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WO2003033533A1 - Antigene de leishmania microfluidise et techniques de fabrication et d'utilisation de celui-ci - Google Patents

Antigene de leishmania microfluidise et techniques de fabrication et d'utilisation de celui-ci Download PDF

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Publication number
WO2003033533A1
WO2003033533A1 PCT/US2001/031894 US0131894W WO03033533A1 WO 2003033533 A1 WO2003033533 A1 WO 2003033533A1 US 0131894 W US0131894 W US 0131894W WO 03033533 A1 WO03033533 A1 WO 03033533A1
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WO
WIPO (PCT)
Prior art keywords
subject
microfluidized
lysate preparation
leishmaniasis
microfluidized lysate
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Application number
PCT/US2001/031894
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English (en)
Inventor
Alan J. Magill
John M. Stiteler
Max Grogl
Edgar D. Rowton
Kenneth H. Eckels
William R. Ballou
Original Assignee
U.S. Army Medical Research And Materiel Command
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Priority to PCT/US2001/031894 priority Critical patent/WO2003033533A1/fr
Priority to CA002463581A priority patent/CA2463581A1/fr
Priority to EP01979743A priority patent/EP1436325A1/fr
Publication of WO2003033533A1 publication Critical patent/WO2003033533A1/fr

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    • 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
    • A61K39/002—Protozoa antigens
    • A61K39/008—Leishmania antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0006—Skin tests, e.g. intradermal testing, test strips, delayed hypersensitivity
    • 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
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • 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

  • This invention relates generally to microfluidized Leishmania lysate preparations.
  • the present invention relates to microfluidized Leishmania lysate preparations for assays and immunogenic compositions.
  • Serology, primary ELISA, IFA and agglutination assays are somewhat useful in the diagnosis of visceral leishmaniasis. Nevertheless, these assays are of little use for diagnosing cutaneous and mucocutaneous leishmaniasis where the antibody liters are low. As a result, the prevention of leishmanial epidemics is greatly hindered and patient management is difficult.
  • the strengths of the leishmanin tests include delayed hypersensitivity as an important feature of all forms of leishmaniasis that maybe measured and that the parasitic antigens elicit a cutaneous delayed-type hypersensitivity response in most individuals with cutaneous leishmaniasis, mucocutaneous leishmaniasis, post kala-azar dermal leishmaniasis, and cured visceral leishmaniasis.
  • the shortcomings of the leishmanin tests include false positive reactions that can be high in areas where there is a background of leishmaniasis, as many individuals in the healthy populations of endemic leishmaniasis areas having no evidence of past infection may show high rates of leishmanin sensitivity.
  • the shortcomings also include cross-reactions with cases of glandular tuberculosis and lepromatous leprosy, cross-reactivity between Leishmania strains as heterologous antigens often give smaller reactions, lack of a universal and standardized Leishmania skin test antigen, unknown sensitizing capacity, unknown stability, unknown dose/response relationships between antigen content and clinical syndrome or infecting parasite, and the standard of care of using Leishmania skin test antigen in many endemic countries is not approved by drug approval agencies such as the U.S. Food and Drug Administration.
  • the present invention generally relates to microfluidized leishmanial antigens and methods of making and using thereof. [12] In some embodiments, the present invention relates to a method of preparing a microfluidized lysate preparation comprising microfluidizing a slurry of at least one
  • the Leishmania parasite through a chamber and disrupting the leishmania parasite with a sudden release of pressure.
  • the method further comprises heat treating the microfluidized lysate preparation.
  • the Leishmania parasite may be E. tropica, L. mexicana, L. guyanensis, L. braziliensis, L. major, L. donovani, L. chagasi, L. amazonensis, L. peruviana, L. panamensis, L. pifanoi, L. infantum, orE aethiopica.
  • the present invention relates to a microfluidized lysate preparation made by microfluidizing a slurry of at least one Leishmania parasite through a chamber and disrupting the leishmania parasite with a sudden release of pressure and heat treating the microfluidized lysate preparation.
  • the Leishmania parasite may be E. tropica, L. mexicana, L. guyanensis, L. braziliensis, L. major, L. donovani, L. chagasi, L. amazonensis, L. peruviana, L. panamensis, L. pifanoi, L. infantum, or L. aethiopica.
  • the present invention relates to a skin test antigen assay for detecting whether a subject has been exposed to a Leishmania parasite or was afflicted with Leishmaniasis comprising administering to the subject an antigenic amount of at least one microfluidized lysate preparation of the present invention and observing any immunogenic response to the microfluidized lysate preparation.
