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Patent US7115363 - Retrovirus capable of causing AIDS, means and methods for detecting it in vitro - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe invention relates to a new class of retroviruses, designated by HIV-2, of which samples have been deposited to the ECACC under numbers 87.01.1001 and 87.01.1002 and to the NCIB under numbers 12.398 and 12.399. It relates also to antigens capable to be obtained from this virus, particularly proteins...http://www.google.com/patents/US7115363?utm_source=gb-gplus-sharePatent US7115363 - Retrovirus capable of causing AIDS, means and methods for detecting it in vitroAdvanced Patent SearchPublication numberUS7115363 B1Publication typeGrantApplication numberUS 08/470,489Publication dateOct 3, 2006Filing dateJun 6, 1995Priority dateJan 22, 1986Fee statusPaidAlso published asUS6037165, US6265149, US6664041, US7341731, US20020051967, US20030170658Publication number08470489, 470489, US 7115363 B1, US 7115363B1, US-B1-7115363, US7115363 B1, US7115363B1InventorsLuc Montagnier, Solange Chamaret, Denise Guetard, Marc Alizon, Fran�ois Clavel, Mireille Guyader, Pierre Sonigo, Francoise Brun-Vezinet, Marianne Rey, Christine Rouzioux, Christine KatlamaOriginal AssigneeInstitut PasteurExport CitationBiBTeX, EndNote, RefManPatent Citations (11), Non-Patent Citations (16), Classifications (33), Legal Events (3) External Links: USPTO, USPTO Assignment, EspacenetRetrovirus capable of causing AIDS, means and methods for detecting it in vitroUS 7115363 B1Abstract The invention relates to a new class of retroviruses, designated by HIV-2, of which samples have been deposited to the ECACC under numbers 87.01.1001 and 87.01.1002 and to the NCIB under numbers 12.398 and 12.399.
wherein the probe comprises an HIV-2 nucleic acid molecule obtained from nucleotides 1�380 of the U3/R region of HIV-2, nucleotides 1�1566 of the gag gene of HIV-2, nucleotides 1114�1524 of the gag gene, nucleotides 1�405 of the gag gene, nucleotides 406�1155 of the gag gene, or nucleotides 1�2673 of the env gene of HIV-2 or a fragment of said nucleic acid molecules;
wherein the insert comprises an HIV-2 nucleic acid molecule obtained from nucleotides 1�380 of the U3/R region of HIV-2, nucleotides 1�1566 of the gag gene of HIV-2, nucleotides 1114�1524 of the gag gene, nucleotides 1�405 of the gag gene, nucleotides 406�1155 of the gag gene, or nucleotides 1�2673 of the env gene of HIV-2 or a fragment of said nucleic acid molecules;
3. The method of claim 1, wherein the hybridization occurs under conditions of 5�SSC, 5� Denhart, 50% formamide, at 42� C., and washing of the resulting hybrid occurs under conditions of 0.1�SSC, 0.1% SDS, at 65� C.
4. The method of claim 2, wherein the hybridization occurs under conditions of 5�SSC, 5� Denhart, 50% formamide, at 42� C. and washing of the resulting hybrid occurs under conditions of 0.1�SSC, 0.1% SDS, at 65� C.
GTGGAAGGCG AGACTGAAAG CAAGAGGAAT ACCATTTAGT TAAAGGACAG GAACAGCTAT ACTTGGTCAG GGCAGGAAGT AACTAACAGA AACAGCTGAG ACTGCAGGGA CTTTCCAGAA GGGGCTGTAA CCAAGGGAGG GACATGGGAG GAGCTGGTGG GGAACGCCTC ATATTCTCTG TATAATATAC CCGCTGCTTG CATTGTACTT CAGTCGCTCT GCGGAGAGGC TGGCAGATTG AGCCCTGGAG GATCTCTCCA GCACTAGACG GATGAGCCTG GGTGCCCTGC TAGACTCTCA CCAGCACTTG GCCGGTGCTG GCAGACGGCC CCACGCTTGC CTGCTTAAAA ACCTTCCTTA ATAAAGCTGC AGTAGAAGCA. 6. The method of any one of claims 1 and 2, wherein said probe encodes the following amino acid sequence:
Met Gly Ala Arg Asn Ser Val Leu Arg Gly Lys Lys Ala Asp Glu Leu Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys His Ile Val Trp Ala Ala Asn Lys Leu Asp Arg Phe Gly Leu Ala Glu Ser Leu Leu Glu Ser Lys Giu Gly Cys Gln Lys Ile Leu Thr Val Leu Asp Pro Met Val Pro Thr Gly Ser Glu Asn Leu Lys Ser Leu Phe Asn Thr Val Cys Val Ile Trp Cys Ile His Ala Glu Glu Lys Val Lys Asp Thr Glu Gly Ala Lys Gln Ile Val Arg Arg His Leu Val Ala Glu Thr Gly Thr Ala Glu Lys Met Pro Ser Thr Ser Arg Pro Thr Ala Pro Ser Ser Glu Lys Gly Gly Asn Tyr Pro Val Gln His Val Gly Gly Asn Tyr Thr His Ile Pro Leu Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Leu Val Glu Glu Lys Lys Phe Gly Ala Glu Val Val Pro Gly Phe Gln Ala Leu Ser Glu Gly Cys Thr Pro Tyr Asp Ile Asn Gln Met Leu Asn Cys Val Gly Asp His Gln Ala Ala Met Gln Ile Ile Arg Glu Ile Ile Asn Glu Glu Ala Ala Glu Trp Asp Val Gln His Pro Ile Pro Gly Pro Leu Pro Ala Gly Gln Leu Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Val Glu Glu Gln Ile Gln Trp Met Phe Arg Pro Gln Asn Pro Val Pro Val Gly Asn Ile Tyr Arg Arg Trp Ile Gln Ile Gly Leu Gln Lys Cys Val Arg Met Tyr Asn Pro Thr Asn Ile Leu Asp Ile Lys Gln Gly Pro Lys Glu Pro Phe Gln Ser Tyr Val Asp Arg Phe Tyr Lys Ser Leu Arg Ala Glu Gln Thr Asp Pro Ala Val Lys Asn Trp Met Thr Gln Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Leu Val Leu Lys Gly Leu Gly Met Asn Pro Thr Leu Glu Glu Met Leu Thr Ala Cys Gln Gly Val Gly Gly Pro Gly Gln Lys Ala Arg Leu Met Ala Glu Ala Leu Lys Glu Val Ile Gly Pro Ala Pro Ile Pro Phe Ala Ala Ala Gln Gln Arg Lys Ala Phe Lys Cys Trp Asn Cys Gly Lys Glu Gly His Ser Ala Arg Gln Cys Arg Ala Pro Arg Arg Gln Gly Cys Trp Lys Cys Gly Lys Pro Gly His Ile Met Thr Asn Cys Pro Asp Arg Gln Ala Gly Phe Leu Gly Leu Gly Pro Trp Gly Lys Lys Pro Arg Asn Phe Pro Val Ala Gln Val Pro Gln Gly Leu Thr Pro Thr Ala Pro Pro Val Asp Pro Ala Val Asp Leu Leu Glu Lys Tyr Met Gln Gln Gly Lys Arg Gln Arg Glu Gln Arg Glu Arg Pro Tyr Lys Glu Val Thr Glu Asp Leu Leu His Leu Glu Gln Gly Glu Thr Pro Tyr Arg Glu Pro Pro Thr Glu Asp Leu Leu His Leu Asn Ser Leu Phe Gly Lys Asp Gln. 7. The method of any one of claims 1 and 2, wherein said probe encodes the following amino acid sequence:
