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WO1999037333A1 - Procede pour eliminer et/ou inactiver des virus - Google Patents

Procede pour eliminer et/ou inactiver des virus Download PDF

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Publication number
WO1999037333A1
WO1999037333A1 PCT/DE1999/000161 DE9900161W WO9937333A1 WO 1999037333 A1 WO1999037333 A1 WO 1999037333A1 DE 9900161 W DE9900161 W DE 9900161W WO 9937333 A1 WO9937333 A1 WO 9937333A1
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WO
WIPO (PCT)
Prior art keywords
particles
viruses
fluid
blood
cells
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.)
Ceased
Application number
PCT/DE1999/000161
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German (de)
English (en)
Inventor
Thomas Schneider
Martin Zeiler
Dietmar Oechsele
Klaus Feifel
Manfred Goos
Ulrich Hengge
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.)
Schenk Filterbau GmbH
Original Assignee
Schenk Filterbau GmbH
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 Schenk Filterbau GmbH filed Critical Schenk Filterbau GmbH
Priority to AU32461/99A priority Critical patent/AU3246199A/en
Publication of WO1999037333A1 publication Critical patent/WO1999037333A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/02—Blood transfusion apparatus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3482—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate by filtrating the filtrate using another cross-flow filter, e.g. a membrane filter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3486—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3693—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits using separation based on different densities of components, e.g. centrifuging
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2205/00—General characteristics of the apparatus
    • A61M2205/75—General characteristics of the apparatus with filters
    • A61M2205/7509—General characteristics of the apparatus with filters for virus

Definitions

  • the present invention relates to a method for removing and / or inactivating viruses, virus-free blood components, a device for removing and / or inactivating viruses from / in a fluid comprising particles and viruses, a method for reducing the vitreiter in body fluids of an animal Organism and an agent for increasing the content of hematopoietic stem and precursor cells in peripheral blood and its use.
  • Viral diseases continue to pose a major threat to human health. In addition to the periodic flu waves, a number of other and often chronic viral diseases have recently been observed. Examples include AIDS, hepatitis C, Ebola, Hanta and cytomegaly infections noted.
  • Virus-free blood plasma or other liquids can be achieved by suitable filtration steps. It has been found that using iodine complexly bound to filter material in the form of crospovidone iodine, as described for example in DE 43 43 236 AI, viruses can be reliably inactivated.
  • the object of the present invention is to provide methods and devices which ensure that in a fluid comprising viruses and particles, the viruses are removed from the fluid or inactivated in the fluid.
  • Another object of the invention is to provide a method for reducing the virus titer in body fluids of an animal organism.
  • the object is achieved by a method for removing and / or inactivating viruses from / in a fluid comprising virus and particles, in which the fluid comprises a cross-linked crospovidone iodine and / or crospovidone hydrogen peroxide matrix and particles embedded therein Filter is guided.
  • the object is achieved by a method for removing and / or inactivating viruses from / in a fluid comprising viruses and particles, in which the particles are separated from the fluid and the particle-free fluid at least once via a matrix and particles of crosslinked embedded therein Crospovidone iodine and / or - 4 -
  • Crospovidone hydrogen peroxide comprehensive filter is performed.
  • virus-free blood components including preparations comprising them, which can be produced by one of the methods according to the invention.
  • the object is achieved by a device for removing and / or inactivating viruses from / in a fluid comprising particles and viruses, the device comprising at least one device for separating the particles from the fluid and a filter device which has at least one matrix and embedded therein particles of cross-linked crospovidone iodine and / or crospovidone hydrogen oxide filter.
  • the object is achieved by using the aforementioned device according to the invention for reducing the virus titer in body fluids of an animal organism and by using it in the methods according to the invention.
  • the object is achieved according to the invention by a means for increasing the content of hematopoietic stem and precursor in peripheral blood, which contains two components intended for sequential administration, the first component comprising an antiviral compound and the second component a combination of comprises at least one antiviral compound and a hematopoietically active compound, and by the use of the agent in the process according to the invention.