  • the Leishmania parasite may be E. tropica, L. mexicana, L. guyanensis, L. braziliensis, L. major, L. donovani, L. chagasi, L. amazonensis, L. peruviana, L. panamensis, L. pifanoi, L. infantum, or E.
  • an immunogenic response indicates that the subject has been exposed to a Leishmania parasite or was afflicted with Leishmaniasis.
  • an induration of about 5 mm or greater observed indicates that the subject has been exposed to a Leishmania parasite or was afflicted with Leishmaniasis.
  • the antigenic amount of the microfluidized lysate preparation comprises about 5 ⁇ g to about 30 ⁇ g of total protein.
  • the microfluidized lysate preparation is administered intradermally to the volar surface of the forearm of the subject.
  • the present invention relates to a kit comprising the microfluidized lysate preparation of the present invention and directions for determining whether a subject has been exposed to a Leishmania parasite or was afflicted with Leishmaniasis.
  • the Leishmania parasite may be E. tropica, L. mexicana, L. guyanensis, L. braziliensis, L. major, L. donovani, L. chagasi, L. amazonensis, L. peruviana, L. panamensis, L. pifanoi, L. infantum, or E. aethiopica.
  • the kit may further comprise at least one pharmaceutical for treating systemic anaphylaxis such as epinephrine, diphenhydramine, and methyl prednisolone.
  • the kit may further comprise at least one pharmaceutical for treating local reactions to the microfluidized lysate preparation such as hydrocortisone, hydrocortisone cream, acetaminophen, or diphenhydramine.
  • the present invention relates to antibodies raised against the microfluidized lysate preparation of the present invention.
  • the present invention relates to a method of determining whether a subject has been exposed to a given Leishmania parasite comprising administering to the subject a panel of antigenic compositions comprising a plurality of microfluidized lysate preparations prepared from a plurality of Leishmania parasites and detecting a presence of an immunogenic reaction that is characteristic to exposure to the given Leishmania parasite.
  • the plurality o ⁇ Leishmania parasites may include at least one parasite belonging to the group consisting of L. tropica, L. mexicana, L. guyanensis, L. braziliensis, L. major, L. donovani, L. chagasi, L. amazonensis, L. peruviana, L. panamensis, L. pifanoi, L. infantum, and E. aethiopica.
  • the present invention relates to a method of immunizing a subject against Leishmaniasis comprising administering to the subject an immunogenic amount of the microfluidized lysate preparation.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the microfluidized lysate preparation and a pharmaceutically acceptable stabilizer such as phenol.
  • the pharmaceutical composition is in the form of a liquid which may be frozen or freeze-dried.
  • the present invention relates to a method for determining post infection of cutaneous leishmaniasis, mucocutaneous leishmaniasis, or post-kala-azar dermal leishmaniasis in a subject comprising administering to the subject an antigenic amount of at least one microfluidized lysate preparation and observing any immunogenic response to the microfluidized lysate preparation.
  • the present invention relates to a method for epidemiologically diagnosing cutaneous leishmaniasis, mucocutaneous leishmaniasis, or post-kala-azar dermal leishmaniasis in a subject comprising administering to the subject an antigenic amount of at least one microfluidized lysate preparation and observing any immunogenic response to the microfluidized lysate preparation.
  • the present invention relates to a method for determining the pattern of present and past leishmaniasis in a- subject comprising administering to the subject an antigenic amount of at least one microfluidized lysate preparation and observing any immunogenic response to the microfluidized lysate preparation.
  • Figure 1 is a flow diagram showing the process for making a Leishmaniasis microfluidized lysate according to the present invention.
  • the present invention allows the detection of post infections and epidemiological diagnosis of all forms of leishmaniasis.
  • the present invention may be used for screening of individuals such as U.S. Service Members who may have been exposed to Leishmania parasites after deployment to Leishmaniasis endemic areas.
  • the present invention may be used in the clinical diagnosis of cutaneous leishmaniasis, mucocutaneous leishmaniasis, and post-kala-azar dermal leishmaniasis in subjects such as U.S. Service Members.
  • the present invention may be used to determine the pattern of past and present infections of leishmaniasis.
  • the present invention provides microfluidized lysate preparations comprising at least one antigen from at least one Leishmania parasite and methods of making and using thereof.
  • the microfluidized lysate preparations of the present invention may be used to elicit induration that is consistent with delayed type hypersensitivity (DTH) in an individual previously infected with or exposed to at least one Leishmania parasite.