Arg Lys Ala Phe Lys Cys Trp Asn Cys Gly Lys Glu Gly His Ser Ala Arg Gln Cys Arg Ala Pro Arg Arg Gln Gly Cys Trp Lys Cys Gly Lys Pro Gly His Ile Met Thr Asn Cys Pro Asp Arg Gln Ala Gly Phe Leu Gly Leu Gly Pro Trp Gly Lys Lys Pro Arg Asn Phe Pro Val Ala Gln Val Pro Gln Gly Leu Thr Pro Thr Ala Pro Pro Val Asp Pro Ala Val Asp Leu Leu Glu Lys Tyr Met Gln Gln Gly Lys Arg Gln Arg Glu Gln Arg Glu Arg Pro Tyr Lys Glu Val Thr Glu Asp Leu Leu His Leu Glu Gln Gly Glu Thr Pro Tyr Arg Glu Pro Pro Thr Glu Asp Leu Leu His Leu Asn Ser Leu Phe Gly Lys Asp Gln. 8. The method of any one of claims 1 and 2, wherein said probe encodes the following amino acid sequence:
Met Gly Ala Arg Asn Ser Val Leu Arg Gly Lys Lys Ala Asp Glu Leu Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys His Ile Val Trp Ala Ala Asn Lys Leu Asp Arg Phe Gly Leu Ala Glu Ser Leu Leu Glu Ser Lys Glu Gly Cys Gln Lys Ile Leu Thr Val Leu Asp Pro Met Val Pro Thr Gly Ser Glu Asn Leu Lys Ser Leu Phe Asn Thr Val Cys Val Ile Trp Cys Ile His Ala Glu Glu Lys Val Lys Asp Thr Glu Gly Ala Lys Gln Ile Val Arg Arg His Leu Val Ala Glu Thr Gly Thr Ala Glu Lys Met Pro Ser Thr Ser Arg Pro Thr Ala Pro Ser Ser Glu Lys Gly Gly Asn Tyr. 9. The method of any one of claims 1 and 2, wherein said probe encodes the following amino acid sequence:
Pro Val Gln His Val Gly Gly Asn Tyr Thr His Ile Pro Leu Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Leu Val Glu Glu Lys Lys Phe Gly Ala Glu Val Val Pro Gly Phe Gln Ala Leu Ser Glu Gly Cys Thr Pro Tyr Asp Ile Asn Gln Met Leu Asn Cys Val Gly Asp His Gln Ala Ala Met Gln Ile Ile Arg Glu Ile Ile Asn Glu Glu Ala Ala Glu Trp Asp Val Gln His Pro Ile Pro Gly Pro Leu Pro Ala Gly Gln Leu Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Val Glu Glu Gln Ile Gln Trp Met Phe Arg Pro Gln Asn Pro Val Pro Val Gly Asn Ile Tyr Arg Arg Trp Ile Gln Ile Gly Leu Gln Lys Cys Val Arg Met Tyr Asn Pro Thr Asn Ile Leu Asp Ile Lys Gln Gly Pro Lys Glu Pro Phe Gln Ser Tyr Val Asp Arg Phe Tyr Lys Ser Leu Arg Ala Glu Gln Thr Asp Pro Ala Val Lys Asn Trp Met Thr Gln Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Leu Val Leu Lys Gly Leu Gly Met Asn Pro Thr Leu Glu Glu Met Leu Thr Ala Cys Gln Gly Val Gly Gly Pro Gly Gln Lys Ala Arg Leu Met Ala Glu Ala Leu Lys Glu Val Ile Gly Pro Ala Pro Ile Pro Phe Ala Ala Ala Gln Gln. 10. The method of any one of claims 1 and 2, wherein said probe encodes the following amino acid sequence:
Met Met Asn Gln Leu Leu Ile Ala Ile Leu Leu Ala Ser Ala Cys Leu Val Tyr Cys Thr Gln Tyr Val Thr Val Phe Tyr Gly Val Pro Thr Trp Lys Asn Ala Thr Ile Pro Leu Phe Cys Ala Thr Arg Asn Arg Asp Thr Trp Gly Thr Ile Gln Cys Leu Pro Asp Asn Asp Asp Tyr Gln Glu Ile Thr Leu Asn Val Thr Glu Ala Phe Asp Ala Trp Asn Asn Thr Val Thr Glu Gln Ala Ile Glu Asp Val Trp His Leu Phe Glu Thr Ser Ile Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Ala Met Lys Cys Ser Ser Thr Glu Ser Ser Thr Gly Asn Asn Thr Thr Ser Lys Ser Thr Ser Thr Thr Thr Thr Thr Pro Thr Asp Gln Glu Gln Glu Ile Ser Glu Asp Thr Pro Cys Ala Arg Ala Asp Asn Cys Ser Gly Leu Gly Glu Glu Glu Thr Ile Asn Cys Gln Phe Asn Met Thr Gly Leu Glu Arg Asp Lys Lys Lys Gln Tyr Asn Glu Thr Trp Tyr Ser Lys Asp Val Val Cys Glu Thr Asn Asn Ser Thr Asn Gln Thr Gln Cys Tyr Met Asn His Cys Asn Thr Ser Val Ile Thr Glu Ser Cys Asp Lys His Tyr Trp Asp Ala Ile Arg Phe Arg Tyr Cys Ala Pro Pro Gly Tyr Ala Leu Leu Arg Cys Asn Asp Thr Asn Tyr Ser Gly Phe Ala Pro Asn Cys Ser Lys Val Val Ala Ser Thr Cys Thr Arg Met Met Glu Thr Gln Thr Ser Thr Trp Phe Gly Phe Asn Gly Thr Arg Ala Glu Asn Arg Thr Tyr Ile Tyr Trp His Gly Arg Asp Asn Arg Thr Ile Ile Ser Leu Asn Lys Tyr Tyr Asn Leu Ser Leu His Cys Lys Arg Pro Gly Asn Lys Thr Val Lys Gln Ile Met Leu Met Ser Gly His Val Phe His Ser His Tyr Gln Pro Ile Asn Lys Arg Pro Arg Gln Ala Trp Cys Trp Phe Lys Gly Lys Trp Lys Asp Ala Met Gln Glu Val Lys Thr Leu Ala Lys His Pro Arg Tyr Arg Gly Thr Asn Asp Thr Arg Asn Ile Ser Phe Ala Ala Pro Gly Lys Gly Ser Asp Pro Glu Val Ala Tyr Met Trp Thr Asn Cys Arg Gly Glu Phe Leu Tyr Cys Asn Met Thr Trp Phe Leu Asn Trp Ile Glu Asn Lys Thr His Arg Asn Tyr Ala Pro Cys His Ile Lys Gln Ile Ile Asn Thr Trp His Lys Val Gly Arg Asn Val Tyr Leu Pro Pro Arg Glu Gly Glu Leu Ser Cys Asn Ser Thr Val Thr Ser Ile Ile Ala Asn Ile Asp Trp Gln Asn Asn Asn Gln Thr Asn Ile Thr Phe Ser Ala Glu Val Ala Glu Leu Tyr Arg Leu Glu Leu Gly Asp Tyr Lys Leu Val Glu Tie Thr Pro Ile Gly Phe Ala Pro Thr Lys Glu Lys Arg Tyr Ser Ser Ala His Gly Arg His Thr Arg Gly Val Phe Val Leu Gly Phe Leu Gly Phe Leu Ala Thr Ala Gly Ser Ala Met Gly Ala Arg Ala Ser Leu Thr Val Ser Ala Gln Ser Arg Thr Leu Leu Ala Gly Tie Val Gln Gln Gln Gln Gln Leu Leu Asp Val Val Lys Arg Gln Gln Glu Leu Leu Arg Leu Thr Val Trp Gly Thr Lys Asn Leu Gln Ala Arg Val Thr Ala Ile Glu Lys Tyr Leu Gln Asp Gln Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln Val Cys His Thr Thr Val Pro Trp Val Asn Asp Ser Leu Ala Pro Asp Trp Asp Asn Met Thr Trp Gln Glu Trp Glu Lys Gln Val Arg Tyr Leu Glu Ala Asn Ile Ser Lys Ser Leu Glu Gln Ala Gln Ile Gln Gln Glu Lys Asn Met Tyr Glu Leu Gln Lys Leu Asn Ser Trp Asp Ile Phe Gly Asn Trp Phe Asp Leu Thr Ser Trp Val Lys Tyr Ile Gln Tyr Gly Val Leu Ile Ile Val Ala Val Ile Ala Leu Arg Ile Val Ile Tyr Val Val Gln Met Leu Ser Arg Leu Arg Lys Gly Tyr Arg Pro Val Phe Ser Ser Pro Pro Gly Tyr Ile Gln Gln Ile His Ile His Lys Asp Arg Gly Gln Pro Ala Asn Glu Glu Thr Glu Glu Asp Gly Gly Ser Asn Gly Gly Asp Arg Tyr Trp Pro Trp Pro Ile Ala Tyr Ile His Phe Leu Ile Arg Gln Leu Ile Arg Leu Leu Thr Arg Leu Tyr Ser Ile Cys Arg Asp Leu Leu Ser Arg Ser Phe Leu Thr Leu Gln Leu Ile Tyr Gln Asn Leu Arg Asp Trp Leu Arg Leu Arg Thr Ala Phe Leu Gln Tyr Gly Cys Glu Trp Ile Gln Glu Ala Phe Gln Ala Ala Ala Arg Ala Thr Arg Glu Thr Leu Ala Gly Ala Cys Arg Gly Leu Trp Arg Val Leu Glu Arg Ile Gly Arg Gly Ile Leu Ala Val Pro Arg Arg Ile Arg Gln Gly Ala Glu Ile Ala Leu Leu *** Gly Thr Ala Val Ser Ala Gly Arg Leu Tyr Glu Tyr Ser Met Glu Gly Pro Ser Ser Arg Lys Gly Glu Lys Phe Val Gln Ala Thr Lys Tyr Gly, wherein *** indicates a stop codon.
