FR2597500A1 - New retrovirus capable of causing AIDS, antigens obtained from this retrovirus and corresponding antibodies and their applications to the diagnosis of AIDS - Google Patents

Abstract

The invention relates to a CEM strain which makes it possible to culture the virus LAV-II. The LAV-II virus-infected CEM cell line has been deposited with the Collection Nationale de Cultures de Micro-organismes (CNCM) of Institut Pasteur under the number I-No. 537 on 24 March 1986.

Description

New retrovirus capable of causing AIDS, antigens obtained from this retrovirus and corresponding antibodies and their ssDp7 ications to the diagnosis of
AIDS.

 The invention relates to a new form of virus, having the capacity to cause lymphadenopathies which can then be relayed by the acquired immunodeficiency syndrome (AIDS). The invention also relates to antigens capable of being obtained from this virus or having properties with it in common. It also relates to the antibodies capable of being provoked against these various antigens.

Finally, the invention relates to applications of these antigens or antibodies to the diagnosis of certain forms of the
SIDA and, with regard to some of them, to the production of immunogenic compositions and of vaccine compositions against this retrovirus.

The isolation of a first retrovirus, for which responsibility for the development of the
SIDA, was the subject of a description in an article by
F. BARRE-SINOUSSI et al of 1983 (Science, vol. 220, n 45-99, 20, p. 868-871. Application to the diagnosis of the presence of antibodies against the virus of certain extracts of the latter, and more particularly of some of its proteins, has been more specifically described in European patent application No. 138.66 ?. This retrovirus is known by the designation LAV. Since other similar strains and variants of LAV have been isolated. for the record those known by the designations
HTLV-III and ARV. The expression ZLAV-I also covers all of these designations and the corresponding viral strains. All the viruses identical or close to the initial isolate "LAV" will be called in this text "LAV type I" or "LAV-I". The new retrovirus which is the subject of this patent and the strains of virus which are close to it, are called LAV type II "or IAV-II.

Each isolate is followed by the first three letters of the name of the patient from whom it was isolated. We can define the set "LAV" against a set of viruses causing either generalized and persistent polyadenopathies, or AIDS and having in vitro a tropism for T4 cells in which the retrovirus induces a cytopathogenic effect. These retroviruses have been shown to be distinct from other human retroviruses already known (HTLV-I and HTLV-II).

 Although there is a fairly large genetic variability of the virus, the different strains isolated to date from American, European, Haitian and African patients have in common antigenic sites conserved on their main proteins: core protein p25 and glycoprotein of envelope gap110 and transmembrane protein gp41-43. This allows the LAV-I prototype strain deposited at the CNCM under the number 1-232 to be used as an antigen strain for the detection of antibodies in all types of patients, whatever their origin. This strain, including the HTLV-III virus isolated by R.C. Gallo et al. cannot be distinguished, is therefore currently used for the detection of antibodies in blood donors and patients by ELISA, Immunofluorescence, the techniques known as "Western Blot" (or immunoblotting) and "RIPA" (Radio Immunoprecipitation Assay ).

However in a serological study carried out on patients born in Guinea-Bissau and hospitalized in
In Portugal, it was observed that some gave sero-negative or very weakly positive reactions by these tests using a LAV-I lysate, while they presented the clinical and immunological signs of AIDS.

 From the lymphocytes cultured in one of these patients1, a retrovirus was isolated, the structure of which in electron microscopy and the profile of the proteins in SDS electrophoresis gel are generally similar to those of LAV. -I. But this new retrovirus, hereinafter called LAV-II, has a lesser kinship, if not a weak kinship with it, both from the point of view of the antigenic homology of its proteins than of the homology of its genetic material.

This new retrovirus or retroviruses having equivalent antigenic and immunological properties, can therefore constitute sources of antigens for the diagnosis of infection by this virus and variants which induce AIDS, which are encountered in particular in patients. Africans or people who have stayed in
Africa.

This virus has been isolated from several patients with
Guinea-Bissau and the Cape Verde Islands, and in particular from the blood, taken under heparin, of a 28-year-old patient, originally from Guinea-Bissau, heterosexual, who had never been transfused and who never was not an addict. Since 1983, he had severe chronic diarrhea, significant weight loss (17 kg) with intermittent fever. Recently he presented Candida and Serratia infections, including esophageal candidiasis, typical of AIDS.

This patient also had anemia, skin anergy, lymphopenia, lymphocyte report
T4 / T8 lymphocytes of 0.15, with a level of T4 lymphocytes less than 100 per serum. His cultured lymphocytes did not respond to stimulation by phyto-hemoglutinin and cencanavalin A. This patient was also diagnosed with recurrent bacteremia due to S.

 enteriditis. cryptosporidioses, AEosoora belli infections and cerebral toxoplasmosis.

