expression systems which are live recombinant carriers such as Sindbis virus, vaccinia virus or other pox viruses, Herpes viruses, Adenoviruses, Salmonella or Mycrobacteria. These infectious agents can then be used directly to immunize the target hosts by generation of immunogen in situ.
  • the vaccines of the invention are administered in a manner consistent with the nature of the vaccine and the nature of the disease and subject. If the recombinantly produced or native immunogens are administered as proteins, they are formulated with conventional excipients for injection or other systemic administration to the host. In addition to injection, formulations may be prepared for other administration methods which include transmucosal or transdermal delivery into the bloodstream. If the immunogens are administered in the form of recombinant DNA expression systems in infectious agents, administration is typically by injection or other mode of conventionally administering the infective agent.
  • the foregoing approach is applicable to the discovery of suitable immunogens for any disease wherein the infectious agents can conveniently be used in the animal model.
  • diseases are those caused by organisms of sufficient size to be retained in a diffusion chamber.
  • the method is applicable to a variety of parasitic diseases including those caused by other filarial nematodes, such as Dipetalonema perstans. Dipetalonema streptocerca,
  • Other disease-causing organisms to which the method is applicable include Strong ⁇ loides spp. , Strongylus spp. , Haemonchus spp. , Trichostrongylus spp. , Ostertagia spp. , Cooperia spp. , Dictyocaulus spp. , Nematodirus spp. , Cyathostominae (small strongyles of horses) , Oesophagosto um spp. , Chabertia ovina, Ancylostoma spp. , Uncinaria spp. , Bunostomum spp. , Filaroides spp.
  • the infectious stage of the agent is determined, if applicable, for identification of the form to be .enclosed in the chamber. Protocols are adjusted to take account of the time required for exertion of the protective effect of the target animal-derived component, such as cells or serum. Using the invention reagent, several immunogens associated with £_. immitis were obtained.
  • One class of such immunogens have molecular weights of 66 kd, 65 kd, 59 kd, 39 kd, 33 kd, 23/24 kd, 22/20.5 kd and 14 kd.
  • the foregoing proteins are produced by L3 and/or L4 larvae. DNAs encoding these proteins can be recovered from cDNA expression libraries prepared from the mRNA of these larval stages by screening for expression with the reagent of the invention. The appropriate cDNA inserts can then be used to produce recombinant immunogen in a suitable expression system which yields practical amounts of the protein or can be ligated into recombinant systems which generate the immunogen in situ, such as the vaccinia virus system.
  • Example l Production of Sera for Passive Transfer Four dogs were immunized with chemically- abbreviated infections, and two dogs served as chemically-treated controls. The dogs were housed in indoor mosquito-free individual cages at a temperature of 22°C and 40-65% humidity. On day 532, post initial immunization, each dog was challenged with 100 L3 D. immitis larvae contained in 5 diffusion chambers, described above. Concomitant with chamber implantation, the dogs were injected subcutaneously with 50 L3 and the infection was allowed to proceed beyond the anticipated pre-patent period. Challenge infections were repeated on day 588 with 100 larvae within diffusion chambers and 30 L3 inoculated subcutaneously.
  • Serum was collected at numerous time points from the immunized dogs, including 554, 588, 602 and 642 days after initial immunization which corresponded to days 22, 56, 77 and 117 after initial challenge.
  • the isolation of serum provides a source of immunoglobulins and soluble factors, but not of cells.
  • Antibody levels were measured to L3 and L4 surface antigens using an indirect fluorescent antibody assay and to L3 and L4 soluble antigens and an excretory- secretory antigen fraction by an indirect ELISA, as described by Grieve et al., (1988) (supra).
  • the sera were pooled and validated as a significant factor in the protective effect in the mouse incubator as described in Example 2.
  • a subcutaneous pocket was formed in male Balb/C BYJ mice approximately 10 weeks old and 20 L3 inoculated into diffusion chambers as described above were implanted into the pocket, along with 0.5 ml of the demonstrated protective serum to be tested. Serum samples were retained for future use.
  • the diffusion chambers were re ⁇ covered two or three weeks later. Living larvae in the chambers were counted and placed into glacial acetic acid followed by 70% ethanol containing 5% glycerin. The ethanol was allowed to evaporate leaving the larvae in glycerin; the larvae were measured using projected images in the Macmeasure image analysis system on a Macintosh computer.
  • experiment 1 used equal portions of serum from individual dogs at each of the three collection points described in Example 1 (days 56, 77 and 117) .
  • experiments 2 and 3 only sera from immune dogs 117 days after initial challenge were used. Control sera were used in all cases; in experiment 2 this was a pool from 12 naive dogs; in experiment 3 from an individual dog.
  • These groups also contained controls which received no serum.
  • the worms were collected and washed twice with wash buffer (PBS/0.1% Triton X-100) and then with extraction buffer (0.05 M Tris/HCl, pH 6.8; 2% CHAPSO; 1 mM PMSF; 1 mM EDTA; 1 mg/1 leupeptin; 1 mg/1 pepstatin) .
  • wash buffer PBS/0.1% Triton X-100
  • extraction buffer 0.05 M Tris/HCl, pH 6.8; 2% CHAPSO; 1 mM PMSF; 1 mM EDTA; 1 mg/1 leupeptin; 1 mg/1 pepstatin
  • Other detergents may be used in place of CHAPSO, including 0.5% Triton X-100, 0.5% CTAB, 2% DOC, or 2% SDS/5% 2-ME/8M urea.
  • the worms are then homogenized 5x for 1 minute each, with 1 minute rest periods, using 250 to 500 ⁇ l for 10,000-20,000 worms (- 500 ⁇ g) .
  • This volume is transferred to an additional tube, and the homogenizer washed with a clean 100-250 ⁇ l of extraction buffer and the wash pooled with the homogenate.
