DE4233646A1 - New HIV-type immune deficiency virus ECACC V 92092318 - Google Patents

Abstract

Immune deficient, HIV-type virus, or its variants, having the essential morphological and immunological properties of the retrovirus deposited as ECACC V92092318 (also designated MVP-5180.91) is new.Also claimed are (1) cDNA (I) corresp. to (part of) the RNA of this virus; (2) recombinant DNA (Ia) contg. (I); (3) antigens (Ag)derivs. from (I) or (Ia), (4) test kits contg. Ag for detecting antibodies and (5) ribonucleic acid coding for the new virus.The specification includes the (practically) complete sequence of the virus; about 9800 bases.

Description

The present invention relates to a new retrovirus the HIV group as well as variants or parts thereof which the essential properties of the virus included. Described becomes a method of growing the retrovirus. The The invention further relates to the extraction of this retrovirus and the use of the virus, its parts or extracts for medical purposes, for diagnostics and for Vaccine production.

Retroviruses, which belong to the so-called HIV group, lead in people infected with it to symptoms of illness, those under the collective term immunodeficiency or AIDS (Acquired Immune Deficiency Syndrome).

Epidemiological studies show that the human Immunodeficiency Virus (HIV) the aetiological agent for the vast majority of AIDS (Acquired Immune Deficiency Represents syndrome) cases. A 1983 from a patient isolated and characterized retrovirus received the Name HIV-1 (Barre-Sinoussi, F. et al., Science 220, 868-871 [1983]). A variant of HIV-1 is described in WO 86/02383 described.  

A second group of human immunodeficiency viruses was developed 1985 identified in West Africa (Clavel, F. et al., Science 233, 343-346 [1986]) and as a human immunodeficiency virus type 2 (HIV-2) denotes (EP-A-0 239 425). HIV-2 retroviruses differ significantly from HIV-1, but also show a relationship to monkeys immunodeficiency virus (SIV-2) on. Like HIV-1, HIV-2 leads to symptoms of AIDS.

Another variant of an immune deficiency retrovirus is in EP-A-0 345 375 and there as HIV-3 retrovirus designated (ANT 70).

Also in Lancet Vol. 340, Sept. 1992, pp. 681-682 the Isolation of another variant immunodeficiency virus described.

It is a characteristic of the human immunodeficiency virus, that they have a high variability that the Comparability of the different isolates clearly complicated. When comparing various HIV-1 isolates occur e.g. B. high variability in some regions of the genome, while other genome areas are comparatively conserved are available (Benn, S. et al. Science 230, 949-951 [1985]). A HIV-2 could also have a much larger polymorphism can be observed (Clavel, F. et al., Nature 324, 691-695 [1986]). Areas have the greatest genetic stability in the gag and pol genes that are structural and encode enzymatically essential proteins; some regions in the env gene as well as the genes (vif, vpr, tat, rev, nef) that are for encoding regulatory proteins indicate a high level Variability. It could also be shown that Antisera against HIV-1 also with gag and pol gene products from HIV-2 cross-reacts, although only little sequence homology Template. The hybridization between these was also of the two viruses little significant, if not very little stringent conditions were used (Clavel, F. et al., Nature 324, 691-695 [1986]).

Due to the wide spread of retroviruses from the HIV group and the fact that between the time of Infection and the time when clear symptoms of pathological changes are recognizable, a period of a few to many years (2-20), it is epidemiologically of great importance, the infection with Retroviruses from the HIV group as early as possible and above all to determine reliably. This doesn't just matter in diagnosing patients who show signs of Have immunodeficiency, but also when checking from blood donors. It has been found that the Use of retroviruses or components thereof of the type HIV-1 or HIV-2 in detection systems none for some sera or only weak detection of antibodies can be, although in the patients of whom the sera originate, signs of immune deficiency occur. With the help of retrovirus according to the invention from the HIV group is in Such evidence is possible in certain cases.

The isolation and characterization of a new human immunodeficiency virus, hereinafter referred to as MVP-5180/91, which in 1991 from peripheral lymphocytes In 1991, 34-year-old female patient from Cameroon was isolated Showed signs of immune deficiency. Geographically this retrovirus from a region in Africa that between West Africa with endemic HIV-2 and HIV-1 virus infection and East Central Africa with almost exclusive HIV-1 spread is localized. Subject of the present So invention is a new HIV group retrovirus, which is designated as MVP-5180/91 and its Variants. The retrovirus MVP-5180/91 was developed by the European Collection of Animal Cell Cultures (ECACC) under number V 920 92 318 according to the terms of the Budapest Treaty deposited.