All of these signs testified to the complex symptoms linked to AIDS "or" ARC "(English abbreviation for
RAIDS Related Complex "), of the type caused by the LAV-I virus. These various observations also met the criteria applied by the Atlanta Center of Disease Control (CDC),
United States.

 The culture of the lymphocytes of these patients and the isolation of the retrovirus were carried out according to the technique already described for the isolation of LAV-I (1) in the article by BARRE-SINOUSSI et al. and European patent application no. 84 401834/0 138 667. These are briefly recalled below. Lymphocytes stimulated for 3 days with phytohemagglutinin (PHA) were cultured in RPM1-16-40 culture medium supplemented with 10 O of fetal calf serum and 10 5M B-mercaptoethanol, interleukin 2 and anti serum - human interferon.

 Virus production was followed by its reverse transcriptase activity. In the culture supernatant, the peak of viral activity appeared between the 14th and the 22nd day, then decreased. The decline and death of cell culture followed. As with LAV-I, sections of lymphocytes infected with LAV-II reveal mature virions and viral particles budding on the surface of infected cells.

The virus was then propagated on cultures of lymphocytes of blood donors, then on continuous lines of leukemic origin, such as HUT 78. It was characterized as being substantially distinct from LAV-I by its antigens and its nucleic acid . The virus has been purified as described in the earlier documents already mentioned. It has been deposited in the "National Collection of
Cultures of Microorganismsw (CNCM) at the INSTITUTE
PASTEUR, December 19, 1985, under nv I-502. In general, the invention relates to any equivalent virus containing structural proteins which have the same immunological properties as those of the LAV-II virus deposited at the CNCM under the number I-502.

 Some characteristics of the antigens and of the nucleic acids, which form part of the composition of LAV-II, result from the tests carried out under the conditions indicated below.

I - ANTIGENS. ESPECIALLY PROTEINS AND GLYCOPRO
TEINES
The virus was metabolically labeled with cysteine and 355-methionine, the infected cells being incubated in the presence of these radioactive amino acids in a culture medium devoid of the corresponding unlabeled amino acid, for a period of 14 to 16 hours. (in particular according to the technique described in the article listed in (21) in the bibliography presented at the end of the description, as regards labeling with 35 S-cysteine. The supernatant is then clarified, then the ultracentrifuged virus one hour at 100,000 g on a 20 s sucrose cushion. The main antigens of the virus are separated by electrophoresis in a polyacrylamide gel (12.5 5) under denaturing conditions (SDS) or in a polyacrylamide gel (10 5) + bis acrylamide (0.13 -.) With SDS (0.1 's in final concentration).

The following colored markers marketed by the company BRL are used as reference molecular weight.
myosin: 200 Kd
phosphorylase B: 97.4 Kd
BSA: 68 Kd
ovalbumin: 43 Kd
chymotrypsin: 25.7 Kd
8 lactoglobulin: 18.4 Kd
lysozyme: 14.3 Kd
Other molecular weight markers have been used in other trials. Reference is made in particular to FIGS. 1a, 1b and 1c which refer to other molecular weight markers known under the letter
M. Antigens are even better distinguished after immunoprecipitation (RIPA) or by immunoblotting (Western blot) using the antibodies present in the patient's serum: their molecular weights determined by their apparent migrations are very close to those of r'AV antigens -I. These molecular weights are expressed in kilodaltons after the corresponding letters Zp or ZgpW, even if in certain cases, when specified, these molecular weights can vary to a certain extent around the indicated value.

 The repetition of the tests made it possible to better define the molecular weights of the LAV-ii antigens. Thus it was found that the molecular weights of the three core proteins, to which we had initially assigned molecular weights of the order of 13,000, 18,000 and 25,000 respectively, had in fact molecular weights closer to the following values: 12,000 , 16,000 and 18,000, respectively. These proteins are hereinafter respectively designated by the abbreviations p12, p16 and p26.

 Similarly, the existence of a transembembrane glycoprotein was quickly recognized, its molecular weight having been assessed at values which could range from 32,000 to 42,000. The repetition of the measurements finally made it possible to specify the molecular weight of this transmembrane glycoprotein: 36,000. In what follows, this glycoprotein is designated by the abbreviation gp36. A major envelope glycoprotein having a molecular weight of the order of 130-140 Kd is constantly observed: this glycoprotein is hereinafter designated by the expression gp140. That said, it should be noted that, in general, the molecular weights are assessed with an accuracy of plus or minus 5, this accuracy may even become a little less for the high molecular weight antigens as we will already have observed for the gap140 or also for a protein which appears to consist of a precursor of gp140, hereinafter designated gp160 (molecular weight of 160 Kd + 10 a).

All of these antigens have been little or not recognized (when labeled) by 355-cysteine by patient sera containing anti-LAV-I antibodies in the detection systems used in the laboratory or by the use of tests using LAV-i lysates, such as those marketed by Diagnostics
Pastor under the brand "ELAVIA". Only the p26 protein was weakly immunoprecipitated by such sera. The envelope protein was not. The serum of the patient infected with the new virus (LAV-II) weakly recognizes a p34 protein of LAV-i. In the detection system used, the other proteins in LAV-I were not recognized.

