JP2865203B2 - A mixture of novel retroviral antigens that cause AIDS - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to human acquired immunodeficiency syndrome (AIDS).
A new drug with the ability to develop a developing lymph node disorder
Pertains to the Russ virus. The present invention also relates to this new class.
Virus-induced antibodies in humans.
Pertains to antigens that can be identified. The invention also relates to these
Pertinent to antibodies elicited by antigens obtained from yeast. The invention further relates to the genomic RAM and sequence correlation of said virus.
Related to cloned DNA sequences having similarity or complementarity. The present invention
The present invention also relates to a method for preparing these cloned DNA sequences. The invention further provides that the cloned DNA sequence encodes
The present invention relates to a polypeptide containing an amino acid sequence to be In addition, the present invention provides several forms of
The use of the antigen of the present invention for in vitro diagnosis of
Retrovirus using some of these antigens
Production of immunogenic and vaccination compositions against Lus
According to. The present invention also provides the antigen for the same purpose as the antigen.
Related to the use of the body, and using certain of these antibodies
Of active ingredients of drugs against human AIDS in various forms
Pertaining to construction. Finally, the invention relates to cloned DNA sequences and the
Polypeptides used as probes for diagnostic kits
Use inventions that include Recognized as the cause of AIDS and known as LAV
Isolation and characterization of the first retrovirus
Already published in 1983 in papers such as F.BARRE-SINOUSSI
(Science, vol. 220, No. 45-49, 20, p. 868
−871). Also, the presence of antibodies against AIDS virus
Certain extracts of the above LAV virus to diagnose;
In particular, the use of certain proteins is a European patent application
No. 138667 describes in detail. Then, like LAV
Similar other strains and several mutants were isolated. this
Examples of these are HTLV-III and ARV.
You. Following the new nomenclature published in Nature in May 1986
The lymph node disorder and AIDS can be induced in humans.
Torovirus has been referred to by the generic name HIV.
This is the "Human Immunodeficiency Virus"
Virus). Depending on the LAV and its variants
The subgroup of retroviruses formed initially
It was designated "LAV Type I" or "LAV-I". In the main
In the following, this subgroup will be referred to as HIV-1.
-1 strain of retrovirus belonging to the virus class
The European patent in LAAV, IDAV-4 and IDAV-2)
LAV when indicating the strain described in application No. 138667
It should be understood that the terminology is maintained. This strain is described below.
LAV in a comparative experiment _BRU Used as of July 1983
Collection of the Pasteur Institute in Paris, France on the 15th
Mationale des Cultures de Micro-Organismes (CNC
M) and accession number I-232. Novel retrovirus which forms the subject of the present invention, and this
Up to the present invention designated "LAV Type II" or "LAV-II"
Related viruses that can grow on human lymphocytes as well as retroviruses
The ills strain is hereinafter referred to as "HIV-2". However, described later
Certain HIV-2 isolates are indicative of isolate donating patients
It should be understood that it is written with three letters. The “HIV-2” group is a human T4 lymphocyte in vitro
Have an affinity for these and when grown on human lymphocytes, these phosphorus
Has a cytopathic effect on papillocytes, systemic and persistent
A virus that causes one form of polydenopathy or AIDS
Can be defined as a group. HIV under the conditions described below
-2 retrovirus is different from HIV-1 type virus
It was proved that. In addition, HIV-2 retrovirus and
And HIV-1 type viruses are both known other
Different from human retroviruses (HTLV-I and HTLV-II)
You. There is a fairly wide genetic variance in the virus,
American, European, Haitian and African to date
Different HIV-1 strains isolated from different patients share a common antigen
Has a part. This site is the main protein,
Protein p25, envelope glycoprotein gp110 and trans
It is retained on the membrane protein gp41-43. This
Therefore, for example, if the archetype LAV strain is
Instead, all HIV-1 classes for all careers
Used as strain of antigen for detection of antibodies to Rus
It is possible to Therefore, this strain is particularly ELISA
Known immunofluorescence, Western blot (
Noin printing) method, RIPA (immunoprecipitation assay)
Detect anti-HIV-1 antibodies in donors and patients using methods
Widely used for. However, HIV-1 lysis of patients from West Africa
According to serologic laboratories conducted on fluids, these
Lysate showed clinical and immunological signs of AIDS
Nevertheless, the serum reaction was negative or very weak positive
I didn't. For the first time, HIV-
2 was isolated. HIV-2 isolated from this source
The structure observed by electron microscope and SDS gel electrophoresis
Protein profile is similar to HIV-1
Was. However, overall, this new retrovirus
HIV-2 is a protein antigenic homology and genetic material
From both homology perspectives, there is little association with HIV-1.
No. This new retrovirus or its equivalent antigen
Retroviruses with sexual and immune properties are
Or the AIDS form first detected by those who have stayed in Africa
Diagnosis of virus or virus variant infection
Configure an antigen source for Typically, this virus is used in transfusion and drug
Collected in the presence of heparin from a 28-year-old heterosexual patient without
From isolated blood. Patient has been quite chronic since 1983
Diarrhea continued, sometimes fever and weight was extremely reduced
(17kg). Later Candida and Serratia Seratia
Infected with AIDS, typical esophageal candidiasis (candidiasi)
s). This patient also has anemia, cutaneous anergy and lymphocytes.
T4 lymphocyte / T8 lymphocyte ratio of 0.15
Damp bulb level is serum mm ^Three Less than 100 per. Culture phosphorus
The papules are phytohemagglutinin and concanavalin A
Did not react violently. This patient also has S.enteriditis
Recurrent bacteremia, cryptosporidioses, war isospo
Infection by Brassica (Isosporabelli) and Brain Toxoplasm
I was suffering from makiosis. These complications are caused by the HIV-1 virus
Type of "AIDS-related syndrome" or ARC (AIDS-Related
Complex). These various findings are
At the Center of Disease Control (CDC) in Atlanta
It also matched the criteria adopted. Culture lymphocytes from patients and isolate retroviruses
In order to achieve this, the BARRE-SINOUSSI paper and European Patent Application No. 8
4 / 401.834-0.138.667.
use. These methods are briefly described below. Fi
Lymphocytes stimulated with tohemagglutinin (PHA) for 3 days
10% fetal bovine serum and 10% ^-Five M β-mercaptoeta
Nol, interleukin-2 and anti- (human interf
Cultivation in RPMI1640 medium supplemented with e.g. Virus production was followed by reverse transcriptase activity.
Peak virus activity in culture supernatant between 14 and 22 days
It has since decreased. Track cell culture decay and death
Was. Observation with an electron microscope shows that HIV is similar to HIV-1.
-2 infected lymphocyte sections are mature pillion
Virus particles are budding on the surface of infected cells.
Cells used to produce cultures of these isolated viruses
The system can be a HUT, CEM or MOLT type cell line, as appropriate.
Or any established phosphorus bearing T4 receptor
Pagocyte cell lines may also be used. The virus is then propagated in donor lymphocyte cultures.
Followed by a continuous cell line of leukemia origin, such as HUT78.
Bred. This antigen and nucleic acid are substantially the same as those of HIV-1.
Characteristic differences were found. Virus
Was purified by the method described in the conventional literature as described above. This
The first isolate of the virus was based on the Budapest Treaty
Deposited with the CNCM on December 19, 1985 under accession number I-502,
After LAV-II MIR. The second isolate is similar
Accession number I on February 21, 1986 based on the Budapest Treaty
Deposited at -532 at the CNCM and named LAV-II ROD. This
These isolates are often simply referred to as MIR or ROD. Generally, the present invention relates to CNCM as I-502 or I-532.
Structure with the same immunological properties as the deposited HIV-2 virus
Any equivalent virus containing proteins (eg, 1986
Accession number I-642 based on the Budapest Treaty on December 19, 2012
HIV-2-IRMO and HIV-deposited under I-643 and I-643, respectively.
2-EHO) and any variants thereof.
I do. The definition of the equivalence criterion will be described later. The present invention also relates to T4 lymphocytes or R4 cells carrying the T4 phenotype.
HIV-2 virus in a permanent cell line derived from a lymphocyte
Or a method for producing a mutant thereof. in this way,
Culture these cell lines pre-infected with HIV-2 virus
Especially when the reverse transcriptase activity level reaches a certain threshold
The amount of virus released into the medium is recovered. For example, a preferred permanent cell line for culturing HIV-2 is
If it is HUT78 cell type. HUT78 strain infected with HIV-2
Was deposited with the CNCM on February 6, 1986 under accession number I-519.
Was. The culture is performed, for example, as follows. Infected HUT78 cells (10 ⁶ / ml) infected normal human lymphocytes (1
0 ⁶ / ml). Medium contains 10% fetal bovine serum
It is a PRMI1640. Cell changes in HUT78 cells after 15-21 days
Observe sexual effects. One week after this observation,
Quantify reverse transcriptase and start virus recovery from supernatant
You. Another cell line suitable for culture is the cell line known as CEM.
Vesicle system. Infection of CEM cells and culture of infected CEM cells are especially
It is performed like Insensitive to T4 lymphocytes pre-infected with HIV-2 virus
Co-culture with stained CEM cells only for the time necessary for CEM infection
You. Then, for example, culture conditions in a suitable medium as described below
To maintain sufficient levels of reverse transcriptase activity in infected cells.
Upon reaching, the produced virus is recovered from the medium. In particular, H from patients referred to hereinafter as ROD in the text
Human T4 lymphocytes and CE previously infected with strain IV-2 for 5 days
M is co-cultivated under the conditions described below. Infected T4 lymph preactivated with phytohemagglutinin
The spheres are 10 days after the start of infection. ⁶ 5 per normal T lymphocyte
000 cpm reverse transcriptase activity. In the supernatant
Culture until the measured reverse transcriptase activity is 100.000 cpm.
Continued. Next, these T4 lymphocytes were converted to CEM cells (3 × 10 ⁶ Feeling
(Stained normal T lymphocytes) and re-incorporated in the following medium:
Cubbed. Medium: 2.92 mg / ml L-glutamine and 10
% Complement-depleted fetal bovine serum and 2 μg / ml polybrene (Poly
brene) with 0.05% anti-interferon alpha serum and 100,000
μg / ml penicillin and 10 μg / ml streptomycin
PRMI1640 containing 10,000 μg / ml neomycin. The medium is changed twice a week. The measured values of reverse transcriptase activity measured in the supernatant are shown below.
Show. 0 days 1,000 (background) 15 days 20,000 21 days 200,000 35 days 1,000,000 CEM cultures infected with HIV-2 virus were obtained on March 24, 1986.
Deposited with the CNCM of the Pasteur Institute on the day under the accession number I-537
Was done. Some of the antigens and nucleic acids involved in the structure of HIV-2
The characteristics were clarified by experiments performed under the following conditions.
Was. In many cases, these characteristics are another type of retro
Comparison with the same properties in irs, especially HIV-1 and SIV
More clear by. Next, the attached drawings will be described. FIGS. 1a, 1b and 1c show HIV-1 and HIV-2
Serum of infected patients and rhesus infected with STLV-III respectively
Cross-sedimentation experiment between monkey serum and HIV-1 virus extract
About. Figures 2a and 2b show the results in SDS polyacrylamide gel.
HIV-1, HIV-2 and STLV-III proteins
5 shows the results of comparison of the electrophoretic mobilities of. FIG. 3 shows the genome arrangement of HIV-1, HIV-2 and STLV-III.
