US20030091985A1 - Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits - Google Patents

Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits Download PDF

Info

Publication number
US20030091985A1
US20030091985A1 US09/988,213 US98821301A US2003091985A1 US 20030091985 A1 US20030091985 A1 US 20030091985A1 US 98821301 A US98821301 A US 98821301A US 2003091985 A1 US2003091985 A1 US 2003091985A1
Authority
US
United States
Prior art keywords
peptide
hiv
immunological properties
groups
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US09/988,213
Inventor
Marc Alizon
Luc Montagnier
Denise Guetard
Francois Clavel
Pierre Sonigo
Mireille Guyader
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut Pasteur de Lille
Original Assignee
Institut Pasteur de Lille
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from FR8600911A external-priority patent/FR2593190B1/en
Priority claimed from FR8601635A external-priority patent/FR2593922B1/en
Priority claimed from FR8601985A external-priority patent/FR2594229B1/en
Priority claimed from US06/835,228 external-priority patent/US4839288A/en
Priority claimed from FR8603881A external-priority patent/FR2596063B1/en
Priority claimed from FR8604215A external-priority patent/FR2597500B1/en
Priority claimed from FR8604556A external-priority patent/FR2596414B1/en
Priority claimed from US07/013,477 external-priority patent/US5079342A/en
Priority claimed from US07/810,908 external-priority patent/US6544728B1/en
Priority to US09/988,213 priority Critical patent/US20030091985A1/en
Application filed by Institut Pasteur de Lille filed Critical Institut Pasteur de Lille
Publication of US20030091985A1 publication Critical patent/US20030091985A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G01N2333/162—HIV-1, HIV-2 env, e.g. gp160, gp110/120, gp41, V3, peptid T, DC4-Binding site
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/974—Aids related test

Definitions

  • the invention relates to cloned DNA sequences analogous to the genomic RNA of a virus known as Lymphadenopathy-Associated Virus II (“LAV-II”), a process for the preparation of these cloned DNA sequences, and their use as probes in diagnostic kits.
  • LAV-II Lymphadenopathy-Associated Virus II
  • the invention relates to a cloned DNA sequence analogous to the entire genomic RNA of HIV-2 and its use as a probe.
  • the invention also relates to polypeptides with amino acid sequences encoded by these cloned DNA sequences and the use of these polypeptides in diagnostic kits.
  • HIV-1 Human Immunodeficiency viruses I
  • LAV-I Lymphadenopathy-Associated Virus I
  • HTLV-III Human T-cell Lymphotropic Virus-III
  • ARV AIDS-Related Virus
  • Lymphadenopathy-Associated Virus II has been described in U.S. application Ser. No. 835,228, which was filed Mar. 3, 1986, and is specifically incorporated herein by reference. Because LAV-II is a second, distinct causative agent of AIDS, LAV-II properly is classifiable as a Human Immunodeficiency Virus II (HIV-2). Therefore, “LAV-II” as used hereinafter describes a particular genus of HIV-2 isolates.
  • HIV-2 is related to HIV-1 by its morphology, its tropism and its in vitro cytopathic effect on CD4 (T4) positive cell lines and lymphocytes
  • HIV-2 differs from previously described human retroviruses known to be responsible for AIDS.
  • the proteins of HIV-1 and 2 have different sizes and their serological cross-reactivity is restricted mostly to the major core protein, as the envelope glycoproteins of HIV-2 are not immune precipitated by HIV-1-positive sera except in some cases where very faint cross-reactivity can be detected. Since a significant proportion of the HIV infected patients lack antibodies to the major core protein of their infecting virus, it is important to include antigens to both HIV-1 and HIV-2 in an effective serum test for the diagnosis of the infection by these viruses.
  • HIV-2 was first discovered in the course of serological research on patients native to Guinea-Bissau who exhibited clinical and immunological symptoms of AIDS and from whom sero-negative or weakly sero-positive reactions to tests using an HIV-1 lysate were obtained. Further clinical studies on these patients isolated viruses which were subsequently named “LAV-II.”
  • LAV-II MIR Collection Nationale des Cultures de Micro-Organismes
  • CNCM Collection Nationale des Cultures de Micro-Organismes
  • I-502 has also been deposited at the British ECA CC under No. 87.001.001 on Jan. 9, 1987.
  • a second LAV-II isolate was deposited at CNCM on Feb. 21, 1986 under Accession No. 1-532 and has also been deposited at the British ECA CC under No. 87.001.002 on Jan. 9, 1987.
  • This second isolate has been subsequently referred to as LAV-II ROD.
  • HIV-2 IRMO No. I-642
  • HIV-2 EHO No. I-643
  • Several additional isolates have been obtained from West African patients, some of whom have AIDS, others with AIDS-related conditions and others with no AIDS symptoms. All of these viruses have been isolated on normal human lymphocyte cultures and some of them were thereafter propagated on lymphoid tumor cell lines such as CEM and MOLT.
  • kits designed to identify HIV-1 infections in the diagnosis of these new patients with HIV-2 disease it has been necessary to devise a new diagnostic kit capable of detecting HIV-2 infection, either by itself or in combination with an HIV-1 infection.
  • the present inventors have, through the development of cloned DNA sequences analogous to at least a portion of the genomic RNA of LAV-II ROD viruses, created the materials necessary for the development of such kits.
  • the present invention relates to the cloned nucleotide sequences homologous or identical to at least a portion of the genomic RNA of HIV-2 viruses and to polypeptides encoded by the same.
  • the present invention also relates to kits capable of diagnosing an HIV-2 infection.
  • a main object of the present invention is to provide a kit capable of diagnosing an infection caused by the HIV-2 virus.
  • This kit may operate by detecting at least a portion of the RNA genome of the HIV-2 virus or the provirus present in the infected cells through hybridization with a DNA probe or it may operate through the immunodiagnostic detection of polypeptides unique to the HIV-2 virus.
  • cloned DNA sequences related to the entire genomic RNA of the LAV-II virus are set forth. These sequences are analogous specifically to the entire genome of the LAV-II ROD strain.
  • kits capable of diagnosing an HIV-2 infection contains the cloned DNA sequences of this invention which are capable of hybridizing to viral RNA or analogous DNA sequences to indicate the presence of an HIV-2 infection.
  • Different diagnostic techniques can be used which include, but are not limited to: (1) Southern blot procedures to identify viral DNA which may or may not be digested with restriction enzymes; (2) Northern blot techniques to identify viral RNA extracted from cells; and (3) dot blot techniques, i.e., direct filtration of the sample through an ad hoc membrane such as nitrocellulose or nylon without previous separation on agarose gel.
  • Suitable material for dot blot technique could be obtained from body fluids including, but not limited to, serum and plasma, supernatants from culture cells, or cytoplasmic extracts obtained after cell lysis and removal of membranes and nuclei of the cells by ultra-centrifugation as accomplished in the “CYTODOT” procedure as described in a booklet published by Schleicher and Schull.
  • the kit contains the polypeptides created using these cloned DNA sequences. These polypeptides are capable of reacting with antibodies to the HIV-2 virus present in sera of infected individuals, thus yielding an immunodiagnostic complex.
  • a vaccinating agent which comprises at least one peptide selected from the polypeptide expression products of the viral DNA in admixture with suitable carriers, adjuvents stabilizers.
  • FIG. 1 generally depicts the nucleotide sequence of a cloned complementary DNA (cDNA) to the genomic RNA of HIV-2.
  • cDNA cloned complementary DNA
  • FIG. 1A depicts the genetic organization of HIV-1, position of the HIV-1 HindIII fragment used as a probe to screen the cDNA library, and restriction map of the HIV-2 cDNA clone, E2.
  • FIG. 1B depicts the nucleotide sequence of the 3′ end of HIV-2.
  • the corresponding region of the HIV-1 LTR was aligned using the Wilbur and Lipman algorithm (window: 10; K-tuple: 7; gap penalty: 3) as described by Wilbur and Lipman in Proc. Natl. Acad. Sci. USA 80: 726-730 (1983), specifically incorporated herein by reference.
  • the U3-R junction in HIV-1 is indicated and the poly A addition signal and potential TATA promoter regions are boxed.
  • the symbols B, H, Ps and Pv refer to the restriction sites BamHI, HindIII, PstI and PvuII, respectively.
  • FIG. 2 generally depicts the HIV-2 specificity of the E2 clone.
  • FIGS. 2A and B specifically depict a Southern blot of DNA extracted from CEM cells infected with the following isolates: HIV-2 ROD (a, c), HIV-2 DUL (b, d), and HIV-1 BRU (e, f). DNA in lanes a, b, f was Pst I digested; in c, d, e DNA was undigested.
  • FIGS. 2C and D specifically depict dot blot hybridization of pelleted virions from CEM cells infected by the HIV-1 BRU (1), Simian Immunodeficiency Virus (SIV) isolate Mm 142-83 (3), HIV-2 DUL (4), HIV-2 ROD (5), and HIV-1 ELI (6).
  • Dot 2 is a pellet from an equivalent volume of supernatant from uninfected CEM.
  • FIG. 2A and C depicts hybridization with the HIV-2 cDNA (E2) and
  • FIG. 2B and D depicts hybridization to an HIV-1 probe consisting of a 9Kb SacI insert from HIV-1 BRU (clone lambda J 19).
  • FIG. 3 generally depicts a restriction map of the HIV-2 ROD genome and its homology to HIV-1.
  • FIG. 3A specifically depicts the organization of three recombinant phage lambda clones, ROD 4, ROD 27, and ROD 35.
  • the open boxes represent viral sequences
  • the LTR are filled
  • the dotted boxes represent cellular flanking sequences (not mapped). Only some characteristic restriction enzyme sites are indicated.
  • ⁇ ROD 27 and ⁇ ROD 35 are derived from integrated proviruses while ⁇ ROD 4 is derived from a circular viral DNA.
  • the portion of the lambda clones that hybridzes to the cDNA E2 is indicated below the maps.
  • a restriction map of the ⁇ ROD isolate was reconstructed from these three lambda clones.
  • restriction sites are identified as follows: B: BamHI; E: EcoRI; H: HindIII; K: KnI; Ps: PstI; Pv: PvuII; S: SacI; X: XbaI.
  • R and L are the right and left BamHI arms of the lambda L47.1 vector.
  • FIG. 3B specifically depicts dots 1-11 which correspond to the single-stranded DNA form of M13 subclones from the HIV-1 BRU cloned genome ( ⁇ J9). Their size and position on the HIV-1 genome, determined by sequencing is shown below the figure.
  • Dot 12 is a control containing lambda phage DNA. The dot-blot was hybridized in low stringency conditions as described in Example 1 with the complete lambda ⁇ ROD 4 clone as a probe, and successively washed in 2 ⁇ SSC, 0.1% SDS at 25° C. (Tm ⁇ 42° C.), 1 ⁇ SSC, 0.1% SDS at 60° C.
  • FIG. 4 generally depicts the restriction map polymorphism in different HIV-2 isolates and shows comparison of HIV-2 to SIV.
  • FIG. 4A specifically depicts DNA (20 ug. per lane) from CEM cells infected by the isolate HIV-2 DUL (panel 1) or peripheral blood lymphocytes (PBL) infected by the isolates HIV-2 GOM (panel 2) and HIV-2 MIR (panel 3) digested with: EcoRI (a), PstI (b), and HindIII (c). Much less viral DNA was obtained with HIV-2 isolates propagated on PBL. Hybridization and washing were in stringent conditions, as described in Example 2, with 10 6 cpm/ml. of each of the E2 insert (cDNA) and the 5 kb. HindIII fragment of ⁇ ROD 4, labelled to 10 9 cpm/ug.
  • FIG. 4B specifically depicts DNA from HUT 78 (a human T lymphoid cell line) cells infected with STLV3 MAC isolate Mm 142-83.
  • HUT 78 a human T lymphoid cell line
  • STLV3 MAC isolate Mm 142-83 The same amounts of DNA and enzymes were used as indicated in panel A. Hybridization was performed with the same probe as in A, but in non-stringent conditions. As described in Example 1 washing was for one hour in 2 ⁇ SSC, 0.1% SDS at 40° C. (panel 1) and after exposure, the same filter was re-washed in 0.1 ⁇ SSC, 0.1% SDS at 60° C. (panel 2). The autoradiographs were obtained after overnight exposition with intensifying screens.
  • FIG. 5 depicts the position of derived plasmids from ⁇ ROD 27, ⁇ ROD 35 and ⁇ ROD 4.
  • the genetic structure of the HIV-2 virus has been analyzed by molecular cloning according to the method set forth herein and in the Examples. A restriction map of the genome of this virus is included in FIG. 4. In addition, the partial sequence of a cDNA complementary to the genomic RNA of the virus has been determined. This cDNA sequence information is included in FIG. 1.
  • a complementary DNA (cDNA) to the HIV-2 genomic RNA initially was cloned in order to provide a specific hybridization probe.
  • an oligo (dT) primed cDNA first-strand was made in a detergent-activated endogenous reaction using HIV-2 reverse transcriptase with virions purified from supernatants of infected CEM cells.
  • the CEM cell line is a lymphoblastoid CD4+ cell line described by G. E. Foley et al. in Cancer 18: 522-529 (1965), specifically incorporated herein by reference.
  • the CEM cells used were infected with the isolate ROD and were continuously producing high amounts of HIV-2.
  • the cDNAs were inserted into the M 13 tg 130 bacteriophage vector.
  • a collection of 10 4 M13 recombinant phages was obtained and screened in situ with an HIV-1 probe spanning 1.5 kb. of the 3′ end of the LAV BRU isolate (depicted in FIG. 1A).
  • Some 50 positive plaques were detected, purified, and characterized by end sequencing and cross-hybridizing the inserts. This procedure is described in more detail in Example 1 and in FIG. 1.
  • the different clones were found to be complementary to the a 3′ end of a polyadenylated RNA having the AATAAA signal about 20 nucleotides upstream of the poly A tail, as found in the long terminal repeat (LTR) of HIV-1.
  • LTR long terminal repeat
  • the LTR region of HIV-1 has been described by S. Wain Hobson et al. in Cell 40: 9-17 (1985), specifically incorporated herein by reference.
  • the portion of the HIV-2 LTR that was sequenced was related only distantly to the homologous domain in HIV-1 as demonstrated in FIG. 1 B. Indeed, only about 50% of the nucleotides could be aligned and about a hundred insertions/deletions need to be introduced. In comparison, the homology of the corresponding domains in HIV-1 isolates from USA and Africa is greater than 95% and no insertions or deletions are seen.
  • E2 The largest insert of this group of M13 clones was a 2 kb. clone designated E2.
  • Clone E2 was used as a probe to demonstrate its HIV-2 specificity in a series of filter hybridization experiments. Firstly, this probe could detect the genomic RNA of HIV-2 but not HIV-1 in stringent conditions as shown in FIG. 2, C and D. Secondly, positive signals were detected in Southern blots of DNA from cells infected with the ROD isolate as well as other isolates of HIV-2 as shown in FIG. 2, A and FIG. 4, A. No signal was detected with DNA from uninfected cells or HIV-1 infected cells, confirming the exogenous nature of HIV-2. In undigested DNA from HIV-2 infected cells, an approximately 10 kb.
  • Lambda phage L47.1 has been described by W. A. M. Loenen et al. in Gene 10: 249-259 (1980), specifically incorporated herein by reference.
  • the genomic library was constructed with a partial Sau3AI restriction digest of the DNA from the CEM cell line infected with HIV-2 ROD .
  • a clone carrying a 9.5 kb. insert and derived from a circular viral DNA was identified as containing the complete genome and designated ⁇ ROD 4.
  • Two other clones, ⁇ ROD 27 and ⁇ ROD 35 were derived from integrated proviruses and found to carry an LTR and cellular flanking sequences and a portion of the viral coding sequences as shown in FIG. 3, A.
  • Plasmid pROD 27-5′ is derived from ⁇ ROD 27 and contains the 5′ 2Kb of the HIV-2 genome and cellular flanking sequences (5′ LTR and 5′ viral coding sequences to the EcoRI site).
  • Plasmid pROD 4.7 contains a HindIII 1.8 Kb fragment from ⁇ ROD 4. This fragment is located 3′ to the fragment subcloned into pROD 4.8 and contains about 0.8 Kb of viral coding sequences and the part of the lambda phage ( ⁇ L47.1) left arm located between the BamHI and HindIII cloning sites.
  • Plasmid pROD 35 contains all the HIV-2 coding sequences 3′ to the EcoRI site, the 3′ L:R and about 4 Kb of cellular flanking sequences.
  • Plasmid pROD 27-5′ and pROD 35 in E. coli strain HB 101 are deposited respectively under No. 1-626 and 1-633 at the CNCM, and have also been deposited at the NCIB (British Collection). These plasmids are depicted in FIG. 5. Plasmids pROD 4-7 and pROD 4-8 in E. coli strain TG1 are deposited respectively under No. 1-627 and 1-628 at the CNCM.
  • pROD 35 is linearized with EcoRI and the EcoRI insert of pROD 27-5′ is ligated in the correct orientation into this site.
  • HIV-2 The relationship of HIV-2 to other human and simian retroviruses was surmised from hybridization experiments. The relative homology of the different regions of the HIV-1 and 2 genomes was determined by hybridization of fragments of the cloned HIV-1 genome with the labelled ⁇ ROD 4 expected to contain the complete HIV-2 genome (FIG. 3, B).
  • HIV-1 and 2 Even in very low stringency conditions (Tm ⁇ 42° C.), the hybridization of HIV-1 and 2 was restricted to a fraction of their genomes, principally the gag gene (dots 1 and 2), the reverse transcriptase domain in pol (dot 3), the end of pol and the Q (or sor) genes (dot 5) and the F gene (or 3′ orf) and 3′ LTR (dot 11).
  • the HIV-1 fragment used to detect the HIV-2 cDNA clones contained the dot 11 subclone, which hybridized well to HIV-2 under non-stringent conditions. Only the signal from dot 5 persisted after stringent washing. The envelope gene, the region of the tat gene and a part of pol thus seemed very divergent.
  • Simian Immunodeficiency Virus also designated Simian T-cell Lymphotropic Virus Type 3, STLV-3
  • STLV-3 Simian Immunodeficiency Virus
  • SIV proteins including the envelope, are immune precipitated by sera from HIV-2 infected patients, whereas the serological cross-reactivity of HIV-1 to 2 is restricted to the core proteins.
  • SIV and HIV-2 can be distinguished by slight differences in the apparent molecular weight of their proteins.
  • HIV-2 is closely related to SIV.
  • the genomic RNA of SIV can be detected in stringent conditions as shown in FIG. 2, C by HIV-2 probes corresponding to the LTR and 3′ end of the genome (E2) or to the gag or pol genes. Under the same conditions, HIV-1 derived probes do not detect the SIV genome as shown in FIG. 2, D.
  • FIG. 4 A depicts examples of such differences for three isolates, all different one from another and from the cloned HIV-2 ROD . It is very likely that these differences at the nucleotide level are accompanied by variations in the amino-acid sequence of the viral proteins, as evidenced in the case of HIV-1 and described by M. Alizon et al. in Cell 46: 63-74 (1986), specifically incorporated herein by reference. It is also to be expected that the various isolates of HIV-2 will exhibit amino acid heterogeneities. See, for example, Clavel et al., Nature 324 (18):691-695 (1986), specifically incorporated herein by reference.
  • the chacterization of HIV-2 will also delineate the domain of the envelope glycoprotein that is responsible for the binding of the surface of the target cells and the subsequent internalization of the virus. This interaction was shown to be mediated by the CD4 molecule itself in the case of HIV-1 and similar studies tend to indicate that HIV-2 uses the same receptor.
  • small homologous domains of the envelopes of the two HIV could represent a candidate receptor binding site. This site could be used to raise a protective immune response against this group of retroviruses.
  • nucleotide sequences which are capable of being used as probes in diagnostic methods to obtain the immunological reagents necessary to diagnose an HIV-2 infection.
  • these sequences may be used as probes in hybridization reactions with the genetic material of infected patients to indicate whether the RNA of the HIV-2 virus is present in these patient's lymphocytes or whether an analogous DNA is present.
  • the test methods which may be utilized include Northern blots, Southern blots and dot blots.
  • One particular nucleotide sequence which may be useful as a probe is the combination of the 5 kb. HindIII fragment of ROD 4 and the E2 cDNA used in FIG. 4.
  • the genetic sequences of the HIV-2 virus may be used to create the polypeptides encoded by these sequences.
  • these polypeptides may be created by expression of the cDNA obtained according to the teachings herein in hosts such as bacteria, yeast or animal cells.
  • These polypeptides may be used in diagnostic tests such as immunofluorescence assays (IFA), radioimmunoassays (RIA) and Western Blot tests.
  • additional diagnostic tests including additional immunodiagnostic tests, may be developed in which the DNA probes or the polypeptides of this invention may serve as one of the diagnostic reagents.
  • additional diagnostic tests including additional immunodiagnostic tests, may be developed in which the DNA probes or the polypeptides of this invention may serve as one of the diagnostic reagents.
  • the invention described herein includes these additional test methods.
  • monoclonal antibodies to these polypeptides or fragments thereof may be created.
  • the monoclonal antibodies may be used in immunodiagnostic tests in an analogous manner as the polypeptides described above.
  • polypeptides of the present invention may also be used as immunogenic reagents to induce protection against infection by HIV-2 viruses.
  • the polypeptides produced by recombinant-DNA techniques would function as vaccine agents.
  • polypeptides of this invention may be used in competitive assays to test the ability of various antiviral agents to determine their ability to prevent the virus from fixing on its target.
  • HIV-2 virions were purified from 5 liters of supernatant from a culture of the CEM cell line infected with the ROD isolate and a cDNA first strand using oligo (dT) primer was synthesized in detergent activated endogenous reaction on pelleted virus, as described by M. Alizon et al. in Nature, 312: 757-760 (1984), specifically incorporated herein by reference.
  • RNA-cDNA hybrids were purified by phenol-chloroform extraction and ethanol precipitation.
  • the second-strand cDNA was created by the DNA polymerase I/RNAase H method of Gubler and Hoffman in Gene, 25: 263-269 (1983), specifically incorporated herein by reference, using a commercial cDNA synthesis kit obtained from Amersham. After attachment of EcoRI linkers (obtained from Pharmacia), EcoRI digestion, and ligation into EcoRI-digested dephosphorylated M13 tg 130 vector (obtained from Amersham), a cDNA library was obtained by transformation of the E. coli TG1 strain. Recombinant plaques (104) were screened in situ on replica filters with the 1.5 kb.
  • HindIII fragment from clone J19 corresponding to the 31 part of the genome of the LAV BRU isolate of HIV-1, 32 P labelled to a specific activity of 10 9 cpm ug.
  • the filters were prehybridized in 5 ⁇ SSC, 5 ⁇ Denhardt solution, 25% formamide, and denatured salmon sperm DNA (100 ug/ml.) at 37° C. for 4 hours and hybridized for 16 hours in the same buffer (Tm ⁇ 42° C.) plus 4 ⁇ 10 7 cpm of the labelled probe (10 6 cpm/ml. of hybridization buffer). The washing was done in 5 ⁇ SSC, 0.1% SDS at 25° C. for 2 hours. 20 ⁇ SSC is 3M NaCl, 0.3M Na citrate.
  • Positive plaques were purified and single-stranded M13 DNA prepared and end-sequenced according to the method described in Proc. Nat'l. Acad. Sci. USA, 74: 5463-5467 (1977) of Sanger et al.
  • DNA was extracted from infected CEM cells continuously producing HIV-1 or 2.
  • the DNA digested with 20 ug of PstI digested with or undigested was electrophoresed on a 0.8% agarose gel, and Southern-transferred to nylon membrane.
  • Virion dot-blots were prepared in duplicate, as described by F. Clavel et al. in Science 233: 343-346 (1986), specifically incorporated herein by reference, by pelleting volumes of supernatant corresponding to the same amount of reverse transcriptase activity. Prehybridization was done in 50% formamide, 5 ⁇ SSC, 5 ⁇ Denhardt solution, and 100 mg./ml. denatured salmon sperm DNA for 4 hours at 42° C.
  • Hybridization was performed in the same buffer plus 10% Dextran sulphate, and 10 6 cpm/ml. of the labelled E2 insert (specific activity 10 9 cpm/ug.) for 16 hours at 42° C. Washing was in 0.1 ⁇ SSC, 0.1% SDS for 2 ⁇ 30 mn. After exposition for 16 hours with intensifying screens, the Southern blot was dehybridized in 0.4 N NaOH, neutralized, and rehybridized in the same conditions to the HIV-1 probe labelled to 10 9 cpm/ug.
  • DNA from the HIV-2 ROD infected CEM (FIG. 2, lanes a and c) was partially digested with Sau3AI. The 9-15 kb. fraction was selected on a 5-40% sucrose gradient and ligated to BamHI arms of the lambda L47.1 vector. Plaques (2 ⁇ 10 6 ) obtained after in vitro packaging and plating on E. coli LA 101 strain were screened in situ with the insert from the E2 cDNA clone. Approximately 10 positive clones were plaque purified and propagated on E. coli C600 recBC. The ROD 4, 27, and 35 clones were amplified and their DNA characterized by restriction mapping and Southern blotting with the HIV-2 cDNA clone under stringent conditions, and gag-pol probes from HIV-1 used under non stringent conditions.
  • GAG gene (546-2111) expresses a protein product having a molecular weight of around 55 Kd and is cleaved into the following proteins:
  • TAT gene (5845-6140 and 8307-8400) is expressed by two exons separated by introns.
  • ART protein (6071-6140 and 8307-8536) is similarly the expression product of two exons.
  • ProSerProThrProAspLys ArgLeuLeuHisGlnThr MetMetAsnGlnLeuLeuIleAlaIleLeuLeuAla CCGTCTCCTACACCAGACAAGTGAGTATGATGAATCAGCTGCTTATTGCCATTTTATTAG . . . . .
  • SerAlacysLeuValTyrCysThrGlnTyrValThrValPheTyrGlyValProThrTrp CTAGTGCTTGCTTAGTATATTGCACCCAATATGTAACTGTTTTCTATGGCGTACCCACGT . 6200 . . . .
  • ValLysGlnIleMetLeuMetSerGlyHisValPheHisSerHisTyrGlnProIleAsn CAGTGAAACAAATAATGCTTATGTCAGGACATGTGTTTCACTCCCACTACCAGCCGATCA . 7100. . . . . LysArgProArgGlnAlaTrpCysTrpPheLysGlyLysTrpLysAspAlaMetGlnGlu ATAAAAGACCCAGACAAGCATGGTGCTGGTTCAAAGGCAAATGGAAAGACGCCATGCAGG . . . . .
  • TrpIleGluAsnLysThrHisArgAsnTyrAlaProcysHisIle TGGATAGAGAATAAGACACACCGCAATTATGCACCGTGCCATATA . . . . LysGlnIleIleAsnThrTrpHisLysValGlyArgAsnValTyr AAGCAAATAATTAACACATGGCATAAGGTAGGGAGAAATGTATAT . . . 1300 LeuProProArgGluGlyGluLeuSerCysAsnSerThrValThr TTGCCTCCCAGGGAACGGGAGCTGTCCTGCAACTCAACAGTAACC . . . . .
  • GlyTyrArgProValPheSerSerProProGlyTyrIleGln *** GGCTATAGGCCTGTTTTCTCTTCCCCCCGGTTATATCCAATAG IleHisIleHisLysAspArgGlyGluProAlaAsnGluGluThr ATCCATATCCACAAGGACCGGGGACAGCCAGCCAACGAAGAAACA . . . 2200 GluGluAspGlyGlySerAsnGlyGlyAspArgTyrTrpProTrp GAAGAAGACGGTGGAAGCAACGGTGGAGACAGATACTGGCCCTGG . . . .
  • GlnAlaThrLysTyrGly CAGGCAACAAAATATGGA . . MetGlyAlaArgAsnSerValLeuArgGlyLysLysAlaAspGlu ATGGGCGCGAGAAACTCCGTCTTGAGAGGGAAAAAAGCAGATGAA . . . . LeuGluArgIleArgLeuArgProGlyGlyLysLysLysTyrArg TTAGAAAGAATCAGGTTACGGCCCGGCGGAAAGAAAAAGTACAGG . . . . .
  • envl (1732-1809) ArgValThrAlaIleGluLysTyr AGAGTCACTGCTATAGAGAAGTAC . .
  • AsnAsn AATAAT env6 (274-330) GluThrSerIleLysProCysValLysLeuThrProLeuCys GAGACATCAATAAACCATGTGTGAAACTAACACCTTTATGT . . 300 . ValAlaMetLysCys GTAGCAATGAAATGC . . env7 (607-660) AsnHisCysAsnThrSerValIle AACCATTGCAACACATCAGTCATC 610 . . ThrGluSerCysAspLysHisTyrTrpAsp ACAGAATCATGTGACAAGCACTATTGGGAT . . .
  • env8 (661-720) AlaIleArgPheArg GCTATAAGGTTTAGA .
  • TyrCysAlaProProGlyTyrAlaLeuLeuArgCysAsnAspThr TACTGTGCACCACCGGGTTATGCCCTATTAAGATGTAATGATACC . . 700 . . env9 (997-1044) LysArgProArgGlnAlaTrpCysTrpPheLysGlyLys AAAAGACCCAGACAAGCATGGTGCTGGTTCAAAGGCAAA 1000 . . .
  • TrpLysAsp TGGAAAGAC env10 (1132-1215) LysGlySerAspProGluValAlaTyrMetTrpThrAsa AAAGGCTCAGACCCAGAAGTAGCATACATGTGGACTAAC . . . . CysArgGlyGluPheLeuTyrCysAsnMetThrTrpPheLeuAsn TGCAGAGGACACTTTTFTFTACTGCAACATGACTTGGTTCCTCAAT . . 1200 . env11 (1237-1305) ArgAsnTyrAlaProCysHisIle CGCAATTATGCACCGTGCCATATA . . .
  • env1, env2, env3 and gag1 are particularly contemnplated for diagnostic purposes, and env4, env5, env6, env7, env8, env9, env10 and env11 are particularly, contemplated as protecting agents.
  • These peptides have been selected in part because of their sequence homology to certain of the envelope and gag protein products of other of the retroviruses in the HIV group.
  • the foregoing peptides may be coupled to a carrier protein by utilizing suitable and well known techniques to enhance the host's immune response.
  • Adjuvants such as calcium phosphate or alum hydroxide may also be added.
  • the foregoing peptides can be synthesized by conventional protein synthesis techniques, such as that of Merrifield.