On the other hand, LAV-II has certain proteins having a certain immunological relationship with similar structural proteins or glycoproteins separable under analogous conditions from a retrovirus recently isolated from captive macaque monkeys, whereas this immunological relationship tends to be erased for other proteins or glycoproteins. The latter retrovirus, which is presumed to be the etiological agent of
AIDS in monkeys, has been designated by the researchers who isolated it (bibliographical references (16-18) below) under the name ZSTLV-IIImac ". It was deposited on February 7, 1986 in the National Collection of
Culture of Micro-Organisms (CNCM) of the Institute
Pastor of Paris under number I-521. For the convenience of language, it will no longer be designated in what follows only by the expression "STLV-III".

 By using the same techniques as those mentioned above, it was found that it was also possible to obtain, from 5TLV-III: - a main protein of the p27 nucleus, having a molecular weight of the order of 27 kilodaltons , - a major envelope glycoprotein, gp140, - a p32 transmembrane protein, which is not observed in RIPA when the virus has previously been labeled with 355-cysteine, but which can be observed in immunoblotting tests (Western blots), in the form of wide bands.

 The major envelope glycoprotein of LAV-II has been shown to be closer to the major envelope glycoprotein of STLV-III than to the major envelope glycoprotein of lav-I.

These observations are essential not only at the molecular weight level: 130-140 kilodaltons for the major glycoproteins of LAV-II and STLV-III, against around 110 for the major envelope glycoprotein of LAVIr but also at the level of immunological properties. since sera taken from patients infected with LAV-II, and more particularly antibodies formed against the gp140 of LAV-II recognize the gp140 of
STLV-III, whereas in similar tests the same sera and the same antibodies of LAV-II do not recognize the gel 10 of LAV-I. But the anti-LAV-I sera which have never reacted with gp140 LAV-II precipitate a 26 Kdal protein labeled with 32S-cysteine, contained in extracts of LAV-Ii.

 The major core protein of LAV-II appears to have an average molecular weight (approximately 26,000) intermediate between that of p25 of LAV-I and p27 of STLV-III.

 Similarly, a major transmembrane protein of LAV-Ii has an average molecular weight, approximately 36,000, between the molecular weight of the corresponding transmembrane protein (approximately 41,000 for p41) and that of the corresponding protein, p32, of STLV-III.

These observations result from tests carried out with viral extracts obtained from LA-II isolated from one of the abovementioned patients. Similar results have been obtained with viral extracts from
LAV-II isolated from the second patient.

In what follows, reference will be made to the drawing in which - FIGS. 1a, 1b and 1c relate to cross immunoprecipitation tests between sera of patients infected respectively with LAV-I and LAV-II and of macaques infected with STLV -III, on the one hand, and viral extracts of LAV-f, on the other hand - Figures 2a and 2b present comparative results as to the electrophoretic mobilities of the proteins of LAV-I, LAV-II and STLV-III , in polyacrylamide-SDS gels - FIG. 3 presents results of cross-hybridization between genomic sequences of LAV-I, LAV-II and
STLV-III, on the one hand, and probes containing different subgenomic sequences of the LAV-I virus, on the other hand.

 In general, the antigens and DNA sequences derived from the genomic DNA of LAV-I used in the comparative tests, the description of which follows, come from the strain of LAV-I deposited at the C.N.C.M. under the number I-232 on July 15, 1983.

Cells infected respectively with LAV-I,
LAV-II and STLV-III were incubated in medium containing 200 µCi / ml cysteine in unlabeled cysteine free medium for 16 hours. The clarified supernatants were centrifuged at 60,000 xg for 90 minutes. The pellets were lysed in RIPA buffer (1), immunoprecipitated with various sera, then subjected to electrophoresis on polyacrylamide gel loaded with sodium dodecylsulfate (SDS-PACE).

 The results observed are illustrated by Figures la, lb and lc.

In FIG. 1a, the immunoprecipitation results observed between a viral extract of
LAV-I obtained from a CEM cell line C1.13 and the following respective sera - anti-LAV positive serum (lane 1), - serum obtained from the first patient mentioned above (lane 2), - serum from a healthy African carrier of anti-LAV antibodies (lane 3), - serum obtained from a macaque infected with STLV-III (lane 4) and - serum of the second patient mentioned above (lane 5).

 In FIG. 1b, the immunoprecipitation results observed between the LAV Xi antigens obtained from the first patient are reported, after prior culture on HUT-78 cells and various sera, more particularly the serum of the first patient mentioned above (band 1 ), the anti-LAV positive serum (lane 2), the serum of the macaque infected with STLV-III (lane 3), the serum of the aforementioned second patient (lane 4).