  • DTH delayed type hypersensitivity
  • the microfluidized lysate preparation is injected intradermally.
  • the present invention also provides a skin test assay for detecting whether a subject has been exposed to a Leishmania parasite or is or has been afflicted with Leishmaniasis comprising at least one microfluidized lysate preparation of at least one Leishmania parasite.
  • the microfluidized lysate of the present invention comprises at least one solubilized antigen of at least one Leishmania parasite.
  • the microfluidized lysate preparation may further comprise a mixture of amino acids, lipids, and carbohydrates.
  • the Leishmania parasite may be specifically selected for a particular reason. For example, an Old World parasite, such as E. tropica strain WR1063, may be used in order to detect E.
  • Leishmania parasites acquired in Southwest Asia.
  • any Leishmania parasite, Old World or New World may be selected by one of ordinary skill in the art, prepared, and used according to the present invention.
  • Examples of a few Leishmania parasites include L. tropica, L. mexicana, L. guyanensis, L. braziliensis, L. major, L. donovani, L. chagasi, L. amazonensis, L. peruviana, L. panamensis, L. pifanoi, L. infantum, L. aethiopica.
  • the L. tropica strain WR1063 used herein was cloned by mechanical-single cell isolation and given the designation, CL1.
  • the microfluidized lysate preparations of the present invention were prepared under current good manufacturing practices (cGMP). Disruption of the promatigotes was accomplished by microfluidization comprising passing a parasite slurry through a chamber and disrupting the cells by the sudden release of pressure. Not only does microfluidization allow the release of membrane embedded antigens, but it provides a reproducible method for providing large volumes of preparations.
  • the microfluidized lysate preparations were heat-treated at about 95 °C in a water bath for about 30 minutes to deactivate the proteolytic enzymes.
  • a strain of vicerotropic leishmania WR1063 was isolated, cloned, and then characterized as L. tropica by isoenzyme analysis.
  • Other Leishmania parasites may be used such as L. mexicana as described in Example 2.
  • a master seed lot and a production ⁇ seed lot of the clone were used to initiate individual bulk production lots of promastigotes.
  • Three bulk lots (about 1 x 10 11 promastigotes each) were pooled in a vial and immediately placed on dry ice. The pooled promastigotes were then thawed by placing the vial in a water bath at about 56 ⁇ 2 °C.
  • the vial was placed at about 4 ⁇ 2 °C to cool. After cooling, the contents of the vial were transferred to a pre-tared 250 ml sterile centrifuge bottle on ice using a 10 cc sterile syringe. Two 0.5 ml samples of the suspended cells were pipetted and placed in a sterile 1.5 ml Nunc cryovial (Fisher Scientific, Pittsburgh, PA) and stored at about -80 ⁇ 10 °C. [31] A micro fluidizer, Model #M-110S was used to extract the E. tropica soluble proteins. The regulator was set and wet ice was put into the cooling jacket of the microfluidizer.
  • the pump was primed by placing the inlet tubing into 500 ml of 0.001 % Tween 80 and 0.9% saline and then opening the air valve. With the regulator at about 100 ⁇ 5 psi, the inlet tubing was inserted into the promastigotes suspension and run through the microfluidizer. Because the cracking pressure fluctuates, an average reading was taken. Cracked cells were collected into the reservoir containing the uncracked cells and cracking continued for about 10 ⁇ 1 minutes. The pre- and post-cracking temperatures were recorded. The 250 ml tube containing the lysed promastigotes was capped and stored at about 4 ⁇ 2 °C.
  • the cracked cells were dispensed into a sterile 250 ml centrifuge bottle and centrifuged at about 3,100 ⁇ 200 rpm (1566 x g) in a Sorvall GSA rotor within a RC-5 Sorvall centrifuge at a time setting of about 30 ⁇ 1 minutes and at a temperature of about 4 ⁇ 2 °C.
  • the bottle was removed and placed in a Class II Biohazard cabinet.
  • the supernatant was poured of into a second sterilized 350 ml centrifuge tube and stored at about 4 ⁇ 2 °C.
  • the pellet was then suspended with about 20 ml of Buffer B comprising, 0.001%) Tween 80 diluted with 0.9%o saline, and vortexed with vortex mixer. Ice was added into the cooling jacket of the microfluidizer as needed. When the regulator was at about 100 ⁇ 5 psi, the inlet tubing was inserted into the suspension and run through the microfluidizer in a continual fashion for about 10 minutes. The cracked cells were collected into a 250 ml centrifuge bottle containing the lysed promastigotes and placed on ice. The pre- and post-cracking temperatures were recorded. Then two 0.5 ml of the cracked cell samples were taken and stored at about -80 ⁇ 10 °C. On ice, the cracked cells and the supernatant were combined and mixed by swirling in the container.
