JP2609448B2 - Viral envelope antigens of lymphadenopathy and acquired immunodeficiency syndrome - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to lymphadenopathy (lymphadenopathy,
And purified immunodeficiency syndrome (hereinafter abbreviated as AIDS) viruses, in particular in the form of purified antigens, methods for producing these antigens, and in particular methods for producing the envelope antigens of these viruses. The invention further relates to a polypeptide encoded by said DNA sequence, with or without glycosylation.

The retrovirus that is an LAS or AIDS agent is FB
ARRE-SINOUSSI et al., Science, 220, 868 (1983). The features are as follows. This retrovirus is T-lymphotropic and its preferred target is Le
composed of u3 cells (or T4 lymphocytes), Mg ⁺
And has a strong affinity for poly (adenylic acid-oligodeoxy-thymidylic acid) (poly (A) -oligo (dT) 12-18). The density in the sucrose gradient is 1.16 to 1.17, the average diameter is 139
Nanometers, the average diameter of the nucleus is 41 nanometers.
The antigen of the virus, particularly protein P25, is immunologically recognized by antibodies contained in serum collected from LAS or AIDS patients. The core protein, protein P25, is HTL
Not recognized immunologically by the proteins p24 of the type VI and type II viruses. The virus also contains HTLV I and HTLV
It does not contain the protein p19 that is immunologically cross-reactive with the protein p19 of II.

This type of retrovirus (sometimes with the generic abbreviation LA
V) is the National Co. of the Pasteur Institute in Paris
Deposit Nos. I-232, I-240 and I-241 with the Collection of Micro-organism Cultures (CNCM). Virus strains similar to LAV in all aspects of morphology and immunology have been isolated in other laboratories. For example, RC
GALLO et al., Science 224,500 (1984) and MGSARN-GADHA
HTLV-I isolated by RAN et al., Science 224,506 (1984).
A retrovirus strain named II, and M.JAY LEVY et al., S
cience, 225, 840-842 (1984). For simplicity of the designation, these viruses and other viruses with comparable morphological and immunological properties will be referred to hereinafter as the generic designation "LAV". For a more detailed description of LAV retroviruses and the like and the use of extracts of these viruses, see 1983.
See European Patent Application, filed Sep. 14, 1984, filed on Sep. 15, 1984 with British Patent Application No. 83 24800 as priority application.

First, the core antigen was AIDS in the test system used at this time.
Or, it was the major antigen of the virus lysate or extract recognized by the serum of the LAS infected patient. Protein p42, which is considered to be composed of the envelope protein, was also detected. Similarly, GALLO et al. Detected a protein, p41, which is also considered a predictable component of the viral envelope.

Methods for producing LAV virus have also been described. In particular, BARRE-SINOUSSI et al., Supra, describe a method for producing virus in T lymphocyte cultures obtained from blood or umbilical cord or bone marrow cells of healthy adult donors. This method has the following main steps, in particular:-after activation by lectin mitogen (initially AIDS
Or transmitting the virus to donor T lymphocytes with a virus suspension obtained from the live supernatant of virus-producing lymphocytes (obtained from a LAS-infected patient);-the presence of anti-α-interferon sheep serum Culturing the TCGF-infected cells underneath; and purifying the produced virus (production generally commences between 9 and 15 days after infection and lasts for 10 to 15 days). The step involves precipitating the virus in polyethylene glycol to produce a first concentration of the virus, and the resulting sample is then subjected to a 20-60% sucrose gradient or (commercially available from Oslo NYEGAARD under the trade name NYCODENZ). Centrifuge in an isotonic gradient of metrizanide and a density of 1.16 to 1.17 or NYCODE in a sucrose gradient.
Recovery of virus by band 1.10-1.11 in NZ gradient.

From a T-type permanent cell line, such as the CEM system, or
B lymphocytes obtained by transforming lymphocytes obtained from healthy donors with Epstein-Barr virus as disclosed in French Patent Application No. 84 07151 on May 9, 4 LA from blastoid cell line
V virus can also be produced. The resulting permanent cell line continuously produces a virus having the essential antigen and morphological characteristics of the LAV virus (indicated by LAV-B in the case of the B lymphoblastoid cell line), provided that A slightly higher density band in sucrose (especially 1.18) is collected). Final purification of the virus may be performed in a NYCODENZ gradient.

Methods for cloning DNA sequences capable of hybridizing to LAS genomic RNA have already been disclosed in UK Patent Application No. 84 23659 on September 19, 1984. In the following, new improvements and common features of the present invention disclosed in the text will be referred to this UK application.

It is an object of the present invention to provide a purified unaltered virus form (or a virus that has been slightly transformed by the purification treatment used) and a method for obtaining said untransformed purified virus.

Another object of the present invention is not only useful for the detection of LAV or related viruses (hereinafter more generally referred to as "LAV virus group") but also more flexible, in particular by immunological methods. It is an object of the present invention to provide an additional novel means effective for detecting a specific portion of genomic DNA of the virus group which expresses a product which cannot be directly detected. It is yet another object of the present invention to provide a polypeptide containing a sequence common to a polypeptide containing an antigenic determinant contained in a protein encoded and expressed by a naturally occurring LAV genome. Yet another object of the present invention is to provide an LA
Means for detecting proteins associated with the V virus, in particular for the diagnosis of AIDS or pre-AIDS (pre-AIDS), or conversely especially patients infected with AIDS or pre-AIDS or more generally asymptomatic LA in carriers or blood-related products
An object of the present invention is to provide a means for detecting an antibody against the V virus or a protein related thereto. Finally, another object of the present invention is to provide an immunogenic polypeptide, more particularly a polypeptide for protection against infection, used for preparing a vaccine composition against AIDS or related syndromes.

The present invention relates to genomic RNA of LAV as described below and
The present invention relates to additional DNA fragments (fragments) capable of hybridizing with additional cDNA variants corresponding to the entire genome of the LAV virus group. Further, the present invention relates to a recombinant DNA comprising the DNA or cDNA fragment.

Unmodified purified LAV retrovirus is sufficient for one or several sufficient amounts of one or several to be visualized when the virus is labeled with 200 microCuries / ³⁵ S-cysteine per ml of unlabeled cysteine-free medium. It is distinguished from conventional retroviruses in that it contains an envelope antigen. These antigens, especially glycoproteins (glycoproteins), are selectively recognized in vitro by the sera of AIDS or LAS infected patients or the sera of asymptomatic carriers of the virus.

Suitable antigens according to the specification obtained from the lysate of this virus (or by light washing of the envelope of the virus) are given by the migration distance of a standard protein of known molecular weight compared to the migration distance in the same migration system. It is a glycoprotein having a molecular weight of about 110,000 daltons to be determined. In particular, comparative measurements were made using the following standard proteins (commercially available from AMERSHAM):-lysozyme-( ¹⁴ C) -methyl (MW: 14300);-carbon dioxide-( ¹⁴ C) -methyl (MW: 30 000); albumin - ⁽¹⁴ C) - methyl (MW: 46000), - bovine serum albumin ⁽¹⁴ C) - methyl (MW: 69000), - phosphorylase ^b-(14 C) - methyl (MW: 92500), - myosin - ( ^The reaction was performed on a 12.5% polyacrylamide gel using ¹⁴ C) -methyl (MW: 200,000) under a voltage of 18 V for 18 hours.

The present invention further provides the serum or L of AIDS or LAS infected patients.
The antigen itself has the ability to be recognized by human serum exposed to the AV virus group or its analogues, in particular antigens having a molecular weight of about 110,000 to 120,000. These antigens are further characterized in that they form a complex with concanavalin A, which is dissociable in the presence of O-methyl-α-D-mannopyranoside. The antigens of the present invention can further bind, for example, other lectins known as "lentil-lectins". Preferred antigens of the invention having a molecular weight of 110,000 are furthermore sensitive to the action of endoglycosidase. By this action, a protein having a molecular weight of about 90,000 is produced from an antigen having a molecular weight of 110,000. The protein can be separated by, for example, an immunoprecipitation method or a separation method using a difference in molecular weight (difference in electrophoresis on a gel). is there.

Preferred antigens of the invention are composed of glycoproteins.

The present invention further relates to a method for producing the virus of the present invention.
This method is essentially different from the above-mentioned conventional method in the final purification step. In particular, the purification step of the method of the present invention is not performed in a gradient, and the fractionation and centrifugation is directly performed on the supernatant of the culture medium of the producing cell. This centrifugation step comprises a first centrifugation step, especially at a centrifugal angular velocity of especially 10,000 rpm, to remove non-viral components, more particularly cellular components, and a higher centrifugation step, especially at 45000 rpm, to sediment the virus itself. A second centrifugation step performed at an angular velocity. In a preferred embodiment, the first centrifugation step at 10000 rpm lasts 10 minutes and the second centrifugation step at 45000 rpm lasts 20 minutes. Of course, these values are merely exemplary, and it should be understood that altering the centrifugation conditions to separate cellular and viral components is within the ability of those skilled in the art.