 Finally, FIG. 1c illustrates the immunoprecipitation results observed between the antigens of an STLV-III isolate obtained from a macaque presenting a monkey AIDS. The sera used and to which bands 1 to 5 refer are the same as those mentioned above in connection with FIG.

 M refers to the markers myosin (200 Kd) galactosidase (130 Kd), bovine serum albumin (69 Kd), phosphorylase B (93 Kd), ovalbumin (46 Kd) and carbonate anhydrase (30 Kd).

 Figures 2a and 2b present the comparative results with regard to the electrophoretic mobilities of the proteins of LAV-i, LAV-II and STLV-III.

 FIG. 2a relates to the tests carried out with extracts of virus labeled with 35S-cysteine, after itsunoprecipitation on SDS-PAGE. The different bands relate to the following virus extracts: virus obtained from patient 1 and immunoprecipitated by the serum coming from the same patient (lane 1), extracted from the same virus iflunoprecipitated with a negative control serum coming from a person not carrying anti-LAV-i or anti-LAV-II antibodies (lane 2), extract of STLV-tII virus immunoprecipitated by a serum from a macaque infected with STLV-III (lane 3), immunoprecipitations observed between extracts of same virus and a negative control serum (lane 4), extracted from LAV-I immunoprecipitated by a serum from a European patient infected with AIDS.

Figure 1b presents the results obtained in Western-blot (immunoblotting) tests. Cell lysates from uninfected or infected HUT-78 cells were electrophoresed on SDS
PAGE, then electrophoretically transferred to a nitrocellulose filter, before being reacted with the serum of the first patient mentioned above (serum diluted to 1 / 1COe).

the nitrocellulose filter was then washed and the detection of fixed antibodies revealed with anti IgG
125 goat humans marked with i.

 The spots observed in bands 1, 2 and 3 relate respectively to agglutination tests between the above-mentioned serum and extracts of uninfected HUT-78 cells (band 1), extracts of HUT-78 cells infected with an LAV virus. -II (lane 2) and extracts of HUT-78 cells infected with STLV-III (lane 3). The numbers appearing in the margins of each of the bands correspond to the approximate molecular weights of the most representative viral proteins (molecular weights in kilodaltons).

Il - NUCLEIOUES ACIDS
The virus RNA, deposited on a filter according to the "spot blot" technique, did not hybridize, under stringent conditions, with the DNA of tAV-I. By "stringent conditions" is meant: the conditions under which the hybridization reaction is carried out by putting the RNA of the
LAV-II in contact with the chosen probe radioactively labeled with P32 (or marked differently), namely at 42 "C in the presence of 50% formamide for 18 hours.

The membrane on which the hybridization reaction was carried out is then washed at 65-C in a buffer containing 0.1 a of SDS and 0.1 X SSC.

 By "non-stringent conditions *" is meant the conditions under which the hybridization reaction is carried out by bringing into contact with the selected probe marked with p32 (od marked otherwise), namely at 42 ° C. in a 2 × SSC buffer. in the presence of formamide 30 0 for 18 hours The washing of the membrane is carried out at 50 ° C. with a buffer containing 0.1 E of SDS and 2 X SSC.

 Hybridization tests were also carried out with a hybridization probe constituted by a recombinant plasmid pBT1 obtained by cloning the DNA of LAV-I from AJ19 (Cell 1985, vol. 40, p.9) in the vector. pUC18. In non-stringent conditions, only a very weak hybridization was observed.

Other probes were used
a) Single-stranded probes of the subgenomic LAV-I DNA produced from subclones of the genome of
LAV-I and inserted into phage M13. The cloned regions concerned the protease gene or the "endonuclease" gene.

 Only a probe of the endonuclease region of LAV-I (nucleotide sequence between bases n 3760 and 4130) gave weak hybridization under non-stringent conditions with tAV-II. The "protease" probe (nucleotide sequence of LAV-I between the bases n '1680 and 1804) did not hybridize, even under non-stringent conditions with tAV-II.

 b) A pRS3 probe constituted by the sequence coding for the “envelope” region of LAV-I (subcloning in pUC18) did not give hybridization under non-stringent conditions with LAV-Ii.

 The "spot blot" technique is also called "dot blot" (spot transfer).

 Additional hybridization results between tÂV-I, LAV-II and STLV-III genomic RNAs, on the one hand, and probes containing different subgenomic sequences of the tAV-I virus, on the other hand, appear in Figure 3.

The supernatants of the various culture media (at a rate of 0.5 to 1 ml for each stain) were centrifuged for 5 minutes at 45,000 rpm; the pellets were resuspended in an NTE buffer containing 0.1% SDS and deposited on a nitrocellulose filter, which was pre-soaked in 2 x SSC medium (0.3 M NaCl, 0.03 sodium citrate M). After baking (for 2 hours at 80 "C), the filters were hybridized with various probes containing genomic subfragments of LAV-I, under non-stringent conditions (30"'of formamide, 5 x SSC, 40 'C), washed with 2 x solution
SSC and containing 0.1 '; of SDS, at 50 .C, then autoradiographed for 48 hours at -70iC with amplification screens.