  • Buffer B comprising, 0.001%
  • the post-cracked cells were immediately centrifuged at about 12,200 ⁇ 200 rpm (about 23,435 to about 25,062 x g) in a Sorval GSA rotor within a RC-5 Sorvall centrifuge for about 30 ⁇ 1 minutes at about 4 ⁇ 2 °C. Then the bottle was removed and placed in BSC, the supernatant was poured off into a sterile 250 ml graduated cylinder and the pellet in the 250 ml bottle was retained.
  • samples were aseptically obtained and assayed for protein content by BCA, endotoxin content LAL (gel-clot), pH, sterility, HPLC, purity by SDS-PAGE, color and appearance.
  • microfluidized lysate preparation was used in a skin test assay for detecting whether a subject had been exposed to a Leishmania parasite, such as L. tropica.
  • a Leishmania parasite such as L. tropica.
  • one of ordinary skill in the art may prepare a microfluidized preparation from more than one Leishmania parasite.
  • the present invention also provides a multivalent microfluidized lysate preparation prepared from at least two different Leishmania parasites.
  • the preparation may comprise the microfluidized lysate of E. tropica, L. mexicana, and L. guyanensis.
  • the skin test assay of the present invention comprises administering to a subject an antigenic amount of a microfluidized lysate of the present invention.
  • the subject is a mammal, more preferably, the subject is human.
  • the microfluidized lysate preparation is administered intradermally, more preferably, the microfluidized lysate preparation is injected into the volar surface of the forearm of the subject. Induration is then measured.
  • induration is measured at least once at about 24 to about 72 hours after the microfluidized lysate preparation was administered. More preferably, induration is measured at about 48 hours after induration as true delayed type hypersensitivity responses in humans are maximal at about 48 hours.
  • an "antigenic amount” is an amount which provides a positive induration response in a subject known to be exposed to a leishmanial parasite. Antigenic amounts range from about 0.01 to about 1.0 ml, preferably about 0.05 to about 0.5 ml, more preferably about 0.1 ml. A positive response indicates previous exposure and sensitization to the antigen has occurred and is recalled, and an antigen specific inflammatory response takes place, thereby indicating that the subject was infected with or had been exposed to at least one Leishmania parasite. A positive response in humans is an induration of about 5 mm or more.
  • the microfluidized lysate preparations of the present invention are safe and immunogenic. Therefore, the present invention also provides immunogenic compositions and vaccines comprising the microfluidized preparations of the present invention.
  • the vaccine or immunogenic compositions of the present invention may be used in combination with an adjuvant, a pharmaceutically acceptable excipient, or both.
  • the present invention also provides methods for immunizing subjects against diseases, infections, or disorders associated with Leishmania parasites, such as Leishmaniasis, all forms of leishmaniases, and diseases as Chagas Disease, and African trypanosomiasis were common crossreacting antigens with immunoprotective properties.
  • the method of preparing the microfluidized lysate preparations as described herein maybe standardized and reproduced to prepare batches of microfluidized lysate preparations having substantially similar characteristics, such as potency and specificity, the microfluidized lysate preparations may be used to purify or screen for ligands which bind to leishmanial antigens or antibodies raised against the microfluidized lysate of the present invention with consistency.
  • the microfluidized lysate preparations of the present invention are suitable for use as immunogens to raise anti-leishmanial antibodies.
  • the antibodies may be prepared by immunizing a suitable subject, e.g., rabbit, goat, mouse or other mammal, with the microfluidized lysate of the present invention by methods standard in the art.
  • the microfluidized lysate preparation may further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.
  • the present invention also provides antibody preparations made by immunizing a suitable subject with a microfluidized lysate preparation of the present invention.
  • the antibodies produced by the subject may be isolated or purified by methods standard in the art.
  • the antibodies raised against the microfluidized lysate of the present invention may be used to isolate leishmanial antigens by methods standard in the art, such as affinity chromatography or immunoprecipitation.
  • the antibodies raised against the microfluidized lysate of the present invention may be used to diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable label or marker. Examples of detectable labels and markers include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelhferone, fluorescem, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and i i l l c aequorin, and examples of suitable radioactive material include I, I, S or H.