  • the object is also achieved by a method for reducing the virus titer in body fluids of an animal organism, in which one of the aforementioned methods according to the invention is used.
  • the particles are cellular blood components.
  • the cellular blood components are selected from the group comprising hematopoietic stem and progenitor cells, erythrocytes, platelets, lymphocytes, monocytes / macrophages and combinations thereof.
  • the cellular components are differentiated cells and / or stem cells.
  • the cellular constituents comprise CD34-positive hematopoietic stem cells.
  • the fluid is blood.
  • viruses are selected from the group consisting of hepatitis B, hepatitis C, cytomalaly, Epstein-Barr viruses and human herpes virus, in particular human herpes virus 6, 7 and 8 and human immunodeficiency virus includes.
  • the invention further proposes that before the particles are passed over the filter, they are fractionated based on their physical, chemical, phenotypic and / or genotypic characteristics. Furthermore, it can be provided that after the particles have been passed over the filter, they are fractionated based on their physical, chemical, phenotypic and / or genotypic characteristics.
  • the particles which can be obtained by one of the methods according to the invention are blood cells and that these are stimulated to multiply and / or differentiate.
  • blood cells are CD34-positive stem cells.
  • the fluid comprising viruses and particles is obtained from a mammalian host organism, and in particular in the mammalian host organism, before the fluid comprising the viruses and particles is withdrawn, the number of hematopoietic stem and precursor cells present in peripheral blood is increased.
  • the number of hematopoietic stem cells present in peripheral blood is increased by administration to the mammalian host organism of an agent for increasing the content of hematopoietic stem and precursor cells in peripheral blood.
  • the mammalian host organism is a human organism.
  • the Particles and / or the fluid are passed several times over the filter.
  • the fluid obtained after the passage through the filter and / or the particles obtained after the passage through the filter are fed to an animal organism.
  • CD34-positive stem cells are supplied to the animal organism.
  • the immune system of the animal organism is destroyed before the supply of cellular blood components to an animal organism.
  • the method according to the invention proposes that the method for reducing the virus titer in body fluids of an animal organism is repeated at intervals.
  • the method according to the invention is particularly preferred if the body fluid is blood.
  • the animal organism is a mammal and in particular a human.
  • An embodiment in which the advantages inherent in the method according to the invention are particularly effective is that the animal organism is chronically ill with a viral disease.
  • the antiviral compound is selected from the group Retrovir ®, ganciclovir and other comprises.
  • the hematopoietically active compound is selected from the group of cytokines, which includes granulocyte colony-stimulating factor G-CSF and others.
  • the first component is intended for administration over a period of 10 to 28 days.
  • the second component is intended for administration over a period of 5 to 10 days.
  • a total of 15 to 38 days is provided for taking the first and second components.
  • the two components are spatially separated and assembled in a packaging unit, the administration units forming the components being able to be removed individually.
  • the administration units are daily units.
  • Retrovir the first component and granulocyte colo- - 9 -
  • the never-stimulating factor G-CSF together with Retrovir is the second component
  • the first component is 9 daily units of Retrovir of 2 g each for oral administration
  • the second component is 5 daily units each consisting of 2 g of Retrovir for oral administration and 5-20 ⁇ g / kg Body weight granulocyte colony stimulating factor G-CSF is.
  • the invention proposes the use of the agent according to the invention in the methods according to the invention.
  • the method according to the invention thus provides that viruses can be removed from a fluid comprising viruses and particles or the viruses contained in a fluid comprising viruses and particles can be inactivated. It is possible for both processes to run simultaneously, one after the other, or even individually.
  • the invention is based on the surprising finding that it is possible to remove or inactivate the viruses by contacting crospovidone iodine and / or crospovidone hydrogen peroxide with a fluid comprising viruses and particles.
  • Crospovidone iodine or crospovidone hydrogen peroxide are understood herein to mean iodine complexes or hydrogen peroxide complexes based on crosslinked PVP (polyvinylpyrrolidone). Crospovidone iodine or crospovidone hydrogen peroxide is present in the filters used in a filter matrix in particulate form. Such filters, which are advantageously designed as depth filters, are in DE 43 43 226 AI and WO 95/16511 - 10 -
  • Said filters can comprise fiber materials as a filter matrix and the content of the crospovidones built into the matrix can be 0.5 to 75% by weight, based on the total weight (dry).