Probes 1-4 are single stranded probes obtained by the "prime cut method" as described in (25). In short, single-stranded fragments from the M13 virus and carrying inserts of
LAV-I subgenomics (30) were ligated to fragments of oligomers (17 nucleotides) derived from M13 (BIOLABS). The complementary strand was then synthesized with the Klenow enzyme in a TM buffer (10 mM Tris, pH 7.5, 10 mM MgCl21) in the presence of dATP, dGTP, dTTP and dCTB labeled with alpha 32Pen (Amersham, 3000 Ci / mMol). The DNA was then digested with the appropriate restriction enzymes, denatured by heat and subjected to electrophoresis on a denaturing polyacrylamide gel (containing 6 S of acrylamide, 8 M urea in a buffer TDE). The gel was then autoradiographed for 5 minutes. The probe was then cut and eluted in 300 mM NaCl buffer, 0.1 s SDS.

Specific activities (AS) of these single-strand probes were estimated at S.108-10 disintegrations per minute / microgram (dpm / ijg).

 The characteristic sequences contained in the various probes were as follows probe 1: nucleotides 990-1070, probe 2: nucleotides 980-1260, probe 3: nucleotides 2170-2240, probe 4: nucleotides 3370-3640.

 The numbers of the above nucleotides are those envisaged in the article referenced in (30).

 Finally, probe 5 consists of a plasmid pUC-18 carrying the EcoR1-Sacl fragment of LAV cloned in AJ19 (31), which was subjected to a nick-translation to obtain an AS of approximately 108 dpm / g.

 The relative arrangements of the subgenic fragments contained in the probes with respect to the whole genome of LAV-I are shown diagrammatically in FIG. 5. The different spots correspond respectively - spot A: viruses obtained from a culture of cells CEM C1.13 infected with LAV-I, - virus stain obtained from HUT-78 cells infected with STLV-III, - spots C and D: isolates respectively obtained from the viruses of the two above-mentioned African patients, - stain E : negative control cell extract obtained from uninfected HUT-78 cells, - spot F: virus obtained from a Zabre patient affected by AIDS and having been cultured on normal T lymphocytes in the presence of TCGF.

 All spots were obtained with an amount of virus corresponding to 25,000 dpm in reverse transcriptase activity, except for spots C: 15,000 dpm.

The observations made were as follows
The genomic RNAs of the two LAV-II isolates, obtained from purified viral particles, did not hybridize with any of the probes under the stringent conditions mentioned above, although the viral particles were isolated and purified from supernatants from cultures of highly infected cells showing high reverse transcriptase activity.

 In the above non-stringent conditions, the following observations were made.

 All the probes hybridized intensively with the genomic RNAs obtained from the control preparations of LAV-I and of another isolate obtained from a patient of Zaire suffering from AIDS.

Two of the probes obtained (nucleotides 990-1070 and 990-1260, both from the GAG region) hybridized slightly with the spots of the extracts of the LÂV-II retroviruses; only one of these two probes (nucleotides 990-1260) also showed slight hybridization with the stain of
STLV-III (Figure 5). As regards the probe containing a fragment of the D01 region (nucleotides 2170-2240), hybridization was observed with STLV-III and, but much weaker, with the RNA of LAV-II. The other probe from the pol region (nucleotides 3370-3640) did not hybridize with any of the LAV-II and STLV-III spots.

Finally, the probe modified by cleavage translation and containing the entire env gene and the LTR (nucleotides 5290-9130) neither hybridized with the RNAs of
STLV-III, nor with those of LAV-iI.

 It will also be noted that another probe, which contained the 5 ′ end of the pol reading frame (corresponding to the protease region) did not hybridize either with the LAV-iI RNAs or with the STLV-III RNAs .

 it therefore also follows from the above that the LAV-II virus appears to be more distant, on a structural level, from the LAV-I virus than it is from STLV-III, from which it nevertheless differs significantly.

The invention also relates, in addition to the virus
LAV-II and its variants, any equivalent virus having immunological characteristics specific to LAV-II.

In general, the invention relates to any virus which, in addition to the properties possessed by LAV-II deposited at the
CNCM, also has the following characteristics.

