DE3743094C2 - - Google Patents

Description

The present invention relates to the retrovirus specified in claim 1 EC ACC No. V 87 111 102 and parts of it with the Antigen properties of the virus. The invention relates to the claims 2 and 3 a method for growing / obtaining this retrovirus as well as the use of the retrovirus, its Parts in diagnostics and in manufacturing vaccines according to claims 4 and 5.

It is known that retroviruses from the HIV group infi with it graced people leads to symptoms that under the collective term AIDS - "Acquired Immune Deficiency Syndrome" - be summarized. One isolated from a patient in 1983 and characterized retrovirus was named HIV 1; its properties proved to be consistent with that Aetiological role of the virus in AIDS diseases. It showed themselves that this retrovirus is extremely variable and in number rich variants exist.

After the discovery of the retroviruses of the HIV 1 type were out Patients isolated further retroviruses, which are a second Type, HIV 2 (S. Brun-Vezinet et al., The Lan cet, January 17, 1987, p. 128; G. Biberfeld et al., The Lancet December 6, 1986, p. 1330; M. Essex et al., Journal of Virologi cal Methods, 17 (1987), 55; and J. Albert, AIDS Research and  Human Retrovirus 3 (1987), 3). The corresponding retroviruses differ significantly from type HIV 1, but have one Relationship to the SIV. Like the HIV 1 virus, HIV leads 2 virus also causes AIDS symptoms.

While for infections with viruses of the type HIV I already verver casual tests are available, the preparation of a HIV 2 infection is still very difficult. The HIV 2 virus shows a low replication rate and is accordingly also difficult to cultivate. In addition, the clinical Gradients are characterized by greater latency. Out for this reason, enzyme immunoassays with HIV 2 antigen are currently only partially available. HIV 1 tests can be used for detection HIV 2 antibodies only if the patient also Has developed antibodies for the core proteins. This is not always the case, and they go in the late stages Antibodies against HIV 2 also return or disappear entirely. In addition, despite the antigenic relationship in this core area by no means all epitopes of the one antigen recognized by the antibody against the other virus. Self under optimal conditions the immunological cross is activity between HIV 1 and HIV 2 not more than 60%, in at whose words "40% are not recognized". Made of safety green A test is required to check the infections of both virusty pen completely covered.

There is therefore a general need for an HIV test with which reliably detects infections from HIV 1- as well as from Have HIV 2 type detected. Such evidence is based of the retrovirus according to the invention from the HIV group possible.

The retrovirus according to the invention from the HIV group was deposited under the deposit number ECACC No. V 87 111 102 at the European Collection of Animal Cell Cultures in Great Britain in accordance with the provisions of the Budapest Treaty. The virus is _hereinafter referred to as HIV _ODS .

The invention is illustrated by the figures purifies. From the pictures shows

Fig. 1 is an electron microscopic representation of the HIV virus _ODS (1 to 40,000);

. Figure 2 illustrates the plaque morphology of HIV _ODS to MT 4 cells;

Fig. 3 is the serological cross-reactivity of HIV-positive sera with _ODS HIV 1 proteins; and

Fig. 4 shows the reactivity of various sera with HIV HIV _ODS -Pro teinen.

The newly isolated virus is an HIV retro virus that gives rise to AIDS symptoms. The virus was isolated from the blood of a patient infected with her West African husband. A virus with, as far as possible, the same properties was isolated from the husband; this received the designation HIV _MDS .

The husband was last in West Africa 11 years ago. From the Anamnesis can be concluded that the infection in Westaf rika has taken place, i.e. 11 years ago or significantly happened earlier. This appears after the characterization virus is also quite possible.

The immunological examination of both patients shows clear symptoms of the first pathological changes. The woman from whom the HIV _ODS virus was isolated had temporary lymphadenopathy. Mitogen stimulation tests were limited in Concanavallin Test A. Antigen stimulation tests with tetanus toxoid and toxoplasma antigen were reduced. The CD 4 cells in the woman were 500 / ul, the CD 4 / CD 8 ratio was 0.9.

