EP2756087A2 - Genomes lentiviraux chimeriques non-integratifs comme vaccins contre hiv-1 - Google Patents

Genomes lentiviraux chimeriques non-integratifs comme vaccins contre hiv-1

Info

Publication number
EP2756087A2
EP2756087A2 EP12756752.7A EP12756752A EP2756087A2 EP 2756087 A2 EP2756087 A2 EP 2756087A2 EP 12756752 A EP12756752 A EP 12756752A EP 2756087 A2 EP2756087 A2 EP 2756087A2
Authority
EP
European Patent Office
Prior art keywords
retrovirus
nucleic acid
hiv
cells
gene
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Withdrawn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
EP12756752.7A
Other languages
German (de)
English (en)
French (fr)
Inventor
Yahia Chebloune
Delphine ALDEBERT
Géraldine ARRODE-BRUSES
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.)
Centre National de la Recherche Scientifique CNRS
Universite Grenoble Alpes
Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Original Assignee
Centre National de la Recherche Scientifique CNRS
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 Centre National de la Recherche Scientifique CNRS filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP2756087A2 publication Critical patent/EP2756087A2/fr
Withdrawn legal-status Critical Current

Links

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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • C12N15/86—Viral vectors
    • 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
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—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
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15034—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
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2740/16134—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
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16234—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
    • C12N2740/16311—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
    • 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
    • C12N2770/00021—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
    • 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
    • C12N2770/00034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Non integrative chimeric lentiviral genomes as innovative vaccines against HIV-1
  • the subject of the present invention is nucleic acids comprising non-integrative chimeric retroviral genomes comprising the 5 'and 3' long terminal repeat (LTP) sequences of the goat lentivirus: arthritis-encephalitis virus Caprine (VAEC, or CAEV for Caprine Arthritis Encephalitis Virus in English) or another retrovirus not integrating into human cells and at least one viral gene of another retrovirus.
  • the invention also relates to a vector comprising such a nucleic acid, an immunogenic or vaccine composition comprising said vector or nucleic acid, and their use for the treatment and / or prevention of infection by a retrovirus or a disease induced by a pathogen.
  • HIV human immunodeficiency virus
  • AIDS Acquired Immunodeficiency Syndrome
  • Africa is the most affected continent, but the infection is growing rapidly in Asia and some Eastern European countries, probably due to lack of means for early detection and lack of treatment. 'infection.
  • many HIV-infected patients in developing countries receive no treatment, and thus contribute to the massive spread of the infection. The economic impact of AIDS will certainly be very important in the coming years.
  • AIDS In Europe, AIDS remains one of the most important communicable diseases, with around 1 million people living with AIDS and more than 20,000 new infections a year in Western and Central Europe; and with approximately 1.5 million people living with AIDS and more than 200 000 new infections per year in Eastern Europe.
  • the development of a prophylactic vaccine that stops this infection remains a priority.
  • the vaccine must induce a CD8 + T lymphocyte response, which is associated with the control of the virus during primary infection, and the presence of which has been shown to be essential for the control of viral load in infected non-human primates ( Jin et al., 1999, J Exp Med, 189: 991-998).
  • CTLs cytotoxic T lymphocytes
  • LTNP long-term non-progressing patients
  • infected with HIV Makedonas et al., 2002, AIDS, 16: 1595-1602
  • CD4 + T cell response is essential for the stimulation and maintenance of the anti-HIV CD8 + T cell response (Kalams et al., 1999, J Virol, 73: 6715- 6720).
  • CD4 + T cells are also essential for setting up and maintaining the antibody-based response produced by B-lymphocytes (LBs).
  • LBs B-lymphocytes
  • DNA vaccination based on viral vectors has never been associated with a development of pathologies, neither in humans nor in animals, and therefore is safer. However, tested in monkeys, these vectors have been shown to be unable to protect animals from experimental infection (Liu et al., 2006, Virology, 351: 444-454, Singh et al., 2005, J Virol, 79: 3419-3428).
  • infectious viral genomes comprising a complete viral genome including the LTRs of CAEV as well as one or two genes of another retroviral genome (Bouzar et al., 2007, Virology, 364 (2): 269-280 Bouzar et al., 2004, Virology, 326 (1): 47-56, Bouzar et al., 2003, Virology, 309 (1): 41-52, Yuhai et al., 2009, Retrovirology, 6 (2). : 22). These genomes have been used to study the mechanisms of pathogenesis induced by highly pathogenic retroviruses in humans and monkeys.
  • the inventors have discovered that the use of the Long Term Repeat (STR or LTR) of the Caprine Arthritis Encephalitis Virus (VAEC), or CAEV, has the potential to improve the expression of vaccinating retroviral genomes and the induction of protective responses against pathogenic retroviruses, while avoiding their integration into the host cells.
  • the inventors have demonstrated that the Caprine Arthritis Encephalitis Virus (CAEV) Repeated Terminal Sequences (LTRs) allow the constitutive expression of the genes associated with them and are not dependent on the CAEV viral tat gene.
  • the CAEV viral tat protein to express the genes of a viral genome to which they are fused, thus allowing strong expression of viral antigens.
  • the inventors have thus developed chimeric genomes, between the lentiviruses of SIV and HIV primates (or HIV in English for Human Immunodeficiency Virus) and CAEV, which have the properties of being non-integrative and non-replicative, while being capable of to perform a replication cycle to express all the HIV and SIV antigens present in the genomes.
  • the inventors have demonstrated that the transfection of these genomes into primate cells (HEK293) allows the expression of all the proteins of the genes present and that these proteins are assembled into viral particles capable of carrying out a single cycle of infection (ie a pseudo-cycle) in target cells, without integration of the viral genome into these target cells.
  • Immunization of humanized NOD / SCID mice with human mononuclear cells demonstrated the presence of strong specific humoral and cellular immune responses against viral antigens.
  • Nucleic acid means the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine, “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine, "DNA molecules”) in single-stranded form or in the form of a double-stranded helix. Double-stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • the term nucleic acid, and in particular DNA or RNA molecule refers only to the primary or secondary structure of the molecule, and is not limited to any particular tertiary forms.
  • this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), viruses, plasmids and chromosomes.
  • DNA molecules e.g., restriction fragments
  • viruses e.g., viruses
  • plasmids e.g., viruses
  • chromosomes e.g., chromosomes.
  • sequences can be described herein in accordance with the normal convention which only gives the sequence in the 5 'to 3' direction along the non-transcribed strand of DNA (That is, the strand having a sequence homologous to the mRNA).
  • a nucleic acid comprising a non-integrative chimeric retroviral genome refers to a nucleic acid which comprises the cis-acting nucleic acid sequences of at least two retroviruses, said nucleic acid not being capable of to integrate into the genome of a host cell.
  • the nucleic acid includes the 5 'and 3' Repeat Terminal Sequences (LTRs) of a first retrovirus, and at least one viral gene of a second retrovirus.
  • LTRs 5 'and 3' Repeat Terminal Sequences
  • retrovirus is meant a virus whose genome consists of an RNA molecule and which comprises a reverse transcriptase, Le. a member of the family Retroviridae.
  • Retroviruses are divided into three genera: oncovirus, lentivirus and spumavirus.
  • Oncoviruses are composed in particular of the following species: murine leukemia virus (MLV), avian leucosis virus (ALV), Rous sarcoma virus (RSR, or RSV in English for Rous Sarcoma Virus), or the simian Mason-Pfizer virus.
