EP1315799A2 - Souches vih attenuees et leur utilisation - Google Patents

Souches vih attenuees et leur utilisation

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Publication number
EP1315799A2
EP1315799A2 EP01982248A EP01982248A EP1315799A2 EP 1315799 A2 EP1315799 A2 EP 1315799A2 EP 01982248 A EP01982248 A EP 01982248A EP 01982248 A EP01982248 A EP 01982248A EP 1315799 A2 EP1315799 A2 EP 1315799A2
Authority
EP
European Patent Office
Prior art keywords
virus
vaccine
hiv
anyone
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.)
Withdrawn
Application number
EP01982248A
Other languages
German (de)
English (en)
Inventor
Jaap Goudsmit
Marion Cornelissen
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.)
Biomerieux BV
Original Assignee
Biomerieux BV
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
Application filed by Biomerieux BV filed Critical Biomerieux BV
Priority to EP01982248A priority Critical patent/EP1315799A2/fr
Publication of EP1315799A2 publication Critical patent/EP1315799A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2740/16061—Methods of inactivation or attenuation

Definitions

  • the present invention relates to the field of immunology, in particular to viruses and more in particular to human immunodeficiency virus.
  • Live-attenuated virus vaccines have been enormously successful. They are widely used to prevent diseases like for instance polio and measles. Until now, however, there is no vaccine against acquired immunodeficiency syndrome (Aids). All over the world much research is being done with human immunodeficiency virus to obtain a suitable vaccine. Although attenuated strains have been obtained, there still remain many safety concerns about either the reversion of attenuated vaccine strains to virulent phenotypes or the induction of fulminant infection in (immunocomprornised) individuals.
  • the present invention discloses the unexpected and important finding that certain non revertant mutations in a human immunodeficiency virus are capable of delaying or diminishing the pathological behavior of said virus for a very long time in vivo.
  • the individual that carried the HIV virus with the mutations described in tables 1 through 4 was relatively healthy with high CD4+ cell counts in the blood. This phenomenon is uncommon in HIV infection where normally a significant drop in CD4+ cell count is observed. In this respect seemed the HIV virus that infected the patient less or even non-pathogenic.
  • the HIV virus was, however, immunogenic as shown by the seroconversion of the individual.
  • FIG. 1 shows the detected amount of HIV-RNA and CD4+ T cells in said patient during the last five years.
  • the present invention discloses an isolated human immunodeficiency virus, comprising at least one non revertant mutation capable of delaying or diminishing the pathological behavior of said immunodeficiency virus when compared to a human immunodeficiency virus not having at least one such a mutation.
  • a virus of the invention is a HIV-1 virus.
  • a non revertant mutation is defined as a mutation which is stable and remains present in the virus over a prolonged period of time.
  • said non revertant mutation is stable and remains present in the virus over a prolonged period of time in a patient.
  • a virus of the invention comprises at least one amino acid sequence as is described in tables 1 and 2.
  • the invention provides a virus of the invention, comprising at least one amino acid sequence as described in table 1 or 2.
  • the invention provides a virus of the invention, comprising at least one amino acid sequence as described in table 1.
  • the invention discloses an isolated virus according to the invention, wherein at least one of said non revertant mutations is located in the gag or pol gene.
  • Important mutations are the 3 amino acid (QAE) and 10 amino acid (QSRPEPTAPP) insertions and the 2 amino acid deletion in the gag gene and the "IPIK” mutation in the pol gene.
  • a virus of the invention may comprise at least one substitution amino acid in an amino acid sequence as described in table 1 or 2.
  • Said substitution amino acid is defined as an amino acid which does not substantially alter the capability of said amino acid sequence of delaying or diminishing the pathological behavior of a virus of the invention when compared to a human immunodeficiency virus not having at least one such a mutation.
  • the invention provides a virus of the invention, which comprises at least one substitution amino acid in at least one amino acid sequence as described in table 1 or 2.
  • a virus of the invention is obtainable by state of the art cloning techniques.
  • a person skilled in the art knows a variety of ways to perform site directed mutagenesis.