In the husband, from whom a similar virus (HIV _MDS ) was isolated, the immunopathological changes were even more pronounced. This is probably due to the earlier infection date when the patient became infected. Lymphadenopathy was detectable. Mitogen stimulation with Pokeweed was missing, with Concanavallin A it was reduced, with phyto-hemagglutinin it was within the normal range. Antigen stimulation with tetanus toxoid and toxoplasma antigen could no longer be triggered. The CD 4 cell count was 300 / µl and the CD 4 / CD 8 ratio was extremely pathological at 0.2.

The newly isolated retrovirus HIV _ODS can be characterized as follows:

• a) The virus has the characteristic HIV form with a cylindrical core and a glycoprotein envelope in the electron microscopic image ( FIG. 1). The virus has a diameter of approximately 100 nm. A "budding" is detectable on the cell membranes. The HIV _ODS can be clearly assigned to the lentiviruses under these morphological criteria.
• b) The virus leads to infections with AIDS symptoms and can accordingly be isolated from patients infected with it will.
• c) HIV _ODS plaques ( FIG. 2) have a small diameter and fuzzy, somewhat washed-out edges. Plaque titration is typically possible. The plaque morphology shows that it is a homogeneous virus population.
• d) The HIV _ODS has structural and functional proteins typical of HIV viruses, such as p17, 24, 32, gp41, p52, p66, gp115 and gp160.
• e) HIV _ODS antigen reacts serologically with both HIV 1 and HIV 2 positive sera. HIV _ODS positive sera react accordingly with HIV 1 and HIV 2 antigen.
• f) HIV _ODS- positive sera react in transmembrane-specific cross-reactions with HIV 1 or HIV 2-gp 41 and the precursor gp 160. A cross-reaction also takes place with the core proteins p18, p24 and the functional proteins p53 and p66.
• g) HIV _ODS is replicated in cultures (monolayers) of primary human macrophages with a very good virus yield, where the viability of the macrophages is significantly less affected than in HIV 1.

HIV _ODS differs from the previously described types of HIV 2, all of which belong to the retroviruses, as follows:

SBL-6669

This virus strain, which was isolated by Albert et al (AIDS Research and Human Retroviruses 3 (1987) (3)), has a transmembrane protein of molecular weight 32,000 which is lacking in HIV _ODS .

HTLV IV

This virus, isolated by Essex et al (Journal of Virological Methods, 17 (1987), 55), also has a transmembrane protein with a molecular weight of 32,000 which does not have HIV _ODS . According to Essex, this virus does not cause any symptoms. In contrast, the newly isolated HIV _ODS virus leads to symptoms of AIDS. The virus designated HTLV IV can no longer be assigned to the HIV class, since it has since been shown that it is practically identical to the SIV and therefore does not constitute an independent isolate.

HIV 2

The HIV _ROD virus isolated by Montagnier et al is not able to induce a glycoprotein-specific cross-reaction with the transmembrane gp41 and the precursor gp160 with HIV 1 serum. The newly isolated HIV _ODS, however, is characterized by such transmembrane-specific cross reactions ( Fig. 3 and 4).

The evaluation of the cross-reactivities of HIV _ODS- positive sera and HIV _ODS proteins with the corresponding HIV 1 and HIV 2 proteins or sera makes it clear that HIV _{ODS is} a new trovirus that is neither HIV 1 nor HIV 2 is to be expected. Based on the immunological findings, it can be established that the relationship to HIV 2 is greater than that of HIV 1 ( FIGS. 3 and 4).

The blots in FIG. 3 show the serological cross-reactivity of HIV _ODS- positive sera with proteins of the HIV 1 virus. For comparison, HIV _MDS - and, as a typical representative of HIV 2, the BS strain with HIV 1-positive sera were also tested.

The blots in the lower part of the figure are coated with HIV 1 antigen. The HIV _ODS serum reacts with the transmembrane protein precursor (gp160 and slightly with the gp41). Otherwise, as expected, there is a reaction with the core protein p 24 and with pol-coded proteins.

The blots in the upper part of the figure are prepared with the HIV _ODS virus antigen. The autologous HIV _ODS serum typically reacts with core, Pol and Env. Proteins, while the HIV 1 reference serum (WHO, HIV 1) only recognizes one gag and one pol coded protein.

The blots in Fig. 4 show the reactivity of various HIV sera with HIV _ODS proteins.

