DE3712334C2 - Process for the cultivation of HIV-2 viruses, cell cultures suitable therefor, and diagnostic agents for the detection of HIV-2 viruses and process for their production - Google Patents

Description

The invention relates to a method for growing HIV-2 viruses, suitable cell cultures as well as diagnostic ones Means for the detection of HIV-2 viruses and methods for their Manufacturing.

AIDS (Acquired Immune Deficiency Syndrome), a self worldwide epidemic spreading disease is caused by HIV viruses (Human Immunodeficiency Virus) - also HTLV III (Human T-cell leukemia virus type III) or LAV (Lymphadenopathy Associated Virus) - caused (Science 220, 859 (1983)). Evidence of this disease, the can have a long latency period Detection of HIV antibodies found in the patient's blood Find. There are several commercial tests of this type now known and routinely used to on the one hand to diagnose AIDS diseases and on the other hand, check blood supplies to prevent spread to prevent the disease from spreading blood.

Recently it has now been found that in addition to the known HIV viruses (hereinafter referred to as HIV-1), and others AIDS-causing viruses occur in certain patients and not or only weakly with the known HIV-1 tests react. These are referred to below as HIV-2. There the risk of infection and illness with HIV-2 as well As serious as with HIV-1, it was diagnosed and diagnosed Prophylaxis necessary, also suitable diagnostics against HIV-2 Find viruses.

The invention is therefore based on the object, means and To provide procedures that allow HIV-2 viruses manufactured and HIV-2 antibodies quickly and safely in corresponding samples can be detected.  

This task is characterized in more detail by the claims measures resolved.

A. Extraction of HIV-2 Viruses 1. Patient selection

Sera from risk patients related to HIV infection were in Western blot, with immunofluorescence and in ver various commercial ELISAs for HIV-1 antibodies tests. Sera that are weak in the test systems mentioned reacted, were then Western Blot for HIV-2 (HTLV IV) Antibody tested. In this way, patients sera are selected that are hardly or not at all in the HIV-1 test not, but reacted clearly in the HIV-2 test.

HIV-2 cultivation

The breeding of HIV-2 viruses can be caused by direct infection a suitable T-lymphocyte culture (e.g. HUT 78, Molt 3, K 37, CEM or HT, as described in WO 85-04897) with the serum of an HIV-2 positive patient. To is such a culture that in about 10⁶ to 10⁷ cells / ml contains a suitable culture medium and with approx. 2 µg / ml Polybrene is spiked with about 10% of the infected serum offset and under CO₂ atmosphere at 37 ° C for so long (1-6 weeks chen) held until syncytia formation and blistered dege generation of cells indicates virus production.

The viruses can be from this culture in a known manner for example by adding sodium to the supernatant chloride / polyethylene glycol and centrifugation of the pre zipitats at about 2000 rpm or by ultracentrifugation be won.

Further cleaning can e.g. B. by centrifugation in Density gradients occur, with the HIV-2 viruses like others Retroviruses have a density of 1.16.  

However, it is more advantageous than infection with serum the blood of HIV-2 infected patients the lymphocytes separate and place them in a suitable culture medium, which phytohemagglutinin (PHA) and interleukin-2 (TCGF-T-cell growth factor) ent for growth stimulation keeps growing about 3 days and then 1-3 times on one Cell culture that is cytopathogenic against HIV for example umbilical cord lymphocytes to pass through before this culture in a dilution of 1:10 to 1: 5 for In the above permanent cell lines is used. The rapidly occurring cytopathogenic effect allows the cultures not quickly identified with HIV-2 and to discard.

Once an appropriate HIV-2 cell culture is obtained is used for further infections and save yourself the detour via patient selection.

The HIV-2 viruses used below are in culture as HUT-78 cells according to Art. 28 Budapest contract with the European Collection of Animal Cell Cultures (PHLS), Porton Down, Salisbury, England filed under number HIY-2 / PEI 402.

B. Detection of HIV-2 antibodies

In any case, the detection of HIV-2 antibodies is based on the specific antibody / antigen reaction with the HIV-2 viruses and the detection of the complexes formed. In addition to the virus itself, the antigen can also be a active fragment, d. H. a special protein from the shell or the core of the virus or virus-containing cells be used.

1. Western blot test

The basics of this method are from Towbin, Proc. Nat. Acad. Sci, USA 76, 4350-4354 (1979). Similarly the HIV-2 viruses are lysed on sodium dodecyl sulfate  (SDS) -containing polyacrylamide gel contains the proteins electrophoretically separated and electrophoretically on a Transfer nitrocellulose layer. After that, on the Nitrocellulose present protein binding sites with Rin saturated serum albumin, the layer dried and in Cut strips parallel to the direction of electrophoresis. When incubated with a test serum are now only the HIV-2 antibodies, which are linked to the HIV-2 virus proteins can react, bound and others contained in the serum Washed out proteins. When incubated with a marked anti-human IgG or IgM antiserum is now stored more in turn to the HIV-2 antibody, so that due to the ternary complex has now been identified can be. As a marker, it is common to use 125-iodine Tyrosine portions of the antibody protein itself too sub and the radioactivity of the complexes thus formed by placing a photo paper on the nitro to measure cellulose strips (RIA method) or the anti body with an easily detectable enzyme (peroxidase; alkaline phosphatase galactoseidase) and to couple Complex by reacting with the appropriate reagents (e.g. H₂O₂ / benzidine derivative; p-nitrophenyl phosphate; Galactoside / Leuco dye) (ELISA method) or the anti bind body to a fluorescent dye and the Fluo evaluate the resence of the complex formed directly (IFA- Method).