Sequence and various subgenomic sequences of the HIV-1 virus
The results of cross-hybridization with the probe
Show. FIG. 4 shows the restriction of cDNA derived from HIV-2 ROD RNA.
Show the map. FIG. 5 shows the E2 fragment of cDNA derived from HIV-2.
3 shows the restriction map of the program. This fragment is HIV-2
Includes a region corresponding to the 3 'LTR region. FIG. 6 shows the nucleotide sequence of a portion of E2. this
The sequence corresponds to the U3 / R region of HIV-2. FIG. 7: On the one hand, the structural elements of HIV-1 (FIG. 7A) and HIV-13 '
Is the E2 region of the HIV-2 cDNA aligned with the region containing the LTR?
The sequences derived from the E2 region of HIV-2 on the other hand
To the sequence taking into account the deletion and insertion of
Shows common nucleotides present in the corresponding sequence of HIV-1
(Fig. 7B). FIG. 8 shows some of the cDNAs derived from HIV-2.
Several clones on phage modified by insertion
(Clone ROD4, ROD27 and ROD35)
You. ROD subcloned in plasmid pUC18
4, sequences from ROD27 and ROD35 are also schematically shown in the figure
Have been. The subcloning sequence is the same as the original ROD4, ROD27 and
And ROD35. FIG. 9 shows (a) 1 extracted from various regions of the complete HIV-1 genome.
One fragment (this fragment is outlined at the bottom of the figure)
HIV-2 cDNA present in ROD4)
And (b) fragment obtained from HIV-2 having the same cDNA
2 shows the relative intensity of hybridization between the two. In general, the HIV-2 antigen described below used in the comparative test
Is the HIV-2 MIR strain deposited at the CNCM under accession number I-502
DNA sequence derived from the genomic DNA of HIV-2
M obtained from the HIV-2 ROD strain deposited under accession number I-532.
It is. I. Proteins and glycoproteins at the time of antigen ³⁵ S] Sistay
And [ ³⁵ [S] methionine for metabolic labeling
In a medium from which the corresponding unlabeled amino acids have been removed,
Incubate infected cells for 14-16 hours in the presence of radioactive amino acids
Was added. [ ³⁵ S] Details about cysteine label
Those listed in reference (21) in the bibliography attached at the end of the book
Method. Next, the clear supernatant is collected and a 20% sucrose
The virus was ultracentrifuged at 100,000 g for 1 hour on a buffer. Strange
Polyacrylamide gel (12.5%) under neutral conditions (SDS)
Or polyacrylamide gel (10%) + bisacrylia
Gel consisting of mid (0.13%) and SDS (final concentration 0.1%)
The major antigen of the virus was separated by electrophoresis in a gel.
The following color markers were used as molecular weight standards. Myosin 200 kd phosphorylase B 97.4 kd BSA 68 kd ovalbumin 43 kd α-chymotrypsin 25.7 kd β-lactoglobulin 18.4 kd lysozyme 14.3 kd In another experiment, another molecular weight marker was used. For example
In particular, in FIGS. 1a, 1b and 1c (indicated by the letter M in the figures)
C) Another molecular weight marker was used. Present in the patient's serum
Immunoprecipitation (RIPA) using existing antibodies or immunoin
Antigen is easy after printing (Western blot)
Can be identified. Apparent measured by apparent mobility
The molecular weight is very close to the molecular weight of the HIV-1 antigen. Generally, in the following description in the text, "p" and / or
The numbers following “gp” indicate the corresponding protein and / or sugar
It corresponds to the approximate molecular weight of the protein divided by 1000.
You. For example, p36 has a molecular weight of about 36,000. But
These molecular weight values are based on the method used to determine the molecular weight.
Variability within 5%, 10% or more depending on
Please understand that. Repeat the experiment to determine the apparent molecular weight of the HIV-2 antigen.
More accurate measurement. Therefore, first about 13,000, 18,000
And 35,000 core proteins, respectively.
In some cases, an apparent molecular weight close to 12,000, 16,000 and 26,000
Had. These proteins will be abbreviated in the text
Shown as p12, p16 and p26. Apparent molecular weight range from 32,000 to 42,000-45,000
Of the protein or glycoprotein estimated to be the value of
The existence of a band can be similarly considered. Repeat measurement
Finally, a band corresponding to an apparent molecular weight of 36,000
Could be defined exactly. In the following description in the text, this van
Is indicated by the abbreviation p36. Another van of 42,000-45,000 (p42)
Is always observed. Probably either p36 or p42
Constituting the ils transmembrane protein
Will be. The main envelope of the order of 130-140 kd molecular weight
Glycoproteins are always observed. This glycoprotein
Hereinafter referred to as gp140 in the text. Generally, the molecular weight is measured with an accuracy of ± 5%.
(High molecular weight 140 ± 10%).
Note that the molecular weight of the antigen may be slightly lower
No. This group of antigens ([ ³⁵ S] Cysteine labeled
Detection), the detection system used in the laboratory
Serum or DIAGNOSTICS P containing anti-HIV-1 antibody
HIV-1 lysis, marketed by ASTEUR under the trademark "ELAVIA"
It is only weakly recognized by tests using liquids. This
Serum immunoprecipitates only p26 protein
Was. The envelope protein did not settle. new
The serum of patients infected with the virus (HIV-2)
The p34 protein was faintly recognized. Use detection system smell
Thus, another HIV-1 protein was not recognized. In contrast, HIV-2 is a rhesus macaque (capti
ve macaque of rhesus species)
Equivalent structural proteins isolated under similar conditions from Torovirus
Some immunological correlation with proteins or glycoproteins
It has certain proteins as shown. This immunological correlation is different
Of proteins and glycoproteins. monkey
This retrovirus is a putative agent of AIDS in Japan
Is a researcher who succeeded in isolation (references (16) to (18))
By STLV-III mac. The following description for convenience
As described above, this retrovirus is simply referred to as STLV-III (or “S
imian Immunodeficiency Virus "
I do. Another retrovirus was isolated from wild green monkeys
STLV-III _AGM Or SIV _AGM Was named. But
Africa, in contrast to the virus present in rhesus monkeys
"STLV-III" present in green monkeys _AGM Is AIDS Thailand
It does not appear to induce any disease of the group. However, for the retrovirus STLV-III mac
Even STLV-III _AGM Against HIV-2 structural protein
And the immunological correlation of glycoproteins is limited
Nucleic acid sequence relationships are limited. Infect humans or monkeys
Differences in retrovirus obtained are first confirmed by experiment
The following facts were found. The HIV-2 virus is described in NLLetvin et al., Science (198
5), STLV- as described by vol. 230, 71-75
III When injected in vivo under conditions that allow mac growth
Does not proliferate chronically in rhesus monkey lymphocytes. Thus, under natural conditions, HIV-2 virus
HIV-2 virus, based on the assumption that
Virus and STLV-III virus isolate.
You. Using the method cited above, the following from STLV-III:
The protein was obtained. -Major core protein on the order of 27 kilodaltons in molecular weight
p27,-the major envelope glycoprotein gp140,-the virus [ ³⁵ S] R when pre-labeled with cysteine
Although not observed in IPA, inom printing
Stern blot) can be observed as a wide band in the test
Probably the transmembrane protein p32. The major envelope glycoprotein of HIV-2 is immunology
From the glycoprotein of the major envelope of HIV-1
Also close to the glycoprotein chamber of the main envelope of STLV-III
It has been found. These findings are important not only in terms of molecular weight but also in terms of immunology.
It is proved from the point. That is, the main types of HIV-2 and STLV-III
Glycoprotein molecular weight is 130-140 kilodalton
In contrast, the molecular weight of the major glycoprotein of HIV-1 is about 110
Kilodaltons and from HIV-2 infected patients
Antibodies formed against collected sera, especially HIV-2 gp140
The body recognizes STLV-III gp140 but recognizes HIV-1 gp110
do not do. However, it did not react with HIV-2 gp140
Anti-HIV-1 serum is present in HIV-2 extracts [ ³⁵ S]
Precipitate the stain-tagged 26 kd protein. The major core proteins of HIV-2 are HIV-1 p25 and STLV
-Has an average molecular weight intermediate to that of p27 (about 26,000). These observations indicate that HIV isolated from one of the patients
Experiments performed using the virus extract obtained from C-2
Obtained by. Similar results were obtained from HI from the second patient.
V-2 extract was also obtained. Cells infected with HIV-1, HIV-2 and STLV-III, respectively
With 200 μCi / ml of unlabeled cysteine-free [ ³⁵ S]
Incubated for 16 hours in medium containing stain. Transparent
The bright supernatant was centrifuged at 60,000 g for 90 minutes. Pellet RIPA
Buffer and immunoprecipitated with various sera.
And polyacrylamide packed with sodium dodecyl sulfate
Degel electrophoresis (SDS-PAGE). The observed results are shown in FIGS. 1a, 1b and 1c. FIG. 1a shows HIV-1 cells obtained from the CEM C1.13 cell line.
View of immunoprecipitation between irs extract and each of the following sera:
The results are shown below. -An anti-HIV-1 positive serum (band 1);-a serum obtained from the first patient (band 2);-a serum of a healthy African anti-HIV-1 antibody carrier (band).
3),-obtained from Rhesus macaques infected with STLV-III
Serum obtained from the second patient (band 4), and-serum obtained from the second patient (band 5). FIG. 1b shows pre-cultured HUT78 cells taken from the first patient.
Between the raised HIV-2 antigen and various sera,
1 patient serum (band 1), anti-HIV-1 positive serum (band 1)
2), serum of Rhesus macaque spp. Infected with STLV-III
(Band 3) and the serum of the second patient (band 4)
The results of the observation of immunoprecipitation during this are shown. Finally, Fig. 1c shows a rhesus macaque with monkey AIDS.
Immunoprecipitation of STLV-III isolates from the genus Bacillus
The result of the observation of falling is shown. Blood used for bands 1-5
Qing is the same as in FIG. 1a. M is myosin (200 kd), galactosidase (130 kd)
d), bovine serum albumin (69 kd), phosphorylase B
(93kd), egg albumin (46kd) and carbonate anhydra
Markers such as ze (30 kd) are shown. 2a and 2b show HIV-1, HIV-2 and STLV-II.
3 shows the results of comparison of the electrophoretic mobilities of the protein I. Figure 2a shows that [ ³⁵ [S] Cysteine-labeled virus extraction
For experiments performed after SDS-PAGE immunoprecipitation of the output.
The different bands relate to the following virus extracts: Patient 1
From the same patient and immunoprecipitated with serum from the same patient
Lus (band 1), possesses anti-HIV-1 or anti-HIV-2 antibody
Not immunoprecipitated with human negative control serum
Virus extract (band 2), STLV-III infected
STLV-III immunoprecipitated with rhesus macaque serum
Virus extract (band 3), same virus extract and shade
Immunoprecipitation observed with sex control sera (van
4) Immunoprecipitation with serum from European AIDS patients
HIV-1 extract. Figure 1b shows a western blot (immuno imprint
3) shows the results obtained in the experiment. Infected or uninfected HU
The cell lysate of T-78 cells was subjected to SDS-PAGE electrophoresis, and
Before reacting with the serum of the first patient (100-fold dilution)
Transfer to nitrocellulose filter by electrophoresis. next
Wash the nitrocellulose filter, ¹²⁵ I-labeled goat anti-hi
The bound antibody visualized by IgG is detected. The spots observed in bands 1, 2 and 3 are the blood
Agglutination reaction between the supernatant and the extract, i.e. uninfected HUT-78 cells
Vesicle extract (band 1), H-2 infected with HIV-2 virus
UT-78 cell extract (band 2) infected with STLV-III
Reaction between the extracted HUT-78 cells (band 3)
Is shown. The numbers in the side margins of the band are most representative
Corresponds to the approximate molecular weight of the protein
T). II Nucleic acid 1. HIV-2 retrovirus RNA Attached to the filter by the "spot blot" method
Ills RNA converts HIV-1 under stringent conditions.