  • Lentiviruses are composed of the following species: Human Immunodeficiency Virus Type 1 (HIV-1), Human Immunodeficiency Virus Type 2 (HIV-1) 2, or HIV-2 for Human Immunodeficiency Virus type 2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV) for Feline Immunodeficiency Virus), the Bovine Immunodeficiency Virus (VIB), the Maedi Visna virus (VMV) of sheep, the Caprine Arthritis Encephalitis Virus (CAEV) or equine infectious anemia virus (VAIE, or EIAV in English for Equine Infectious Anemia Virus).
  • the spumavirus can be HFV.
  • the retrovirus when the retrovirus is HIV-1, it can be of any serogroup, for example serogroup M (serotype AD, FH, J, K), O, N or P.
  • the retrovirus when the retrovirus is HIV-2, it can be of any serogroup, for example serogroup A or B.
  • viral gene is meant a gene present in a retroviral genome.
  • the viral gene can be the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu or env gene.
  • the "gag” gene for "group specifies antigen” encodes the Gag precursor polyprotein that is cleaved to provide the basic structural proteins of retroviruses, which are capsid proteins, nucleocapsid proteins, and matrix proteins.
  • the HIV Gag protein is the precursor of the p24 capsid protein, the p6 and p7 nucleocapsid proteins, and the p17 matrix protein.
  • the gag gene is the gag gene of HIV-1 (NCBI gene ID ⁇ 55030, updated August 20, 201 1), HIV-2 (NCBI gene ID No. 14900001, updated Aug. 27, 201 1), SIV (NCBI gene ID No. 956108, updated August 27, 201 1) or FIV (NCBI gene ID No. 1489988, updated August 20, 201 1).
  • the "pol” gene codes for reverse transcriptase, integrase and protease.
  • the pol gene is the pol gene of HIV-1 (NCBI gene ID No. 155348, updated 27 August 201 1), HIV-2 (NCBI gene ID No. 1490001, implemented update 27 August 201 1), FIV (NCBI gene ID No. 1489989, update 27 August 201 1) or SIV (NCBI gene ID No. 956107, updated August 20, 201 1).
  • the "viral” or “viral infectivity factor” gene encodes a protein required for the production of infectious virions.
  • the bright gene is the live gene of HIV-1 (NCBI gene ID No.
  • HIV-2 NCBI gene ID No. 1724712, updated January 21, 2010
  • FIV NCBI gene ID No. 1724709, updated February 7, 2010
  • SIV NCBI gene ID ⁇ 490005, updated January 21
  • the "vpr” gene codes for the viral R protein, which plays an important role in the G2 phase cell cycle arrest and in the regulation of the transport of the cytoplasmic pre-integration complex to the nucleus, the viral replication.
  • the vpr gene is the vpr gene of HIV-1 (NCBI gene ID No. 155807, updated August 7, 2011), HIV-2 (NCBI gene ID ⁇ 724718, updated January 21, 2010) or SIV (NCBI gene ID No. 956112, updated January 15, 2011).
  • the "vpx” gene codes for the viral protein X and is related to the vpr gene.
  • the vpx gene is the gene vpxdu HIV-2 (NCBI gene ID No. 1724714, updated March 19, 2011) or SIV (NCBI gene ID ⁇ 490006, updated July 16 2011).
  • the “nef” gene encodes myristylated 27- to 25-kDa protein, called the Negative Regulatory Factor, which plays a key role in the depletion of CD4 cells in vivo.
  • the nef gene is the nef gene of HIV-1 (NCBI gene ID 56110, updated 7 August 2011), HIV-2 (NCBI gene ID No. 1724715, updated 19 March 2011) or SIV (NCBI gene ID No. 1490008, updated July 2, 2011).
  • the tat gene encodes an 86- to 101-amino acid protein called Trans-Activator of Transcription that increases the transcription rate of the retroviral genome.
  • the gene / ai is the tat gene of HIV-1 (NCBI gene ID No. 155871, updated August 20, 2011), HIV-2 (NCBI gene ID ⁇ 724713, updated February 7, 2010) or SIV (NCBI gene ID No. 956113, updated February 7, 2010).
  • the gene “rev” encodes a protein called "Regulator of Virion Expression” that allows the export of viral RNA from the nucleus to the cytoplasm.
  • the rev gene is the rev gene of HIV-1 (NCBI gene ID No. 155908, updated on August 7, 2011), HIV-2 (NCBI gene ID No. 1724716, updated day of May 21st
  • the "vpu” gene encodes a protein called "Viral Protein U” that is involved in viral budding and enhancement of virion release.
  • the vpu gene is the vpu gene of HIV-1 (NCBI gene ID No. 155945, updated on August 7, 2011) or SIV (NCBI gene ID No. 2828723, updated from January 21, 2010).
  • the "env” gene encodes the gp160 precursor protein which is processed and cleaved to give the gp120 and gp41 envelope proteins.
  • the gene eni / is the env gene of HIV-1 (NCBI gene ID No. 155971, updated on August 7, 201 1), HIV-2 (NCBI gene ID ⁇ 724717). June 18, 201 1), FIV (NCBI gene ID ⁇ 489987, updated June 18, 201 1) or SIV (NCBI gene ID No. 1490007, updated June 18, 201 1).
  • CAEV Caprine Arthritis Encephalitis Virus
  • LTRs Terminal Sequences
  • the invention thus relates to a nucleic acid comprising a non-integrative chimeric retroviral genome, wherein said chimeric retroviral genome comprises:
  • LTR Terminal Sequences
  • a first retrovirus said first retrovirus being a lentivirus, such as the Arthritis-Encephalitis Caprine Virus (CAEV), the Maedi Visna Virus (VMV) mutton, equine infectious anemia virus (EIAV), or an oncovirus or spumavirus, and
  • CAEV Arthritis-Encephalitis Caprine Virus
  • VMV Maedi Visna Virus
  • EIAV equine infectious anemia virus
  • an oncovirus or spumavirus an oncovirus or spumavirus
  • At least one viral gene of a second retrovirus said second retrovirus not being the first retrovirus.
  • the LTR sequences used are preferably derived from a retrovirus that does not integrate into the genome of a "host" or "patient", said host or patient being a host or patient in the genome of which the second and / or third retroviruses can integrate.
  • the first retrovirus is CAEV
  • said host or patient is not a goat but may be a man, a monkey, a cat, or a horse
  • the first retrovirus is the EIAV
  • said host or patient is not a horse but can be a man, a monkey, a cat, or a sheep
  • the first retrovirus is the VMV
  • said host or patient is not a sheep but can be a man, a monkey, a cat, or a horse.
  • the "first retrovirus” is the Caprine Arthritis Encephalitis Virus or CAEV.
  • CAEV is a type retrovirus Goat lentivirus similar to the human immunodeficiency virus (HIV), but does not cause AIDS-like pathology in its host.
  • “Repeated Terminal Sequences” denotes a sequence allowing the control of transcription, that is to say comprising an enhancer, a promoter, one or more transcription initiation signal (s). , one or more transcription termination signal (s), one or more polyadenylation signal (s).
  • the CAEV LTRs comprise an enhancer, a promoter and a transcription initiation signal as well as a transcription termination signal and a polyadenylation signal.
  • the 5 'and 3' LTRs of CAEV are identical and comprise or consist of the sequence SEQ ID NO: 3.
  • VMV Maedi Visna Virus
  • EIAV Equine Lentivirus LTRs are used.