Such a modification of the purification process produces a virus preparation that allows for easier isolation of the antigen as compared to a virus preparation purified by conventional methods. In any case, the virus ultimately obtained by the method of the invention is readily recognized by the serum of a patient or a human exposed to a LAV virus or morphologically or antigenically similar strains.

The antigens of the invention can be obtained from the virus by lysing (or other suitable treatment) the virus in the presence of any suitable detergent and collecting and separating the free antigen. Lysis of the virus is advantageously performed in the presence of aprotinin or any other agent suitable for inhibiting the action of proteases. The antigen of the invention can thus be separated by any method known per se, for example by separating the proteins by using different electrophoresis in a given gel, whereby the desired protein is In an electrophoresis step under conditions well set for molecular weight, the protein is isolated from regions of the gel where it is normally found. On the other hand, lectins,
In particular, a lysate (lysate) of the virus using the affinity for concanavalin A or lentil-lectin
Can be separated from the antigen of the present invention. The lectin used is preferably immobilized on a solid support such as, for example, a crosslinked polymer derived from agarose and sold under the trade name SEPHAROSE. After washing the immobilized antigen with a suitable buffer, the antigen can be eluted in any suitable manner, especially with a solution of O-methyl-α-D-mannopyranoside.

As a more sufficient method for purifying these antigens, a patient's serum known to have an effective antibody against the protein, a concentrated antibody preparation (polyclonal antibody), or more specifically, a molecular weight of 110,000, hereinafter abbreviated as gp110 Immunoprecipitation may be performed using a monoclonal antibody against the antigen of the present invention.

Other features of the present invention will become apparent in the following description of the virus and antigen of the present invention, particularly the isolation of the envelope antigen of the virus, with reference to the accompanying drawings. The first
The figure is a figure obtained from a photographic copy of the gel strip used to perform the electrophoresis of the lysed extract of T lymphocytes infected with the LAV suspension and of the uninfected (control) T lymphocytes, respectively. The figure shows the complete LAV genome (clone λJ1
9), FIG. 3 shows the complete sequence of the LAV virus genome, FIGS. 4a-4e and FIG. 5 show some of the three possible reading phases of LAV genomic RNA and appear in each of said reading phases FIG. 6 is a schematic explanatory diagram showing an open reading frame (ORF), and FIG. 6 is a schematic explanatory diagram of a terminal long repeat (LTR) of LAV.

I-Virus and antigen production T lymphocytes obtained from healthy donors and infected with LAV1 under the conditions described by F. BARRE-SINOUSSI et al., Or from leukemia patients, and infected with LAVI in vitro as well. CEM cells
Cultures were maintained in microcurie's medium containing ³⁵ S-cysteine and no unlabeled cysteine.
Infected lymphocytes were cultured in non-denaturing media so that the desired antigen was not degraded. Thereafter, a first centrifugation at 10,000 rpm is performed on the supernatant of the medium to remove non-viral components.
Run for 10 minutes, then 45000 rpm to sediment the virus
A second centrifugation was performed for 20 minutes. Next, especially F.BARR
The virus pellet was lysed by detergent in the presence of aprotinin (5%) under the conditions described in E-SINOUSSI et al.

As a control, the same treatment was performed with lymphocytes collected from a healthy donor.

Each lysate was then immunoprecipitated with serum from AIDS or LAS infected patients. Similar immunoprecipitations were performed on sera from healthy donors or other infected patients. next
The medium was electrophoresed in an SDS-polyacrylamide gel.

The results are shown in FIG. Gel strips numbered 1-6 were obtained from samples labeled with ³⁵ S-cysteine. Strips numbered 7-10 indicate the results observed with infected or uninfected lymphocyte samples labeled with ³⁵ S-methionine. Further, the strip M corresponds to the migration distance of the standard protein, and the molecular weight of the standard protein is shown in the right part of the drawing.

Labeled viral proteins are indicated on the left side of the figure.

Columns 7-10 show the specific protein p25 of LAV labeled with ³⁵ S-methionine. This protein is not present in strips 8-10 corresponding to the results obtained with samples from healthy lymphocytes.

Rows 3 and 5 correspond to the results observed in samples obtained from infected lymphocytes labeled with ³⁵ S-cysteine. Proteins p25 and p18, characteristic core proteins of LAV, and also the glycoprotein gp11, which is also specific for LAV
0 also appears. Although not very noticeable, an image corresponding to protein p41 (molecular weight about 41000) also appears in each sample.

The virus of the present invention and the antigen of the present invention can be precipitated with a lectin, particularly concanavalin A, or immobilized on a Sepharose-concanavalin A column.
In particular, purification of the envelope glycoprotein can be performed as follows. This fixation can be carried out by contacting a lysate of the LAV virus dissolved in a particularly suitable buffer with Concanavalin A bound to Sepharose. Preferred buffer compositions are shown below.

Tris 10 mM NaCl 0.15 M CaCl ₂ 1 mM MgCl ₂ 1 mM Once the detergent 1% pH 7.4 fixation available under the trade name TRITON is obtained, Sepharose-Concanavalin A is washed with a buffer having the same composition. However, reduce the TRITON concentration to 0.1%. Then dissolved in washing buffer
Elute with a 0.2 M solution of O-methyl-α-D-mannopyranoside.

Performing immunoprecipitation using an antibody contained in the serum of an AIDS-infected patient or a polyclonal antibody obtained from a serum derived from an animal previously sensitized with the `` unmodified '' virus of the present invention or the glycoprotein. May further concentrate the protein. The complex can then be dissociated with a solution having a sufficient ionic salt content to recover the protein. Preferably, the antibody preparation is immobilized on an insoluble carrier such as Sepharose B by a method known per se.

A monoclonal antibody produced by a hybridoma previously prepared for gp110 can also be used. These monoclonal antibodies and the hybridomas producing the monoclonal antibodies also form part of the present invention.

Hereinafter, methods for producing and selecting a monoclonal antibody against gp110 glycoprotein will be described.

Immunization of mice A group of 6-8 week old Balb / c mice was used. One group was sensitized with the glycoprotein-containing virus and another group was sensitized with purified glycoprotein gp110. The sensitization procedure was common to all mice, with 10 mg in the presence of complete Freund's adjuvant on day 0, incomplete Freund's adjuvant on day 14, and in the absence of adjuvant on days 28 and 42. The antigen preparation was injected. The first three injections were in peritoneal tissue and the fourth was intravenous.

Fusion and Hybrid Cultures Azaguanine resistant non-secreting myeloma mutant cells 5.53 P3 × 63 Ag8 are used which are themselves derived from the MOPC-21 cell line. On day 45, Fazekas de st-GROTH and SCHEIDEGGER
Is fused with immunized mouse splenocytes in the presence of polyethylene-glycol 4000 using the method of Using the same culture method
Placed in a 24-cup plate (known as COSTAR)
Hybrids are selected in RPMI 16-40 "HAT" medium.

96 in the presence of a “feeder cell” layer of isogenic thymocytes
Antibody-producing hybridomas of appropriate specificity are cloned in a cup plate. Production clones are selected in this way and then expanded in 24-cup plates, also in the presence of thymocytes. When confluence appears in one of the cups, it is injected into the peritoneal tissue of balb / c mice that had been injected with PRISTANE 8 days ago and / or maintained in liquid medium.

Demonstration of Anti-LAV Antibodies Characterization of antibody-producing clones of appropriate specificity can be achieved by using five different methods. In the first step, antibody-producing hybrids are measured in an ELISA test to prove the presence of mouse immunoglobulin in the supernatant. This first
Anti-LAV supernatant with antibodies to viral components from selection
Either use an ELISA test to prove the presence of the antibody or by immunofluorescence on virus-producing human cells. Finally, the supernatant is analyzed by radioimmunoprecipitation of the cysteine-labeled virus and the Western-Blot method on the virus preparation. These methods can determine the specificity of an anti-LAV antibody.

Results Cells from the different fusions are cultured in 648 cups. According to these microscopic observations, most of these cups contain single hybrid clones that can grow in "HAT" selection medium. More than 50% of these produce antibodies that give a positive response in an ELISA antiviral test. The most representative fusions are tested by the Western-Blot method and several fusions are subcloned taking into account their specific reactivity in antiviral ELISA and their behavior under culture conditions. Particularly selected hybrids produce antibodies that selectively recognize the viral glycoprotein gp110 having a molecular weight of about 110 KD. All subcloned fusions yield antibody-producing clones. After expression, this clone is injected into isogenic mice. Analysis of the specificity of the antibodies present in different peritoneal fluids confirms the specificity of said ascites antibody for gp110.

The resulting monoclonal antibody can be used to purify a protein containing an antigenic site also contained in gp110. Therefore, the present invention also relates to such a purification method.
This method should be used to ensure that no uncontrolled separation of gp110 occurs during the purification of the virus prior to lysis, provided that the virus lysate or T lymphocyte lysate or other
It is also advantageously used for LAV producing cells and the like. Of course, this method can also be used with any solution containing a protein or polypeptide or glycoprotein that contains an antigenic site that is contained in gp110 or normally an envelope protein and recognized by a monoclonal antibody.
In order to carry out this method, it is advantageous to immobilize the monoclonal antibody on a solid support, which is preferably adapted for affinity chromatography. For example, these monoclonal antibodies were purchased from the Swedish company PHARMCIA AG
Is fixed to a three-dimensional mesh-like agarose lattice commercially available under the trade name SEPHAROSE by, for example, a cyanogen bromide method.