Did not hybridize with DNA. "Stringent conditions" refer to HIV-2 RNA ³² P
Radioactively labeled (or otherwise labeled)
Probe the hybridization reaction with the probe
It means the processing conditions to be performed later. Hybridization
Is a membrane, especially 50% formamide in 0.1% SDS / 5 × SSC
Performed at 42 ° C for 18 hours in the presence of (vol / vol) aqueous solution
You. Next, the membrane in which the hybridization reaction has occurred is added to the membrane for 0.
Wash at 65 ° C in buffer containing 15% SDS and 0.1 x SSC
I do. "Non-stringent conditions" refers to hybridization conditions.
Means the processing conditions under which the solution reaction and the washing are performed.
I want to be understood. That is, 5 × containing 30% formamide
SSC buffer in 0.1% SDS ³² P label (or other method)
Contact with the selection probe labeled at) for 18 hours at 42 ° C.
And hybridization with 0.1% SDS
Wash the membrane at 50 ° C in a buffer containing
U. HIV derived from λJ19 (Cell, 1985, vol. 40, p. 9)
-1 DNA was cloned into the vector pUC18.
Hybridization consisting of the damaged tissue plasmid pBT1
Perform hybridization experiments using probes
Was. Under non-stringent conditions, HIV-2 RNA
Weak hybridization between cloned DNA from HIV-1
Only observations were made. Another probe containing the cloned sequence of HIV-1 was also used.
Was. (A) Phas produced from a subclone of the HIV-1 genome
Single strand of HIV-1 subgenomic DNA inserted into M13
probe. The cloned region is the protease gene or
Associated with the nuclease gene. Endonuclease of HIV-1 under non-stringent conditions
One probe in the lease region (between bases 3760 and 4130)
Only nucleotide sequence of HIV-2)
I got an isomerization. "Protease" probe (salt
HIV-1 nucleotide sequence between groups 1680 and 1804)
HIV-2 and hybrida under stringent conditions
Did not (B) HIV-1 (subcloned in pUC18)
A probe consisting of a sequence encoding the "envelope" region
PRS3 interacts with HIV-2 under non-stringent conditions.
Did not hybridize. Spot blots can also be used as dot blots
Known (transition by spot). Genomic RNA of HIV-1, HIV-2 and STLV-III and HIV-
1 with probes containing various subgenomic sequences of the virus
The result of the hybridization between them is shown in FIG. On various media (0.5-1 ml per spot)
The supernatant was centrifuged at 45,000 rpm for 5 minutes. Pellets 0.1% SDS
Resuspend in NTE buffer containing
Attached to Luther. The filter was placed in 2 × SSC medium (0.3
M NaCl, 0.03M sodium citrate)
Was. (2 hours at 80 ° C) After baking, filter out HIV
Various probes containing -1 genomic subfragments
Both are under non-stringent conditions (30% formamide,
5 × SSC, 40 ° C), containing 0.1% SDS
Wash with 2xSSC at 50 ° C, with intensifying screen
Autoradiographed at -70 ° C for 48 hours. Probes 1 to 4 are the same as those described in (25)
G is a single-stranded probe obtained by the “G” method. This method is simplified
Simply, the subgenomic HIV-derived from the M13 virus
The single-stranded fragment carrying one insert (30) was converted to M13 (B
IOLADS) oligomer fragment (17 nuclei)
Reotide). Next, in the α position ³² P-labeled dAT
Presence of P, dGTP, dTTP and dCTB (Amersham, 3000 Ci / mmol)
In the presence, TM buffer (10 mM Tris, pH 7.5, 10 mM MgC
l _Two The complementary strand was synthesized with the KIenow enzyme in ()). Next, the DNA
Digested with appropriate restriction enzymes, heat denatured, denatured polyacrylic
Amide gel (6% acrylamide in TDE buffer, 8M
(With urea). Then run the gel for 5 minutes
I ran it on a tradograph. Next, cut the probe and
Eluted in 0 mM NaCl, 0.1% SDS buffer. These one
The specific activity (SA) of the strand probe was determined and ⁸ −10 ⁹ Collapse
Amount / min / microgram (dpm / μg) was obtained. The characteristic sequences present in the various probes are shown below. Probe 1: nucleotides 990-1070, probe 2: nucleotides 980-1260, probe 3: nucleotides 2170-2240, probe 4: nucleotides 3370-3640. The nucleotide numbers are based on reference (30). Finally, probe 5 was used to clone the HIV clone into λJ19 (31).
Plasmid pUC18 carrying EcoR I-Sac I fragment
Consists of This is processed by Nick Translation
About 10 SA ⁸ dpm / μg was obtained. Complete subgenomic fragments present in the probe
The relative configuration to HIV-1 is shown in FIG. Various sports
The codes correspond to the following, respectively. Spot A: culture of CEM C1.13 cells infected with HIV-1
Spot B: obtained from HUT-78 cells infected with STLV-III
Viruses, spots C and D: each of the two African patients
Isolate obtained from ills, spot E: negative control obtained from uninfected HUT-78 cells.
Rolled cell extract, Spot F: Presence of TCGF from a Zaire AIDS patient
Virus cultured in normal T lymphocytes in the presence. All spots except spot C show reverse transcriptase activity
A virus load corresponding to 25,000 dpm was obtained. spot
C corresponded to 15,000 dpm. The following observations were made: High reverse transcriptase activity from the culture supernatant of highly infected cells
Virus particles have been isolated and purified.
Genotype of two HIV-2 isolates obtained from irs particles
RNA can be purified under any of the above stringent conditions.
And did not hybridize. The following observations were made under the non-stringent conditions.
Was done. All probes are control HIV-1 preparations
Of genomic RNA obtained from E. coli and Zaire AIDS patients
Strongly hybridizes with genomic RNA obtained from the isolate
did. Two of the probes obtained (both in the gag region of HIV-1)
Nucleotides 990-1070 and 990-1260 obtained from
Are spots from the HIV-2 retrovirus extract
Slightly hybridized. These two probes
Only one (nucleotides 990-1260) has STLV-III
And hybridized slightly (Fig. 3). pol territory
Region (nucleotides 2170-2240)
Regarding lobes, the hybrid type with STLV-III was observed.
Detected or, to a lesser extent, hives with HIV-2 RNA
Redislanding was also observed. Another probe in the pol region
Reotide 3370-3640) is either HIV-2 or STLV-III.
Did not hybridize with this spot. Finally, HIV-
2 complete env genes and LTR (nucleotides 5290-9130)
Probes containing STLV-III RNA and RIV-2 RNA
Neither did it hybridize. Also noteworthy is the HIV-1 (protease region
Another) containing less than 5 'of the pol reading frame
The probe is also compatible with both HIV-2 RNA and STLV-III RNA.
Is not to hybridize. Therefore, from the above results, the HIV-2 virus is structurally
It is considered closer to STLV-III than HIV-1 from the ground. Only
However, HIV-2 is quite different from STLV-III.
This is the result of various observations on the infectivity of HIV-2 virus.
Supports the fruit. That is, against the monkey of the HIV-2 virus.
Infection capacity is virtually zero, but the STLV-III virus
Show obvious infectivity to monkeys of the same species. HIV-2 restriction map and genomic RNA sequence or this genome
CDNA restriction maps and sequences obtained from RNA
Can be easily estimated. Because of HIV deposited at CNCM
-2 strains have these restriction maps and
This is because it provides the genetic material necessary for determining the sequence of the peptide.
The genomic restriction map of one of the HIV-2 isolates of the present invention
The conditions to be determined and the number of cDNAs from these genomes
The conditions for determining the arrangement of such portions will be described later. FIG. 4 shows a retrovirus representative of the HIV-2 retrovirus.
3 shows a restriction map of the viral genome. Figure 5 shows this cD
Figure 3 shows a restriction map of a substantial fragment of NA. last
Next, a portion of this fragment was sequenced. FIG. 6 shows this sequence and the many restriction sites it contains.
Is shown. Clone complete cDNA or clone clone of this cDNA
Specific hybridization probe
Can be used as 2) cDNAs derived from HIV-2 RNA and the cDNA
The conditions for obtaining this cDNA are described below. In the first stage of this cDNA production, HIV-2 reverse transcriptase
Purified pyrions obtained from the supernatant of infected CEM cells using
Allow the detergent to carry out an intrinsic reaction activated by the detergent.
Depending on the oligo (dT) or
Produces an initiator cDNA strand. CEF cell line is GEFol
ey et al., Lymph described in Cancer 18: 522-529 (1965).
It was a blastoid CD4 + cell line. This document is included in this specification.
Shall be rare. ROD isolate these used CEM cell lines
, A significant amount of HIV-2 is produced continuously
It turned out to be. (Nucleotide and cell DNA poly
After synthesizing the second strand (in the presence of merase), the double-stranded cDNA
It was inserted into the bacterial phage vector M13 TG130. Ten ^Four Pairs
A phage library of exchanged M13 phage was obtained and
Processed by in situ screening with -1 probe
Was. The HIV-1 probe was used to detect the cDNA derived from the RAN of the LAV isolate.
Includes a 1.5 kb fragment obtained from the 3 'end (7th
See Figure A). About 50 positive plaques were detected.
Purify the insert for cross-hybridization and termination
And its properties were determined. By this processing procedure, LTR (S. Wain Hobson et al., Cell 4
0: 9-17 (1985) “long terminal repeat”
Abbreviation) Substantially complementary to the 3 'end of polyadenylated RNA in the region
Of various clones containing specific sequences (see above)
Is incorporated herein by reference). Relevant M13 that hybridizes to the 3'LTR region of HIV-1
The largest insert in the group of clones is about E2, designated E2.
This is a 2 kb clone. Same as 3 'LTR clone of HIV-1
Similarly, clone E2 is nucleated upstream from the poly (A) end.
AATAAA signal and HIV- located approximately 20 otide apart
3 ′ LTR region corresponding to two 3 ′ LTR regions. part
After sequencing, the 3 'LTR region of HIV-2 was
It was found that it was not closely related to the homology region. Figure 5 shows the E2 flag integrated into plasmid pSPE2.
3 shows a restriction map of a segment (a narrow rectangular area). this is
It contains the R region portion and U3 region of HIV-2. The figure shows the R region and U
It does not show the boundary with the three areas. The sequence of the E2 part is shown in FIG. FIG. 6 also shows that
The location of the restriction site is also indicated. 3 'LTR of HIV-1 and HIV-2
FIG. 7 shows that the regions are not closely related to each other. Real
In this case, about 50% of the two LTR sequences have some insertions and deletions.
(Only about 50% of the sequence
Homology). In contrast, Americans and Africans
Corresponding regions of various isolates of HIV-1 in humans may be inserted or deleted.
Shows over 95% sequence homology without loss. Using clone E2 as a specific probe for HIV-2
And a clone obtained from HIV-2 present in another clone.
Rows were identified on the hybridization filters. This probe can also be used under stringent conditions for HIV-
2 genomic RNA is detected. This probe is also
CEM or similar cells infected with an abscess or another HIV-2 isolate
Vesicular DNA can be detected by the so-called Southern blot method. Non-infected
CDNA derived from cells or HIV-1 infected cells and this probe
Under the same stringent conditions as
No signal was detected in the test. this
These results indicate that HIV-2 is more exogenous than HIV-1.