  • viruses are removed or inactivated, but not damage to the particles themselves, especially if the particles are cellular blood components and especially lymphocytes or hematopoietic stem and precursor cells.
  • the filter material can be provided with a sufficient pore size, which leads to an enlargement of the filter surface, which is available for contacting the fluid comprising viruses and particles.
  • fluid is understood in particular to be blood, but also lymph fluid.
  • Blood is also to be understood here to mean whole blood, blood mixed with anticoagulants or otherwise treated, blood plasma, blood plasma fractions, expanded whole blood and blood plasma and the like.
  • blood also includes the blood to be observed under the various pathological conditions, such pathological conditions not only the cellular component (s) of the blood but also its soluble, i.e. can include plasmatic component (s).
  • aqueous fluid should also - 11 -
  • whole blood can then also be fed directly to a treatment with a filter comprising particulate crospovidone iodine and / or crospovidone hydrogen, thus enabling reliable removal and / or inactivation of viruses.
  • a filter comprising particulate crospovidone iodine and / or crospovidone hydrogen, thus enabling reliable removal and / or inactivation of viruses.
  • the methods according to the invention open up new possibilities in the processing of whole blood, blood plasma containing particles, cellular blood components, blood plasma fractions and the like.
  • the fluid is conditioned via the filter in a suitable manner for the pass step.
  • suitable anticoagulants as are known to the person skilled in the art, for example by adding ACD or heparin.
  • viruses to be removed or inactivated from / in a fluid comprising particles and viruses, the particles being separated from the fluid first and then the particles, optionally suspended in a fluid, on the one hand and the particle-free one Fluid, on the other hand, can be further processed separately in a suitable manner, wherein further treatment can also consist in non-treatment.
  • at least one of the two fractions mentioned is treated with the crosslinked particulate crospovidone iodine and / or crospovidone hydrogen peroxide to remove and / or inactivate the viruses, preferably the particle-free fluid, for example the plasma free of cellular blood components.
  • Particle and fluid are typically separated by differential centrifugation.
  • the particles are blood cells
  • devices such as apheresis centrifuges and the like are typically used, particularly since they are able to remove and possibly enrich large quantities of nucleated cells from the peripheral blood.
  • the filter comprising particulate crospovidone iodine and / or crospovidone hydrogen peroxide
  • Virus-free is intended to include the physical absence of viruses as well as the absence of biologically active viruses, i.e. that, for example, the virus capsid or parts thereof are still present even after the methods according to the invention have been carried out, but the virus is no longer biologically active. Furthermore, the term also includes the fact that no virus or viral activity is no longer detectable, or at least below a defined titer or level of biological, i.e. viral, activity is reduced.
  • the methods according to the invention are not limited to fluid comprising particles and viruses obtained directly from an animal organism, but can of course also be applied to made-up (blood) preparations, so that the fluid generally has a liquid phase shows how, for example, buffer solutions and the particles can represent only a single cell type - 13 -
  • CD34-positive hematopoietic stem and progenitor cells are of great importance as cellular components because they are able to largely build up a functional immune system as a result of differentiation and multiplication. Accordingly, in the therapy of lymphotropic viruses after destruction of the differentiated cells in the patient's body carrying the viruses, the patient's immune system can be rebuilt by implantation of corresponding hematopoietic stem cells or derivatives derived therefrom. In the case of AIDS in particular, the special stem and progenitor cells are of central importance since they do not have the CD4 molecule responsible for a specific interaction of the HI virus with the cell surface.
  • the fluid is blood in many advantageous embodiments of the invention, it is not so limited. This can also involve blood which has already been processed differently or products obtained therefrom or corresponding preparations which still have a virus content.
  • the methods according to the invention can also be used for a subsequent treatment of such preparations if, for example, it has been proven after processing that the preparations are virus-positive.