2. ELISA method (enzyme linked immunoadsorbent assay)

For this test, the wells are one Microtiter plate made of polystyrene with about 0.5 µg protein lysate of HIV-2 viruses or a corresponding amount of the viruses in coated with a buffer solution and by drying or precipitation fixed with alcohol or acetone. Additional binding sites for protein are saturated by bovine serum albumin, before the test serum is added, so that only HIV-2 protein antibodies to the appropriate antigens is bound. By reacting with an enzyme-labeled anti-human IgG or IgM antiserum then becomes a detectable ternary immune complex formed (see above). By  it is the binding of the antigen to the carrier surface possible between the individual incubation steps Wash out excess reagent and thereby disrupt the to avoid next reaction. The color reaction of the ternary complex becomes after a defined time stopped and the result usually with a scanner evaluated.

Instead of the microtiter plates, it is also possible to use the Virus antigens on the wall of a cuvette or on beads bind from polystyrene and so the test arrangement in itself modify in a known manner.

Instead of a polystyrene surface, which is known Protected proteins can also use other suitable materials be used.

It is also possible, instead of the simple described "Sandwich" method marking over several "intermediate antibody "to fix or the test serum a defined Add amount of a labeled HIV-2 antigen and so to make a "competitive" test. For more variations is referred to the numerous publications of these methods grasslands.

3. IFA (Immunofluorescence assy)

In this test, cells containing HIV-2 viruses are directly in a buffer solution (approx. 50 µl) on slide brought, dried and fixed with alcohol / acetone. The In this state, tests can be stored at -20 ° C. Approx. 20 µl test serum 1:10 in PBS is used to carry out the test (phosphate buffered saline) added and 1 hour at 37 ° C incubated, washed and with a dilute solution of Fluorescein-conjugated anti-human IgG or IgM stained and evaluated the fluorescence under a microscope.  

The above methods are only intended to provide general insight enable. Methods known to those skilled in the art for cell growth and Propagation of viruses as well as known and for other immune tests Appropriate methods should therefore also be used by of the invention.

With simultaneous use of HIV-2 and Known HIV-1 virus preparations can be used in particular for Examination of preserved blood suitable means of detection of both Create viruses.

In the following examples, preferred configurations are as dergend without the invention is to be limited thereto.  

example 1 Production of HIV-2 viruses

From 10 ml of heparinized whole blood from an HIV-2 positive patient the mononuclear cells were ge over a Ficoll gradient cleans. The purified mononuclear cells were used for 3 days PHA stimulated and with 10% Interleukin 2 RPMI 1640 (20% FCS) cultured. There was a three-time passage on the navel cord lymphocytes that have been enriched in the manner mentioned above. Then there was a cell-bound transfer to the permanent T-lymphoma line HUT 78. These cultures were permanently treated with 2 µg / ml Polybrene kept. After about 4 weeks of cell cultivation there was a typical cytopathic effect for the HIV group with syncytia formation and subsequent blistering degeneration of the cell len. Evidence of the virus production now beginning was over performed a reverse transcriptase detection.

For this test, 30 ml of cell culture supernatant at 100,000 G Centrifuged for one hour and then the pellet in the RT assay tested. The RT test gave 40,000 CPM with a background from 2000 CPM.

The Western blot, carried out with in the manner described above obtained antigen, gave the typical band pattern of HIV-2.

Example 2 Immunofluorescence test

HIV-2 infected and uninfected T lymphocytes (HUT 78) who washed twice in PBS and in PBS on 4-6 × 10⁷ cells / ml re suspended. 2 µl (approx. 10⁵ cells) of the infected and Uninfected cells are separated on masked, defatted Slides applied and ge overnight at room temperature dries.  

One pair of slides each with 20 µl test and con troll serum in a dilution in PBS of 1:20 and 1:40 opened and incubated at 37 ° C for 30 min in a humid chamber. Then it is washed twice with PBS for 10 minutes, dried, with 20 μl FITC-labeled anti-human IgG applied, again for 30 min Incubated at 37 ° C and washed. The cells stained this way Preserved with 50% glycerin in PBS and with a fluorine escence microscope (Leitz HBO 200; 300x magnification) rated.