Was confirmed. For viral DNA that is not integrated
The approximately 10 kb species that is estimated to respond is
It was detected as the main component of undigested DNA. Viral DNA circle
Another DNA with an apparent size of 6 kb estimated to correspond to the shape
was detected. Another part of the HIV-2 genome was also identified. For this reason
A genomic library is constructed in phage lambda L47
did. Phage lambda L47.1 can be obtained from Gene 10
249-259 (1980). This reference is
Included in this specification. CEM cells infected with HIV-2 ROD after digestion with the enzyme Sau3A I
Flag obtained by digesting DNA from strain
A genomic library was constructed by the above method. About 2 × 1 clones containing the labeled E2 insert of HIV-2 cDNA
0 ⁶ Individual recombinant plaques were screened in situ. Ten
Recombinant phages were detected and purified on plaques. this
Restriction maps of three of the phages are
Southern blot with F2 insert under stringent conditions
Hybridization ability and non-stringency
Southern blot with HIV-1 subgenomic probe under conditions
), Which are characterized by the ability to hybridize.
Was. Derived from whole circular viral DNA containing the complete HIV-2 genome
A clone containing the 9.5 kb insert was identified. this
Was named "Lambda ROD4". Built-in profile
Lambda ROD27 and two other clones derived from
Lambda ROD35 and the LTR sequence of the viral coding sequence
Cell DNA sequence. Figure 8 shows the sequence of each clone.
Show. Lambda clone fragment into plasmid vector
Subcloned in pUC18. λROD4, λROD27 and
λROD35 fragment and in the above plasmid vector
The respective subclones are also shown in FIG. The following sub-color
Was obtained. -A cell sequence adjacent to the 5.2 kb region of the HIV-2 genome (EcoRI
5 'LTR and 5' coding viral sequence around the site)
PROD27-5 'derived from lambda ROD27 containing. -Due to lambda ROD4 containing a Hind III fragment of about 5 kb
Coming pROD4.8. This fragment is located in the center of the HIV-2 genome.
Corresponding to the part. PROD2 with HIV-2 inserts overlapping each other
7-5 'and pROD4.8. PRO containing the 1.8 kb HindIII fragment of lambda ROD4
D4.7. This fragment is subcloned in pROD4.8
Placed in three directions for the fragment
0.8 kb coding viral sequence and phage lambda
BamHI and HindIII clips on the left arm of (Lambda L47.1)
And the portion existing between the roning sites. -The entire HIV-2 coding sequence in three directions to the EcoR I site
And the 3 'LTR and a contiguous nucleotide sequence of about 4kb of cellular origin
Including pROD35. PROD27-5 'and pROD35 present in E. coli HB101 are 1
Accession numbers I-626 and I-63 to the CNCM on November 21, 986, respectively.
Deposited at 3. PROD4.7 and pROD4.8 present in E. coli TG1 were 1986 1
Called CNCM on January 21 with accession numbers I-627 and I-628, respectively.
Entrusted. The complete HIV-2 ROD genome whose restriction map is shown in FIG.
And pROD35 and pROD27-5 'previously linearized with EcoRI
Was rebuilt. The EcoRI insert of pROD27-5 'was inserted into pROD35.
It bound to the EcoRI site in the correct orientation. HIV-2 and other human or monkey retroviruses
The degree of correlation between
And tested. Between various regions of the HIV-1 and HIV-2 genome
Is derived from the cloned HIV-1 genome
Fragment and radiolabeled lambda ROD4 derived flag
Determined by the hybridization test
Was. These fragments (numbers) for the HIV-1 genome
The relative positions of Nos. 1 to 11) are shown in the lower part of FIG. Even under very weak stringent conditions (42 ° C)
The HIV-1 genome and HIV-2 genome are the respective gag genes
(Spots 1 and 2), pol reverse transcriptase region (spot
3), pol terminal region, Q (or sor) gene (spot
5) and F (or 3'orf) gene and 3 'LTR (sport
Hybridized only at level 11). HIV-2
HIV-1 plasmid used to detect the first cDNA clone
Segment corresponds to the subclone of spot 11,
Hives relatively well with HIV-2 under stringent conditions
Redisize. The signal obtained from spot 5 is
The only signal that survives a lingent wash
It is. Envelope gene, tat gene region and pol part
The minutes seem to vary greatly. These data and
The sequence obtained from the LTR (Fig. 3) shows that HIV-2 (less
(With respect to its envelope)
Indicates that there is no HIV-2 is more closely related to SIV than HIV-1 (MDDanie
et al., Science 228: 1201-1204 (1985).
Included in the description). All SIV proteins, including envelope proteins
Quality is immunoprecipitated by the serum of HIV-2 infected patients
However, the serological cross-reactivity between HIV-1 and HIV-2 was
Limited to protein. However, SIV and HIV-
2 means the difference in molecular weight of each protein
Therefore, it can be identified. With regard to nucleotide sequences, HIV-2 is still SIV
Closely related. Furthermore, the characterization of HIV-2 allows the target cell surface
Binding to Virus and Virus Internalization
Region of the envelope glycoprotein that induces
It is possible to specify. The interaction involves the CD4 molecule itself
Occur through. HIV-1 and HIV-2 share the same receptor
Probably used. Therefore, there is a large gap between the HIV-1 and HIV-2 env genes.
Although there are differences, these two forms of HIV envelope
The limited homologous area of the loop is T4 lymphocytes
May be considered a component that binds to the common receptor
it can. The action of these sites prevents HIV virus
Of peptides to elicit a protective immune response in developing humans
Is formed. With the genetic material of the virus or provirus carrier
To form a probe in the hybridization reaction,
In particular, to detect the presence of HIV-2 viral RNA in lymphocytes
The preferred sequence is a 5 kb Hind III with ROD4 and E2 cDNA.
Nucleotide sequence obtained by fragment engagement
including. The experiment may be performed by any method, especially
Southern Blot, Southern Blot and Dot Blot
Method can be used. Furthermore, the identification of some parts of the retroviral genome
And cDNA species derived from HIV-2 retroviral genome
Examples of the production of multiple recombinant DNAs containing different parts
The characteristics of the present invention will be more apparent from the following description.
Would. However, the present invention is not limited to the description of these examples.
Absent. EXAMPLES Example 1 DNA Probe Used in HIV-2 Diagnostic Kit cDNA Complementary to Genomic RNA Obtained from Purified Pillion
Was prepared in the following manner. 48 hours CEM cells infected with HIV-2 ROD isolate of HIV-2
The supernatant obtained after interculturing was ultracentrifuged. European mentioned above
Method and substance described in Patent Application No. 84 / 401.234-0138667
Centrifugation pellet containing pyrion using the same method
Centrifuge on a sucrose gradient to form a new centrifuge pellet.
Was. Activated with detergent using purified HIV-2 preparation
CDNA was synthesized using the endogenous reaction performed. Briefly, virion preparations were combined with 50 mM Tris-HCl and 5 mM
mM MgCl _Two And 10 mM DTT and 0.025% Triton detergent
(Product name TRITON) and 4 kinds of deoxynucleotides of 50μM each
Dotriphosphate and oligo (dT) initiator
Was added to the reaction mixture. Keep the reaction at 37 ° C for 90 minutes
Was. Phenol converts proteins present in the first reaction medium
Extract, RNase, E. coli DNA polymerase 1 and 4
For 1 hour at 15 ° C in the presence of deoxynucleotides
The second DNA strand was synthesized by maintaining at 22 ° C. for 1 hour. These two
T4 DNA polymerase acts on heavy chain cDNA to create blunt ends
Formed. All reagents used in this procedure were commercially available.
(AMERSHAM cDNA kit)
Used for (1) In the presence of T4 DNA ligase (a commercial product of BIOLABS)
(Linker) containing EcoR I site (Pharmaci
a commercially available product) to the blunt end of the cDNA, and (2)
Digest these linkers with restriction endonuclease EcoRI
(3) Gel filtration on AcA 34 (LKB-IBF) (trade name: UL
Linker flag by TROGEL gel column)
To the M13TG130 vector cleaved with EcoRI.
Insert cDNA. After transformation of the E. coli TG1 strain,
I got a library. About 10 ^Four Obtained recombinant M13 plaque
Was done. Recombinant M13 clone containing HIV-2 cDNA from cDNA library
Plaque hybridization to select
Method used. M13 plaque DNA to nitrocellulose
Transfer to a filter and filter the LAV (or HI) described in the European patent application.
V) Subgeno from the "lambda J19" clone of the virus
Hybridized with the HIV-1 probe. This pro
Is about 1500 base pairs (bp) of HIV-1 DNA?
Comprising an insert. This insert contains the env gene
R-segment in reading frame and 3 'LTR of HIV-1
Bound by two Hind III restriction sites in the This
The probe of 3) is composed of the 3 'end of the env gene,
Includes 3 segments and a part of the R segment of the LTR.
It was 1500 base pairs (bp) long. A probe containing a 1.5 kb Hind III insert was ligated (AMERSHA
Initiator and Klenow DNA polymer (using M kit)
Incubate at 15 ° C for 4 hours in the presence of Rase I
By that, ³² P−] dCTP and −dTTP (3000 Ci × 10
^-3 Mol). Library cDNA clones
Low stringency conditions for hybridization tests
Performed overnight. Hybridization medium used
Solution at 37 ° C in 5x SSC, 5x Denhart and 25% form
Amide and 100μg / ml denatured salmon sperm DNA and labeled probe
(Specificity 10 ⁹ 2 × 10 at cpm / μg ⁷ cpm)
Was. Filter using three solutions of the following composition in order
Was washed three steps. First stage washing: 5 × SSC, 0.1% SDS, 25 ° C., 4 × 15 minutes Second stage washing: 2 × SSC, 0.1% SDS, 42 ° C., 2 × 30 minutes Third stage washing: 0.1 × SSC, 0.1 % SDS, 65 ° C, 2 x 30 minutes
while. Autoradiography of filters after each washing step
To After the first washing step, several positive clones are detected,
It was still detected after the second stage wash. However the second
All signals disappeared after three washing steps. this
Indicates that the positive clone is not closely related to the HIV-1 genome, but
Be sufficient to make the choice despite that
Is shown. Subculture positive clones and re-attach to plaques
And the same stringent conditions as the first-stage cleaning
Re-hybridized with the same probe under these conditions. these
Most of the clones were still positive. Fully human DNA under moderately stringent conditions
Using a probe, 5x SSC, 5x Denhart and 40%
Hybridization in muamide, then 1 × SSC at 50 ° C.,
Clones were selected by washing with 0.1% SDS.
The previous positive clone was not detected at all, and
It did not correspond to the cDNA of the reverse DNA or ribosomal RNA. The positive M13 recombinant clone was cultured in a fluid medium and
The characteristics were determined as follows. (1) Insert size M13 single-stranded type DNA was extracted from each clone, and M1
Second using the 317-mer initiator sequence and the Klenow enzyme
Formed a chain. The insert is excised with EcoR I (BOEHRINGER)
Analyzed by agarose gel electrophoresis. Most inserts
Contains 200-600 and 200 bp, but only clone E2.1
Was exceptionally about 2 kbp in length. (2) Analysis of nucleotide sequence Sa described in Proc. Natl. Acad. Sci. 74: 5463-7 (1977)
partial clones using dideoxy method such as nger
Were sequenced. This document is included in this specification
And Various independent clones have similar nucleotide sequences
, But the poly (A) chain at the 3 'end is an exception
The length. These results are based on these cDNA
Indicates that the loan was derived from an RNA template. Detailed distribution
According to the column analysis, a cDNA clone containing the 3 'end of the HIV-2 genome was obtained.