 The preferential target of the LAV-II retrovirus is constituted by human Leu 3 cells (or T4 lymphocytes) and for "immortalized" cells derived from these T4 lymphocytes, for example the cells of the HUT-78 lineages considered in the context of this application . In other words, he has a specific tropism ~ for these cells. it can be cultivated in permanent lines of the HUT type or expressing the T4 protein. It is not infectious for T8 lymphocytes. It is cytoxic to human T4 lymphocytes. It has a reverse transcriptase activity requiring the presence of Mg 2+ ions and having a strong affinity for poly (adenylateoligodeoxy-thymidylase) (poly (A) -oligo (dT) 12-18). It has a density of 1.16 in a sucrose gradient. It has an average diameter of 140 nanometers and a core with an average diameter of 41 nanometers. The lysates of this virus contain a p26 protein which does not immunologically cross with the p24 protein of the HTLV-I virus or of the HTLV-II virus. These lysates also contain a p16 protein which is not recognized immunologically by the p19 protein of HTLV -I or HTLV-II in RIPA radioimmunoprecipitation tests (abbreviation of the English expression Zradioimmunoprecipitation assay "). They also contain an envelope glycoprotein having a molecular weight of the order of 130,000-140,000 which does not cross immunologically with the gap110 of LAV-I, but which in contrast crosses immunologically with the envelope glycoprotein gp140 of STLV-III. These lysates also contain a precursor of this glycoprotein having a molecular weight of the order of 160,000. still contain a transmembrane glycoprotein, markable by 355-cysteine, having a molecular weight of the order of 36,000. The genomic RNA of LAV-II does not hybridize with the genomic RNA of LAV-I under stringent conditions. Under non-stringent conditions, it does not hybridize, neither with the nucleotide sequence derived from LAV-I and containing the env gene and the LTR which adjoins it, more particularly with the nucleotide sequence 5290-9130 of tAV-I, nor with sequences from the Pol region of the LAV-I genome, in particular with the nucleotide sequence 2170-2240.

Under non-stringent conditions, it hybridizes weakly with nucleotide sequences of the LAV-I region, in particular the nucleotide sequences 990-1070 and 990-1260.

 The restriction map and the sequence of the genomic RNA or of the cDNAs obtained from these genomic RNAs are accessible to those skilled in the art, as soon as the LAV-II strain deposited at the C.N.C.M. may, after appropriate multiplication, place in its hands the genetic material necessary for the determination of these restriction maps and nucleotide sequences. it should be noted that any virus capable of inducing AIDS and whose genomic RNA is capable of hybridizing under the usual conditions with that of the viral strain deposited at the C.N.C.M. (or with a cDNA or fragment of cDNA derived from the genomic RNA of LAV-II deposited at the CNCM) must be considered as an equivalent of LAV-II. This is particularly so when these equivalent viruses are characterized by properties essentials substantially analogous to those mentioned above for the LAV-II strain deposited at the CNCM

 The above notion of equivalence also extends to viruses, the extracts or lysates of which contain antigens of the above-mentioned type and which lead to cross-immunological reactions with the antigens of LAV-II deposited at the C.N.C.M.

The invention also relates to a process for producing the LAV-II virus in permanent cell lines derived from T4 lymphocytes, for example of the HUT 78 cell type (line deposited at the CNCM under the number I-519 on February 6, 1986), this process consisting in cultivating these lines previously infected with the tAV-ii virus, and in recovering the quantities of virus released in the culture medium. The prior infection can in particular be carried out as follows
The HUT 78 cells (106 / ml) are cocultured with normal infected human lymphocytes (106 / ml). The culture medium is RPMI 1640 with 10% fetal calf serum. After 15 to 21 days, a cytopathogenic effect of the HUT 78 cells is observed. The reverse transcriptase is assayed one week after this observation, in the culture supernatant. We can then start to recover the virus from this supernatant.

 The invention also relates to each of the antigens, in particular proteins and glycoproteins in the purified state, as they can be obtained from LAV-II. One speaks of purified proteins or glycoproteins * when these proteins or glycoproteins lead respectively only to single bands in electrophoresis on polyacrylamide gel, in particular under the experimental conditions which have been indicated above. Any suitable method of separation and / or purification to obtain each of them can be used, as an example of a technique that can be used, that described by RC MONTELARO et al., J. of Virology, June 1982, pp. 1029-1038.

 In general, the invention relates to all antigens, in particular proteins, glycoproteins or polypeptides having immunological properties equivalent to those of LAV-II. Two antigens are said to be "equivalent" in the context of this description, as soon as they are recognized by the same antibodies, in particular antibodies which can be isolated from a serum obtained from a patient who has had an infection. with an LAV-iI, or as soon as they meet the conditions of "immunological equivalence" indicated below.

 Among the equivalent polypeptides, proteins or glycoproteins, fragments of the above antigens (or ppeptides reconstituted by chemical synthesis) must be classified, since they give rise to cross immunological reactions with the antigens from which they are derived. In other words, the invention relates to any polypeptide having, in common with the above antigens, identical or similar epitopes, capable of being recognized by the same antibodies. The latter type of polypeptides include the expression products of corresponding sequences of the DNAs coding for the corresponding polypeptide sequences.