  • microfluidized lysate preparation, antibodies, or antigens of the present invention may be incorporated into a pharmaceutical composition suitable for administration.
  • Such compositions typically comprise the microfluidized lysate preparation, antibodies, or antigens of the present invention and a pharmaceutically acceptable carrier.
  • pharmaceutical compositions of the present invention comprise an antigenic amount of the microfluidized lysate preparation or antigen or a therapeutically effective amount of antibodies raised against the microfluidized lysate preparation, and an inert, pharmaceutically acceptable carrier or diluent.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.
  • the pharmaceutical compositions of the present invention include sterile saline comprising 0.4%> phenol.
  • the antigenic activity of the microfluidized lysate of the present invention may be measured by any of the methods available to those skilled in the art, including in vitro and in vivo assays. Examples of suitable assays for activity measurements are provided herein. Properties of the microfluidized lysate, such as protein content, endotoxin content, pH, sterility, purity, and color, may be assessed, for example, by methods standard in the art. Other pharmacological methods may also be used to determine the efficacy of the microfluidized lysate as antigenic compositions.
  • LSTA -Lg (BPR-2334-RS) was conducted as follows. A parasite specimen was obtained from an active dermal lesion from a human subject otherwise healthy with unremarkable medical history. The subject tested negative in screening with viral/STD panel (HIV, HTLV, CMV, HCV, HBV and syphilis). The subject's travel history was determined.
  • FBS Fetal Bovine Serum
  • FBS Type JJ The Fetal Bovine Serum (FBS) Type JJ, was mycoplasma and bovine virus tested, screened for bacteriophage, tested for endotoxin, tested for AVA, cell culture tested, heat-inactivated, and from a non BSE country (Gibco Life Technologies, Grand Island, NY; Fetal Bovine Serum, Qualified, Origin-United States, Catalogue Number 26140, Lot Number 1016982).
  • the promastigotes cultured from this specimen, (strain WR2334) were determined by isoenzymes to be L. guyanensis. No known viral contaminants, such as LRV1-4, were detected in the promastigotes.
  • a clonal line, WR2334 Clone D2 (WR2334-D2) was established through serial endpoint dilution and microscopic visualization of a single promastigote. This source seed stock of this clone was then expanded in culture, aliquoted and cryopreserved in liquid nitrogen. Sterility and morphology were determined by conventional methods.
  • LSTA-Lg (BPR-377-00, Lot No. 0705) was manufactured under defined cGMP conditions from cryopreserved aliquots of WR2334-Clone D2, by culturing and expansion to roller bottles in appropriate medium.
  • the cultured promastigotes were harvested by centrifugation, washed, suspended in cryopreservation medium, aliquoted, and stored in liquid nitrogen. Sterility and morphology were determined by conventional methods.
  • LSTA-Lg (BPR-377-00, Lot No. 0705) was manufactured under defined cGMP conditions from cryopreserved aliquots of WR2334-Clone D2 Master Cell Bank, by culturing and expansion to roller bottles in appropriate medium. The cultured promastigotes were harvested by centrifugation, washed, suspended in cryopreservation medium, aliquoted, and stored in liquid nitrogen. Sterility and morphology were determined by conventional methods.
  • BPR-383-00, Lot No. 0707 was produced. Specifically, bulk promastigotes were cultured in Schneider's Drosophila Medium (SDM), supplemented with 20%> fetal bovine serum (heat inactivated). The inoculum was initiated in flasks and when the culture set point was reached, it was transferred to roller bottles and expanded. Promastigotes were harvested by centrifugation when a sufficient quantity was been obtained. The culture was maintained in healthy, log-growth phase throughout the production run, including the time of harvest. Harvested promastigotes were centrifuged and washed 5 times.
  • SDM Schneider's Drosophila Medium
  • fetal bovine serum heat inactivated
  • the pellets were reconstituted in sterile saline USP and manually filled in 5.0 mis aliquots at 109 promastigotes/ml.
  • the vials were labeled, stored and frozen at -80 °C for subsequent production and purification steps. Sterility and morphology were determined by conventional methods.
  • Lg (BPR-389-00, Lot No. 0716) was purified. Specifically, cryopreserved vials were thawed and processed through a microfluidizer (MFL#1). The material was centrifuged at 3,100 rpm for 30 minutes and the supernatant was saved. The pellet was resuspended and microfluidized again (MFL#2). The material was centrifuged at 12,200 rpm for 30 minutes and the two supernatants combined. The pooled supernatants were placed in a 0.22 micron sterile filter unit (500 ml) and filtered. The filtrate was then heat-treated at 90 °C for 15-20 minutes to inactivate parasite proteases.