The loan proved not to be closely related to HIV-1. (3) Hybridization of HIV-2 genomic RNA and DNA
(A) Production of genomic RNA of HIV-2 The infection supernatant was centrifuged (50,000 rpm, 30 minutes). Sedimentary
Pellets in 10 mM Tris pH 7.5, 1 mM EDTA, 0.1% SDS
Resuspended. One of the insert clones, F1.1, was labeled and
Genomic RN of various virus isolates by blot analysis
Used as a hybridization probe with A
Was. The dot blot method includes the following steps. (I) In 20 × SSC (3M NaCl, 0.3 sodium citrate)
Sample (HIV-2) on nitrocellulose membrane soaked in advance
Lysate) and let it dry in air.
Baking for 2 hours at (° C)
Conduct an action. This hybridization is strongly stringent.
(5 × SSC, 5 × Denhart, 50% horror)
Muamide, 42 ° C). Next, wash at 0.1 × SSC, 0.1% SDS, 65 ° C
Was cleaned. Under these conditions, the probe
Two different types of HIV, including the origin LAV-II ROD
-2 hybridized with spots of isolate. ST
LV-III mac (simian T lymphotropic virus (also as SIV)
Publicly known), type III rhesus macaque (macaque)
Weak hybridization at the spot formed by
Signal was detected and the HIV-1 isolate was hybridized.
No ligation was detected. ³² Clone E2.1 containing 2 kb insert as P-labeled probe
When performing a Southern blot experiment using
Hybridization between DNA and DNA from uninfected cells
Not detectable, but under strong stringent conditions
Bands were detected in isolated cells infected with HIV-2. H
IV-2 is a polymorphism equivalent to HIV-1 at the restriction map level
Is shown. Southern blot for whole cell DNA of infected cells
, Two types of signals are detected. That is, (1)
In the case of the virus in the integrated form, the molecular weight MW is about 20 kb
A signal was detected in the above DNA fraction, and (2) genome
Low molecular weight (9,10kb) for viruses that are not incorporated into DNA
A signal is detected in the fractions. These properties are highly specific for retroviruses.
You. Performed using STLV-III (SIV-3) of infected cells
Simultaneous experiments have shown that simian retrovirus can become HIV-2
It was confirmed that they were not closely related (signal was weak)
It is only detected under stringent conditions). this
These experiments show that the probe can specifically detect HIV-2.
Indicates that (4) cDNA of HIV-2 in bacterial plasmid vector
Subcloning Select positive M13 clone E2.1,
Subcloned in Recombinant M13 (TG130) phage
The DNA of E2 was replaced with the HIV-2 genome (obtained from HIV-2 ROD).
Single stranded DNA containing a 2 kb insert (M-
13-ROD-E2). Melto this insert
n, DA, 357 Nucleic Acid Res. 12: 035-7056 (1984)
Was transferred to the plasmid pSP65 described in 17-mer initiator sequence (AMERSHAM) and 4 nuclei
Presence of Leotide A, C, T, G and DNA polymerase I (Klenow)
The second strand was constructed in vitro in the presence. EcoR I insert into EcoR
I Excised by digestion, purified on agarose gel, EcoR
Ligated to pSP65 previously digested with I. Use binding mixture
To transform Escherichia coli DH1 strain to make ampicillin resistant
Recombinants were selected based on this. 50 μg / g of the identified recombinant
Culture in LB medium (Luria medium) containing ml of ampicillin
Was. These plasmids were purified and correctly inserted
The presence of the fragment was monitored. One of the resulting clones was named pSPE2,
On May 5th with accession number I-595 to CNCM, Paris, France
Entrusted. Insertion derived from HIV-2 cDNA and inserted into the probe
The input is a nucleotide as defined above that corresponds to a part of E2.
Included in the array. Example II c complementary to DNA complementary to genomic RNA of HIV-2 virion
DNA cloning HI from cell supernatant 5 from CEM strain infected with ROD isolate
V-2 virions were purified. Detergent activated endogenous
Sedimentation in the presence of oligo (dT) initiator using reaction
The first strand of cDNA was synthesized by contact with the purified virus. This
These include Alizon, Nature 312 , 757-760 (1984)
It was used. RNA / cDNA hybrid to phenol / clo
Extracted with a mixture of chloroform and precipitated with ethanol
Therefore, it was purified. Gubler and Hoffma
n () in the presence of DNA polymerase I / RNase H
Produced. The description of the document is included in this specification.
You. Using the components of a commercial cDNA synthesis kit by AMERSHAM
Blunt ends in double-stranded cDNA in the presence of DNA polymerase T4
Formed. A commercially available EcoR I adapter by PHARMACIA (linker
-) Was ligated to the end of the cDNA. CD modified in this way
After digestion in the presence of EcoR I, NA
It is more commercially available and is itself pre-digested with EcoR I.
Inserted into the dephosphorylated phage vector M13tg130
Was. CDNA band was obtained after transformation of E. coli TG1 strain.
Was. The above 1.5 kb Hind III flag obtained from HIV-1
With clone J19 containing the fragment
Plaque (10 ^Four )
Was screened in situ. Filter with 5 × SSC, 5 × DENHARDT solution, 25%
Lumaldechent and denatured salmon sperm DNA (100 μg / ml)
Prehybridization at 37 ° C for 4 hours in the presence of
Next, an additional labeled probe (4 × 10 ⁷ cpm) in the presence of the same
Hybridization (Tm-42 ° C) for 16 hours. ⁶ cpm
/ ml final hybridization buffer solution
Obtained. The plate was washed with a 5 × SSC, 0.1% SDS solution at 25 ° C. for 2 hours (20
× SSC is a solution of 3M NaCl and 0.3M sodium citrate
Will be understood to correspond). Positive puller
Purified to prepare M13 single-stranded DNA, and analyzed by the method of Sanger et al.
The ends were sequenced. DNA of HIV-1 and HIV-2 infected cells and HIV-
1. HIV-2 and SIV virion RNA and HIV-2 clone
Hybridization with probe derived from cDNA
Infectious CEM that continuously produces HIV-1 and HIV-2 respectively
DNA was extracted from the cells. These two types of retro wheels
Digest DNA samples with 20 μg of Pst I in some cases.
Do not digest in some cases, 0.8% agarose gel electricity
It was electrophoresed and transferred to a nylon membrane by the Southern method. 0.
NTE buffer with 1% SDS and same reverse transcriptase activity
A small amount of the infected supernatant collected in advance was immersed in 2 × SSC solution.
Attached to the pickled nitrocellulose. 50% formamide, 5 × SSC, 5 × Denhart and 100 mg /
4 hours at 42 ° C in a solution containing 2 ml of denatured salmon sperm DNA.
Rehybridization was performed. In the same buffer
10% dextran sulfate and 10 ⁶ cpm / ml E2 labeled insert
(Specific activity 10 ⁹ cpm / μg) at 42 ° C.
Hybridization was performed for hours. Then 0.1 × SS
Wash twice with C and 0.1% SDS solution for 30 minutes each.
Was. Expose to an intensifying screen for 16 hours and store in 0.4N NaOH.
Dehybridize and neutralize the xanthos, under the same conditions
Under 10 ⁹ Again with the HIV-1 probe labeled with cpm / μg
Hybridized. Example III The Complete DNA of HIV-2 Provirus in Phage Lambda
Cloning CE infected with HIV-2 ROD (FIG. 2, bands a and c)
M cell DNA is partially digested with Sau3A I. 9-15 kb fraction
Was selected on a 5-40% sucrose gradient and the lambda L47.1 vector
BamHI arm. In vitro package and large
Plaques obtained after attachment to Enterobacteriaceae strain LA101 (2 × 10 ⁶ )
Was hybridized with the E2 cloned cDNA insert.
In-situ screening was performed by the About 10 sun
Sex clones are purified on plaques and transformed with E. coli C600 recBC
Propagated. Clone random ROD4, ROD27 and ROD35
Amplified. Create each restriction map and use the Southern method to HIV
Together with the -2 cDNA clone under stringent conditions.
After hybridization, the gal-pol
Hybridized under non-stringent conditions with lobes
Perform DNA characterization by performing
became. FIG. 8 shows the obtained three recombinant phages ROD4 and ROD27.
And the structure of ROD35. The elongated rectangle in these figures shows the first infected CEM.
Corresponds to the sequence of the provirus obtained from the DNA of the cell.
You. The white part corresponds to the retroviral sequence. Shaded area
The minutes correspond to parts of the cellular DNA. The black part is the virus
Corresponding to the LTR in the sequence. Cells indicated by letters L and R were used for cloning.
Corresponds to arm out of lambda L47.1 phage vector
You. Some restriction sites are also shown. Especially B is Ba
mH I, E is EcoR I, H is Hind III, K is Kpn I, Ps is Pst
I and Pv are Pvu II, S is Sac I and X is Xba I site. These viral sequences have an intersection with the E2 sequence. E2
These determined by hybridization with
Are also shown. ROD4 is derived from circular viral DNA. ROD27 and RO
D35 is derived from a provirus integrated into the cellular DNA structure
Be led. Finally, the insert subcloned under the above conditions
And the corresponding ROD4, ROD27 and ROD35 sequences.
The relative position of the insert is also shown. The inserts shown are, in particular, plasmids pROD27-5 ', pROD27.
35-3 ', pPOD4.6, pROD4.8 and pROD4.7 inserts.
You. FIG. 9 shows M containing nucleic acids derived from the complete LAV genome.
Different parts of single-stranded DNA from 13 subclones?
Between the obtained subfragments 1 to 11 and ROD4, respectively.
The relative intensity of the resulting hybridization spot is shown.
You. Complete LAV genome (determined based on sequencing)
The relative positions of these various fragments with respect to
Shown at the bottom. Spot 12 is a phage lambda control
Control spots produced using roll DNA
Corresponding. C in the spot transfer (dot blot) method
The hybridization experiments included the complete HIV-2 cDNA.
Lambda ROD4 recombinant as a probe
Performed under stringent conditions. Then the following article
Were sequentially washed. 2 x SSC, 0.1% SDS, 25 ° C (Tm-42
° C), 1 × SSC, 0.1% SDS, 60 ° C (Tm-20 ° C), 0.1% ×
SSC, 0.1% SDS, 60 ° C (Tm-3 ° C). The spots shown are obtained after overnight exposure on the radiograph.
Was. Example IV In Vitro Diagnostic Assay for the Presence of HIV-2 Virus in Biological Media
Materials and Methods Subjects Subjects from Egas Moniz Hospital, Lisbon, September 1985 to September 1986
HIV-2 infection among inpatients or outpatients during the month
Patients were selected. For this choice, people from Africa or
Shows HIV-1 serum from all those who have stayed in Africa
(Immunofluorescence-IFA- or ELISA) and HIV-2 (IFA)
Antibody tests for both were performed. Serologically HIV-2
The following tests were performed only on subjects in which infection was detected. Virus isolation HIV isolation was performed on 12 subjects as described above.
Was. That is, the peripheral blood lymphocytes (PBL) of the subject are punctured with PHA.