The LAV-II virus has proven to be usable as a source of antigens for the detection of antibodies in all people who have come into contact with the virus.
LAV-II. The different LAV-II antigens have been recognized by sera from other patients of
Guinea-Bissau with ARC, or asymptomatic people whose antibodies immunoprecipitate proteins from
LAV-II
In general, the invention relates to any composition which can be used for the diagnosis of the presence in a biological fluid serum, in particular of persons having been brought into contact with LAV-II, of antibodies against at least one of the antigens of LAV-II. This composition can be applied to the selective diagnosis of the corresponding variety of AIDS, using diagnostic techniques, as described in the European patent application cited above, except that extracts, lysates or purified antigens of LAV-II are used instead of those of LAV-I. In this regard, the invention relates more particularly to compositions containing at least one of the proteins p12, p16, p26, in the case of internal proteins, or at least one of the glycoproteins gp36, gp40 or gap160. Mention will be made, as examples of compositions, of those which simultaneously contain: - p26 and gp36, - p26, gp36, gp140 and, where appropriate, gp160, - p12, p16 and p26, - p16, p26 and gp 140 ....

 it goes without saying that these compositions are only examples. In particular, the invention relates to viral extracts or lysates containing all of these proteins and / or glycoproteins or any fractions from which one or more of the above proteins or glycoproteins have previously been separated.

It should also be understood that the expression compositions containing a protein or glycoprotein of this virus ", should not be understood as limited to extracts or lysates of the LAV-II virus
The invention also relates to compositions combining proteins and / or glycoproteins of a tAV-II, with proteins and / or glycoproteins of an LAV-I, for example
- either proteins of the core of LAV-I and tAV-Ii, in particular p25 of LAV-I and p26 of LAV-II, or else p18 of LAV-I and p16 an LAV-II,
- either LAV-I envelope glycoproteins and LAV-II envelope glycoproteins, in particular LAV-I gp110 and LAV-Ii gp140, or LAV gp42 and gp36 from LAV-iI,
- or naturally mixtures of proteins and / or glycoproteins of an LAV-I and proteins and / or glycoproteins of an LAV-ii.

 Such compositions, used for diagnosis, therefore allow operations for diagnosing AIDS or the symptoms associated with it, which extend over a wider spectrum of causative agents. it goes without saying that the use for the operations of diagnosis of compositions which contain only proteins and / or glycoproteins of LAV-II is nonetheless useful for more selective diagnoses of the category of retrovirus which can be held responsible for the disease.

The invention also relates to DNAs or fragments of DNAs, more particularly DNAs and fragments of cloned DNAs obtained from RNA or from cDNAs derived from RNA of the LAV-II retrovirus. The invention also particularly relates to all equivalent DNAs, in particular any DNA having sequence homologies with the DNA of the
LAV-II, in particular with the sequences coding for the env and Dol regions of the LAV-II strain deposited at the
CNCM, at least equal to 70 0, preferably 90
In general, it will also be said that the invention relates to any equivalent DNA (or RNA) capable of hybridizing with the DNA or RNA of LAV-II in the spot blot technique "under non-stringent conditions such as as defined above.

 The invention likewise relates to sera capable of being produced in animals by inoculation therewith with LAV-ii. The invention therefore relates more particularly to polyclonal antibodies more specifically directed against each of the antigens1, in particular proteins or glycoproteins of the virus. It also relates to the monoclonal antibodies which can be produced by conventional techniques, these monoclonal antibodies being directed more specifically respectively against the various proteins of LAV-II.

 These polyclonal or monoclonal antibodies can be used in different applications. We will mainly mention their use to neutralize the corresponding proteins, or even inhibit the infectivity of the whole virus. They can also be used, for example, to demonstrate viral antigens in biological preparations or to carry out purification operations of the corresponding proteins and / or glycoproteins, for example by their use in affinity chromatography columns.

it is understood that, in general, the technical literature available (in particular that of which the bibliographical references are recalled within the framework of the present description) as regards LAV-I and the virus called HTLV-III must be considered as part of the present invention, since the techniques described by this literature apply under conditions similar to the isolation of the virus
LAV-II or equivalent viruses, for obtaining from these viruses their various constituents (in particular proteins, glycoproteins, polypeptides, nucleic acids).

We can also call on the lessons of this technical literature with regard to the application of the various constituents concerned, in particular to diagnostic operations of the corresponding forms of ALS or AIDS.

 The present invention relates more particularly to a method of in vitro diagnosis of AIDS, which comprises bringing a serum or another biological medium coming from a patient who is the subject of the diagnosis into contact with a composition containing one at least LAV-II proteins or glycoproteins, or an extract or lysate of the virus, and the detection of the immunological reaction. Examples of such compositions have been indicated above.

 Preferred methods involve, for example, ELISA or immunofluorescence immunoenzymatic reactions. The titrations can be measurements by direct or indirect immunofluorescence or direct or indirect immunoenzymatic assays.

 Thus the present invention also relates to virus extracts (either from an extract of one or more LAV-II only, or from a mixture of extracts originating from one or more LAV-II, on the one hand , and one or more LAV-I, on the other hand), these extracts being marked. Any suitable type of marker can be used: enzymatic, fluorescent, radioactive, etc.