The isolation and characterization of a first retrovirus, known as LAV, whose responsibility in the development of AIDS had been recognized, formed the subject of a description in a paper by F. BARRE-SINOUSSI et al. already in 1983 (Science, vol. 220, No. 45�99, 20, p. 868�871). Application of some extracts of this virus, and more especially of some of its proteins, to the diagnosis of the presence of antibodies against the virus was described more especially in European Patent Application no. 138.667. Since then, other similar strains and some variants of LAV have been isolated. Examples which may be recalled are those known by the names HTLV-III and ARV.
To apply the new rules of nomenclature published in Nature in May 1986, the retroviruses capable of inducing in man the abovementioned lymphadenopathies and AIDS will be given the overall designation �HIV�, an abbreviation of the term �Human Immunodeficiency Virus�. The subgroup of retroviruses formed by LAV and its variants was initially designated by the terms �LAV type I� or �LAV-I�. The latter subgroup will be designated hereinafter HIV-1, it being understood that the term LAV will still be retained to denote that strain, among the strains of retrovirus (in particular LAAV, IDAV-4 and IDAV-2) belonging to the HIV-1 virus class which are described in the abovementioned European Patent Application 138,667, which was used in the comparative experiments described later, namely LAVBRU, which was deposited with the Collection Nationale des Cultures de Micro-organismes (CNCM) (National Collection of Micro-organism Cultures) of the Institut Pasteur de Paris, France, on 15th Jul. 1983 under no. I-232.
The new retrovirus which forms the subject of the present patent and the virus strains which are related to it and which are, like it, capable of multiplying in human lymphocytes, formerly known as. �LAV type II� or �LAV-II�, are henceforward known as �HIV-2�, it being understood that the designations of certain HIV-2 isolates described later will be followed by three letters which refer to the patients from whom they were isolated.
The �HIV-2� group can be defined as a group of viruses having in vitro a tropism for human T4 lymphocytes, and which have a cytopathogenic effect with respect to these lymphocytes when they multiply therein, and then either cause generalized and persistent polyadenopathies or one of the forms of AIDS. The HIV-2 retroviruses have proved to be different from the HIV-1 type viruses under the conditions mentioned later. Like these latter viruses, they are different from the other human retroviruses which are already known (HTLV-I and HTLV-II).
Although there is fairly wide genetic variability in the virus, the different HIV-1 strains isolated to date from American, European, Haitian and African patients have antigenic sites in common conserved on their principal proteins, i.e. core protein p25, envelope glycoprotein gp110 and transmembrane protein gp41-43. This relationship makes it possible, for example, for the prototype LAV strain to be used as a strain of antigens for detecting antibodies against all HIV-1 class viruses, in all people who carry them, regardless of their origin. This strain is hence currently used for detecting anti-HIV-1 antibodies in blood donors and patients, in particular by immunofluorescence and in particular by the technique known as ELISA, �Western Blot� (or immuno-imprinting) and �RIPA�, an abbreviation for Radioimmunoprecipitation Assay.
This combination of signs was evidence of �complex symptoms linked with AIDS� or �ARC� (abbreviation for �AIDS-Related Complex�), of the type caused by HIV-1 virus. These various observations were also in conformity with the criteria applied by the Center of Disease Control (CDC) of Atlanta, USA.
In particular, co-culturing was carried out, under the conditions specified below, of human T4 lymphocytes which had been infected five days beforehand with a strain of HIV-2 virus originating from a patient hereinafter designated �ROD�, on the one hand, and CEM, on the other hand.
The infected T4 lymphocytes, activated beforehand with phytohaemagglutinin, proved to possess a reverse transcriptase activity of 5,000 cpm/106 normal T lymphocytes three days after the beginning of the infection. Culturing was continued until the measured reverse transcriptase activity reached 100,000 cpm in the supernatant. These T4 lymphocytes were then placed in contact with CEM cells (3�106 infected normal T lymphocytes) and reincubated in the following culture medium: RPMI 1640 containing 2.92 mg/ml of L-glutamine, 10% of decomplemented foetal calf serum, 2 ug/ml of Polybrene, 0.05% of anti-interferon-alpha serum, 100,000 ug/ml of penicillin, 10 ug/ml of streptomycin and 10,000 ug/ml of neomycin.