  • the present invention also discloses a method for obtaining a virus according to the invention, comprising providing a wild type human immunodeficiency virus with at least one non revertant mutation capable of delaying or diminishing the pathological behavior of said immunodeficiency virus when compared to a human immunodeficiecy virus not having at least one such a mutation.
  • a virus strain of the invention can be isolated by randomly collecting human immunodeficiency strains and selecting for strains comprising sequence similarities to a virus according to the invention.
  • sequence similarity is meant that the isolated strains comprise at least one same mutation as a virus according to the invention, said mutation being capable of delaying or diminishing the pathological behavior of said isolated virus when compared to a human immunodeficiency virus not having at least one such a mutation.
  • Said isolated virus may contain additional mutations.
  • Said additional mutation may also be involved in the delaying or diminishing of the pathological behavior of said isolated virus when compared to a human immunodeficiency virus not having at least one such a mutation. Said additional mutation may render said isolated virus even more attenuated.
  • the invention provides a method for obtaining a virus of the invention comprising collecting a certain number of strains, sequencing at least part of said strains, comparing obtained sequences with sequences of virus according to the invention, and isolating strains comprising sequence similarities to a virus according to the invention.
  • said strain is amplified before sequencing in said method.
  • a method of the invention is particularly useful for obtaining an attenuated virus according to the invention. Therefore, in another aspect the invention provides a virus obtainable by a method according to the invention.
  • a virus of the invention may be used to prepare a vaccine. If administered to an immunocompetent individual, said individual will develop antibodies against HIV. Said antibodies give the individual at least a partial protection against more virulent strains.
  • the invention provides a virus according to the invention for use as a vaccine.
  • a virus of the invention is preferably processed further.
  • the mutations described in tables 1 through 4, or a selection thereof can be used for the design of a safe live attenuated HIV vaccine.
  • the same mutations can be used in vaccines composed of dead virus, virus without replicatable nucleic acid or protein sub units.
  • the present invention provides a use of a virus according to the invention for the preparation of a vaccine.
  • said vaccine will specifically at least partly provide an individual with protection against Aids.
  • the invention discloses a use of a virus according to the invention for the preparation of a vaccine for Aids.
  • the invention discloses a vaccine comprising virus according to the invention.
  • a vaccine of the invention is particularly useful for prophylaxis of Aids. Therefore, the present invention provides a method for, at least in part, prophylaxis of Aids, comprising administering a vaccine according to the invention to an individual.
  • a person skilled in the art is capable of identifying a virus of the invention in an individual.
  • Mutations comprised by a virus of the invention can be used as target sequences for diagnostic assays to discriminate HIV sequences with and without the mutations from tables 1 through 4. Diagnostics capable of identifying these mutations may play a role in assessing the life expectancy of infected individuals, whereas these mutations or a subset thereof indicates a better quality of life and a longer disease free period compared to other HIV viruses. Therefore, another embodiment of the invention discloses a method for identifying a virus of the invention in an individual, comprising collecting a sample comprising virus or parts thereof, from said individual. and detecting strains comprising sequence similarities to a virus of the invention.
  • said sample is a plasma, serum or blood sample.
  • Virus may be collected from an individual by collecting blood samples comprising peripheral blood monocytic cells (PBMC).
  • PBMC peripheral blood monocytic cells
  • another embodiment discloses a method of the invention, wherein said virus is collected by isolating peripheral blood monocytic cells from said individual.
  • Sequence similarities are defined as before in this description.
  • a person skilled in the art is able to determine sequence similarities. For instance, he/she is able to detect a virus of the invention using antibodies with a binding specificity for one or more of the stable mutations of said virus.
  • a person skilled in the art can detect sequence similarities by sequencing collected virus from an individual. Techniques of sequencing are well known in the art.