  • the tube is rocked 4 hours-overnight and centrifuged at 12,000g for 10 minutes.
  • the supernate is harvested and the pellet is washed once with extraction buffer and saved for additional extractions if desired.
  • the combined total volume of extract is less than 1 ml and about 20 ng of protein is solubilized per L4 larva used.
  • the procedure for L3 is identical, except that the .wash buffer is PBS without detergent.
  • the extracts were subjected to polyacrylamide gel electrophoresis and tested with portions of the serum shown to be protective in the murine model.
  • pooled canine sera which had been shown to stunt larval growth as described in Example 2 were used as the immuno- reactant in the Western blots, the results were as shown in Figure 1.
  • the 39 kd band shown in Figure 1 is separated from a 45 kd band when a second dimension is added to the electrophoresis.
  • This 45 kd protein is not immunoreactive.
  • the serum is specifically immunoreactive with a 39 kd protein present in the L4 larval stage. This protein has a pi of about 5.
  • Control serum shows no immunoreactivity with this protein. Reactivity to the 39 kd molecule is present in immune dogs, but not in control dogs. Sera from dogs with microfilaremic infection or amicrofilaremic infection do not recognize this molecule.
  • the proteins associated with the larval stages were also metabolically labeled using S-35 methionine; or the surfaces were labeled, prior to extraction, with 1-125 or with biotin.
  • S-35 methionine the radiolabeled amino acid was added to the parasites after 48 hrs in culture according to the method of Abraham, D., et al., J Parasitol (1987) 23:377-383.
  • 1-125 the method of Mok, M. , et al., Molec Biochem Parasitol (1988) 3.1:173-182, was used.
  • a transient band represented by 65.3 kd was recognized by 3 of 4 immune dogs.
  • passive transfer of the earliest immune dog serum which showed uniform responses to the 39 kd protein i.e., the day-142 immune serum shown in Figure 3
  • recoveries of intact larvae were only 58.3% in the case of immune serum compared to 65.8% for controls.
  • a 39 kd protein which reacted with sera from all immune dogs but not with sera from naive cohorts.
  • the protein is shown to be present in Western blots obtained from L4 soluble antigen and solubilized L4 larval pellets and is shown to be present, although apparently to a lesser degree, in L3.
  • This protein appears to be absent from adult ]D_. immitis and the microfilariae. It is clearly a distinct protein from the p35 protein described by Scott, A.L., et al., Acta Tropica (1990) (supra) , and is relatively acidic, having a pi of approximately 5.
  • a 14 kd immunogen is detected with immune dog serum using Western blots and immunoprecipitation employing S-35 and iodine-labeled components.
  • the protein is detected with immune dog serum, but not by serum from controls.
  • Additional proteins detected are of 66 kd and 23/24 kd.
  • L3 and L4 Another potential source of protective antigens in parasitic diseases are excretory/secretory products which are associated with various stages of the parasite.
  • the transition between L3 and L4 involves excretion/ secretion of a number of proteins which are harvested as follows: Larvae are cultured at 250-400/ml, washed at 48 hr and cultured an additional 4 days. The worms are then settled out and the supernate collected. This is filtered through a 0.45 ⁇ m filter and protease inhibitors added as in L4 solubilization. The ES is then concen ⁇ trated and buffer exchanged by ultrafiltration over a 10 kd membrane (A icon Centriprep-10 and/or Centricon- 10).
  • the final buffer is 0.05 M Tris/HCl pH 6.8 with protease inhibitors. Yields may be app. 5 ng/larvae. Final volume frequently 150-250 ⁇ l.
  • This extract referred to as DILEX, was prepared using larvae which were metabolically labeled with S-35 methionine and tested with respect to immune and control sera from dogs. The immune serum was that obtained on day 554 post immunization as set forth in Example 3.
  • lane 1 shows molecular weight standards
  • lane 2 the i munoprecipitates from immune dog
  • lane 3 from control dog
  • lane 4 bead control
  • lane 5 DILEX itself.
  • Genomic and cDNA expression libraries in ⁇ ZapII (Short, J.M., et al., Nucleic Acids Res (1988) 16:7583- 7600) , a derivative of ⁇ gtll, were prepared from total genomic DNA, or L4 or L3 larval stage mRNA's, respectively, using standard procedures (Short Protocols in Molecular Biology (1989) Ausubel, M.F., et al. , eds.) Screening of these libraries with pooled immune dog sera permits identification of clones which contain candidate antigens. The clones identified as immunoreactive with the immune serum provide a source of DNA encoding desired proteins which can conveniently be produced as fusion proteins in E___ coli.
  • the DNA inserts are recovered from the ⁇ ZapII phagemid by digesting with EcoRI and purified using agarose gel electrophoresis.
  • the purified DNA is ligated into the expression vector pGEX-3X such that when the plasmid is expressed in EL. coli the protein encoded by the DNA insert produces a fusion protein with glutathione-S-transferase. This procedure is described in detail by Smith, D.B., et al., Gene (1988) 67:31-40. Plasmids containing the DNA inserts are transformed into __]____ coli and successful transformants are grown in the presence of IPTG.
  • the induced fusion protein is purified from the lysate by affinity chromatography with glutathione-beads as described by Smith et al. (supra) .
  • Recombinant peptides derived from any of a variety of expression systems are used to immunize dogs for the purpose of obtaining specifically reactive blood components.
  • Recombinant antigens are administered to dogs with or without adjuvant by the subcutaneous, intramuscular, intradermal or intravenous routes.
  • blood is collected from dogs by routine venipuncture.
  • Serum is collected from coagulated blood and used directly or stored frozen prior to use.
  • Leukocytes are collected from anticoagulant-treated blood by density gradient centrifugation and used directly or stored by freezing at l°C/minute with storage in liquid nitrogen.

