Classifications

• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
• • 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
  • G01N33/56966—Animal cells
• • 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
  • G01N33/56966—Animal cells
  • G01N33/56972—White blood cells

Definitions

• T4 lymphocytes specific for the LAV virus their process for obtaining and their application to the production of this virus.
• the invention relates to populations of T4 lymphocytes specific for the LAV virus, to methods for obtaining these populations and to their use for the production of this virus.
• the invention also relates to an in vitro diagnostic method for studying the course of the disease due to infection with the LAV virus and / or discrimination between asymptomatic carriers of anti-LAV antibodies and people with lymphadenopathy syndromes (ALS) or suffering from para-AIDS or AIDS proper, that is to say, the disease known under the full name of "acquired immunodeficiency syndrome".
• ALS lymphadenopathy syndromes
• T4 extends to all cells carrying the T4 phenotype.
• T4 cells will be considered in the context of the present application to extend both to non-permanent human lymphocytes and to cells belonging to permanent lines carrying the above-mentioned T4 phenotype. , such as lines known under the designations CEM, H9, HUT, MOLT, etc.
• the LAV virus, the etiologic agent of the above-mentioned diseases, and various variants of this virus have been completely identified in the European patent application filed on September 14, 1984 in the name of INSTITUT PASTEUR and under the title "Antigens , means and method for the prognosis of diag ⁇ ly phadénopathie and syndrome immunodépres- sion acquired "under the priority of UK patent application No. 83 24800 filed 15 September 1983. for example, we can cite the LAV virus strains deposited in the National Collection of Micro- Cultures organizations of the Pasteur Institute of Paris under the numbers 1-232, 1-240, 1-241 or 1-502. We also know that similar viruses have been isolated in the United States of America.
• LAV virus also called HIV (English abbreviation of "Human Immunodeficiency Virus”) according to the nomenclature rules recently adopted to designate LAV viruses and all viruses with equivalent properties.
• T4 lymphocytes also known as "3Leu cells" form the main target of the virus.
• These lymphocytes are recognizable by monoclonal antibodies, for example those sold by the company Ortho Pharmaceutical Cor ⁇ poration under the designation 0KT4. Nal monoclo ⁇ these antibodies have been described more particularly in European Patent Application No. 18 794 filed on 25 April 1980. It has also been determined that these monoclonal antibodies re ⁇ knew more particularly a molecular weight of 62,000 dalton protein (C. Terhorst et al. Science (1980), 209, 520-521). This protein has been called “T4 antigen” by the above authors. It will hereinafter also be designated as "T4 protein". In what follows, the monoclonal antibodies specific for this protein will be called "anti-T4 antibodies”.
• One of the aims of the present invention is to provide the means which allow this discrimination between people carrying anti-LAV antibodies, but in fact still still asymptomatic and people already sick.
• T4 lymphocytes are infectable by the LAV virus (Science, 1984, 225, p. 59-63, Klatzmann et al). But until now it was not established that T4 lymphocytes not infected with HIV (LAV) in TJ tests of the type which had been carried out by KLAZMANN et al or other researchers had structural elements , at the very least phenotypes allowing them to be distinguished from those which had been • infected. 5
• LAV HIV
• the invention is based on the discovery that in fact T4 lymphocytes susceptible to infection by an HIV actually had characteristics distinct from T4 lymphocytes which were not. Those cells which are infectable will be called T4X cells in the following 0, while the cells also carrying the T4 phenotype, but which are not infectious and are not even recognized by the aforementioned virus, will be called T4Y cells.
• T4X cells in the following 0, while the cells also carrying the T4 phenotype, but which are not infectious and are not even recognized by the aforementioned virus, will be called T4Y cells.
• the invention has also made it possible to show that the T4X cells and the T4Y cells seem to differ from the receptor for the virus. In fact, as will be seen in the remainder of this description, the T4X cells, once separated from the T4Y cells, form populations which can be infected "at 100 V by the virus.
• the method according to the invention for separating T4X cells T4Y which takes advantage of this discovery, is characterized in that it comprises a stage of incubation of an initial population of T4 cells in the presence of inactivated LAV virus, which has been shown to be able to form a complex with the cells.
• T4X A second step in the process consists in separating the T4X cells on which the inactivated virus is fixed, from the T4Y cells which have proved not to form a complex with the inactivated virus.
• an additional stage of incubation in the absence of an inactivated virus is necessary.
• the method according to the invention takes advantage of two additional properties of T4X lymphocytes. They are recognizable both by the virulent LAV virus and by the inactivated forms of this virus.
• T4X cells on which had previously been fixed inactivated LAV virus (including inactivated virus by heat, for example by treatment at a temperature of 56 ° C ⁇ 3 ° C), are maintained in in incubation in the absence of virus, internalization seems to occur, since after a certain time, the T4X cells regain their ability to fix, again, an inactivated or virulent virus.
• a first type of application is concerned with the in vitro diagnosis of the absence or on the contrary of the presence of the infection, in particular when the latter has not manifested itself by apparent clinical symptoms.
• the invention also aims to allow monitoring of the progress of the infection in progress.
• a second type of application is concerned with obtaining populations of substantially homogeneous T4X cells, usable in particular for the production of large quantities of virus or as cell culture - a particularly sensitive test for the study of cytopathogenicity - of new isolates of HIV or, in the case where this cytopathogenicity has been recognized, for the study of the capacity of substances presumed to have - an antiviral effect of inhibiting the capacity of the infectious virus to infect these cell cultures - tests.
• compositions containing HIV antigens in particular from biological samples, for example from human sera
• detection after incubation of the mixture formed during and under conditions suitable for allowing infection, of any infectious activity within the test culture.
• T4X lymphocytes it may not be necessary to carry out the terminal incubation step of T4X lymphocytes in the absence of inactivated virus. This is in particular the case if, to fix the virus on its cellular receptor, a virus which is both inactivated and labeled is used. Thanks to the labeling, it then becomes possible to count, within an initial population of T4 lymphocytes, the relative proportions of T4X cells and T4Y cells that the initial population contained, by means of any appropriate detection device or adapted to the type of marking used.
• a suitable device consists, for example, of a cell sorter if it is desired to separate the T4X from the T4Y.
• a preferred embodiment of this method consists in using the LAV virus labeled with fluorescein and a fluorescence microscope.
• a variant of the process consists in using LAV coupled to an enzyme (peroxidase, ⁇ -galactosidase, alkaline phosphatase, etc.) by a bifunctional agent which does not modify the recognition of the cellular receptor by the viral antigen (glutaraldehyde or diaminobenzidine for example) and revealed the "LAV-T4X" complex by well-known immunoenzymatic techniques such as Elisa, taking advantage of the modification of the radiation absorption properties of a substrate after hydrolysis of the latter by the enzyme coupled to the LAV virus.
• an enzyme peroxidase, ⁇ -galactosidase, alkaline phosphatase, etc.
• a bifunctional agent which does not modify the recognition of the cellular receptor by the viral antigen (glutaraldehyde or diaminobenzidine for example) and revealed the "LAV-T4X" complex by well-known immunoenzymatic techniques such as Elisa, taking advantage of the modification of the radiation absorption properties
• T4X lymphocytes represent in a healthy subject approximately 10 to 15% of the initial population of T4 lymphocytes.
• experience shows that the relative proportion of T4X cells vis-à-vis T4Y cells is modified in people testifying to clinical signs characteristic of ALS or AIDS.
• any other mode of separation of the T4X lymphocytes retaining the inactivated virus can be used, for example when the latter is not itself labeled.
• the lymphocytes on which the inactivated virus is fixed can be detected by anti-LAV antibodies.
• the detection of these antibodies can be direct, in particular if they are labeled with a fluorescent, radioactive or enzyme label, or indirect, for example using labeled anti-immunoglobulins.
• the invention therefore provides a method for in vitro measurement of the T4X / T4Y lymphocyte ratio from any biological sample, in particular from blood originating from a person subjected to the diagnosis. Determining such a ratio makes it possible, even in persons carrying anti-LAV antibodies, to ensure that in principle these persons carrying antibodies are not threatened in the short term by the disease, in other words, do not have ALS or AIDS. On the contrary, if the ratio is changed, it can be assumed that the disease is developing. In such a case, the invention further enables the determination of the stage of development of the disease and, if the measurements are repeated over time, of its evolution, in particular in correlation with the variations in this ratio in the direction a decrease.
• the invention therefore provides means allowing the desired real discrimination between asymptomatic people and already sick people.
• the invention therefore also provides new parameters which can effectively be substituted for the old ones, as regards the recognition of the installation of the disease.
• T4 / T8 lymphocyte ratio could hardly be used for this screening, due to its small relative variation, at least until a very advanced stage of the disease
• monitoring of the T4X / T4Y ratio allows now a close monitoring of the evolution of the disease, therefore the choice of the most effective therapy.
• Yet another method of measurement comprises, for example, the study of the variation in the proportions of lymphocytes which, within an initial population of T4 lymphocytes which have been brought into contact beforehand with inactivated virus, are recognized. by anti-T4 antibodies.
• This test takes advantage of the disturbance in the recognition property possessed by anti-T4 antibodies for T4 lymphocytes, when the latter have previously been exposed to the LAV virus. It has in fact been observed that, if a population of T4 lymphocytes is incubated in the presence of anti-T4 antibodies, these tend to bind to all the T4 cells. The binding of anti-T4 antibodies is reduced when T4 lymphocytes are previously incubated in the presence of LAV virus.
• T4X lymphocytes and T4Y lymphocytes it is possible, by respective counts of T4X lymphocytes and T4Y lymphocytes to determine the T4X / T4Y ratio mentioned above.
• T4X lymphocytes after separation of the T4X lymphocytes, in particular under the conditions indicated below and internalization of the inactivated virus in the T4X lymphocytes, it is possible to assess their relative proportion by bringing them into contact with a preparation of inactivated LAV virus and labeled, for example with fluorescein, then measure the intensity of fluorescence emitted which is correlated to the number of T4X lymphocytes present.
• T4X lymphocytes also "control" the manifestations of certain functions of the T4Y lymphocytes, if we judge by the observed inhibition of the expression of the phenotype T4 and the IL-2 receptor (called "TAC”), of all T4 lymphocytes in people already with advanced AIDS.
• TAC phenotype T4 and the IL-2 receptor
• the invention also applies to the separation of T4X and T4Y lymphocytes, as indicated above.
• Such a method is more particularly interesting to implement on populations of T4 cells belonging to permanent lines (CEM, H9, etc.) in order to obtain permanent T4X cells all infectable by the LAV virus, so that virus production yields can be amplified.
• a preferred embodiment of this method for obtaining T4X cells comprises bringing a population of T4 lymphocytes into contact with a fixed inactivated virus on a solid support. This contacting is then carried out in a heterogeneous medium consisting of a fluid phase containing the initial population of T4 cells and a solid phase retaining the inactivated virus.
• the T4X cells are first fixed on the solid support by the intermediary of the virus, which allows the separation of the two phases. After internalization of the inactivated virus, an at least partial "dropout" of the T4X lymphocytes from the solid support is then observed. These T4X lymphocytes can then be collected.
• the solid support of this process preferably consists of magnetic latex beads or agarose polyacrylamide beads of the type described in French patent application no . 75 36889 published under no . 2 334 106 in the name of 1 ' PASTOR INSTITUTE.
• magnetic latex beads the separation is easily carried out using a magnet.
• a preliminary incubation of the initial population of T4 cells is carried out in the presence of inactivated virus, then these T4 lymphocytes are brought into contact with anti-antibody -T4, or the like, fixed on a suitable insoluble support.
• T4Y cells whose receptors for anti-T4 antibodies are not masked by the inactivated LAV virus, therefore bind to the solid support by means of anti-T4 antibodies, while T4X cells are bound to the LAV virus. inactivated remain in sus ⁇ pension in the fluid.
• T4X cells After separation of the two phases, in particular by simple decantation and internalization of the virus inactivated by T4X cells, a population of T4X cells is obtained, all of which are infectable by the LAV virus.
• This population of T4X cells also has the characteristic that essentially all of its lymphocytes carry a receptor for the virulent or inactivated LAV, a receptor which appears distinct from the receptors for the monoclonal antibodies 0KT4, since these do not distinguish between the T4X and T4Y lymphocytes between them.
• the invention also relates to a process for the production of the LAV virus by infection of populations essentially made up of T4X cells maintained in culture and by recovery of the viruses produced by these cells.
• It also relates to a sensitive method for detecting the infectious nature or not of a biological preparation presumed to contain LAV or HIV antigens, for example from a sample of a biological fluid (for example serum) originating from a carrier of anti-LAV antibodies, this process consisting in bringing this sample into contact with said population of T4X, in carrying out the incubation of the mixture over time and under conditions allowing possible infection of these T4X lymphocytes by a LAV or HIV virus, and to detect possible infection of T4X lymphocytes, for example by the manifestation of reverse transcriptase activity in the event of a positive response and, if necessary, the complete destruction over time of this population of T4X.
• a biological fluid for example serum
• this process can be implemented under similar conditions either with a viral preparation whose character infec ⁇ vis-à-vis human lymphocytes must be determined, or with a preparation of retroviruses whose infectious nature for humans had already been recognized, but this in the presence of active principles of possible drugs, the possible ability of which to inhibit the infectious action of this retrovirus to be tested.
• the viral preparation may be that which had been obtained from a patient affected (or likely to be affected) by AIDS, in order to determine which of the drugs which could be the more efficient in order to get at least one remission of the development of the disease in the affected patient.
• the unbound FITC was then removed by gel filtration on a molecular sieve of Sephadex G25 from Pharmacia or Biorad (which retains the isothiocyanate).
• the virus thus labeled is then inactivated by treatment at a temperature of 56 "C ⁇ 1 * C.
• T4 cells previously fractionated using specific monoclonal antibodies.
• LAV-FITC at 2.5 mg / ml for 60 minutes at 0 + 4 * C. After three washes with cold PBS, the cells are then observed using a fluorescence microscope and used for sorting .
• Table I summarizes the results obtained. It can be seen that in a population of previously fractionated T4 lymphocytes, only 10% are recognized by FITC-LAV. However, less than 0.5% of a population of fractionated T8 cells are labeled with this virus. Similarly, we find that B lymphocytes and onocytes are not or are poorly recognized by FITC-LAV, also confirming that these cells have very few receptors for the virus.
• the cells latex beads contact is made in 7 incubating 10 T4 cells with 200 mcg of latex having fixed the virus for 15 minutes at 0 + 4 ° C in a PBS solution containing 5% fetal calf serum, 1 % biotic anti- Q (penicillin, streptomycin, neomycin mixture, marketed by Gibco) and 0.1% sodium azide. Then using a magnet, the beads on which the T4X cells are fixed are separated from the supernatant containing the population T Y.
• T4 lymphocytes 7,210 T4 (T4) lymphocytes are centrifuged for 10 minutes at
• the pellet is taken up in 2 ml of PBS containing 5% FCS,
• the cells were incubated with 0.75 g of purified virus, sterile filtered and previously inactivated for 30 minutes at 56 ° C; Incubation takes place at 4 ° C for 10 minutes (cells and virus should be at 4 ° C upon contact) the cells are then centri ⁇ Fugees 2 times at 450 g for 10 minutes at 4 ° C and the cu ⁇ batch is taken up in 4 ml of PBS 5% FCS. iii) Separation of cells:
• T4X cells T4X cells. It is also possible to recover the T4Y cells, for example after washing 4 times in PBS containing 1% of FCS by syringe jets. The cells fixed on the 0KT4 are detached, these cells then forming the desired population of T4Y lymphocytes.

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• 1986
  • 1986-01-15 FR FR8600515A patent/FR2592893A1/fr not_active Withdrawn
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