Blots 1, 3 and 4 are coated with HIV _ODS virus antigen, and blot 2 with HIV _MDS (the strain grown by the husband). The patient sera with the antibodies against HIV _ODS or HIV _MDS react with the respective autologous antigens in the same way (regarding all essential proteins). An analogous behavior is shown by an HIV-2 antiserum (BS) carried as a control. In contrast, the HIV 1 control serum (WHO reference HIV 1) only reacts with the core and the Pol protein.

Like other viruses in the HIV group, HIV _ODS shows great variability. Cultivates (isolates) obtained from different patients are therefore no longer genetically identical; however, they all have the same antigen properties in every case. The present invention thus comprises retroviruses or isolates which have the essential antigen properties of the deposited virus HIV _ODS , ECACC V 87 111 102.

The invention also includes portions of such retroviruses and extracts with the essential antigenic properties of HIV _ODS as deposited.

In enzyme immunoassays in which the HIV _{ODS was used} as an antigen, 30 HIV 1-positive sera showed a positive seroreaction (90%) in 27 cases and a positive reaction in 18 HIV 2-positive sera in all cases. There was also a positive reaction in all cases with 5 HIV 2 sera, in which a superinfection with another HIV virus was also assumed. HIV-negative control sera showed no reaction.

Enzyme immunoassays based on HIV _{ODS are} able to indicate - in addition to HIV _ODS infections - infections of HIV 1 - as of HIV 2 type with high reliability.

The peculiarity of the new virus is that it does so probably with sera from HIV 1 patients as well as with sera from HIV 2-patient leads to positive reactions.

The invention further relates to a method for breeding / harvesting new virus. The virus gets infected from it Lymphocytes with PHA-stimulated donor lymphocytes after Ent removal of the T suppressor cells (CD 8+) in the presence of Inter leukin-2 and anti-alpha interferon grown and on T 4-positive cells increased in the same way. The distance the suppressor cells are made by magnetobeads.

The HIV-ODS virus can infi from peripheral blood cells (PBL) graced people by coculturing with pH-stimulated spenes derlymphozyten be grown. It could be done in the local laboratory are shown that the cultivation from primary Monozy infected patients is possible. The cultivation  takes place in RPMI medium 1640 with 20% FCS, interleukin-2, Anti-alpha interferon and polybrene. Crucial for the success of cultivation is the prior to cocultivation Removal of suppressor cells by means of immunoadsorption Magnetobeads. The appearance of characteristic multinu Smaller syncytia indicates the start of virus reproduction there; detection is via the virus-specific reverse Transcriptease and electron microscopy.

The virus can be found in vitro on all T4-positive cells engage, with different yields depending on the cell line stand. A culture approach is recommended for propagation primary human lymphocytes, which in principle are the same Treated as in the cultivation described above will.

In the manner described in this way, after filtering through filters of 0.2 μ cell-free culture supernatants are obtained as a stock virus with titers of 1 × 10 ⁵ PFU / ml, frozen in liquid nitrogen.

The further propagation of the new virus according to the above described This method is characterized by particularly high virus yields and labeled accordingly highly concentrated preparations. This and its properties leave the new virus as very suitable for the production of vaccines against HIV infections appear.

example Plaque titration of HIV _ODS on MT 4 cells

Materials: 6-fold cluster plates, high-speed centrifuge tube (50 ml), low-speed centrifuge, CO ₂ incubator.

Reagents: Poly-L-lysine (MW 90,000), agarose, RPMI 1640, fetal calf serum (FCS), aqua bidest.

Cells: HTLV 1-transformed MT 4 cells.

Buffer: PBS with 150 mM NaCl
2.7 mM KCl
6.5 mM Na ₂ HPO ₄ × 2 H ₂ O
1.9 mM KH ₂ PO₄

Media and solutions

Single and double concentrated RPMI 1640, poly-L-lysine Solution: 50 µg / ml Aqua bidest, 1.2% agarose in Aqua bidest. It is heated to boiling in a water bath for 10 minutes and then equal volumes of agarose and RPMI 1640 (2 ×) are mixed. FCS is added at a final concentration of 20%.