  • MFL#1 microfluidizer
  • the final filtered and heat- treated bulk was formulated and the protein concentration adjusted to about 0.35 mg per ml.
  • the lot was bottled and stored at 4 °C.
  • the bulk material was tested for the following characteristics: Visual Inspection: Color, Appearance; Protein Content: micro- BCA (prior to phenol addition); Purity/Identity: SDS-PAGE, HPLC; and Quality: Sterility; Rabbit Pyrogen, Endotoxin (LAL), pH.
  • the final product of L. guyanensis (WR2334-Clone D2) Leishmania Skin Test
  • LSTA-Lg (BPR-390-00, Lot No. 0717) was placed in vials. Specifically, the formulated, bulk LSTA-Lg was aseptically filled in 1.0 ml aliquots into 10 ml Type I glass vials, closed with 20 mm rubber closures and stored at 4°C.
  • a microfluidizer, Model #M- 11 OS was washed and autoclaved per the manufacturer's directions.
  • the regulator was set and wet ice was put into the cooling jacket of the microfluidizer.
  • the pump was primed by placing the inlet tubing into 500 ml of 0.001%) Tween 80 and 0.9% saline and then opening the air valve. With the regulator at 100 ⁇ 5 psi, the inlet tubing was inserted into the promastigotes suspension and run through the microfluidizer. Because the cracking pressure fluctuates, an average reading was taken. Cracked cells were collected into the reservoir containing the uncracked cells and cracking continued for 10 ⁇ 1 minutes. The pre- and post-cracking temperatures were recorded. The 250 ml tube containing the lysed promastigotes was capped and stored at 4 ⁇ 2 °C.
  • the cracked cells were dispensed into a sterile 250 ml centrifuge bottle and centrifuged at 3,100 ⁇ 200 rpm (1566 x g) in a Sorvall GSA rotor within a RC-5 Sorvall centrifuge at a time setting of 30 ⁇ 1 minutes and at a temperature of 4 ⁇ 2 °C.
  • the bottle was removed and placed in a Class U Biohazard cabinet.
  • the supernatant was poured of into a second sterilized 350 ml centrifuge tube and stored at 4 ⁇ 2 °C.
  • the pellet was then suspended with 20 ml of Buffer B, comprising 0.001% Tween 80 diluted with 0.9% saline, and vortexed with vortex mixer.
  • Ice was added into the cooling jacket of the microfluidizer as needed.
  • the inlet tubing was inserted into the suspension and run through the microfluidizer in a continual fashion for 10 minutes.
  • Cracked cells were collected into a 250 ml centrifuge bottle containing the lysed promastigotes and placed on ice. The pre- and post-cracking temperatures were recorded. Then two 0.5 ml of the cracked cell samples were taken and stored at -80 ⁇ 10 °C. On ice the cracked cells and the supernatant were combined and mixed by swirling in the container.
  • the post-cracked cells were immediately centrifuged at 12,200 ⁇ 200 rpm (23,435 - 25,062 x g) in a Sorval GSA rotor within a RC-5 Sorvall centrifuge for 30 ⁇ 1 minutes at 4 ⁇ 2 °C. Then the bottle was removed and placed in BSC, the supernatant was poured off into a sterile 250 ml graduated cylinder and the pellet in the 250 ml bottle was retained.
  • the bulk clarified promastigotes lysate was tightly sealed and stored at 4 ⁇ 2 °C until treated with heat. 25%> glycerol was added to the bulk clarified promastigotes lysate to give a final concentration of 1% > and then mixed by swirling. The total weight of the in process bulk was determined. Then the bulk clarified promastigotes lysate was incubated for 30 ⁇ 2 minutes using a water bath at 93 ⁇ 2 °C. After heat treatment, the bulk was cooled at 4 ⁇ 2 °C. Aseptically two 0.5 ml aliquots were taken and retained at -80 ⁇ 10 °C. One 1.0 ml sample was taken for protein concentration testing by the Bio-Rad method (Bio-Rad Laboratories, Hercules, CA) and for SDS-PAGE.
  • samples were aseptically obtained and assayed for protein content by BCA, endotoxin content LAL (gel-clot), pH, sterility, HPLC, purity by SDS-PAGE, color and appearance.
  • FIG. 1 is a flow diagram of process for making the Leishmaniasis microfluidized lysate of the present invention.