I�Antigens, in Particular Proteins and Glycoproteins
It is generally specified that, in the text which follows, the numbers which follow the designations �p� and/or �gp� correspond to the approximate molecular weights of the corresponding proteins and/or glycoproteins, divided by 1000. For example, p36 has a molecular weight of the order of 36,000. It is, however, understood that these molecular weight values can vary within a range which can reach 5%, 10% or even more, depending on the techniques used for the determination of these molecular weights.
The same considerations apply to the existence of protein or glycoprotein bands whose apparent molecular weights were assessed at values which could range from 32,000 to 42,000�45,000. Repetition of the measurements finally enabled a band corresponding to an apparent molecular weight of 36,000 to be precisely defined. In the text which follows, this band is designated by the abbreviation p36. Another band at 42,000�45,000 (p42) is consistently observed also. One or other of p36 or p42 probably constitutes a transmembrane glycoprotein of the virus.
A major envelope glycoprotein having a molecular weight of the order of 130�140 kd is consistently observed this glycoprotein is designated hereinafter by the term gp140.
It is appropriate to note that, in general, the molecular weights are assessed with an accuracy of �5%, this accuracy even being capable of becoming a little lower for antigens of high molecular weight, as was fournd for gp140 (molecular weight of 140�10%). This group of antigens (when they are labelled with [35 S]cysteine is only faintly recognized, if at all, by sera of patients containing anti-HIV-1 antibodies in the detection systems used in the laboratory or by the use of tests employing HIV-1 lysates, such as those marketed by DIAGNOSTICS PASTEUR under the name �ELAVIA�. Only the p26 protein was weakly immunoprecipitated by such sera. The envelope protein was not precipitated. The serum of the patient infected with the new virus (HIV-2) faintly recognizes a p34 protein of HIV-1. In the detection system used, the other HIV-1 proteins were not recognized.
In contrast, HIV-2 possesses some proteins which show some immunological relationship with comparable structural proteins or glycoproteins, separated under similar conditions from a retrovirus recently isolated from captive macaques of the rhesus species, whereas this immunological relationship tends to become obliterated for other proteins or glycoproteins. This latter retrovirus, which is presumed to be the etiological agent of AIDS in monkeys, was designated by the investigators who isolated it [bibliographic references (16�18) below] by the name �STLV-IIImac�. For convenience of reference, it will be designated in the text which follows simply by the term �STLV-III� (or alternatively by the term SIV, an abbreviation for �Simian Immunodeficiency Virus�).
Another retrovirus, designated �STLV-IIIAGM� or SIVAGM), has been isolated in wild green monkeys. However, in contrast to the virus present in rhesus monkeys, the presence of �STLV-IIIAGM� does not appear to induce an AIDS-type disease in African green monkeys.
The HIV-2 virus does not multiply in chronic fashion in the lymphocytes of rhesus monkeys when it has been injected in vivo and under working conditions which permit the development of the STLV-IIImac virus, as have been described by N. L. Letvin et al., Science (1985), vol. 230, 71�75.
These findings apply not only in respect of the molecular weights, 130�140 kilodaltons for the major glycoproteins of HIV-2 and STLV-III compared with approximately 110 for the major envelope glycoprotein of HIV-1, but also in respect of the immunological properties, since sera drawn from patients infected with HIV-2, and more especially antibodies formed against the HIV-2 gp140, recognize the STLV-III gp140 whereas, in comparable experiments, the same sera and the same antibodies to HIV-2 do not recognize the HIV-1 gp110. However, anti-hiv-2 do not recognize the HIV-1 gp110. However, anti-HIV-1 sera which have never reacted with the HIV-2 gp140s precipitate a [35S]cysteine-labelled 26 kd protein present in extracts of HIV-2.
II�Nucleic Acids
1/ The RNAs of the HIV-2 retrovirus The RNA of the virus, deposited on a filter according to the �spot blot� technique, did not hybridize, under stringent conditions, with the DNA of HIV-1.
By �stringent conditions�, there are understood the conditions under which the hybridization reaction between the RNA of the HIV-2 and the chosen probe, radio-actively labelled with 32P (or labelled in a different manner), followed by the washing of the probe, are carried out. The hybridization, on a membrane, is carried out at 42� C. in the presence of an aqueous solution particularly of 50% formamide (volume/volume) in 0.1% SDS/5�SSC for 18 hours. The membrane on which the hybridization reaction has been carried out is then washed at 65� C. in a buffer containing 0.15% of SDS and 0.1�SSC.
By �non-stringent conditions�, there are understood the conditions under which the hybridization reaction and the washing are carried out. The hybridization is carried out by bringing into contact with the chosen probe, labelled with 32P (or otherwise labelled), namely at 42� C. in a 5�SSC buffer, 0.1% SDS, containing 30% of formamide for 18 hours. The washing of the membrane is carried out at 50� C. with a buffer containing 0.1� � of SDS and 2�SSC.
a/ single-stranded probes of subgenomic DNA of HIV-1, produced from subclones of the HIV-1 genome and inserted in phage M13. The cloned regions related to the protease gene or the �endonuclease� gene.
Only one probe of the endonuclease region of HIV-1 (nucleotide sequence between bases nos. 3760 and 4130) gave a weak hybridization under non-stringent conditions with HIV-2. The �protease� probe (HIV-1 nucleotide sequence between bases nos. 1680 and 1804) did not hybridize even under non-stringent conditions with HIV-2.
b/ A probe pRS3, consisting of the sequence coding for the �envelope� region of HIV-1 (subcloning in pUC18) did not give hybridization under non-stringent conditions with HIV-2.
The �spot blot� technique is also known as �dot blot� (transfer by spots).
The supernatants of the different culture media (in the proportion of 0.5 to 1 ml for each spot) were centrifuged for 5 minutes at 45,000 revolutions per minute; the pellets were resuspended in an NTE buffer containing 0.1% of SDS and deposited on a nitrocellulose filter. The latter was pre-soaked in a 2�SSC medium (0.3 M NaCl, 0.03 M sodium citrate). After baking (for 2 hours at 80� C.), the filters were hybridized with various probes contraining genomic sub-fragments of HIV-1, under non-stringent conditions (30% formamide, 5�SSC, 40� C.), washed at 50� C. with a 2�SSC solution containing 0.1% of SDS and then autoradiographed for 48 hours at −70� C. with enhancing screens.
The probes 1�4 are single-stranded probes obtained by the �prime cut� method as described in (25). Briefly, the single-stranded fragments originating from the M13 virus and bearing subgenomic HIV-1 inserts (30) were ligated to oligomeric fragments (17 nucleotides) originating from M13 (BIOLABS). The complementary strand was then synthesized with Klenow enzyme in a TM buffer (10 mM Tris, pH 7.5, 10 mMMgCl2) in the presence of dATP, dGTP, dTTP and dCTB, labelled with 32P at the alpha-position (Amersham, 3000 Ci/mmol). The DNA was then digested with the appropriate restriction enzymes, heat denatured and subjected to electrophoresis on a denaturing polyacrylamide gel (containing 6% of acrylamide, 8 M urea in a TDE buffer). The gel was then autoradiographed for 5 minutes. The probe was then cut out and eluted in a 300 mM NaCl, 0.1% SDS buffer. Specific activities (SA) of these single-stranded probes were estimated at 5�108�109 disintegrations per minute/microgram (dpm/ug).
nucleotides 990�1070,
nucleotides 980�1260,
nucleotides 2170�2240
nucleotides 3370�3640.