method Plaque assay

• 1. There will be a series of dilutions of the viral inoculum in simply concentrated RPMI 1640.
• 2. 1.5 times in each well of the 6-fold cluster plates Given poly-L-lysine solution.
• 3. Incubate for one hour at room temperature.
• 4. Each well is 3 times at room temperature with 0.5 ml PBS washed.
• 5. Into each well is then added 1 ml of MT 4 cells (prewashed 3 times with PBS at room temperature, approximately 5 x 10 ⁶ cells / ml). The cluster plates are shaken gently.
• 6. Cell adsorption over 20 min at room temperature.
• 7. Wash the cell lawn gently three times with 2 ml PBS Room temperature. The cluster plates are after adding the PBS  gently shaken. After the last wash it becomes complete aspirated.
• 8. Virus adsorption over 60 min at 37 ° C and 5% CO ₂ .
• 9. Complete suction of the inoculum.
• 10. Add 1 ml of agarose supernatant. 10 min incubation at Room temperature without moving the trays.
• 11. Incubation at 37 ° C in the CO ₂ incubator.
• 12. On the 3rd day after the incubation, the cells are washed with 1 ml Feeded agarose supernatant.
• 13. The plaques are best on the 6th day after incubation recognizable. The plaque diameter is then about 2 mm.

RT assay

Materials: 96-fold microtiter plates (with flat bottom for tissue cultures), shaking device for microtiter plates, repeating pipette, Transstar 96 pipettor, bio-freeze ampoules, DEAE paper (0.45 µm), ³² P-plexiglass protective screen, incubation plate, X-ray film holder and intensifying screens, Kodak XAR film.

Reagents: Tris, dithioerythreithol (DTE), KCl, MgCL ₂ × 6 H ₂ O, Triton X-100, methyl- (1 ′, 2′- ³ H) -TTP (100 Ci / mmol) template primer (Poly rA. Oligo dT _10-15 ), Na ₂ HPO ₄ , ethanol 100%, Triton X-100

Stock solutions: 50 mM MgCl ₂
750 mM KCl
250 mM Tris-HCl pH 8.0
0.5% Triton X-100

DTE: 50 mM (freshly made)
Methyl- (1 ′, 2′- ³ H) -TTP (100 Ci / mmol): 1 mCi / ml poly rA. oligo dT _10-15 (5 A ₂₆₀ / ml): 100 µl + 900 µl Aqua bidest (sufficient for one microtiter plate)

Test procedure

• 1. Reaction mixture per test (in the following order pipetted into each well of the microtiter plate at 4 ° C): RT-mix (5 ×): 10 µl
  DTE (50 mM): 10 µl
  Poly rA. oligo dT _10-15 : 10 µl
  Methyl- (1 ′, 2′- ³ H) -TTP 10 µl (10 µCi)
  Test sample 10 µl The test sample means the cell culture supernatant or samples such as sera, cerebrospinal fluid, etc.; the latter must be concentrated by ultracentrifugation at 100,000 g _av for 1 h.
• 2. Rotation of the microtiter plates on a microtiter plate shaker and incubation for 15 min on ice water.
• 3. Incubation for 60 min at 37 ° C on an incubation plate.
• 4. Aspirate 25 µl of the incubation mixtures using a Transstar 96 pipettor and direct transfer to dry DEAE paper.
• 5. 6 times washing the DEAE paper for 5 min each in 100 ml of 0.5 M Na ₂ HPO ₄ .
• 6. Wash twice for 1 min each in 100 ml of double-distilled water.
• 7. Wash twice for 1 min each in 100 ml of ethanol Drying on a gel dryer under vacuum.
• 8. Autoradiography overnight.

Claims (5)

1. Retrovirus ECACC No. V 87 111 102 and parts of which with its antigen properties.   2. A method for cultivating / obtaining the retrovirus according to claim 1, characterized in that the virus from infected lymphocytes with PHA-stimulated Donor lymphocytes after prior removal of T suppressor cells (CD 8) in the presence of interleukin-2 and anti-alpha Interferon grown and on T 4 positive cells in the same Way is increased. 3. The method according to claim 2, characterized in that removal of the T suppressor cells by magnetobeads he follows. 4. Use of the retrovirus, its parts according to claim 1 for the detection of HIV infections and AIDS. 5. Use of the retrovirus, its parts according to claim 1 for the manufacture of a vaccine against HIV infections and AIDS.