Therefore, the present invention further relates to a method for separating the relevant antigen. In the method, an antigen mixture (for example, a virus lysate or extract) containing the antigen of interest is brought into contact with an affinity column carrying the monoclonal antibody, and the polypeptide, protein or glycoprotein selectively recognized by the monoclonal antibody is contacted. Is selectively immobilized in a suitable buffer, especially a solution of suitable ionic strength, such as a salt solution, preferably an ammonium acetate solution, which is prepared by lyophilizing a preparation or solution acidified in a glycine buffer at pH 2-4 or the same pH. The monoclonal antibody is recovered by dissociating the antigen-antibody complex using no residue, and the eluted polypeptide, protein or glycoprotein is recovered.

It will also be apparent that the present invention further relates to polypeptide fragments having a lower molecular weight and comprising an antigenic site that can be recognized by the same monoclonal antibody.
It will also be apparent to one of skill in the art that once a monoclonal antibody that recognizes the gp110 glycoprotein is available, the method of the invention can be applied to smaller peptide sequences or fragments containing a common antigenic site or epitope. In order to obtain a fragment having a smaller size, a known method may be used.
For example, in such a method, the original polypeptide is cleaved by an enzyme capable of cleaving a larger polypeptide at a specific site. Examples of such proteins include enzymes of Staphylo-coccus aureus V8, α-chymotrypsin, “mouse submandibular gland protease” marketed by BOEHRINGER, Vibrio alginolytic
Us chemovar iophagus collagenase, etc., which specifically recognizes the peptides Gly-Pro, Gly-Ala, etc.

Further, by cloning a fragment excised from cDNA composed of the genome of the LAV variant, a polypeptide or fragment of the envelope antigen of the virus can be obtained.

FIG. 2 and FIG. 3 are restriction maps of such a cDNA consisting of a total of 9.1-9.2 kb. The polypeptide encoded by the cDNA fragment was present in the region from the KpnI site (position 6100) to the BgIII site (position 9150) in the restriction map shown in FIG. The presence of a characteristic site of an envelope antigen, such as a LAV virus, in any expressed polypeptide (in a suitable host cell previously transformed with the corresponding fragment or a vector containing said fragment) may be determined by any suitable immunochemistry. Can be detected by strategic means.

The present invention more specifically relates to a polypeptide encoded by a cDNA fragment described below. The present invention further provides
V cDNA variants corresponding to the retroviral genome,
(From the 5 'end to the 3' end) pertains to the nucleic acid fragment itself which is characterized by including a series of restriction sites in the order described below.

In addition, a continuous site of the entire LAV genome (refer to the restriction map of λ J19 in FIG. 1) is shown below as coordinates for the Hind III site of R region (selected as coordinate 1). Coordinates are ± 200b
It is estimated with an accuracy of p.

Hind III 0 Sac I 50 Hind III 520 Pst I 800 Hind III 1 100 BgI II 1 500 Kpn I 3 500 Kpn I 3 900 Eco RI 4 100 Eco RI 5 300 Sal I 5 500 Kpn I 6 100 Bgl II 6 500 Bgl II 7 600 Hind III 7 850 Bam HI 8 150 Xho I 8 600 Kpn I 8 700 Bgl II 8 750 Bgl II 9 150 Sac I 9 200 Hind III 9 250 Another DNA variant according to the invention optionally has a coordinate of approximately 5550
Contains additional Hind III.

FIG. 1 shows a more detailed restriction map of the complete DNA (λ J19).

Figures 4a-4e show more detailed nucleotide sequences of preferred DNAs of the present invention.

The invention further relates to other preferred DNA fragments and polypeptide sequences (glycosylated or non-glycosylated) as described below.

Sequencing of LAV Sequencing and determination of particularly important sites cited in UK Patent No. 84
This was performed on a phage recombinant corresponding to λ J19 disclosed in 23659. A method for preparing this recombinant is disclosed in that application. All recombinant phage DNAs of clone λ J19 (disclosed in the aforementioned application) were purified by DEININGER (1983), An
Sonicated according to the protocol of alytical Biochem. The Klenow reaction was maintained at 16 ° C. for 12 hours to repair the DNA. The DNA is electrophoresed on a 0.8% agarose gel,
DNA of ~ 600 bp in size was excised, electroeluted and precipitated. The DNA was resuspended (in 10 mM Tris, pH 8, 0.1 mM EDTA) and T4 DNA- and RNA-ligase (Maniatis T et al. (198
2), Molecular Cloning, Cold S
M13mp8 RF DNA (cleaved with restriction enzyme SmaI and subjected to alkaline phosphorylation (phosphatase treatment)) using Spring Harbor Laboratory. Further studies were performed using the E. coli TGI strain. This strain has the following genotype:

Δlac pro, supE, thi.F′traD36, proAB, lacI ^q , ZΔ
M15, r ^- This E. coli TGI strain has the property of facilitating the recognition of recombinants. The blue color of cells transfected with a plasmid that has not been recombined with a fragment of LAV DNA does not change. In contrast, cells transfected with the recombinant plasmid containing the LAV DNA fragment form white colonies. The method used is disclosed in Gene (1983), 26 , 101.

This strain was subjected to the Hanahan method (Hanahan D (1983), J. Mol. Bi
ol. 166, 557) with the ligation mix. Cells were plated out on trypton-agarose plates containing IPTG and X-gal in soft agarose. White plaques were picked and screened or screened directly in situ using nitrocellulose filters. These DNAs contain the pUC18 H of λ J19.
Hybridized with the nick translated DNA insert of the ind III subclone. As a result, the plasmid or subclone of λ identified in the following table could be isolated. Also noteworthy in this table are the names of each plasmid, plus the "Coll" from the Institute of Pasteur, Paris, France.
ection Nationale des Cultures de Micro-organismes
(CNCM) ", the accession number of the cell culture of the E. coli TGI strain containing the corresponding plasmid deposited. The TGI cell line that has not been transformed has accession number I.
Deposited with the CNCM at -364. All these deposit dates are 1
November 15, 984. The size of the corresponding insert from the LAV genome is also indicated.

Table Essential characteristics of the recombinant plasmid-pJ19-1 plasmid (I-365) 0.5 kb Hind III-Sac I-Hind III-pJ19-17 plasmid (I-367) 0.6 kb Hind III-Pst I-Hind III-pJ19 -6 plasmid (I-366) 1.5 kb Hind III (5 ') BamHI Xho I Kpn I Bgl II Sac I (3') Hind III-pJ19-13 plasmid (I-368) 6.7 kb Hind III (5 ') Bgl II Kpn I Kpn I Eco RI Eco RI Sal I Kpn I Bgl II Bgl II Hind III (3 ') positive hybridized M13 phage plate was grown for 5 hours to extract single-stranded DNA.

Dideoxy method and Sanger et al. (Sanger et al. (1977), Pro
c. Natl. Acad. Sci., USA, 74 , 5463 and the M13 cloning and sequencing handbook, AMERSHAM (1983)). 17
-Mer oligonucleotide primer α- ³⁵ SdATP (400C
i / mmol, AMERSHAM) and 0.5X-5X buffer gradient gel (Bigg
en MD et al. (1983), Proc. Natl. Acad. Sci., USA, 50 , 3
963). Staden's program (Staden R.
(1982), Nucl. Acids Res. 10 , 4731) and read the gel into a computer. All suitable references and methods are described in the AMERSHAM M13 Cloning and Sequencing Handbook.

The complete DNA sequence of λ J19 (also referred to as LAV-Ia) is shown in FIGS. 4-4e.

The sequence was reconstructed from the sequence of the phage λ J19 insert. Numbering (as described below) begins with an experimentally located capsite. Important genetic elements, major open reading frames and their expected products are shown, along with a Hind III cloning site. Potential glycosylation sites in the env gene are shown with a line at the top. P25gag of NH ₂ − determined by protein microsequencing
The terminal sequence is boxed.

Each nucleotide was sequenced an average of 5.3 times. Both strands (strands) were determined for 85% of the sequences and the remainder were sequenced at least twice from independent clones.
The base composition is T22.2%, C17.8%, A35.8%, G244.2%, and G + C42%. The dinucleotide GC was usually very poorly shown (0.9%) in eukaryotic cell sequences (Bird, 1980).

5 and 6 are schematic diagrams showing the length of a continuous reading frame corresponding to a continuous reading phase (indicated by numbers 1, 2, and 3 on the left side of FIG. 5). The relative position of these open reading frames (ORFs) to the nucleotide structure of the LAV genome is indicated by a number indicating the position of each of the corresponding nucleotides in the DNA sequence. The vertical line corresponds to the position of the corresponding end reading codon.

The following genes and DNA fragments can be identified in the various reading frames shown. Next, the proteins or glycoproteins encoded by the genes and fragments will be described.