Co-culture with vigorous, normal human PHA-stimulated PBL
Maintained in the presence of leukin-2 (IL-2). Of culture
The presence of cytotoxicity and the reverse transcriptase activity (RT) in the supernatant
Monitored. Immunofluorescence assay (IFA) IFA slides were prepared as follows. HIV-2 infection
Washed MOLT-4 cells twice with PBS and IFA glass slide
Sink in layers (10 ^Four Cells / well), air-dried, cold aceto
Fixed. Dilute these cells 10-fold for IFA
Reacted with the test serum for 45 minutes at 37 ° C, washed, dried,
Fluorescein-conjugated goat anti-human IgG, A, M (100-fold rare
) At 37 ° C for 30 minutes. After washing, cells are
Counterstain with 90% Evans blue, 90% glycerol, 10%
The cells were sealed in PBS and observed with a fluorescence microscope. ELISA Commercially available test serum ELAVIA (Pasteur Diagnostics) or A
Test serum of several subjects for HIV-1 antibody using B-BOTT
Tested. Radioimmunoprecipitation Assay (RIPA) HIV-1 or HIV-2 infected CEM cells ³⁵ S Sistay
(200 μCi / ml) for 16 hours. Collect supernatant
Collect, pellet virus particles, RIPA buffer
(Tris-HCl 50 mM pH7.5, NaCl 150 mM, EDTA 1 mM, 1
% Triton X100, sodium deoxycholate 0.1
%, SDS 0.1%). 10 for each reaction ^Five against cpm
50 μl of lysate at the corresponding dilution is added to 5 μl of test serum and 4 μl.
The reaction was carried out at 18 ° C. for 18 hours. Immune complexes were isolated from Sepharose-Pro
Bind to TEIN A (PHARMACIA), wash and boil for 2 minutes
Eluted by raising. Next, the eluted antigen was
Analyze by midgel electrophoresis and autoradiography.
Was. Dot blot hybridization Pellet virus isolated from subject's PBL,
Tris-HCl 10mM pH7.5, NaCl 10m, EDTA 1mM, SDS 0.
Lysed to 5%. Each lysate corresponding to 50,000 cpm RT activity
1 μ was attached to nitrocellulose. Strong string
Hybridization and washing under gentle conditions
became. Hybridization is performed in 6 × SSC, 5 × Den
hart, wash in 50% formamide at 42 ° C for 18 hours, wash
Was performed at 65 ° C. in 0.1 × SSC, 0.1% SDS. Specific activity 10
⁸ cpm / μg ³² P-labeled HIV-1 and HIV-2 probes
used. HIV-1 probe is LAV _BRU Complete Geno of the isolate
HIV-2 probe is LAV-2 _ROD From the isolate
It was derived from a 2 kb cDNA. Results Subjects Population Serologically and / or virologically indicative of HIV-2 infection
The test was performed on 30 subjects with weather. 12 men and 18
She was a woman. The ages range from 11 to 55 with an average age of 35.
You. Everyone except one has stayed in West Africa for several years.
is there. 26 are Guinea-Bissau residents
Yes, they are Cape Verde Islands
I'm from. One from Angola on Cape Verde Island
An 11 year old boy who has lived for several years. Only one in the test population
Westerners have lived in Zaire for eight years and have stayed completely in West Africa
A 40-year-old Portuguese man who has never done anything. Clinical Findings 17/30 had AIDS according to CDC criteria. this
The main symptom of these patients is chronic diarrhea, often
Along with this, he lost more than 10 kg in one year. 10 people
Diarrhea was associated with intestinal opportunistic infection in 7 patients, with 7 patients
So the only pathogen is the war Isospora belli
Yes, one has only Cryptosporidium, and two have both
Had one of the pathogens. Intestinal opportunistic pathogens in three patients
No body was detected. 8 out of 17 AIDS patients
Was diagnosed with esophageal candidiasis. Six AIDS patients call
He showed a sucker syndrome. Two people have been diagnosed with pulmonary tuberculosis
Another unidentified Mycobacterium has been detected
Was. Pulmonary aspergillosis (asperogillosi) in two patients
s) and one had tuberculosis. Another two AIDS cases
Patients have pneumonitis episodes whose pathogen cannot be identified.
Repeat the sword, one patient
She had Pneumocystis pneumonia. This is only postmortem examination
It turned out. 4 out of 17 AIDS patients are Kaposi meat
Three had this on the skin only, but one had
Postmortem findings revealed sporadic visceral lesions in the patient.
Central nervous system disorders were observed in two AIDS patients. One person
Is a cerebral lymphoma and one is a subacute encephalitis of unknown cause
You. Four patients show signs of AIDS-related syndrome (ARC)
Was. Two patients have diffuse lymph node damage with persistent fever
One person has chronic diarrhea with weight loss and one person has a tracheal
Recurrent episode of pneumonia and multiple lymph node damage
Was. Of the remaining nine patients, six are associated with HIV infection
No symptoms seemed to occur, one had only pulmonary tuberculosis, and one had depression
Only one person had cerebral syphilis due to diffuse disseminated lymph node damage.
Seven patients died during the 12-month trial, and all died
AIDS. Serologic studies for each patient against both HIV-1 and HIV-2 antibodies
At least one serum test was performed. Serum of all patients
All were positive by HIV-2 antibody test with IFA. So
Of these, 21 were tested for HIV-2 antibody by RIPA.
Was. All sera are high in a virus called gp140
Quantitative envelope glycoprotein sedimented clearly.
The sera of 16 of them also contain the major core protein p26
And only one serum is called another p16
Reactor protein. Serum cross-reactive HIV-1 anti-
The presence of the body was examined. The IFA test was performed on 24 sera. 1
2 sera were negative, 10 sera were weakly reactive and 2
One serum was positive. 21 sera tested by ELISA
Was. Sixteen sera were negative and five sera were positive. Most
Subsequently, antibody tests for HIV-1 protein in 11 sera were performed.
The survey was performed at RIPA. Three sera are viral proteins
Did not react at all with only two sera, pol gene product p34
And the five sera react with the major core protein p25
I responded. The two sera were HIV-1 envelope glycoproteins.
Reacted weakly with dendritic gp110. These two sera and IFA
Alternatively, all sera positive for HIV-1 antibody test by ELISA are RIPA
Has a strong reactivity with gp140 of HIV-2, which is HIV-1
It is an indicator of the presence of HIV-2 rather than infection. Inspection group
Only one patient who did not enter serologicly became HIV-1
It was infected and not infected with HIV-2. This patient is central
A 21-year-old woman from Africa (San Tome Island)
AIDS patients. Virus isolation Retroviruses from peripheral blood lymphocytes in 12 patients
An isolation test was performed. Existence and culture of typical cytopathic effects
The presence of a peak of particle-related reverse transcriptase activity in the
HIV was isolated from 11 samples. All 11 isolates were dot blot hybridized
And was identified as HIV-2 by the alternative method. 10 isolates
Virus dots are hybridized under stringent conditions.
Clone HIV-2c by redidation and washing
Strongly hybridizes with HIV-2 probe derived from DNA
Under the same conditions.
Did not hybridize at all. One isolate
Only weakly hybridizes with the HIV-2 probe and HIV-1
And did not hybridize. Immunological test Identification of helper T cells (CD4 +) in 13 patients
The number of circulating lymphocytes was measured and helper T cells and
The ratio to the Lesser T cells was calculated. Of these patients
Of the 11 had AIDS. That is, the average of helper T cells
Absolute count is 243 + 300 / μ, helper vs. support
The average presser ratio was 0.25 + 0.15. AR clinically
In one patient showing C, the number of helper T lymphocytes was 240 /
In μ, the ratio was 0.18. I have brain syphilis and I have HIV
Helper T in another patient who shows no signs of relapse syndrome
The lymphocyte count was 690 / μ and the ratio was 0.82. Discussion Whether you have AIDS or ARC in this study or
30 West Africans apparently do not show HIV-related syndrome
The subject has demonstrated HIV-2 infection. From this result
It was concluded that HIV-2 is a major disease in West African AIDS patients.
It is considered to be an etiological agent. In our patients
Depending on the serological and virological profiles observed
HIV-2 infection is often associated with HIV-1 infection
Did not. HIV-1 and HIV-2 are antigenically and genetically large.
Similar CD4− + T phosphorus despite differences
Has affinity for papocytes, has a similar cytopathic effect, and has a similar
Form, common immunity among several constituent proteins
Has a reactive epitope. Nishi who was infected with HIV in this study
All African subjects are HIV-2 infected and have HIV-1 sensation
It turned out that there was no dye. Therefore a new virus
HIV-2 may be the leading cause of AIDS in West Africa. HIV-2 AIDS Syndrome is HIV-1 in Central Africa
It was no different from the related AIDS syndrome. Most common
A common symptom is chronic diarrhea with considerable weight loss.
Many originate in the war Isospora and / or Cryptosporidium
Cause. Other opportunistic infections such as candidiasis, my
Cobacteriosis (including Mycobacterium tuberculosis) and Toxoplasma
The disease was similar to HIV-1 related African AIDS.
Was. The most common complication among Western AIDS patients
Lini pneumocystis pneumonia observed in only one case during study
No Cryptococcal meningitis was detected. However, observed in HIV-2 infected AIDS patients
Immune abnormalities are similar to those for HIV-1 related AIDS. 30 people who had serum antibodies reactive with HIV-2 antigen
Only 7 of all subjects can be detected by IFA or ELISA
HIV-1 specific antibodies. In RIPA these seven people
All subjects share strong immune response epitopes
Major core protein p25 (HIV-1) or p26 (HIV-
It had an antibody reactive with 2). 5 subjects
All of these five were HIV-1 negative by ELISA
And 3 are borderline and 2 are negative in IFA
Met. Can some subjects not be fully assayed?
However, nine subjects had HIV-1 specific IFA and / or EL
Positive or negative ISA response to HIV-2 virus
It had serum antibodies to aprotein p26. this
Their findings are relevant to HI used in Africa and other areas.
It is important to include HIV-2 antigen in V serum tests
And Isolate retrovirus from peripheral lymphocytes of 11 patients
did. In all cases, the virus was propagated within 2 weeks
Presence of reverse transcriptase activity in culture supernatant and existence of cytopathic effect
The characteristics were determined by the location. However, this
Cytopathic effects vary considerably from isolate to isolate
is there. In some isolates, significant
Large numbers of fused cells have been formed, and in another isolate
Small fusion cells are formed only a little and the viability of the culture
Has little effect. RNA obtained from all but one isolate is strong
HIV-2 showing the 3 'end of the genome under stringent conditions
Probes clearly derived from cDNA clones
It was found that it hybridized. These isolates
Hybridizes completely with the HIV-1 probe under the same conditions
Did not. This means that these subjects are infected
Prove that all things belong to the same type of virus.
Only one isolate was very weakly hybridized with HIV-1
I did it. However, this virus is HIV-2
Isolated from patients with serum antibodies that react with all of the antigens
Was. The invention further relates to the HIV-2 virus and its variants only.