  • Example 3 Skin Test Antigen Assay in 10 Subjects [62] A microfluidized lysate preparation of L. tropica prepared according the Example
  • the microfluidized lysate preparation further included Tween-80® and dextran.
  • the subjects received four doses two weeks apart of 0.1 ml of the microfluidized lysate preparation in escalating concentrations.
  • the first dose had 0.25 ⁇ g
  • the second dose had 2.5 ⁇ g
  • the third dose had 8.0 ⁇ g
  • the fourth dose had 25.0 ⁇ g of total protein. 25.0 ⁇ g of the preparation was found to be safe in six of the 10 subjects.
  • One subject developed clear rhinorrhea and nasal pruritus within minutes of receiving the first dose. Four hours later, transient urticaria at the test and control-diluent sites occurred.
  • Phenol as a preservative was tested in 15 Leishmania na ⁇ ve human subjects and was found to be safe as a single injection at doses of 0.38 ⁇ g, 3.8 ⁇ g, and 38.0 ⁇ g.
  • a subject is deemed to be clinically diagnosed with an active Leishmaniasis infection upon the demonstration of motile promastigotes in aspirate cultures or microscopic Leishmania amastigotes in samples obtained from lesions obtained from the subject.
  • the parasite may be visualized by conventional methods.
  • a subject is deemed to be clinically healed upon 100% reepitheliazation of the ulcer.
  • Antigen 40 CFU/ml, MSTA®, Connaught Labs Inc. (Swiftwater, PM), and Candida albicans skin test antigen, Candin®, Allermed Laboratories, Inc. (San Diego, CA), before receiving the preparations.
  • Subj ects who have a history of atopy, allergic reactions, or asthma will be excluded from the study including those who are allergic to phenol, pharmaceutical detergents or glycol. Also excluded are those subjects taking steroids, antihistamines, cimetidine, and immunosupressants, as well as those who had a splenectomy or have an active medical or psychiatric conditions that may increase the risks associated with participation in the study or interfere with the interpretation of study results. Pregnant or nursing subjects will be excluded. Subjects having active cutaneous Leishmaniasis with scars, i.e. possible re-infections and residivant Leishmaniasis will be excluded. Subjects having been immunized within 4 weeks prior to the start of the study will be excluded as well as those having anergy on delayed type hypersensitivity testing (less than about 5 mm of induration).
  • Primary endpoints are the occurrence of local or systemic reactions to the skin or the occurrence of non-specific immune responses to the skin tests.
  • the microfluidized lysate preparations are considered safe if there are no clinically significant local or systemic reactions in the healthy and healed subjects.
  • the micro fluizided lysate preparations will also be considered safe if no severe reactions are determined in active leishmania subjects.
  • Secondary endpoints are the size of the induration which accompanies the delayed type hypersensitivity response (potency); the percentage of subjects with Leishmaniasis in which the preparation induced a positive response about 5 mm or greater delayed type hypersensitivity reaction (sensitivity), the percent of healthy subjects in which the preparation fails to induce a positive delayed type hypersensitivity reaction (specificity).
  • microfluidized lysate preparations are first tested in healthy subjects, followed by healed subjects, and followed by visceral or cutaneous subjects.
  • the same subject is administered three different preparations, E. mexicana, L. tropica, and E. guyanensis, at a given concentration simultaneously.
  • This study allows the direct comparison of the three preparations and reduces the number of subjects in the study.
  • the study is a dose escalation study which tests three dose levels of the microfluidized lysate preparations. Three cohorts of 20 subjects per clinical group are used. The dose of each preparation escalates for each cohort.
  • Intradermal injections of saline and a 1:100 dilution of the diluent are administered as controls concomitantly with the preparations.
  • 20 healthy subjects (Cohort 1) are administered microfluidized lysate preparations having total protein concentrations of 5 ⁇ g (0.1 ml ID). If no clinically significant reaction is observed within two days after administration in Cohort 1, Cohort 2 is administered preparations having 15.0 ⁇ g total protein (0.1 ml ID). If no significant reaction is observed within two days after administration in Cohort 2, Cohort 3 is administered preparations having 30.0 ⁇ g total protein (0.1 ml ID). Each subject is followed for two days to assess local and systemic reactions.
  • the controls are 0.1 ml saline and a 1:100 dilution of the microfluidized lysate preparation diluent comprising 0.001% Tween-80, 1% glycerol, 0.4%> phenol diluted with 0.9% saline stored at 4 ⁇ 2 °C.