1) “gag gene” (or ORF-gag) “gag gene” encodes a core protein.

Near the 5 'end of gag: gag orf, the first "typical" start codon (Koza) in gagorf and from the cap site
k, 1984) (336 positions). The precursor protein consists of 500 amino acids. 55kd for calculated molecular weight 55841
Gag precursor polypeptide. The N-terminal amino acid sequence of the major core protein p25 is encoded by a nucleotide sequence starting at position 732 (see FIG. 5a). This clearly forms a link between the cloned LAV genome and the immunologically characterized LAV p25 protein. The protein encoded 5 'of the sequence encoding p25 is rather hydrophilic. Its calculated molecular weight of 14866 matches the molecular weight of gag protein p18. gag
The 3 'portion of the region probably encodes a retroviral nucleic acid binding protein (NBP). In fact, HTLV-1 (Seiki et al., 19
83) and RSV (Schwartz et al., 1983) as well as all NBP
(Orozlan etc., 1984) common basic units Cys-X ₂ -Cys-X
_8-9- Cys overlap observed (nucleotide 1 of LAV sequence
509 and 1572). Consistent with its function, the putative NBP is extremely basic (17%, Arg + Lys).

In particular, the genomic fragment (ORF-gag) that appears to encode a core antigen including p25, p18 and p13 is identified at nucleotide position
It appears to be located between 312 (starting at 5 'CTA GCG GAG 3') and nucleotide position 1835 (ending with CTCG TCA CAA 3 '). The structure of the peptide or protein encoded by the ORF portion is considered to be the structure corresponding to phase 2.

The methionine amino acid "M" encoded by ATG at positions 336-338 is likely to be the starting methionine of the gag protein precursor. ORF-gag termination and therefore ga
The end of the g protein appears to be at position 1835.

Amino acid sequence Pro-Ile-Val-Gln-Asn-Ile-Gln-G
p25 thought to start from ly-Gln-Met-Val-His-
The beginning of the protein appears to be encoded by the nucleotide sequence CCTATA ..., starting at position 732.

Therefore, the present invention more specifically relates to the following DNA sequences.

-A DNA sequence from nucleotide 336 to nucleotide 1336 which appears to encode a protein p55 which is thought to contain the amino acid sequence corresponding to the amino acid sequence of the core proteins p18 and p25 of the LAV virus-nucleotide 7 which appears to encode protein p25
DNA sequence from 32 to about 1300 nucleotides-DNA sequence from about nucleotide 1371 to nucleotide about 1650 likely to encode protein p13-Nucleotide 3 likely to encode protein p18
DNA sequence from 36 to about nucleotide 611.

The invention furthermore relates to the amino acid structures encoded by said fragments, in particular the proteins p13, p18, p25 and p55 or the polypeptides, having a structure corresponding to the structure obtained by direct translation of the DNA sequence or fragment specifically defined above A purified polypeptide having the formula: These peptide sequences are evident from FIG. 4a. More particularly, the present invention relates to purified polypeptides having a peptide sequence equal to or equivalent to that encoded by a DNA sequence extending from the following nucleotide positions:

-336-1650 (p55) -336-611 (p18) -1373-1650 (p13) -732-1300 (p25) -732-1300 (p25) p13, p18 and p25 are all derived from the same precursor, p55 It should be pointed out that this is possible.

The invention further relates to polypeptide fragments encoded by the corresponding DNA fragment of the gag reading frame. Particularly hydrophilic peptides in the gag reading frame are identified as described below. The peptides are defined starting with the amino acid 1 = Met encoded by ATG starting at 336-338 in the LAV DNA sequence and are numbered according to the order in the gag sequence (FIG. 3a). The first and second numbers for each peptide indicate the N-terminal and C-terminal amino acids, respectively.

 These hydrophilic peptides contain the following amino acids:

Amino acids 12-32, i.e.Glu-Leu-Asp-Arg-Trp-Glu-Lys-Ile-Arg-Leu-Arg-Pro-Gly-Gly-Lys-Lys-Lys-Tyr-Lys-Leu-Lys amino acids 37-46 That is, Ala-Ser-Arg-Glu-Leu-Glu-Arg-Phe-Ala-Val-amino acids 49-79, i.e.Gly-Leu-Leu-Glu-Thr-Ser-Glu-Gly-Cys-Arg-Gln-Ile- Leu-Gly-Gln-Leu-Gln-Pro-Ser-Leu- Gln-Thr-Gly-Ser-Glu-Glu-Leu-Arg-Ser-Leu-Tyr- amino acids 88-153 or Val-His-Gln-Arg -Ile-Glu-Ile-Lys -Asp-Thr-Lys-Glu-Ala-Leu-Asp-Lys-Ile-Glu-Glu-Glu -Gln-Asn-Lys-Ser-Lys-Lys-Lys-Ala-Gln -Gln-Ala-Ala -Ala-Asp-Thr-Gly-His-Ser-Ser-Gln-Val-Ser-Gln-Asn -Tyr-Pro-Ile-Val-Gln-Asn-Ile-Gln-Gly-Gln -Met-Val -His-Gln-Ala-Ile-Ser-Pro-Arg-Thr-Leu-Asn- Amino acids 158-165 or Val-Val-Glu-Glu-Lys-Ala-Phe-Ser
-Amino acids 178-188 ie Gly-Ala-Thr-Pro-Gln-Asp-Leu-Asn -Thr-Met-Leu- Amino acids 200-220 ie Met-Leu-Lys-Glu-Thr-Ile-Asn-Glu-Glu -Ala-Ala-Glu-Trp-Asp-Arg-Val-His-Pro-Val-His-Ala- amino acids 226-234, i.e.Gly-Gln-Met-Arg-Glu-Pro-Arg-Gly-Ser- amino acid 239 -264 or Thr-Thr-Ser-Thr-Leu-Gln-Glu-Gln -Ile-Gly-Trp-Met-Thr-Asn-Asn-Pro-Pro-Ile-Pro-Val- Gly-Glu-Ile-Tyr -Lys-Arg- amino acids 288-331, i.e.Gly-Pro-Lys-Glu-Pro-Phe-Arg-Asp-Tyr-Val-Asp-Arg-Phe-Tyr-Lys-Thr-Leu-Arg-Ala-Glu- Gln-Ala-Ser-Gln-Glu-Val-Lys-Asn-Trp-Met-Thr-Glu -Thr-Leu-Leu-Val-Gln-Asn-Ala-Asn-Pro-Asp-Cys-Lys-amino acid 352 -361 i.e.Gly-Val-Gly-Gly-Pro-Gly-His-Lys-Ala-Arg-amino acid 377-390 i.e.Met-Met-Gln-Arg-Gly-Asn-Phe-Arg -Asn-Gln-Arg- Lys-Ile-Val-amino acids 399-432 or Gly-His-Ile-Ala-Arg-Asn-Cys-Arg-Ala-Pro-Arg-Lys-Lys-Gly-Cys-Trp-Lys-Cys-Gly-Lys -Glu-Gly-His-Gln-Met-Lys-Asp-Cys-Thr-Glu-Arg-GLn-Ala-Asn- amino acids 437-484 or Ile-Trp-Pro-Ser-Tyr-Lys-Gly-Arg- Pro-Gly-Asn-Phe-Leu-Gln-Ser-Arg-Pro-Glu-Pr o -Thr-Ala-Pro-Pro-Glu-Glu-Ser-Phe-Arg-Ser-Gly-Val -Glu-Thr-Thr-Thr-Pro-Ser-Gln-Lys-Gln-Glu-Pro-Ile- Asp-Lys-Glu-Leu-Tyr-amino acids 492-498 or Leu-Phe-Gly-Asn-Asp-Pro-Ser- The invention further relates to any combination of these peptides.

2) "pol gene" (i.e. ORF-pol) pol: This reverse transcriptase gene can encode a protein with up to 1,003 amino acids (calculated MW = 113629).
The first methionine codon is the origin of the reading frame (starting point)
Since this is the 92nd triplet from, this protein may be translated from the spliced messenger RAN, thus forming the gag-pol polyprotein precursor.

The pol coding region is the only region in which significant homology (homology) has been found with other retroviral protein sequences. Currently three homology regions (domains) have been defined, the first of which consists of a very short region containing 17 amino acids (starting at 1856). The homology region is located within the p15gag ^RSV protease (Dittm
ar and Moelling 1978), the polypeptide encoded by the open reading frame is located between gag and pol of HTLV-1 (FIG. 5) (Schwartz et al., 1983, Seiki et al., 1983).
Year). Thus, this first region may correspond to a conserved sequence within the viral protease. Alternative positions of the region within the three genomes are not likely to be important. This is because retroviruses express the gag-pol polyprotein precursor by splicing or other mechanisms (Schwartz et al., 1983, Seiki et al., 1983). The second and longest homology region (starting at 2048) probably represents the reverse enzyme core sequence. In the region of 250 amino acids, with only minimal insertions or deletions, the amino acid identity of LAV is 38% for RSV, 25% for HTLV-I, M
21% of oMu LV (Schinnick et al., 1981);
TLV-I and RSV show 38% identity within the same region. The third homology region is located at the 3 'end of the pol reading frame,
Corresponds to a portion of the pp32 peptide of RSV with exonuclease activity (Misra et al., 1982). Again HTLV
The homology to the corresponding RSV sequence is greater than to -1.

