EP0349541A1 - Mit antigenischen molekülen geladene liposome von intrazellparasiten - Google Patents

Mit antigenischen molekülen geladene liposome von intrazellparasiten

Info

Publication number
EP0349541A1
EP0349541A1 EP19880901530 EP88901530A EP0349541A1 EP 0349541 A1 EP0349541 A1 EP 0349541A1 EP 19880901530 EP19880901530 EP 19880901530 EP 88901530 A EP88901530 A EP 88901530A EP 0349541 A1 EP0349541 A1 EP 0349541A1
Authority
EP
European Patent Office
Prior art keywords
liposomes
sensitized
ppd
agents
egg
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19880901530
Other languages
English (en)
French (fr)
Inventor
Franz Legros
Jean-Marie Ruysschaert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite Libre de Bruxelles ULB
Original Assignee
Universite Libre de Bruxelles ULB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universite Libre de Bruxelles ULB filed Critical Universite Libre de Bruxelles ULB
Publication of EP0349541A1 publication Critical patent/EP0349541A1/de
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • 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
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/586—Liposomes, microcapsules or cells
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers

Definitions

  • the present invention is based on the sensitization of liposomes to antigenic molecules of intracellular parasites and therefore opens new applications of such sensitized liposomes, as means of detection of Ig (lytic or non-lytic) against these infectious antigens in sera or other body fluids (pleural, peritoneal, bronchoalveolar lavage) and as vaccination agents capable of eliciting immunity (cellular, but also humoral) against the infecting agent.
  • the aim is more specifically to provide means of diagnosis and immunization for tuberculosis, but the invention is of course not limited to this application, as will appear below. .
  • liposomes for various pharmacological applications, in particular as vectors of substances with medicinal activity.
  • Antigenic molecules natural or haptenic, have also been exposed on the surface of liposomes. These so-called sensitized liposomes have been used either for the detection of immunoglobulins in sera.
  • immunized animals either as adjuvants of immunity likely to increase humoral, but also cellular immunization.
  • the invention relates specifically as new agents which can be used for the abovementioned purposes (immunoassay and vaccination) on liposomes sensitized to specific antigenic molecules of intracellular parasites.
  • These liposomes are preferably of the so-called “multilamellar” (MLV) type with or without cholesterol.
  • MLV multilamellar
  • multilamellar liposomes with cholesterol having Egg Pc / Ch ratios between 4/1 and 4/4, the latter ratio or values close to this being very particularly preferred.
  • sensitized liposomes Also provided are methods of producing sensitized liposomes. Two operating variants are available for this purpose:
  • the two types of sensitized liposomes are capable of fixing immunoglobulins raised against antigenic molecules.
  • the type of sensitization of the liposomes to a mixture of immunoglobulins corresponds to variable quantities of selected antigenic molecules which are exposed on the surface of the liposomes and to differences in the exposure and the affinity of these antigens.
  • the repeated administration, subcutaneous or intraperitoneal, of the two types of liposomes causes the appearance of a delayed hypersensitivity reaction, witness of a cell type immunity, in the guinea pig.
  • the preparation is left to stand for 30 min. at room temperature.
  • Liposomes of different compositions and sensitized according to the two methods are exposed to a sheep antiserum raised against H37Rv, radioiodinated and unlabeled.
  • the preparation and radioiodination of this antiserum is described in the Applicant's patent application filed the same day for: "Method for rapid detection of infections with intracellular germs, in particular Mycobacterium tuberculosis or Mycobacterium leprae and kit suitable for this effect".
  • Competition curves are obtained between an amount of 125 I antiserum and different dilutions of the unlabeled antiserum (from 1/10 to 1/10 000).
  • the proportion of radioiodinated molecules attached to liposomes-PPD and the dissociation constant of the molecules are measured.
  • the liposomal composition which fixes the greatest number of radioiodinated molecules with the greatest affinity seems to be Egg Pc / Ch 4: 4 with direct encapsulation of PPD.
  • Liposomes consisting of Egg PC and Ch (4: 3), containing calcein and sensitized to PPD by the two methods indicated above (encapsulation and contacting), presented an immune lysis (calcein release and appearance fluorescence measurable in the medium) in the presence of anti-BCG rabbit serum (Dakopatts) or anti Mycobacterium tuberculosis sheep serum and complement from rabbit serum. This reaction was not observed in the presence of a serum known to have no antimycobacterial antibodies or in the absence of complement.