  • viruses can be applied to all virus forms, ie DNA as well as RNA viruses, enveloped and non-enveloped viruses, virions, prions and similar biological systems.
  • viruses mentioned herein also include their various vario- and seroty- - 14 -
  • the particles contained in a fluid are fractionated before or after passage through a filter containing particulate crospovidone iodine or crospovidone hydrogen peroxide, a combination of both steps of course can take place within the scope of the invention.
  • the fractionation can be based on the most diverse characteristics of the particles.
  • Physical features are to be understood here, inter alia, to mean the size, surface charge and shape, chemical features, inter alia, the composition of the cytoplasmic membrane or layers based thereon and also associated or connected molecules which occur both naturally, on a pathological process or on a technical one Processing or modification step can be based.
  • Phenotypic characteristics are also to be understood in the broadest sense as the physical and chemical characteristics mentioned, as well as those that are based on the specific biological and biochemical equipment of the particles, especially when it comes to cellular blood components. These include various surface markers, enzymatic activities or detection systems coupled to the surface markers.
  • Phenotypic features are also intended to include immunological features herein. With regard to the genotypic characteristics, those are to be understood that are based on the genetic makeup of the respective cells. In particular, this includes the detection and labeling of certain cell populations using - 15 -
  • the lymph cells obtained using the methods described herein can be propagated or differentiated ex vivo using known culture and differentiation techniques. Certain cell populations, which are therapeutically advantageous, can thus be selectively expanded compared to the normal distribution in the blood. This is particularly advantageous when it comes to hematopoietic stem and progenitor cells, such as CD34-positive hematopoietic stem cells.
  • CD34-positive hematopoietic stem cells are of central importance, especially in AIDS, because due to the lymphotrophic behavior of the virus, a reconstitution of the immune system is ultimately only possible on the basis of non-infected cells, as the HI virus itself as a result of the reproductive cycle would be excreted further if the viruses were removed from the plasma.
  • the CD4 molecule which is not expressed on the surface of CD34-positive stem cells, is essentially responsible for the uptake of HI viruses. Accordingly, these hematopoietic stem cells, increased and possibly differentiated, can form the basis for a reconstitution of a patient's immune system after the virus has been removed from the body, for example using the methods according to the invention, and the lymphatic cells carrying it have also been removed or destroyed .
  • Hj CD s > ⁇ ra P ra PJ 0 ⁇ CD ⁇ ⁇ P ra P ⁇ tr ⁇ ⁇ P ⁇ n ⁇ - LQ ⁇ ⁇ to ⁇ d Hj P ⁇ Hj Hj rr PJ ⁇ tr P ) ⁇ Hj
  • H ⁇ rt ⁇ LQ d 3 d P ⁇ CD 3 d P )
  • P ⁇ p d PJ ra Di ⁇ ⁇ - P Di ⁇ - O 3 ⁇ HS ⁇ tr d rt ⁇ - ⁇ d ra P ⁇ Hi PPPP ) 3 o ⁇ rt rt ⁇ HJ Hi Hj ra> d P Di P rart P 3 Di LQ ⁇ - ⁇ ! W ⁇ ⁇ ⁇ Hj ⁇ 1
  • Hi PJ_ PJ ⁇ ⁇ - ⁇ py 1 ⁇ P ⁇ - 0 Hj Hj ⁇ - ⁇ - ra Di ⁇ ⁇ ⁇ P ⁇ i
  • CD LQ d LQ P ⁇ - ⁇ - ⁇ ⁇ ⁇ rt N LQ tr r) tr ⁇
  • the plasma portion and the particulate portion are separated, the blood plasma being subjected to filtration using a filter comprising crospovidone iodine or hydrogen and the viruses contained therein being removed or inactivated, and the particulate fraction optionally being further treated separately.
  • the further treatment of the particulate fraction can be designed in such a way that individual cell populations are selectively removed on the basis of their phenotypic, chemical, physical and biological properties and then expanded or differentiated. Subsequently, parts of these cells or cell populations and / or parts of the plasma, possibly also both components completely, can be returned to the animal, i.e. attributed in particular to the human organism.
  • the proportion of stem cells can be increased using the agent described here and thus the yield of stem cells and / or differentiated can be specifically increased in the first steps of the method according to the invention outlined above.
  • a certain amount of blood is withdrawn from the patient suffering from a viral disease and treated as described above before this, or parts thereof, are returned to him.
  • Connection and / or inactivation of viruses from / in a fluid comprising particles and viruses is or will be connected, in which the process described above is carried out and the blood treated in this way, optionally in the form of components or parts, is immediately returned to the body.
  • the method according to the invention for reducing the virus titer has to be repeated at intervals, the frequency and extent being essentially influenced by the course of the disease and by the type of viral disease.
  • suction can be exerted from the plasmatic compartment onto the non-plasmatic compartments of the patient suffering from a viral disease, with the result that there is a therapeutically positive, increased flushing out of the viruses from the non-plasmatic compartment and A new equilibrium between the virus titer in the plasmatic and in the non-plasmatic compartment comes at a lower level.
  • the patient has hematopoietic stem cells
  • this also applies very particularly to the method according to the invention for reducing the virus titer, it being possible in a further sub-step in this regard that the immune system of the animal organism is destroyed or conditioned before the virus-free blood components are administered in such a way that it starts from the supplied hematopoietic stem and progenitor cells, it is possible to rebuild the immune system, or parts thereof, without this being impaired by viral activities.
  • FIG. 1 represents a schematic representation of the device for removing and / or inactivating viruses from / in a fluid comprising particles and viruses, from which further advantages and features of the underlying invention result.
  • HIV human immunodeficiency virus
  • ren components of the blood separated In the present case, an apheresis centrifuge was used, in which the blood cell separation is based on differential centrifugation and which allows large amounts of nucleated cells from the peripheral blood to be enriched.
  • Suitable devices are, for example, Fenwal CS 3000 (company Baxter) or Haemonetics model V50 (company Haemonetics) or COBE Spectra (company COBE). Such devices allow the processing of 10 to 25 1 blood volume in 1 to 3 hours.
  • the blood introduced into the apheresis device was treated with anticoagulants to prevent the blood from coagulating during apheresis. After the apheresis is complete, the plasmatic components and the cellular components can be processed further separately.
  • boundary conditions of the centrifugation apparatus used for cell extraction must be individually adapted to the respective blood batch and depend not only on the cell population to be obtained but also on the respective rheological properties of the blood batch to be specifically treated. However, the setting of the said boundary conditions is daily practice for the person skilled in the art.
  • the blood plasma obtained from the apheresis is then fed to a treatment with a depth filter, which is shown schematically in FIG. 1.
  • plasma is passed through a biocidal filter layer or alternatively through a biocidal filter - 23 -
  • the filter layers typically consist of suitable carrier materials such as cellulose or polyolefins or other suitable substances or a mixture thereof as well as auxiliaries and additives.
  • suitable carrier materials such as cellulose or polyolefins or other suitable substances or a mixture thereof as well as auxiliaries and additives.
  • the PVPP iodine which acts as an active agent, is inertly incorporated into the carrier matrix.
  • the viruses are inactivated when they pass through the filter system, while the blood plasma passes through the filter without damage.
  • the functional diagram of a typical biocidal filtration is shown in FIG. 1 using the example of a filtration with two layers.
  • virus-containing plasma enters the filter configured with layers 2 and 3 at the entry point 1 before it emerges again at the exit point 4 as virus-inactivated plasma.
  • the thickness and diameter of the layers can be adapted to the particular application, so that the capacity required for the plasma volume to be filtered is available.
  • the layers are suitably arranged in a holder and can be fixed in order to enable an optimal filtration process.
  • This functional part can then be poured into a sheathing made of a suitable plastic that is usually used for the storage or conveyance of blood plasma, such as, for example, used in bags for storing blood plasma or in dialysis tubes.
  • the sheathing is additionally provided with connecting hoses and at the end with a suitable coupling for connection to cannulas, collecting containers etc.
  • PJ Hj PJ ⁇ ⁇ Hj 3 d PJ X rt P - y ⁇ - P): • ra P ⁇ - rt ü f ⁇ ISI UI ⁇ - rt ⁇ Hj Hi ⁇ - 3 ⁇ - ⁇ rt ⁇ D. ⁇ ⁇ ⁇ CD ⁇ N ffi Hj CD tr d UI
  • Acetic acid washed before cell aggregates were removed through a sterile 30 ⁇ m nylon filter. The cells were then resuspended in 300 ⁇ l of this buffer solution per 10 8 white blood cells and subjected to treatment with the filter apparatus, the layer thickness and the filter base area depending on the cell type to be filtered (lymphocytes: approximate diameter 9 to 12 ⁇ m and monocytes / macrophages: approximate diameter 12 to 16 ⁇ m) was selected.
  • HIV-DNA was detected using conserved gag primers using a nested PCR reaction (nested PCR).
  • nested PCR The HCH2 cell line, which contains one HIV genome per cell, served as a positive control. The following primers were used:
  • 5 'gag687 AGAACCAAGGGGAAGTGACATAGCA and 3' gag992: TTACAATCTGGGTTCGCATTTTGG and 3 'gagl047: TGCTGTCATCATTTCTTCTAGTGT and 5' LTR768: GCGGAGGCTAGAAGGAGAGAG.
  • the 5'LTR768 and 3'gagl047 were used for the primary amplification under the following reaction conditions: 2 minutes at 94 ° C. (1 cycle), 50 ° C. for 2 minutes and 72 ° C. for 2 minutes (per cycle). Then 39 cycles of 1 minute at 94 ° C, 1 minute at 50 ° C and 1 minute at 72 ° C were connected. An amplificate of 1068 base pairs was found.
  • the secondary reaction was carried out with 10 ⁇ l of the DNA (template) of the first reaction formed using the primers 5'gag687 and 3'gag992. The individual cycles were identical to those described above, the total number of cycles was 30.
  • the analysis of the DNA fragments was carried out in a 1% agarose gel by means of ethyl bromide staining.
  • Sections of the HIV-1 reverse transcriptase gene were amplified by mononuclear cells prior to treatment in cell culture and at different times during the culture. - 27 -
  • RT-PCR was used using the following primers: 5'2536: CACTT-TAAATTTTCCCAT and 3'3388: ACATAATTGCCTTACTTTAATC.
  • a magnesium concentration of 1.25 mM and 50 cycles under the above reaction conditions was used.
  • An 825 base pair DNA piece was amplified and sequenced using the T7 polymerase method in order to detect possible mutations in the reverse transcriptase.
  • HIV patients were exposed to a conditioning regimen consisting of 2 g of Retrovir per os for 14 days before the apheresis treatment and 10 ⁇ g / kg body weight of granulocyte colony-stimulating factor G -CSF exists for the last 5 days before the apheresis treatment.
  • a conditioning regimen consisting of 2 g of Retrovir per os for 14 days before the apheresis treatment and 10 ⁇ g / kg body weight of granulocyte colony-stimulating factor G -CSF exists for the last 5 days before the apheresis treatment.
  • the HIV-positive blood obtained in this way was separated into blood plasma and cellular blood components, as described in Example 1, and the viruses were removed or inactivated. - 28 -
  • the CD34-positive stem cells were then selectively isolated from the mixture of white blood cells of the peripheral blood obtained by specific antigen-antibody-mediated immune reactions on the surface of the cells.
  • a commercially available monoclonal antibody eg clone UBEND / 10 or HP10A-2
  • clone UBEND / 10 or HP10A-2 was used for this purpose, which recognizes an epitope of the 115 kDa transmembrane glycoprotein (CD34). This is expressed on 2-4% of the cells present in the bone marrow and has been identified as the earliest marker of the hematopoietic stem cells. This procedure is also known as positive selection.
  • the population of white blood cells (shown below for ⁇ 2 ⁇ 10 9 mononuclear cells) was mixed with 100 ⁇ l of a block reagent (eg F c fragment of the immunoglobulin G; concentration 1 mg / ml).
  • a block reagent eg F c fragment of the immunoglobulin G; concentration 1 mg / ml.
  • F c fragment of the immunoglobulin G concentration 1 mg / ml
  • the cells were placed on the column in a concentration of 5 ⁇ 10 8 white blood cells and separated from the run by means of pregnancy.
  • the column was then washed four times with 500 ⁇ l PBS with 0.5% human serum albumin and 5 mM EDTA.
  • the column was then removed from the magnetic separator and placed in a small sterile tube under a sterile workbench and eluted with 1 ml of the above buffer. This process was repeated again with another column prepared - 29 -
  • the cell filtrate of the first run which contained all leukocytes except the CD3-positive stem cells, can in principle be (re) infused into the patient.
  • the eluate obtained after the treatment with the CD34 antibody column was then incubated in the sense of a negative selection with a monoclonal antibody (GK1.5) against the CD4 antigen, which was coupled with paramagnetic microbeads and via the magnetic column of CD4 positive cells separated. This was necessary to remove all of the early progenitor cells bearing the CD4 antigen from the cell mixture, since CD4 acts as the main receptor molecule for HIV.
  • the purified CD34-positive stem cells thus obtained were then centrifuged for 5 minutes at 1500 rpm and in RPMI 1640 / IMDM 10% (250 ml ISCOVE's modified Dulbecco's medium; 250 ml RPMI 1640; 50 ml 10% human AB serum ; 5 ml of an IM sodium pyruvate solution, 5 ml of a 2 mM L-glutamic acid solution; 5 ml of a 100 U / ml penicillin solution and 0.1 mg / ml streptomycin; 500 ⁇ l of a 5 x 10 5 molar solution of 2-mercaptoethanol) resuspended. These cells were propagated in the cell culture medium mentioned above with the addition of stem cell factor, IL-1, IL-3 and IL-6 in plastic culture bottles with 0.3% agar.
  • the preparation comprising CD34-positive hematopoietic stem cells obtained in this way was examined for the effectiveness of the removal or inactivation of the HI viruses by means of the HIV detection tests listed in Example 1 and no HIV viruses could be detected anymore. - 30 -
  • CD34-positive cells After about 2 weeks of cell culture, the number of CD34-positive cells used could be increased by a factor of 30 to 50 (typically to 5 x 10 - 5 x 10 10 ). After this period, an aliquot of the cells was subjected to a comprehensive FACS analysis (fluorescence-activated cell sorting). For this purpose, 2 ⁇ 10 5 CD34-positive cells with the corresponding antibody, which was conjugated with fluorescein isothiocyanate (FITC) or phyco-erythrin (PE), were used. Antibodies against were used - 31 -
  • CD5 the following cellular antigens of hematopoietic differentiation: CD5, CD7, CD10, CD19, CD20, CD13, CD14, CD15, CD16, CD33, CD38, CD45RA, CD45R0, CD71 and HLADR.
  • the corresponding isotypic controls were carried out as a negative control. After 20 minutes incubation at 4 ° C, the cells were washed with PBS 1% bovine serum albumin and 50,000 cell events were evaluated.
  • CD34-positive progenitor studies were carried out.
  • 3000 or 10,000 CD34-positive cells / ml were incubated in ISCOVE's methyl cellulose medium with 2 IU erythropoietin / ml and 50 ⁇ l PHA-stimulated human conditioned medium with 1% fetal calf serum. 250 ⁇ l each of this solution were incubated in 24 perforated plates for 14 days at 37 ° C. and 5% CO 2 .
  • CFU-E The colony forming units of the erythroid line
  • CFU-GM the granulocyte / macrophages
  • the lymphocyte function was tested in vitro using functional tests of the hematopoietic CD34-positive stem cells obtained from HIV-containing blood. For this purpose, 10 cells / ml in 2 ml of RIO medium in 24 perforated plates were stimulated with either anti-CD3 (clone 12F6) in the concentration of 0.1 ⁇ g / ml or PHA (5.0 ⁇ g / ml). After 6-48 hours of incubation, the supernatant for cytokines was examined in the interferon- ⁇ and interleukin 4 ELISA according to the manufacturer's instructions.