Figures 4a to 4c also show a DNA fragment extending from nucleotide position 1631 (starting at 5'TTT TTT ... 3 ') to nucleotide position 5162, which is thought to correspond to the pol gene. The polypeptide structure of the corresponding polypeptide is considered to be the structure corresponding to phase 1. This stops at position 4639 (5'G GAT GAG
GAT3 'ends. These genes are thought to encode a viral polymerase or reverse transcriptase.

3) Envelope gene (i.e. ORF-env) The env: env open reading frame has a possible initiator methionine codon very close to the beginning (eighth triplet). If so, the molecular weight of the putative env protein (861 amino acids, calculated MW = 97376) is consistent with the size of the LAV glycoprotein (110 Kd and 90 Kd after glycosidase treatment). There are 32 potential N-glycosylation sites (Asn-X-Ser / Thr), which are shown in outline in FIGS. 4d and 4e.
One of the interesting features of env is the extremely large number of Trp residues at both ends of the protein.

A DN thought to encode an envelope protein
The A sequence corresponds to nucleotide position 5746 (5'AAA GAG GAG
A ... starting from 3 ') to nucleotide position 8908 (...
A ACT AAA GAA 3 '). The polypeptide structure of the sequence of the envelope protein corresponds to the structure read according to the "Phase 3" reading phase.

It is believed that env transcription is initiated at the level of the ATG codon at positions 5767-5769.

There are three hydrophobic regions unique to retroviral envelope proteins corresponding to the signal peptide (encoded by nucleotides 5815-5850 bp), the second region (7315-7350 bp), and the transmembrane segment (7831-7809 bp) ( Seiki et al., 1983). Second hydrophobic region (7
Before (315-7350 bp), there is an Arg + Lys-rich section, which may represent a site for proteolytic cleavage, by analogy with other retroviral proteins. Appear to form a membrane binding protein with the outer envelope polypeptide (Seiki et al., 1983, Kiyokawa et al., 1984). LAV
A notable feature of the envelope protein sequence is that the segment encoding the transmembrane protein has an unusual length (150 residues). This env protein shows no homology to any sequence in the Protein Data Bank. The small amino acid pattern common to the transmembrane proteins of all leukemia-inducing retroviruses (Cianciolo et al., 1984) does not exist in LAVenv.

The invention more particularly relates to a DNA sequence extending from nucleotide 5767 to nucleotide 7314, which is considered to encode gp110 (envelope glycoprotein of the LAV virus having a molecular weight of about 110,000 daltons), which starts at about nucleotides; A polypeptide backbone of a glycoprotein sequence corresponding to a glycoprotein sequence of approximate molecular weight believed to be 90,000 daltons and later found to be 55,000. gp110
The polypeptide resulting from the complete removal of the sugar residues of
Obtained by appropriate glycosidase treatment of gp110.

The present invention further provides a purified polypeptide having the amino acid structure (or polypeptide backbone) of gp110 and gp90, respectively, which corresponds to the linear translation of the DNA sequences and fragments (FIGS. 4d and 4e) identified more clearly above. Also involved.

The invention also relates to a polypeptide comprising a neutralizing epitope.

The location of the neutralizing epitope is further evident in FIG. 4d.
More specifically, refer to the three-letter group indicated by an overline included in the amino acid sequence of the envelope protein (reading phase 3). These are generally of the formula Asn
-X-Ser or Asn-X-Thr. X is any other amino acid. Thus, the molecular weight of the first protein product or polypeptide backbone of the env glycoprotein is greater than 91,000. These groups usually appear to carry glycosylated residues. Asn-X-Ser and Asn-X having these added glycosylated residues
-Thr residues constitute the hydrophilic region of the protein, and the normal conformation of the protein
Appears to be exposed to the outside of this conformation. Therefore, they are considered epitopes that can be used effectively in vaccine compositions.

Thus, the invention more particularly relates to peptide sequences that are included in the env protein and can be excised (or have the same amino acid structure) from this protein. These peptide sequences have a size not to exceed 200 amino acids.

Preferred peptides of the present invention (hereinafter a, b, c, d, e, f) are considered to correspond to the peptides encoded by the nucleotide sequences spanning the following positions, respectively.

a) from about 6171 to about 6276 b) from about 6336 to about 6386 c) from about 6466 to about 6516 d) from about 6561 to about 6696 e) from about 6936 to about 7006 f) from about 7611 to about 7746 Other hydrophilic peptides in the frame include those described below. These peptides are defined starting at amino acid 1 = lysine encoded by AAA at positions 5746-5748 in the LAV DNA sequence (FIGS. 4d and 4e) and are numbered according to the order relative to the terminal sequence. The first and second numbers for each peptide indicate the N-terminal and C-terminal amino acids of that peptide.

These hydrophilic peptides include the following.

Amino acids 8-23, i.e.Met-Arg-Val-Lys-Glu-Lys-Tyr-Gln-His-Leu-Trp-Arg-Trp-Gly-Trp-Lys- amino acids 63-78, i.e.Ser-Asp-Ala- Lys-Ala-Tyr-Asp-Thr-Glu-Val-His-Asn-Val-Trp-Ala-Thr-amino acids 82-90, i.e.Val-Pro-Thr-Asp-Pro-Asn-Pro-Gln-Glu- Amino acids 97-123, namely Thr-Glu-Asn-Phe-Asn-Met-Trp-Lys-Asn-Asp-Met-Val-Glu-Gln-Met-His-Glu-Asp-Ile-Ile-Ser-Leu- Trp-Asp-Gln-Ser-Leu-amino acids 127-183, i.e.Val-Lys-Leu-Thr-Pro-Leu-Cys-Val-Ser-Leu-Lys-Cys-Thr-Asp-Leu-Gly-Asn- Ala-Thr-Asn-Thr-Asn-Ser-Ser-Asn-Thr-Asn-Ser-Ser-Ser-Gly-Glu-Met-Met-Met-Glu-Lys-Gly-Glu-Ile-Lys-Asn- Cys- Ser-Phe-Asn-Ile-Ser-Thr-Ser-Ile-Arg-Gly-Lys-Val-Gln-Lys-amino acids 197-201, i.e.Leu-Asp-Ile-Ile-Pro-Ile-Asp- Asn-Asp-Thr-Thr- amino acids 239-294, i.e.Lys-Cys-Asn-Asn-Lys-Thr-Phe-Asn-Gly-Thr-Gly-Pro-Cys-Thr-Asn-Val-Ser-Thr- Val -Gln-Cys-Thr-His-Gly-Ile-Arg-Pro-Val-Val-Ser-Thr -Gln-Leu-Leu-Leu-Asn-Gly-Ser-Leu-Ala-Glu-Glu-Glu- Val-Val-Ile-Arg-Ser-Ala-Asn-Phe-Thr-Asp-Asn-Ala-Lys-amino acids 300-327 That is, Leu-Asn-Gln-Ser-Val-Glu-Ile -Asn-Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser -Ile-Arg-Ile-Gln-Arg-Gly -Pro-Gly-Arg- amino acids 334-981, i.e.Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys-Asn-Ile-Ser-Arg-Ala-Lys-Trp-Asn- Ala -Thr-Leu-Lys-Gln-Ile-Ala-Ser-Lys-Leu-Arg-Glu-Gln -Phe-Gly-Asn-Asn-Lys-Thr-Ile-Ile-Phe-Lys-Gln-Ser- Ser-Gly-Gly-Asp-Pro-amino acids 397-424, namely Cys-Asn-Ser-Thr-Gln-Leu-Phe-Asn-Ser-Thr-Trp-Phe-Asn-Ser-Thr-Trp-Ser- Thr-Glu -Gly-Ser-Asn-Asn-Thr-Glu-Gly-Ser-Asp- amino acids 466-500, i.e.Leu-Thr-Arg-Asp-Gly-Gly-Asn -Asn-Asn-Asn-Gly- Ser-Glu-Ile-PheArg-Pro-Gly-Gly -Gly-Asp-Met-Arg-Asp-Asn-Trp-Arg-Ser-Glu-Leu-Tyr-Lys-Tyr-Lys-Val- amino acids 510-523 I.e., Pro-Thr-Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg- amino acids 551-577, i.e.Val-Gln-Ala-Arg-Gln-Leu-Leu- Ser-Gly-Ile-Val-Gln-Gln-Gln-Asn-Asn-Leu-Leu-Arg -Ala-Ile-Glu-Ala-Gln-Gln-His-Leu- amino acids 594-603, i.e.Ala-Val- Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln- amino acids 621-630, i.e. Pro-Trp-Asn-A la-Ser-Trp-Ser-Asn-Lys-Ser- amino acids 657-679, i.e.Leu-Ile-Glu-Glu-Glu-Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu- Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-amino acids 719-758, i.e.Arg-Val-Arg-Gln-Gly-Tyr-Ser-Pro-Leu-Ser-Phe-Gln-Thr-His- Leu-Pro-Thr-Pro-Arg -Gly-Pro-Asp-Arg-Pro-Glu-Gly-Ile-Glu-Glu-Glu-Gly -Gly-Glu-Arg-Asp-Arg-Asp-Arg-Ser- Ile-amino acids 780-803, i.e.Tyr-His-Arg-Leu-Arg-Asp-Leu-Leu-Leu-Ile-Val-Thr-Arg-Ile-Val-Glu-Leu-Leu-Gly-Arg-Arg- Gly-Trp-Glu- The present invention also relates to any combination of these peptides.