Two of the probes obtained (nucleotides 990�1070 and 990�1260, both originating from the gag region of HIV-1) hybridized slightly with the spots from extracts of the HIV-2 retroviruses; only one of these two probes (nucleotides 990�1260) also showed slight hybridization with the STLV-III spot (FIG. 3). As regards the probe containing a fragment of the pol region (nucleotides 2170�2240), hybridization was observed with STLV-III and, albeit much more weakly, with the RNA of HIV-2. The other probe of the pol region (nucleotides 3370�3640) did not give hybridization with any of the HIV-2 and STLV-III spots.
Lastly, the probe modified by nick translation and containing the entire env gene and the LTR (nucleotides 5290�9130) of HIV-2 did not hybridize either with the RNAs of STLV-III or with those of HIV-2.
This sequence, and a number of the restriction sites which it contains, are shown in FIG. 6. The cloned whole cDNA�or cloned fragments of this cDNA�can themselves be used as specific hybridization probes.
The first stage of manufacture of this cDNA comprised the production of an oligo(dT) serving as a primer or of an initiator cDNA strand, by carrying out an endogenous reaction activated by a detergent, using the reverse transcriptase of HIV-2, on purified virions obtained from supernatants of infected CEM cells. The CEM cell line was a lymphoblastoid CD4+ cell line described by G. E. Foley et al. in Cancer 18: 522�529 (1965), which is considered to be incorporated herein by reference. These CEM cells used are infected with an ROD isolate, which was shown to produce substantial amounts of HIV-2 continuously.
This procedure enabled different clones to be isolated, containing sequences approximately complementary to the 3′ end of the polyadenylated RNA of the LTR [abbreviation for �long terminal repeat� of HIV-1, described by S. Wain Hobson et al. in Cell 40: 9�17 (1985)] region, considered to be incorporated herein by reference.
This probe also detects the genomic RNA of HIV-2 under stringent conditions. It likewise permits detection, by the so-called �Southern blot� method on the DNA of CEM or similar cells infected with an ROD isolate or with other HIV-2 isolates. No signal is detected under the same stringency conditions in tests of hybridization of this probe with cDNAs originating from uninfected cells or from cells infected with HIV-1. These results confirmed the exogenous nature of HIV-2 with respect to HIV-1. An approximately 10 kb species, probably corresponding to the non-integrated viral DNA, was detected as a principal constituent in the undigested DNA of cells infected with HIV-2. Anoter DNA having an apparent size of 6 kb, possibly corresponding to a circular form of the viral DNA, was also detected.
The other portions of the HIV-2 genome were also identified. For this purpose, a genome library was constructed in phage lambda L47. Phage lambda L47.1 has been described by W. A. M. Loenen et al. in Gene 10 249�259 (1980), which publication is considered to be incorporated herein by reference.
Approximately 2�106 recombinant plaques were screened in situ with a clone containing the labelled E2 insert of the HIV-2 cDNA. Ten recombinant phages were detected on plaques and purified. The restriction maps of three of these phages, characterized by their capacity for �Southern blot� hybridization with the E2 insert under stringent conditions, as well as with subgenomic probes of HIV-1 under non-stringent conditions.
A clone bearing a 9.5 kb insert and derived from the whole circular viral DNA, containing the complete HIV-2 genome, was identified. It was designated �Lambda ROD 4�. The other two clones, Lambda ROD 27 and Lambda ROD 35, derived from integrated proviruses, bear LTR sequences of the viral coding sequences and adjacent cell DNA sequences. The different sequences are shown in FIG. 8.
pROD 27�5, derived from Lambda ROD 27, contains a 5.2 kb region of the HIV-2 genome and adjacent cell sequences (5′ LTR and 5′ coding viral sequence around an EcoRI site);
Even under very low stringency conditions (Tm-42� C.), the HIV-1 and HIV-2 genomes hybridize only at the level of their respective gag genes (spots 1 and 2), reverse transcriptase regions in pol (spot 3), end regions of pol, Q (or sor) genes (spot 5) and F (or 3′ orf) genes and 3′ LTR (spot 11). The HIV-1 fragment used for detecting the first cDNA clones of HIV-2 corresponds to the subclone of spot 11, which hybridizes relatively well with HIV-2 under non-stringent conditions. A signal originating from spot 5 is the only one which persists after stringent washing. The envelope gene, the tat gene region and part of pol appear to be highly divergent. These data, as well as the sequence obtained with LTR (FIG. 3), demonstrate that HIV-2 is not (at all events, as regards its envelope) a variant of HIV-1.
It is observed that HIV-2 is more closely related to SIV [described by M. D. Daniel et al in Science 228 1201�1204 (1985)], which must be considered to be incorporated herein by reference] than it is to HIV-1.
Advantageous sequences for forming probes in hybridization reactions with the genetic material of patients carrying viruses or proviruses, in particular for detecting the presence of HIV-2 virus RNA in their lymphocytes, contain a nucleotide sequence resulting from the combination of 5 kb HindIII fragments of ROD 4 and E2 cDNA. The experiments can be carried out by all methods, in particular by the �Northern blot�, �Southern blot� and �dot blot� techniques.
In summary, the virion preparation was added to a reaction mixture containing 50 mM Tris-HCL, 5 mM MgCl2, 10 mM DTT, 0.025% of the detergent marketed under the name TRITON, and 50 μM of each of the 4 deoxynucleoside triphosphates and an oligo(dT) initiator. The reaction was carried out for 90 minutes at 37� C.
After extraction with phenol of the proteins present in the first reaction medium, the second cDNA chain was synthesized in the presence of RNAse, E. coli DNA polymerase 1 and the 4 deoxynucleotides, for 1 hour at 15� C. and 1 hour at 22� C. Blunt ends were created on this double-stranded cDNA by the action of T4 DNA polymerase. All the reagents for this procedure are commercially available (AMERSHAM cDNA kit) and were used as recommended by the supplier.
To select, in the cDNA library, recombinant M13 clones containing the HIV-2 cDNA, the technique of plaque hybridization was used. The DNA of the M13 plaques was transferred to nitrocellulose filters and hybridized with subgenomic HIV-1 probes derived from the �lambda J19� clone of an LAV (or HIV) virus decribed in the European patent application. This probe contained an insert consisting of a portion having an approximate length of 1500 base pairs (bp) of HIV-1 DNA. This insert was bounded by two HindIII restriction sites, respectively, inside the open reading frame of the �env� gene and in the R segment of the 3′ LTR end of HIV-1. This probe contained the 3′ end of the env gene, the whole F gene, the U3 segment and a portion of the R segment of the LTR, having an approximate length of 1500 base pairs (bp).