  • another embodiment of the invention discloses a method according to the invention, wherein said sequence similarities are detected by sequencing.
  • sequence similarities between an isolated strain and a virus of the invention there are other possibilities to detect sequence similarities between an isolated strain and a virus of the invention.
  • One possibility is for example hybridization with probes comprising at least one sequence of virus according to the invention.
  • yet another embodiment of the invention provides a method according to the invention, wherein said sequence similarities are detected by hybridization with probes comprising at least one sequence of virus according to the invention.
  • a person skilled in the art can think of other possibilities to detect sequence similarities between an isolated strain and a virus of the invention. If another way of detecting is used in a method of the invention, it is still within the scope of the present invention.
  • the method is build up of the following steps.
  • lOx PCR buffer II 500mM KCl, lOOmM Tris-HCl, pH8.3; included in kit
  • JZH2R primer 5 A- GCT ATC ATC ACA ATG GAC NNN NNG , 3 A
  • JZH1 primer 5A- GCT ATC ATC ACA ATG GAC , 3 ⁇
  • RNAse-H (lU/ ⁇ l; Boehringer Mannheim,; 786357).
  • Standard dilution rate as input for the amplification is 10 times (10 ⁇ l GAT product + 90 ⁇ l Baker water) or 100 times (10 ⁇ l GAT product + 990 ⁇ l Baker water). Usually a dilution rate of 100 times generates the best results. Therefore first the 100 times dilution is used for amplification. If the result is not satisfactory an additional amplification on the 10 times dilution is done.
  • PBMC peripheral blood monocytic cells
  • PBMC peripheral blood monocytic cells
  • Cryop eserved PBMC were thawed and washed with culture medium (Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, recombinant interleukin-2 (20 U/ml, PROLEUKIN; Chiron Benelux BV) and antibiotics (penicillin (100 U/ml) and streptomycin (100 ⁇ g/ml)) to remove residual DMSO.
  • culture medium Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, recombinant interleukin-2 (20 U/ml, PROLEUKIN; Chiron Benelux BV) and antibiotics (penicillin (100 U/ml) and streptomycin (100 ⁇ g/ml)
  • serial dilutions of HIV-1 infected PBMC (0.5.10 4 to 4.10 4 per well) were cocultivated with 2 to 3 days phytohaemagglutinin (PHA) stimulated healthy donor PBMC (10 5 per well) in a final volume of 200 ⁇ l culture medium for 28 days.
  • PHA phytohaemagglutinin
  • Cells were resuspended and were transferred to 96-well plates containing fresh healthy donor PHA- stimulated PBMC (10 5 per well) and further cultured in a volume of 200 ⁇ l.
  • virus stocks were grown in 25 ml culture flasks. Cell free supernatants of these viral cultures were aliquotted and stored at -70 °C. Viruses obtained using this procedure were considered to be clonal if less than one third of the wells of a cell dilution were positive for ⁇ 24.
  • Figure 1 The detected amount of HIV RNA and CD4+ T cells in a patient that carried HIV viruses with the mutations described in tables 1 through 4.
  • amino acid numbering is according to the numbering of the amino acid sequences of the HIV-1 consensus B sequences of the different HIV-1 genes in the Los Alamos database (http:/hiv-web.lanl.gov, Human Retroviruses and AIDS 1999: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Kuiken CL, Foley B, Hahn B,
  • amino acid numbering is according to the numbering of the aminoacid sequences of the HIV-1 consensus B sequences of the different HIV-1 genes in the Los Alamos database (http://hiv-web.lanl.gov. Human Retroviruses and AIDS 1999: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Kuiken CL, Foley B, Hahn B, Korber B, McCutchan F, Marx PA, Mellors JW, Mullins Jl, Sodroski J, and Woiinksy S, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM))
  • amino acid numbering is according to the numbering of the aminoacid sequences of the HIV-1 consensus B sequences of the different HIV-1 genes in the Los Alamos database (http://hiv-web.lanl.gov, Human Retroviruses and AIDS 1999: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Kuiken CL, Foley B, Hahn B, Korber B, McCutchan F, Marx PA, Mellors JW, Mullins Jl, Sodroski J, and Woiinksy S, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM))
  • amino acid numbering is according to the numbering of the aminoacid sequences of the HIV-1 consensus B sequences of the different HIV-1 genes in the Los Alamos database (http://hiv-web.lanl.gov. Human Retroviruses and AIDS 1999: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Kuiken CL, Foley B, Hahn B, Korber B, McCutchan F, Marx PA, Mellors JW, Mullins Jl, Sodroski J, and Woiinksy S, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM))