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Abstract

Les cellules, le sérum ou des fractions de ceux-ci prélevés sur des hôtes exposés, notamment caractérisés par une capacité à assurer une protection contre des infections, constituent des agents de triage permettant d'identifier des antigènes destinés à être utilisés dans des vaccins protecteurs. Les matières biologiques prélevées sur des hôtes natifs exposés peuvent être validées in vivo chez un hôte sans rapport par leur capacité à détruire ou à neutraliser l'agent infectieux. La validation est effectuée par l'implantation de l'agent infectieux dans une enveloppe de membrane chez un hôte animal auquel on a transmis le réactif de triage potentiel. Ledit réactif potentiel détruisant ou neutralisant avec succès l'agent infectieux peut alors être utilisé afin de trier des antigènes produits par des banques d'expression d'ADNc ou dans des extraits des agents infectieux afin d'identifier les composants de vaccins efficaces. Ce procédé a permis d'identifier des immunogènes potentiels contre la Dirophilaria immitis, notamment un de 39 kd.
PCT/US1992/000848 1991-02-12 1992-01-30 Reactifs et procedes d'identification de vaccins WO1992013560A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0635058A4 (fr) * 1991-11-12 1994-12-07 Univ Colorado State Res Found Vaccin a base de protease anti-dirofilaria immitis.
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.
US5569603A (en) * 1994-03-08 1996-10-29 Heska Corporation Dirofilaria immitis GP29 proteins, nucleic acid molecules and uses thereof
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
US5639876A (en) * 1991-02-12 1997-06-17 Heska Corporation Nucleic acid molecules encoding novel parasitic helminth proteins
US5977306A (en) * 1991-02-12 1999-11-02 Heska Corporation Parasitic helminth P39 proteins, and uses thereof
US6114142A (en) * 1991-02-12 2000-09-05 Heska Corporation Parasitic helminth nucleic acid molecules/and uses thereof
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
WO2001007615A1 (fr) * 1999-07-27 2001-02-01 Heska Corporation Proteines diag2 d'helminthe parasite, molecules d'acide nucleique et leurs utilisations