But not humans with immunological properties specific to HIV-2
Includes all equivalent viruses that infect. The present invention
Shows only the characteristics of the HIV-2 virus deposited at the CNCM
But not all viruses with the following characteristics:
You. The preferred target for the HIV-2 retrovirus is human Leu3 cells
(Or T4 lymphocytes) and those derived from these T4 lymphocytes
Established "immortalized" cells, such as those described herein
It consists of the HUT78 cells described above. In other words,
Torovirus also has specific affinity for these cells
One. The retrovirus is a HUT expressing the T4 protein, C
It is cultured in EM, MOLT or a permanent strain of the same type. The leto
The rovirus does not infect T8 lymphocytes. The retro virus
Is cytotoxic to human T4 lymphocytes. T4 lymphocytes
The cytopathic effect of the HIV-2 virus on HIV is particularly
Proven by the appearance of The retrovirus is Mg ₂₊ I
Has reverse transcriptase activity that requires the presence of
Rate oligodeoxy thymidylate (poly (A) -origin)
Go (dT), with strong affinity for 12-18). The leto
Rovirus has a density of about 1.16 in a sucrose gradient. The
Retroviruses have an average diameter of about 140 nm and a core average diameter of about 4
1 nm. The solubility of this virus is HTLV-1 virus
With the p24 protein of the virus or HTLV-2 virus
Contains no p26 protein. Therefore, these p26 proteins
The molecular weight of the quality is the molecular weight of the corresponding p25 protein of HIV-1
Slightly larger (by only about 1,000), the corresponding p2 of SIV
7 Slightly higher than the molecular weight of the protein (also about 1,000
Ke) small. The HIV-2 lysate is further purified by RIPA (radioimmu
Abbreviation of noprecipitation assay) HTLV-1 or HTL
P16 that is not immunorecognized by the p19 protein of V-2
Contains protein. These further have a molecular weight of 130,000-140,0
Containing envelope glycoproteins of order 00
The protein does not cross-react with gp110 of HIV-1 but STLV-
Cross-reacts with the gp140 envelope glycoprotein of III.
The lysate also contains 36,000 and 42,000-45,000
Has a molecular weight of ³⁵ S) Can be labeled with cysteine
Including proteins or glycoproteins. HIV-2 Genome R
NA binds to HIV-1 genomic RNA under stringent conditions.
Do not hybridize. The RNA is induced from HIV-1 and
v A nucleotide sequence containing a gene and an LTR adjacent to the gene.
Do not hybridize with the column under non-stringent conditions.
No. The RNA is, in particular, the nucleotide sequence 5290-91 of HIV-1.
30 and the sequence of the pol region of the HIV-1 genome, in particular nucleotides
Do not hybridize to any of the sequences 2170-2240.
No. The RNA comprises the nucleotide sequence of the HIV-1 region,
Nucleotide sequences 990-1070 and 990-1260 and non-stringent
Hybridizes weakly under gent conditions. The ability to infect humans and trigger one of the forms of AIDS
It has essential properties as described above and is deposited with CNCM.
HIV-2 virus strain (or derived from its genomic RNA)
CDNA or cDNA fragment) and stringent conditions
Any RNA with genomic RNA that can hybridize
Understand that Torovirus can be considered equivalent to HIV-2
Let's do it. The present invention further relates to each antigen obtained from HIV-2, particularly
The present invention relates to a protein and a glycoprotein in a manufactured state. "Purification"
The terms protein or glycoprotein respectively refer to
Under polyacrylamide gel electrophoresis under various experimental conditions
Means a protein or glycoprotein that produces a band
You. Any suitable separation and / or purification to obtain each antigen
A manufacturing method may be used. RCMO as an example of a method that can be used
NTELARO et al., J. of Virology June 1982, pp1029-1038
There is a method described in. The present invention generally relates to HIV-2 derived antigens
All antigens having immune properties equivalent to the immune properties of the compound,
Especially for proteins, glycoproteins or polypeptides
You. In this case, the two antigens are the same antibody, especially HIV-2
Is recognized by antibodies isolated from the serum of the patient
Or if two antigens have the “immunological equivalence” condition described below
Two antigens are considered "equivalent" when satisfying
It is. Fragments of the antigen (or reconstructed by chemical synthesis)
Peptide) was the antiserum that was the source of the fragment.
Equivalent polypeptide, protein
It may be considered a protein or a glycoprotein. Paraphrase
Thus, the present invention relates to the same
Or any port sharing a similar epitope with the antigen.
It also includes a polypeptide. Polypep belonging to this type
The tide is the DNA that encodes the corresponding polypeptide sequence.
This is the expression product of the corresponding sequence. HIV-2 virus is all that came into contact with the HIV-2 virus
Used as an antigen source to detect antibodies in humans
It turned out to be. The present invention generally relates to biofluid serum, particularly HIV-2.
In the contacted human serum, at least the HIV-2 antigen
A set that can be used to diagnose the presence of an antibody against one
According to the adult. This composition is described in the aforementioned European Patent Application
HIV-2 instead of HIV-1 in diagnostic methods such as
Various corresponding extracts using extracts, lysates or purified antigens
It can be used to selectively diagnose AIDS. to this
In this context, the present invention is particularly directed to proteins that are internal proteins.
At least one of p12, p16, p26 or p36 or gp140.
The composition. The following proteins are examples of these
There are compositions that include at the same time. -P26 and gp36 -26, p36 and gp140 -p12, p16 and p26 -p16, p26 and gp140 and the like. Obviously these compositions are only some examples
Will. In particular, the invention relates to this group of proteins and
Virus extract or lysate containing glycoprotein and / or glycoprotein
Or separation of one or more of said proteins or glycoproteins
Pertains to all fractions used. The present invention also relates to a protein and / or glycoprotein of HIV-2.
Protein and / or HIV-1 protein and / or glycoprotein
The present invention relates to a composition containing a combination with a protein. This combination
For example, the core protein of -HUV-1 or HIV-2
Quality, especially p-1 of HIV-1 and p26 of HIV-2, or p-1 of HIV-1
18 and p16 of HIV-2, and-the envelope glycoprotein of HIV-1 and the HIV-2
Velocity glycoproteins, especially gp110 and HIV-2 of HIV-1
Gp140, or p42 of HIV-1 and p36 or p42 of HIV-2
-P45, -HIV-1 protein and / or glycoprotein and HIV-
Mixture with protein 2 and / or glycoprotein 2
Good. Such diagnostic compositions are those that cover a wide range of etiological agents.
It provides a means for diagnosing AIDS or AIDS-related syndrome. To tell
Soon, HIV-2 protein and / or glycoprotein
The use of a composition containing only proteins as a diagnostic tool
The type of retrovirus can be more selectively diagnosed. The invention also relates to DNA or DNA fragments, more particularly
Contains clones derived from HIV-2 retroviral RNA
Pertaining to DNA fragments obtained from DNA and cDNA. Book
The invention is particularly directed to all equivalent DNA, especially to the DNA of HIV-2.
Sequence homology, especially HIV-2 bacteria deposited at CNCM
Sequence homology with the sequence encoding the env region and pol region of the strain
At least 50%, preferably 70%, more preferred
Or DNA having a range of up to 90%. Book in general
The invention is based on non-stringent conditions as described above.
HIV-2 DNA or RNA and hybrid by "spot blot" method
It refers to any equivalent DNA (or RNA) that can be rediscovered.
I can. The present invention also relates to a method of inoculating an animal with HIV-2 and
It relates to serum that can be produced. Therefore, more specifically, the present invention
Are the respective antigens of the virus, especially the proteins or
The present invention relates to a polyclonal antibody specific to a glycoprotein. Ma
Various HIV-2 proteins that can be produced by conventional methods
Respectively related to monoclonal antibodies. These polyclonal or monoclonal antibodies are
Can be used in various applications. The main application is the corresponding tongue
Neutralization of protein or inhibition of infectivity of whole virus.
Also, for example, for the detection of viral antigens in biological products.
Or, for example, affinity chromatography
The corresponding protein and / or glycoprotein
It may be used to carry out a purification treatment of a substance. Generally, the viruses named HIV-1 and HTLV-III
Technical literature available on the
Reference) uses the technology described in these references for the HIV-2 virus.
Alternatively, it can be used under the same conditions for isolating an equivalent virus, and
First, various components (especially proteins,
To produce glycoproteins, polypeptides and nucleic acids)
As long as they can be used under similar conditions.
And The use of these various components, especially LAS or
Is related to the use of AIDS as a diagnostic tool
Even the teachings of these technical documents can be used. The invention particularly relates to a method for in vitro diagnosis of AIDS. The
The method should be based on the serum or other organism of the subject to be diagnosed.
The medium is used for HIV-2 protein or glycoprotein
At least one or an extract or lysate of the virus
The immune response is detected by contact with the composition. Such composition
Examples have been described above. Preferred methods are, for example, ELISA or immunofluorescence type
Including the immunoenzymatic reaction. The titer is direct or indirect immunofluorescence
For light or direct or indirect immunoenzyme assays
Can be measured more. Therefore, the present invention also relates to virus extraction (one or more HI
Extract of V-2 virus alone or one or more types of HIV-2
Irs extract and one or more HIV-1 virus extracts
Mixture). These extracts are labeled.
Appropriate labels such as enzymes, fluorescence, radioactivity, etc. may be used. Such titration includes, for example, the following process. -A specific amount of the extract of the invention or said composition
Attach to wells of the interplate. -Mainly containing antibodies whose presence is to be detected in vitro
Serum is introduced at increasing dilutions into the wells. -Incubate the microtiter plate. -Carefully place the microtiter plate in a suitable buffer.
Wash. -Human immunoglobules in wells of microtiter plates
A labeled antibody specific for phosphorus is introduced. At least a specific wave
Substrate is changed so that the radiation absorption of the substrate changes in the long band.
Label with hydrolyzable selection enzyme. -The hydrolysis of the substrate, preferably by comparison with a control.
Detect the degree of degradation as a measure of disease latency or disease
You. The present invention also relates to the diagnostic assembly or kit.
You. The kit may comprise, for example, a radiolabel, an enzyme label or an immunofluorescent label.
Extracts of said type of virus or more highly purified
-Anti-human immunoglobulin or protein A (preferably
Bound to a water-insoluble carrier such as agarose beads);-a lymphocyte extract obtained from a healthy human;-a buffer and optionally a substrate for visualizing the labeling substance.
And From the above description, the present invention has been described using the aforementioned probe.
HIV-2 virus or its variants performing the various steps described
It will be appreciated that it encompasses methods for diagnosing heterologous strains. The stage
Are characteristics that demonstrate the characteristic characteristics of the HIV-2 virus.
It is performed in a fixed order. The present invention naturally covers the blood of a suspected carrier of the HIV-2 virus.
HIV-2 in a sample of clear or another biological fluid or tissue
CDNA or its probe as a probe to diagnose the presence or absence of ills
Related to the use of fragments (or recombinants containing them)
You. These probes (radioactive, enzyme, fluorescent labels, etc.)
Preferably). HIV-2 wi
For performing diagnostic methods for mutants of Rus or HIV-2
One particularly advantageous feature of the probe is the HIV-2 virus.
Contains all or part of the cDNA complementary to the genome
Or in particular in the various clones identified above.
Include existing fragments. In particular, the clone
HIV-2 cDNA present in E2, more particularly
3 'end (LTR) and / or 5' end of HIV sequence of lawn E2
Contains the end sequence or the env region of the HIV-2 virus cDNA
cDNA is advantageous. The diagnostic method and the diagnostic kit for HIV-2 virus of the present invention
Probes used in the probe are limited to forward probes
Not. Conversely, HIV-2 virus or HIV-2 mutants or
Is a nucleic acid obtained from the genome of a structurally related virus.
Otide sequences that can generate one of the forms of AIDS
Detect HIV-2 antibodies in human biological fluids
Includes the entire nucleotide sequence. However, first
Nucleotide sequence obtained from human infectious HIV-2
The use of rows is of course preferred. Detection may be performed by any known method. In particular,
Serum or other biological media such as cerebrospinal fluid, saliva, etc.