The probe containing the 1.5 kg HindIII insert was labelled with [32P]-dCTP and -dTTP (3000 Ci�10−3 mole) by incubating the probe in the presence of initiators and Klenow DNA polymerase I for 4 hours at 15� C. (using an AMERSHAM kit). The tests of hybridization of the cDNA clones of the library were performed overnight under low stringency conditions, in a solution of a hybridization medium containing 5�SSC, 5� Denhart, 25% of formamide, 100 μg/ml of denatured salmon sperm DNA and the labelled probe (2�107 cpm with a specificity of 109 cpm/μg) at 37� C. The filters were subjected to three washing stages, successively in the presence of the three solutions whose compositions are stated as follows:
Washing no. 1: 5�SSC, 0.1% SDS, at 25� C. for 4�15 minutes
Washing no. 2: 2�SSC, 0.1% SDS, at 42� C. for 2�30 minutes
Washing no. 3:0.1�SSC, 0.1% SDS, at 65� C. for 2�30 minutes Each washing is followed by autoradiography of the filters.
The clones were also selected using a total human DNA probe under conditions of moderate stringency and by hybridization in 5�SSC, 5� Denhart and 40% formamide, followed by washing in 1�SSC, 0.1% SDS at 50� C. None of the previously positive clones was detected, and consequently did not correspond to specific repeated DNA or to the cDNA of the ribosomal RNA.
Several clones were partially sequenced using the dideoxy method of Sanger et al., described in Proc. Natl. Acad. Sci. 74:5463�7 (1977), which forms part of the present description. Various independent clones contained similar nucleotide sequences, with the exception of the poly(A) chains at their 3′ ends, the lengths of which were different. These results demonstrate that these cDNA clones were derived from the RNA template. Detailed sequence analysis of these cDNA clones, including the 3′ end of the HIV-2 genome, showed a limited relationship with HIV-1.
An infected supernatant was centrifuged (50,000 revolutions, 30 minutes). The pellet of the deposit was resuspended in 10 mM Tris pH 7.5, 1 mM EDTA, 0.1% SDS. One of the insertion clones, F1.1, was labelled and used as a probe for hybridization with the genomic RNA of different viral isolates, according to the �dot-blot� technique.
The �dot-blot� technique comprised the following stages:
(i) Depositing the sample (HIV-2 lysate) in spots on a nitrocellulose membrane soaked beforehand in 20�SSC (3 M NaCl, 0.3 M sodium citrate) and dried in the air, (ii) baking the membrane for 2 hours at 80 C, and (iii) performing the hybridization.
This hybridization was performed under high stringency conditions (5�SSC, 5� Denhart, 50′ formamide at 42� C.). It was followed by washing in 0.1�SSC, 0.1% SDS at 65� C. Under these conditions, the probe hybridizes strongly to the spots originating from two independent isolates of HIV-2, including LAV-II ROD, from which the cloned cDNA originated. A weak hybridization signal was detected with the spot formed by STLV-III mac [Simian T-lymphotropic Virus (also known as �SIV�), type III macaque], and no hybridization was detected with the HIV-1 isolates.
The �Southern blot� experiments, employing the clone E2.1 containing the 2 kb insert as a 32P-labelled probe, did not reveal any hybridization with the DNAs of uninfected cells, but detected bands in detached cells infected with HIV-2, under high stringency conditions. HIV-2 shows polymorphism at levels of its restriction map which are equivalent to those of the restriction maps of HIV-1. With the complete cellular DNA of infected cells, two types of signal are detected by the �Southern blot� method: (1) in DNA fractions having molecular weights MW of approximately 20 kb and more, in the case of integrated forms of the virus, and (2) in the fractions of lower MW (9,10 kb), in the case of the virus not integrated in the genome.
The positive M13 clone, E 2.1, was selected and subcloned in a plasmid vector. The DNA of the recombinant M 13 (TG 130) phage E 2 was purified in the form of a single-stranded DNA (M−13-ROD-E2) containing the 2 kb insert containing the 3′ portion of the HIV-2 genome (obtained from HIV-2 ROD). This insert was transferred to plasmid pSP65, described by Melton, D. A., in 357 Nucleic Acid Res. 12:035�7056 (1984).
HIV-2 virions were purified from 5 liters of a culture supernatant from a CEM line infected with a ROD isolate. A first strand of cDNA was synthesized in contact with sedimented purified virus, in the presence of an oligo(dT) initiator and employing an endogenous reaction activated by a detergent, according to the technique described by Alizon et al., Nature 312, 757�760 (1984). The RNA/cDNA hybrids were purified by extraction with a phenol/chloroform mixture and by precipitation with ethanol. The second strand of cDNA was produced in the presence of DNA polymerase I/RNAse H, according to the method described by Gubler and Hoffman ( ). The description in this paper is considered to be incorported herein by reference.
The filters were prehybridized in the presence of a medium containing 5�SSC, 5� DENHARDT solution, 25% formaldehyde and denatured salmon sperm DNA (100 micro-grammes/ml), at 37� C. for 4 hours, and then hybridized for 16 hours in the same buffer (Tm-42� C.) in the presence of additional labelled probe (4�107 cpm), to provide a final hybridization buffer solution containing 106 cpm/ml.
Washing was carried out with a 5�SSC, 0.1% SDS solution at 25� C. for 2 hours (it being understood that 20�SSC corresponds to a 3 M NaCl and 0.3 M sodium citrate solution). The plaques which responded positively were purified and the M13 single-stranded DNAs were prepared and their ends sequenced according to the method of Sanger et al.
The DNAs were extracted from infected CEM cells continuously producing HIV-1 and HIV-2, respectively. DNA samples of these two retroviruses, digested in some cases with 20 μg of PstI, and undigested in other cases, were subjected to electrophoresis on 0.8% agarose gel and transferred by the �Southern� method to a nylon membrane. Small volumes of infected supernatant, taken up in an NTE buffer containing 0.1% of SDS and having the same reverse transcriptase activity, were deposited on nitrocellulose which had been soaked beforehand in a 2�SSC solution.
A prehybridization was carried out in a solution containing 50� of formamide, 5�SSC, 5� Denhart and 100 mg/ml of denatured salmon sperm DNA, for 4 hours at 42� C. A hybridization was performed in the same buffer, to which 10% of dextran sulphate and 106 cpm/ml of E2 labelled insert (specific activity 109 cpm/μg) had been added, for 16 hours at 42� C. Two washings were then carried out with a 0.1�SSC, 0.1% SDS solution for 30 min each. After exposure for 16 hours to an intensifying screen, the Southern spot is dehybridized in 0.4 N NaOH, neutralized, and rehybridized under the same conditions with the HIV-1 probe labelled with 109 cpm/μg.
Example III Cloning in Phase Lambda of the Complete DNA of the HIV-2 Provirus
The DNA of CEM cells infected with HIV-2 ROD (FIG. 2, bands a and c) is partially digested with Sau3AI. The 9�15 kb fraction was selected on a 5�40% sucrose gradient and ligated to the BamHI arm of the lambda L47.1 vector. The plaques (2�106) obtained after in vitro packaging and deposition on E. coli strain LA 101 were screened in situ by hybridization with the insert of the E2 cloned cDNA. Approximately 10 positive clones were purified on plaques and propagated in E. coli C600 recBC. The clones lambda ROD 4, ROD 27 and ROD 35 were amplified, and their DNAs characterized by drawing up their restriction maps and by hybridization by Southern's method with the cDNA clone of HIV-2 under stringent conditions and with the gag-pol probes of HIV-1 under non-stringent conditions.
