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WO1996036699A1 - Souches non pathogenes de vih-1 et procedes pour les detecter - Google Patents

Souches non pathogenes de vih-1 et procedes pour les detecter Download PDF

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Publication number
WO1996036699A1
WO1996036699A1 PCT/AU1996/000301 AU9600301W WO9636699A1 WO 1996036699 A1 WO1996036699 A1 WO 1996036699A1 AU 9600301 W AU9600301 W AU 9600301W WO 9636699 A1 WO9636699 A1 WO 9636699A1
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seq
hiv
nef
pathogenic
strain
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PCT/AU1996/000301
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English (en)
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Dale Alan Mcphee
Nicholas John Deacon
Alison Louise Greenway
John Mills
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Actract Pty. Limited
Macfarlane Burnet Syndicate No.1 Pty. Ltd.
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Priority to AU56400/96A priority Critical patent/AU5640096A/en
Publication of WO1996036699A1 publication Critical patent/WO1996036699A1/fr

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    • 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
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • 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
    • 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
    • C12N2740/16311—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
    • 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
    • C12N2740/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates generally to non-pathogenic strains of HIN-1 and methods for detecting same.
  • the methods of the present invention are particularly useful in determining the likelihood of an individual who is seropositive for HIN-1 developing AIDS or AIDS-related symptoms.
  • the present invention further contemplates therapeutic compositions for the treatment and prophylaxis of AIDS and ALDS-related disorders.
  • Another aspect of the present invention is directed to strains of HIN-1 capable of synthesising a modified ⁇ ef protein or having a wild-type ⁇ ef protein modified after synthesis thereby rendering those strains of HIN- 1 substantially non-pathogenic.
  • Genomic nucleotide sequences of HIN-1 strains referred to herein are represented by their corresponding D ⁇ A sequence.
  • Exemplary viral isolates referred to herein as "C18” and “C98” were deposited at the PHLS Centre for Applied Microbiology and Research, European Collection of Animal Cell Cultures (ECACC), Division of Biologies, Porton Down, Salisbury, Wiltshire SP4 OJG. C18 was deposited on 17 October, 1994 under Provisional Accession Number V94101706 and C98 was deposited on 31 October, 1994 under Provisional Accession Number V941031169. Viral isolate "C54” was deposited at ECACC on 10 March, 1995 under Provisional Accession Number V95031022.
  • HIV-1 Human Immunodeficiency Virus type 1 (HIV-1) (Barre-Sinoussi et al, 1983). Infection by HIV-1 is generally characterised by progressive immune system damage (Teeuwsen et al, 1990; Clerici et al, 1989) leading to opportunistic infections, malignancies or wasting syndrome that constitute clinically-defined AIDS (Busch et al, 1991 ; Klaslow et al, 1990).
  • HIV-1 Human Immunodeficiency Virus type 1
  • AZT is not an innocuous compound and AZT, metabolic products thereof or impurities therein can cause a number of side effects which limit long term treatment with the drug. Furthermore, AZT resistant isolates have been reported during treatment. Clearly, therefore, a need exists to develop alternative strategies in preventing, treating and diagnosing HIV-1 infection.
  • HTN-1 infection The initial phases of HTN-1 infection are summarised by Levy (1993) as involving attachment, fusion and nucleocapsid entry. These phases have been the traditional foci in research into development of antiviral strategies.
  • the molecular events at the virus genomic level have also been the subject of intense scientific research with an aim being the development of a live attenuated vaccine as a possible approach for the treatment or prophylaxis of HIV- 1 infection.
  • There is a high variable rate of progression from initial HIV-1 infection to AIDS which reflects a rapidly changing pathogen and variable immune response of the host to infection (Sheppard et al, 1993). With regards to the latter, HIV-1 can be considered as a heterogenous group of viruses differing at the genetic level with concomitant variable pathogenicity.
  • HIV-1 strains can differ in their capacity to kill cells. Furthermore, it appears that HIV-1 strains evolve in a host after infection and that the evolution varies depending on the tissues infected by the virus.
  • the major sites in the genome apparently responsible for biological and pathological variation are the highly variable envelope region (Cheng-Mayer et al, 1991; Shioda et al, 1992; Hwang, et al 1991; Sullivan et al, 1993; Groenink et al, 1993) and the viral regulatory regions such as Tat (Leguern et al, 1993).
  • the genetic complexity of the HIN- 1 group of viruses together with their variable pathogenicity, are major difficulties in the development of live vaccines, genetic vaccines or component vaccines.
  • Kestler et al (1991) found that the nef gene is required for full pathogenic potential in SIV. However, such is the complexity of HIV- 1 and the variability of immune responses between individuals let alone different species, that it is far from clear whether He/deleted strains of HIV- 1 would behave similarly to nef deleted strains of SIV-1. There is a need, therefore, in order to investigate the possibility of a «e/deleted FUN-l strain as a vaccine candidate, to identify individuals infected with such modified viruses.
  • the present invention arose in part from an investigation of the immunological profiles of those individuals infected with the non-pathogenic HIN-1 strains.
  • a non-pathogenic strain of HTV-l is indicated by a particular deletion in the nef coding region which results in an altered immunological profile for the expressed protein.