Example 3 ELISA test

5 mg / ml of highly purified HIV-2 viruses are mixed 1: 1 with a solution of 1% Triton X-100 in 5 mM sodium carbonate buffer pH 9.6 and Warmed to 56 ° C for 30 minutes to inactivate the viruses. The Sus Pension is cooled to room temperature, 1: 500 with 50 mM Na trium carbonate buffer diluted pH 9.6, in ELISA plastic wells filled and kept at 4 ° C. for 16 hours in order to transfer the antigens to the Ge bind the wall of the tank. Then the rest of the solution is poured out and the wells washed with distilled water and dried. Now the wells with 200 ul of a solution of 0.1% Rin derserum albumin and 2% rabbit serum in 50 mM sodium carbonate buffer pH 9.6 filled, incubated for 1 hour at 37 ° C and with 0.05% Tween-20 washed in PBS before 100 µl test solution (serum 1: 100 diluted with PBS + 005% Tween) and 90 min 37 ° C is incubated. Residues of the test serum are replaced by two Wash with 0.05% Tween-20 and 0.5% rabbit serum in PBS and 2 washes in 0.5% rabbit serum in PBS removed. After that 100 µl of a rabbit anti-human IgG solution is conjugated with peroxidase 1: 1000 in 0.05% Tween-20 in PBS, 60 min. incubated at 37 ° C and washed 4 times as before. Then will a solution of 10 mg o-phenylenediamine and 10 µl H₂O₂ (30% in Phosphate buffer pH 6.8) added, kept at 20 ° C. for 30 minutes and stopped the reaction with 2 N sulfuric acid. The reaction will measured using the yellow color at 486 nm.  

Example 4 Western blot test

150 mg of HIV-2 viruses are in a solution of 350 ul buffer (2% SDS, 2% glycerin, 0.1 M Tris-HCl pH 6.8, 20 mM PMSF (Phenylmethylsulfonyl fluoride), 0.2% bromophenol blue, 0.5% β-mercaptoethanol) lysed on an SDS polyacrylamide gel plate 16 cm × 16 cm and applied with a voltage of 180 V. Virus proteins separated electrophoretically. After a period of approx. A nitrocellulose filter is placed for 4 hours and the protein bands with a voltage of 60 V in 3.5 min electrophoretically transfer. The nitrocellulose filter is made with 1% BSA (Bovine serum albumin) and 1% gelatin soaked in PBS, pH 7.4, dried and cut into strips 0.5 cm wide. For The strips are used with a buffer solution 1 of 10% fetal calf serum, 1% BSA and 0.1% Triton X-100 in PBS 90 min moistened at 20 ° C and then 1 strip each with a 1: 100 dilution of the test serum in the above buffer 1 incubated. The incubation period depends on the Temperature. 90 min at 37 ° C, 120 min at 20 ° C and 16 hours at 4 ° C have proven themselves.

After incubation, the strips are 3 times 15 minutes with the puf fer 1 before washing with a 1: 1000 dilution of anti-human IgG - antiserum, peroxidase - conjugated in 2.5 ml Buffer solution 1 were incubated at 20 ° C for 60 min. Excess antiserum is 3 times 15 min with buffer solution 1 and Washed 3 times 15 min with PBS, pH 7.4.

The strips are stained with a solution of 50 mg 3.3- Diaminobenzidine and 50 µl hydrogen peroxide in 100 ml PBS, pH 7.4, Incubated for 20 min at 20 ° C. The reaction is carried out by 3 times 15 min Wash stopped with distilled water, the strips dried and evaluated photometrically. It shows up with the HIV-2 antibody-positive sera, the bands characteristic of HIV-2 at 26kd, 32kd, 51kd, 55kd, 65kd, 130kd. With the cross test with known HIV-1 positive sera result in no or only one weak reaction against p26 (gag) and / or yp 32 (env).  

All three of the above tests can also be used in the same way a mixture of HIV-1 and HIV-2 preparations are carried out, with a simultaneous test for both antibodies. Of course, only in the Western blot test by different bands differentiate.

Claims (8)

1. A method for obtaining HIV-2 viruses, characterized in that a culture of permanent T-lymphocyte cells with serum or leucocytes of an HIV-2 infected patient or an HIV-2 infected cell culture is added and grown under suitable culture conditions and after formation of a sufficient virus concentration from the culture, the viruses or virus-containing cells are isolated in the usual way. 2. The method according to claim 1, characterized in that in the serum or HIV-2 viruses contained in leucocytes of infected patients or passaged and enriched several times in lymphocyte cultures, on which they have a strong cytopathic effect before they are on a permanent cell culture be transmitted. 3. The method according to claim 1 or 2, characterized in that the permanent cell culture is HUT 78, Molt 3, CEM or HT. 4. Permanent T-lymphocyte cell culture containing HIV-2 viruses. 5. Cell culture according to claim 4, characterized in that it is a HAT 78 culture is known as HIV-2 PEI 402 at PHLS is deposited. 6. Containing agents for the detection of HIV-2 antibodies HIV-2 virus preparations according to any one of claims 1-3 and HIV-2 Virus / HIV-2 Antibody Detection Reagents Complexes. 7. Composition according to claim 6, for use in a Western blot, ELISA or immunofluorescence methods. 8. Composition according to claim 6 or 7, characterized in that for simultaneous detection of HIV-1 antibodies an HIV-1 virus is included.