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Communicable Diseases (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un virus de l'immunodéficience humaine isolé, contenant au moins une forme mutante non réversée capable de retarder ou d'atténuer le comportement pathologique dudit virus de l'immunodéficience, contrairement à un virus de l'immunodéficience humaine ne présentant pas au moins une forme mutante de ce type. Un virus selon la présente invention peut être utilisé pour préparer un vaccin contre le SIDA. Ce virus peut également être utilisé pour réaliser des analyses diagnostiques sur des patients porteurs du VIH.
EP01982248A 2000-09-08 2001-09-05 Souches vih attenuees et leur utilisation Withdrawn EP1315799A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP01982248A EP1315799A2 (fr) 2000-09-08 2001-09-05 Souches vih attenuees et leur utilisation

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP00203116 2000-09-08
EP00203116 2000-09-08
EP01982248A EP1315799A2 (fr) 2000-09-08 2001-09-05 Souches vih attenuees et leur utilisation
PCT/EP2001/010244 WO2002020571A2 (fr) 2000-09-08 2001-09-05 Souches vih attenuees et leur utilisation

Publications (1)

Publication Number Publication Date
EP1315799A2 true EP1315799A2 (fr) 2003-06-04

Family

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EP (1) EP1315799A2 (fr)
AU (1) AU2002213882A1 (fr)
WO (1) WO2002020571A2 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6270464B1 (en) 1998-06-22 2001-08-07 Artemis Medical, Inc. Biopsy localization method and device
WO2001051661A2 (fr) 2000-01-13 2001-07-19 Amsterdam Support Diagnostics B.V. Systeme universel d'amplification de l'acide nucleique pour acides nucleiques d'un prelevement
WO2004081225A2 (fr) 2003-03-07 2004-09-23 Rubicon Genomics, Inc. Amplification et analyse d'un genome entier et de bibliotheques de transcriptomes entiers generees par un procede de polymerisation d'adn
US8206913B1 (en) 2003-03-07 2012-06-26 Rubicon Genomics, Inc. Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process
WO2005090607A1 (fr) 2004-03-08 2005-09-29 Rubicon Genomics, Inc. Procedes et compositions pour la generation et l'amplification de bibliotheques d'adn pour la detection et l'analyse sensible de methylation d'adn
WO2007018601A1 (fr) 2005-08-02 2007-02-15 Rubicon Genomics, Inc. Compositions et methodes de traitement et d'amplification d'adn consistant a utiliser plusieurs enzymes dans une seule reaction
WO2007018602A1 (fr) 2005-08-02 2007-02-15 Rubicon Genomics, Inc. Isolement des ilots cpg au moyen d'un procede de segregation thermique et de selection-amplification enzymatique
AU2006323930B2 (en) * 2005-12-07 2012-06-21 Janssen Sciences Ireland Uc Methods, plasmid vectors and primers for assessing HIV viral fitness
US9512493B2 (en) * 2011-08-24 2016-12-06 Grifols Therapeutics Inc. Compositions, methods, and kits for nucleic acid hybridization
WO2016172465A1 (fr) * 2015-04-24 2016-10-27 The Johns Hopkins University Compositions et procédés se rapportant à la caractérisation de réservoirs proviraux

Family Cites Families (1)

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Publication number Priority date Publication date Assignee Title
WO1994017825A1 (fr) * 1993-02-05 1994-08-18 The Regents Of The University Of California Mutants de genes multiples du virus d'immunodeficience humaine utilises dans un vaccin

Non-Patent Citations (1)

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Title
See references of WO0220571A3 *

Also Published As

Publication number Publication date
WO2002020571A3 (fr) 2003-03-13
AU2002213882A1 (en) 2002-03-22
WO2002020571A2 (fr) 2002-03-14

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