  • one aspect of the present invention is directed to an isolated strain of HIN-1 or a biological source thereof, said HIN-1 having the following characteristics: (i) is substantially non-pathogenic in human subjects; and
  • (ii) carries a modified nef gene which encodes a nef gene product substantially immunologically non-interactive with antibodies to amino acids 162 to 177 of ⁇ ef in wild-type HI V-l.
  • H-V-I ⁇ L ⁇ Amino acids of 162-177 of wild-type H-V-I ⁇ L ⁇ strain (Myers et al, 1994) [hereinafter referred to as "HIV-I ⁇ L ⁇ "] are as follows: Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro Glu [SEQ ID NO: 1].
  • the present invention relates in part to this particular amino acid sequence (SEQ ID NO: l) from HIV- ln ⁇ or from the functionally equivalent region of other pathogenic strains of HIV- 1.
  • HIV-l an isolated strain of HIV-l or a biological source thereof, said HIV-l having the following characteristics: (i) is substantially non-pathogenic in human subjects; and (ii) encodes an Nef protein or portion thereof which is interactive with wild-type HIV-l Nef antibodies but which is substantially non-interactive with antibodies to amino acids 162 to 177 of wild-type HIV-l Nef protein.
  • Still another aspect of the present invention relates to an isolated strain of HIV- 1 or a biological source thereof which is substantially non-pathogenic in human subjects and which is substantially incapable of directing synthesis of a Nef protein or portion thereof comprising amino acids 162 to 177 of wild-type HtV-1 Nef protein.
  • an isolated strain of HIN-1 or a biological source thereof said HIN-1 being substantially non-pathogenic in humans and comprising a mutation in its genome, corresponding to amino acids 162 to 177 of wild-type HIN-1 ⁇ ef, such that these amino acids are substantially not represented in a ⁇ ef protein or derivative thereof produced by said isolated HIN-1 strain, or insufficient of the amino acid sequence to induce an immune response to that region of ⁇ ef.
  • the genomic mutation in the non-pathogenic strain of HIV-l is a mutation in one or more of nucleotides 9271 to 9317 relative to HIV- 1 ⁇ 3 or in a functionally equivalent region in another pathogenic strain of HIV-l.
  • a non-pathogenic strain of HIV-l comprising a genome which is substantially incapable of hybridising under low stringent conditions at 42 °C a nucleic acid molecule comprising that sequence of nucleotides which encodes all or part of amino acids 162 to 177 of wild-type HIN-l.
  • the nucleic acid molecule is a synthetic oligonucleotide.
  • Alternative stringency conditions such as medium or high may also be employed.
  • the present invention provides non-pathogenic HIV-l isolate C 18 deposited at the ECACC on 17 October, 1994 under Provisional Accession Number V94101706. 7 -
  • the present invention provides non-pathogenic HIV-l isolate C98 deposited at the EC ACC on 31 October, 1994 under Provisional Accession Number V941031169.
  • the present invention provides non-pathogenic HIV-l isolate C54 deposited at ECACC on 10 March, 1995 under Provisional Accession No. V95031022.
  • HTV-1 isolates or biological sources thereof are referred to herein as D36, C49, C64 and C124.
  • a non-pathogenic strain of HIV-l is a strain which, at the clinical level, does not lead to the development of AIDS, at least within the median time of 6-10 years following infection with HIV-l .
  • a non-pathogenic strain of HIV-l is considered not to alter CD4+ cell counts or ⁇ 2 -microglobulin concentrations in the infected individual.
  • a non-pathogenic strain of HIV-l may not alter CD8+ and CD3+ cell counts and would not alter lymphocyte counts.
  • CD4+:CD8+ ratios also remain unchanged relative to normal non-infected individuals. Furthermore, generally, a non-pathogenic strain of HIV-l does not induce p24 antigenaemia. A non-pathogenic HIV-l of the present invention is generally still infectious but those infected with the virus remain free of symptoms for at least 6-10 years after infection.
  • a laboratory classified non-pathogenic strain of HIV-l may be determined at any time after infection.
  • the term "non-pathogenic” is not to be considered as a strain that is never pathogenic under any conditions as this might depend on the host individual, the level of immune responsiveness in that individual and the extent or otherwise of other, for example, immune compromising disorders. Accordingly, a "non-pathogenic" HIV-l isolate of the present invention may also be considered a "low virulent" strain of the virus.
  • a non-pathogenic strain of HIV-l as contemplated herein may be isolated from an asymptomatic individual or may be derived from a pathogemc strain by mutation.
  • an example of a pathogenic strain is HIV-I NL ⁇ as described by Myers et al (1994).
  • the non-pathogenic nature of the HIV-l strains of the present invention is conveniently evidenced by the cohort of seven individuals comprising one donor and six recipients which have remained free of symptoms or signs of HIV-l infection for greater than the median time of 6-10 years.
  • the individuals of the cohort are seropositive and virus positive for HIV-l following infection with the virus as determined by Western blot analysis and genetic analysis (e.g. using PCR techniques).
  • a seropositive individual is one showing reactivity to at least one HIV-l glycoprotein (such as but not limited to gp 41-45, gpl20, gpl ⁇ O) and at least three other virus-specific bands by immunoblot.
  • a virus positive individual is an individual containing virus nucleic acid sequence as determined by, for example, genetic analysis.
  • a non-pathogenic HIV-l isolate is also a strain of HIV-l which:
  • the immune response is to a glycoprotein, for example, gp41-45, gpl20 and/or gpl60.
  • the cytokine monitored is an interleukin, such as IL-2.
  • the recall antigen is influenza or tetanus toxoid.
  • a non-pathogenic HIV-l isolate is also one which: (iv) does not substantially alter proliferative responses or cytokine production to allogeneic mononuclear cells.
  • Reference to a "wild-type HIV-l” is meant to include reference to architypal pathogenic HIN-1 NL43 (Myers etal, 1994).
  • Reference to a biological source includes blood or blood-related products or components such as lymphocytes, plasma, tissue fluid and tissue extracts.
  • Reference herein to a low stringency at 42 °C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridisation, and at least about 1M to at least about 2M salt for washing conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions.
  • medium stringency which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions
  • high stringency which includes and encompasses from at least about 31% v/v to at least about 50% v/v form
  • the non-pathogenic strain of HTN-1 carries a mutation in the nef gene such that a ⁇ ef protein is not produced or a modified ⁇ ef protein is produced substantially not carrying amino acids 162-177 or a portion thereof of wild-type HIV- l ⁇ ef.
  • a “mutation” is considered herein to include a single or multiple nucleotide substitution, deletion and/or addition. Most preferred mutations are single or multiple deletions of at least one, most preferably at least ten and even more preferably at least twenty contiguous nucleotides from a region corresponding to amino acids 162-177 of the ⁇ ef protein.
  • a viral isolate which: (i) is interactive to antibodies to a glycoprotein from HIV-l such as at least one of gp41- 45, gp 120 and/or gp 160; (ii) is substantially non-pathogenic in human subjects; and
  • (iii) carries a deletion mutation of at least ten nucleotides in a region corresponding to all or part of amino acids 162 to 177 encoded by the nef gene of a pathogenic strain of HIV- 1.
  • the nucleotide sequence of the nef gene in wild-type HIV-I NL ⁇ is as follows:
  • nucleotide sequence encoding amino acids 162-177 of wild-type HIV-I HL ⁇ Nef is as follows:
  • Exemplary decanucleotide deletions in nef include the following:
  • TACACCCTGT [SEQ ID NO:14] ACACCCTGTG [SEQ ID NO:15] CACCCTGTGA [SEQ ID NO:16] ACCCTGTGAG [SEQ ID NO:17]
  • CTGTGAGCCT [SEQ ID NO:20] TGTGAGCCTG [SEQ ID NO:21]
  • GAGCCTGCAT [SEQ ID NO:24] AGCCTGCATG [SEQ ID NO:25] GCCTGCATGG [SEQ ID NO:26] CCTGCATGGA [SEQ ID NO:27] CTGCATGGAA [SEQ ID NO:28] TGCATGGAAT [SEQ ID NO:29]
  • the present invention extends to any or all single or multiple nucleotide deletions to a contiguous series of at least ten nucleotides from the region of the we/gene corresponding to all or part of amino acids 162 to 177 and which render the strain substantially non-pathogenic.
  • the deletions may encompass the entire gene or parts thereof and may represent a single deletion or two or more deletions.
  • the non-pathogenic HIV-l isolates of the present invention comprise a nucleotide sequence at a region corresponding to the wild- type nef gene which is non-identical to SEQ ID NO:3, and where the non-identity is at least the 5%, more preferably at least the 10% and even more preferably at least the 20% level.
  • the present invention contemplates a viral isolate which: (i) is reactive to antibodies to a glycoprotein from HIV-l such as at least one of gp41-45, gp 120 and/or gp 160;
  • CAGCTTGTTA [SEQ ID NO:6] AGCTTGTTAC [SEQ ID NO:7]
  • CCCTGTGAGC [SEQ ID NO :18] CCTGTGAGCC [SEQ ID NO :19] CTGTGAGCCT [SEQ ID NO :20] TGTGAGCCTG [SEQ ID NO .21]
  • the subject HIV-l isolate is non-pathogenic as hereinbefore defined.
  • reference herein to "a deletion" includes reference to a contiguous or non-contiguous series of two or more deletions.
  • the non-pathogenic isolate may carry a single decanucleotide deletion or may carry more than one decanucleotide deletion. Where it carries multiple deletions these may all correspond to a contiguous sequence or be from different parts of the nef gene. Furthermore, the terminal end portions of a deletion may lie within a decanucleotide as defined above. It is emphasised that the present invention extends to analogous sequences from other pathogenic strains of HIV-l which might carry nef genes with a slightly altered sequence relative to HIV- 1 ⁇ 3 .
  • the non-pathogenic isolates of the present invention may further comprise a mutation in the LTR region of the viral genome. Such a mutation may be in the form of a single or multiple nucleotide deletions, additions and/or substitutions to a single contiguous region of the LTR or in two or more non-contiguous regions of the LTR.
  • Yet another aspect of the present invention provides an infectious molecular clone comprising genetic sequences derived from the non-pathogenic HIV-l isolates as hereinbefore described and includes genetic sequences encoding major structural proteins such as gag, env and pol. Infectious molecular clones are particularly useful as genetic compositions capable of "infecting" host cells without need of viral coat.
  • the infectious molecular clones of the present invention may also be derived from pathogenic HIV-l strains rendered non-pathogenic as hereindescribed.
  • a method of attenuating a pathogenic strain of HIV-l comprising inducing a mutation in the nef gene to generate a non-pathogenic HIV-l strain as hereinbefore described.
  • Preferred mutations result in a modified Nef protein carrying a deletion of amino acids 162 to 177.
  • the mutations may also constitute substitutions and/or insertions of heterologous nucleic acid molecules in the nef region encoding amino acids 162 to 177 such as the incorporation of a sense (i.e. co- suppression), antisense or ribozyme molecule.
  • a method for screening for a compound capable of disrupting transcription of nef at or about nucleotides 9271 to 9317 comprising contacting a compound to be tested with a genetic construct capable of expressing the nef gene and screening translation products for disruption at a portion of Nef corresponding to amino acids 162 to 177.
  • a further related embodiment contemplates a method for screening for a compound capable of binding to and/or disrupting function of amino acids 162 to 177 of Nef, said method comprising contacting a compound to be tested with recombinant Nef and identifying compounds which interact with amino acids 162 to 177 or in close proximity thereto.
  • non-pathogenic HIV-l strains of the present invention includes reference to components, parts, fragments and derivatives thereof including both genetic and non-genetic material.
  • the non-pathogenic HIV-l strains may be in isolated from or resident in peripheral blood mononuclear cells (PBMCs) or other like cells where the genome of the HIV-l strains is integrated as DNA from said HIV-l strains such as proviral DNA.
  • PBMCs peripheral blood mononuclear cells
  • the present invention extends to recombinant virus such as from (or resident in) prokaryotes or eukaryotes as well as in the form of infectious molecular clones.
  • the present invention provides for the non-pathogenic HIV-l isolate, genomic material therefrom, complementary proviral DNA, molecular infectious clones, recombinant viral particles or genetic sequences therefrom or cells expressing same or blood cells carrying proviral DNA or to any mutants, derivatives, components, fragments, parts, homologues or analogues of the foregoing.
  • Another aspect of the present invention contemplates a synthetic peptide comprising a sequence of amino acids as defined in SEQ ID NO: 1 or a part or a fragment thereof.
  • the synthetic peptide comprises at least four, preferably at least five, more preferably at least six and even more preferably at least seven or more of the amino acids as defined in SEQ ID NO: 1.
  • the synthetic peptides may comprise chemically modified amino acids or structurally, functionally and/or stereochemically equivalent substitute amino acids. Such synthetic peptides are useful in diagnostic protocols or in therapeutic volums, such as in generating antibodies to that particular region of the Nef protein.
  • non-pathogenic HIV-l strains of the present invention are particularly useful in the development of therapeutic compositions, therapeutic molecules and/or diagnostic reagents. With regards to the former, the non-pathogenic HIV-l strain may be considered as a live attenuated vaccine where individuals carrying DNA derived from said non-pathogenic HIV-l strain such as proviral DNA in target cells are protected from infection by a corresponding pathogenic strain.
  • vaccine is used in its broadest sense as a therapeutic composition or molecule which prevents or reduces HIV-l infection or risk of infection or which ameliorates the symptoms of infection. It may involve the stimulation of an immune response or may involve blocking HIN-1 cells receptors and/or the use of genetic compositions, for example, to introduce ribozymes or antisense molecules to HTN- 1 directed genetic sequences or to prepare infectious molecular clones. For convenience, all such compositions are referred hereinafter to "therapeutic compositions”.
  • the present invention contemplates a method for inhibiting or reducing the risk of infection by a pathogenic strain of FflN- 1 , said method comprising administering to a subj ect a non-pathogenic HIV-l as hereinbefore defined in an amount effective to infect target cells and to generate target cells carrying proviral D ⁇ A from said non-pathogemc HIN-1. More particularly, the present invention contemplates a method for inhibiting or reducing productive infection of an individual by a pathogenic strain of HIV-l, said method comprising administering to a subject a non-pathogenic isolate of HIV-l in an amount effective to infect target cells and to generate target cells carrying proviral D ⁇ A from said non-pathogenic HIV-l .
  • productive infection as used in the specification and claims herein is meant the infection of a cell or cells by a pathogenic strain of HIV-l which leads ultimately to the symptomology of AIDS or AIDS related diseases.
  • infection of an individual by a non-pathogenic strain of HIV-l would not lead to productive infection.
  • ⁇ on pathogenic HIV-l strains generally replicate to a sufficient extent to protect cells against challenge by virulent or pathogenic strains.
  • the methods of the present invention are also applicable prophylactically (i.e. to prevent de novo infection) or therapeutically (i.e. to reduce or slow disease progression).
  • a method for treating a human subject infected with a pathogenic strain of HIN-1 comprising administering to said subject an effective amount of a non-pathogenic isolate of HIN-1 which encodes a modified ⁇ ef protein which is substantially non-interactive to antibodies to amino acids 162 to 177 to wild type HIV-l ⁇ ef.
  • a further embodiment contemplates a method of treating a condition in a human, said condition associated with or induced by infection by HIV-l, said method comprising administering to said human an effective amount of a therapeutic agent capable of disrupting transcription of the nef gene or translation of the ⁇ ef product or activity of the ⁇ ef product.
  • Still another embodiment provides a therapeutic agent wherein the therapeutic agent targets amino acids 162 to 177 on ⁇ ef or nucleotides 9271 to 9317 of nef.
  • the present invention further provides a method for vaccinating an individual against the development of AIDS or AIDS related diseases, said method comprising administering to said individual a non-pathogenic isolate of HIV-l in an amount effective to infect target cells and to generate target cells carrying proviral D ⁇ A from said non-pathogenic HIV-l.
  • the term "vaccinating” should not be taken as hmiting the invention to the prevention of HIV-l infection by solely immunological means.
  • the term “vaccinating” includes any means of preventing productive infection of an individual by pathogenic HIN- 1.
  • the non-pathogenic HIN- 1 strains according to these aspects of the invention are generally defined as encoding a modified ⁇ ef protein which is substantially non-interactive to antibodies to amino acids 162 to 177 of wild- type HIN ⁇ ef.
  • a therapeutic composition as hereinbefore defined is administered.
  • the non-pathogenic isolate may be administered inter alia as an isolated viral preparation or via infected blood cells.
  • Another aspect of the invention provides a therapeutic composition for inhibiting or reducing the risk of infection by a pathogenic strain of HIV-l said therapeutic composition comprising a non-pathogenic strain of HIV-l or genetic sequences derived therefrom as hereinbefore described and optionally one or more pharmaceutically acceptable carriers and/or diluents.
  • the therapeutic compositions comprise the synthetic peptides comprising an amino acid sequence as set forth in SEQ ID NO: l or a part, fragment or homologue thereof.
  • a vaccine would generate high titre antibodies to a specific region of Nef protein.
  • the therapeutic composition of the present invention is generally suitable for intravenous, intraperitoneal, intramuscular, intramucosal (e.g. nasal spray, respiratory spray) or other forms of parenteral administration.
  • the therapeutic composition might also be administered via an implant or rectally or orally.
  • the non- pathogenic HIV-l strain may also contain one or more other mutations to further reduce the risk of reversion to virulence and/or to insert a genetic sequence capable of providing directly or indirectly an identifiable signal, having further anti-HTV-1 properties and/or immunostimulatory or cell regulatory properties.
  • the non-pathogenic HIV-l isolate in the therapeutic composition may comprise additional genetic material capable of directing expression of antisense nucleotide sequences to inhibit expression of one or more proteins encoded by a pathogenic strain of HIV-l.
  • sense co-suppression may be employed. Preferred sense or antisense molecules would reduce expression of the nef gene and in particular would target the nucleotide sequence encoding amino acids 162 to 177 of Nef.
  • the non-pathogenic HIV-l strain may be considered as a targeting agent to introduce genetic constructs capable of reducing expression of one or more HIV-l proteins or polypeptides.
  • a viral isolate which: (i) is genetically or immunologically related to a pathogenic strain of HIV-l ; (ii) is substantially non-pathogenic in human subjects; and (iii) comprises a nucleotide sequence which directs expression of a mRNA molecule capable of inhibiting, reducing or otherwise down-regulating translation and expression of Nef.
  • the nucleotide sequence reduces levels of amino acids 162 to 177 in Nef.
  • the above aspect relates to use of antisense technology.
  • the present invention extends, however, to use of ribozymes and/or co-suppression to achieve the same results.
  • the present invention provides a means for designing therapeutic compositions directed to inhibiting expression of a nef gene in a pathogenic HIV-l strain (such as contemplated above) as well as developing a therapeutic regime aimed at inhibiting the activity of the nef gene product.
  • the present invention provides a therapeutic composition comprising a molecule capable of inhibiting the intracellular activity of the nef gene product, said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents.
  • the inhibition affects amino acids 162 to 177 of Nef.
  • the molecule contemplated by the above aspect of the subject invention may be a protein, polypeptide, peptide, chemical compound, sugar moiety or derivative of the nef gene product.
  • the molecule will need to be capable of entering an infected cell.
  • the molecule may be encoded by a second nucleotide sequence on a targeting vector.
  • the molecule may be a nucleic acid molecule capable of targeting the nef gene or a portion thereof which encodes amino acids 162 to 177 of Nef.
  • the deletion mutants of the present invention may result in a modified nef gene product either having no readily discernable activity or having activity different to the naturally occurring Nef protein.
  • a mutant nef gene product if a mutant nef gene product is produced, it will generally have a lower molecular weight than the naturally occurring Nef protein and will have a different overall amino acid sequence.
  • it will be immunologically distinguishable from wild-type Nef in that it will be substantially non-interactive with antibodies to amino acid 162 to 177 of wild-type Nef. This provides, therefore, for a means for diagnosing individuals with benign HIV-l infection by, for example, assaying for a modified Nef protein or screening for a modified nef ' gene sequence.
  • a method for determining the pathogenicity of an HIV-l strain after said HIN-1 strain infects cells of an individual comprising contacting a biological sample from said individual with an effective amount of an antibody specific to a protein from a non-pathogenic strain of HIN- 1 (as hereinbefore defined) for a time and under conditions sufficient to form an antibody-modified ⁇ ef protein complex and then detecting said complex.
  • the antibody is specific to amino acids 162 to 177 and a non-pathogenic HIN-1 strain would be substantially non-interactive to such an antibody.
  • the present invention contemplates a method for determining the pathogenicity of an HIV-l strain after said HIN-1 strain infects cells of an individual, said method comprising contacting a biological sample from said individual with an effective amount of an antibody specific to amino acids 162 to 177 of wild-type HIV-l ⁇ ef then detecting the presence of a complex between said antibody and ⁇ ef wherein the substantial absence of binding is indicative of the H1N-1 strain being non-pathogenic.
  • a method for determining whether an individual is infected with a pathogenic strain of HIN-1 comprising contacting a biological sample from said individual with a binding effective amount of an antibody specific for amino acids 162 to 177 of wild type HIV-l ⁇ ef and detecting the presence or absence of binding of said antibody wherein the absence of binding of said antibody is indicative of a non- pathogenic strain of HIN-1.
  • the presence or absence of said complex would be indicative of a modified we/gene product and of the non-pathogenicity of the strain of HIV-l.
  • the biological sample is a sample likely to contain the modified nef gene product such as tissue extract or cell extract of an infected cell.
  • suitable biological fluid would include serum, whole blood, lymph and mucosal secretion amongst other fluids.
  • an assay could be based on the inability for a we/specific antibody to bind to a modified nef protein.
  • the term "contacting" includes "mixing".
  • a modified Nef molecule in biological fluid can be detected using a wide range of immunoassay techniques such as those described in US Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These methods may be employed for detecting antibody interaction with a Nef protein or derivative thereof or for screening for the substantial absence of binding such as an antibody to amino acids 162 to 177 of Nef.
  • a modified Nef product-specific antibody is immobilised onto a solid substrate to form a first complex and the sample to be tested for the presence of a modified Nef product brought into contact with the bound molecule.
  • a second modified nef protein antibody labelled with a reporter molecule capable of producing a detectable signal
  • a reporter molecule capable of producing a detectable signal
  • this assay includes a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody, or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and then added simultaneously to the bound antibody.
  • simultaneous assay in which both sample and labelled antibody are added simultaneously to the bound antibody
  • reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and then added simultaneously to the bound antibody.
  • the antibodies used above may be monoclonal or polyclonal.
  • the antibody is specific to amino acids 162 to 177 of Nef and absence of antibody binding to a modified Nef protein would be indicative of a non- pathogenic strain of HIV-l.
  • the solid substrate is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the molecule to the insoluble carrier.
  • reporter molecule is meant a molecule which, by its chemical nature, produces an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecule in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes).
  • an enzyme immunoassay an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to one skilled in the art.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, ⁇ -galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. It is also possible to employ fluorogenic substrates, which yield a fluorescent product.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescent compounds may be chemicall coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illuminatio with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the ligh energy, inducing a state of excitability in the molecule, followed by emission of the light at characteristic colour visually detectable with a light microscope.
  • the fluorescen labelled antibody is allowed to bind to the first antibody-hapten complex. After washing of the unbound reagent, the remaining complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method.
  • other reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed. It will be readily apparent to the skilled technician how to vary the procedure to suit the required purpose. It will also be apparent that the foregoing can be used to label a modified ne/product and to use same directly in the detection of, for example, circulatory antibodies specific to said modified «e/product.
  • the Nef protein or part thereof or a peptide corresponding to all or part of SEQ ID NO: 1 is immobilised onto a solid support and biological fluid potentially containing antibodies to Nef or regions thereof brought into contact with the immobilized molecules. After a suitable incubation period, the presence or absence of antibody binding to the immoblized molecule is determined.
  • a method of determining whether an individual is infected with a pathogenic strain of HIV-l comprising immobilising a peptide comprising all or a portion of SEQ ID NO: 1 and contacting said immobilised peptide with a blood or serum sample from said individual and screening for antibody binding to said immobilised peptide, wherein the substantial absence of antibody binding is indicative of a non-pathogenic strain of HIV-l.
  • Genetic assays may also be conducted to screen for abberations in the «e/gene and in particular the region encoding amino acids 162 to 177.
  • Such a genetic assay may be by Southern or Northern blot analysis, PCR analysis or the like using oligonucleotides specific to a deleted region of a nef gene and/or LTR region.
  • a method for determining the pathogenicity of an HIN-1 strain after said HIV-l strain infects cells of an individual comprising determining directly or indirectly the presence of a mutation such as a deletion mutation in the genome of said HIV-l wherein the presence of a such a mutation is indicative of the presence of a non-pathogenic strain of HIV-l .
  • a method of determining whether an individual is infected with a pathogenic strain of HIN-1 comprising contacting D ⁇ A putatively containing HIV-l D ⁇ A from said individual with an oligonucleotide probe specific for nucleotides 9271 to 9317 of wild-type HIV-l and screening for the substantial absence of hybridisation wherein said substantial absence of hybridisation is indicative of a non-pathogenic strain of HIV-l .
  • Preferred oligonucleotide probes encompass a decanucleotide selected from those set forth in SEQ ID ⁇ O:4 to SEQ ID NO:42, inclusive.
  • the deletion mutation may result in the genome being unable to synthesise a polypeptide or protein from a pathogenic strain of HIV-l or may direct the synthesis of a truncated form of said polypeptide or protein.
  • the mutation may also lead to altered expression of a polypeptide detectable by, for example, decreased synthesis of a particular protein, such as the nef gene product.
  • said non-pathogenic HIV-l carries a deletion in its genome of at least 10 nucleotides resulting in production of a modified Nef protein which does not comprise amino acids 162 to 177.
  • kits for the diagnosis of infection by pathogenic strains of HIV-l or for determining the pathogenicity of infecting virus would be in compartmental form each comprising one or more suitable reagents for conducting the assay.
  • Figure 1 shows reactivity of sera from LTP individuals (la); HIV-l-ve individuals (lbi, lbii); individuals with autoimmune disease (A/HIN-1) (lbiii); LT ⁇ P1 (lc) and LT ⁇ P2 (Id) with full length Nef 27 derived from H-V-l ⁇ .
  • Figure 2a shows reactivity of sera from LTP individuals against Nef-derived peptides.
  • Synthetic peptides corresponding to amino acid residues 1 to 19 (i), 20 to 36 (ii), 44 to 65 (iii); 72 to 83 (iv), 89 to 97 (v); 109 to 114 (vi), 121 to 135 (vii), 162 to 177 (viii), 164 to 186 (ix) and 187 to 206 (x) of HIN- 1 ⁇ 3 Nef were coated onto wells of 96-well microtitre plates at a concentration of 500 ng/well.
  • Sera (titrated from 1:300 to 1;100,000) from the LTP individuals were then incubated with the immobilised peptides and the presence of antibodies in the sera which recognise the Nef-derived peptide were detected using a biotin-streptavidin HRP system with o-phenylenediamine as substrate. Absorbance was measured using a titertek plate reader at wavelengths of 630 and 450 nm.
  • Figure 2b shows reactivity of sera from LTNPl individuals against Nef-derived peptides.
  • Synthetic peptides corresponding to amino acid residues 1 to 19 (i), 20 to 36 (ii), 44 to 65 (iii), 72 to 83 (iv), 89 to 97 (v), 109 to 1 14 (vi), 121 to 135 (vii), 162 to 177 (viii), 164 to 186 (ix) and 187 to 206 (x) of HTV-I NL ⁇ Nef were coated onto wells of 96-well microtitre plates at a concentration of 500 ng/well.
  • Sera (titrated from 1 :300 to 1 : 100,000) from the LTNPl individuals were then incubated with the immobilised peptides and the presence of antibodies in the sera which recognise the Nef-derived peptides were detected using a biotin-streptavidin HRP system with o-phenylenediamine as substrate. Absorbance was measured using a titertek plate reader at wavelengths of 630 and 450 nm.
  • Figure 2c shows reactivity of sera from LTNP2 individuals against Nef-derived peptides.
  • Synthetic peptides corresponding to amino acid residues 1 to 19 (i), 20 to 36 (ii), 44 to 65 (iii), 72 to 83 (iv), 89 to 97 (v), 109 to 114 (vi), 121 to 135 (vii), 162 to 177 (viii), 164 to 186 (ix) and 187 to 206 (x) of HIV- 1 ⁇ 3 Nef were coated onto wells of 96-well microtitre plates at a concentration of 500 ng/well.
  • Sera (titrated from 1 :300 to 1 : 100,000) from the LTNP2 individuals were then incubated with the immobilised peptides and the presence of antibodies in the sera which recognise the Nef-derived peptides were detected using a biotin-streptavidin HRP system with o-phenylenediamine as substrate. Absorbance was measured using a titertek plate reader at wavelengths of 630 and 450 nm. *
  • HIV-l infected blood a non-pathogenic HIV-l strain was isolated from a recipient of HIV-l infected blood.
  • the recipient is designated "C18".
  • Other recipients are defined as “C54” and “C98”.
  • the donor is identified herein as "D36”.
  • the place of isolation may be indicated after the abbreviation of "HIV”.
  • C18 and C98 Exemplary viral isolates referred to herein as "C18” and “C98” were deposited at the PHLS Centre for Applied Microbiology and Research, European Collection of Animal Cell Cultures (EC ACC), Division of Biologies, Porton Down, Salisbury, Wiltshire SP4 O G. C18 was deposited on 17 October, 1994 under Provisional Accession Number V94101706 and C98 was deposited on 31 October, 1994 under Provisional Accession Number V941031169. Another isolate "C54” was deposited at ECACC on 10 March, 1995 under Provisional Accession Number V95031022.
  • viruses were isolated by the following procedure:
  • HIV negative donor PBMC were stimulated by culture in RPMI 1640 containing 10% v/v fetal calf serum (FCS), 15 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 0.1%) w/v sodium bicarbonate with 100 IU/ml penicillin and 100 ⁇ g/ml streptomycin with the addition of 10 ⁇ g/ml PHA (Wellcome, Temple Hill, Dartford,
  • PBMC PBMC on days 7, 14 and 21.
  • Virus production was assayed for by cell-free reverse transcriptase activity ( ⁇ eate et al, 1987) or p24 activity (Abbott Diagnostics assay).
  • Serum samples were obtained from seven HlV+ve individuals, D-36, C-124, C-98, C-64, C-18, C-49 and C-54. Individuals C124, C-98, C-64, C18, C49 and C-54 (recipients) were infected through units of blood or blood products from donor D36, over a 2-year period. Long-term follow-up of the six recipients and the donor, shows persistent long term asymptomatic infection. This group is referred to herein as the long term non-progressor 1 (LT ⁇ P1) cohort. Members of this cohort have been infected for an average of 11 years (10.75 to 14 years) and it was established that the donor had been infected since April, 1981 (Learmont et al, 1992).
  • LTP1 long term non-progressor 1
  • Peptide Synthesis Peptides corresponding to amino acid residues 1 to 19, 20 to 36, 44 to 65, 72 to 83, 89 to 97, 10 109 to 114, 121 to 136, 162 to 177, 164 to 186 and 187 to 206 of HIV-l nef (HIN- 1 ⁇ ) were synthesised using standard t-Boc chemistry and purified by high pressure chromatography as described elsewhere (Fecondo et al, 1993).
  • PBS-Tween PBS containing 0.05% v/v Tween 20
  • any remaining available sites on the wells blocked by incubation of 150 ⁇ l of gelatine (1% w/v) in PBS for 1 h at 37°C.
  • 50 ⁇ l of serum diluted in PBS/BSA (1% w/v) was added to the wells and incubated for
  • LTNPl long term progressors
  • LT ⁇ P2 long term non-progressors
  • Nef-derived peptides Recognition of Nef-derived peptides by sera Recognition of synthetic peptides, which correspond to amino acid sequences of Nef, by the LTNPl cohort was assessed. Various peptides were assessed to detect those antigenic epitopes of Nef protein recognised by these individuals. Peptides corresponding to amino acid sequences 1 to 19; 20 to 36; 44 to 65; 72 to 83; 89 to 97; 109 to 114; 121 to 135; 162 to 177; 164 to 186 and 187 to 206 of HIV-l ⁇ 3 Nef protein were used to screen sera for the presence of specific antibodies. All sera from the LTP group recognised all Nef-derived peptides tested (Figure 2a(i)-(x)).
  • Sera from patients with autoimmune disease displayed only low background non-specific recognition. Normal sera from HIN-l-ve individuals tested to date also displayed only background activity.
  • Sera from the LT ⁇ Pl and LT ⁇ P2 groups also showed significant reactivity against Nef peptides corresponding to Nef amino acid sequences 1-19, 20-36, 44-65, 72-83, 89-97, 109-114, 121- 135, 164-86 and 187-206 ( Figure 2b(i)-(x) and c(i)-(x)).
  • MOSIER DE GULIZIA RJ, MACISAAC PD, TORBETT BE, LEVY JA (1993) Science 260: 689-692.
  • TEEUWSEN VJP SIEBELINK KHJ, DE WOLF F, GOUDSMIT J, UYTDEHAAG FGCM, OSTERHAUS ADME (1990) AIDS 4: 11 -81.
  • MOLECULE TYPE DNA
  • MOLECULE TYPE DNA
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO:33 TGGAATGGAT 10

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Abstract

La présente invention concerne généralement des souches non pathogènes de VIH-1 et des procédés pour les détecter. Les procédés de la présente invention sont particulièrement utiles pour déterminer la probabilité qu'a un individu séropositif VIH-1 de développer le SIDA ou des symptômes liés au SIDA. La présente invention concerne en outre des compositions thérapeutiques pour le traitement et la prévention du SIDA et des troubles liés au SIDA. Un autre aspect de la présente invention concerne des souches de VIH-1 capables de synthétiser une protéine Nef modifiée ou comportant une protéine Nef sauvage qui est modifiée après synthèse, ce qui rend ces souches de VIH-1 sensiblement non pathogènes.
PCT/AU1996/000301 1995-05-17 1996-05-17 Souches non pathogenes de vih-1 et procedes pour les detecter WO1996036699A1 (fr)

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WO1995021912A1 (fr) * 1994-02-14 1995-08-17 The Macfarlane Burnet Centre For Medical Research Limited Souches non pathogenes de vih-1

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021912A1 (fr) * 1994-02-14 1995-08-17 The Macfarlane Burnet Centre For Medical Research Limited Souches non pathogenes de vih-1

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
J. VIROL., Vol. 69(1), (Jan. 1995), X. HUANG et al., "Characterisation of Nef Sequences in Long-Term Survivors of Human Immunodeficiency Virus Type 1 Infection", pages 93-100. *
SCIENCE, Vol. 258, (18 December 1992), M.D. DANIEL et al., "Protective Effects of a Live Attenuated SIV Vaccine with a Deletion in the Nef Gene", pages 1938-1941. *
THE NEW ENGLAND J. MEDICINE, Vol. 332(4), (26 Jan. 1995), F. KIRCHHOFF et al., "Absence of Intact Nef Sequences in a Long-Term Survivor with Non-Progressive HIV-1 Infection", pages 228-232. *

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