4) Other ORFs The present invention further relates to DNA sequences which provide an open reading frame defined as ORF-Q, ORF-R and 1,2,3,4,5. The relative positions of these frames are shown in particular in FIGS. 2 and 3.

 These ORFs have the following positions.

ORF-Q phase 1 Start 4554 End 5162 ORF-R 〃 2 〃 8325 〃 8972 ORF-1 〃 1 〃 5105 〃 5392 ORF-2 〃 2 〃 5349 〃 5591 ORF-3 １ 1 〃 5459 〃 5692 ORF-4 〃 2 595 5595 〃 5849 ORF-5 〃 1 〃 8042 〃 8355 ORF Q and FLAV genome (+) strand (chain) is composed of core structural protein (gag), reverse transcriptase (pol) and envelope protein (env). It was found to contain the encoding statutory retroviral gene and two additional open reading frames (orf) (Table 1). These two frames are hereinafter referred to as Q and F. LAV gene structure 5 'LTR-gag-pol-Q
The -env-F-3 'LTR is unique. The pol and env genes overlap with each other in all replication competent retroviruses, whereas in LAV these genes are four smaller (<100
Triplet) orf Q before orf (192 amino acids)
Separated by orf F (206 amino acids) is env
Has a feature that it slightly overlaps the 3 'end of the LTR and is half coded by the U3 region of the LTR.

Such a structure clearly distinguishes LAV from previously sequenced retroviruses (FIG. 2). (-)
Strands are clearly non-coding strands. The extra Hind III site (relative to λ J19) of the LAV clone λ J81 is clearly a non-coding region between Q and env (positions 5166-574).
5). A sequence that differs from the HindIII recognition sequence by a single base (underlined) (AAGCC
T) starts at position 5501. It is expected that many of the restriction site polymorphisms between the various isolates will map to this region. Clone λ J81 is also described in British Patent Application No. 84 23659, filed September 15, 1984.

Q and F Table 1 shows the nucleotide positions at the ends of these frames.

The position of orf Q is unprecedented in the retroviral structure, and orf F is unique in that half is encoded by the U3 element of the LTR.
Both orfs are "strong" initiator codons (Kozak 1
984) near its 5 'end and can encode a protein of 192 amino acids (calculated MW = 22487) and a protein of 206 amino acids (calculated MW = 23316), respectively. Both of these putative proteins are hydrophilic (pQ49% polar, 15.1% Arg + Lys; pF46% polar, 11% Arg +
Lys) and therefore do not appear to bind directly to the membrane. The functions of these putative proteins pQ and pF cannot be predicted because no homology is found when screening protein sequence data banks. orf F and HTLV−
There is no detectable homology between one pX protein. In addition, the hydrophobic / hydrophilic profiles of both are completely different. It is already known that retroviruses can introduce cellular genes, especially proto-oncogenes (Weinberg 1982). We orf
It is believed that Q and F represent exogenous genetic material and do not represent any trace of cellular DNA. Because (I) LAV DNA does not hybridize to the human genome under stringent (stringent) conditions (Alizon
Et al., 1984), (II) codon usage for orf Q and F is g
This is because they are comparable to the ag, pol and env genes (data not shown).

FIG. 6 schematically shows the structure of the reconstructed LTR and the flanking elements of the virus. This LTR is 638 bp in length and exhibits the usual characteristics (Chen and Barker, 1984).
Year): (I) its border is defined by an inverted repeat (5 'ACTG) containing a conserved TG dinucleotide (Temin, 1
(II) 981); (II) There is a tRNA primer binding site (PBS) adjacent to the 5 'LTR that is complementary to ▲ tRNA ^lys ₃ （(Raba et al., 1).
979); (III) There is a complete 15 bp polyprint lacto adjacent to the 3 'LTR. Following the other three polyprint lacts found between nucleotides 8200-8800, the PBS
The sequence complementary to the sequence immediately before is not followed. The boundaries of the U5, R and U3 elements were determined as follows. U5 with PBS,
It is located between the polyadenylation site established from the sequence at the 3 'end of the LAV cDNA primed with oligo (dt) (Al
izon et al., 1984). Therefore, the length of U5 is 84 bp. R +
U5 length was determined by synthesizing tRNA-primed LAV cDNA. After alkaline hydrolysis of the primer, the length of R + U5 was 181 ± 1 bp. Thus, the length of R is 97 bp, and the position of the capping site at its 5 'end can be determined. U3 has a length of 456 bp. L
AV LTR is a characteristic regulatory element, a polyadenylation signal sequence AATAAA 19 bp from the R-U5 junction and ATATAAG which appears to be a 2 bp TATA box 5 'of the cap site.
And an array. There are no other direct repeats in the LTR. Interestingly, the LAV LTR has some similarities to the mouse mammary tumor virus (MMTV) LTR (Donehower et al., 1981). For example, both LTRs can be used as primers for (-) strand synthesis.
^{Use lys} ₃ ▼. In contrast, all other exogenous mammalian retroviruses currently known use tRNA ^pro (Chen and Barker, 1984). In addition, these LT
R has a very similar polyprint craft (LAV
AAAAAGAAAGGGGGG for MMTV, AAAAAGAGAAAAAGGGG for MMTV
G). Virus (+) strand synthesis can be intermittent. Because the polyprint tracts on either side of the U3 element of the 3 'LTR are 4331-4 at the 3' end of the orf pol
This is because 336 exactly overlaps. In addition, MMTV and LAV
It differs from the others in that the U3 element can code orf. In the case of MMTV, U3 includes the entire orf, but in the case of LAV
Contains 110 codons in the 3 'half of orf F of U3.

The long repeat sequence (LTR) at the end of the LAV is shown in FIG. As described above, this LTR was reconstructed from the λ J19 sequence by juxtaposing the sequence adjacent to the Hind III cloning site.

LAV DNA clone pLAV7 primed with oligo (dT)
5 (Alizon et al., 1984) sequencing excludes the possibility of a cluster of Hind III sites in the R region of LAV. The LTR is bounded by inverted repeats (IR). The viral elements on both sides of the LTR are both the 5 'LTR tRNA primer binding site (PBS) and the 3' LTR
Has been described as a polyprint rack (PU). The putative TATA box, cap site, polyadenylation signal (AATAAA) and polyadenylation site (CAA) are also shown. The position of open reading frame F (648 nucleotides) is indicated above the LTR illustration.

The LTR (Long Terminal Repeat) can also be defined as extending between position 8560 and position 160 (the tip extends over position 9097/1).
Located at 9097 and leads to the beginning of the sequence below due to the LTR structure of the retrovirus.

Table 1 summarizes the position and size of the virus reading frame. The nucleotide coordinates are the first triplet, the first methionine start codon (Met), and the stop codon (stop).
And the first base. The number of amino acids and the calculated molecular weight (MW) are calculated for the unmodified precursor product from the first methionine to the end, excluding pol. The size and MW of pol indicate the size and MW of the entire orf.

The invention more particularly relates to all the aforementioned specific DNA fragments corresponding to the open reading frame. One skilled in the art can obtain all of these DNA fragments by, for example, cutting the entire DNA corresponding to the complete genome of the LAV species by partial or total digestion with appropriate restriction enzymes, and then recovering the desired fragment. Please understand that you can do it.
The various DNAs described above can also be used as sources for suitable fragments. The following techniques can be used to isolate the fragments contained in the aforementioned plasmids and used for DNA sequencing.

Of course, other methods may be used. One example is 9/1984
It is described in UK Patent Application No. 8423659, filed on March 19, 1992. Below are a few examples of methods that can be used.

a) DNA is transfected into mammalian cells with a suitable selectable marker by various methods, such as calcium phosphate precipitation, polyethylene glycol, protoplast fusion, and the like.

b) A DNA fragment corresponding to the gene was
Cloning into an expression vector for yeast or mammalian cells and purifying the resulting protein.

c) Provirus to generate fusion polypeptide
The DNA is "shot-gun treated" (fragmented) and introduced into a prokaryotic expression vector. Recombinants producing fusion proteins that can act as antigens can be identified simply by screening these recombinants using antibodies against the LAV antigen.

The invention further relates to a corresponding microorganism or cell, especially a eukaryotic cell such as yeast, for example saccharomyces cerevisiae, or a higher eukaryotic cell, especially a mammalian cell, comprising any of the above DNA sequences. It also relates to DNA recombinants, especially modified vectors, which are adapted for light conversion and suitable for expressing said DNA sequence in a corresponding microorganism or cell. A common method of this kind was 1984 11
This is described in the aforementioned British Patent Application No. 8429099, filed on Mar. 16, 2016.