  • This test makes it possible to diagnose all infectious diseases by demonstrating serum antibodies using liposomes carrying antigens from the infecting microorganism.
  • liposomes sensitized by antigens are capable of playing a role in cellular mechanisms of immunity in vivo, either by imitating the infectious agent (bilayer of phosholipid carrying immunogenic molecules of this agent) and by being captured by it.
  • the local macrophages of the infected host either by imitating those macrophages which have "processed” the infectious agent and which have exposed some of its antigenic molecules on their surface.
  • the antigenic molecules by which the liposome has been sensitized are presented to the lymphocytes, recognized by receptors and the mechanisms of cellular and humoral immunity can be stimulated.
  • the sensitized liposome can constitute the vehicle for a new type of vaccination, since it represents an almost natural model of presentation of antigenic molecules to the immune system.
  • the liposome-PPD must be able to induce a positive reaction of delayed hypersensitivity (positive intradermoreaction) in animals previously sensitized by the B.C.G.
  • the liposomes-PPDs deposited in the dermis are phagocytosed by macrophages and that the antigens of which these liposomes were carriers are then presented to and call sensitized lymphocytes, thus causing the appearance of the characteristic erythema and induration. of the positive intradermoreaction. This conclusion therefore allows the use of the liposome as a vehicle for vaccination.
  • the liposome-PPD contains only lipids (Egg Pc, Ch) and proteins extracted from Mycobacteria, to the exclusion of all mycobacteria and its genetic material.
  • the technique can be applied as much to the "Old Tuberculin", which counts the proteins of the bacteria as well as its surface polysaccharides and glycolipids whose general and aspecific immunogenicity character is well known, as to specific antigenic molecules of Mycobacterium tuberculosrs
  • a liposome sensitized to the "Old Tuberculin” therefore imitates at best the phospholipid bilayer of the mycobacterial membrane carrying the proteins, polysaccharides and lipids of this intracellular infectious agent.
  • Cryptococcus neoformans Cryptococcosis
  • Candida albicans (mainly): Candidiasis
  • opportunistic germ diabetic patients, treated with corticosteroids, with broad spectrum antibiotics - first cutaneous or mucous membrane involvement (skin, mouth, esophagus, vagina, respiratory tract); systemic dissemination possible (preferred locations: kidneys, eyes, meninges, skin and myocardium)
  • Histoplasma capsulatum Histoplasmosis (USA) - pulmonary involvement with important general signs
  • Salmonella bacteria Salmonellosis
  • Brucella (am, melitensis, abortus): Brucellosis - generalized chronic granulomatous disease (spleen, bone, etc.)
  • Chlamydiae 9. Chlamydiae (Bedsoniae) Psittacosis
  • Mycobacteria - tuberculosis
  • bovis tuberculosis
  • the same liposome sensitized by the antigens of a given parasite can be used both for vaccination and for detection of Ig (lytic or non-lytic) in human serum.
  • Encapsulation of an AIDS virus extract can be done using octylglucoside.
  • the method of preparation below is given by way of non-limiting example.
  • the preparation of the liposomes is carried out here in two stages: a) preparation of monolamellar liposomes by the injection method, b) fixation in the liposomal bilayer of the HTLV3 extract by its molecules with hydrophobic component after formation of a complex glycoprotein -detergent and "fluidification" of liposomes, a) Preparation of liposomes
  • the liposomes are then separated from planar liquids by filtration on a Sephadex-G75 column previously equilibrated with buffer (TP).
  • TP buffer
  • b) Fixation of the HTLV34 Extract on the Liposomes The HTLV3 was extracted with octylglucoside (final concentration 7.5 mM). Liposomes are thinned with 1 mM octylglucoside. The two solutions are then mixed in proportions of 500 ⁇ g of HTLV3 / ⁇ M extract of lipids. This means that 0.375 ⁇ m of lipids are added to 137 ⁇ g of HTLVS extract.
  • the extract-detergent complex and the fluidized liposomes are dialyzed, first against a gradient of decreasing octylglucoside concentration (from 7.5 to 0 mM) to form liposomes carrying HTLV3 molecules, then against the buffer (TP) containing 1 mM NaN 3 .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Public Health (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Dispersion Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • AIDS & HIV (AREA)
  • Mycology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)
EP19880901530 1987-01-15 1988-01-13 Mit antigenischen molekülen geladene liposome von intrazellparasiten Pending EP0349541A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
LU86736 1987-01-15
LU86736A LU86736A1 (fr) 1987-01-15 1987-01-15 Liposomes sensibilises a des molecules antigeniques de parasites intracellulaires,procede pour les preparations et leurs applications comme moyens de diagnostic et comme agents d'immunisation

Publications (1)

Publication Number Publication Date
EP0349541A1 true EP0349541A1 (de) 1990-01-10

Family

ID=19730849

Family Applications (2)

Application Number Title Priority Date Filing Date
EP19880901530 Pending EP0349541A1 (de) 1987-01-15 1988-01-13 Mit antigenischen molekülen geladene liposome von intrazellparasiten
EP88870002A Ceased EP0277930A1 (de) 1987-01-15 1988-01-13 Gegen antigene Moleküle von Intrazellparasiten sensitive Liposome, Verfahren zu deren Herstellung und deren Anwendung als Diagnosemittel und als Immunisationsmittel

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP88870002A Ceased EP0277930A1 (de) 1987-01-15 1988-01-13 Gegen antigene Moleküle von Intrazellparasiten sensitive Liposome, Verfahren zu deren Herstellung und deren Anwendung als Diagnosemittel und als Immunisationsmittel

Country Status (4)

Country Link
EP (2) EP0349541A1 (de)
JP (1) JPH02502007A (de)
LU (1) LU86736A1 (de)
WO (1) WO1988005307A1 (de)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1708350A1 (ru) * 1988-04-15 1992-01-30 Оренбургский Государственный Медицинский Институт Штамм BacILUS SUвтILIS - носитель радионуклидов
IT1238343B (it) * 1989-10-16 1993-07-13 Cesalpino Andrea Fond Procedimento per la preparazione di vaccini capaci di generare non solo la risposta immune dei linfociti t helper,ma anche un'efficace risposta di linfociti t citotossici,e vaccini con queste caratteristiche
DK0555333T3 (da) * 1990-10-19 1996-04-29 Univ Florida Kunstige virushylstre
US5252348A (en) * 1990-10-19 1993-10-12 Univ. Of Florida Research Foundation, Inc. Artificial viral envelopes
US5753258A (en) * 1990-10-19 1998-05-19 University Of Florida Artificial viral envelopes

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4117113A (en) * 1974-06-25 1978-09-26 National Research Development Corporation Immunological preparations
US4199565A (en) * 1979-03-26 1980-04-22 Merck & Co., Inc. Liposome particle containing viral or bacterial antigenic subunit
US4261975A (en) * 1979-09-19 1981-04-14 Merck & Co., Inc. Viral liposome particle
DE3173713D1 (en) * 1980-09-05 1986-03-20 Frappier Armand Inst Formation of an immunosome exclusively made of viral antigens reconstituted on an artificial membrane
NL8202527A (nl) * 1982-06-22 1984-01-16 Univ Utrecht Vaccins tegen door bacterien met kapsel-polysacchariden verwekte ziekten en werkwijze voor het bereiden daarvan.
EP0203676B1 (de) * 1985-04-19 1992-01-29 The Wistar Institute Of Anatomy And Biology Impfstoff für die Erzeugung einer gegen ein Virus schützenden immunogenen T-Zellen-Antwort
US4663161A (en) * 1985-04-22 1987-05-05 Mannino Raphael J Liposome methods and compositions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8805307A1 *

Also Published As

Publication number Publication date
WO1988005307A1 (fr) 1988-07-28
LU86736A1 (fr) 1988-08-23
JPH02502007A (ja) 1990-07-05
EP0277930A1 (de) 1988-08-10

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