Like HIV-1 and HIV-2, the invention grows MVP-5180/91 in the following cell lines HUT 78, Jurkat cells, C8166- Cells and MT-2 cells. Isolation and propagation of  Viruses are described in the book "Viral Quantitation in HIV Infection, Editor Jean-Marie Andrieu, John Libbey Eurotext, 1991 " described in detail. The ones described there Working methods become the subject of reference by reference Disclosure of the present application made.

Furthermore, the virus according to the invention has one magnesium-dependent reverse transcriptase, but not is dependent on manganese. This represents another commonality to the viruses HIV-1 and HIV-2.

To better understand the differences of the MVP-5180/91 virus according to the invention for the retroviruses HIV-1 and HIV-2 should initially briefly build the immune deficiency causing retroviruses. Inside the The RNA is located in a cone-shaped core, the virus is composed of protein subunits that the Name p 24 (p for protein). This inner core is surrounded by a protein shell that consists of the protein p 17 is built (outer core) and one Glycoprotein envelope is surrounded by lipids that are made up of of the host cell, the transmembrane protein gp 41, and contains the coat protein 120 (gp 120). This gp 120 can then binding to the CD-4 receptors of the host cells come in.

As far as is known, the RNA of the HIV virus has the following gene regions - to put it simply - at what is termed long terminal repeats (LTR), and the following gene regions gag, pol, env and nef. The gene gag encodes, inter alia, for the core (core) proteins, p 24 and p 17, the gene pol encodes, inter alia, for reverse transcriptase, RNAse H and integrase and the gene env encodes for the glycoproteins gp 41 and gp 120 the virus envelope. The gene nef codes for a protein with a regulatory function. A schematic arrangement of the genome of retroviruses of the HIV type is shown in FIG. 1.

A distinction between the retroviruses HIV-1 and HIV-2 is possible, inter alia, by testing viral antigen with a monoclonal antibody, which is commercially available as a test kit from Abbott (HIVAG-1 monoclonal), and against (HIV-1) p 24 is directed. It is known that the reverse transcriptase content is approximately the same in the HIV-1 and HIV-2 virus types. Therefore, if the absorbance (E 490 nm) obtained by the antigen-antibody reaction and the reverse transcriptase content is plotted in dilutions of the digested viruses, a graphic similar to that in FIG. 2 is obtained. It is found that, in relation to the reverse transcriptase content in HIV-1, there is a very high binding affinity for p 24 with the monoclonal antibody used. For HIV-2, on the other hand, there is only a very low binding affinity for p 24 when using the monoclonal antibody, again based on the content of reverse transcriptase. If these measurements are carried out for MVP-5180/91, the curve is pretty much midway between the curve of HIV-1 and HIV-2, ie the binding affinity of the monoclonal antibody against MVP-5180/91 p 24 is against HIV -1 reduced. FIG. 2 shows this situation schematically, where RT means reverse transcriptase and the protein p 24 is used as antigen (Ag) against which the monoclonal antibody which is present in the test kit commercially available from Abbott is directed.

A very versatile system of genetic engineering has become the so-called PCR (polymerase chain reaction), the ones required to carry out the method Components can be purchased. With this Method it is possible to amplify DNA sequences if DNA regions of the sequence to be amplified are known are. Short complementary DNA fragments are then required (Oligonucleotides = primer) can be synthesized a short range of the ones to be amplified Attach nucleic acid sequence. For carrying out the test  HIV nucleic acids are brought together with the primers in a reaction mixture which additionally contains a polymerase and Contains nucleotide triphosphates. The polymerization (DNA Synthesis) is carried out for a certain time and then the nucleic acid strands are separated by heating. To Cooling then starts the polymerization again. If it So the retrovirus according to the invention is an HIV-1 or HIV-2 virus, then the nucleic acid would have to can be amplified using primers which are conserved within the known sequences of the Viruses HIV-1 and HIV-2. Such primers are in part previously described (Laure, F. et al., Lancet ii, (1988) 538-541 for pol 3 and pol 4 or Ou C.Y. et al., Science 239 (1988) 295-297 for sk 38/39, sk 68/69).

It has now been found that when using certain primer pairs which have the following sequence:

or a combination of pol3 and pol4 with

and with the PCR with nested primer amplificates of the MVP 5180/91 DNA were obtained.

No or only weak amplicons compared to HIV-1, which may be due to contamination obtained with the following primer sequences:

Compared to HIV-1 weak amplicons, which, however, the same intensity as the HIV-2 isolate used (MVP-11971/87) were obtained with

A widely used method for the detection of HIV antibodies are the so-called Western blot (immunoblot). The viral proteins become gel electrophoretic separated and then transferred to a membrane. The one with the Proteins are then transferred to proteins Sera related to the patient to be examined brought. Provided antibodies against the viral proteins are present, they bind to the proteins. After washing only specific antibodies against viral remain Proteins. The antibodies are then combined with anti-antibodies visualized that are regularly coupled with an enzyme that catalyzes a color reaction. In this way can visualize the bands of viral proteins become.

The virus MVP-5180/91 according to the invention shows the Viruses HIV-1 and HIV-2 two significant in Western blot significant differences. The HIV-1 shows regularly a strong band which can be assigned to the protein p 24 and a very weak, often barely visible gang, that the Protein p 23 is assigned. HIV-2 has a strong band that is assigned to the protein p 25 and sometimes one weak band attributable to protein p 23. in the The MVP-5180 / 91- according to the invention differs from this Virus two approximately equally strong bands on the proteins correspond to p 24 and p 25.  

Another significant difference is in the Bands that are assigned to the reverse transcriptase. HIV-1 shows a band (p 53) that of reverse transcriptase corresponds and a band (p 66) that of the reverse Transcriptase associated with the RNAse H corresponds. At HIV-2 corresponds to the reverse transcriptase to the protein p 55 and, when linked to RNAse H, protein p 68. In contrast, the MPV-5180/91 according to the invention has a band on the protein p 48, that of the reverse transcriptase and a band at the protein p 60, which is the Reverse transcriptase in conjunction with RNAse H corresponds.

From these results it can be concluded that the Reverse transcriptase of the MVP-5180/91 a molecular weight that is between about 3 and about 7 kilodaltons smaller than that of reverse transcriptase from HIV-1 or HIV-2. The Reverse transcriptase from MPV-5180 thus shows Molecular weight that is between about 4,500 daltons and is about 5,500 daltons smaller than reverse transcriptase of HIV-1 or HIV-2.

It was found that with the help of the invention Virus MVP-5180/91 anti-env antibodies in sera from German Patients who show signs of immunodeficiency are weak can be detected, but the sera are strong react if instead of the virus of the invention HIV-1 virus is used. This stronger detection reaction was mainly localized in the gp 41 protein. Both Trials were juxtaposed with serum panels once come from German patients and from African patient with signs of immunodeficiency come.

The characteristics indicated above characterize such Virus variants, the MVP-5180/91 according to the invention correspond. So if from heparinized donor blood, that comes from people who show signs of immunodeficiency  and preferably come from Africa, immunodeficiency viruses can be isolated, then this way virus according to the invention or variants thereof are obtained.

Since the virus was isolated, which mentioned the above Has properties, the cloning of a cDNA the following way: The virus is made from a correspondingly large amount of culture (about 1 l) precipitated and taken up in phosphate buffered saline. Then pelleting is carried out using a (20%) sucrose Pillow. The virus pellet can be in 6 M guanidinium chloride 20 mM dithiothreitol and 0.5% Nonidet P 40 suspended become. CsCl becomes up to a concentration of 2 molar added and the solution containing the broken virus is applied to a cesium chloride cushion. Then the viral RNA pelleted by centrifugation, dissolved, with Phenol extracted and with ethanol and lithium chloride precipitated. With the help of an oligo (dT) primer, the Synthesis of the first strand of cDNA on the viral RNA or Parts of it done. The synthesis with the addition of Reverse transcriptase can be done at Using a commercially available kit. For the Synthesis of the second strand becomes the RNA strand of the RNA / DNA hybrids digested with RNase H and the second strand synthesized using E. coli DNA polymerase I. With Using T4 DNA polymerase can then blunt ends generated and these with suitable linkers for Restriction interfaces are connected. To Restriction digestion with the appropriate Restriction endonuclease is the cDNA fragment from a Agarose gel isolated and with a previously appropriate cut vector ligated. The vector with the cDNA insert can then be used to transform competent E. coli cells used because of. The colonies obtained are then expanded Membranes transferred, lysed and denatured and finally by hybridization with digoxigenin or biotin labeled nucleic acid found. After genetic engineering Preparation of the corresponding cDNA is an isolation of the  desired DNA fragments that originate from the retrovirus, possible. By incorporating these fragments into suitable ones Expression vectors can then be the desired protein or Protein fragment can be expressed and used for diagnostic Tests are used.

As an alternative to the specified method, this can Immunodeficiency virus cloned using PCR technology using the primers given above can.

Using the isolated sequence, immunodominant epitopes (Peptides) are assembled and synthesized.

In the diagnostic tests, a serum sample is taken of the investigator brought together with the protein chains of one or more proteins or glycoproteins (the can be expressed in eukaryotic cell lines) or Parts of it that come from MVP-5180/91. Preferred Test procedures include immunofluorescence or immuno-enzymatic test methods (e.g. Elisa, immunoblot) a.

In the case of immunoenzymatic tests (ELISA), for example Antigen from MVP-5180/91 or a variant thereof is bound to the walls of microtiter plates. The dosage used depends on the test system and the treatment of the microtiter plates. Then will be serum or serum dilutions that depend on the investigating person come into the holes of the Given microtiter plates. After a certain Incubation period, the plate is washed and specific Immune complexes are detected by antibodies that specifically bind human immunoglobulins and the beforehand with an enzyme, for example Horseradish peroxidase, alkaline phosphatase, etc. linked or with enzyme labeled antigen. This Enzymes can turn a colorless substrate into a highly colored one  The product can then transform and change in color strength the presence of specific anti-HIV antibodies be read. Another way of using of the virus according to the invention in test systems is the Use in Western blots.

Even if the production of vaccines against Immunodeficiency disorders prove to be extremely difficult proves, this virus or parts of it, d. H. immunodominant epitopes and inducers of cellular Immunity, or genetically engineered antigens to Development and manufacture of vaccines used become.

example 1

The immunodeficiency virus MVP-5180/91 according to the invention was from the blood of a patient with signs of immunodeficiency isolated. Peripheral mononuclear cells were used for this purpose (peripheral blood lymphocytes, PBL) and peripheral Lymphocytes from the blood (PBL) of a non-HIV-infected Donors stimulated with phytohemaglutinin and in culture held. The usual medium RPMI was used for this 1640 with 10% fetal calf serum. The cultural conditions are described in Landay A. et al., J. Inf. Dis., 161 (1990) pp. 706-710. The formation of Giant cells under the microscope. The production of HIV Viruses were detected using the p 24 antigen of Abbot's commercially available test. A another test to determine the growth of the virus was the Test using particle-bound reverser Transcriptase (Eberle J., Seibl R., J. Virol. Methods in press). The growth of the viruses was therefore based on the enzymatic activities in the culture supernatant one to determined twice a week to increase virus production monitor. Once a week there were new ones Donor lymphocytes added.  

After HIV virus replication was found, fresh peripheral blood lymphocytes (PBL) healthy donor not infected with HIV with the supernatant the first culture infected. This step was repeated and then with the supernatant H 9 and HUT became 78 cells infected. In this way it was a permanent production of the immunodeficiency virus possible. The virus was found in the ECACC deposited under number V 920 92 318.

Example 2

The so-called is currently used to detect HIV infections Western blot or immunoblot is a standard procedure. According to the one in J. Virol. Meth. 15 (1987) pp. 11-23 by Gürtler et al. The procedure described was different sera examined. Here sera from German patients compared to those sera from African Patients were obtained. Here were the following Get results:

The results presented above show that an out German patients isolated HIV-1 type virus if it is used to detect HIV infections, may not produce clear results if the patient has been infected with a virus that MVP-5180/91 according to the invention corresponds. It will be there assumed that with the help of the virus according to the invention such viruses can be detected that at least approximately 85% homology, based on the total genome, to the exhibit virus according to the invention.  

Example 3

Further Western blots were carried out in accordance with the procedure given in Example 2. The results are shown in the attached FIG. 3. In this test, the viral protein of the immunodeficiency virus MVP-5180/91 according to the invention and once the viral protein of an HIV-1 type virus (MVP-899) were separated by gel electrophoresis and then transferred to cellulose filters. These filter strips were incubated with the sera from different patients and then the specific antibodies were visualized by a color reaction. The left half of the figure with the heading MVP-5180 shows the immunodeficiency virus according to the invention. The right half of the figure shows a virus isolated from a German donor (MVP-899), which is an HIV-1 virus.

The individual filter strips were then incubated with the sera from different patients. Looking at FIG. 3, the same sera (from German patients) were implemented with two filter strips, the numbers 8 and 26; 9 and 27; 10 and 28; 11 and 29; 12 and 30; 13 and 31; 14 and 32; 15 and 33 and 16 and 34 denote the same sera. Sera from African patients were used in the Western blots with the numbers 17 and 18. The numbers on the right hand side indicate the approximate molecular weights in thousands (KD).

FIG. 3 clearly shows that sera from German patients with the immunodeficiency virus according to the invention react in the Western blot with the gp 41 only very weakly. In contrast, sera from African patients react very strongly with the immunodeficiency virus according to the invention. Fig. 3 therefore makes clear that such immunodeficiency infections can be detected using the immunodeficiency virus according to the invention, which provide for use of an HIV-1 or HIV-2 virus only in question, that is not clearly positive results. This possibility of detection can have far-reaching diagnostic significance, since in cases in which only questionable results are obtained in the Western blot, it cannot be determined with certainty whether it is an infection with an immunodeficiency virus. However, if such questionable results of an infection with a virus of the type according to the invention can be assigned with the help of the immunodeficiency virus according to the invention, then this represents a significant diagnostic advance.

Claims (13)

1. Immunodeficiency virus of the HIV group or variants of this Virus that is the essential morphological and immunological properties of the at the European Collection of Animal Cell Cultures (ECACC) under the No. V 920 92 318 deposited retrovirus with the Designation MVP-5180/91. 2. immunodeficiency virus according to claim 1, characterized characterized as being one of the reverse transcriptase has a corresponding protein band in the Western blot, the 3-7 Kilodalton is smaller than the corresponding band of viruses HIV-1 and / or HIV-2. 3. immunodeficiency virus according to one of claims 1 or 2, characterized in that this retrovirus with a monoclonal antibodies directed against the protein p 24 has less reactivity, based on the reverse Transcriptase activity than the HIV-1 virus and more Activity, based on the activity of the reverse Transcriptase, as HIV-2. 4. Immunodeficiency virus according to one of the previous ones Claims, characterized in that with his Transmembrane protein gp 41 antigen-antibody reactions good are detectable with sera from Africa Patients and that with the gp 41 only a minor or no antigen-antibody reaction with sera from Germany-born patients can be proven. 5. Immunodeficiency virus according to one of the above Claims, characterized in that there is an RNA sequence has about that with the RNA of the deposited virus 85% or more, based on the entire genome, is homologous.   6. cDNA which is complementary to the RNA or parts thereof, at the European Collection of Animal Cell Cultures (ECACC) under number V 920 92 318 Immunodeficiency virus MVP-5180/91. 7. Antigen, which using the cDNA according to claim 6 was made or using the Amino acid structure derived from its cDNA can. 8. Antigen that according to the immunodeficiency virus Claim 1 was made. 9. Test kit for the detection of antibodies against immune deficiency causative viruses, characterized in that antigen is used according to claim 7 or 8. 10. Test kit according to claim 9, characterized in that it's a western blot. 11. Test kit according to claim 9, characterized in that it's an Elisa test or a fluorescent antibody Proof test is. 12. Use of the immunodeficiency virus according to one of the Claims 1 to 5, the cDNA according to claim 6 and / or the Antigen according to claim 7 or 8 for the detection of Retroviruses that cause immunodeficiency. 13. Use of the retrovirus according to one of claims 1 to 5, the cDNA according to claim 6 and / or the antigen according to Claim 7 or 8 for the production of vaccines.