The hybridization experiments in the spot transfer (dot blot) method were carried out under the low stringency conditions of Example II using, by way of a probe, the lambda ROD 4 recombinant containing the total (DNA of HIV-2. The washings were then carried out successively under the following conditions: 2�SSC, 0.1% SDS at 25� C. (Tm-42� C.), 1�SSC, 0.1% SDS at 60� C. (Tm-20� C.) and 0.1%�SSC, 0.1% SDS at 60� C. (Tm-3� C.).
Example IV In Vitro Diagnostic Test for the Presence of HIV-2 Virus in a Biological Medium
HIV-2-infected patients were recruited among individuals visiting the Egas Moniz Hospital in Lisbon either for hospitalization or for consultation, between September 1985 and September 1986. For this selection, all individuals of African origin or having stayed in Africa underwent a serum test for antibodies against both HIV-1 (Immunofluorescence�IFA�and/or ELISA) and HIV-2 (IFA). Only those patients who were proved serologically to be infected with HIV-2 were included in the study.
IFA slides were prepared as follows: HIV-2-infected MOLT-4 cells were washed twice in PBS and layered onto IFA glass slides (104 cells/well), air dried and fixed with cold acetone. For IFA these cells were reacted with a 1/10 dilution of the test serum for 45 minutes at 37� C., washed, dried, and reacted with a fluorescein-conjugated goat antihuman IgG, A, M (1/100 diluted) for 30 minutes at 37� C. After washing, cells were counterstained in 0.006% Evans blue, mounted in 90% glycerol, 10% PBS and examined under a fluorescence microscope.
HIV-1 or HIV-2 infected CEM cells were cultured in the presence of 35S cysteine (200 microCi/ml) for 16 hours. The supernatant was collected, viral particles were pelleted and lysed in RIPA buffer (Tris-HCL 50 mM pH 7.5, NaCl 150 mM, EDTA 1 mM, 1% Triton X100, sodium deoxycholate 0.1%, SDS 0.1%). For each reaction, 50 microlitres of a dilution of lysate corresponding to 105 cpm was reacted with 5 microlitres of test serum for 18 hours at 4� C. Immune complexes were bound to Sepharose-protein A (PHARMACIA), washed, and eluted by boiling for 2 minutes. Eluted antigens were then analysed by SDS-polyacryl-amide gel electrophoresis and autoradiography.
Virus isolated from patients' PBLs were pelleted and lysed in Tris-HCL 10 mM pH 7.5, NaCl 10 mM, EDTA 1 mM, SDS 0.5%. One microliter of each lysate, corresponding to 50,000 cpm of RT activity, was deposited onto nitro-cellulose. Hybridization and washing were conducted in high stringency conditions: hybridization in 6�SSC, 5� Denhart, 50% formamide, for 18 hours at 42� C.; and washing in 0.1�SSC, 0.1% SDS at 65� C. W. used HIV-1 and HIV-2 probes, 32P labelled to a specific activity of 108 cpm/microgram. The HIV-1 probe corresponds to the complete genome of the LAVBRU isolate, and HIV-2 probe was derived from a 2 kilobases cDNA clone from LAV-2ROD isolate.
Thirty patients with serological and/or virologic evidence of HIV-2 infection were studied. They were 12 males and 18 females. The mean age was 35, ranging 11�55. All patients, except one, have stayed for several years in West Africa: 26 were born and living in Guinea-Bissau and 2 were originating from the Cape Verde Islands. One patient was an 11-year old boy from Angola who had lived in the Cape Verde islands for several years. The only European in the study population was a 40 year-old Portuguese man who had lived for 8 years in Zaire, and denied any stay in West Africa.
DISCUSSION In this study, we demonstrated HIV-2 infection in 30 West African patients presenting either with AIDS, ARC or with no apparent HIV-related symptoms. The results nevertheless permit the conclusion that HIV-2 must be considered to be a major aetiological agent of AIDS in West African patients. The serological and virologic profiles that we observed indicate that HIV-2 infection was not often associated with HIV-1 infection in our patients. Despite important antigenic and genetic differences, HIV-1 and HIV-2 display similar tropism for CD4+ T lymphocytes, similar cytopathic effects, similar morphology, and share common immunoreactive epitopes in some of their constitutive proteins. Since all West African patients with HIV infection in this study were found to be infected with HIV-2 and none of them with HIV-1, the new virus HIV-2 may be the major cause of AIDS in West Africa.
The preferred target for the HIV-2 retrovirus consists of human Leu 3 cells (or T4 lymphocytes) and and �immortalized� cells derived from these T4 lymphocytes, for example cells of the HUT 78 lines dealt with in the context of this patent application. In other words, it has a specific tropism for these cells. It can be cultured in permanent lines of the HUT, CEM, MOLT or similar type, expressing the T4 protein. It is not infectious for T8 lymphocytes. It is cytotoxic for the human T4 lymphocytes. The cytopathogenic nature of HIV-2 viruses with respect to T4 lymphocytes manifests itself, in particular, by the appearance of multinucleated cells. It has a reverse transcriptase activity which requires the presence of Mg2+ ions and has a strong affinity for polyadenylate oligodeoxythymidylate (poly(A)-oligo(dT) 12�18). It has a density of approximately 1.16 in a sucrose gradient. It has a mean diameter of 140 nanometers and a core having a mean diameter of 41 nanometers. The lysates of this virus contain a p26 protein which does not cross immunologically with the p24 protein of HTLV-1 virus or HTLV-II virus. These p26 proteins hence have a molecular weight which is slightly higher (by approximately 1000) than the corresponding p25 proteins of HIV-1 and slightly lower (again, of the order of approximately 1000 lower) than the corresponding p27 proteins of the SIV. The lysates of HIV-2 contain, in addition, a p16 protein which is not immunologically recognized by the p19 protein of HTLV-1 or of HTLV-II in RIPA (abbreviation for radioimmunoprecipitation assay) experiments. They contain, in addition, an envelope glycoprotein having a molecular weight of the order of 130,000�140,000, which does not cross immunologically with the gp 110 of HIV-1 but which, on the other hand, crosses immunologically with the gp 140 envelope glyco-protein of STLV-III. These lysates also contain proteins or glycoproteins which can be labelled with (35S)cysteine, having molecular weights, respectively, of the order of 36,000 and 42,000�45,000. The genomic RNA of HIV-2 does not hybridize with the genomic RNA of HIV-1 under stringent conditions. Under non-stringent conditions, it does not hybridize with nucleotide sequence derived from HIV-1 and containing the env gene and the LTR adjacent to it. In particular, it does not hybridize with the nucleotide sequence 5290�9130 of HIV-1, nor with sequences of the pol region of the HIV-1 genome, in particular with the nucleotide sequence 2170�2240. under non-stringent conditions, it hybridizes weakly with nucleotide sequences of the HIV-1 region, in particular the nucleotide sequences 990�1070 and 990�1260.