The present invention more particularly relates to modified DNA recombinant vectors which are modified by the aforementioned DNA sequences and which can convert higher eukaryotic cells, especially mammalian cells, into light.
All of the sequences are such that the first expressed nucleotide codon corresponds to the first triplet of the DNA sequence as defined above, under the direct control of a promoter contained in the vector and identified by the cellular polymerase. Preferably, they are arranged. The invention therefore furthermore relates to corresponding DNA fragments which can be obtained from the LAV genome or the corresponding cDNA by any suitable method.
As a method therefor, for example, the LAV is preferably surrounded by a restriction enzyme at the level of a restriction site surrounding each of the fragments and close to the opposite end of these fragments.
Cleavage of the genomic or cDNA, recovery and identification of the desired fragments depending on their size, and, if necessary, checking the restriction map or nucleotide sequence of these fragments (or carrying the polypeptide encoded by said DNA fragment) Reaction with a monoclonal antibody specifically acting on the epitope to be treated), and, if necessary, for the purpose of controlling the desired nucleotide sequence at the end of the DNA fragment or vice versa.
A method of treating the ends of a fragment with an exonuclease, such as Bal 31, for the purpose of repairing the fragment with an enow enzyme and possibly linking the fragment to a synthetic polynucleotide fragment designed to reconstruct the nucleotide termini of the fragment There is.
These fragments can then be inserted into any of the vectors described above, and the corresponding polypeptides can be expressed by cells transformed with the vector. The corresponding polypeptide can then be recovered from the transformed cells, if necessary, after lysis of the cells, and purified by methods such as electrophoresis. Of course, any conventional technique may be used to perform these operations.

The present invention further relates to a cloned probe that can start from any DNA fragment of the present invention,
Thus, any recombinant DNA containing such a fragment, in particular a prokaryotic or eukaryotic cell, which can be amplified and carries the said fragment is also concerned.

When used as a molecular hybridization probe-by labeling the cloned DNA fragment-with a radionucleotide or a fluorescent reagent-
In body fluids, in blood products (eg anti-hemophilia factors such as factor VIII concentrates) and in vaccines, ie hepatitis B vaccines
LAV virion RNA can be detected directly. It has already been found that whole virus can be detected in the culture supernatant of LAV-producing cells. One suitable method for performing this detection is to immobilize the virus on a support (carrier), such as a nitrocellulose filter, disrupt the virions (radioactive or "cold" fluorescent labels, or Hybridization (with an enzyme label) with the labeled probe. This type of method has already been developed for hepatitis B virus in peripheral blood (SCOTTO J. et al. He.
patology (1983), 3,379-384).

The probe of the present invention further provides for rapid screening of genomic DNA derived from tissue of a patient exhibiting symptoms of LAV to detect whether proviral DNA or RNA is present in host and other tissues. Can also be used to do.

One method that can be used for such screening comprises the following steps. DNA is extracted from the tissue and this D
The NA is cut with a restriction enzyme, the fragment is subjected to electrophoresis, Southern blotting of the genomic DNA from the tissue is performed, and then hybridized with the labeled cloned LAV proviral DNA.
Hybridization may be carried out on the spot.

Lymph and lymphoid tissues and non-lymphoid tissues of humans, primates and other mammalian species can also be screened for the presence of other evolutionarily related retroviruses. Again, the method described above can be used. However, hybridization and washing are performed under non-stringent conditions.

The DNA or DNA fragment of the present invention may be used for diagnostic purposes in LA
It can also be used to express V virus antigens.

The present invention is generally chemically synthesized, isolated from a viral sample, or expressed by various DNAs of the present invention, particularly transformed with an appropriate vector that has been previously modified with the corresponding DNA. Later these DNA
The ORFs or fragments thereof relate to the polypeptide itself, including all expressed in a suitable host, particularly a prokaryotic or eukaryotic host.

In general, the invention relates to any polypeptide fragment (or molecule, in particular a glycoprotein having the same polypeptide backbone as the above-mentioned polypeptides) having an epitope specific to the LAV protein or glycoprotein.
Also, the N-terminus and C-terminus of the polypeptide or molecule are each free, or, independently of each other, usually linked to the N-terminus or C-terminus of the larger polypeptide or glycoprotein of the LAV virus It is covalently linked to amino acids other than amino acids. These amino acids are themselves free or belong to another polypeptide sequence. The invention particularly relates to hybrid polypeptides containing any of the above-identified epitope-bearing polypeptides, which are usually recombined (recombined) with other polypeptide fragments that are foreign to the LAV protein, The heterologous polypeptide fragment is large enough to increase the immunogenicity of the epitope-bearing polypeptide, but the fragment itself is immunogenically inactive, or It does not prevent sex.

Hybrid polypeptides such as those described above, which can include up to 150, and optionally up to 250 amino acids, are typically
The expression product of a vector originally containing a nucleic acid sequence that can be expressed in a suitable host under the control of a suitable promoter or replicon, said nucleic acid sequence comprising:
It has been previously modified by inserting a DNA sequence encoding an epitope-bearing polypeptide into the nucleic acid sequence.

Particularly, the epitope-bearing polypeptide in which the N-terminal and C-terminal amino acids are free can be chemically synthesized by a technique known in the field of protein chemistry.

Peptide synthesis in homogeneous solutions and on solid phases is known.

In this regard, “Methoden der organischen” edited by E. WUNSCH
Chemie (Method of Organic Chemistry) ", vol.15-I & II, THIEME, S
The method of synthesis in homogeneous solution described by Houbenweyl in Tuttgart, 1974 can be used.

In this synthesis method, two consecutive amino acids are continuously condensed in an appropriate order, or a previously obtained or formed continuous peptide fragment containing a plurality of aminoacyl residues is continuously condensed in an appropriate order. Consisting of: Among the reactive groups of the above aminoacyl residue or fragment, all those other than the carboxyl group and the amino group involved in the formation of the peptide bond must be protected in advance. However, the carboxyl group is advantageously activated prior to the formation of the peptide bond by methods well known in the art of peptide synthesis. Alternatively, in other cases, for example, 1-ethyl-3- (3-dimethyl-
The coupling reaction may be effected using a carbodiimide-type conventional coupling reagent such as (aminopropyl) -carbodiimide. When the amino acid residue used has an additional amine group (for example, lysine) or an acid function (for example, glutamic acid), the functional group may be a carbobenzoxy group or a t-butyloxycarbonyl group if it is an amine group. And if it is a carboxyl group, t-
Can be protected with a butyl ester group. Similar procedures are valid for the protection of other reactive groups. For example, SH groups (such as cysteine) can be protected with acetamidomethyl or paramethoxybenzyl groups.

In the case of sequential synthesis in which amino acids are linked one by one, synthesis involves first condensing the C-terminal amino acid with the amino acid corresponding to the adjacent aminoacyl group in the desired sequence, and then sequentially condensing the amino acid to the N-terminal amino acid as described above. It is preferable to realize this. Another preferred technique that can be used is "solid phase peptide sy
nthesis "(J. Am. Chem. Soc., 45, 2149-2154).

In the Merrifield method, the first C-terminal amino acid of the chain is fixed to a suitable porous polymer resin by its carboxyl group, wherein the amino group of the amino acid is protected, for example, by a t-butyloxycarbonyl group.

After the first C-terminal amino acid is immobilized on the resin as described above, washing the resin with an acid, i.e., trifluoroacetic acid if the protecting group for the amine group is a t-butyloxycarbonyl group. To remove.

Next, the carboxyl group of the second amino acid to provide the second aminoacyl group of the desired peptide sequence is coupled to the amine group from which the protecting group of the C-terminal amino acid immobilized on the resin has been removed. Preferably, the carboxyl group of the second amino acid is activated, for example, with dicyclohexylcarbodiimide, and the amine group of the amino acid is, for example, t
-Protected with a butyloxycarbonyl group. Thus, a first portion of the desired peptide chain, including the first two amino acids, is obtained. The protecting group for the amine group is removed as in the previous case. Thus, a desired peptide can be obtained by sequentially fixing the aminoacyl groups.

If the peptide chain formed as described above has various side-chain groups, the protecting groups of the side-chain groups can then be removed. The desired peptide thus obtained can be separated from the resin, for example with hydrofluoric acid, and finally recovered in pure form from the acid solution by conventional procedures.

Epitope or antigenic determinant
covalently linking the peptide sequence to a physiologically acceptable non-toxic carrier molecule to increase its in vivo immunogenicity, particularly for those that are minimally peptide sequences having inant) May need to be coupled or "coupled."

Examples of carrier molecules or polymeric carriers that can be used to realize a conjugate according to the invention include natural proteins such as tetanus toxoid, ovalbumin, serum albumin, hemocyanin and the like. For example, synthetic polymer carriers such as polylysine or poly (DL-alanine) -poly (L-lysine) can be used.

Other types of usable polymeric carriers, usually having a molecular weight above 20,000, are known from the literature.

The conjugate is described in “Infect. And Immunity”, 33, 193-198 (19
81) as described by Frantz and Robertson or alternatively in Applied and Environmental Micro
−biology, October 1981, Vol.42, n ゜ 4,611−614
It can be synthesized by known methods as described by auffman.

As an example, the following coupling agents can be used. : Glutaraldehyde, ethyl chloroformate,
Water-soluble carbodiimide, [N-ethyl-N '(3-dimethylaminopropyl) carbodiimide, HCl], diisocyanate, bis-diazobenzidine, dichloro and trichloro-s-triazine, cyanogen bromide, benzoquinone,
And “Scand. J. Immunol.”, 1978, Vol. 8, p. 7-23 (Avra
meas, Ternynck, Guesdon).

Any coupling method can be used to link one or more reactive groups on the peptide to one or more reactive groups on the carrier. Further, the bond is formed by coupling the carboxyl group of the peptide to the carrier in the presence of a coupling agent of the type used for protein synthesis, ie, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, N-hydroxybenzotriazole and the like. Advantageously, it is realized between an amine group and vice versa. When the carrier is hemocyanin, for example, BOQUET, P.et
al., Molec. Immunol., 19 , 1441-1549 (1982), it is also possible to bond the amine groups of the peptide and the carrier with glutaraldehyde.

The immunogenicity of an epitope-bearing peptide can be enhanced by oligomerizing the peptide in the presence of, for example, glutaraldehyde or any other suitable coupling agent (coupling agent). In particular, the present invention relates to water-soluble immunogenic oligomers obtained as described above,
Pertains to those containing 1010 monomer units.

Glycoproteins, proteins and polypeptides of the invention (hereinafter collectively referred to as "antigens") may be obtained in purified form from LAV virus samples, or-especially with respect to peptides-by chemical synthesis, Or A
Antibodies in biological media, especially biological fluids such as human or animal serum, from the perspective of being able to diagnose IDS
Useful in methods of detecting the presence of a LAV antibody.

The invention particularly relates to an in vitro diagnostic method using an envelope glycoprotein (or a polypeptide having an epitope of said glycoprotein) to detect said antibody in human serum having an anti-LAV antibody. Other polypeptides-especially those having a core protein epitope-can also be used.

Preferred embodiments of the method according to the present invention include:-a predetermined amount of one or more of the above-mentioned antigens is placed in a cup of a titration microplate;-a diluted biological fluid or serum to be diagnosed in the cup. -Increasing the dilution;-incubating the microplate;-carefully washing the microplate with a suitable buffer;-adding a specifically labeled antibody against blood immunoglobulin into the cup. And detecting the formed antigen-antibody complex indicating the presence of the LAV antibody in the biological fluid.

The labeling of the anti-immunoglobulin antibody is advantageously carried out by an enzyme selected from among the enzymes capable of hydrolyzing the substrate, said substrate undergoing a change in its radiation absorption at least within a given wavelength range. By detecting the substrate, preferably in comparison to a control, the potential risk or presence of the disease can be determined.

Thus, the preferred method is enzyme-linked immunosorbent assay or fluorescent antibody-based detection, especially by ELISA technique. The titration may be a measurement by a fluorescent antibody method or a direct or indirect enzyme immunoassay. In this way, the antibody in the investigated serum can be titrated quantitatively.

The invention also relates to a diagnostic kit itself for the in vitro detection of antibodies against the LAV virus, which kit comprises any of the polypeptides defined herein and all the biology required to carry out a diagnostic assay. And chemical reagents and equipment. A preferred kit contains all the reagents necessary to perform an ELISA assay. Thus, a preferred kit comprises, in addition to any of the polypeptides described above, a suitable buffer and an anti-human immunoglobulin, wherein the anti-human immunoglobulin is labeled with an immunofluorescent molecule or an enzyme. A preferred kit in this last case also comprises a substrate which can be hydrolyzed by the enzyme, the substrate exhibiting a signal at least at a given wavelength, in particular an altered radiation absorption, wherein the signal is Indicates the presence of the antibody in it.

The present invention relates to any antigen, i.e. the polypeptides disclosed above, the whole antigen, in particular the purified gp110, together with a suitable pharmaceutically or physiologically acceptable carrier.
It also relates to a vaccine composition having an active ingredient consisting of a fragment or immunogenic fragment, a fusion polypeptide or an oligopeptide.

A first class of preferred active ingredients is the gp110 immunogen.

Other preferred active ingredients to be considered in the art are
Asn-X-Ser and Asn-X-, as defined above, consisting of peptides containing less than 250, preferably less than 150 amino acid units, as can be inferred for the complete genome of LAV. Consists of a peptide containing one or more residues selected from Ser. Peptides that can be preferably used for the production of vaccine components are the peptides (a) to (f) proposed above. As one non-limiting example, the dosage of the vaccine composition may be appropriate in vivo, ie, an amount effective to produce antibodies in a host, particularly a human host. A suitable dosage range is 10 to 500 micrograms / kg of polypeptide, protein or glycoprotein, for example 50 to 100 micrograms / kg.

The various peptides according to the invention can also be used for the production of antibodies of their own, preferably monoclonal antibodies specific for each of the various peptides. For the production of hybridomas that secrete the above monoclonal antibodies, conventional production and screening methods are used. The above monoclonal antibodies, which are themselves part of this invention, confirm and further determine the relative proportions of various polypeptides or proteins in biological specimens containing LAV or related viruses, especially specimens obtained from humans. Very useful to do.

The present invention further relates to a host (prokaryotic or eukaryotic cell) capable of expressing the above DNA fragment, which is transformed by the above-described recombinant.

Finally, the invention also relates to a vector for the transformation of eukaryotic cells of human origin, in particular lymphocytes whose polymerase can identify the LTR of LAV. This vector is particularly characterized in that the LTR of LAV is present in the vector.
R is active as a promoter that places the protein of interest under its own control to allow efficient transcription and translation of the DNA insert encoding the protein in a suitable host.

Naturally, the invention also applies to certain variants of the genome belonging to the retrovirus that can be considered as equivalent to LAV as well as the corresponding DNA fragment (ORF) with substantially equivalent properties.

It is to be understood that each of the claims below includes any equivalent of a product (glycoprotein, polypeptide, DNA, etc.), in which case the equivalent means any of the above claims. A product or polypeptide that is distinguished from, for example, one or more amino acids by a given polypeptide as defined above, but that has substantially the same immunological or immunogenic properties as said given polypeptide. . DNA
The same equivalent rule applies to
Is understood to be associated with equivalent rules for the polypeptide encoded by.

In addition, all documents referred to in the present specification and all patent applications and patents not specifically specified in the present specification but closely related to the patent applications and patents specifically indicated in the present specification are described in the present specification. It is understood that it should be considered to be included as a reference.

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Continuation of the front page (31) Priority claim number 85501473 (32) Priority date January 21, 1985 (33) Priority claim country United Kingdom (GB) Referee number Hei 6-19805 (72) Inventor Montenier, Liyuksk France , 92350 · Le Presi Rovanson, Riu Dou Malaburi, 21 (72) Inventor Criyust, Bernard France, 75012 Paris, Riu Dou Madagascar, 7 (72) Inventor Ciamale, Solanjiu France , 75015 Paris, Lille-le-Club, 324 (72) Inventor Clavuel, François, France, 75016 Paris, Lille-de-Las-Opsion, 83 (72) Inventor Cielman, Jean-Claude, France, 78310, Elan Cour, Clos Delgan 2 (72) Inventor Bar-Sinusi, Françoise France, 130130 It ツ sie-Rém-Rinault, Rille-Deluvin, 50 (72) Inventor Rizon, Marc France, 75005 Paris, Riuille du You Cardinal Lemoine, 71 (72) Inventor Sonigo, Pierre France, 75015 Paris, Riu Guille Tambert, 23 (72) Inventor Stoile, Col France, 92320-Chatateillon, Veila Downeys, 4 Bis (72) Inventor Dano, Olivier France, 75015 Paris, Place Lolle, 1 (72) Inventor Vuan-Obson, Simon France, 78180 Montigny-les-Bretonneux, Liuux-Jiann-dou-la-Fontaine, 3 (56) Reference Science, 224 (1984) p. 503 -505

Claims (5)

(57) [Claims] 1. A purified product which contains a polypeptide backbone of a glycoprotein having a molecular weight of about 110,000 and which can be recognized by human serum containing an antibody against the LAV virus envelope, said polypeptide backbone comprising: The product as described above, which is encoded by a nucleic acid fragment extending between nucleotide Nos. 5767 and 7314 of FIG. 4 attached to the specification. 2. The purified product of claim 1, which is a purified glycoprotein. 3. The purified product according to claim 2, which forms a complex with concanavalin A and the complex can be dissociated in the presence of O-methyl-α-D-mannopyranoside. 4. The method according to claim 2, which binds to a lectin.
Purified product. 5. The purified product according to any one of claims 1 to 4, which is also sensitive to the action of endoglycosidase.