Abstract

A method for diagnosing an HIV-2 (LAV-II) infection and a kit containing reagents for the same is disclosed. These re-agents include cDNA probes which are capable of hybridizing to at least a portion of the genome of HIV-2. In one embodiment, the DNA probes are capable of hybridizing to the entire genome of HIV-2. These reagents also include polypeptides encoded by some of these DNA sequences.

Description

  • This application is a continuation-in-part of U.S. patent application Ser. No. ______ of Alizon et al. for “Cloned DNA Sequences Related to the Entire Genomic RNA of Human Immunodeficiency Virus II (HIV-2), Polypeptides Encoded by these DNA Sequences and Use of these DNA Clones and Polypeptides in Diagnostic Kits,” filed Jan. 16, 1987, which is a continuation-in-part of U.S. patent application Ser. No. 931,866 filed Nov. 21, 1986, which is a continuation-in-part application of U.S. patent application Ser. No. 916,080 of Montagnier et al. for “Cloned DNA Sequences Related to the Genomic RNA of the Human Immunodeficiency Virus II (HIV-2), Polypeptides Encoded by these DNA Sequences and Use of these DNA Clones and Polypeptides in Diagnostic Kits,” filed Oct. 6, 1986 and U.S. patent application Ser. No. 835,228 of Montagnier et al. for “New Retrovirus Capable of Causing AIDS, Antigens Obtained from this Retrovirus and Corresponding Antibodies and their Application for Diagnostic Purposes,” filed Mar. 3, 1986. The disclosures of each of these predecessor applications are expressly incorporated herein by reference.[0001]
    • BACKGROUND OF THE INVENTION
  • The invention relates to cloned DNA sequences analogous to the genomic RNA of a virus known as Lymphadenopathy-Associated Virus II (“LAV-II”), a process for the preparation of these cloned DNA sequences, and their use as probes in diagnostic kits. In one embodiment, the invention relates to a cloned DNA sequence analogous to the entire genomic RNA of HIV-2 and its use as a probe. The invention also relates to polypeptides with amino acid sequences encoded by these cloned DNA sequences and the use of these polypeptides in diagnostic kits. [0002]
  • According to recently adopted nomenclature. as reported in Nature, May 1986, a substantially-identical group of retroviruses which has been identified as one causative agent of AIDS are now referred to as Human Immunodeficiency viruses I (HIV-1). This previously-described group of retroviruses includes Lymphadenopathy-Associated Virus I (LAV-I), Human T-cell Lymphotropic Virus-III (HTLV-III), and AIDS-Related Virus (ARV). [0003]
  • Lymphadenopathy-Associated Virus II has been described in U.S. application Ser. No. 835,228, which was filed Mar. 3, 1986, and is specifically incorporated herein by reference. Because LAV-II is a second, distinct causative agent of AIDS, LAV-II properly is classifiable as a Human Immunodeficiency Virus II (HIV-2). Therefore, “LAV-II” as used hereinafter describes a particular genus of HIV-2 isolates. [0004]
  • While HIV-2 is related to HIV-1 by its morphology, its tropism and its in vitro cytopathic effect on CD4 (T4) positive cell lines and lymphocytes, HIV-2 differs from previously described human retroviruses known to be responsible for AIDS. Moreover, the proteins of HIV-1 and 2 have different sizes and their serological cross-reactivity is restricted mostly to the major core protein, as the envelope glycoproteins of HIV-2 are not immune precipitated by HIV-1-positive sera except in some cases where very faint cross-reactivity can be detected. Since a significant proportion of the HIV infected patients lack antibodies to the major core protein of their infecting virus, it is important to include antigens to both HIV-1 and HIV-2 in an effective serum test for the diagnosis of the infection by these viruses. [0005]
  • HIV-2 was first discovered in the course of serological research on patients native to Guinea-Bissau who exhibited clinical and immunological symptoms of AIDS and from whom sero-negative or weakly sero-positive reactions to tests using an HIV-1 lysate were obtained. Further clinical studies on these patients isolated viruses which were subsequently named “LAV-II.”[0006]
  • One LAV-II isolate, subsequently referred to as LAV-II MIR, was deposited at the Collection Nationale des Cultures de Micro-Organismes (CNCM) at the Institut Pasteur in Paris, France on Dec. 19, 1985 under Accession No. I-502 and has also been deposited at the British ECA CC under No. 87.001.001 on Jan. 9, 1987. A second LAV-II isolate was deposited at CNCM on Feb. 21, 1986 under Accession No. 1-532 and has also been deposited at the British ECA CC under No. 87.001.002 on Jan. 9, 1987. This second isolate has been subsequently referred to as LAV-II ROD. Other isolates deposited at the CNCM on Dec. 19, 1986 are HIV-2 IRMO (No. I-642) and HIV-2 EHO (No. I-643). Several additional isolates have been obtained from West African patients, some of whom have AIDS, others with AIDS-related conditions and others with no AIDS symptoms. All of these viruses have been isolated on normal human lymphocyte cultures and some of them were thereafter propagated on lymphoid tumor cell lines such as CEM and MOLT. [0007]
  • Due to the sero-negative or weak sero-positive results obtained when using kits designed to identify HIV-1 infections in the diagnosis of these new patients with HIV-2 disease, it has been necessary to devise a new diagnostic kit capable of detecting HIV-2 infection, either by itself or in combination with an HIV-1 infection. The present inventors have, through the development of cloned DNA sequences analogous to at least a portion of the genomic RNA of LAV-II ROD viruses, created the materials necessary for the development of such kits. [0008]
    • SUMMARY OF THE INVENTION
  • As noted previously, the present invention relates to the cloned nucleotide sequences homologous or identical to at least a portion of the genomic RNA of HIV-2 viruses and to polypeptides encoded by the same. The present invention also relates to kits capable of diagnosing an HIV-2 infection. [0009]
  • Thus, a main object of the present invention is to provide a kit capable of diagnosing an infection caused by the HIV-2 virus. This kit may operate by detecting at least a portion of the RNA genome of the HIV-2 virus or the provirus present in the infected cells through hybridization with a DNA probe or it may operate through the immunodiagnostic detection of polypeptides unique to the HIV-2 virus. [0010]
  • Additional objects and advantages of the present invention will be set forth in part in the description which follows, or may be learned from practice of the invention. The objects and advantages may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims. [0011]
  • To achieve these objects and in accordance with the purposes of the present invention, cloned DNA sequences related to the entire genomic RNA of the LAV-II virus are set forth. These sequences are analogous specifically to the entire genome of the LAV-II ROD strain. [0012]
  • To further achieve the objects and in accordance with the purposes of the present invention, a kit capable of diagnosing an HIV-2 infection is described. This kit, in one embodiment, contains the cloned DNA sequences of this invention which are capable of hybridizing to viral RNA or analogous DNA sequences to indicate the presence of an HIV-2 infection. Different diagnostic techniques can be used which include, but are not limited to: (1) Southern blot procedures to identify viral DNA which may or may not be digested with restriction enzymes; (2) Northern blot techniques to identify viral RNA extracted from cells; and (3) dot blot techniques, i.e., direct filtration of the sample through an ad hoc membrane such as nitrocellulose or nylon without previous separation on agarose gel. Suitable material for dot blot technique could be obtained from body fluids including, but not limited to, serum and plasma, supernatants from culture cells, or cytoplasmic extracts obtained after cell lysis and removal of membranes and nuclei of the cells by ultra-centrifugation as accomplished in the “CYTODOT” procedure as described in a booklet published by Schleicher and Schull. [0013]
  • In an alternate embodiment, the kit contains the polypeptides created using these cloned DNA sequences. These polypeptides are capable of reacting with antibodies to the HIV-2 virus present in sera of infected individuals, thus yielding an immunodiagnostic complex. [0014]
  • To further achieve the objects of the invention, a vaccinating agent is provided which comprises at least one peptide selected from the polypeptide expression products of the viral DNA in admixture with suitable carriers, adjuvents stabilizers. [0015]
  • It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate one embodiment of the invention and, together with the description, serve to explain the principles of the invention.[0016]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 generally depicts the nucleotide sequence of a cloned complementary DNA (cDNA) to the genomic RNA of HIV-2. [0017]
  • FIG. 1A depicts the genetic organization of HIV-1, position of the HIV-1 HindIII fragment used as a probe to screen the cDNA library, and restriction map of the HIV-2 cDNA clone, E2. [0018]
  • FIG. 1B depicts the nucleotide sequence of the 3′ end of HIV-2. The corresponding region of the HIV-1 LTR was aligned using the Wilbur and Lipman algorithm (window: 10; K-tuple: 7; gap penalty: 3) as described by Wilbur and Lipman in Proc. Natl. Acad. Sci. USA 80: 726-730 (1983), specifically incorporated herein by reference. The U3-R junction in HIV-1 is indicated and the poly A addition signal and potential TATA promoter regions are boxed. In FIG. 1B, the symbols B, H, Ps and Pv refer to the restriction sites BamHI, HindIII, PstI and PvuII, respectively. [0019]
  • FIG. 2 generally depicts the HIV-2 specificity of the E2 clone. [0020]
  • FIGS. 2A and B specifically depict a Southern blot of DNA extracted from CEM cells infected with the following isolates: HIV-2[0021] ROD (a, c), HIV-2DUL (b, d), and HIV-1BRU (e, f). DNA in lanes a, b, f was Pst I digested; in c, d, e DNA was undigested.
  • FIGS. 2C and D specifically depict dot blot hybridization of pelleted virions from CEM cells infected by the HIV-1[0022] BRU(1), Simian Immunodeficiency Virus (SIV) isolate Mm 142-83 (3), HIV-2DUL (4), HIV-2ROD (5), and HIV-1ELI (6). Dot 2 is a pellet from an equivalent volume of supernatant from uninfected CEM. Thus, FIG. 2A and C depicts hybridization with the HIV-2 cDNA (E2) and FIG. 2B and D depicts hybridization to an HIV-1 probe consisting of a 9Kb SacI insert from HIV-1 BRU (clone lambda J 19).
  • FIG. 3 generally depicts a restriction map of the HIV-2 ROD genome and its homology to HIV-1. [0023]
  • FIG. 3A specifically depicts the organization of three recombinant phage lambda clones, [0024] ROD 4, ROD 27, and ROD 35. In FIG. 3A, the open boxes represent viral sequences, the LTR are filled, and the dotted boxes represent cellular flanking sequences (not mapped). Only some characteristic restriction enzyme sites are indicated. λROD 27 and λROD 35 are derived from integrated proviruses while λROD 4 is derived from a circular viral DNA. The portion of the lambda clones that hybridzes to the cDNA E2 is indicated below the maps. A restriction map of the λROD isolate was reconstructed from these three lambda clones. In this map, the restriction sites are identified as follows: B: BamHI; E: EcoRI; H: HindIII; K: KnI; Ps: PstI; Pv: PvuII; S: SacI; X: XbaI. R and L are the right and left BamHI arms of the lambda L47.1 vector.
  • FIG. 3B specifically depicts dots 1-11 which correspond to the single-stranded DNA form of M13 subclones from the HIV-1[0025] BRU cloned genome (λJ9). Their size and position on the HIV-1 genome, determined by sequencing is shown below the figure. Dot 12 is a control containing lambda phage DNA. The dot-blot was hybridized in low stringency conditions as described in Example 1 with the complete lambda λROD 4 clone as a probe, and successively washed in 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.) and exposed overnight. A duplicate dot blot was hybridized and washed in stringent conditions (as described in Example 2) with the labelled lambda J19 clone carrying the complete HIV-1BRU genome. HIV-1 and HIV-2 probes were labelled the same specific activity (108 cpm/g.).
  • FIG. 4 generally depicts the restriction map polymorphism in different HIV-2 isolates and shows comparison of HIV-2 to SIV. [0026]
  • FIG. 4A specifically depicts DNA (20 ug. per lane) from CEM cells infected by the isolate HIV-2[0027] DUL (panel 1) or peripheral blood lymphocytes (PBL) infected by the isolates HIV-2GOM (panel 2) and HIV-2MIR (panel 3) digested with: EcoRI (a), PstI (b), and HindIII (c). Much less viral DNA was obtained with HIV-2 isolates propagated on PBL. Hybridization and washing were in stringent conditions, as described in Example 2, with 106 cpm/ml. of each of the E2 insert (cDNA) and the 5 kb. HindIII fragment of λROD 4, labelled to 109 cpm/ug.
  • FIG. 4B specifically depicts DNA from HUT 78 (a human T lymphoid cell line) cells infected with STLV3 MAC isolate Mm 142-83. The same amounts of DNA and enzymes were used as indicated in panel A. Hybridization was performed with the same probe as in A, but in non-stringent conditions. As described in Example 1 washing was for one hour in 2× SSC, 0.1% SDS at 40° C. (panel 1) and after exposure, the same filter was re-washed in 0.1× SSC, 0.1% SDS at 60° C. (panel 2). The autoradiographs were obtained after overnight exposition with intensifying screens. [0028]
  • FIG. 5 depicts the position of derived plasmids from [0029] λROD 27, λROD 35 and λROD 4.
    • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • Reference will now be made in detail to the presently preferred embodiments of the invention, which, together with the following examples, serve to explain the principles of the invention. [0030]
  • The genetic structure of the HIV-2 virus has been analyzed by molecular cloning according to the method set forth herein and in the Examples. A restriction map of the genome of this virus is included in FIG. 4. In addition, the partial sequence of a cDNA complementary to the genomic RNA of the virus has been determined. This cDNA sequence information is included in FIG. 1. [0031]
  • Also contained herein is data describing the molecular cloning of the complete 9.5 kb genome of HIV-2, data describing the observation of restriction map polymorphism between different isolates, and an analysis of the relationship between HIV-2 and other human and simian retroviruses. From the totality of these data, diagnostic probes can be discerned and prepared. [0032]
  • Generally, to practice one embodiment of the present invention, a series of filter hybridizations of the HIV-2 RNA genome with probes derived from the complete cloned HIV-1 genome and from the gag and pol genes were conducted. These hybridizations yielded only extremely weak signals even in conditions of very low stringency of hybrization and washing. Thus, it was found to be difficult to assess the amount of HIV-2 viral and proviral DNA in infected cells by Southern blot techniques. [0033]
  • Therefore, a complementary DNA (cDNA) to the HIV-2 genomic RNA initially was cloned in order to provide a specific hybridization probe. To construct this cDNA, an oligo (dT) primed cDNA first-strand was made in a detergent-activated endogenous reaction using HIV-2 reverse transcriptase with virions purified from supernatants of infected CEM cells. The CEM cell line is a lymphoblastoid CD4+ cell line described by G. E. Foley et al. in Cancer 18: 522-529 (1965), specifically incorporated herein by reference. The CEM cells used were infected with the isolate ROD and were continuously producing high amounts of HIV-2. [0034]
  • After second-strand synthesis, the cDNAs were inserted into the M 13 tg 130 bacteriophage vector. A collection of 10[0035] 4 M13 recombinant phages was obtained and screened in situ with an HIV-1 probe spanning 1.5 kb. of the 3′ end of the LAVBRU isolate (depicted in FIG. 1A). Some 50 positive plaques were detected, purified, and characterized by end sequencing and cross-hybridizing the inserts. This procedure is described in more detail in Example 1 and in FIG. 1.
  • The different clones were found to be complementary to the a 3′ end of a polyadenylated RNA having the AATAAA signal about 20 nucleotides upstream of the poly A tail, as found in the long terminal repeat (LTR) of HIV-1. The LTR region of HIV-1 has been described by S. Wain Hobson et al. in Cell 40: 9-17 (1985), specifically incorporated herein by reference. The portion of the HIV-2 LTR that was sequenced was related only distantly to the homologous domain in HIV-1 as demonstrated in FIG. 1 B. Indeed, only about 50% of the nucleotides could be aligned and about a hundred insertions/deletions need to be introduced. In comparison, the homology of the corresponding domains in HIV-1 isolates from USA and Africa is greater than 95% and no insertions or deletions are seen. [0036]