 Such titrations include, for example - the deposit of determined quantities of the extract or targeted compositions in accordance with the present invention in the wells of a titration microplate - the introduction into these wells of increasing dilutions of the serum to be diagnosed e - incubation of the microplate - careful washing of the microplate with an appropriate buffer - introduction into the microplate wells of labeled antibodies specific for human immunoglobulins, the labeling being carried out by an enzyme chosen from those which are capable hydrolyze a substrate so that the latter then undergoes a modification of its absorption of radiation, at least in a band of determined wavelengths and - the detection, preferably in a comparative manner with respect to a control, of the importance of substrate hydrolysis as a measure of potential risk or actual presence of the disease.

 The present invention also relates to kits or “kits” for the above diagnosis, which comprise - an extract or a more purified fraction of the types of virus indicated above, this extract or fraction being labeled, for example so radioactive, enzymatic or immunofluorescence - human anti-immunoglobulins or protein A (advantageously fixed on a support insoluble in water, such as agarose beads) - an extract of lymphocytes obtained from a person in good health - tampons and, if necessary, substrates for viewing the marking.

 The invention naturally also relates to the use of cDNAs or their fragments (or of recombinants containing them) as probes, for the diagnosis of the presence or not of LAV-II virus in samples of sera or of other liquids or biological tissues obtained from patients suspected of being carriers of the LAV-II virus. These probes are preferably also labeled (radioactive, enzymatic, fluorescent markers, etc.). Detection can be carried out in any way known per se, in particular by bringing these probes into contact with either the nucleic acids obtained from the cells contained in these sera or other biological media, for example cerebrospinal fluids, saliva, etc. , either with these media themselves as soon as their nucleic acids have been made accessible to hybridization with these probes, and this under conditions allowing hybridization between these probes and these nucleic acids and by detection of hybridization possibly produced. The above diagnosis involving hybridization reactions can also be carried out using mixtures of probes respectively originating from a LAV-I and a LAV-II, since it is not necessary to differentiate between the type of LAV virus you are looking for.

 As goes without saying and as it moreover already results from the foregoing, the invention is in no way limited to those of its modes of application and embodiments which have been more especially envisaged; on the contrary, it embraces all its variants; in particular those which are also indicated in the claims which follow and which, as such, must be considered as incorporated - or reincorporable - into the present description.

 It is further specified that in the above the numbers which followed the indications p- and / or "gap" correspond to the approximate molecular weights of the corresponding proteins and / or glycoproteins divided by 1000. For example the gp36 has a molecular weight of the on the order of 36,000. it is understood, however, that these molecular weight values can vary within a range of up to 5 ×, depending on the techniques used for determining these molecular weights.

 it is mentioned that the cytopathogenic nature of the LAVII virus with regard to lymphocytes is characterized by the appearance of multi-nucleated cells.

 The LAV II virus can be cultivated in permanent lines carrying the T4 phenotype, in particular in cells known under the designation CEM.

 The infection and then the culture of the infected CEM cells can be carried out as follows. The coculture of T4 lymphocytes previously infected with the LAV II virus and of uninfected cells of the CEM line is carried out for the time necessary for the infection of the CEMs. The culture conditions are then further continued in an appropriate medium, for example that described below and, when the reverse transcriptase activity of the infected cells has reached a sufficient level, the virus produced is recovered from the culture medium. .

 In particular, a co-culture was carried out, under the conditions specified below, of human T4 lymphocytes which had been infected five days previously with a strain of LAV II virus originating from a patient designated hereinafter "Rod", on the one hand and CEM, on the other hand.

 The infected T4 lymphocytes, previously activated by phytoglutinin, were found to have a reverse transcriptase activity of 5000 cpm / 106 normal T lymphocytes three days after the start of the infection. The culture was continued until the measured reverse transcriptase activity reached 100,000 cpm in the supernatant. These T4 lymphocytes were then brought into contact with CEM cells (3.106 CEM cells per 1.106 infected normal T lymphocytes) in the following culture medium: RPMI 1640 containing 2.92 mg / ml of L glutamine, 10P of fetal calf serum decomplemented, 2 µg / ml polybrene, 0.05% anti-interferon-alpha serum, 10,000,000 u / ml penicillin, 10 mcg / ml streptomycin and 10,000 mcg / ml neomycin. The culture medium was brought to the temperature of 56 ° C for 30 minutes.

 The culture medium is changed twice a week.

The measures of reverse transcriptase activity measured in the supernatant were as follows:
- on day 0: 100,000
- on day 15: 20,000
- on day 21: 200,000
- on day 35: 1,000,000
The culture of CEM infected with the LAV II virus was deposited in the National Collection of Culture of Microorganisms of the INSTITUT PASTEUR (CNCM) under the n "I-537 on March 24, 1986

Claims (30)

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