  • 0.1 ml of a microfluidized lysate preparation is injected intradermally into alcohol-cleansed volar surface of the forearm under the supervision of a physician who will have medication and equipment to treat anaphylaxis.
  • the diameter of the induration, erythema, or both will be measured in millimeters at about 30 minutes after administration to detect immediate IgE hypersensitivity, and about 48 hours later to detect delayed type hypersensitivity by outlining the indurated border with a ballpoint pen and transferring to paper damped with 70%> ethanol for permanent record. The largest diameter and its perpendicular diameter are measured and averaged.
  • the Sokal method maybe used. See Montenegro, J. (1926) Archives of Dermatology and Syphilology 13:187-194. The placement of each preparation and control for each subject are randomized and recorded. Skin test readers are unaware of the placements. Subjects are monitored closely.

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Abstract

La présente invention concerne des préparations de lysat microfluidisé de parasites leishmania et des techniques de fabrication de celles-ci. Cette invention concerne aussi des techniques d'utilisation de ces préparations de lysat microfluidisé dans des dosages d'antigène de test cutané ainsi que des kits comprenant ces préparations de lysat microfluidisé. Ces préparations de lysat microfluidisé sont fabriquées selon de bonnes pratiques de fabrication courantes et peuvent donc être normalisées. Ces préparations peuvent être produites de façon uniforme.
PCT/US2001/031894 2001-10-12 2001-10-12 Antigene de leishmania microfluidise et techniques de fabrication et d'utilisation de celui-ci WO2003033533A1 (fr)

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CA002463581A CA2463581A1 (fr) 2001-10-12 2001-10-12 Antigene de leishmania microfluidise et techniques de fabrication et d'utilisation de celui-ci
EP01979743A EP1436325A1 (fr) 2001-10-12 2001-10-12 Antigene de leishmania microfluidise et techniques de fabrication et d'utilisation de celui-ci

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0293827A2 (fr) * 1987-06-01 1988-12-07 Yeda Research And Development Company, Ltd. Essai pour leishmaniose
WO1998035045A2 (fr) * 1997-02-12 1998-08-13 Corixa Corporation Antigenes leishmania utilises dans le traitement et le diagnostic de la leishmaniose

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0293827A2 (fr) * 1987-06-01 1988-12-07 Yeda Research And Development Company, Ltd. Essai pour leishmaniose
WO1998035045A2 (fr) * 1997-02-12 1998-08-13 Corixa Corporation Antigenes leishmania utilises dans le traitement et le diagnostic de la leishmaniose

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"M-110S Microfluidizer", MICROFLUIDICS CORPORATION, 1998, XP002202508, Retrieved from the Internet <URL:http://www.microfluidicscorp.com/mi_e_110s.htm> [retrieved on 20020618] *
DE LUCA P M ET AL: "Evaluation of the stability and immunogenicity of autoclaved and nonautoclaved preparations of a vaccine against American tegumentary leishmaniasis", VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 17, no. 9-10, 5 March 1999 (1999-03-05), pages 1179 - 1185, XP004158241, ISSN: 0264-410X *
LEWIS R.: "New Disrupters help cell biologists retrieve products", THE SCIENTIST, vol. 24, no. 18, 13 December 1993 (1993-12-13), pages 18, XP002202507, Retrieved from the Internet <URL:http://www.the-scientist.com/yr1993/dec/tools_931213.html> [retrieved on 20020618] *
REED S G ET AL: "SELECTION OF A SKIN TEST ANTIGEN FOR AMERICAN VISCERAL LEISHMANIASIS", AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, vol. 35, no. 1, 1986, pages 79 - 85, XP002202015, ISSN: 0002-9637 *
SCOTT P ET AL: "VACCINATION AGAINST CUTANEOUS LEISHMANIASIS IN A MURINE MODEL I. INDUCTION OF PROTECTIVE IMMUNITY WITH A SOLUBLE EXTRACT OF PROMASTIGOTES", JOURNAL OF IMMUNOLOGY, vol. 139, no. 1, 1987, pages 221 - 227, XP002202016, ISSN: 0022-1767 *
STITELER J M ET AL: "ODS Leishmania skin test, MFL-LSTA(R2): Stability of the cGMP product in the guinea pig animal model.", AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, vol. 62, no. 3 Supplement, March 2000 (2000-03-01), 49th Annual Meeting of the American Society of Tropical Medicine and Hygiene;Houston, Texas, USA; October 29-November 02, 2000, pages 310, XP008004176, ISSN: 0002-9637 *

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