  • Lymphocyte proliferation was performed at least 10 days after the last stimulation.
  • allogeneic mononuclear cells of the peripheral blood of HIV-negative donors irradiated with 30 G ⁇ were used in RPMI-1640 medium with 10% human AB serum in a cell density of 10 6 cells / ml.
  • 0.1 ml was added to the wells of a 96-well plate with a round bottom and 0.1 ml of cultured lymphocytes to be tested (10 6 cells / ml) or with medium alone.
  • PHA 10 ⁇ g / ml
  • the cells were incubated for 5 days and incubated with radiolabelled thymidine (1 ⁇ Ci / well) overnight.
  • the stimulation indices represented the ratio of counts in stimulated cultures divided by counts in unstimulated cultures. - 33 -
  • B-lymphocyte cell lines were established from patient blood by immortalization with the Epstein-Barr virus. These were infected with recombinant vaccinia viruses that express either the E. coli Lac Z gene or the HIV genes (gag, pol and env) of HIV-1 IIIB. To activate the natural killer function or the lymphokine To investigate killer function, K562 and Daudi cells were used as target cells. The target cells were labeled with sodium chromate, which contained radioactive chromium-51.
  • the effector cells were either autologous mononuclear cells of the peripheral blood or the cultured and propagated lymphocytes (especially CD8 lymphocytes) and were added to the microtiter plates in a ratio of 50: 1, 25: 1, 12.5: 1 and 6.25 : 1 added.
  • the controls included medium and target cells alone to determine the spontaneous release of the radioactive chromium without specific lysis.
  • the maximum lysis was determined by treating the target cells with detergents. After 4-5 hours of incubation at 37 ° C., the cell supernatants were harvested and the number of counts was determined in a ⁇ counter.
  • CD34-positive stem cells obtained in this way have not been damaged in terms of their differentiability as a result of the method according to the invention - 34 -

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Abstract

L'invention concerne un procédé pour éliminer et/ou inactiver des virus hors d'un/dans un fluide comprenant des virus et des particules. Selon ce procédé, le fluide passe à travers un filtre comprenant une matrice et des particules de crospovidone-iode réticulé et /ou de crospovidone-peroxyde d'hydrogène, qui sont enrobées dans ladite matrice.
PCT/DE1999/000161 1998-01-22 1999-01-22 Procede pour eliminer et/ou inactiver des virus Ceased WO1999037333A1 (fr)

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DE19802375A DE19802375A1 (de) 1998-01-22 1998-01-22 Verfahren zur Entfernung und/oder Inaktivierung von Viren

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0370656A1 (fr) * 1988-11-09 1990-05-30 Schering Corporation Traitement de la suppression médullaire associée avec le SIDA
WO1995016511A1 (fr) * 1993-12-17 1995-06-22 Basf Aktiengesellschaft Filtre a lit profond pour l'extermination de micro-organismes et l'inactivation de virus, et son utilisation
US5609864A (en) * 1990-09-04 1997-03-11 Shanbrom; Edward Preservation of blood, tissues and biological fluids
WO1997021488A1 (fr) * 1995-12-11 1997-06-19 Dendreon Corporation Composition pour separation de cellules, trousse et procede associes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0370656A1 (fr) * 1988-11-09 1990-05-30 Schering Corporation Traitement de la suppression médullaire associée avec le SIDA
US5609864A (en) * 1990-09-04 1997-03-11 Shanbrom; Edward Preservation of blood, tissues and biological fluids
WO1995016511A1 (fr) * 1993-12-17 1995-06-22 Basf Aktiengesellschaft Filtre a lit profond pour l'extermination de micro-organismes et l'inactivation de virus, et son utilisation
WO1997021488A1 (fr) * 1995-12-11 1997-06-19 Dendreon Corporation Composition pour separation de cellules, trousse et procede associes

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