  • the VMV 5 'LTR comprises or consists of the sequence in position 1 to 161 of NCBI Reference Sequence NC_001452.1 (updated December 8, 2008).
  • the VMV 3 'LTR comprises or consists of the sequence at position 9106 through 9202 of the NCBI Reference Sequence NC_001452.1 (updated December 8, 2008).
  • the 5 'LTR of the ElAV comprises or consists of the sequence in position 61 to 381 of the NCBI NC_001450 Sequence of Sequence (updated on 1 March 2010).
  • the 3 'LTR of the ElAV comprises or consists of the sequence in position 7269 to 8289 of the NCBI NC_001450 Sequence of Sequence (updated on 1 March 2010).
  • the LTRs of an oncovirus such as murine leukemia virus (MLV), avian leukemia virus (ALV), Rous sarcoma virus (RSV), or simian Mason-Pfizer virus, or the LTRs of a spumavirus, such as HFV are used.
  • the 5 'LTR of the MLV comprise or consist of the sequence at position 1 to 210 of the NCBI Reference Sequence NC_001702.1 (updated February 5, 201 1).
  • the MLV 3 'LTR comprises or consists of the sequence at position 5735 to 8135 of the NCBI Reference Sequence NC_001702.1 (updated February 5, 201 1).
  • the 5 'LTR of the ALV consists of or consists of the sequence at position 1 to 594 of the NCBI NC_0151 Reference Sequence 16.1 (updated April 18, 201 1).
  • the 3 'LTR of the ALV consists of or consists of the sequence at position 5338 to 7489 of the NCBI NC_0151 Reference Sequence 16.1 (updated April 18, 201 1).
  • the RSV 5 'LTR consists of or consists of the sequence in position 22 to 102 of NCBI Reference Sequence NC_001407.1 (updated December 8, 2008).
  • the RSV 3 'LTR comprises or consists of the sequence at position 9058 to 9292 of the NCB Sequence NC_001407.1 (updated December 8, 2008).
  • the simian Mason-Pfizer virus 5 'LTR comprises or consists of the sequence at position 26 to 123 of the NCBI Reference Sequence NC_001550.1 (updated December 8, 2008).
  • the simian Mason-Pfizer virus 3 'LTR comprises or consists of the sequence at position 7573 to 781 1 of NCBI Reference Sequence NC_001550.1 (updated December 8, 2008).
  • the 5 'LTR of the HFV consists of or consists of the sequence at position 1 to 1760 of the NCBI Reference Sequence NC_001364.1 (updated 22 April 2009).
  • the 3 'LTR of the HFV consists of or consists of the sequence at position 1 1487 to 13246 of the NCBI Reference Sequence NC_001364.1 (updated 22 April 2009).
  • CAEV Caprine Arthritis Encephalitis Virus
  • LTRs Terminal Sequences
  • the "second retrovirus” is different from the first retrovirus. It can be an oncovirus, a lentivirus or a spumavirus.
  • the second retrovirus is not CAEV.
  • the second retrovirus is an oncovirus, such as murine leukemia virus (MLV), avian leukosis virus (ALV), Rous sarcoma virus (RSV), or simian Mason-Pfizer virus.
  • a lentivirus such as the human immunodeficiency virus type 1 (HIV-1), the human immunodeficiency virus type 2 (HIV-2), the simian immunodeficiency virus (SIV), the feline immunodeficiency virus (FIV) or equine infectious anemia virus (EIAV), or a spumavirus, such as HFV.
  • the second retrovirus is HIV-1, HIV-2, SIV or IVF.
  • said at least one viral gene of said second retrovirus is selected from gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes.
  • said chimeric retroviral genome comprises at least two, three (e.g., gag, pol, vif, or gag, pol, env genes), four, five, six, seven, eight, nine, ten genes. viral of said second retrovirus.
  • said chimeric retroviral genome comprises the iai gene of said second retrovirus.
  • said chimeric retroviral genome comprises the set of gag, pol, vif, vpx, vpr, nef, tat, rev, and vpu genes of said second retrovirus.
  • said chimeric retroviral genome comprises the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and S IV, HIV-1, HIV-2 or FIV genes.
  • said chimeric retroviral genome comprises or consists of the sequence of the SIV retroviral genome (SEQ ID NO: 4), the retroviral genome of HIV-1 (SEQ ID NO: 2), the retroviral genome of HIV-1. 2 (SEQ ID NO: 5), or retroviral genome of FIV (SEQ ID NO: 6).
  • the chimeric retroviral genomes of SIV, HIV-1, HIV-2 and FIV are respectively schematically represented in FIGS. 1 to 4.
  • said chimeric retroviral genome further comprises at least one viral gene.
  • a third retrovirus said third retrovirus not being the first retrovirus, i.e., being different from said first retrovirus.
  • said third retrovirus is not CAEV.
  • Said "third retrovirus" may be chosen from one of the retroviruses as defined above.
  • said second retrovirus and third retrovirus are different.
  • Said second retroviruses and third retroviruses may or may not be of different kinds, for example said second and third retroviruses may each be an oncovirus, a lentivirus, or a spumavirus, or said second and third retroviruses may be respectively (i) a lentivirus and a spumavirus, or conversely a spumavirus and a lentivirus, (ii) an oncovirus and a lentivirus, or conversely a lentivirus and an oncovirus, or (iii) a spumavirus and an oncovirus, or conversely an oncovirus and a spumavirus.
  • said chimeric retroviral genome comprises at least one viral gene of a second retrovirus and at least one viral gene of a third retrovirus
  • said second retroviruses and third retroviruses are each a lentivirus, and preferably, said lentivirus is selected among HIV-1, HIV-2, SIV, IVF or EIAV.
  • the second retrovirus and the third retrovirus are lentiviruses of different species, serogroup, or serotype.
  • said third retrovirus is HIV-2, or when said second retrovirus is serogroup M HIV-1, said third retrovirus is HIV-1 serogroup O, or when said second retrovirus is HIV-1 serogroup M and serotype 1, said third retrovirus is HIV-1 serogroup M and serotype B.
  • said at least one viral gene of said third retrovirus is selected from gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes.
  • said chimeric retroviral genome further comprises at least two, three, four, five, six, seven, eight, nine or ten viral genes of said third retrovirus, preferably including the tat gene.
  • said chimeric retroviral genome further comprises all the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of said third retrovirus, that is to say that the said chimeric retroviral genome comprises the set of gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of said second retrovirus and the set of gag, pol, vif, vpx, vpr, nef genes, tat, rev, vpu and env of said third retrovirus.
  • Said retroviral chimeric genome may therefore comprise a viral gene of said second retrovirus and nine viral genes of said third retrovirus, or two viral genes of said second retrovirus and eight viral genes of said third retrovirus, or three viral genes of said second retrovirus and seven viral genes of said third retrovirus. , or four viral genes of said second retrovirus and six viral genes of said third retrovirus, or five viral genes of said second retrovirus and five viral genes of said third retrovirus, or six viral genes of said second retrovirus and four viral genes of said third retrovirus, or seven viral genes said second retrovirus and three viral genes of said third retrovirus, or eight viral genes of said second retrovirus and two viral genes of said third retrovirus, or nine viral genes of said second retrovirus and a viral gene of said third retrovirus.
  • said chimeric retroviral genome may therefore comprise the gag gene of said second retrovirus and the pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of said third retrovirus; or the gag and pol genes of said second retrovirus and the genes vif, vpx, vpr, nef, tat, rev, vpu and env of said third retrovirus; or the gag, pol, vif genes of said second retrovirus and the vpx, vpr, nef, tat, rev, vpu and env genes of said third retrovirus; or the gag, pol, vif, vpx genes of said second retrovirus and the vpr, nef, tat, rev, vpu and env genes of said third retrovirus; or gag, pol, alive, vpx, vpr of said second retrovirus and the genes
  • said chimeric retroviral genome comprises the gag, pol, vif, vpx and vpr genes of said second retrovirus and the nef, tat, rev, vpu and env genes of said third retrovirus.
  • said chimeric retroviral genome comprises the gag, pol, vif, vpx and vpr genes of SIV and the nef, tat, rev, vpu and env genes of HIV-1, or conversely the gag, pol genes, vivid, vpx e ⁇ vpr d HIV-1 and the genes nef, tat, rev, vpu and env of SIV.
  • said chimeric retroviral genome comprises the gag, pol, vif, vpx and HIV-1 genes and the nef, tat, rev, vpu and env genes of HIV-2, or conversely the gag genes.
  • said chimeric retroviral genome comprises or consists of the sequence SEQ ID NO: 7 (a schematic representation of this chimeric retroviral genome is in FIG. 5).
  • said pol gene when the pol gene is present in the chimeric retroviral genome, said pol gene is a pol gene deleted sequences encoding integrase (in).
  • said pol gene is deleted from the sequence SEQ ID NO: 8 (SIV integrase sequence), SEQ ID NO: 9 (HIV-1 integrase sequence), SEQ ID NO: 10 ( HIV-2 integrase sequence), or SEQ ID NO: 1 1 (sequence of the FIV integrase).
  • said retroviral genome comprises or consists of the sequence SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the chimeric retroviral genomes comprising or consisting of the sequence SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are respectively diagrammatically shown in Figures 6 to 10.
  • the invention also relates to a vector comprising a nucleic acid according to the invention.
  • vector refers to an extrachomosomal element by which a DNA or RNA sequence (ie, a "foreign” gene) can be introduced into a host cell, thereby transforming the host and allowing expression ( ie transcription and translation) of the introduced sequence.
  • the extrachromosomal element can be a self-replicative sequence, a phage sequence or a nucleotide sequence, a single or double-stranded DNA or RNA, a plasmid, a cosmid.
  • a vector typically contains the DNA of a transmissible agent, into which a foreign DNA is inserted and a selection marker.
  • restriction enzymes which cleave DNA at specific sites (specific nucleotide groups), called Restriction sites.
  • the foreign DNA is inserted at one or more restriction sites of the DNA vector, and then transported by the vector into a host cell with the DNA of the transmissible agent.
  • a segment or DNA sequence comprising an added or inserted DNA, such as a vector may also be referred to as a "DNA construct".
  • a common type of vector is a "plasmid", which is usually an autonomous double-stranded DNA molecule, usually of bacterial origin, that can easily accept additional (foreign) DNA and can easily be introduced into a host cell. appropriate.
  • the vector comprises a selection marker, such as an antibiotic resistance gene, and a nucleic acid according to the invention.
  • the resistance gene is a resistance gene to ampicillin or kanamycin.
  • nucleic acids and / or the vector according to the invention can be used to transform or transfect a host cell or organism, i.e. for the expression of the chimeric retroviral genome according to the invention.
  • host cell refers to any cell of any organism that is selected, modified, transformed, transfected, transduced, cultured, or used or manipulated in any way for the production of a substance by the cell, for example for the expression of a gene, a DNA sequence, a protein, a virion by the cell.
  • the host cell is a mammalian cell. Suitable host cells include, but are not limited to, HEK293 cells, human CD4 + T lymphocyte CEMx174 and M8166, human CD4 + T cells, human CD8 + T cells, mononuclear cells of human blood.
  • the transformation of the host cell or organism with the nucleic acid and / or the vector according to the invention can be carried out according to the standard techniques known to those skilled in the art, for example by transfection, electroporation, microinjection, DEAE-Dextran transduction, cell fusion, calcium phosphate precipitation, or use of gene gun, or a DNA vector transporter (see for example, Wu et al., 1992, J Biol Chem 267: 963- 967, Wu et al., 1988, J Biol Chem 263: 14621-14244, Hartmut et al., Canadian Patent Application No. 2,012,311, published Mar. 15, 1990). Immunogenic or vaccine composition and uses thereof
  • the nucleic acid or the vector according to the invention may be used for immunogenic or vaccinal purposes.
  • the invention also relates to an immunogenic or vaccine composition
  • an immunogenic or vaccine composition comprising a nucleic acid or a vector according to the invention.
  • vaccine refers to prophylactic or therapeutic vaccination.
  • immunogenic or vaccine composition is meant a composition for inducing an immune response against a retrovirus as defined above.
  • immune response is meant a response involving T cells, for example CD4 + and CD8 + T cells, and B cells.
  • the immunogenic or vaccine composition according to the invention is monovalent, i.e. it allows an immune response against a single retrovirus, for example against HIV-1 or HIV-2.
  • the immunogenic or vaccine composition according to the invention is multivalent, ie it allows an immune response against several retroviruses, for example against HIV-1 and HIV-2 or several pathogens, for example against HIV-1 and HCV (Hepatitis C Virus).
  • the vaccinating vector expresses the antigens of the one and the other pathogenic agent.
  • the immunogenic or vaccine composition according to the invention is polyvalent.
  • Such an immunogenic or vaccine composition can be obtained by combining several immunogenic or vaccine monovalent compositions according to the invention.
  • the immunogenic or vaccine composition may further comprise at least one other vaccine, ie an attenuated live virus, an inactivated virus or a subunit, against another virus, such as a sexually transmitted virus, such as for example hepatitis B, hepatitis C virus or papillomavirus.
  • the immunogenic or vaccine composition according to the invention comprises a pharmaceutically acceptable vehicle.
  • a “pharmaceutically acceptable carrier” refers to any vehicle in which the immunogenic or vaccine composition according to the invention can be formulated. This includes a saline solution such as a phosphate buffered saline. In general, a diluent or vehicle is chosen depending on the mode and route of administration, and according to standard pharmaceutical practice.
  • a pharmaceutically acceptable carrier includes, without limitation, ion exchangers, aluminum, aluminum stearate, lecithin, self-emulsifying drug delivery systems such as D-oc-tocopherol polyethylene glycol 1000 succinate, surfactants used in dosage form pharmaceutical such as Tweens or other polymeric delivery matrices, serum proteins such as human albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, glyceride mixtures of saturated fatty acids of plants, water, salts or electrolytes, such as protamine sulphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene polymer blocks and wool grease.
  • ion exchangers aluminum, aluminum stearate, lecithin,
  • Cyclodextrins such as A-, B-, and g-cyclodextrins, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2-and 3-hydroxypropyl-b-cyclodextrins, or other solubilized derivatives may also be advantageously used for to improve the delivery of the compositions according to the invention.
  • compositions according to the invention may further contain an adjuvant.
  • Any adjuvant or mixture of pharmaceutically acceptable adjuvants conventionally used in the field of vaccines can be used for this purpose.
  • suitable adjuvants are aluminum salts such as aluminum hydroxide or aluminum phosphate and DC-Chol.
  • Any adjuvant or mixture of pharmaceutically acceptable adjuvants conventionally used in the field of vaccines can be used for this purpose.
  • suitable adjuvants include aluminum salts such as aluminum hydroxide or aluminum phosphate and DC-Chol.
  • compositions according to the invention may contain adjuvant genes, that is to say genes that express proteins that will act as adjuvants by increasing the immunogenicity of the expressed viral proteins.
  • adjuvant genes genes that express proteins that will act as adjuvants by increasing the immunogenicity of the expressed viral proteins.
  • genes that encode cytokines such as interleukins (II) [IL-2, IL12, IL-15, ... or GM-CSF (granulocyte-macrophage colony-stimulating factor)].
  • cytokines such as interleukins (II) [IL-2, IL12, IL-15, ... or GM-CSF (granulocyte-macrophage colony-stimulating factor)].
  • II interleukins
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • nucleic acid, the vector, the immunogenic or vaccine composition according to the invention can be administered orally, by inhalation, or parenterally (in particular by intradermal, subcutaneous, intravenous, intramedullary or intramuscular injection).
  • parenteral route the nucleic acid, the vector, the immunogenic or vaccine composition according to the invention may be in the form of injectable solutions and suspensions, packaged in ampoules or flasks.
  • Forms for parenteral delivery are generally obtained in mixing the nucleic acid, the vector, the immunogenic or vaccine composition according to the invention with buffers, emulsifiers, stabilizers, preservatives, solubilizing agents.
  • these mixtures can then be sterilized and packaged in the form of intradermal, subcutaneous, intravenous, intramedullary or intramuscular injections.
  • buffers based on organic phosphate salts as a buffer.
  • emulsifying agents include methylcellulose, acacia, sodium carboxymethylcellulose.
  • stabilizers include sodium sulphite, sodium metasulfite, and examples of preservatives include sodium p-hydroxybenzoate, sorbic acid, cresol and chlorocresol.
  • the nucleic acid, the vector, the immunogenic or vaccine composition may also be in freeze-dried form.
  • the vaccine DNA solution can be injected directly or administered by electroporation in vivo using a commercial electroporator or encapsulated in liposomes or nanoparticles or using any in vivo transfection method that allows for better introduction of the vaccine DNA. in the cells of the vaccinated host.
  • the invention relates to a nucleic acid according to the invention, a vector according to the invention and / or an immunogenic or vaccine composition according to the invention for their use in the prevention and / or treatment of an infection. by a retrovirus.
  • prevention or “prevention” is meant the inhibition of a retroviral infection, ie preventing the retrovirus from causing an infection, or preventing the spread of the retrovirus within an infected subject or subject to the other.
  • treating or “treatment” is meant limiting the severity of the disease, preventing recurrent infections, i.e. limiting the reactivation of latent or persistent infections, and alleviating the symptoms of retrovirus infections.
  • the retrovirus is as defined above, and most preferably the retrovirus is HIV-1, HIV-2 or FIV.
  • patient refers to a human or non-human mammal, or a bird.
  • the patient is a primate, a murine (mouse), a feline (cat), a canine, an equine (a horse), a bird, a human, including women, men, adults and children.
  • the present invention also relates to a method of vaccination or treatment of a subject requiring it comprising the administration of a prophylactically or therapeutically effective amount of a nucleic acid according to the invention, or a vector according to the invention, or an immunogenic or vaccine composition according to the invention.
  • a “prophylactically or therapeutically effective amount” refers to a quantity of nucleic acid, vector or immunogenic or vaccine composition for conferring a therapeutic or prophylactic effect on the subject being treated.
  • the therapeutic effect may be objective (i.e. measurable by tests or markers) or subjective (i.e. the subject gives an indication of an effect or an effect).
  • An effective amount may vary from about 0.01 ⁇ g Kg to 5000 ⁇ g Kg, alternatively from about 0.1 to 1000 ⁇ g Kg, alternatively from about 1 to 500 ⁇ g Kg.
  • the actual amounts will also vary depending on the route. administration, the use or otherwise of in vivo transfection method, the size and weight of the subject, as well as the possibility of co-use with other agents.
  • the mode of administration of the nucleic acid according to the invention, or of the vector according to the invention, or of the immunogenic or vaccine composition according to the invention can be carried out intravenously, under cutaneous, intradermal, intramedullary, or intramuscular.
  • Figure 1 Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 2.
  • Figure 2 Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 4.
  • Figure 3 Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 5.
  • Figure 4 Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 6.
  • Figure 5 Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 7.
  • Figure 6 Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 1.
  • Figure 7 Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 12.
  • Figure 8 Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 13.
  • Figure 9 Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 14.
  • Figure 10 Schematic representation of the nucleic acid encoded by the sequence SEQ ID NO: 15.
  • Figure 11 Schematic representation of the CAEV genome construction, pSHIV KU2 plasmid, pCA-LTR-SHIV KU2 IN- and pA4SHIV KU2 -
  • FIG 12 Evaluation of the number of T cells secreting IFN- ⁇ Balb / c mice. Spleen cells of BALB / c immunocompetent mice that were immunized with pA4SHIV KU 2 and pCA-LTR-SHIV KU 21 N- and stimulated with the Gag, Env peptides and the Tat + Rev + Nef peptide pool. The number of spots has been calculated for 1 million PBMCs.
  • Figure 13 Evaluation of the number of human T cells secreting IFN- ⁇ in immunized NOD / SCID-hu mice.
  • Spleen cells from immunodeficient mice reconstituted by human blood mononuclear cells and immunized with pA4SHIV KU 2 or pCA-LTR-SHIV KU 2I N- or pSHIV KU 2 are stimulated by the Gag, Env peptides and by the Tat + Rev peptide pool + Nave.
  • the number of spots was calculated for 1 million PBMCs and normalized to 20%.
  • Figure 14 Representation of CA-LTR-SHIV KU 2-IN- vector preparation.
  • Figure 15 Schematic representation of the vector CA-LTR-SHIV KU 2-
  • Figure 16 Schematic representation of the vector CA-LTR-SHIV KU 2-IN-.
  • Figure 17 Representation of the preparation of the vector CAL-HIV-IN-.
  • Figure 18 Schematic representation of the CAL-HIV-IN- vector.
  • SEQ ID NO: 1 represents the sequence of a chimeric retroviral genome comprising the LTRs of the SAEV and the genome of the SHIV deleted sequences encoding the integrase.
  • SEQ ID NO: 2 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the genome of HIV-1.
  • SEQ ID NO: 3 represents the CAEV LTR sequence.
  • SEQ ID NO: 4 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the genome of SIV.
  • SEQ ID NO: 5 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the genome of HIV-2.
  • SEQ ID NO: 6 represents the sequence of a chimeric retroviral genome comprising the CAEV LTRs and the FIV genome.
  • SEQ ID NO: 7 represents the sequence of a chimeric retroviral genome comprising the CAEV LTRs and the SHIV genome.
  • SEQ ID NO: 8 represents the sequence of the SIV integrase.
  • SEQ ID NO: 9 represents the sequence of the integrase of HIV-1.
  • SEQ ID NO: 10 represents the sequence of the integrase of HIV-2.
  • SEQ ID NO: 11 represents the sequence of the FIV integrase.
  • SEQ ID NO: 12 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the genome of HIV-1 deleted from the sequences encoding integrase.
  • SEQ ID NO: 13 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the genome of HIV-2 deleted from the sequences encoding integrase.
  • SEQ ID NO: 14 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the genome of FIV deleted sequences coding for integrase.
  • SEQ ID NO: 15 represents the sequence of a chimeric retroviral genome comprising the LTRs of CAEV and the SIV genome deleted of the sequences encoding integrase.
  • SEQ ID NO: 16 represents the sequence of the vector pCA-LTR-SHIV K u2-
  • SEQ ID NO: 17 represents the sequence of the vector pCA-LTR-SHIV KU 2-IN-.
  • SEQ ID NO: 18 represents the sequence of the vector CAL-HIV-IN-.
  • the CA-LTR-SHIV K U2 vector contains the Simian and Human Immunodeficiency Virus (SHIV) genome deleted from the SIV LTRs and replaced by the CAEV LTRs.
  • SHIV contains a chimeric genome composed of that of the VIS-mac239 in which the tat, env and SIV genes were deleted and replaced by the HIV-1 vpu, tat, env and rev genes.
  • the vector therefore carries the SIV vp, vpx, gag, pol, SIV genes and the tat, rev, vpu and env genes of HIV-1 under the transcriptional control of the 5 'and 3' LTRs of CAEV.
  • the pol gene has been deleted from the coding sequences of the integrase (in).
  • the non-deleted pCA-LTR-SHIV KU 2 vaccinating vector of the integrase coding sequences consists of the sequence SEQ ID NO: 16 (FIG. 15).
  • the pCA-LTR-SHIV vector KU 2 -IN-, deleted coding sequences of the integrase consists of the sequence SEQ ID NO: 17 ( Figure 16).
  • the construction of the CA-LTR-SHIV vector KU 2-IN- was carried out as follows (FIG. 14).
  • the SHIV- K U2 vector was digested with EcoR1 and Nar1, and then the 0.8 kb LTR fragment was removed.
  • the vector CAEV-pBSCA was then digested with EcoR1 and Nar1 and the 0.5 kb LTR fragment was purified. Both fragments were then ligated.
  • the SHIV-1 LTRCA vector was then digested with Stu1 and Aval and the 0.8 kb LTR fragment removed.
  • the 3 'LTR of CAEV was amplified with Stu1 and Aval primers, the PCR products were digested with Stu1 and Aval and the 0.5 kb LTR fragment was purified. Both fragments were ligated to generate CAL-SHIV K u2- Finally digestion with Kpn1 and Acc1 in the pol gene was performed to remove 314 bp of the SHIV integrase gene to generate CAL-
  • Plasmids pSHIV KU 2 and pA4SHIV KU 2 are plasmids used as controls. Their constructions have been described in many publications (Liu ZQ et al., 2006, Ramakrisna Hegde et al., 2005). 1.2 Production of vaccine DNA
  • Bacteria E. co // 'K12 (JM109) containing the plasmid are being pre-cultured in 5 ml of LB medium containing 0.05 mg / ml kanamycin and incubated overnight at 30 ° C with stirring at 150 rev / min (rpm). From the pre-culture, the bacterial suspension is diluted 100 000th in LB medium, then 50 ⁇ l of the dilution are spread on the surface of the agar / LB / Kanamycin contained in a petri dish which is then incubated at 32 ° C overnight.
  • the isolated colonies grown on the agar of the petri dish are inoculated in 5 ml of LB liquid medium containing 0.05 mg / ml of kanamycin and cultured with stirring at 150 rpm at 30 ° C overnight.
  • a fraction (1 ml) of the culture is used for rapid extraction of the DNA using the Macherey-Nagel Mini-prep kit or Qiagen, according to the recommended protocol, and the extracted DNA is separated on a gel. agarose at 1% to check its quality.
  • the bacteria corresponding to the DNA deemed satisfactory are used to seed the 1 L cultures which are cultured under the same conditions as above, for the isolation of DNA in maxirieparation.
  • Bacteria cultured with stirring (150 rpm) overnight are pelleted by centrifugation (4000 g, 4 ⁇ C, 15 min) and the pellet is resuspended in 8 ml of resuspension buffer (Tris-HCl 50mM pH8). , EDTA 10mM).
  • the cells are then lysed by addition of 8 ml of alkaline lysis buffer (200 mM NaOH, 1% SDS). to release the plasmid DNA.
  • the lysate is neutralized by addition of 8 ml of neutralization buffer (3M potassium acetate pH 5.5). The mixture is then incubated for 5 min on ice and then centrifuged 15 minutes at 15 000 g at 4 q C.
  • the solution containing the DNA is transferred to a pre-equilibrated column to retain the plasmid DNA.
  • the column is washed three times with the wash buffer and the DNA is then eluted and precipitated with isopropanol.
  • the precipitated pellet of DNA is obtained by centrifugation (30 min, 15,000 g at 4 ° C).
  • the DNA is then washed with 2 ml of 70% ethanol and centrifuged for 10 min at 4 ⁇ ⁇ to 15 000 g to remove excess impurities and salts and the pellet is dried and resuspended in a suitable volume of ultrapure water. .
  • the concentration of the DNA solution is then determined spectrophotometrically at a wavelength ⁇ equal to 260 nm and the quality of the plasmid is verified by electrophoretic migration on a 1% agarose gel.
  • the size of the plasmid and the integrity of the plasmid are verified on agarose gel after digestion with Bam H1 and Eco R1 restriction enzymes, for example.
  • the cell lines were obtained from the National Institutes of Health
  • the cells are cryopreserved in 10% dimethylsulfoxide (DMSO), at -170% in liquid nitrogen. They are thawed and then cultured in culture flasks.
  • DMSO dimethylsulfoxide
  • HEK293T cells (Immortalized Human Embryonic Kidney 293) are a permanent line of human embryonic kidney cells. They are used because they are very easy to transfect, with very high transfection efficiencies that can reach 100%. The lentiviral genome is strongly expressed in these cells and the proteins assemble into infectious particles and their co-culture with the indicator cells (CEM or M8166) allows the formation of typical syncytia.
  • the HEK293T cells are adherent, grown in monolayer on the surface of the flasks in MEM medium supplemented with 10% fetal calf serum (FCS), 1% penicillin 5,000 Unit / ml streptomycin 5,000 ⁇ g / ml and 1% gentamycin 10 mg / ml.
  • FCS fetal calf serum
  • the cells are maintained at 37 ⁇ C in a humid atmosphere of 5% C0 2.
  • the culture medium is changed every three days. To carry out subcultures, the nutrient medium is removed, the cells are washed with PBS / EDTA and incubated for 1 minute at 37 ° C in the presence of 0.5% trypsin-EDTA 0.01%. After detachment of the cells, a volume of MEM medium is immediately added to the cells and then the cells are homogenized and transferred to new flasks.
  • CEMx174 and M8166 cells are human CD4 + T cell lines that are permissive to human and simian lentivirus infection and form typical cytopathic effects (CPE). They are non-adherent and are cultured in RPMI medium supplemented with 10% FCS, 1% penicillin-streptomycin and 1% gentamycin. The cells are maintained at 37 ⁇ C in a humid atmosphere of 5% C0 2. The medium is changed every three days by performing a centrifugation step at 1500 g for 5 minutes. The pellet is then resuspended by suctions and successive upsets in a suitable volume of culture medium.
  • CPE cytopathic effects
  • ExGen500 (Euromedex, France) is composed of a cationic polymer based on linear polyethylenimine. This polymer has a very high cationic charge density for forming complexes with DNA by ionic bonds. These ExGen500 / DNA complexes are then able to interact with the plasma membranes of generally anionic cells (interaction via sulphated proteoglycans). This results in endocytosis of the complexes by the cells and their transport to endosomes / lysosomes. By its ability to protonate at acidic pH, ExGen500 buffers the acidic vesicle medium thus preventing degradation of the transfected DNA. This property also causes osmotic shock, which allows the DNA to be released into the cytoplasm of the cell. ExGen500 then promotes the transport of DNA to the nucleus and prevents its degradation by cytoplasmic nucleases.
  • plasmid DNA Five ⁇ g of the plasmid DNA are added to 350 ⁇ l of 150 mM NaCl solution and 15 ⁇ l of ExGen 500. The mixture is incubated for 40 minutes at room temperature. The mixture is then added to the flasks containing HEK-293T coated with freshly renewed medium.
  • CEMx174 Infection of CEMx174 and amplification of viral stock
  • the transfected HEK-293T DNA pCA-LTR-SHIV KU2 -in- or pSHIV KU2 are being co-culture with CEMx174, and from 48 hours the CEMx174 develop signs of infection resulting the formation of ECP that result from the fusion of CEMx174 to form syncitia.
  • Infected CEMx174 are transferred to a new flask in the presence of fresh CEMx174 to amplify the virus that is harvested from the supernatant.
  • the viral stock is collected from 48 hours using a syringe, then the cell debris is removed by passing through a 0.22 ⁇ diameter filter before being put in tubes that are stored. at -80 'C.
  • An aliquot (10-100 ⁇ ) of the virus supernatant is used to inoculate the target cells to evaluate its infectivity by cytopathic development or by detection of expression of marker genes.
  • the supernatant containing virus was diluted every ten (serial dilutions) in medium to obtain dilutions of 10 ⁇ 1-10 ⁇ 6 which are used to inoculate quadriplate in wells containing 1 .10 5 cells per well in 0, 5 to 1 ml of RPMI medium in a 24-well plate.
  • the cells thus inoculated are incubated at 37 ° C. and 5% CO 2 and maintained by changing the medium every 3 days. They are observed regularly for the development of ECP.
  • Samples of HEK293T cells transfected with pCA-LTR-SHIV KU 2-IN-, pSHIV KU 2 or A4SHIV KU 2 are fixed in a solution of 2.5% glutaraldehyde diluted in a cacodylate buffer (0.1 M sodium cacodylate). . They are then post-fixed at 4 ° C in cacodylate buffer containing 1% Osmium tetraoxide (OsO4) for 60 minutes. The samples are then incubated overnight at 4 ⁇ C in the dark in uranyl acetate pH 4. The samples were then immersed in baths successive 10 minutes of ethanol diluted to 30%, 60%, 90% and 100% respectively.
  • a cacodylate buffer 0.1 M sodium cacodylate
  • OsO4 Osmium tetraoxide
  • the samples are immersed for two hours in a 50/50 mixture of pure ethanol and epoxy resin (8 ml of DDSA, 7 ml of MNA, 13 ml of Epoxy). The samples are then placed for two hours in pure epoxy resin before being included in Beem capsules and polymerized for 48 hours at 60 ° C.
  • Ultrafine sections of these areas are made using a diamond knife using an ultra microtome. These 70 nm thick sections are deposited on copper grids to be observed at a voltage of 80kV using a Jeol 1200 EX transmission ME.
  • mice The 6 week old mice are irradiated with a dose of 120 Centigray of gamma radiation for 50 seconds.
  • mice Humanization of mice with PBMCs of human blood
  • mice Whole blood collected on sodium citrate is centrifuged (2000g, 10 min, 20 ° C) to recover the white cell layer between plasma and red cells.
  • the cells are diluted 3 times in PBS / EDTA, gently deposited over a Ficoll cushion (lymphocyte separation medium) and then centrifuged for 45 minutes at 2000g at 20 ° C.
  • the PBMC are recovered, washed several times in PBS / EDTA and resuspended in PBSxl then 50.10 6 PBMC in 0.1 ml are injected for each mouse intraperitoneally. Immunization of mice
  • the SCID-hu mice are injected intramuscularly (IM) with 50 .mu.g of DNA pCA-LTR-SH IV KU2 -in-, pSHIV KU2 or pA4SHIV 2- KU BALB / Those aged 6-8 weeks are directly immunized by IM injection with 100 ⁇ g of each of the DNAs.
  • IM intramuscularly
  • This test is based on the detection of antibodies against viral antigens that are attached to the bottom of wells of the 96-well plate.
  • the sera to be tested (recovered at different post-immunization times), the positive and negative controls are deposited in the wells.
  • the anti-HIV antibodies possibly present are fixed on the viral antigens.
  • a secondary detection acid is added which carries a biotin molecule which interacts with streptavidin coupled to the HPRO enzyme.
  • the antigen / Ac complexes formed will then be detected by adding the substrate of the enzyme, TMB, which will give rise to a color reaction.
  • the coloration is translated into optical density by reading with an ELISA reader photometer.
  • the reagents and products are brought to room temperature.
  • the negative and positive controls (1 ⁇ ) provided in the kit, the blanks (1 ⁇ ) and the serum samples 10 and 50 ⁇ are deposited in the wells in a final volume of 10 ⁇ .
  • the plate is incubated for 30 minutes at 37 ° C, then rinsed with the wash solution.
  • the solution of secondary Ac diluted to one hundredth is added (100 ⁇ ) in all the wells except in the whites.
  • the plate is then covered with parafilm and incubated for 20 minutes at 37 ⁇ ⁇ .
  • a solution A and B of TMB is added after a washing step and the plate is incubated for 15 min at laboratory temperature.
  • 100 ⁇ l of H 2 SO 4 to 2 N are added per well. The reading of the plate is made at 450 nm.
  • This technique is based on the ability of seroneutralizing antibodies (sero-N) to inhibit the infection of virus-sensitive cells.
  • seroneutralizing antibodies sero-N
  • a constant amount of infectious virus is contacted with serial dilutions of the serum to be tested, then the mixture is inoculated into a microplate-permissive cell culture and incubated at 5 days.
  • the virus is most often a cytopathogenic strain, and therefore the absence or reduction in the number of PCEs translates the presence of Ac into the tested serum.
  • the SHIV K U2 viral stock is diluted 1/1 in RPMI (the volume of virus supernatant is determined per 100 TCID 50 , which corresponds to the dilution of virus for which 50% of the wells have syncitia) and the sera recovered from the mice.
  • NOD / SCID controls humanized and vaccinated with pCA-LTR-SHIV KU 2-IN- or pSHIV KU 2 are diluted in the same medium (at dilutions 10, 20, 40, 80, 160 and 320).
  • the diluted virus and serum dilutions were mixed in a 96 well plate (1 ⁇ / well) and the mixture was incubated 1 h at 4 ⁇ .
  • This test aims to highlight and evaluate the proportion of T lymphocytes (LT) that secrete IFN- ⁇ in specific response to antigenic stimulation.
  • mice are deeply anesthetized before the whole blood and the spleen are removed.
  • the spleens of the mice are put in RPMI medium in ice.
  • the blood in dry tubes is used to isolate the serum.
  • the splenocytes are isolated following grinding of the spleen in a petri dish between a pair of slides in the presence of PBS and 1% EDTA.
  • the cells are washed twice in PBS / EDTA (2000g centrifugation, 5 min at 20 ° C.) in order to purify and enrich in splenocytes. Before inoculating the wells with the cells, the plate is washed with PBS and incubated for 30 min with PBS + 10% FCS at laboratory temperature.