The invention also relates to each of the antigens, in particular proteins and glycoproteins in the purified state, such as may be obtained from HIV-2. Reference to �purified� proteins or glycoproteins implies that these proteins or glycoproteins lead, respectively, only to single bands in polyacrylamide gel electrophoresis, in particular under the experimental conditions which have been described above. Any suitable method of separation and/or purification for obtaning each of these can be used. By way of example of a technique which can be employed, that describes by R. C. MONTELARO et al., J. of Virology, June 1982, pp. 1029�1038, will be mentioned.
The invention relates generally to all antigens, in particular proteins, glycoproteins or polypeptides, originating from an HIV-2 and having immunological properties equivalent to those of these antigenic compounds of HIV-2. Two antigens are said to be �equivalent�, in the context of this account, inasmuch as they are recognized by the same antibodies, in particular antibodies which can be isolated from a serum obtained from a patient who has been infected with an HIV-2, or inasmuch as they meet the conditions for �immunological equivalence� stated below.
or envelope glycoproteins of an HIV-1 and envelope glycoproteins of an HIV-2, in particular the gp 110 of HIV-1 and the gp 140 of HIV-2, or alternatively the p42 of HIV-1 and the p36 or p42�45 of HIV-2,
The invention also relates to the DNAs or DNA fragments, more especially cloned DNAs and DNA fragments, obtained from the RNA and cDNAs derived from the RNA of the HIV-2 retrovirus. The invention also relates especially to all equivalent DNAs, in particular any DNA possessing sequence homologies with the DNA of HIV-2, especially with the sequences which code for the env and pol regions of the strain of HIV-2 deposited with the CNCM, equal at least to 50%, preferably to 70% and still more advantageously to 90%. It will also be stated generally that the invention relates to any equivalent DNA (or RNA) capable of hybridizing with the DNA or RNA of HIV-2 in the �spot blot� technique, under non-stringent conditions as defined above.
HIV-2 Mir and HIV-2ROD have also been deposited in the �National Collection of Animal Cell Cultures� (ECACC) in Salisbury (Great-Britain) on Jan. 9, 1987, under accessing numbers 87 011001 and 87 011002 respectively.
Moreover, plasmids pROD35 and pROD27.5 have been deposited at the �National Collection of Industrial Bacteria� (NCIB) in Aberdeen (Great-Britain) on Jan. 9, 1987 under accessing numbers 12 398 and 12 399 respectively.
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Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS4629783Aug 19, 1985Dec 16, 1986Genetic Systems CorporationPolypeptidesUS4839288Mar 3, 1986Jun 13, 1989Institut PasteurRetrovirus capable of causing AIDS, antigens obtained from this retrovirus and corresponding antibodies and their application for diagnostic purposesUS5051496 *Jan 16, 1987Sep 24, 1991Institut PasteurPolypeptides encoded by DNA fragments for detection of AIDSUS5055391Jan 3, 1990Oct 8, 1991Institut PasteurMethod and kit or detecting antibodies to antigens of human immunodeficiency virus type 2 (hiv-2)US5066782Jan 10, 1990Nov 19, 1991Institut PasteurRetrovirus capable of causing AIDS, means and method for detecting it in vitroUS5079342 *Feb 11, 1987Jan 7, 1992Institut PasteurDiagnosis of AIDSUS5223423Mar 31, 1989Jun 29, 1993United States Of AmericaCharacterization of replication competent human immunodeficiency type 2 proviral clone hiv-2sbl/isyUS5268265May 17, 1991Dec 7, 1993Institut PasteurImmunological complex comprising an antigen of Simian Immunodeficiency Virus (SIV) and an antibody against human immunodeficiency virus type 2 (HIV 2), and method and kit for detecting antibodies to HIV-2 reactive with antigens of SIVUS5306614Dec 20, 1991Apr 26, 1994Institut PasteurMethods and kits for diagnosing human immunodeficiency virus type 2(HIV-2)US5670309Feb 7, 1994Sep 23, 1997Johnson & JohnsonMethods and diagnostic kits for the detections of HIV-2-specific antibodies employing polypeptides obtained from the simian immunodeficiency virusWO1985004897A1Apr 23, 1985Nov 7, 1985Us HealthMethod and cell line for continuous production of retroviruses (htlv-iii) related to aids* Cited by examinerNon-Patent CitationsReference1 *Ausubel, F., 1987, "Current Protocols in Molecular Biology," John Wiley & Sons, New York, pp. 6.3.5-6.3.6 and 2.9.7-2.9.10.2Chakrabarti et al., Sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses, Nature 328:543-547 (1987).3Clavel et al., 1986 Science 233:343-346.4Clavel et al., 1986, Nature 324:691-695.5Clavel et al., Molecular cloning and polymorphism of the human immune deficiency virus type 2, Nature 324:691-695 (1986).6 *Claven et al, Natrue, v. 324, Dec. 1986, pp. 691-695.7 *Guyader et al, Naure, v. 326, Apr. 16, 1987, pp. 662-669.8Guyader et al., Genome organization and transactivation of the human immunodeficiency virus type 2, Nature 326:662-669 (1987).9Keller et al., Amino Acid Changes in the Fourth Conserved Region of Human Immunodeficiency Virus Type 2 Strain HIV-2<SUB>ROD </SUB>Envelope Glycoprotein Modulate Fusion, J. Virol. 67(10) :6253-6258 (1993).10 *Stryer, Biochemistry, 1975, W.H. Freeman and Company, San Francisco, pp. 761-763.11Stryer, Biochemistry, pp. 761-763 (W.H. Freeman and Company, San Francisco 1975).12 *Wahl et al., 1987, "Molecular Hybridization of Immobilized Nucleic Acids: Theoretical Concepts and Practical Considerations," Meth. Enzymol. 152:399-407.13Wain-Hobson et al., Nucleotide Sequence of the AIDS Virus, LAV, Cell 40:9-17 (1985).14 *Wallace et al., 1987, "Oligonucleotide Probes for the Screening of Recombinant DNA Libraries," Meth. Enzymol. 152:432-442.15Wang et al., Detection of antibodies to human T-lymphotropic virus type III by using a synthetic peptide of 21 amino acid residues corresponding to a highly antigenic segment of gp41 envelope protein, Proc. Natl. Acad. Sci. USA 83:6159-6163 (1986).16 *Wong-Staal, F., "Human Immunodeficiency Viruses and Their Replication", in Virology, Second Edition, Fields et al., eds., Raven Press, Ltd., 1990, pp. 1530-1535.* Cited by examinerClassifications U.S. Classification435/5, 424/188.1, 536/24.3, 536/23.72, 424/208.1International ClassificationG01N33/53, A61K38/00, C12N7/00, C12Q1/68, A61K39/00, A61K39/21, C07K14/16, C07K7/06, G01N33/569, C07H21/02, C12Q1/70Cooperative ClassificationC12N7/00, C07K14/005, C12N2740/16222, A61K38/00, A61K39/00, G01N2333/162, G01N2469/20, G01N33/56988, C12Q1/703, C12N2740/16021, C07K7/06, C12N2740/16122European ClassificationG01N33/569K2, C12Q1/70B2B, C12N7/00, C07K7/06, C07K14/005Legal EventsDateCodeEventDescriptionMar 15, 2014FPAYFee paymentYear of fee payment: 8Mar 17, 2010FPAYFee paymentYear of fee payment: 4Apr 15, 2008CCCertificate of correctionRotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services