Contact probe with nucleic acid obtained from existing cells
Or hybridizable with probe
The probe itself is brought into contact with the medium itself containing the nucleic acid
You. This contact results in a hybrid between these probes and the nucleic acid.
It is performed under conditions that can cause the isomerization. What happens next
The detected hybridization is detected. HIV virus
If type identification is not required, hybridization
The diagnosis including the response to HIV-1 or HIV-2, respectively.
It may be performed using the obtained mixture of probes. Generally, the serum of a suspected carrier of the HIV-2 virus or
HIV-2 virus in other fluid or tissue samples
Alternatively, a method for diagnosing the presence or absence of a mutant thereof includes the following steps:
No. (1) Prepare a labeled probe. (2) The cell in the sample of the suspected carrier on the appropriate membrane
The DNA is contacted with the labeled probe,
At least one hybridization under
Do. (3) Stringent conditions for hybridization
Wash the membrane with a solution that maintains (4) Detection of the presence of HIV-2 virus by immunodetection
Put out. According to another preferred embodiment of the method of the present invention,
Hybridization under non-stringent conditions
Membrane under conditions adapted to no hybridization conditions
Is washed. The invention particularly relates to a GA comprising each of the following nucleotide sequences:
Includes viral RNA corresponding to cDNA with G and ENV genes
The present invention relates to the HIV-2 virus. These sequences correspond to the cDNA pair corresponding to the genomic HIV-2 ROD.
Obtained by sequencing the response region. These arrays are
Matches the amino acid they encode. As described above, the present invention provides that the RNA has similar properties.
In particular, the corresponding GAG and ENV sequences of HIV-2 ROD
Above, preferably 70%, more preferably 90% or more
GAG and ENV regions containing sequences with otide sequence homology
For all HIV-2 viruses with More specifically, the present invention provides that the structure is also included in GAGRODN.
CDNA plasmids encoding p16, p26 and p12, respectively.
Pertaining to mentament. The invention is particularly directed to:-from nucleotide 1 (encoding p16) to the nucleotide
Sequence of nucleotide 405-nucleotide 406 (encoding p26)
Sequence of nucleotides 1155-nucleotides from nucleotide 1156 (encoding p12)
Pertains to the sequence of Pide 1566. In addition, the present invention particularly relates to nucleotide 1 contained in ENVR.
CDNA encoding gp140 spanning from nucleotide 2574 to
Pertaining to fragments. The present invention further relates to the nucleotide sequence
Genetic code that does not include the amino acid sequence modification
Nucleotide by nucleotide substitution using degeneracy
The nucleotide sequence is different from the nucleotide sequence. Similarly, the present invention provides an amino acid sequence corresponding to the amino acid sequence described above.
Proteins or glycoproteins with no acid sequences and equivalents
Affects the overall immune properties of said peptide
By adding, substituting or deleting amino acids that do not give
The present invention relates to a peptide obtained from the above peptide. The invention particularly relates to amino acids encoded by ENVRN
The present invention relates to an envelope glycoprotein having a sequence. The present invention also relates to 10-500, preferably 50
HIV- formulated to administer a dosage unit of ~ 100 mg.
2 virus envelope antigens, especially HIV-2 virus
An immunogenic composition comprising gp140. Finally, the present invention relates to the protein (p12, p16 or p2
6) or a protein having the structure of gp140 or
A method for producing a predetermined portion of the protein. The method
Now, a cell host that expresses the insert in the vector is suitable.
Said vector which can be selected to transform said cell host
The nucleic acid sequence corresponding to
The selected host is transformed with the modified vector
The cell host transformed by the promoter is cultured and expressed.
The recovered protein is recovered and purified. Expression product of nucleic acid sequence derived from HIV-1 genome
1985 for producing a peptide or protein consisting of
Published in European Patent Application No. 85 905513.9 filed on October 18, 2016
Using the peptide or the peptide derived from HIV-2.
Can also be applied to protein production. Europe
The description of the state patent application is specifically incorporated herein by reference.
Shall be HIV-2 protein molecular weight (MP) to HIV-1 molecular weight
It is shown below in comparison with. HIV-2 Mir and HIV-2 ROD were announced on January 9, 1987 by Sali.
sbury 「National Collection of Animal Cel」
l Cultures "(ECACC), accession number 87 011001 and
Accepted at 87 011002. In addition, plasmids pROD35 and pROD27.5 were purchased in January 1987.
Aberdeen (UK) “National Collection of I
ndustrial Bacteria ”(NCIB) with accession numbers 12 398, respectively.
And deposited at 12 399. All references mentioned herein are included in the present invention.
Shall be BIBLIOGRAPHIE 1-F. Barr-Sinoussi et al., Science 220, 868 (198
3). 2-L. Montagnier et al., In: Human Tcell leukemia
Orlym-phoma viruses (Gallo, RC, Essex, ME, Gross, L., ed
s.) Cold Spring Harbor Laboratory, New York, 363 (19
84). 3-M.Popovic, MGSarngadharan, E.Read, RCGallo, Sc
ience 224,497 (1984). 4-J. Levy et al., Science 225, 840 (1984). 5-P. Sonigo et al., Cell 42, 369 (1985). 7-JWCurran et al., Science 229, 1352 (1985). 8-A. Ellrodt et al., Lancet i, 1383 (1984). 9-P. Piot et al., Lancet ii, 65 (1984). 10-F. Brun-Vezinet et al., Science 226,453 (198
Four). 11-N. Clumeck et al., N. Engl. J. Med. 313, 182 (198
Five). 12-ABRabson and MAMartin, Cell 40,477 (198
Five). 13-S, Benn et al., Science 230,949 (1985). 14-M. Alizon, Manuscript in preparation. 15-F. Brun-Vezient, Unpublished data. 16-MD Daniel et al., Science 228, 1201 (1985). 17-PJ Kanki et al., Science 228, 1199 (1985). 18-NLLetwin et al., Science 230, 71 (1985). 19-A. Gatzar et al., Blood 55, 409 (1980). 20-D. Klatzmann et al., Science 225, 59 (1984). 21-L. Montagnier et al., Virology 144, 283 (1985). 22-JS Allan et al., Science 228, 1091 (1985). 23-F.CHIVel, Manuscript in preparation.24-F-diMarzo Veronese et al., Science 229,1402
(1985). 25-VSKalyanaraman et al., Science 218, 751 (198
2). 26-ISYChen, J. McLaughlin, JCGasson, SCClark a
nd DWGolde, Nature 305, 502 (1983). 27-F. Barin et al., Lancet ii, 1387 (1985). 28-PJ Kanki, J. Alroy, M. Essex, Science 230, 951 (198
Five). 29-H. Towbin et al., Proc. Natl. Acad. Sci. USA 76, 4350
(1979). 30-S. Wain-Hobson, P. Sonigo, O. Danos, S. Cole et M. Al
izon, Cell 40, 9 (1985). 31-M. Alizon et al., Nature 312, 757 (1984).

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12Q 1/68 C12Q 1/70 1/70 C12N 15/00 A // (C12P 21/00 C12R 1:92) (31) Priority Claim number 86/01635 (32) Priority date February 6, 1986 (33) Priority claim country France (FR) (31) Priority claim number 86/01985 (32) Priority date February 13, 1986 ( 33) Priority claiming country France (FR) (31) Priority claiming number 835228 (32) Priority date March 3, 1986 (33) Priority claiming state United States (US) (31) Priority claiming number 86/03881 (32) Priority date March 18, 1986 (33) Priority country France (FR) (31) Priority number 86/04215 (32) Priority date March 24, 1986 (33) Priority country France (FR) (31) Priority claim number 91080 (32) Priority date October 6, 1986 (33) Priority country United States (US) (31) Priority number 933184 (32) Priority date November 21, 1986 (33) Priority country United States (US) (72) Inventor Getar, Denise, France, 75015 Paris, Lille-en-Serme Payens, 4b. Inventor Arizon, Marc France, 75005 Paris, Lille de France Yu Cardinal Lemoanne, 71 (72) Inventor Claver, François France, 75016 Paris, Liuilleux-de-Lassonpsion, 83 (72) Inventor Guilleyadere, Mireille France, 75016 Paris, Square -Rocamadour, 2 (72) Inventors Sonigo, Pierre, France, 75015 Paris, Lille-Guille Tambelle, 23 (72) Inventors Blanc-Vesinet, Francois, 75005 Paris , Boulevard Saint-Giermain, 24 (72) Rei, Marianne, France, 75004 Paris, Lille deux-Temples, 10 (72) Inventor Rigiu, Christine France, 75015 Paris, Lille deux Dansig, 21 (72) Inventor Katrama , Christine France, 75004 Paris, Lille d'eau Giarant, 8

Claims (1)

(57) [Claims] Infectious for human T4 lymphocytes, accession number I-50
2, an HIV-2 retrovirus or a variant thereof having the essential morphological and immunological properties of the retrovirus deposited at the CNCM at I-532, I-642 or I-643, comprising: Have properties, ie: (a) preferably target human Leu3 cells (or T4 lymphocytes) or permanent cell lines derived from said T4 lymphocytes; (b) against infected human T4 lymphocytes (C) reversal that has strong affinity for polyadenylate oligodeoxythymidylate (poly (A) -oligo (dT) 12-18) and requires the presence of Mg ²⁺ ions (D) the lysate further contains the HTLV-1 retrovirus gp110
Approximately 130,000-140,000 which does not immunologically cross-react with
(E) that the genomic RNA is HIV-stringent under stringent conditions.
No hybridization with any of the genomic RNA, ENV gene, or LTR. (F) Genomic RNA is H under non-stringent conditions.
A mixture of antigenic proteins or glycoproteins of an HIV-2 retrovirus or a mutant thereof, characterized by weakly hybridizing with the nucleotide sequence of the GAG region of the IV-1 genome. 2. 2. The mixture according to claim 1, wherein the mixture consists of a whole extract or lysate of the retrovirus. 3. Claims include an HIV-2 retrovirus antigen that is immunologically recognized by an antibody against the human HIV-2 retrovirus and has the immunological properties of an HIV-2 p12 protein having a molecular weight of about 12,000 daltons represented by the following amino acid sequence: 2. The mixture according to range 1. 4. Claims include an HIV-2 retrovirus antigen which is immunologically recognized by an antibody against the human HIV-2 retrovirus and has the immunological properties of the HIV-2 p16 protein having a molecular weight of about 16,000 daltons represented by the following amino acid sequence: 2. The mixture according to range 1. 5. Claims include an HIV-2 retrovirus antigen which is immunologically recognized by an antibody against the human HIV-2 retrovirus and has the immunological properties of an HIV-2 p26 protein having a molecular weight of about 26,000 daltons represented by the following amino acid sequence: 2. The mixture according to range 1. 6. HIV- having a molecular weight of about 36,000 daltons that is immunologically recognized by an antibody against the human HIV-2 retrovirus
2. The mixture according to claim 1, comprising an antigen of the HIV-2 retrovirus having the immunological properties of the 2p36 protein. 7. HIV- having the immunological properties of the HIV-2 p42 protein having a molecular weight of about 42,000 to 45,000 daltons that is immunologically recognized by an antibody against the human HIV-2 retrovirus
2. The mixture according to claim 1, comprising two retroviral antigens. 8. An HIV-2 retrovirus antigen which is immunologically recognized by an antibody against the human HIV-2 retrovirus and has the immunological properties of the HIV-2 gp140 protein having a molecular weight of about 130,000 to 140,000 daltons represented by the following amino acid sequence: The mixture of claim 1 comprising: