WO1988008449A1

WO1988008449A1 - Hiv related human retrovirus strain with cloned nucleotide sequence and applications thereof

Info

Publication number: WO1988008449A1
Application number: PCT/SE1988/000218
Authority: WO
Inventors: Jan Albert; Gunnel Biberfeld; Eva Maria FENYÖ; Erling Norrby
Original assignee: Statens Bakteriologiska Laboratorium (Sbl)
Priority date: 1987-04-28
Filing date: 1988-04-28
Publication date: 1988-11-03
Also published as: AU1711088A; SE8701765D0; SE8701765L

Abstract

The present invention pertains to the isolation of a HIV related human retrovirus with antigenic similarities with HTLV-IV and LAV-II. The entire genome of the virus is disclosed. A method for large scale production of this virus and of viral antigens and a method for detection of antibodies directed against HIV related human retroviruses, as well as identification of viral antigenes which can be used for production of polyclonal and monoclonal antibodies and active immunisation, are disclosed.

Description

HIV RELATED HUMAN RETROVIRUS STRAIN WITH CLONED NUCLEOTIDE SEQUENCE AND APPLICATIONS THEREOF

Human immunodeficiency virus (HIV) (for a definition, see Reference 1 in reference list below) is a unique pathogen in man. The virus causes persistent infections which in many cases lead to development of the acquired immunodeficiency syndrome (AIDS). In these individuals opportunistic infections, malignancies and neurological syndromes relentlessly lead to death. Currently no therapeutic or prophylactic measures are available. The combat against the insidious spreading of HIV therefore has to rely on containment via epidemiological interventions. For this purpose a precise serological identification of healthy as well as diseased virus carriers is an absolute prerequisite.

HIV is a nontransforming retrovirus with a genomic structure similar to that of members of the lentivirus subfamily (References 2, 3). The envelope of lentiviruses contains a small and poorly glycosylated transmembrane protein and a large heavily glycosylated external protein (References 4-7). Both these proteins show an antigenic lability (Reference 8). The differences in polypeptide composition of env gene products between strains of HIV involve up to 30% of the araino acids (References 9, 10). The impact of this variation requires evaluation in two different contexts. One of these concerns the use of the envelope protein or parts thereof as antigen in serological tests and the other the use of the same kind of products for active immune prophylaxis. Antibodies to the glycoproteins are identified in essentially all infected persons, whereas antibodies to the core proteins tend to disappear with advancing disease (References 5-7). Although the large external glycoprotein of HIV isolates shows extensive antigenic variations sera from infected persons generally identify gp120 of HTLV-IIIB and other representative HIV strains (References 5-7). Exceptions to this rule are certain virus isolates from West Africa. Thus virus isolated from apparently healthy individuals in Senegal (HTLV-IV) (Reference 11) and from AIDS patients in Guinea Bissau and Cape Verde (LAV-II) (Reference 12) have glycoproteins that are antigenically distinct from the envelope proteins of strains isolated in North America, Europe and Central Africa. Further differences are found in the electrophoretic mobility of some of the structural components.

Infection with HIV related human retroviruses has so far mainly been demonstrated among patients from West Africa. However, it is reasonable to expect that these viruses will spread to other parts of the world. It will therefore be increasingly important to serologically identify not only persons infected with currently prevailing HIV strains, but also individuals exposed to HIV related human retroviruses of West African origin. Many persons infected with these viruses will be identified in HIV ELISA because of the cross reactions between the core proteins. However, this may not always be the case since e.g. both of the patients from whom LAV-II was isolated were negative in HIV ELISA (Reference 12). Thus there is a great need for serological tests which identify antibodies against HIV related viruses.

The task to develop a vaccine protective against AIDS has become further complicated by the identification of at least partly pathogenic HIV related human retroviruses in West Africa with unique glycoprotein immunogenicity. The possibility to identify neutralizing epitopes in the envelope shared between HIV and HIV related human retroviruses seems small. A future envelope vaccine may therefore have to be bivalent. The present invention consists of

1. Isolation of a HIV related human retrovirus with antigenic similarities with HTLV-IV and LAV-II.

2. A method for large scale production of this virus and of viral antigens.

3. A method for detection of antibodies directed against HIV related human, retroviruses.

4. Identification of viral antigens which can be used for production of polyclonal and monoclonal antibodies and active immunisation.