  • The largest insert of this group of M13 clones was a 2 kb. clone designated E2. Clone E2 was used as a probe to demonstrate its HIV-2 specificity in a series of filter hybridization experiments. Firstly, this probe could detect the genomic RNA of HIV-2 but not HIV-1 in stringent conditions as shown in FIG. 2, C and D. Secondly, positive signals were detected in Southern blots of DNA from cells infected with the ROD isolate as well as other isolates of HIV-2 as shown in FIG. 2, A and FIG. 4, A. No signal was detected with DNA from uninfected cells or HIV-1 infected cells, confirming the exogenous nature of HIV-2. In undigested DNA from HIV-2 infected cells, an approximately 10 kb. species, probably corresponding to linear unintegrated viral DNA, was principally detected along with a species with an apparent size of 6 kb., likely to be the circular form of the viral DNA. Conversely, rehybridization of the same filter with an HIV-1 probe under stringent conditions showed hybridization to HIV-1 infected cells only as depicted in FIG. 2, B. [0037]
  • To isolate the remainder of the genome of HIV-2, a genomic library in lambda phage L47.1 was constructed. Lambda phage L47.1 has been described by W. A. M. Loenen et al. in Gene 10: 249-259 (1980), specifically incorporated herein by reference. The genomic library was constructed with a partial Sau3AI restriction digest of the DNA from the CEM cell line infected with HIV-2[0038] ROD.
  • About 2×10[0039] 6 recombinant plaques were screened in situ with labelled insert from the E2 cDNA clone. Ten recombinant phages were detected and plaque purified. Of these phages, three were characterized by restriction mapping and Southern blot hybridization with the E2 insert and probes from its 3′ end (LTR) or 5′ end (envelope), as well as with HIV-1 subgenomic probes. In this instance, HIV-1 probes were used under non-stringent conditions.
  • A clone carrying a 9.5 kb. insert and derived from a circular viral DNA was identified as containing the complete genome and designated [0040] λROD 4. Two other clones, λROD 27 and λROD 35 were derived from integrated proviruses and found to carry an LTR and cellular flanking sequences and a portion of the viral coding sequences as shown in FIG. 3, A.
  • Fragments of the lambda clones were subcloned into a plasmid vector p UC 18. [0041]
  • Plasmid pROD 27-5′ is derived from [0042] λROD 27 and contains the 5′ 2Kb of the HIV-2 genome and cellular flanking sequences (5′ LTR and 5′ viral coding sequences to the EcoRI site).
  • Plasmid p ROD 4-8 is dervied from [0043] λROD 4 and contains the about 5 Kb HindIII fragment that is the central part of the HIV-2 genome.
  • Plasmid pROD 27-5′ and p ROD 4.8 inserts overlap. [0044]
  • Plasmid pROD 4.7 contains a HindIII 1.8 Kb fragment from [0045] λROD 4. This fragment is located 3′ to the fragment subcloned into pROD 4.8 and contains about 0.8 Kb of viral coding sequences and the part of the lambda phage (λL47.1) left arm located between the BamHI and HindIII cloning sites.
  • [0046] Plasmid pROD 35 contains all the HIV-2 coding sequences 3′ to the EcoRI site, the 3′ L:R and about 4 Kb of cellular flanking sequences.
  • Plasmid pROD 27-5′ and [0047] pROD 35 in E. coli strain HB 101 are deposited respectively under No. 1-626 and 1-633 at the CNCM, and have also been deposited at the NCIB (British Collection). These plasmids are depicted in FIG. 5. Plasmids pROD 4-7 and pROD 4-8 in E. coli strain TG1 are deposited respectively under No. 1-627 and 1-628 at the CNCM.
  • To reconstitute the complete HIV-2 ROD genome, [0048] pROD 35 is linearized with EcoRI and the EcoRI insert of pROD 27-5′ is ligated in the correct orientation into this site.
  • The relationship of HIV-2 to other human and simian retroviruses was surmised from hybridization experiments. The relative homology of the different regions of the HIV-1 and 2 genomes was determined by hybridization of fragments of the cloned HIV-1 genome with the labelled [0049] λROD 4 expected to contain the complete HIV-2 genome (FIG. 3, B). Even in very low stringency conditions (Tm −42° C.), the hybridization of HIV-1 and 2 was restricted to a fraction of their genomes, principally the gag gene (dots 1 and 2), the reverse transcriptase domain in pol (dot 3), the end of pol and the Q (or sor) genes (dot 5) and the F gene (or 3′ orf) and 3′ LTR (dot 11). The HIV-1 fragment used to detect the HIV-2 cDNA clones contained the dot 11 subclone, which hybridized well to HIV-2 under non-stringent conditions. Only the signal from dot 5 persisted after stringent washing. The envelope gene, the region of the tat gene and a part of pol thus seemed very divergent. These data, along with the LTR sequence obtained (FIG. 1, B), indicated that HIV-2 is not an envelope variant of HIV-1, as are African isolates from Zaire described by Alizon et al., Cell 40:63-74 (1986).
  • It was observed that HIV-2 is related more closely to the Simian Immunodeficiency Virus (SIV) than it is to HIV-1. This correlation has been described by F. Clavel et al. in C.R. Acad. Sci. (Paris) 302: 485-488 (1986) and F. Clavel et al. in Science 233: 343-346 (1986), both of which are specifically incorporated herein by reference. Simian Immunodeficiency Virus (also designated Simian T-cell [0050] Lymphotropic Virus Type 3, STLV-3) is a retrovirus first isolated from captive macaques with an AIDS-like disease in the USA. This simian virus has been described by M. D. Daniel et al. in Science 228: 1201-1204 (1985), specifically incorporated herein by reference.
  • All the SIV proteins, including the envelope, are immune precipitated by sera from HIV-2 infected patients, whereas the serological cross-reactivity of HIV-1 to 2 is restricted to the core proteins. However SIV and HIV-2 can be distinguished by slight differences in the apparent molecular weight of their proteins. [0051]
  • In terms of nucleotide sequence, it also appears that HIV-2 is closely related to SIV. The genomic RNA of SIV can be detected in stringent conditions as shown in FIG. 2, C by HIV-2 probes corresponding to the LTR and 3′ end of the genome (E2) or to the gag or pol genes. Under the same conditions, HIV-1 derived probes do not detect the SIV genome as shown in FIG. 2, D. [0052]
  • In Southern blots of DNA from SIV-infected cells, a restriction pattern clearly different from HIV-2[0053] ROD and other isolates is seen. All the bands persist after a stringent washing, even though the signal is considerably weakened, indicating a sequence homology throughout the genomes of HIV-2 and SIV. It has recently been shown that baboons and macaques could be infected experimentally by HIV-2, thereby providing an interesting animal model for the study of the HIV infection and its preventive therapy. Indeed, attempts to infect non-human primates with HIV-1 have been successful only in chimpanzees, which are not a convenient model.
  • From an initial survey of the restriction maps for certain of the HIV-2 isolates obtained according to the methods described herein, it is already apparent that HIV-2, like HIV-1, undergoes restriction site polymorphism. FIG. 4 A depicts examples of such differences for three isolates, all different one from another and from the cloned HIV-2[0054] ROD. It is very likely that these differences at the nucleotide level are accompanied by variations in the amino-acid sequence of the viral proteins, as evidenced in the case of HIV-1 and described by M. Alizon et al. in Cell 46: 63-74 (1986), specifically incorporated herein by reference. It is also to be expected that the various isolates of HIV-2 will exhibit amino acid heterogeneities. See, for example, Clavel et al., Nature 324 (18):691-695 (1986), specifically incorporated herein by reference.
  • Further, the chacterization of HIV-2 will also delineate the domain of the envelope glycoprotein that is responsible for the binding of the surface of the target cells and the subsequent internalization of the virus. This interaction was shown to be mediated by the CD4 molecule itself in the case of HIV-1 and similar studies tend to indicate that HIV-2 uses the same receptor. Thus, although there is wide divergence between the env genes of HIV-1 and 2, small homologous domains of the envelopes of the two HIV could represent a candidate receptor binding site. This site could be used to raise a protective immune response against this group of retroviruses. [0055]
  • From the data discussed herein, certain nucleotide sequences have been identified which are capable of being used as probes in diagnostic methods to obtain the immunological reagents necessary to diagnose an HIV-2 infection. In particular, these sequences may be used as probes in hybridization reactions with the genetic material of infected patients to indicate whether the RNA of the HIV-2 virus is present in these patient's lymphocytes or whether an analogous DNA is present. In this embodiment, the test methods which may be utilized include Northern blots, Southern blots and dot blots. One particular nucleotide sequence which may be useful as a probe is the combination of the 5 kb. HindIII fragment of [0056] ROD 4 and the E2 cDNA used in FIG. 4.
  • In addition, the genetic sequences of the HIV-2 virus may be used to create the polypeptides encoded by these sequences. Specifically, these polypeptides may be created by expression of the cDNA obtained according to the teachings herein in hosts such as bacteria, yeast or animal cells. These polypeptides may be used in diagnostic tests such as immunofluorescence assays (IFA), radioimmunoassays (RIA) and Western Blot tests. [0057]
  • Moreover, it is also contemplated that additional diagnostic tests, including additional immunodiagnostic tests, may be developed in which the DNA probes or the polypeptides of this invention may serve as one of the diagnostic reagents. The invention described herein includes these additional test methods. [0058]
  • In addition, monoclonal antibodies to these polypeptides or fragments thereof may be created. The monoclonal antibodies may be used in immunodiagnostic tests in an analogous manner as the polypeptides described above. [0059]
  • The polypeptides of the present invention may also be used as immunogenic reagents to induce protection against infection by HIV-2 viruses. In this embodiment, the polypeptides produced by recombinant-DNA techniques would function as vaccine agents. [0060]
  • Also, the polypeptides of this invention may be used in competitive assays to test the ability of various antiviral agents to determine their ability to prevent the virus from fixing on its target. [0061]
  • Thus, it is to be understood that application of the teachings of the present invention to a specific problem or environment will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein. Examples of the products of the present invention and representative processes for their isolation and manufacture appear above and in the following examples. [0062]
    • EXAMPLES Example 1 Cloning of a cDNA Complementary to Genomic RNA From HIV-2 Virions
  • HIV-2 virions were purified from 5 liters of supernatant from a culture of the CEM cell line infected with the ROD isolate and a cDNA first strand using oligo (dT) primer was synthesized in detergent activated endogenous reaction on pelleted virus, as described by M. Alizon et al. in Nature, 312: 757-760 (1984), specifically incorporated herein by reference. RNA-cDNA hybrids were purified by phenol-chloroform extraction and ethanol precipitation. The second-strand cDNA was created by the DNA polymerase I/RNAase H method of Gubler and Hoffman in Gene, 25: 263-269 (1983), specifically incorporated herein by reference, using a commercial cDNA synthesis kit obtained from Amersham. After attachment of EcoRI linkers (obtained from Pharmacia), EcoRI digestion, and ligation into EcoRI-digested dephosphorylated M13 tg 130 vector (obtained from Amersham), a cDNA library was obtained by transformation of the [0063] E. coli TG1 strain. Recombinant plaques (104) were screened in situ on replica filters with the 1.5 kb. HindIII fragment from clone J19, corresponding to the 31 part of the genome of the LAVBRU isolate of HIV-1, 32P labelled to a specific activity of 109cpm ug. The filters were prehybridized in 5× SSC, 5× Denhardt solution, 25% formamide, and denatured salmon sperm DNA (100 ug/ml.) at 37° C. for 4 hours and hybridized for 16 hours in the same buffer (Tm −42° C.) plus 4×107 cpm of the labelled probe (106 cpm/ml. of hybridization buffer). The washing was done in 5× SSC, 0.1% SDS at 25° C. for 2 hours. 20× SSC is 3M NaCl, 0.3M Na citrate. Positive plaques were purified and single-stranded M13 DNA prepared and end-sequenced according to the method described in Proc. Nat'l. Acad. Sci. USA, 74: 5463-5467 (1977) of Sanger et al.
    • Example 2 Hybridization of DNA from HIV-1 and HIV-2 Infected Cells and RNA from HIV-1 and 2 and SIV Virons with a Probe Derived From an HIV-2 Cloned cDNA
  • DNA was extracted from infected CEM cells continuously producing HIV-1 or 2. The DNA digested with 20 ug of PstI digested with or undigested, was electrophoresed on a 0.8% agarose gel, and Southern-transferred to nylon membrane. Virion dot-blots were prepared in duplicate, as described by F. Clavel et al. in Science 233: 343-346 (1986), specifically incorporated herein by reference, by pelleting volumes of supernatant corresponding to the same amount of reverse transcriptase activity. Prehybridization was done in 50% formamide, 5× SSC, 5× Denhardt solution, and 100 mg./ml. denatured salmon sperm DNA for 4 hours at 42° C. Hybridization was performed in the same buffer plus 10% Dextran sulphate, and 10[0064] 6 cpm/ml. of the labelled E2 insert (specific activity 109 cpm/ug.) for 16 hours at 42° C. Washing was in 0.1× SSC, 0.1% SDS for 2×30 mn. After exposition for 16 hours with intensifying screens, the Southern blot was dehybridized in 0.4 N NaOH, neutralized, and rehybridized in the same conditions to the HIV-1 probe labelled to 109 cpm/ug.
    • Example 3 Cloning in Lambda Phage of the Complete Provirus DNA of HIV-2
  • DNA from the HIV-2[0065] ROD infected CEM (FIG. 2, lanes a and c) was partially digested with Sau3AI. The 9-15 kb. fraction was selected on a 5-40% sucrose gradient and ligated to BamHI arms of the lambda L47.1 vector. Plaques (2×106) obtained after in vitro packaging and plating on E. coli LA 101 strain were screened in situ with the insert from the E2 cDNA clone. Approximately 10 positive clones were plaque purified and propagated on E. coli C600 recBC. The ROD 4, 27, and 35 clones were amplified and their DNA characterized by restriction mapping and Southern blotting with the HIV-2 cDNA clone under stringent conditions, and gag-pol probes from HIV-1 used under non stringent conditions.
    • Example 4 Complete Genomic Sequence of the ROD HIV-2 Isolate
  • Experimental analysis of the HIV-2 ROD isolate yielded the following sequence which represents the complete genome of this HIV-2 isolate. Genes and major expression products identified within the following sequence are indicated by nucleotides numbered below: [0066]
  • 1) GAG gene (546-2111) expresses a protein product having a molecular weight of around 55 Kd and is cleaved into the following proteins: [0067]
  • a) p 16 (546-950) [0068]
  • b) p 26 (951-1640) [0069]
  • c) p 12 (1701-2111) [0070]
  • 2) polymerase (1829-4936) [0071]
  • 3) Q protein (4869-5513) [0072]
  • 4) R protein (5682-5996) [0073]
  • 5) X protein (5344-5679) [0074]
  • 6) Y protein (5682-5996) [0075]
  • 7) Env protein (6147-8720) [0076]
  • 8) F protein (8557-9324) [0077]
  • 9) TAT gene (5845-6140 and 8307-8400) is expressed by two exons separated by introns. [0078]
  • 10) ART protein (6071-6140 and 8307-8536) is similarly the expression product of two exons. [0079]
  • 11) LTR:R (1-173 and 9498-9671) [0080]
  • 12) U5 (174-299) [0081]
  • 13) U3 (8942-9497) [0082]
  • It will be known to one of skill in the art that the absolute numbering which has been adopted is not essential. For example, the nucleotide within the LTR which is designated as “1” is a somewhat arbitrary choice. What is important is the sequence information provided. [0083]
    GGTCGCTCTGCGGAGAGGCTGGCAGATTGAGCCCTGGGAGGTTCTCTCCAGCACTAGCAG
             .         .         .         .         .         .
    GTAGAGCCTGGGTGTTCCCTGCTAGACTCTCACCAGCACTTGGCCGGTGCTGGGCAGACG
             .         .         .       100         .         .
    GCCCCACGCTTGCTTGCTTAAAAACCTCTTAATAAAGCTGCCAGTTAGAAGCAAGTTAAG
             .         .         .         .         .         .
    TGTGTGCTCCCATCTCTCCTAGTCGCCGCCTGGTCATTCGGTGTTCACCTGAGTAACAAG
             .       200         .         .         .         .
    ACCCTGGTCTGTTAGGACCCTTCTTGCTTTGGGAAACCGAGGCAGGAAAATCCCTAGCAG
             .         .         .         .         .      300
    GTTGGCGCCTGAACAGGGACTTGAAGAAGACTGAGAAGTCTTGGAACACGGCTGAGTGAA
             .         .         .         .         .         .
    GGCAGTAAGGGCGGCAGGAACAAACCACGACGGAGTGCTCCTAGAAAGGCGCGGGCCGAG
             .         .         .       400         .         .
    GTACCAAAGGCAGCGTGTGGAGCGGGAGGAGAAGAGGCCTCCGGGTGAAGGTAAGTACCT
             .         .         .         .         .         .
    ACACCAAAAACTGTAGCCGAAAGGGCTTGCTATCCTACCTTTAGACAGGTAGAAGATTGT
             .       500         .         .         .         .
         MetGlyAlaArgAsnSerValLeuArgGlyLysLysAlaAspGluLeuGluArgIle
    GGGAGATGGGCGCGAGAAACTCCGTCTTGAGAGGGAAAAAAGCAGATGAATTAGAAAGAA
             .         .         .         .         .       600
     ArgLeuArgProGlyGlyLysLysLysTyrArgLeuLysHisIleValTrpAlaAlaAsn
    TCAGGTTACGGCCCGGCGGAAAGAAAAAGTACAGGCTAAAACATATTGTGTGGGCAGCGA
             .         .         .         .         .         .
      LysLeuAspArgPheGlyLeuAlaGluSerLeuLeuGluSerLysGluGlyCysGlnLys
    ATAAATTGGACAGATTCGGATTAGCAGAGAGCCTGTTGGAGTCAAAAGAGGGTTGTCAAA
             .         .         .       700         .         .
      IleLeuThrValLeuAspProMetValProThrGlySerGluAsnLeuLysSerLeuPhe
    AAATTCTTACAGTTTTAGATCCAATGGTACCGACAGGTTCAGAAAATTTAAAAAGTCTTT
             .         .         .         .         .         .
      AsnThrValCysValIleTrpCysIleHisAlaGluGluLysValLysAspThrGluGly
    TTAATACTGTCTGCGTCATTTGGTGCATACACGCAGAAGAGAAAGTGAAAGATACTGAAG
             .       800         .         .         .         .
      AlaLysGlnIleValArgArgHisLeuValAlaGluThrGlyThrAlaGluLysMetPro
    GAGCAAAACAAATAGTGCGGAGACATCTAGTGCCAGAAACAGGAACTGCAGAGAAAATGG
             .         .         .         .         .       900
      SerThrSerArgProThrAlaProSerSerGluLysGlyGlyAsnTyrProValGlnHis
    CAAGCACAAGTAGACCAACAGCACCATCTAGCGAGAAGGGAGGAAATTACCCAGTGCAAC
             .         .         .         .         .         .
      ValGlyGlyAsnTyrThrHisIleProLeuSerProArgThrLeuAsnAlaTrpValLys
    ATGTAGGCGGCAACTACACCCATATACCGCTGAGTCCCCGAACCCTAAATGCCTGGGTAA
             .         .         .       1000         .         .
      LeuValGluGluLysLysPheGlyAlaGluValValProGlyPheGlnAlaLeuSerGlu
    AATTAGTAGAGGAAAAAAAGTTCGGGGCAGAAGTAGTGCCAGGATTTCAGGCACTCTCAG
             .         .         .         .         .         .
    GlyCysThrProTyrAspIleAsnGlnMetLeuAsnCysValGlyAspHisGlnAlnAla
    AAGGCTGCACGCCCTATGATATCAACCAAATGCTTAATTGTGTGGGCGACCATCAAGCAG
             .       1100         .         .         .         .
      MetGlnIleIleArgGluIleIleAsnGluGluAlaAlaGluTrpAspValGlnLisPro
    CCATGCAGATAATCAGGGAGATTATCAATGAGGAAGCAGCAGAATGGGATGTCCAACATC
             .         .         .         .         .       1200
      IleProGlyProLeuProAlaGlyGlnLeuArgGluProArgGlySerAspIleAlaGly
    CAATACCAGGCCCCTTACCAGCGGGGCAGCTTAGAGAGCCAAGGGGATCTGACATAGCAG
             .         .         .         .         .         .
      ThrThrSerThrValGluGluGlnIleGlnTrpMetPheArgProGlnAsnProValPro
    GGACAACAAGCACAGTAGAAGAACAGATCCAGTGGATGTTTAGGCCACAAAATCCTGTAC
             .         .         .       1300         .         .
    ValGlyAsnIleTyrArgArgTrpIleGlnIleGlyLeuGlnLysCysValArgMetTyr
    CAGTAGGAAACATCTATAGAAGATGGATCCAGATAGGATTGCAGAAGTGTGTCAGGATGT
             .         .         .         .         .         .
      AsnProThrAsnIleLeuAspIleLysGlnGlyProLysGluProPheGlnSerTyrVal
    ACAACCCGACCAACATCCTAGACATAAAACAGGGACCAAAGGAGCCGTTCCAAAGCTATG
             .       1400         .         .         .         .
    AspArgPheTyrLysSerLeuArgAlaGluGlnThrAspProAlaValLysAsnTrpMet
    TAGATAGATTCTACAAAAGCTTGAGGGCAGAACAAACAGATCCAGCAGTGAAGAATTGGA
             .         .         .         .         .       1500
      ThrGlnThrLeuLeuValGlnAsnAlaAsnProAspCysLysLeuValLeuLysGlyLeu
    TGACCCAAACACTGCTAGTACAAAATGCCAACCCAGACTGTAAATTAGTGCTAAAAGGAC
             .         .         .         .         .         .
      GlyMetAsnProThrLeuGluGluMetLeuThrAlaCysGlnGlyValGlyGlyProGly
    TAGGGATGAACCCTACCTTAGAAGACATGCTGACCGCCTGTCAGGGGGTAGGTGGGCCAG
             .         .         .       1600         .         .
      GlnLysAlaArgLeuMetALaGluAlaLeuLysGluValIleGlyProAlaProIlePro
    GCCAGAAAGCTAGATTAATGGCAGAGGCCCTGAAAGAGGTCATAGGACCTGCCCCTATCC
             .         .         .         .         .         .
      PheAlaAlaAlaGlnGlnArgLysAlaPheLysCysTrpAsnCysGlyLysGluGlyHis
    CATTCGCAGCAGCCCAGCAGAGAAAGGCATTTAAATGCTGGAACTGTGGAAAGGAAGGGC
             .       1700         .         .         .         .
      SerAlaArgGlnCysArgAlaProArgArgGlnGlyCysTrpLysCysGlyLysProGly
    ACTCGGCAAGACAATGCCGAGCACCTAGAAGGCAGGGCTGCTGGAAGTGTGGTAAGCCAG
             .         .         .         .         .       1800
                                ThrGlyArgPhePheArgThrGlyProLeuGly
      HisIleMetThrAsnCysProAspArgGlnAlaGlyPheLeuGlyLeuGlyProTrpGly
    GACACATCATGACAAACTGCCCCAGATAGACAGGCAGGTTTTTTAGGACTGGGCCCTTGGG
             .         .         .         .         .         .
     LysGluAlaProGlnLeuProArgGlyProSerSerAlaGlyAlaAspThrAsnSerThr
      LysLysProArgAsnPheProValAlaGlnValProGlnGlyLeuThrProThrAlaPro
    GAAAGAAGCCCCGCAACTTCCCCGTGGCCCAAGTTCCGCAGGCGCTGACACCAACAGCAC
             .         .         .       1900         .         .
     ProSerGlySerSerSerGlySerThrGlyGluIleTyrAlaAlaArgGluLysThrGlu
      ProValAspProAlaValAspLeuLeuGluLysTyrMetGlnGlnGlyLysArgGlnArg
    CCCCAGTGGATCCAGCAGTGGATCTACTGGAGAAATATATCCAGCAAGGGAAAAGACAGA
             .         .         .         .         .         .
     ArgAlaGluArgGluThrIleGlnGlySerAspArgGlyLeuThrAlaProArgAlaGly
      GluGlnArgGluArgProTyrLysGluValThrGluAspLeuLeuHisLeuGluGlnGly
    GAGAGCAGAGAGAGAGACCATACAAGGAAGTGACAGAGGACTTACTGCACCTCGAGCAGG
             .       2000         .         .         .         .
     GlyAspThrIleGlnGlyAlaThrAsnArgGlyLeuAlaAlaProGlnPheSerLeuTrp
      GluThrProTyrArgGluProProThrGluAspLeuLeuHisLeuAsnSerLeuPheGly
    GGGAGACACCATACAGGGAGCCACCAACAGAGGACTTGCTGCACCTCAATTCTCTCTTTG
             .         .         .         .         .       2100
     LysArgProValValThrAlaTyrIleGluGlyGlnProValGluValLeuLeuAspThr
      LysAspGln
    GAAAAGACCAGTAGTCACAGCATACATTGAGGGTCAGCCAGTAGAAGTCTTGTTAGACAC
             .         .         .         .         .         .
     GlyAlaAspAspSerIleValAlaGlyIleGluLeuGlyAsnAsnTyrSerProLysIle
    AGGGGCTGACGACTCAATAGTAGCAGGAATAGAGTTAGGGAACAATTATAGCCCAAAAAT
             .         .         .       2200         .         .
     ValGlyGlyIleGlyGlyPheIleAsnThrLysGluTyrLysAsnValGluIleGluVal
    AGTAGGGGGAATAGGGGGATTCATAAATACCAAGGAATATAAAAATGTAGAAATAGAAGT
             .         .         .         .         .         .
     LeuAsnLysLysValArgAlaThrIleMetThrGlyAspThrProIleAsnIlePheGly
    TCTAAATAAAAAGGTACGGGCCACCATAATGACAGGCGACACCCCAATCAACATTTTTGG
             .       2300         .         .         .         .
     ArgAsnIleLeuThrAlaLeuGlyMetSerLeuAsnLeuProValAlaLysValGluPro
    CAGAAATATTCTGACAGCCTTAGGCATGTCATTAAATCTACCAGTCGCCAAAGTAGAGCC
             .         .         .         .         .       2400
     IleLysIleMetLeuLysProGlyLysAspGlyProLysLeuArgGlnTrpProLeuThr
    AATAAAAATAATGCTAAAGCCAGGGAAAGATGGACCAAAACTGAGACAATGGCCCTTAAC
             .         .         .         .         .         .
     LysGluLysIleGluAlaLeuLysGluIleCysGluLysMetGluLysGluGlyGlnLeu
    AATAAAAATAATGCTAAAGCCAGGGAAAGATGGACCAAAACTGAGACAATGGCCCTTAAC
             .         .         .         .         .         .
     LysGluLysIleGluAlaLeuLysGluIleCysGluLysMetGluLysGluGlyGlnLeu
    AAAAGAAAAAATAGAAGCACTAAAAGAAATCTGTGAAAAAATGGAAAAAGAAGGCCAGCT
             .         .         .       2500         .         .
     GluGluAlaProProThrAsnProTyrAsnThrProThrPheAlaIleLysLysLysAsp
    AGAGGAAGCACCTCCAACTAATCCTTATAATACCCCCAGATTTGCAATCAAGAAAAAGGA
             .         .         .         .         .         .
     LysAsnLysTrpArgMetLeuIleAspPheArgGluLeuAsnLysValThrGlnAspPhe
    CAAAAACAAATGGAGGATGCTAATAGATTTCAGAGAACTAAACAAGGTAACTCAAGATTT
             .       2600         .         .         .         .
     ThrTluIleGlnLeuGlyIleProHisProAlaGlyLeuAlaLysLysArgArgIleThr
    CACAGAAATTCAGTTAGGAATTCCACACCCAGCAGGGTTGGCCAAGAAGAGAAGAATTAC
             .         .         .         .         .       2700
     ValLeuAspValGlyAspAlaTyrPheSerIleProLeuHisGluAspPheArgProTyr
    TGTACTAGATGTAGGGGATGCTTACTTTTCCATACCACTACATGAGGACTTTAGACCATA
             .         .         .         .         .         .
     ThrAlaPheThrLeuProSerValAsnAsnAlaGluProGlyLysArgTyrIleTyrLys
    TACTGCATTTACTCTACCATCAGTGAACAATGCAGAACCAGGAAAAAGATACATATATAA
             .         .         .       2800         .         .
     ValLeuProGlnGlyTrpLysGlySerProAlaIlePheGlnHisThrMetArgGlnVal
    AGTCTTGCCACAGGGATGGAAGGGATCACCAGCAATTTTTCAACACACAATGAGACAGGT
             .         .         .         .         .         .
     LeuGluProPheArgLysAlaAsnLysAspValIleIleIleGlnTyrMetAspAspIle
    ATTAGAACCATTCAGAAAAGCAAACAAGGATGTCATTATCATTCAGTACATGGATGATAT
             .       2900         .         .         .         .
     LeuIleAlaSerAspArgThrAspLeuGluHisAspArgValValLeuGlnLeuLysGlu
    CTTAATAGCTAGTGACAGGACAGATTTAGAACATGATAGGGTAGTCCTGCAGCTCAAGGA
             .         .         .         .         .       3000
     LeuLeuAsnGlyLeuGlyPheSerThrProAspGluLysPheGlnLysAspProProTyr
    ACTTCTAAATGGCCTAGGATTTTCTACCCCAGATGAGAAGTTCCAAAAAGACCCTCCATA
             .         .         .         .         .         .
     HisTrpMetGlyTyrGluLeuTrpProThrLysTrpLysLeuGlnLysIleGlnLeuPro
    CCACTGGATGGGCTATGAACTATGGCCAACTAAATGGAAGTTGCAGAAAATACAGTTGCC
             .         .       3100         .         .         .
     GlnLysGluIleTrpThrValAsnAspIleGlnLysLeuValGlyValLeuAsnTrpAla
    CCAAAAAGAAATATGGACAGTCAATGACATCCAGAAGCTAGTGGGTGTCCTAAATTGGGC
             .         .         .         .         .         .
     AlaGlnLeuTyrProGlyIleLysThrLysHisLeuCysArgLeuIleArgGlyLysMet
    AGCACAACTCTACCCAGGGATAAAGACCAAACACTTATGTAGGTTAATCAGAGGAAAAAT
             .       3200         .         .         .         .
     ThrLeuThrGluGluValGlnTrpThrGluLeuAlaGluAlaGluLeuGluGluAsnArg
    GACACTCACAGAAGAAGTACAGTGGACAGAATTACCAGAAGCAGAGCTAGAAGAAAACAG
             .         .         .         .         .       3300
     IleIleLeuSerGlnGluGlnGluGlyHisTyrTyrGlnGluGluLysGluLeuGluAla
    AATTATCCTAAGCCAGGAACAAGAGGGACACTATTACCAAGAAGAAAAAGAGCTAGAAGC
             .         .         .         .         .         .
     ThrValGlnLysAspGlnGluAsnGlnTrpThrTyrLysIleHisGlnGluGluLysIle
    AACAGTCCAAAAGGATCAAGAGAATCAGTGGACATATAAAATACACCAGGAAGAAAAAAT
             .         .         .       3400         .         .
     LeuLysValGlyLysTyrAlaLysValLysAsnThrHisThrAsnGlyIleArgLeuLeu
    TCTAAAAGTAGGAAAATATGCAAAGGTGAAAAACACCCATACCAATGGAATCAGATTGTT
             .         .         .         .         .         .
     AlaGlnValValGlnLysIleGlyLysGluAlaLeuValIleTrpGlyArgIleProLys
    AGCACAGGTAGTTCAGAAAATAGGAAAAGAAGCACTAGTCATTTGGGGACGAATACCAAA
             .       3500         .         .         .         .
     PheHisLeuProValGluArgGluIleTrpGluGlnTrpTrpAspAsnTyrTrpGlnVal
    ATTTCACCTACCAGTAGAGAGAGAAATCTGGGAGCAGTGGTGGGATAACTACTGGCAAGT
             .         .         .         .         .       3600
     ThrTrpIleProAspTrpAspPheValSerThrProProLeuValArgLeuAlaPheAsn
    GACATGGATCCCAGACTGGGACTTCGTGTCTACCCCACCACTGGTCAGGTTAGCGTTTAA
             .         .         .         .         .         .
     LeuGluGlnThrThrAsnGlnGlnAlaGluLeuGluAlaPheAlaMetAlaLeuThrAsp
    ACTAGAGCAAACTACCAATCAGCAAGCAGAACTAGAAGCCTTTGCGATGGCACTAACAGA
             .       3800.         .         .         .         .
     SerGlyProLysValAsnIleIleValAspSerGlnTyrValMetGlyIleSerAlaSer
    CTCGGGTCCAAAAGTTAATATTATAGTAGACTCACAGTATGTAATGGGGATCAGTCCAAG
             .         .         .         .         .       3900
     GlnProThrGluSerGluSerLysIleValAsnGlnIleIleGluGluMetIleLysLys
    CCAACCAACAGAGTCAGAAAGTAAAATAGTGAACCAGATCATAGAAGAAATGATAAAAAA
             .         .         .         .         .         .
     GluAlaIleTyrValAlaTrpValProAlaHisLysGlyIleGlyGlyAsnGlnGluVal
    GGAAGCAATCTATGTTGCATGGGTCCCAGCCCACAAAGGCATAGGGGGAAACCAGGAAGT
             .         .         .       4000         .         .
     AspHisLeuValSerGlnGlyIleArgGlnValLeuPheLeuGluLysIleGluProAla
    AGATCATTTAGTGAGTCAGGGTATCAGACAAGTGTTGTTCCTGGAAAAAATAGAGCCCGC
             .         .         .         .         .         .
     GlnGluGluHisGluLysTyrHisSerAsnValLysGluLeuSerHisLysPheGlyIle
    TCAGGAAGAACATGAAAAATATCATAGCAATGTAAAAGAACTGTCTCATAAATTTGGAAT
             .       4100         .         .         .         .
     ProAsnLeuValAlaArgGlnIleValAsnSerCysAlaGlnCysGlnGlnLysGlyGlu
    ACCCAATTTAGTGGCAAGGCAAATAGTAAACTCATGTGCCCAATGTCAACAGAAAGGGGA
             .         .         .         .         .       4200
     AlaIleHisGlyGlnValAsnAlaGluLeuGlyThrTrpGlnMetAspCysThrHisLeu
    AGCTATACATGGGCAAGTAAATGCAGAACTAGGCACTTGGCAAATGGACTGCACACATTT
             .         .         .         .         .         .
     GluGlyLysIleIleIleValAlaValHisValAlaSerGlyPheIleGluAlaGluVal
    AGAAGGAAAGATCATTATAGTAGCAGTACATGTTGCAAGTGGATTTATAGAAGCAGAAGT
             .         .         .       4300         .         .
     IleProGluGluSerGlyArgGlnThrAlaLeuPheLeuLeuLysLeuAlaSerArgTrp
    CATCCCACAGGAATCAGGAAGACAAACAGCACTCTTCCTATTGAAACTGGCAAGTAGGTG
             .         .         .         .         .         .
     ProIleThrHisLeuHisThrAspAsnGlyAlaAsnPheThrSerGlnGluValLysMet
    GCCAATAACACACTTGCATACAGATAATGGTGCCAACTTCACTTCACAGGAGGTGAAGAT
             .       4400         .         .         .         .
     ValAlaTrpTrpIleGlyIleGluGlnSerPheGlyValProTyrAsnProGlnSerGln
    GGTAGCATGGTGGATAGGTATAGAACAATCCTTTGGAGTACGTTACAATCCACAGAGCCA
             .         .         .         .         .       4500
     GlyValValGluAlaMetAsnHisHisLeuLysAsnGlnIleSerArgIleArgGluGln
    AGGAGTAGTAGAAGCAATGAATCACCATCTAAAAAACCAAATAAGTAGAATCAGAGAACA
             .         .         .         .         .         .
     AlaAsnThrIleGluThrIleValLeuMetAlaIleHisCysMetAsnPheLysArgArg
    GGCAAATACAATAGAAACAATAGTACTAATGGCAATTCATTGCATGAATTTTAAAAGAAG
             .         .         .       4600         .         .
     GlyGlyIleGlyAspMetThrProSerGluArgLeuIleAsnMetIleThrThrGluGln
    GGGGGGAATAGGGGATATGACTCCATCAGAAAGATTAATCAATATGATCACCACAGAACA
             .         .         .         .         .         .
     GluIleGlnPheLeuGlnAlaLysAsnSerLysLeuLysAspPheArgValTyrPheArg
    AGAAGGCAGAGATCAGTTGTGGAAAGGACCTGGGGAACTACTGTGGAAAGGAGAAGGAGC
             .       4700         .         .         .         .
     GluGlyArgAspGlnLeuTrpLysGlyProGlyGluLeuLeuTrpLysGlyGluGlyAla
    AGAAGGCAGAGATCAGTTGTGGAAAGGACCTGGGGAACTACTGTGGAAAGGAGAAGGAGC
             .         .         .         .         .       4800
     ValLeuValLysValGlyThrAspIleLysIleIleProArgArgLysAlaLysIleIle
    AGTCCTAGTCAAGGTAGGAACAGACATAAAAATAATACCAAGAAGGAAAGCCAAGATCAT
             .         .         .         .         .         .
     ArgAspTyrGlyGlyArgGlnGluMetAspSerGlySerHisLeuGluGlyAlaArgGlu
           Met GluGluAspLysArgTrpIleValValProThrTrpArgValProGlyArg
    CAGAGACTATGGAGGAAGACAAGAGATGGATAGTGGTTCCCACCTGGAGGGTGCCAGGGA
             .         .         .       4900         .         .
     AspGlyGluMetAla
      MetGluLysTrpHisSerLeuValLysTryLeuLysTyrLysThrLysAspLeuGluLys
    GGATGGAGAAATGGCATAGCCTTGTCAAGTATCTAAAATACAAAACAAAGGATCTAGAAA
             .         .         .         .         .         .
      ValCysTyrValProHisHisLysValGlyTrpAlaTrpTrpThrCysSerArgValIle
    AGGTGTGCTATGTTCCCCACCATAAGGTGGGATGGGCATGCTGGACTTGCAGCAGGGTAA
               .       5000         .         .         .         .
        PheProLeuLysGlyAsnSerHisLeuGluIleGlnAlaTyrTrpAsnLeuThrProGlu
      TATTCCCATTAAAAGGAAACAGTCATCTAGAGATACAGGCATATTGGAACTTAACACCAG
               .         .         .         .         .       5100
        LysGlyTrpLeuSerSerTyrSerValArgIleThrTrpTyrThrGluLysPheTrpThr
      AAAAAGGATGGCTCTCCTCTTATTCAGTAAGAATAACTTGGTACACAGAAAAGTTCTGGA
               .         .         .         .         .         .
        AspValThrProAspCysAlaAspValLeuIleHisSerThrTyrPheProCysPheThr
      CAGATGTTACCCCAGACTGTGCAGATGTCCTAATACATAGCACTTATTTCCCTTGCTTTA
               .         .         .       5200         .         .
        AlaGlyGluValArgArgAlaIleArgGlyGluLysLeuLeuSerCysCysAsnTyrPro
      CAGCAGGTGAAGTAAGAAGAGCCATCAGAGGGGAAAAGTTATTGTCCTGCTGCAATTATC
               .         .         .         .         .         .
        ArgAlaHisArgAlaGlnValProSerLeuGlnPheLeuAlaLeuValValValGlnGln
    CCCGAGCTCATAGAGCCCAGGTACCGTCACTTCAATTTCTGGCCTTAGTGGTAGTGCAAC
               .       5300          .         .         .         .
       MetThrAspProArgGluThrValProProGlyAsnSerGlyGluGluThrIleGly
      AsnAspArgProGluArgAspSerThrThrArgLysGlnArgArgArgAspTyrArgArg
    AAAATGACAGACCCCAGAGAGACAGTACCACCAGGAAACAGCCGCGAAGAGACTATCGGA
             .         .         .         .         .       5400
    GluAlaPheAlaTrpLeuAsnArgThrValGluAlaIleAsnArgGluAlaValAsnHis
      GlyLeuArgLeuAlaLysGlnAspSerArgSerHisLysGlnArgSerSerGluSerPro
    GAGGCCTTCGCCTGGCTAAACAGGACAGTAGAAGCCATAAACAGAGAAGCAGTGAATCAC
             .         .         .         .         .         .
    LeuProArgGluLeuIlePheGluValTrpGlnArgSerTrpArgTyrTrpHisAspGlu
      ThrProArgThrTyrPheProGlyValAlaGluValLeuGluIleLeuAla
    CTACCCCGAGAACTTATTTTCCAGGTGTGGCAGACGTCCTGGAGATACTGGCATGATGAA
             .         .         .       5500         .         .
    GlnGlyMetSerGluSerTyrThrLysTyrArgTyrLeuCysIleIleGlnLysAlaVal
    CAAGGGATGTCAGAAAGTTACACAAAGTATAGATATTTGTGCATAATACAGAAAGCACTG
             .         .         .         .         .         .
    TyrMetHisValArgLysGlyCysThrCysLeuGlyArgGlyHisGlyProGlyGlyTrp
    TACATGCATGTTAGGAAAGGGTGTACTTGCCTGGGGACGGGACATGGGCCAGGAGGGTGG
             .       5600         .         .         .         .
    ArgProGlyProProProProProProProGlyLeuVal
                                             MetAlaGluAlaProThrGlu
    AGACCAGGGCCTCCTCCTCCTCCCCCTCCAGGTCTGGTGTAATGGCTGAAGCACCAACAG
             .         .         .         .         .       5700
      LeuProProValAspGlyThrProLeuArgGluProGlyAspGluTrpIleIleGluIle
    AGCTCCCCCCGGTGGATGGGACCCCACTGAGGGAGCCAGGGGATGAGTGGATAATAGAAA
             .         .         .         .         .         .
      LeuArgGluIleLysGluGluAlaLeuLysHisPheAspProArgLeuLeuIleAlaLeu
    TCTTGAGAGAAATAAAAGAAGAAGCTTTAAAGCATTTTGACCCTCGCTTGCTAATTGCTC
             .         .         .       5800         .         .
                            MetGluThrPreLeuLysAlaProGluSerSerLeu
      GlyLysTyrIleTyrThrArgHisGlyAspThrLeuGluGlyAlaArgGluLeuIleLys
    TTGGCAAATATATCTATACTAGACATGGAGACACCCTTGAAGGCGCCAGAGAGCTCATTA
             .         .         .         .         .         .
    LysSerCysAsnGluProPheSerArgThrSerGluGlnAspValAlaThrGlnGluLeu
      ValLeuGlnArgAlaLeuPheThrHisPheArgAlaGlyCysGlyHisSerArgIleGly
    AAGTCCTGCAACGAGCCCTTTTCACGCACTTCAGAGCACTTCACAGCAGGATGTGGCCACTCAAGAATTG
             .       5900         .         .         .         .
    AlaArgGlnGlyGluGluIleLeuSerGlnLeuTyrArgProLeuGluThrCysAsnAsn
      GlnThrArgGlyGlyAsnProLeuSerAlaIleProThrProArgAsnMetGln
    GCCAGACAAGGGGAGGAAATCCTCTCTCAGCTATACCGACCCCTAGAAACATGCAATAAC
             .         .         .         .         .       6000
    SerCysTyrCysLysArgCysCysTyrHisCysGlnMetCysPheLeuAsnLysGlyLeu
    TCATGCTATTGTAAGCCATGCTGCTACCATTGTCAGATGTGTTTTCTAAACAAGGGGCTC
             .         .         .         .         .         .
    GlyIleCysTyrGluArgLysGlyArgArgArgArgThrProLysLysThrLysThrHis
              MetAsnGluArgAlaAspGluGluGlyLeuGlnArgLysLeuArgLeuIle
    GGGATATGTTATGAACGAAAGGGCAGACGAAGAAGCACTCCAAAGAAAACTAAGACTCAT
             .         .         .       6100         .         .
    ProSerProThrProAspLys
     ArgLeuLeuHisGlnThr
                               MetMetAsnGlnLeuLeuIleAlaIleLeuLeuAla
    CCGTCTCCTACACCAGACAAGTGAGTATGATGAATCAGCTGCTTATTGCCATTTTATTAG
             .         .         .         .         .         .
      SerAlacysLeuValTyrCysThrGlnTyrValThrValPheTyrGlyValProThrTrp
    CTAGTGCTTGCTTAGTATATTGCACCCAATATGTAACTGTTTTCTATGGCGTACCCACGT
             .       6200         .         .         .         .
      LysAsnAlaThrIleProLeuPheCysAlaThrArgAsnArgAspThrTrpGlyThrIle
    GGAAAAATGCAACCATTCCCCTCTTTTGTGCAACCAGAAATAGGGATACTTGGGGAACCA
             .         .         .         .         .       6300
      GlnCysLeuProAspAsnAspAspTyrGlnGluIleThrLeuAsnValThrGluAlaPhe
    TACAGTGCTTGCCTGACAATGATGATTATCAGGAAATAACTTTGAATGTAACAGAGGCTT
             .         .         .         .         .         .
    AspAlaTrpAsnAsnThrValThrGluGlnAlaIleGluAspValTrpHisLeuPheGlu
    TTGATGCATGGAATAATACAGTAACAGAACAAGCAATAGAAGATGTCTGGCATCTATTCG
             .         .         .       6400         .         .
      ThrSerIleLysProCysValLysLeuThrProLeuCysValAlaMetLysCysSerSer
    AGACATCAATAAAACCATGTGTCAAACTAACACCTTTATGTGTAGCAATGAAATGCAGCA
             .         .         .         .         .         .
      ThrGluSerSerThrGlyAsnAsnThrThrSerLysSerThrSerThrThrThrThrThr
    GCACAGAGAGCAGCACAGGGAACAACACAACCTCAAAGAGCACAAGCACAACCACAACCA
             .       6500         .         .         .         .
      ProThrAspGlnGluGlnGluIleSerGluAspThrProcysAlaArgAlaAspAsnCys
    CACCCACAGACCAGGAGCAAGAGATAAGTGAGGATACTCCATGCGCACGCGCAGACAACT
             .         .         .         .         .       6600
      SerGlyLeuGlyGluGluGluThrIleAsncysGlnPheAsnMetThrGlyLeuGluArg
    GCTCAGGATTGGGAGAGGAAGAAACGATCAATTGCCAGTTCAATATGACAGGATTAGAAA
             .         .         .         .         .         .
      AspLysLysGlnTyrAsnGluThrTrpTyrSerLysAspValValCysGluThrAsn
    GAGATAAGAAAAAACAGTATAATGAAACATGGTACTCAAAAGATGTGGTTTGTGAGACAA
             .         .         .       6700         .         .
      AsnSerThrAsnGlnThrGlnCysTyrMetAsnHisCysAsnThrSerValIleThrGlu
    ATAATAGCACAAATCAGACCCAGTGTTACATGAACCATTGCAACACATCAGTCATCACAG
             .         .         .         .         .         .
      SerCysAspLysHisTyrTrpAspAlaIleArgPheArgTyrCysAlaProProGlyTyr
    AATCATGTGACAAGCACTATTGGGATGCTATAAGGTTTAGATACTGTGCACCACCGGGTT
             .       6800         .         .         .         .
      AlaLeuLeuArgCysAsnAspThrAsnTyrSerGlyPheAlaProAsnCysSerLysVal
    ATGCCCTATTAAGATGTAATGATACCAATTATTCAGGCTTTGCACCCAACTGTTCTAAAG
             .         .         .         .         .       6900
      ValAlaSerThrCysThrArgMetMetGluThrGlnThrSerThrTrpPheGlyPheAsn
    TAGTAGCTTCTACATGCACCAGGATGATGGAAACGCAAACTTCCACATGGTTTGGCTTTA
             .         .         .         .         .         .
      GlyThrArgAlaGluAsnArgThrTyrIleTyrTrpHisGlyArgAspAsnArgThrIle
    ATGGCACTAGAGCAGAGAATAGAACATATATCTATTGGCATGGCAGAGATAATAGAACTA
             .         .         .       7000         .         .
      IleSerLeuAsnLysTyrTyrAsnLeuSerLeuHisCysLysArgProGlyAsnLysThr
    TCATCAGCTTAAACAAATATTATAATCTCAGTTTGCATTGTAAGAGGCCAGGGAATAAGA
             .         .         .         .         .         .
      ValLysGlnIleMetLeuMetSerGlyHisValPheHisSerHisTyrGlnProIleAsn
    CAGTGAAACAAATAATGCTTATGTCAGGACATGTGTTTCACTCCCACTACCAGCCGATCA
             .       7100.         .         .         .         .
      LysArgProArgGlnAlaTrpCysTrpPheLysGlyLysTrpLysAspAlaMetGlnGlu
    ATAAAAGACCCAGACAAGCATGGTGCTGGTTCAAAGGCAAATGGAAAGACGCCATGCAGG
             .         .         .         .         .       7200
      ValLysGluThrLeuAlaLysHisProArgTyrArgGlyThrAsnAspThrArgAsnIle
    AGGTGAAGGAAACCCTTGCAAAACATCCCAGGTATAGAGGAACCAATGACACAAGGAATA
             .         .         .         .         .         .
      SerPheAlaAlaProGlyLysGlySerAspProGluValAlaTyrMetTrpThrAsnCys
    TTAGCTTTGCAGCGCCAGGAAAAGGCTCAGACCCAGAAGTAGCATACATGTGGACTAACT
             .         .         .       7300         .         .
      ArgGlyGluPheLeuTyrCysAsnMetTHrTrpPheLeuAsnTrpIleGluAsnLysThr
    GCAGAGGAGAGTTTCTCTACTGCAACATGACTTGGTTCCTCAATTGGATAGAGAATAAGA
             .         .         .         .         .         .
      HisArgAsnTyrAlaProCysHisIleLysGlnIleIleAsnThrTrpHisLysValGly
    CACACCGCAATTATGCACCGTGCCATATAAAGCAAATAATTAACACATGGCATAAGGTAG
             .       7400         .         .         .         .
      ArgAsnValTyrLeuProProArgGluGlyGluLeuSerCysAsnSerThrValThrSer
    GGAGAAATGTATATTTGCCTCCCAGGGAACGGGAGCTGTCCTGCAACTCAACAGTAACCA
             .         .         .         .         .       7500
      IleIleAlaAsnIleAspTrpGlnAsnAsnAsnGlnThrAsnIleThrPheSerAlaGlu
    GCATAATTGCTAACATTGACTGGCAAAACAATAATCAGACAAACATTACCTTTAGTGCAG
             .         .         .         .         .         .
      ValAlaGluLeuTyrArgLeuGluLeuGlyAspTyrLysLeuValGluIleThrProIle
    AGGTGGCAGAACTATACAGATTGGAGTTGGGAGATTATAAATTGGTAGAAATAACACCAA
             .       7600         .         .         .         .
      GlyPheAlaProThrLysGluLysArgTyrSerSerAlaHisGlyArgHisThrArgGly
    TTGGCTTCGCACCTACAAAAGAAAAAAGATACTCCTCTGCTCACGGGAGACATACAAGAG
             .         .         .         .         .         .
      ValPheValLeuGlyPheLeuGlyPheLeuAlaThrAlaGlySerAlaMetGlyAlaAla
    GTGTGTTCGTGCTAGGGTTCTTGGGTTTTCTCGCAACAGCAGGTTCTGCAATGGGCGCGG
             .       7700         .         .         .         .
      SerLeuThrValSerAlaGlnSerArgThrLeuLeuAlaGlyIleValGlnGlnGlnGln
    CGTCCCTGACCGTGTCGGCTCAGTCCCGGACTTTACTGGCCGGGATAGTGCAGCAACAGC
             .         .         .         .         .       7800
      GlnLeuLeuAspValValLysArgGlnGlnGluLeuLeuArgLeuThrValTrpGlyThr
    AACAGCTGTTGGACGTGGTCAAGAGACAACAAGAACTGTTGCGACTGACGGTCTGGGGAA
             .         .         .         .         .         .
      LysAsnLeuGlnAlaArgValThrAlaIleGluLysTyrLeuGlnAspGlnAlaArgLeu
    CGAAAAACCTCCAGGCAAGAGTCACTGCTATAGAGAAGTACCTACAGGACCAGGCGCGGC
             .         .         .       7900         .         .
      AsnSerTrpGlyCysAlaPheArgGlnValCysHisThrThrValProTrpValAsnAsp
    TAAATTCATGGGGATGTGCGTTTAGACAAGTCTGCCACACTACTGTACCATGGGTTAATG
             .         .         .         .         .         .
      SerLeuAlaProAspTrpAspAsnMetThrTrpGlnGluTrpGluLysGlnValArgTyr
    ATTCCTTAGCACCTGACTGGGACAATATGACGTGGCAGGAATGGGAAAAACAAGTCCGCT
             .       8000         .         .         .         .
      LeuGluAlaAsnIleSerLysSerTrpAspIlePheGlyAsnTrpPheAspLeuThrSer
    TGTATGAACTACAAAAATTAAATAGCTGGGATATTTTTGGCAATTGGTTTGACTTAACCT
             .         .         .         .         .       8100
      TyrGluLeuGlnLysLeuAsnSerTrpAspIlePheGlyAsnTrpPheAspLeuThrSer
    TGTATGAACTACAAAAATTAAATAGCTGGGATATTTTTGGCAATTGGTTTGACTTAACCT
             .         .         .         .         .         .
      TrpValLysTyrIleGlnTyrGlyValLeuIleIleValAlaValIleAlaLeuArgIle
    CCTGGGTCAAGTATATTCAATATGGAGTGCTTATAATAGTAGCAGTAATAGCTTTAAGAA
             .         .         .       8200         .         .
      ValIleTyrValValGlnMetLeuSerArgLeuArgLysGlyTyrArgProValPheSer
    TAGTGATATATGTAGTACAAATGTTAAGTAGGCTTAGAAAGGGCTATAGGCCTGTTTTCT
             .         .         .         .         .         .
                               SerIleSerThrArgThrGlyAspSerGlnPro
                             AsnProTyrProGlnGlyProGlyThrAlaSerGln
      SerProProGlyTyrIleGlnGlnIleHisIleHisLysAspArgGlyGlnProAlaAsn
    CTTCCCCCCCCGGTTATATCCAACAGATCCATATCCACAAGGACCGGGGACAGCCAGCCA
             .       8300         .         .         .         .
    ThrLysLysGlnLysLysThrValGluAlaThrValGluThrAspThrGlyProGlyArg
     ArgArgAsnArgArgArgArgTrpLysGlnArgTrpArgGlnIleLeuAlaLeuAlaAsp
      GluGluThrGluGluAspGlyGlySerAsnGlyGlyAspArgTyrTrpProTrpProIle
             .         .         .         .         .       8400
     SerIleTyrThrPheProAspProProAlaAspSerProLeuAspGlnThrIleGlnHis
      AlaTyrIleHisPheLeuIleArgGlnLeuIleArgLeuLeuThrArgLeuTyrSerIle
    TAGCATATATACATTTCCTGATCCGCCAGCTGATTCGCCTCTTGACCAGACTATACAGCA
             .         .         .         .         .         .
     LeuGlnGlyLeuThrIleGlnGluLeuProAspProProThrHisLeuProGluSerGln
      GysArgAspLeuLeuSerArgSerPheLeuThrLeuGlnLeuIleTyrGlnAsnLeuArg
    TCTGCAGGGACTTACTATCCAGGAGCTTCCTGACCCTCCAACTCATCTACCAGAATCTCA
             .         .         .       8500         .         .
     ArgLeuAlaGluThr                     MetGlyAlaSerGlySerLysLys
      AspTrpLeuArgLeuArgThrAlaPheLeuGlnTyrGlyCysGluTrpIleGlnGluAla
    GAGACTGGCTGAGACTTAGAACAGCCTTCTTGGAATATGGGTGCGAGTGGATCCAAGAAG
             .         .         .         .         .         .
    HisSerArgProProArgGlyLeuGlnGluARgLeuLeuArgLalArgAlaGlyAlaCys
      PheGlnAlaAlaAlaArgAlaThrArgGluThrLeuAlaGlyAlaCysArgGlyLeuTrp
    CATTCCAGGCCGCCGCGAGGGCTACAAGAGAGACTCTTGCGGGCGCGTGCAGGGGCTTGT
             .       8600         .         .         .         .
    GlyGlyTyrTrpAsnGluSerGlyGlyGluTyrSerArgPheGlnGluGlySerAspArg
      ArgValLeuGluArgIleGlyArgGlyileLeuAlaValProArgARgIleArgGlnGly
    GCAGGGTATTGGAACGAATCGGGAGGGGAATACTCGCGGTTCCAAGAAGGATCAGACAGG
             .         .         .         .         .       8700
    GluGlnLysSerProSerCysGluGlyArgGlnTyrGlnGlnGlyAspPheMetAsnThr
      AlaGluIleAlaLeuLeu
    GAGCAGAAATCGCCCTCCTGTGAGGGACGGCAGTATCAGCAGGGAGACTTTATGAATACT
             .         .         .         .         .         .
    ProTrpLysAspProAlaAlaGluArgGluLysAsnLeuTyrArgGlnGlnAsnMetAsp
    CCATGGAAGGACCCAGCAGCAGAAAGGGAGAAAAATTTGTACAGGCAACAAAATATGGAT
             .         .         .       8800         .         .
    AspValAspSerAspAspAspAspGlnValArgValSerValThrProLysValProLeu
    GATGTAGATTCAGATGATGATGACCAAGTAAGAGTTTCTGTCACACCAAAAGTACCACTA
             .         .         .         .         .         .
    ArgProMetThrHisArgLeuAlaIleAspMetSerHisLeuIleLysThrArgGlyGly
    AGACCAATGACACATAGATTGGCAATAGATATGTCACATTTAATAAAAACAAGGGGGGGA
             .       8900         .         .         .         .
    LeuGluGlyMetPheTyrSerGluArgArgHisLysIleLeuAsnIleTyrLeuGluLys
    CTGGAAGGGATGTTTTACAGTGAAAGAAGACATAAAATCTTAAATATATACTTAGAAAAG
             .         .         .         .         .       9000
    GluGluGlyIleIleAlaAspTrpGlnAsnTyrThrHisGlyProGlyValArgTyrPro
    GAAGAAGGGATAATTGCAGATTGGCAGAACTACACTCATGGGCCAGGAGTAAGATACCA
             .         .         .         .         .         .
    MetPhePheGlyTrpLeuTrpLysLeuValProValAspValProGlnGluGlyGluAsp
    ATGTTCTTTGGGTGGCTATGGAAGCTAGTACCAGTAGATGTCCCACAAGAAGGGGAGGAC
             .         .         .       9100         .         .
    ThrGluThrHisCysLeuValHisProAlaGlnThrSerLysPheAspAspProHisGly
    ACTGAGACTCACTGCTTAGTACATCCAGCACAAACAAGCAAGTTTGATGACCCGCATGGG
             .         .         .         .         .         .
    GluThrLeuValTrpGluPheAspProLeuLeuAlaTyrSerTyrGluAlaPheIleArg
    GAGACACTAGTCTGGGAGTTTGATCCCTTGCTGGCTTATAGTTACGAGGCTTTTATTCGG
             .       9200         .         .         .         .
    TyrProGluGluPheGlyHisLysSerGlyLeuProGluGluGluTrpLysAlaArgLeu
    TACCCAGAGGAATTTGGGCACAAGTCAGGCCTGCCAGAGGAAGAGTGGAAGGCGAGACTG
             .         .         .         .         .       9300
    LysAlaArgGlyIleProPheSer
    AAAGCAAGAGGAATACCATTTAGTTAAAGACAGGAACAGCTATACTTGGTCAGGGCAGGA
             .         .         .         .         .         .
    AGTAACTAACAGAAACAGCTGAGACTGCAGGGACTTTCCAGAAGGGGCTGTAACCAAGGG
             .         .         .       9400         .         .
    AGGGACATGGGAGGAGCTGGTGGGGAACGCCCTCATATTCTCTGTATAAATATACCCGCT
             .         .         .         .         .         .
    AGCTTGCATTGTACTTCGGTCGCTCTGCGGAGAGGCTGGCAGATTGAGCCCTGGGAGGTT
             .       9500         .         .         .         .
    CTCTCCAGCAGTAGCAGGTAGAGCCTGGGTGTTCCCTGCTAGACTCTCACCAGCACTTGG
             .         .         .         .         .       9600
    CCGGTGCTGGGCAGACGGCCCCACGCTTGCTTGCTTAAAAACCTCCTTAATAAAGCTGCC
             .         .         .         .         .         .
    AGTTAGAAGCA
             .
    • Example 5 Sequences of the Coding Regions for the Envelope Protein and GAG Product of the ROD HIV-2 Isolate
  • Through experimental analysis of the HIV-2 ROD isolate, the following sequences were identified for the regions encoding the env and gag gene products. One of ordinary skill in the art will recognize that the numbering for both gene regions which follow begins for convenience with “1” rather than the corresponding number for its initial nucleotide as given in Example 4, above, in the context of the complete genomic sequence. [0084]
  • Envelope Sequence [0085]
    MetMetAsnGlnLeuLeuIleAlaIleLeuLeuAlaSerAlaCys
    ATGATGAATCAGCTGCTTATTGCCATTTTATTAGCTAGTGCTTGC
             .         .         .         .
    LeuValTyrCysThrGlnTyrValThrValPheTyrGlyValPro
    TTAGTATATTGCACCCAATATGTAACTGTTTTCTATGGCGTACCC
        .         .         .         .         .
    ThrTrpLysAsnAlaThrIleProLeuPheCysAlaThrArgAsn
    ACGTGGAAAAATGCAACCATTCCCCTGTTTTGTGCAACCAGAAAT
           100         .         .         .
    ArgAspThrTrpGlyThrIleGlnCysLeuProAspAsnAspAsp
    AGGGATACTTGGGGAACCATACAGTGCTTGCCTGACAATGATGAT
        .         .         .         .         .
    TyrGlnGluIleThrLeuAsnValThrGluAlaPheAspAlaTrp
    TATCAGGAAATAACTTTGAATGTAACAGAGGCTTTTGATGCATGG
             .       200         .         .
    AsnAsnThrValThrGluGluAlaIleGluAspValTrpHisLeu
    AATAATACAGTAACAGAACAAGCAATAGAAGATGTCTGGCATCTA
        .         .         .         .         .
    PheGluThrSerIleLysProCysValLysLeuThrProLeuCys
    TTCGAGACATCAATAAAACCATGTGTCAAACTAACACCTTTATGT
             .         .       300         .
    ValAlaMetLysCysSerSerThrGluSerSerThrGlyAsnAsn
    GTAGCAATGAAATGCAGCAGCACAGAGAGCAGCACAGGGAACAAC
        .         .         .         .         .
    ThrThrSerLysSerThrSerThrThrThrThrThrProThrAsp
    ACAACCTCAAAGAGCACAAGCACAACGACAACCACACCCAGAGAC
             .         .         .       400
    GlnGluGlnGluIleSerGluAspThrProCysAlaArgAlaAsp
    CAGGAGCAAGAGATAAGTGAGGATACTCCATGCGCACGCGCAGAC
        .         .         .         .         .
    AsnCysSerGlyLeuGlyGluGluGluThrIleAsnCysGlnPhe
    AACTGCTCAGGATTGGGAGAGGAAGAAACGATCAATTGCCAGTTC
             .         .         .         .
    AsnMetThrGlyLeuGluARgAspLysLysLysGlnTyrAsnGlu
    AATATGACAGGATTAGAAAGAGATAAGAAAAAAACAGTATAATGAA
      500         .         .         .         .
    ThrTrpTyrSerLysAspValValCysGluThrAsnAsnSerThr
    ACATGGTACTCAAAAGATGTGGTTTGTGAGACAAATAATAGCACA
             .         .         .         .
    AsnGlnThrGlnCysTyrMetAsnHisCysAsnThrSerValIle
    AATCAGACCCAGTGTTACATGAACCATTGCAACACATCAGTCATC
        .       600         .         .         .
    ThrGluSerCysAspLysHisTyrTrpAspAlaIleArgPheArg
    ACAGAATCATGTGACAAGCACTATTGGGATGCTATAAGGTTTAGA
             .         .         .         .
    TyrCysAlaProProGlyTyrAlaLeuLeuArgCysAsnAspThr
    TACTGTGCACCACGGGTTATGCCCTATTAAGATGTAATGATACC
        .        .       700         .         .
    AsnTyrSerGlyPheAlaProAsncysSerLysValValAlsSer
    AATTATTCAGGCTTTGCACCCAACTGTTCTAAAGTAGTAGCTTCT
             .         .         .         .
    ThrCysThrArgMetMetGluThrGlnThrSerThrTrpPheGly
    ACATGCACCAGGATGATGGAAACGCAAACTTCCACATGGTTTGGC
        .         .         .       800        .
    PheAsnGlyThrArgAlaGluAsnArgThrTyrIleTyrTrpHis
    TTTAATGGCACTAGAGCAGAGAATAGAACATATATCTATTGGCAT
             .         .         .         .
    GlyArgAspAsnArgThrIleIleSerLeuAsnLysTyrTyrAsn
    GGCAGAGATAATAGAACTATCATCAGCTTAAACAAATATTATAAT
        .         .         .         .       900
    LeuSerLeuHisCysLysArgProGlyAsnLysThrValLysGln
    CTCAGTTTGCATTGTAAGAGGCCAGGGAATAAGACAGTGAAACAA
             .         .         .         .
    IleMetLeuMetSerGlyHisValPheHisSerHisTyrGlnPro
    ATAATGCTTATGTCAGGACATGTGTTTCACTCCCACTACCAGCCG
        .         .         .         .         .
    IleAsnLysArgProArgGlnAlaTrpCysTrpPheLysGlyLys
    ATCAATAAAAGACCCAGACAAGCATGGTGCTGGTTCAAAGGCAAA
          1000         .         .         .
    TrpLysAspAlaMetGlnGluValLysThrLeuAlaLysHisPro
    TGGAAAGACGCCATGCAGGAGGTGAAGACCCTTGCAAAACATCCC
        .         .         .         .         .
    ArgTyrArgGlyThrAsnAspThrArgAsnIleSerPheAlaAla
    AGGTATAGAGGAACCAATGACACAAGGAATATTAGCTTTGCAGCG
             .      1100         .         .
    ProGlyLysGlySerAspProGluValAlaTyrMetTrpThrAsa
    CCAGGAAAAGGCTCAGACCCAGAAGTAGCATACATGTGGACTAAC
        .         .         .         .         .
    CysArgGlyGluPheLeuTyrCysAsnMetThrTrpPheLeuAsn
    TGCAGAGGAGAGTTTCTCTACTGCAACATGACTTGGTTCCTCAAT
            .         .      1200         .
    TrpIleGluAsnLysThrHisArgAsnTyrAlaProcysHisIle
    TGGATAGAGAATAAGACACACCGCAATTATGCACCGTGCCATATA
        .         .         .         .         .
    LysGlnIleIleAsnThrTrpHisLysValGlyArgAsnValTyr
    AAGCAAATAATTAACACATGGCATAAGGTAGGGAGAAATGTATAT
             .         .         .      1300
    LeuProProArgGluGlyGluLeuSerCysAsnSerThrValThr
    TTGCCTCCCAGGGAACGGGAGCTGTCCTGCAACTCAACAGTAACC
        .         .         .         .         .
    SerIleIleAlaAsnIleAspTrpGlnAsnAsnAsnGlnThrAsn
    AGCATAATTGCTAACATTGACTGGCAAAACAATAATCAGACAAC
             .         .         .         .
    IleThrPheSerAlaGluValAlaGluLeuTyrArgLeuGluLeu
    ATTACCTTTAGTGCAGAGGTGGCAGAACTATACAGATTGGAGTTG
     1400         .         .         .         .
    GlyAspTyrLysLeuValGluIleThrProIleGlyPheAlaPro
    GCAGATTATAAATTGGTAGAAATAACACCAATTGGCTTCGCACCT
    ThrLysGluLysArgTyrSerSerAlaHisGlyArgHisThrArg
    ACAAAAGAAAAAAGATACTCCTCTGCTCACGGGAGACATACAAGA
        .      1500         .         .         .
    GlyValPheValLeuGlyPheLeuGlyPheLeuAlaThrAlaGly
    GGTGTGTTCGTGCTAGGGTTCTTGGGTTTTCTCGCAACAGCAGGT
             .         .         .         .
    SerAlaMetGlyAlaArgAlaSerLeuThrValSerAlaGluSer
    TCTGCAATGGGCGCTCGAGCGTCCCTGACCGTGTCGGCTCAGTCC
        .         .      1600         .         .
    ArgThrLeuLeuAlaGlyIleValGlnGlnGluGlnGluLeuLeu
    CGGACTTTACTGGCCGGGATAGTGCAGCAACAGCAACAGCTGTTG
             .         .         .         .
    AspValValLysArgGlnGlnGluLeuLeuArgLeuThrValTrp
    GACGTGGTCAAGAGACAACAAGAACTGTTGCGACTGACCGTCTGG
        .         .         .      1700         .
    GlyThrLysAsnLeuGlnAlaArgValThrAlaIleGluLysTyr
    GGAACGAAAAACCTCCAGGCAAGAGTCACTGCTATAGAGAAGTAC
             .         .         .         .
    LeuGlnAspGlnAlaArgLeuAsnSerTrpGlyCysAlaPheArg
    CTACAGGACCAGGCGCGGCTAAATTCATGGGGATGTGCGTTTAGA
        .         .         .         .      1800
    GlnValCysHisThrThrValProTrpValAsnAspSerLeuAla
    CAAGTCTGCCACACTACTGTACCATGGGTTAATGATTCCTTAGCA
             .         .         .         .
    ProAspTrpAspAsnMetThrTrpGlnGluTrpGluLysGluVal
    CCTGACTGGGACAATATGACGTGGCAGGAATGGGAAAAACAAGTC
        .         .         .         .         .
    ArgTyrLeuGluAlaAsnIleSerLysSerLeuGluGlnAlaGln
    CGCTACCTGGAGGCAAATATCAGTAAAAGTTTAGAACAGGCACAA
          1900         .         .         .
    IleGlnGlnGluLysAsnMetTyrGluLeuGlnLysLeuAsnSer
    ATTCAGCAAGAGAAAAATATGTATGAACTACAAAAATTAAATAGC
        .         .         .         .         .
    TrpAspIlePheGlyAsnTrpPheAspLeuThrSerTrpValLys
    TGGGATATTTTTGGCAATTGGTTTGACTTAACCTCCTGGGTCAAG
             .      2000         .         .
    TyrIleGlnTyrGlyValLeuIleIleValAlaValIleAlaLeu
    TATATTCAATATGGAGTGCTTATAATAGTAGCAGTAATAGCTTTA
        .         .         .         .         .
    ArgIleValIleTyrValValGlnMetLeuSerArgLeuArgLys
    AGAATAGTGATATATGTAGTACAAATGTTAAGTAGGCTTAGAAAG
             .         .      2100         .
    GlyTyrArgProValPheSerSerProProGlyTyrIleGln***
    GGCTATAGGCCTGTTTTCTCTTCCCCCCCCGGTTATATCCAATAG
    IleHisIleHisLysAspArgGlyGluProAlaAsnGluGluThr
    ATCCATATCCACAAGGACCGGGGACAGCCAGCCAACGAAGAAACA
             .         .         .      2200
    GluGluAspGlyGlySerAsnGlyGlyAspArgTyrTrpProTrp
    GAAGAAGACGGTGGAAGCAACGGTGGAGACAGATACTGGCCCTGG
        .         .         .         .         .
    ProIleAlaTyrIleHisPheLeuIleArgGlnLeuIleArgLeu
    GCGATAGCATATATACATTTCCTGATCCGCCAGCTGATTCGCCTC
             .         .         .         .
    LeuThrArgLeuTyrSerIleCysArgAspLeuLeuSerArgSer
    TTGACCAGACTATACAGCATCTGCAGGGACTTACTATCCAGGAGC
     2300         .         .         .         .
    PheLeuThrLeuGluLeuIleTyrGlnAsnLeuArgAspTrpLeu
    TTCCTGACCCTCCAACTCATCTACCAGAATCTCAGAGACTGGCTG
             .         .         .         .
    ArgLeuArgThrAlaPheLeuGlnTyrGlyCysGluTrpIleGln
    AGACTTAGAACAGCCTTCTTGCAATATGGGTGCGAGTGGATCCAA
        .      2400         .         .         .
    GluAlaPheGlnAlaAlaAlaArgAlaThrArgGluThrLeuAla
    GAAGCATTCCAGGCCGCCGCGAGGGCTACAAGAGAGACTCTTGCG
             .         .         .         .
    GlyAlaCysArgGlyLeuTrpArgValLeuGluArgIleGlyArg
    GGCGCGTGCAGGGGCTTGTGGAGGGTATTGGAACGAATCGGGACG
             .         .      2500         .         .
    GlyIleLeuAlaValProArgARgIleArgGlnGlyAlaGluIle
    GGAATACTCGCGGTTCCAAGAAGGATCAGACAGGGAGCAGAAATC
             .         .         .         .
    AlaLeuLeu***GlyThrAlaValSerAlaGlyArgLeuTyrGlu
    GCCCTCCTGTGAGGGACGGCAGTATCAGCAGGGAGACTTTATGAA
        .         .         .      2600         .
    TyrSerMetGluGlyProSerSerArgLysGlyGluLysPheVal
    TACTCCATGGAAGGACCCAGCAGCAGAAAGGGAGAAAAATTTGTA
             .         .         .         .
    GlnAlaThrLysTyrGly
    CAGGCAACAAAATATGGA
        .         .
    MetGlyAlaArgAsnSerValLeuArgGlyLysLysAlaAspGlu
    ATGGGCGCGAGAAACTCCGTCTTGAGAGGGAAAAAAGCAGATGAA
             .         .         .         .
    LeuGluArgIleArgLeuArgProGlyGlyLysLysLysTyrArg
    TTAGAAAGAATCAGGTTACGGCCCGGCGGAAAGAAAAAGTACAGG
        .         .         .         .         .
    LeuLysHisIleValTrpAlaAlaAsnLysLeuAspArgPheGly
    CTAAAACATATTGTGTGGGCAGCGAATAAATTGCACAGATTCGGA
           100         .         .         .
    LeuAlaGluSerLeuLeuGluSerLysGluGlyCysGlnLysIle
    TTAGCAGAGAGCCTGTTGGAGTCAAAAGAGGGTTGTCAAAAAATT
        .         .         .         .
    LeuThrValLeuAspProMetValProThrGlySerGluAsnLeu
    CTTACAGTTTTAGATCCAATGGTACCGACAGGTTCAGAAAATTTA
             .       200         .         .
    LysSerLeuPheAsnThrValCysValIleTrpCysIleHisAla
    AAAAGTCTTTTTAATACTGTCTGCGTCATTTGGTGCATACAGGCA
        .         .         .         .         .
    GluGluLysValLysAspThrGluGlyAlaLysGlnIleValArg
    GAAGAGAAAGTGAAAGATACTGAAGGAGCAAAACAAATAGTGCGG
             .         .       300         .
    ArgHisLeuValAlaGluThrGlyThrAlaGluLysMetProSer
    AGACATCTAGTGGCAGAAACAGGAACTGCAGAGAAAATGCCAAGG
        .         .         .         .
    ThrSerArgProThrAlaProSerSerGluLysGlyGlyAsnTyr
    ACAAGTAGACCAACAGCACCATCTAGCGAGAAGGGAGGAAATTAC
            .         .         .       400
    ProValGlnHisValGlyGlyAsnTyrThrHisIleProLeuSer
    CCAGTGCAACATGTAGGCGGCAACTACACCCATATACCGCTGACT
        .         .         .         .         .
    ProArgThrLeuAsnAlaTrpValLysLeuValGluGluLysLys
    CCCCGAACCCTAAATGCCTGGGTAAAATTAGTAGAGGAAAAAAAG
             .         .         .         .
    PheGlyAlaGluValValProGlyPheGlnAlaLeuSerGluGly
    TTCGGGGCAGAAGTAGTGCCAGGATTTCAGGCACTCTCAGAAGGC
      500         .         .         .         .
    CysThrProTyrAspIleAsnGlnMetLeuAsnCysValGlyAsp
    TGCACGCCCTATGATATCAACCAAATGCTTAATTGTGTGGGCGAC
             .         .         .         .
    HisGlnAlaAlaMetGlnIleIleArgGluIleIleAsnGluGlu
    CATCAAGCAGCCATGCAGATAATCAGGGAGATTATCAATGAGGAA
        .       600         .         .         .
    AlaAlaGluTrpAspValGlnHisProIleProGlyProLeuPro
    GCAGCAGAATGGGATGTGCAACATCCAATACCAGGCCCCTTACCA
             .         .         .         .
    AlaGlyGlnLeuArgGluProArgGlySerAspIleAlaGlyThr
    GCGGGGCAGCTTAGAGAGCCAAGGGGATCTGACATAGCAGGGACA
        .         .       700         .         .
    ThrSerThrValGluGluGlnIleGlnTrpMetPheArgProGln
    ACAAGCACAGTAGAAGAACAGATCCAGTGGATGTTTAGGCCAGAA
    AsnProValProValGlyAsnIleTyrArgArgTrpIleGlnIle
    AATCCTGTACCAGTAGGAAACATCTATAGAAGATGGATCCAGATA
        .         .         .       800         .
    GlyLeuGlnLysCysValARgMetTyrAsnProThrAsnIleLeu
    GGATTGCAGAAGTGTGTCAGGATGTACAACCCGACCAACATCCTA
             .         .         .         .         .
    AspIleLysGlnGlyProLysGluProPheGlnSerTyrValAsp
    GACATAAAACAGGGACCAAAGGAGCCGTTCCAAAGCTATGTAGAT
        .         .         .         .      900
    ArgPheTyrLysSerLeuArgAlaGluGlnThrAspProAlaVal
    AGATTCTACAAAAGCTTGAGGGCAGAACAAACAGATCCAGCAGTG
             .         .         .         .
    LysAsnTrpMetThrGlnThrLeuLeuValGlnAsnAlaAsnPro
    AAGAATTGGATGACCCAAACACTGCTAGTACAAAATGCCAACGCA
        .         .         .         .         .
    AspCysLysLeuValLeuLysGlyLeuGlyMetAsnProThrLeu
    GACTGTAAATTAGTGCTAAAAGGACTAGGGATGAACCCTACCTTA
          1000         .         .         .
    GluGluMetLeuThrAlacysGlnGlyValGlyGlyProGlyGln
    GAAGAGATGCTGACCGCCTGTCAGGGGGTAGGTGGGCCAGGCCAG
        .         .         .         .         .
    LysAlaArgLeuMetAlaGluAlaLeuLysGluValIleGlyPro
    AAAGCTAGATTAATGGCAGAGGCCCTGAAAGAGGTCATAGGACCT
             .      1100         .         .
    AlaProIleProPheALaAlaAlaGlnGlnArgLysAlaPheLys
    GCCCCTATCCCATTCGCAGCAGCCCAGGAGAGAAAGGCATTTAAA
        .         .         .         .         .
    CysTrpAsnCysGlyLysGluGlyHisSerAlaArgGlnCysArg
    TGCTGGAACTGTGGAAAGGAAGGGCACTCGGCAAGACAATGCCGA
             .         .       1200         .
    AlaProArgARgGlnGlyCysTrpLysCysGlyLysProGlyHis
    GCACCTAGAAGGCAGGGCTGCTGGAAGTGTGGTAAGCCAGGACAC
        .         .         .         .         .
    IleMetThrAsnCysProAspArgGlnAlaGlyPheLeuGlyLeu
    ATCATGACAAACTGCCCAGATAGACAGGCAGGTTTTTTAGGACTG
             .         .         .       1300
    GlyProTrpGlyLysLysProArgAsnPheProValAlaGlnVal
    GGCCCTTGGGGAAAGAAGCCCCGCAACTTCCCCGTGGCCCAAGTT
        .         .         .         .         .
    ProGlnGlyLeuThrProThrAlaProProValAspProAlaVal
    CCGCAGGGGCTGACACCAACAGCACCCCCAGTGGATCCAGCAGTG
             .         .         .         .
    AspLeuLeuGluLysTyrMetGlnGlnGlyLysArgGlnArgGlu
    GATCTACTGGAGAAATATATGCAGCAAGGGAAAAGACAGAGAGAG
     1400         .         .         .         .
    GlnArgGluArgProTyrLysGluValThrGluAspLeuLeuHis
    CAGAGAGAGAGACCATACAAGGAAGTGACAGAGGACTTACTGCAC
             .         .         .         .
    LeuGluGlnGlyGluThrProTyrArgGluProProThrGluAsp
    CTCGAGCAGGGGGAGACACCATACAGGGAGCCACCAACAGAGGAC
        .       1500         .         .         .
    LeuLeuHisLeuAsnSerLeuPheGlyLysAspGln
    TTGCTGCACCTCAATTCTCTCTTTGGAAAAGACCAG
             .         .         .
    • Example 6 Peptide Sequences Encoded by the ENV and GAG Genes
  • The following coding regions for antigenic peptides, identified for convenience only by the nucleotide numbers of Example 5, within the env and gag gene regions are of particular interest. [0086]
    envl (1732-1809)
                          ArgValThrAlaIleGluLysTyr
                          AGAGTCACTGCTATAGAGAAGTAC
                                  .         .
    LeuGluAspGlnAlaArgLeuAsnSerTrpGlyCysAlaPheArg
    CTACAGGACCAGGCGCGGCTAAATTCATGGGGATGTGCGTTTAGA
        .         .         .         .      1   
    GlnValCys
    CAAGTCTGC
    env2 (1912-1983)
                          SerLysSerLeuGluGlnAlaGln
                          AGTAAAAGTTTAGAACAGGCACAA
                                  .         .
    IleGlnGlnGluLysAsnMetTyrGluLeuGlnLysLeuAsnSer
    ATTCAGCAAGAGAAAAATATGTATGAACTACAAAAATTAAATAGC
     1940          .         .         .         .
    Trp
    TGG
    env3 (1482-1530)
    Pro ThrLysGluLysArgTyrSerSerAlaHisGlyArgHisThrArg
    CCT ACAAAAGAAAAAAGATACTCCTCTGCTCACGGGAGACATACAAGA
            .      1500         .         .         .
    env4 (55-129)
             CysThrGlnTyrValThrValPheTyrGlyValPro
             TGCACCCAATATGTAACTGTTTTCTATGGCGTACCC
                   .         .         .         .
    ThrTrpLysAsnAlaThrIleProLeuPheCysAlaThr
    ACGTGGAAAAATGCAACCATTCCCCTCTTTTGTGCAACC
          100         .         .
    env5 (175-231)
                                            AspAsp
                                            GATGAT
                                                 .
    TyrGluGluIleThrLeuAsnValThrGluAlaPheAspAlaTrp
    TATCAGGAAATAACTTTGAATGTAACAGAGGCTTTTGATGCATGG
            .       200         .         .     
    AsnAsn
    AATAAT
    env6 (274-330)
       GluThrSerIleLysProCysValLysLeuThrProLeuCys
       GAGACATCAATAAACCATGTGTGAAACTAACACCTTTATGT
             .         .       300         .
    ValAlaMetLysCys
    GTAGCAATGAAATGC
        .         .
    env7 (607-660)
                         AsnHisCysAsnThrSerValIle
                         AACCATTGCAACACATCAGTCATC
                          610         .         .
    ThrGluSerCysAspLysHisTyrTrpAsp
    ACAGAATCATGTGACAAGCACTATTGGGAT
             .         .         .
    env8 (661-720)
                                  AlaIleArgPheArg
                                  GCTATAAGGTTTAGA
                                            .
    TyrCysAlaProProGlyTyrAlaLeuLeuArgCysAsnAspThr
    TACTGTGCACCACCGGGTTATGCCCTATTAAGATGTAATGATACC
        .         .       700         .        .
    env9 (997-1044)
          LysArgProArgGlnAlaTrpCysTrpPheLysGlyLys
          AAAAGACCCAGACAAGCATGGTGCTGGTTCAAAGGCAAA
          1000         .         .          .
    TrpLysAsp
    TGGAAAGAC
    env10 (1132-1215)
          LysGlySerAspProGluValAlaTyrMetTrpThrAsa
          AAAGGCTCAGACCCAGAAGTAGCATACATGTGGACTAAC
                 .       .         .         .
    CysArgGlyGluPheLeuTyrCysAsnMetThrTrpPheLeuAsn
    TGCAGAGGACACTTTTFTFTACTGCAACATGACTTGGTTCCTCAAT
             .         .       1200        .
    env11 (1237-1305)
                         ArgAsnTyrAlaProCysHisIle
                         CGCAATTATGCACCGTGCCATATA
                            .         .         .
    LysGlnIleIleAsnThrTrpHisLysValGlyArgAsnValTyr
    AAGCAAATAATTAACACATGGCATAAGGTAGGGAGAAATGTATAT
             .         .         .      1300
    GAG1 (991-1053)
    AspCysLysLeuValLeuLysGlyLeuGlyMetAsnProThrLeu
    GACTGTAAATTAGTGCTAAAAGGACTAGGGATGAACCCTACCTTA
          1000         .         .         .
    GluGluMetLeuThrAla
    GAAGAGATGCTGACCGCC
  • Of the foregoing peptides, env1, env2, env3 and gag1 are particularly contemnplated for diagnostic purposes, and env4, env5, env6, env7, env8, env9, env10 and env11 are particularly, contemplated as protecting agents. These peptides have been selected in part because of their sequence homology to certain of the envelope and gag protein products of other of the retroviruses in the HIV group. For vaccinating purposes, the foregoing peptides may be coupled to a carrier protein by utilizing suitable and well known techniques to enhance the host's immune response. Adjuvants such as calcium phosphate or alum hydroxide may also be added. The foregoing peptides can be synthesized by conventional protein synthesis techniques, such as that of Merrifield. [0087]
  • It will be apparent to those skilled in the art that various modifications and variations can be made in the processes and products of the present invention. Thus, it is intended that the present application cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. For convenience in interpreting the following claims, the following table sets forth the correspondence between codon codes and amino acids and the correspondence between three-letter and one-letter amino acid symbols. [0088]
               DNA CODON      AMINO ACID 3 LET.  AMINO ACID 1 LET.
    --------------------------------------------------------------
    :   : \2:  T   C   A   G  :  T   C   A   G  :  T   C   A   G  :
    : 1 : 3\:                 :                 :                 :
    --------------------------------------------------------------
    :   : T : TTT TCT TAT TGT : PHE SER TYR CYS :  F   S   Y   C  :
    : T : C : TTC TCC TAC TGC : PHE SER TYR CYS :  F   S   Y   C  :
    :   : A : TTA TCA TAA TGA : LEU SER *** *** :  L   S   *   *  :
    :   : G : TTG TCG TAG TCG : LEU SER III TRP :  L   S   *   W  :
    --------------------------------------------------------------
    :   : T : CTT CCT CAT CGT : LEU PRO HIS ARG :  L   P   H   R :
    : C : C : CTC CCC CAC CGC : LEU PRO HIS ARG :  L   P   H   R  :
    :   : A : CTA CCA CAA CGA : LEU PRO GLN ARG :  L   P   Q   R  :
    :   : G : CTG CCG CAG CCG : LEU PRO GLN ARG :  L   P   Q   R  :
    --------------------------------------------------------------
    :   : T : ATT ACT AAT AGT : ILE THR ASN SER :  I   T   N   S  :
    : A : C : ATC ACC AAC AGC : ILE THR ASN SER :  I   T   N   S  :
    :   : A : ATA ACA AAA AGA : ILE THR LYS ARG :  I   T   K   R  :
    :   : G : ATG ACG AAG AGG : MET THR LYS ARG :  M   T   K   R  :
    --------------------------------------------------------------
    :   : T : GTT GCT GAT GGT : VAL ALA ASP GLY :  V   A   D   G  :
    : G : C : GTC GCC GAC GGC : VAL ALA ASP GLY :  V   A   D   G  :
    :   : A : GTA GCA GAA GGA : VAL ALA GLY GLY :  V   A   E   G  :
    :   : G : GTG GCG GAG GGG : VAL ALA GLU GLY :  V   A   E   G  :
    --------------------------------------------------------------
    3 Letter 1 Letter CODONS
    ALA A GCT GCC CGA GCG
    ARG R CGT CGC CGA CGG AGA AGG
    ASN N AAT AAC
    ASP O GAT GAC
    CYS C TGT TGC
    GLN Q CAA CAG
    GLU E GAA CAG
    GLY G GGT GGC CGA CCG
    HIS H CAT CAC
    ILE I ATT ATC ATA
    LEU L CTT CTC CTA CTG TTA TTG
    LYS K AAA AAG
    MET M ATG
    PHE F TTT TTC
    PRO P CCT CCC CCA CCG
    SER S TCT TCC TCA TCG AGT AGC
    THR T ACT ACC ACA ACG
    TRP W TGG
    TYR Y TAT TAC
    VAL V GTT GTC GTA GTG
    *** * TAA TAG TGA

Claims (43)

What is claimed is:
1. A method for diagnosing an HIV-2 infection which comprises:
(a) contacting genetic DNA or RNA from a body sample obtained from a person suspected of having an HIV-2 infection with a DNA probe derived from at least a portion of the genome of the HIV-2 virus; and
(b) determining whether a hybridized complex is created.
2. The method of claim 1 wherein said body sample is selected from the group consisting of tissue, blood cells, cells and body fluids.
3. The method of claim 1 wherein the presence of the hybridized complex is determined by a process selected from the group consisting of Southern blot, Northern blot and dot blot.
4. The method of claim 1 wherein the cDNA probe is analogous to the entire genome of the HIV-2 virus.
5. A DNA probe capable of hybridizing to the entire genome of the HIV-2 virus.
6. A method for diagnosing an HIV-2 infection which comprises:
(a) contacting sera obtained from a patient suspected of having an HIV-2 infection with a polypeptide expression product of a DNA segment derived from the genome of the HIV-2 virus; and
(b) determing whether an immunocomplex is formed.
7. The method of claim 6 wherein the formation of the immunocomplex is determined by a process selected from the group consisting of radioimmunoassays (RIA), radioimmunoprecipitation assays (RIPA), immunofluoresence assays (IFA), enzyme-linked immunosorbent assays (ELISA) and Western blots.
8. A process for detecting the presence of a virus selected from the group consisting of LAV-II, HIV-2, STLV-III and other viruses which form complexes with LAV-II reagents comprising:
(a) contacting DNA or RNA from a sample suspected of containing viral genetic material with a DNA probe derived from a portion of the genome of the HIV-2 virus; and
(b) determining whether a hybridized complex is created.
9. A peptide selected from the group consisting of env1, env2, env3, env4, env5, env6, env7, env8, env9, env10, env11 and gag1.
10. A kit for diagnosing an HIV-2 infection by the method of claim 6 and comprising env1, env2, env3 and gag1 peptides as the polypeptide expression product.
11. A vaccinating agent comprising at least one peptide selected from the group consisting of env4, env5, env6, env7, env8, env9, env10 and env11 in admixture with suitable carriers.
12. A peptide having common immunological properties with the peptide structure of the envelope glycoprotein of a virus of the HIV-2 class, said peptide having no more than 40 amino acid residues.
13. A peptide according to claim 12 having either of the following formulas:
XR--A-E-D-YL-DQ--L-WGC-----CZ    XA-E-D-YL-DZ
in which X and Z are OH or NH2 or, to the extent that the immunological properties of the natural peptides lacking these groups shall not be essentially modified, the groups having from one to five amino acid residues, and each of the hyphens corresponding to an aminoacyl residue chosen from those which permit the conservation for the peptide characterized above of the immunological properties of either of the following peptide sequences:
RVTAIEKYLQDQARLNSWGCAFRQVC    AIEKYLQDQ
14. A peptide according to claim 12 having either of the following formulas:
X--E--Q-QQEKN--EL--L---Z      XQ-QQEKNZ
in which X and Z are OH or NH2 or, to the extent that the immunological properties of the natural peptides lacking these groups shall not be essentially modified, the groups having from one to five amino acid residues, and each of the hyphens corresponding to an aminoacyl residue chosen from those which permit the conservation for the peptide characterized above of the immunological properties of either of the following peptide sequences:
SLEQAQIQQEKNMYELQKLNSW      QIQQEKN
15. A peptide according to claim 12 characterized as having either of the following formulas
XEL--YK-V-I-P-G-APTK-KR-----Z     XYK-V-I-P-G-APTK-KRZ
in which X and Z are OH or NH2 or, to the extent that the immunological properties of the natural peptides lacking these groups shall not be essentially modified, the groups having from one to five amino acid residues, and each of the hyphens corresponding to an aminoacyl residue chosen from those which permit the conservation for the peptide characterized above of the immunological properties of either of the following peptide sequences:
ELGDYKLVEITPIGFAPTKEKRYSSAH     YKLVEITPIGFAPTKEK
16. A peptide according to claim 12 characterized as having either of the following formulas:
X----VTV-YGVP-WK-AT--LPCA-Z     XVTV-YGVP-WK-ATZ
in which X and Z are OH or NH2 or, to the extent that the immunological properties of the natural peptides lacking these groups shall not be essentially modified, the groups having from one to five amino acid residues, and each of the hyphens corresponding to an aminoacyl residue chosen from those which permit the conservation for the peptide characterized above of the immunological properties of one of the following peptide sequences:
CTQYVTVFYGVPTWKNATIPLFCAT     VTVFYGVPTWKNAT EKLWVTVYYGVPVWKEATTTLFCAS     VTVYYGVPVWKEAT
17. A peptide according to claim 16 characterized as having one of the following formulas:
CTQYVTVFYGVPTWKNATIPLFCAT     VTVFYGVPTWKNAT EKLWVTVYYGVPVWKEATTTLFCAS     VTVYYGVPVWKEAT EDLWVTVYYGVPVWKEATTTLFCAS     VTVYYGVPVWKEAT DNLWVTVYYGVPVWKEATTTLFCAS     VTVYYGVPVWKEAT
18. A peptide according to claim 12 characterized as having either of the following formulas:
X---QE--L-NVTE-F--W-NZ         XL-NVTE-FZ
in which X and Z are OH or NH2 or, to the extent that the immunological properties of the natural peptides lacking these groups shall not be essentially modified, the groups having from one to five amino acid residues, and each of the hyphens corresponding to an aminoacyl residue chosen from those which permit the conservation for the peptide characterized above of the immunological properties of one of the following peptide sequences:
DDYQEITL-NVTEAFDAWNN        L-NVTE PNPQEVVLVNVTENFNMWKN        LVNVTE
19. A peptide according to claim 18 characterized as having one of the following formulas:
DDYQEITL-NVTEAFDAWNN        L-NVTEAF PNPQEVVLVNVTENFNMWKN        LVNVTENF PNPQEIELENVTEGFNMWKN        LENVTEGF PNPQEIALENVTENFNMWKN        LENVTENF
20. A peptide according to claim 12 characterized as having one of the following formulas:
XL---S-KPCVKLTPLCV--KZ       XKPCVKLTPLCVZ     XS-KPCVKLTPLCVZ
in which X and Z are OH or NH2 or, to the extent that the immunological properties of the natural peptides lacking these groups shall not be essentially modified, the groups having from one to five amino acid residues, and each of the hyphens corresponding to an aminoacyl residue chosen from those which permit the conservation for the peptide characterized above of the immunological properties of one of the following peptide sequences:
ETSIKPCVKLTPLCVAMK DQSLKPCVKLTPLCVSLK     KPCVKLTPLCV   SLKPCVKLTPLCV
21. A peptide according to claim 20 characterized as having one of the following formulas:
ETSIKPCVKLTPLCVAMK DQSLKPCVKLTPLCVSLK DQSLKPCVKLTPLCVTLN      PCVKLTPLC
22. A peptide characterized as having either of the following formulas:
X---N-S-IT--C-Z    XN-S-ITZ
in which X and Z are OH or NH2 or, to the extent that the immunological properties of the natural peptides lacking these groups shall not be essentially modified, the groups having from one to five amino acid residues, and each of the hyphens corresponding to an aminoacyl residue chosen from those which permit the conservation for the peptide characterized above of the immunological properties of one of the following peptide sequences:
NHCNTSVITESCD    NTSVIT TSCNTSVITQACP    NTSAIT
23. A peptide according to claim 22 characterized as having one of the following formulas:
NHCNTSVITESCD    NTSVIT TSCNTSVITQACP    NTSVIT INCNTSVITQACP    NTSVIT INCNTSAITQACP    NTSAIT
24. A peptide according to claim according to claim 12 characterized as having the following formula:
XYC-P-G-A-L-C-N-TZ
in which X and Z are OH or NH2 or, to the extent that the immunological properties of the natural peptides lacking these groups shall not be essentially modified, the groups having from one to five amino acid residues, and each of the hyphens corresponding to an aminoacyl residue chosen from those which permit the conservation for the peptide characterized above of the immunological properties of either of the following peptide sequences:
YCAPPGYALLRC-NDT YCAPAGFAILKCNNKT
25. A peptide according to claim 24 characterized as having one of the following formulas:
YCAPPGYALLRC-NDT YCAPAGFAILKCNNKT
YCAPAGFAILKCNDKK YCAPAGFAILKCRDKK
26. A peptide according to claim 12 characterized as having the following formula:
X------A-C------W--Z
in which X and Z are OH or NH2 or, to the extent that the immunological properties of the natural peptides lacking these groups shall not be essentially modified, the groups having from one to five amino acid residues, and each of the hyphens corresponding to an aminoacyl residue chosen from those which permit the conservation for the peptide characterized above of the immunological properties of either of the following peptide sequences:
NKRPRQAWCWFKG-KWKD N--MRQAHCNISRAKWNA
27. A peptide according to claim 26 characterized as having one of the following formulas:
NKRPRQAWCWFKG-KWKT N--MRQAHCNISRAKWNA D--IRRAYCTINETEWDK I--IGQAHCNISRAQWSK
28. A peptide according to claim 12 characterized as having either of the following formulas:
X-G-DPE------NC-GEF-YCN-----NZ             XNC-GEF-YCNZ
in which X and Z are OH or NH2 or, to the extent that the immunological properties of the natural peptides lacking these groups shall not be essentially modified, the groups having from one to five amino acid residues, and each of the hyphens corresponding to an aminoacyl residue chosen from those which permit the conservation for the peptide characterized above of the immunological properties of one of the following peptide sequences:
KGSDPEVAYMWTNCRGEFLYCNMTWFLN             NCRGEFLYCN -GGDPEIVTHSFNCGGEFFYCNSTQLFN             NCGGEFFYCN
29. A peptide according to claim 28 characterized as having one of the following formulas:
KGSDPEVAYMWTNCRGEFLYCNMTWFLN             NCRGEFLYCN -GGDPEIVTHSFNCGGEFFYCNSTQLFN             NCGGEFFYCN -GGDPEITTHSFNCRGEFFYCNTSKLFN             NCRGEFFYCN -GGDPEITTHSFNCGGEFFYCNTSGLFN             NCGGEFFYCN
30. A peptide according to claim 12 characterized as having either of the following formulas:
X-----C-IKQ-I------G---YZ      XC-IKQ-IZ
in which X and Z are OH or NH2 or, to the extent that the immunological properties of the natural peptides lacking these groups shall not be essentially modified, the groups having from one to five amino acid residues, and each of the hyphens corresponding to an aminoacyl residue chosen from those which permit the conservation for the peptide characterized above of the immunological properties of one of the following peptide sequences:
RNYAPCHIKQIINTWHKVGRNVY      CHIKQII TITLPCRIKQFINMWQEVGKAMY      CRIKQFI
31. A peptide according to claim 30 characterized as having one of the following formulas:
RNYAPCHIKQIINTWHKVGRNVY      CHIKQII TITLPCRIKQFINMWQEVGKAMY      CRIKQFI SITLPCRIKQIINMWQKTCKAMY      CRIKQII NITLQCRIKQIIKMVAGR-KAIY      CRIKQII
32. The antigenic peptide gag1 characterized as having the following formula:
XNCKLVLKGLGMNPTLEEMLTAZ
in which X and Z are OH or NH2 or, to the extent that the immunological properties of the natural peptides lacking these groups shall not be essentially modified, the groups having from one to five amino acid residues, and each of the hyphens corresponding to an aminoacyl residue chosen from those which permit the conservation for the peptide characterized above of the immunological properties of the following peptide sequence:
XNCKLVLKGLGMNPTLEEMLTA
33. An antigenic composition containing at least one gag1 peptide according to claim 32 or at least an oligomer of this peptide, characterized as having the capacity to be recognized by human biological fluids such as serum containing anti-HIV-2 antibodies and under appropriate conditions anti-HIV-1 antibodies.
34. An antigenic composition containing at least one peptide according to claims 13, 14 or 15, or at least an oligomer of the peptide, characterized in that the peptide specifically recognizes the presence of anti-HIV-2 antibodies.
35. An immunogenic composition containing at least one peptide according to any one of the claims 16-31 or at least an oligomer of the peptide or the peptide conjugated with a carrier molecule, in association with an acceptable pharmaceutical vehicle for the production of vaccines, the composition characterized in that it induces antibody production against the peptide in sufficient quantities to form an effective immunocomplex with the entire HIV-2 retrovirus and its corresponding proteins.
36. An immunogenic composition according to claim 35 further comprising peptides having formulas corresponding to the envelope glycloprotein sequences of HIV-1 and HIV-2 which have an amino acid homology greater than 50%.
37. An immunogenic composition according to either of claims 35 or 36 having at least one peptide or at least an oligomer of the peptide or the peptide conjugated with a carrier molecule, the composition coresponding to a peptide chosen from the group consisting of Env4, Env5, Env6 and Env10.
38. A procedure for the in vitro diagnosis of HIV-2 infections in a biological fluid, comprising:
contacting the biological fluid with at least one peptide according to claims 12, 13, 14, 15 or 32, or a conjugate of the peptide with a carrier molecule;
detecting the eventual presence in the biological fluid of an antigen-antibody complex by physical or chemical methods.
39. The diagnostic procedure of claim 38, wherein the detection step is performed by a test selected by the group consisting of enzyme-linked immuno absorbent assay (ELISA), immunofluoresence assay (IFA), radioimmunoassay (RIA), and radioimmunoprecipitation assay (RIPIA).
40. A kit for the in vitro diagnosis of an HIV-2 infection in a biological fluid comprising:
a peptide composition containing a peptide according to claims 12, 13, 14, 15 or 32, or a mixture of such peptides, or a conjugate of such peptides with a carrier molecule;
an appropriate reaction environment for the production of an antigen-antibody complex;
one or more reagents adapted for the detection of the formation of antigen-antibody complexes; and
a biological fluid as a reference sample having no antibodies recognized by said peptide composition.
41. An protein selected from the group described in Example 4 consisting of p 16, p 26, p 12, polymerase, Q protein, R protein, X protein, Y protein, env protein, F protein, TAT, ART, U5 and U3.
42. A kit for diagnosing an HIV-2 infection by the method of claim 6 and comprising as the polypeptide expression product a protein of claim 41.
43. A vaccinating agent comprising at least one protein of claim 41 in association with appropriate carriers.
US09/988,213 1986-01-22 2001-11-19 Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits Abandoned US20030091985A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US09/988,213 US20030091985A1 (en) 1986-01-22 2001-11-19 Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits

Applications Claiming Priority (22)

Application Number Priority Date Filing Date Title
FR8600911A FR2593190B1 (en) 1986-01-22 1986-01-22 NEW RETROVIRUS THAT MAY CAUSE AIDS, ANTIGENS OBTAINED FROM THIS RETROVIRUS AND CORRESPONDING ANTIBODIES AND THEIR APPLICATIONS TO AIDS DIAGNOSIS
FR8600911 1986-01-22
FR8601635A FR2593922B1 (en) 1986-02-06 1986-02-06 NEW RETROVIRUS THAT MAY CAUSE AIDS, ANTIGENS OBTAINED FROM THIS RETROVIRUS AND CORRESPONDING ANTIBODIES AND THEIR APPLICATIONS TO AIDS DIAGNOSIS
FR8601635 1986-02-06
FR8601985A FR2594229B1 (en) 1986-02-13 1986-02-13 NEW LAV-II TYPE RETROVIRUS LIKELY TO CAUSE AIDS, ANTIGENS OBTAINED THEREFROM AND CORRESPONDING ANTIBODIES AND THEIR APPLICATIONS TO AIDS DIAGNOSIS
FR8601985 1986-02-13
US06/835,228 US4839288A (en) 1986-01-22 1986-03-03 Retrovirus capable of causing AIDS, antigens obtained from this retrovirus and corresponding antibodies and their application for diagnostic purposes
FR8603881 1986-03-18
FR8603881A FR2596063B1 (en) 1986-03-18 1986-03-18 NEW RETROVIRUS THAT MAY CAUSE AIDS, ANTIGENS OBTAINED FROM THIS RETROVIRUS AND CORRESPONDING ANTIBODIES AND THEIR APPLICATIONS TO AIDS DIAGNOSIS
FR8604215 1986-03-24
FR8604215A FR2597500B1 (en) 1986-03-24 1986-03-24 NEW RETROVIRUS THAT MAY CAUSE AIDS, ANTIGENS OBTAINED FROM THIS RETROVIRUS AND CORRESPONDING ANTIBODIES AND THEIR APPLICATIONS TO AIDS DIAGNOSIS
FRBF86.04.556 1986-03-28
FR8604556A FR2596414B1 (en) 1986-03-28 1986-03-28 HYBRIDOMAS OBTAINED FROM TRANSGENIC ANIMAL LYMPHOCYTES CARRYING A GENE EXPRESSING A SPECIFIED PROTEIN AND PROCESS FOR THE PREPARATION OF SUCH PROTEIN FROM SUCH HYBRIDOMAS
US91608086A 1986-10-06 1986-10-06
US93318486A 1986-11-21 1986-11-21
US07/003,764 US5051496A (en) 1986-01-22 1987-01-16 Peptides related to human immunodeficiency virus II (HIV-2)
US07/013,477 US5079342A (en) 1986-01-22 1987-02-11 Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits
US75236891A 1991-09-03 1991-09-03
US07/810,908 US6544728B1 (en) 1986-01-22 1991-12-20 Methods and kits for diagnosing human immunodeficiency virus type 2 (HIV-2), proteins of HIV-2, and vaccinating agents for HIV-2
US08/214,221 US5580739A (en) 1986-01-22 1994-03-17 Peptides of human immunodeficiency virus type 2 (HIV-2) and in vitro diagnostic methods and kits employing the peptides for the detection of HIV-2
US08/468,424 US6355789B1 (en) 1986-01-22 1995-06-06 Cloned dna sequences related to the entire genomic rna of human immunodeficiency virus ii (hiv-2), polypeptides encoded by these dna sequences and use of these dna clones and polypeptides in diagnostic kits
US09/988,213 US20030091985A1 (en) 1986-01-22 2001-11-19 Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US08/468,424 Continuation US6355789B1 (en) 1986-01-22 1995-06-06 Cloned dna sequences related to the entire genomic rna of human immunodeficiency virus ii (hiv-2), polypeptides encoded by these dna sequences and use of these dna clones and polypeptides in diagnostic kits

Publications (1)

Publication Number Publication Date
US20030091985A1 true US20030091985A1 (en) 2003-05-15

Family

ID=27581623

Family Applications (6)

Application Number Title Priority Date Filing Date
US08/214,221 Expired - Lifetime US5580739A (en) 1986-01-22 1994-03-17 Peptides of human immunodeficiency virus type 2 (HIV-2) and in vitro diagnostic methods and kits employing the peptides for the detection of HIV-2
US08/466,706 Expired - Lifetime US6048685A (en) 1986-01-22 1995-06-06 In vitro diagnostic assay employing HIV-2 antigens for the detection of HIV-2 specific antibodies
US08/468,424 Expired - Lifetime US6355789B1 (en) 1986-01-22 1995-06-06 Cloned dna sequences related to the entire genomic rna of human immunodeficiency virus ii (hiv-2), polypeptides encoded by these dna sequences and use of these dna clones and polypeptides in diagnostic kits
US08/466,707 Expired - Lifetime US6162439A (en) 1986-01-22 1995-06-06 Human immunodeficiency virus type 2 (HIV-2) polypeptides and methods of producing them
US08/468,774 Expired - Lifetime US5770703A (en) 1986-01-22 1995-06-06 Nucleic acids encoding peptides of the envelope region of HIV-2 and peptides, polypeptides, and methods for producing the peptides and polypeptides of the HIV-2 envelope gene
US09/988,213 Abandoned US20030091985A1 (en) 1986-01-22 2001-11-19 Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits

Family Applications Before (5)

Application Number Title Priority Date Filing Date
US08/214,221 Expired - Lifetime US5580739A (en) 1986-01-22 1994-03-17 Peptides of human immunodeficiency virus type 2 (HIV-2) and in vitro diagnostic methods and kits employing the peptides for the detection of HIV-2
US08/466,706 Expired - Lifetime US6048685A (en) 1986-01-22 1995-06-06 In vitro diagnostic assay employing HIV-2 antigens for the detection of HIV-2 specific antibodies
US08/468,424 Expired - Lifetime US6355789B1 (en) 1986-01-22 1995-06-06 Cloned dna sequences related to the entire genomic rna of human immunodeficiency virus ii (hiv-2), polypeptides encoded by these dna sequences and use of these dna clones and polypeptides in diagnostic kits
US08/466,707 Expired - Lifetime US6162439A (en) 1986-01-22 1995-06-06 Human immunodeficiency virus type 2 (HIV-2) polypeptides and methods of producing them
US08/468,774 Expired - Lifetime US5770703A (en) 1986-01-22 1995-06-06 Nucleic acids encoding peptides of the envelope region of HIV-2 and peptides, polypeptides, and methods for producing the peptides and polypeptides of the HIV-2 envelope gene

Country Status (1)

Country Link
US (6) US5580739A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7255996B2 (en) 2003-12-19 2007-08-14 Gen-Probe Incorporated Compositions, methods and kits for detecting the nucleic acids of HIV-1 and HIV-2
US9547029B1 (en) 2008-09-18 2017-01-17 Luc Montagnier System and method for the analysis of DNA sequences

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6541609B2 (en) 1985-11-14 2003-04-01 President And Fellows Of Harvard College HIV-2 peptides
US4839288A (en) * 1986-01-22 1989-06-13 Institut Pasteur Retrovirus capable of causing AIDS, antigens obtained from this retrovirus and corresponding antibodies and their application for diagnostic purposes
US6544728B1 (en) * 1986-01-22 2003-04-08 Institut Pasteur Methods and kits for diagnosing human immunodeficiency virus type 2 (HIV-2), proteins of HIV-2, and vaccinating agents for HIV-2
US6265149B1 (en) * 1986-01-22 2001-07-24 Institut Pasteur In vitro diagnostic methods and kits for the detection of HIV-2-specific antibodies
JP4166940B2 (en) * 1997-08-01 2008-10-15 バイオ−ラッド ラボラトリーズ，インコーポレイティド Synthetic antigens for detection of antibodies immunoreactive with HIV virus
US6228128B1 (en) 1997-11-10 2001-05-08 Charlotte Johansen Antimicrobial activity of laccases
US6984486B1 (en) 1999-02-19 2006-01-10 J. David Gladstone Institutes Synthetic peptide of regulatory virus protein R (VPR) of human immunodeficiency virus type 1 (HIV-1) and the utilization thereof
WO2000049038A2 (en) 1999-02-19 2000-08-24 Ulrich Schubert Synthetic peptide of regulatory virus protein r (vpr) of human immunodeficiency virus type 1 (hiv-1) and the utilization thereof
US7306919B1 (en) 2001-10-31 2007-12-11 Thornthwaite Jerry T Antigen-antibody cancer recognition system
KR100451565B1 (en) * 2002-04-17 2004-10-08 유지창 Protein for Binding to NC Protein of HIV-1
EP1800126B1 (en) * 2004-09-08 2014-12-17 The United States of America as represented by the Secretary of Health and Human Services, NIH Compositions and methods for the detection of hiv-1/hiv-2 infection
US8262657B2 (en) * 2007-08-16 2012-09-11 Nutek Orthopaedics, Inc. External fixation apparatus with adjustable pin clamping means and convergent bone pins
US9487837B2 (en) 2008-10-06 2016-11-08 Morehouse School Of Medicine Exosome-mediated diagnosis of hepatitis virus infections and diseases
US10545149B2 (en) * 2008-10-06 2020-01-28 Morehouse School Of Medicine Detection of HIV-related proteins in urine
WO2011119920A2 (en) 2010-03-25 2011-09-29 Oregon Health & Science University Cmv glycoproteins and recombinant vectors
CA2832109C (en) 2011-06-10 2021-07-06 Oregon Health & Science University Cmv glycoproteins and recombinant vectors
CA2789539A1 (en) 2011-09-12 2013-03-12 International Aids Vaccine Initiative Immunoselection of recombinant vesicular stomatitis virus expressing hiv-1 proteins by broadly neutralizing antibodies
EP2586461A1 (en) 2011-10-27 2013-05-01 Christopher L. Parks Viral particles derived from an enveloped virus
ES2631608T3 (en) 2012-06-27 2017-09-01 International Aids Vaccine Initiative Env-glycoprotein variant of HIV-1
US20150065381A1 (en) 2013-09-05 2015-03-05 International Aids Vaccine Initiative Methods of identifying novel hiv-1 immunogens
EP2873423B1 (en) 2013-10-07 2017-05-31 International Aids Vaccine Initiative Soluble hiv-1 envelope glycoprotein trimers
US10174292B2 (en) 2015-03-20 2019-01-08 International Aids Vaccine Initiative Soluble HIV-1 envelope glycoprotein trimers
EP3072901A1 (en) 2015-03-23 2016-09-28 International Aids Vaccine Initiative Soluble hiv-1 envelope glycoprotein trimers

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4629783A (en) * 1985-04-29 1986-12-16 Genetic Systems Corporation Synthetic antigen for the detection of AIDS-related disease
US4839288A (en) * 1986-01-22 1989-06-13 Institut Pasteur Retrovirus capable of causing AIDS, antigens obtained from this retrovirus and corresponding antibodies and their application for diagnostic purposes
US5079342A (en) * 1986-01-22 1992-01-07 Institut Pasteur Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits
US5670309A (en) * 1987-04-16 1997-09-23 Johnson & Johnson Methods and diagnostic kits for the detections of HIV-2-specific antibodies employing polypeptides obtained from the simian immunodeficiency virus

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4652599A (en) * 1984-04-23 1987-03-24 The United States Of America As Represented By The Department Of Health And Human Services Method of continuous production of retroviruses (HTLV-III) from patients with AIDS and pre-AIDS using permissive cells
US5306614A (en) * 1986-01-22 1994-04-26 Institut Pasteur Methods and kits for diagnosing human immunodeficiency virus type 2(HIV-2)
US5066782A (en) * 1986-01-22 1991-11-19 Institut Pasteur Retrovirus capable of causing AIDS, means and method for detecting it in vitro
DE3879881D1 (en) * 1987-11-16 1993-05-06 Hoffmann La Roche RECOMBINANT HIV-2 POLYPEPTIDE.

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4629783A (en) * 1985-04-29 1986-12-16 Genetic Systems Corporation Synthetic antigen for the detection of AIDS-related disease
US4839288A (en) * 1986-01-22 1989-06-13 Institut Pasteur Retrovirus capable of causing AIDS, antigens obtained from this retrovirus and corresponding antibodies and their application for diagnostic purposes
US5079342A (en) * 1986-01-22 1992-01-07 Institut Pasteur Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits
US5670309A (en) * 1987-04-16 1997-09-23 Johnson & Johnson Methods and diagnostic kits for the detections of HIV-2-specific antibodies employing polypeptides obtained from the simian immunodeficiency virus

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7255996B2 (en) 2003-12-19 2007-08-14 Gen-Probe Incorporated Compositions, methods and kits for detecting the nucleic acids of HIV-1 and HIV-2
US7666600B2 (en) 2003-12-19 2010-02-23 Gen-Probe Incorporated Cross-reactive primers for amplifying the nucleic acids of HIV-1 and HIV-2
US20100190149A1 (en) * 2003-12-19 2010-07-29 Gen-Probe Incorporated CROSS-REACTIVE HYBRIDIZATION PROBE FOR DETECTING HIV-1 AND HIV-2 NUCLEIC ACIDS IN THE p31 GENE SEQUENCE
US8318432B2 (en) 2003-12-19 2012-11-27 Gen-Probe Incorporated Cross-reactive hybridization probe for detecting HIV-1 and HIV-2 nucleic acids in the P31 gene sequence
US9547029B1 (en) 2008-09-18 2017-01-17 Luc Montagnier System and method for the analysis of DNA sequences
US9910013B1 (en) 2008-09-18 2018-03-06 Luc Montagnier System and method for the analysis of DNA sequences

Also Published As

Publication number Publication date
US6048685A (en) 2000-04-11
US6355789B1 (en) 2002-03-12
US5580739A (en) 1996-12-03
US6162439A (en) 2000-12-19
US5770703A (en) 1998-06-23

Similar Documents

Publication Publication Date Title
US5051496A (en) Peptides related to human immunodeficiency virus II (HIV-2)
US5079342A (en) Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits
US6544728B1 (en) Methods and kits for diagnosing human immunodeficiency virus type 2 (HIV-2), proteins of HIV-2, and vaccinating agents for HIV-2
US6518015B1 (en) Peptide comprising the sequence CAFRQVC and methods using it
US6355789B1 (en) Cloned dna sequences related to the entire genomic rna of human immunodeficiency virus ii (hiv-2), polypeptides encoded by these dna sequences and use of these dna clones and polypeptides in diagnostic kits
US5545726A (en) Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits
US6979535B2 (en) Human immunodeficiency virus type 2 (HIV-2) env polypeptide and diagnostic assays
JP2611106B2 (en) A new retrovirus that causes AIDS
US5066782A (en) Retrovirus capable of causing AIDS, means and method for detecting it in vitro
US6296807B1 (en) Kits for the detection of human immunodeficiency virus type 2 (HIV-2) antigens
US5306614A (en) Methods and kits for diagnosing human immunodeficiency virus type 2(HIV-2)
US7115363B1 (en) Retrovirus capable of causing AIDS, means and methods for detecting it in vitro
US5830641A (en) In vitro diagnostic assays for the detection of HIV-1 or HIV-2 employing viral-specific antigens and antibodies
US5858651A (en) Nucleotide sequences of human immunodeficiency virus type 2 (HIV-2), probes of HIV-2, and methods of using these probes
US5268265A (en) Immunological 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 SIV
US6429306B1 (en) Nucleic acids of a human immunodeficiency virus type 2 (HIV-2)
US20030186219A1 (en) Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits
CA1338323C (en) Retrovirus capable of causing aids, means and methods for detecting it in vitro

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION