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WO1993012252A1 - Procede de mise a l'epreuve d'agents antiviraux et animaux d'experience utilises dans ce procede - Google Patents

Procede de mise a l'epreuve d'agents antiviraux et animaux d'experience utilises dans ce procede Download PDF

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Publication number
WO1993012252A1
WO1993012252A1 PCT/NL1992/000226 NL9200226W WO9312252A1 WO 1993012252 A1 WO1993012252 A1 WO 1993012252A1 NL 9200226 W NL9200226 W NL 9200226W WO 9312252 A1 WO9312252 A1 WO 9312252A1
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WO
WIPO (PCT)
Prior art keywords
experimental animals
cells
immune system
mice
administered
Prior art date
Application number
PCT/NL1992/000226
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English (en)
Inventor
Dirk Willem Van Bekkum
Wim Huppes
Original Assignee
Nederlandse Organisatie Voor Toegepastnatuurwetenschappelijk Onderzoek Tno
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 Nederlandse Organisatie Voor Toegepastnatuurwetenschappelijk Onderzoek Tno filed Critical Nederlandse Organisatie Voor Toegepastnatuurwetenschappelijk Onderzoek Tno
Publication of WO1993012252A1 publication Critical patent/WO1993012252A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/02—Breeding vertebrates
    • 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
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2

Definitions

  • the invention relates to a method for testing antiviral agents that are directed against an immunosuppressive virus, in which the effect is determined that the agent to be tested has on experimental animals which have been transplanted with cells of a donor immune system that are capable of being infected by the immunosuppressive virus and have been infected with the immunosuppressive virus .
  • HIV human immunodeficiency virus
  • the HIV infection in humans has a course of months to years, so that a quick evaluation of the substances to be tested is not well possible.
  • modified cancer cell lines with a 'reporter gene 1 have been developed. Such a gene gives a change of the cells (for instance blue coloring after addition of substrate) if they are infected by the virus. So far, this method has been operational only with cancer cells that do not originate from the immune system, see e.g. the leading article of Emilie in AIDS 4, p 791, 1990. This is a great disadvantage because different types of cells react to different activating signals.
  • in vitro tests One particular disadvantage of in vitro tests is that the data obtained therewith do not reflect the actual interaction between immune system and virus. This may lead to false-positive and false-negative results.
  • In vivo methods where, in contrast with in vitro methods, cells in live beings are studied) with type-specific virus. There are analogously operating, yet dissimilar im unodeficient viruses to the human one, which are specific for, respectively, the cat (FIV) , the rhesus monkey (SIV) and the bovine (BIV) . Tests with these viruses on the corresponding animal can provide first insights into the agents examined, but cannot simply be extrapolated and applied to the human situation.
  • RNA genetically transmittable material
  • MAIDS urine acquired immunodeficiency syndrome
  • MLV murine leukemia virus
  • fetal liver and/or lymph gland and/or thymus are transplanted under the capsule of a kidney of a so-called SCID mouse ("severe combined immune deficient"), followed by an injection with the virus into the transplant.
  • SCID mouse severe combined immune deficient
  • mice After the treatment of these mice with antiviral substances, such as AZT (azidothymidine) , the effect thereof can be measured on the basis of the decrease of the number of HIV-infected human cells.
  • antiviral substances such as AZT (azidothymidine)
  • both methods result in a not very active human immune system in the SCID mice, which is not' a good reflection of the human situation.
  • the two systems also have the disadvantage that the extent to which the human cells develop varies for a reason which has not been clarified yet.
  • the McCune system moreover has the disadvantage that the introduction of the pieces of tissue into mice can be cumbersome and time-consuming and is not always successful.
  • the Mosier system has the disadvantage that it is cumbersome to demonstrate the human cells occurring with a low frequency (approximately 0.1%) as well as to demonstrate the HIV in these cells. This last is done with a PCR (Polymerase Chain Reaction: a method whereby the DNA amount is artificially amplified) which is susceptible to error.
  • the invention relates to a method in which a foreign immune system is transplanted into a small experimental animal, which, as a result, dies within two weeks from Graft- versus-Host Disease, unless an immunosuppressive virus is administered at the same time.
  • the Graft-versus-Host Disease develops as a result of a rejection reaction of the donor immune system against the receptor experimental animal.
  • the agents administered which prevent or combat infection with immunosuppressive viruses, will cause the experimental animal to die nonetheless, because the transplanted immune system is then insufficiently suppressed by the virus, so that the rejection reaction will occur.
  • the invention relates to a quick method for testing, in (preferably small) experimental animals, antiviral agents that are directed against immunosuppressive viruses. It is possible to test agents that block the spread of the virus in the body (such as soluble CD4 and vaccines/antibodies) as well as agents that inactivate the virus (such as pharmaceuticals and anti-sense constructs) . Because small experimental animals are used and the duration of the treatment is short, only a small amount of the substance to be tested is needed. As a consequence, the method is moreover useful for the investigation of larger numbers of agents. Human lymphocytes can be obtained readily in large amounts from a blood bank. The method is in principle ethically acceptable because the blood cells, unlike the other components of the donor blood, are not used in medicine (on account of the danger of Graft-versus-Host Disease or on account of the induction of undesired immunization) .
  • the animal experiment according to the invention occurs according to a procedure which, unlike the existing alternatives, is simple to carry out. Also, an activated immune system is studied, which is more in accordance with reality than are the existing alternatives. Moreover, it is not yet possible to obtain results that are of comparable quality without using experiments on animals.
  • the invention provides a method for testing antiviral agents that are directed against an immunosuppressive virus, in which the effect is determined that the agent to be tested has on experimental animals which have been transplanted with cells of a donor immune system that are capable of being infected by the immunosuppressive virus and have been infected with the immunosuppressive virus, this method being characterized in that experimental animals are used having an immune system disturbed to such an extent that they, upon transplantation with cells of a donor immune system, can develop a fatal graft-versus-host reaction, and these experimental animals are transplanted with so large a dose of cells of the donor immune system that the fatal graft-versus- host reaction develops except in those experimental animals that have at the same time been infected with the immunosuppressive virus.
  • mice such as mice, rats, hamsters, guinea pigs and rabbits, and, more particularly, that mice are used with a SCID or a XID mutation, or normal mice of 2-6 weeks of age.
  • the experimental animals are treated with agents such as gamma-rays, X-rays or neutron rays, cytostatics or antibodies against lymphocytes, these agents disturbing the immune system of the experimental animal.
  • agents such as gamma-rays, X-rays or neutron rays, cytostatics or antibodies against lymphocytes, these agents disturbing the immune system of the experimental animal.
  • a very suitable method for instance in mice, is to treat the experimental animals with approx. 9 Gy gamma radiation.
  • the experimental animals are transplanted with type-specific bone marrow cells. Thus, a serious deterioration of the condition of the experimental animals can be prevented.
  • the cells of a donor immune system to be administered are human lymphocytes. It is further preferred that, for instance in mice, the cells of a donor immune system are administered in an amount of at least 10 7 cells, preferably at least 10 8 cells, per experimental animal.
  • the immunosuppressive virus to be administered is HIV.
  • the invention further provides experimental animals which have been transplanted with cells of a donor immune system that are capable of being infected by an immunosuppressive virus and have been infected with the immunosuppressive virus, characterized in that the experimental animals have an immune system disturbed to such an extent that they, upon transplantation with cells of a donor immune system, can develop a fatal graft-versus-host reaction and have been transplanted with so large a dose of cells of the donor immune system that the fatal graft-versus- host reaction could have occurred if the experimental animals had not at the same time been infected with the immunosuppressive virus.
  • lymphocytes (2) use is made of small experimental animals (1) , lymphocytes (2) and immunosuppressive viruses (3) .
  • the test is preferably carried out with laboratory mice.
  • Various mouse strains can be used for the test according to the invention. Even normal mice may be used, provided they are of very young age, because new-born mice still have an immature immune system that does not yet produce antibodies against type-foreign lymphocytes. Instead of normal mice, however, the known SCID or XID ("X-linked Immune
  • lymphocytes can be isolated from the blood, the spleen, the bone marrow, the thymus and the lymph glands of the experimental animal to be investigated (rhesus monkey, cat, bovine, mouse, and the like) or of man.
  • the lymphocytes can be amplified in a culture bottle or be temporarily stored . in a tube (by freezing the cells and storing them at extremely low temperatures) .
  • the test is preferably carried out with human lymphocytes .
  • the immunosuppressive virus can be introduced as a culture fluid into which the virus has been secreted by a virus producing cell line, or by means of infected cells.
  • the study is preferably done with HIV. Because FIV and BIV are not infectious to man, situations are conceivable where working with these viruses is advantageous. So far, SIV has .not been demonstrated to be infectious to man. MLV could be used as well.
  • the method according to the invention preferably comprises a treatment of the small experimental animals with a high dose of total body irradiation (X-rays, neutron rays or, preferably, gamma-rays) and/or with high doses of cytostatics (for instance endoxan) or antibodies against lymphocytes, such as ATG (anti-thymocytes globulin) , so as to suppress their own defense and to make room within the organs for the transplant.
  • cytostatics for instance endoxan
  • lymphocytes such as ATG (anti-thymocytes globulin)
  • the experimental animals preferably receive a transplantation of their type of bone marrow. Further, the lymphocytes to be infected are administered. A previously determined amount of these lymphocytes is given, which, in control experiments, ha-s resulted in Graft-versus-Host Disease. Directly after, the immunosuppressive virus is administered.
  • Figure 1 is a graph of the effect of an infection with HIV (the HTLV- ⁇ IIIb strain) on the development of the Graft-versus-Host Disease in CBA/N mice.
  • HIV the HTLV- ⁇ IIIb strain
  • the number of mice that are still alive is plotted against the number of days elapsed after the transplantation with human lymphocytes and the injection w ' ith HIV.
  • mice with the XID mutation were used, belonging to the CBA/N mouse strain which has a deficient production of antibody against some body-foreign substances, including human blood cells. These mice were treated with a dose of 9 Gy gamma radiation so as to destroy their own immune system. This could only be done from the twenty-first day after birth. Postponement of this treatment is not desirable, because, as the mouse grows older, and hence bigger, more human immune cells are required to induce the Graft-versus-Host Disease. Given the above-mentioned high irradiation dose, the mice required a supporting bone marrow transplantation. To that end, the mice had 500,000 type-specific bone marrow cells administered into a vein.
  • mice 32 CBA/N mice were weaned on day 21 after birth and subsequently irradiated with 9 Gy gamma on a Cesium 137 source with a dosing rate of 0.87 Gy per minute. Eight mice served as controls for the irradiation. They received no further treatment and died from the consequences of the irradiation around day 12 after the treatment . The other mice received a supporting bone marrow transplantation four hours after the irradiation. Eight mice served as controls for this transplantation and were still alive after a month of observation. The other 16 mice had 200 million human PBL injected into the abdominal cavity five hours after irradiation.
  • mice Six hours after irradiation, eight mice had 0.25 ml sterile culture fluid administered into the abdominal cavity (see diagram) . To the other eight mice, 0.25 ml HIV III-B culture fluid was administered, likewise into the abdominal cavity. This volume contains approximately 25,000 infectious Units. Of the mice that had the supernatant with the HIV III-B injected, two were still alive after a month and the others lived two or three days longer than the respective mice that did not have the virus injected (see Figure 1) . The difference in survival was significant.
  • a further advantage of the method is that, in addition to a test result in the form of yes/no prevention of Graft- versus-Host Disease, also a quantitative assessment can be obtained about the extent of antiviral effect of the agent to be investigated by measuring the number of transplanted human lymphocytes that are infected with HIV. That assessment can be realized with a fluorescence cytometry method (FCM) , which is set for the detection in cells that contain HIV, see the standard operating procedure (SOP) described hereinbelow.
  • FCM fluorescence cytometry method
  • PBS phosphate-buffered salt solution of the composition: 8.2 g/1 NaCl 1.9 g/1 Na2HP0 .2H 2 0 0.3 g/1 aH 2 P0 4 .2H 2 0 pH 7.4 NPBI, cat. no. D1011