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU660662B2 (en) * 1991-02-08 1995-07-06 Progenics Pharmaceuticals, Inc. CD4-gamma2 and CD4-IgG2 chimeras

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US4842999A (en) * 1986-08-11 1989-06-27 Adi Diagnostics Inc. Canine heartworm vaccine and diagnostic test

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US4842999A (en) * 1986-08-11 1989-06-27 Adi Diagnostics Inc. Canine heartworm vaccine and diagnostic test

Non-Patent Citations (5)

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Title
American Journal of Tropical Medicine Hygiene, Volume 39, No. 4, issued 1988, GRIEVE et al., "Induction of Protective immunity in Dogs to infection with Dirofilaria immitis using Chemically-Abbreviated Infections", pages 373-379, see pages 376-378. *
Journal of Parasitology, Volume 74, No. 2, issued 1988, ABRAHAM et al., "Active and Passive Immunization of Mice against Larval Dirofilaria immitis", pages 275-282, see entire document. *
Parasitology, Volume 99, issued 1989, DELVES et al., "Neurosecretory-like Material in 3rd- and 4th-stage Dirofilaria immitis larvae (Nematoda: Filarioidea)", pages 99-104, see entire article. *
Parasitology, Volume 99, issued 1989, IBRAHIM et al., "Antigen Shedding from the Surface of the Infective Stage larvae of Dirofilaria immitis", pages 89-97, see entire article. *
See also references of EP0571536A4 *

Cited By (16)

* 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
US6114142A (en) * 1991-02-12 2000-09-05 Heska Corporation Parasitic helminth nucleic acid molecules/and uses thereof
US5977306A (en) * 1991-02-12 1999-11-02 Heska Corporation Parasitic helminth P39 proteins, and uses thereof
US5639876A (en) * 1991-02-12 1997-06-17 Heska Corporation Nucleic acid molecules encoding novel parasitic helminth proteins
EP0635058A4 (fr) * 1991-11-12 1994-12-07 Univ Colorado State Res Found Vaccin a base de protease anti-dirofilaria immitis.
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
EP0680316A4 (fr) * 1993-01-12 1997-12-17 Paravax Inc Nouvelles proteines parasitaires helminthiques.
US5866126A (en) * 1994-03-08 1999-02-02 Heska Corporation Dirofilaria immitis GP29 antibodies and uses thereof
EP0749312A4 (fr) * 1994-03-08 1999-04-28 Heska Corp PROTEINES Gp29 DE -i(DIROFILARIA IMMITIS), MOLECULES D'ACIDE NUCLEIQUE ET LEURS UTILISATIONS
US5618532A (en) * 1994-03-08 1997-04-08 Heska Corporation Dirofilaria immitis Gp29 proteins and uses thereof
US5569603A (en) * 1994-03-08 1996-10-29 Heska Corporation Dirofilaria immitis GP29 proteins, nucleic acid molecules and uses thereof
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
WO2001007615A1 (fr) * 1999-07-27 2001-02-01 Heska Corporation Proteines diag2 d'helminthe parasite, molecules d'acide nucleique et leurs utilisations
US6392017B1 (en) 1999-07-27 2002-05-21 Heska Corporation Parasitic helminth DiAg2 proteins and uses thereof

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