I. Isolation of the HIV related human retrovirus

We have isolated a new HIV related human retrovirus strain from lymphocytes of a West African woman. The strain has been designated SBL-6669-85 (SBL-6669 for short). A sample of said strain, integrated in and produced by U937 clone 2 cells, has been deposited in accordance with the Budapest Convention at The European Collection of Animal Cell Cultures, Porton Down, Salisbury, SP4 OJG, U.K. with the filing No. 87042402. Our SBL-6669 material is a stable isolate which can be cultivated in several host cell lines and can be used in assays for the defection of antibody to HIV related human retroviruses and frequently also HIV in samples of body fluids.

Accordingly the present invention provides HIV related human retrovirus SBL-6669.

We have found that SBL-6669 can be maintained for prolonged periods of time in both the T-cell line HUT 78 (Reference 14) and the monocyclic cell line U937 clone 2 (Reference 15) and a further feature of the present invention comprises U937 clone 2 cells harbouring our SBL-6669 virus. Serum collected from the patient from whom SBL-6669 virus was obtained reacted with HTLV-IIIB antigens in enzyme linked immunosorbent assay (ELISA), but the confirmatory WB test failed to show reactivity against gp41 and gpl20 of HTLV-IIIB although gag and pol gene products (p24, p55, p34) were identified. Ficoll-Paque separated PBMC (peripheral blood mononuclear cells) from the patient was cocultivated with PHA stimulated PBMC from a healthy seronegative donor. Particle associated reverse transscriptase (RT) activity (Reference 13) was observed within 2 weeks. Cocultivation of PBMC with HUT 78 cells yielded a continuously virus producing cell line showing typical multinucleated giant cells. This line has continued to produce virus for more than 6 months. By cell free infection have also other cell lines, among them U937 clone 2 , been infected.

SBL-6669 was shown to be a probable member of the lentivirus family by:

a) possessing a reverse transscriptase activity with a preference for Mg cations

b) the morphology of the viruses visualised by electron microscopy.

SBL-6669 was shown to differ from the prototype HIV strain HTLV-IIIB by the absence of cross-reactions between the glycoproteins of the two viruses.

SBL-6669 exhibited properties closely related to the recently identified HIV related human retroviruses, LAV-II and HTLV-IV, as evidenced by complete cross-reaction in RIPA and Western blotting. However, SBL-6669 differs from HTLV-IV and LAV-II by the size of the structural proteins as determined by RIPA and Western blotting.

Our SBL-6669 material was compared biologically and immunologically with LAV-II and HTLV-IV. HTLV-IV was obtained from Dr. M. Essex. Serum from a healthy Gambian immigrant to Sweden was used as HTLV-IV reference serum. This serum had been shown by Dr. Essex (personal communication) to react with envelope and core proteins of HTLV-IV. LAV-II (LAV type II ROD, CNCM No. 1-532) and LAV-II reference serum (No. 5473) was obtained from Dr. L. Montagnier, Institute Pasteur, Paris, France. HTLV-IIIB was provided by Dr. R. Gallo, National Cancer Institute, Bethesda, Md and a selected high titer HTLV-IIIB positive serum from a healthy homosexual man was used as HIV reference serum.

Comparison of the in vitro growth characteristics of the three West African virus isolates showed that LAV-II and SBL-6669 readily infected and replicated in the human T-cell lines HUT 78, CEM and Jurkat as well as in the monocytoid cell lines U937 clone 16 and U937 clone 2 (Reference 15). In contrast we have not been able to productively infect any of these cell lines with HTLV-IV. In the studies of HTLV-IV we have therefore exclusively worked with the HTLV-IV infected HUT 78 cells originally received from Dr. Essex. All viruses generated multinucleated cells, although to varying degrees. However, cell lysis was only observed in SBL-6669 and LAV-II infected cultures.

The approximate molecular size of major proteins of HTLV-IV, LAV-II and SBL-6669 was determined in RIPA (Fig. 2) and WB (Fig. 3). Table 1 lists the approximate size of corresponding proteins of HTLV-IV, LAV-II, SBL-6669 and HTLV-IIIB. The external glycoprotein had an approximate molecular size of 120 kD in all four virus strains. Although we observed small interstrain size variation these were not as pronounced as the previously reported differences between LAV-I and LAV-II (Reference 12). The presumed transmembrane glycoprotein was only observed effectively in WB tests. In all three West African virus strains a 30-35 kD broad band was observed. A similar band was previously observed in LAV-II and HTLV-IV (References 11, 12) and it was interpreted to be a candidate transmembranous protein. However, the present study showed a 41 kD protein in addition to the 32 kD protein in SBL-6669 (Fig. 2a).

Certain interstrain variations in the size of the 30-35 kD protein were observed among the West African virus isolates (Fig. 3a). More distinct variations were seen in the size of the core proteins of these viruses (Fig. 2). The differences between LAV-II och SBL-6669 proteins were limited, but distinct.

The immunological relationship between the four virus isolates was studied by cross-serological analysis in RIPA and WB tests (Figs. 2 and 3). All sera of West African origin gave identical reactions in these tests. They all identified the external glycoprotein (Fig. 2), the presumed transmembrane glycoprotein (Fig. 3), and the core proteins of the three West African virus isolates as well as the core proteins of HTLV-IIIB. However, none of the sera specific for the three West African viruses identified the two envelope proteins of HTLV-IIIB. One additional serum of West African origin has been tested with identical result (data not shown). Three HIV reference sera (one included in Fig. 1) identified all HTLV-IIIB proteins and the core proteins of the West African virus isolates, but failed to recognize the envelope proteins of HTLV-IV, LAV-II and SBL-6669. We have, however, observed that some HIV positive sera, other than those used in our study, could detect the transmembrane glycoprotein of HTLV-IV in WB tests. The cross reactivity between the West African viruses and HTLV-IIIB appeared to be greater for the core protein equivalent to p24 of HTLV-IIIB than for the protein equivalent to p19 (Fig. 2).

Production of SBL-6669 virus and antigens

According to a further feature of the present invention we provide a method of production of SBL-6669 virus and antigens. These antigens can among other things be used for detection of antibodies which react with SBL-6669. The basis for the production of SBL-6669 is that we have succeeded to persistently infect certain tumour cell lines. U937 clone 2 cells and HUT 78 cells have become stable producers of virus. Other T4⁺ positive cell lines such as Jurkat and .CEM can also be persistently infected. Thus our SBL-6669 virus is well suited for in vitro production of virus in several cell lines and other cell cultures.

An alternative method for the production of SBL-6669 virus is to infect the cell line U937 clone 16. This cell line is highly sensitive to the cytopathic effect of the virus and is eventually killed, but very large amounts of virus are produced during a short period of time before the cells are killed.

When using the SBL-6669 virus material as antigens in ELISA and Western blotting we first purified the virus by centrifugation onto a sucrose cushion, and this method will be described further in the examples. When needed purification of the antigens to a higher degree can be achieved.

By use of affinity chromatography in which viral antigens are either specifically bound by antibodies or glycoproteins bound by lecitins a purified antigen can be obtained. With this method it is not necessary to start with free virions since viral antigens can be purified from lysed infected cells as well as from culture supernatant, into which the external glycoprotein is shedded.

A third possible method for the production of SBL-6669 and SBL-6669 derived proteins is to construct recombinant vectors comprising the complete or part of the genome of SBL-6669 and transfect this vector into suitable bacteria, fungi or cells. One way of constructing the recombinant vector is briefly to isolate unintegrated or integrated viral DNA from SBL-6669 infected cells, to cleave the latter form of DNA with a restriction enzyme to obtain a provirus, and to insert said virus into a suitable plasmid. Another way is to isolate virion RNA or mRNA from SBL-6669 infected cells, form double stranded cDNA from said RNA and insert said double stranded cDNA into a phage lambda to form a recombinant DNA molecule, removing cDNA from said molecules and inserting cDNA into a suitable plasmid. In both cases the resulting plasmid is transfected into a suitable bacteria, such as E. coli, fungi or cells.

Still another method to produce SBL-6669 antigens is to synthesize synthetic peptides where the amino acid sequence has been deduced from the primary nucleotide sequence of SBL-6669.

SBL-6669 virus or antigens prepared in any of the ways mentioned above can be used for several purposes as will be shown below.

Use of SBL-6669 virus and antigens

a) Serology

According to a further feature of the invention we provide serological methods for assaying a biological sample for antibody to SBL-6669 viruses by ELISA, Western blotting and radioimmuno precipitation assay. In the ELISA a semi-purified SBL-6669 antigen (see Examples 2 and 3) is bound to an inert support, preferably polystyrene microtiter plates. The plates can be stored for several months. The sample to be tested, for instance serum, is then added. Antibodies specifically directed against SBL-6669 antigen are bound and can be detected by the addition of a labelled antihuman Ig G antibody.

Alternatively a competitive ELISA can be performed where the SBL-6669 antigen is bound to the inert support via antibodies specifically directed against SBL-6669. The sample to be tested is added together with labelled antibodies known to specifically bind to SBL-6669, and test serum competes with the labelled antibodies for the binding sites of the immobilized antigen.

Because of the need of confirming ELISA results with a more specific method we have developed a Western blotting test and a RIPA.

In the Western blotting a semi-purified SBL-6669 is. prepared in the same way as the antigen used in the ELISA (see Example 2). The viral proteins are subjected to acrylamide electrophoresis and then transferred electrophoretically to nitrocellulose which is cut into strips. By incubating the strips with samples to be tested antibodies directed against SBL-6669 are bound to the strips. The bound antibodies are then visualized (see Example 4).

In RIPA viral proteins are labelled by a radioisotope. Either lysed labelled virus infected cells of labelled virions can be used. We have found that best results are obtained by labelling U937 clone 16 cells at the peak of infection and preparing a cell lysate. At the peak of infection almost no production of cellular proteins occurs and hence a fairly pure antigen is obtained although a cell lysate is used. The labelled antigen is incubated with. the sample to be tested and the formed immuno complexes bound to Protein A Sepharose. After washing the immuno complexes are dissolved and the remaining viral antigens are separated by SDS-polyacrylamide electrophoresis whereafter the labelled viral antigens are detected by autoradiography (see Example 5).

In the examples antigen was prepared by radiolabelling of persistently infected HUT 78 cells. This was done to allow comparison with HTLV-IV which we were only able to grow in HUT 78 cells. The virion produced from the HUT 78 cells were lysed and used as RIPA antigen. However, it is more convenient to use a cell lysate in serodiagnosis. To prepare the radiolabelled cell lysate labelling is performed as described in Example 5, however, in this case the radiolabelled cells are collected by centrifugation, washed three times in PBS and lysed in RIPA buffer. The cell lysate is clarified by centrifugation at 15 000 g for 15 minutes.

Both the RIPA and the Western blotting performed according to the instructions in the examples have proven highly specific and sensitive.

b) The use of SBL-6669 antigens in a vaccine against AIDS

A future vaccine against AIDS will probably have to include not only HIV antigens but also HIV related human retrovirus antigens since there is limited if any cross-reaction between the envelope proteins of the two virus groups. The present invention provides SBL-6669 antigens which may prove useful for active immunisation. Since a vaccine for maximum safety should be completely free from virus nucleic acid the strategy will be to use purified virus components, polypeptides expressed in bacteria, fungi or cells transformed with a recombinant vector containing cDNA from SBL-6669 or to use a synthetic SBL-6669 derived polypeptide.

c) Production of polyclonal and monoclonal antibodies against SBL-6669

By injecting SBL-6669 antigens prepared in any of the described ways in an animal an immune response is evoked. Antibodies are produced against a variety of epitopes of the different viral proteins and polyclonal anti-SBL-6669 sera are obtained. Monoclonal antibodies against SBL-6669 can be produced by fusing antibody synthesizing cells from the immunized animal with a suitable cell line by subsequent cloning and identification of antibody secreting clones. These monoclonal and polyclonal antibodies can be used in a variety of ways. One is bonding SBL-6669 antigen to the inert support of the competitive ELISA. Another is to detect SBL-6669 virus or antigens in samples such as body fluids. Basically such an antigen capture test comprises a solid phase where antibodies, human, polyclonal animal or monoclonal are bound to an inert support. The test material is added in a liquid phase and viral antigens, if present, are captured by the antibodies of the solid phase. Bound antigen is detected by the addition of antibodies against the antigen, these antibodies are either directly labelled or detected by a second labelled antibody.

Description of the drawings

Fig. 1 is an electron micrography showing the virus SBL-6669 (14 000 × 7.1).

Fig. 2 shows a cross characterisation of SBL-6669, HTLV-IV, LAV-II and HTLV-IIIB by RIPA. The method used is described in Example 5.

Figs. 3a and 3b show a cross characterisation of SBL-6669, HTLV-IV, LAV-II and HTLV-IIIB by Western blotting. The method used in described in Example 4.

Fig. 4 shows in the form of a table the primary nucleotide sequence and deduced amino acid sequence of proteins of HIV-2 SBL/ISY which is a recombinant cDNA clone-which represents the complete genome of the virus SBL-6699 (=SBL-6689-85). The proviral DNA was obtained from a genomic library constructed from DNA of HUT-78 cells infected with SBL-6669-85 virus using the lambda-phage vector EMBL-3. In the table on Fig. 4 the uppermost row on each page and every fourth row of capitals therebelow discloses the nucleotide base sequence with the capitals A, C, G and T resp. in the DNA, and the three rows below every base sequence row discloses the amino acids, denoted with capitals, and stop codons denoted with asterisks, into which the various DNA base triplets translate. In the amino acid rows the open reading frames are marked with underlining (partially overlapping). Together with the underlining the extension of various genes are also marked on Fig. 4. The clone of Fig. 4 gave infectious virus.

Thus protection is requested for the entire genome disclosed on Fig. 4 and for parts thereof, especially those parts marked by underlining in the figure, and corresponding to various genes as marked on the figure, such as the gag gene (corresponding to nucleotides 547 to 2106 on Fig. 4), the pol gene (nucleotides 1827 to 4931) and the env gene (nucleotides 6144 to 8682), the corresponding amino acid sequences and parts thereof and various products derived therefrom, or use thereof, such as clones prepared by the recombinant vector method, HIV test devices and methods, etc., as is also disclosed or mentioned elsewhere in this text.

Example 1

Virus SBL-6669 was isolated from peripheral blood mononuclear cells (PBMC) from a West African woman with clinical and immunological signs of immuno-deficiency although not fulfilling the criteria for AIDS. The PBMC from the patient was obtained from 30 ml of heparinised blood which was separated by Ficoll-Paque according to the manufacturers' recommendations. 5 × 10⁶ PBMC was cocultured with 5 × 10⁶

PBMC from a healthy sero-negative blood donor. The cells were cultured in RPMI-1640 supplemented by 10% fetal calf serum

(Gioco), 10% T-cell growth factor (Cellular Products), 5 μg/ml hydrocortisone, Streptomycin and penicillin. Particle associated reverse transcriptase activity was transiently observed in the culture during the second and third week of culture. 1 × 10⁶ cells from this culture were cocultivated with 3 × 10⁶ HUT 78 cells on day 3. The lymphocytes disappeared gradually over the course of 1 month. The HUT 78 cells became chronically infected with SBL-6669 virus and continued to produce virus for more than 6 months. The HUT 78 cells do not require addition of T-cell growth factor or hydrocortisone. By cell free infection (50 × 10³ cpm reverse transcriptase activity per 1 × 10⁶ cells) we have infected

U937 clone 2 cells with SBL-6669 virus. Like the HUT 78 cells these U937 clone 2 cells are chronically infected, grow without T-cell growth factor and have produced virus for several months.

SBL-6669 is antigenically closely related to the HIV related human retroviruses LAV-II and HTLV-IV but more distantly related to HTLV-IIIB (see Figs. 2 and 3).

Example 2

Preparation of antigen for ELISA and Western blotting.

Antigen was prepared from SBL-6669 virus grown on CEM and HUT 78 cells. The cells were sedimented and the culture media were concentrated 5- to 10-fold by ultrafiltration in a hollow fibre cassette system (Minisette ^TM, Filtron Scandinavia HB) with filters excluding molecules up to 300 kD. The concentrate was clarified twice by centrifugation at 10 000 g for 15 minutes. The virions were then pelleted at 16 000 g for 16-18 h, suspended in STE-buffer (0.1 M NaCl, 0.01 M

Tris-HCl, pH 7.5, 0.001 M EDTA) and further purified by running through 30% sucrose at 20 000 g for 6 h. The virions were resuspended in 1 ml/STE-buffer with the addition of 0.25%

Triton × 100 per 1 1 culture media. The recovery was 1-2 mg protein/1.

Example 3

Enzyme linked immunosorbent assay (ELISA) for detection of antibodies against HTLV-IV and LAV antigen were performed according to the following protocol. Microtiter plates (Nunc microtiter modules, code No. 4-64394) were coated with

100 μl/well of purified virus antigens diluted to about

5 μg/ml in 0.05 M carbonate buffer, pH 9.6 and left over night at +4°C. The plates were washed three times in phosphate buffered saline (PBS) and three times in distilled water.

100 μl of sera diluted 1/100 in PBS containing 20% fetal calf serum and 0.5% Tween 20 ( PBS-FCS-Tween) were added to each well and incubated 1-2 h at room temperature. After washes as above 100 μl alkaline phosphatase conjugated swine anti-human IgG (Orion Diagnostica) diluted 1/100 in PBS-FCS-Tween were added. After 1-2 h incubation at room temperature the plates were washed as above 100 μl of substrate (p-nitrophenyl phosphate) in 10% diethanolamine buffer pH 9.8 were added to each well. The reaction was allowed to take place for 30 minutes at room temperature and then stopped by adding 50 μl of 3 M sodium hydroxide to each well. Absorbance was read at 405 nm with a Titretek multiscanner (Flow Laboratories). The cut off value was determined as three times the absorbance value of a negative standard serum. Values between two and three times the control serum were suggested as border line values.

Sera from West African patients with suspected tuberculosis were tested for antibodies against SBL-6669 virus with ELISA:

26 positive of 125 tested = 20.8%

50 Swedish blood donors were negative.

The 26 samples that were positive in ELISA as well as a majority of the negative samples have been confirmed by Western blotting tests.

Example 4

Fig. 3 (a and b).

Western blot analysis of viral antigens with human sera.

HTLV-IIIB virions were obtained as a gift from Dr. R.C. Gallo (National Cancer Institute, Bethesda). HTLV-IV, SBL-6669 and LAV-II antigens were prepared from infected HUT 78 cells. Virus from clarified supernatant was concentrated by centrifugation at 13 000 rpm over night followed by centrifugation through 30% sucrose in a SW 28 rotor (Beckman) at 13 000 rpm over night. The virus pellet was resuspended in STE buffer (0.1 M NaCl, 0.01 M Tris-HCl, pH 7.5, 0.001 M EDTA) and stored at -70°C. Before use the virus preparation was diluted in Laemmli sample buffer and boiled for 2 minutes. The viral proteins were separated by SDS-polyacrylamide gel electrophoresis (12% gels) and were then electrophoretically (100 V for three hours) transferred onto nitrocellulose. The nitrocellulose sheet was cut into strips and incubated with 5% dry milk in phosphate buffered saline (PBS) for 30 minutes. Each strip was incubated over night at +4°C with human serum diluted 1:100 in PBS-BSA-T (PBS containing 0.1% bovine serum albumin and 0.1% Tween). After 3 washes the strips were incubated with the following reagents (from Amersham); 1) biotinylated sheep anti-human Ig, 2) streptavidin, 3) biotinylated horseradish peroxidase. The strips were washed after each incubation step. The colour was developed by o-dianisidine (Sigma, 25 mg in 100 ml Tris-HCl supplemented with 0.01% H₂O₂). Indicated human sera were reacted with the following antigens: A: HTLV-IIIB, B: HTLV-IV, C: SBL-6669, D: LAV-II.

Example 5

Fig. 2.

Immunoprecipitation of metabolically labelled viral proteins by human sera. Virus (HTLV-IV, SBL-6669, LAV-II and HTLV-IIIB) propagated in HUT 78 cells was radioactively labelled by incubation for 12-14 hours in medium containing 35 S-cystein (150 μCi/ml). Clarified (10 000 g, 10 minutes) supernatants were centrifuged at 60 000 g for 75 minutes. The pellet was lysed in RIPA buffer (140 mM NaCl, 1 mM dithiotreitol, 10 mM Tris, pH 8.0, 0.035% phenyl-methyl-sulfonylfluoride and 0.5% NP 40). Aliqouts (15-50 μl ) of the lysates were precipitated for 14 hours at 4°C by 4 μl of serum. The immuno complexes were bound to 6 μg Protein A-Sepharose (Pharmacia, Sweden), washed four times in washing buffer (three times in 0.5 M NaCl, 0.001 M EDTA, 0.02 M Tris-HCl, pH 7.6, 1% sodium deoxycholate and 30% sucrose and one time in 0.01 M Tris-HCl, pH 7,6), resuspended in sample buffer (3% SDS, 1 M urea and 3% ß-mercaptoethanol in 1 M Tris-H₃PO₄, pH 6.8) and boiled at 100°C for 3 minutes. Immunoprecipitates were analysed by SDS-polyacrylamide gel electrophoresis on 12% gels with 2.25% stacking gel. Each serum was reacted with the following antigens: A: HTLV-IIIB, B: HTLV-IV, C: SBL-6669, D: LAV-II. C ¹⁴ labelled molecular weight markers

(phosphorylase b 97 000; bovine serum albumin 69 000; ovalbumin 46 000; carbonic anhydrose 30 000; lactoglobulin A

18 500) (lanes marked No. 1) were included in each gel. The apparently weak reactivity of LAV-II serum against HTLV-IIIB antigens was due to a loss of material during the preparation procedure of the experiment used for illustration.

TABLE 1

Approximate size of core and envelope proteins of HTLV-IIIB and of corresponding proteins of HTLV-IV, SBL-6669 and LAV-II.

HTLV-IIIB p19 p24 gp41 gp120

HTLV-IV 17 kD 27 kD 30-34 kD 120 kD

SBL-6669 17.5 kD 26 kD 32 kD (41 kD)* 120 kD

LAV-II 18 kD 26 kD 32-35 kD 120 kD

*Two bands were seen in WB tests.

The disclosure of the following references 1 to 16 is included into this text by reference.

References

1. Coffin, J. et al., Science 232, 697 (1986).

2. Gonda, M.A. et al., Science 227, 173-177 (1985).

3. Wain-Hobson, S., Sonigo, P., Danos, O., Cole, S. and Alizon, M., Cell 40, 9-17 (1985).

4. Sarngadharan, M.G., Popovic, M., Bruch, L., Schupbach, J. and Gallo, R.C., Science 224, 506-508 (1984).

5. Montagnier, L. et al., Virology 144, 283-289 (1985).

6. Allan, J.S. et al., Science 228, 1091-1094 (1985).

7. Barin, F. et al., Science 228, 1094-1096 (1985).

8. Hahn, B.H. et al., Science 232, 1548-1553 (1986).

9. Alizon, M., Wain-Hobson, S., Montagnier, L. and Sonigo, P., Cell 46, 63-74 (1986).

10. Starcich, B.R. et al, Cell 45, 637-648 (1986).

11. Kanki, P.J. et al., Science 232, 238-243 (1986).

12. Clavel, F. et al., Science 233, 343-346 (1986).

13. Åsjö, B. et al., Lancet II, 660-662 (1986).

14. Gazdar, A. et al., Blood 55, 409-417 (1980).

15. Sundström, C. and Nilsson, K., Int. J. Cancer 17, 565-577 (1976).

16. Devare, S.G. et al., Proc. Natl. Acad. Sci. U.S.A. 83, 5718-5722 (1986) .

Claims

1. A human immunodeficiency virus (HIV) related human retrovirus strain, characterized by exhibiting the RIPA (Radio Immuno Precipitation Assay) analysis characteristics shown on Fig. 2 and the Western blotting characteristics shown on Fig. 3a and 3b resp. when treated in the way defined in the specification and/or when compared with one or more of the virus types HTLV-IV, LAV-II and HIV, or especially virus comprising a proviral DNA sequence shown in Fig. 4 or a corresponding RNA sequence or degenerates thereof or a part thereof or a sequence or part thereof which give essentially the same analytical or physiological effects or have essentially the same effects, and including mutants or strains derived thereof having essentially the same characteristics.

2. A virus strain according to claim 1, characterized by exhibiting in RIPA and Western blotting analysis under the conditions stated in the specification proteins and glycoproteins with the approximate size values 17.5 kD, 26 kD,

32 kD, 41 kD, 120 kD.

3. A virus strain according to claim 1 or 2 , characterized in that said virus is integrated into and produced by U937 clone 2 cells denoted U937 clone 2/SBL-6669/85 deposited at The European Collection of Animal Cell Cultures, Porton Down, Salisbury, SP4 OJG, GB according to the rules of the Budapest Convention with the deposition No. 87042402.

4. A virus strain according to any of the preceding claims, characterized by being cultured in cells of cell cultures, preferably in human T-cell lines of the types HUT 78, Jurkat and CEM, or the monocytoid cell lines U937, clone 2 and clone 16.

5. Cells, preferably human T-cell lines of the types HUT 78, Jurkat, CEM, or the monocytoid cell lines U937, clone 2 and clone 16, characterized by being infected with a HIV related human retrovirus strain according to any of the preceding claims.

6. A recombinant vector comprising cDNA representing a virus strain according to any of claims 1-5 and complete virus or parts thereof produced by bacteria, fungi or cells transformed by such a vector.

7. A cDNA clone (HIV-2 SBL/ISY) according to claim 6, characterized by having the primary nucleotide sequence disclosed in Fig. 4, or a degenerate or a part thereof, especially a clone having capacity to give production of infectious virus particles.

8. Proteins or peptides produced by a recombinant vector according to claim 6 or a cDNA clone according to claim 7 or with an amino acid sequence identical with or derived from the deduced amino acid sequence of the proteins of HIV-2 SBL/ISY (disclosed on Fig. 4), especially proteins encoded by the gag gene (corresponding to nucleotides 547-2106 of Fig. 4), the pol gene (nucleotides 1827-4931) and the env gene (nucleotides 6144-8682), and sequences which at more than 90% and especially more than 95% of the positions are identical with any of said sequences, and the generates thereof, or which give essentially the same reactions, especially analysis reactions, or physiological effects.

9. Synthetically produced proteins and peptides, characterized in that the amino acid sequence is derived from the primary nucleotide sequence of a virus according to any of claims 1-5, especially the primary nucleotide sequence of HIV-2 SBL/ISY disclosed on Fig. 4 or a part thereof, or a degenerate thereof.

10. Proteins and glycoproteins which have been purified and recovered, preferably by affinity chromatography, from virus or products according to any of claims 1-9 or cells infected with such virus or from culture medium containing parts of such virus.

11. A process for producing the virus according to any of the preceding claims by infecting susceptible cells, preferably human T-cell lines of the types HUT 78, Jurkat and CEM, or the monocytoid cell lines U937, clone 2 and clone 16, characterized in that said cells are infected by cocultivation with cells infected with the virus according to any of the preceding claims or by cell free infection with said virus.

12. The use of virus or proteins and peptides derived therefrom according to any of the preceding claims, especially an env gene derived protein or peptide, as an antigen for assay of the presence of infection with HIV related human retrovirus or HIV, or for preparing assay devices for assay of the presence thereof or vaccine against said infections.

13. The use of virus or proteins and peptides derived therefrom according to any of the preceding claims for preparing polyclonal or monoclonal antibodies against virus, especially for indicating the presence of HIV related human retrovirus or part thereof in a sample, e.g. in serum, by an antigen capture test.

14. A process for detecting antibodies which specifically bind to antigenic parts of the virus or proteins and peptides derived therefrom according to any of the preceding claims in body fluids from persons infected with said virus or with related human retrovirus, characterized by bringing the virus or proteins and peptides derived therefrom according to any of the preceding claims into contact with a sample suspected to contain such antibodies and measuring the quantity of antigen-antibody complex formed, e.g. with RIPA, Western blotting, ELISA or indirect immunofluorescence methods.