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
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  • Hematology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

Agents antiviraux dirigés contre un virus immunosuppresseur et éprouvés sur des animaux d'expérience auxquels on a greffé les cellules d'un système immunitaire donneur, ces cellules pouvant être infectées par le virus immunosuppresseur, et étant infectés par celui-ci. Les animaux d'expérience utilisés ont un système immunitaire perturbé à un degré tel que, lors de la greffe des cellules du donneur, ils sont susceptibles de connaître une réaction mortelle du greffon contre l'hôte. On leur a greffé une dose suffisamment importante de cellules du donneur pour que la réaction mortelle du greffon contre l'hôte se produise chez tous les animaux d'expérience à l'exception de ceux qui sont également infectés par le virus immunosuppresseur.
PCT/NL1992/000226 1991-12-18 1992-12-15 Procede de mise a l'epreuve d'agents antiviraux et animaux d'experience utilises dans ce procede WO1993012252A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL9102122A NL9102122A (nl) 1991-12-18 1991-12-18 Werkwijze voor het testen van antivirale middelen; daarvoor bruikbare proefdieren.
NL9102122 1991-12-18

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WO1993012252A1 true WO1993012252A1 (fr) 1993-06-24

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PCT/NL1992/000226 WO1993012252A1 (fr) 1991-12-18 1992-12-15 Procede de mise a l'epreuve d'agents antiviraux et animaux d'experience utilises dans ce procede

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AU (1) AU3268693A (fr)
NL (1) NL9102122A (fr)
WO (1) WO1993012252A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994005779A1 (fr) * 1992-09-09 1994-03-17 O'brien, Caroline, Jane Procede de verification de reponse immune individuelle a un anticorps a l'aide d'une souris a scid
WO1995021246A3 (fr) * 1994-02-02 1995-09-14 Victor Smit Procede de production de virus et de cellules resistantes aux virus en vue de la mise a l'epreuve de substances antivirales a l'aide d'animaux de laboratoire
WO1995021243A3 (fr) * 1994-02-02 1995-09-14 Victor Smit Procede de production in vivo de produits et cellules immunologiques, et de mise a l'epreuve in vivo de substances immunomodulatrices
FR2729973A1 (fr) * 1995-01-26 1996-08-02 Centre Nat Rech Scient Procede de criblage de composes anti-viraux

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0322240A2 (fr) * 1987-12-23 1989-06-28 The Board Of Trustees Of The Leland Stanford Junior University Mammifères immunocompromis chimériques et leur utilisation
WO1990003177A1 (fr) * 1988-09-22 1990-04-05 The United States Of America, Represented By The Secretary, United States Department Of Commerce Modele animal pour le diagnostic et le test de vaccins ou d'agents therapeutiques contre le sida

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0322240A2 (fr) * 1987-12-23 1989-06-28 The Board Of Trustees Of The Leland Stanford Junior University Mammifères immunocompromis chimériques et leur utilisation
WO1990003177A1 (fr) * 1988-09-22 1990-04-05 The United States Of America, Represented By The Secretary, United States Department Of Commerce Modele animal pour le diagnostic et le test de vaccins ou d'agents therapeutiques contre le sida

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994005779A1 (fr) * 1992-09-09 1994-03-17 O'brien, Caroline, Jane Procede de verification de reponse immune individuelle a un anticorps a l'aide d'une souris a scid
WO1995021246A3 (fr) * 1994-02-02 1995-09-14 Victor Smit Procede de production de virus et de cellules resistantes aux virus en vue de la mise a l'epreuve de substances antivirales a l'aide d'animaux de laboratoire
WO1995021243A3 (fr) * 1994-02-02 1995-09-14 Victor Smit Procede de production in vivo de produits et cellules immunologiques, et de mise a l'epreuve in vivo de substances immunomodulatrices
FR2729973A1 (fr) * 1995-01-26 1996-08-02 Centre Nat Rech Scient Procede de criblage de composes anti-viraux

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Publication number Publication date
NL9102122A (nl) 1993-07-16
AU3268693A (en) 1993-07-19

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