  • the cells ( 5 ⁇ 10 5 splenocytes) are inoculated in each well, and are inoculated with peptide pools of the Gag, Env, Tat, Rev and Nef proteins at a final concentration of 2 ⁇ g / ml.
  • Positive controls CD3-2 included in the kit
  • negative controls are added to the test.
  • the plate is covered with an aluminum pouch and incubated at 37 ° C for 19 hours.
  • the cells and peptides are washed, then the biotinylated anti-IFN- ⁇ monoclonal Ab (7-b6-biotin) is added, and the plate is covered and incubated for 2 hours at room temperature.
  • Streptavidin diluted in PBS containing 0.5% FCS is added (1 ⁇ M / well) and the plate is incubated for 1 hour at room temperature.
  • the TMB, developer substrate, is added later after another washing step, then the plate is washed and dried after emergence of blue spots. The reading of the plate is done with the binocular magnifier at magnification * 40.
  • the criteria for positivity of a well are determined for each condition by calculating the average of the number of spots of the duplicates, as well as the standard deviations.
  • the number of spots is calculated for 1 million PBMC and is normalized to 20% for the data obtained in NOD / SCID mice.
  • the test is considered to be positive if the value of the average of the spots is greater than 10 spots per million PBMCs which corresponds to the average of spots obtained with the culture controls.
  • splenocytes and PBMCs are labeled with CFSE (1 ⁇ g ml) for 10 min at 37 ⁇ C, then the cells are washed with 1 X PBS to remove excess.
  • the labeled cells are seeded in deep wells of 96 well plates at 2.10 s / well in 1 ml of AIM V medium, then stimulated with the different peptide pools (Gag, Env and Tat + Rev + Nef) at a rate of of 2 ⁇ g / ml in the presence of anti-CD49 and CD28 co-stimulation mAbs.
  • Cells without peptides are used as a negative control and cells supplemented with phytohemagglutinin (PHA) 2 ⁇ g / ml are used as a positive control.
  • the cells are cultured for 5 days (37 ° C. under humidity) and then restimulated with the same peptide pools for 6 hours before labeling them.
  • the cells are harvested by centrifugation (2000g, 5 min, 4 ° C), resuspended in 100 ⁇ l of PBS and first labeled with surface Ab (CD3, CD4 and CD8) [Pacific Blue anti-human CD3 (5 ⁇ l) ), Anti-human CD4 PE (10 ⁇ l) and APC / Cy7 anti-human CD8 (10 ⁇ l) for 30 min at room temperature.
  • the cells are then centrifuged and washed with 1 X PBS and then fixed and permeabilized in 100 ⁇ l of Cytofix Cytoperm BD.
  • the cells are then incubated for 20 minutes at 4 ⁇ C with Ac "anti-human IFN- ⁇ PE-Cy7" and "Alexa Fluor 647 anti-human Granzyme A” (5 ⁇ ) for cytoplasmic markings.
  • the cells are finally washed with PBS and fixed with 4% PFA prior to acquisition and flow cytometric analysis.
  • An LSRII flow cytometer from BD connected to BD FACSDiva6 software was used. This instrument can measure up to 13 fluorescence parameters and two physical parameters such as FSC size (Forward Scatter) and SSC (Side Scatter) complexity or granulosity.
  • the instrument is equipped with three lasers. The blue laser emitting at 488nm can independently excite several fluorochromes (FITC, PE, PE-Cy7). The red laser emitting at 633nm can excite the APC and APC-Cy7 fluorochromes. And finally the purple laser that emits at 405nm can excite the Pacific Blue fluorochrome. 1.7. Immunization of macaque
  • a total of 12 cynomolgus macaques are used in this study. Six macaques constitute the control group and the six others the vaccinated group. The animals were immunized by a single double injection of DNA by the intramuscular route (4 mg / animal) and 1 mg / animal by electroporation (EP).
  • PBMC peripheral blood mononuclear cells
  • the electrophoretic profile also demonstrates the presence of a single DNA band around 14,000 bp (which in theory is 13,739 bp) corresponding to the plasmid.
  • the electrophoretic profile obtained after separation of 0.5 ⁇ g of DNA shows the absence of an episome and shows three bands of high molecular weight DNA, corresponding to the circular, wound and supercoiled forms of the plasmid pCA-LTR- SHIV KU 2-IN-.
  • the purity and quantitative evaluation of the plasmid DNAs of our two preparations were verified by spectrophotometry.
  • the values of the measurements of the absorbance at wavelengths 230, 260 and 280 were used to determine the ratios 260/280 which were 1.75 and 1.82 and 260/230 of 2.04 and 1.92 respectively. a satisfactory quality of our DNA.
  • the DNA concentrations are respectively 545 ⁇ g ml and 765 ⁇ g / ml.
  • the profile of the digestion of the plasmid with EcoR1 reveals the presence of two bands of approximately 5000 and 7500 bp resulting from the cuts at the two EcoRI sites, three bands with Bam H 1 of 2400 bp, 4900 bp and 7400 bp resulting from the cuts at the two Bam H sites 1, and a band with Sph 1 located at 10 000pb resulting from the cut at the single site Sph1.
  • An additional 2400 bp band is also observed with BamH 1 digestion and it would result from the episome.
  • the HEK293T cells were transfected with a control plasmid pCG-GFP expressing GFP, the plasmid pSH IV KU 2, and then with the plasmid pCA-LTR-SH IV KU 2-IN-. Cells transfected with the plasmid pCG-GFP made it possible to estimate the transfection efficiency by evaluating the number of GFP + cells.
  • EK293T H cells transfected with pCA-LTR-SH IV K u2-IN- or pSH IV K u2 produce virions that induce typical syncitia
  • these cells were co-cultured with CEMx174, followed by appearance of CPE was followed by observation under the microscope. Both co-culture produced characteristic ECPs.
  • the recovered supernatant containing the CA-LTR- SH IV KU 2-IN- was inoculated once, then a second time to M81 66 in culture.
  • the first inoculation produced CPE from 48 hours and after 76 hours they were more numerous.
  • the supernatant of these infected cells was recovered and inoculated again with M81 66.
  • no CPE is visible either at 48 or 76 hours postinoculation.
  • the presence of typical ECP suggests that plasmid DNA is replicated in HEK293T cells and produced virions CA-LTR-SHIV KU 2-IN-.
  • the PCEs of the first infection are indicative of the infectivity of the M8166 human CD4 + LT line by the particles produced, and their absence during the second infection is indicative of the productive replication deficiency in these cells.
  • Serum samples from immunized mice taken at approximately 1 month post-immunization are examined for the presence of Ac that binds specifically to the virus antigens using a commercial ELISA.
  • the results of this analysis are summarized in Table 1 (below). These results show that about half of the samples from mice immunized with the pCA-LTR-SHIV KU 2-IN- DNA, like those immunized with SHIV KU 2 DNA, have lower levels of positive ( 44% and 37% respectively) in contrast to those from mice immunized with pA4SHIV KU 2 (50%).
  • These results demonstrate the ability of pCA-LTR-SHIV K u2-IN- vaccine DNA to induce humoral responses in NOD / SCID-hu mice.
  • Sera of SCID-hu mice vaccinated with the selected pCA-LTR-SHIV K u2-IN- or SHIV KU 2 plasmids are those whose OD has been found positive by ELISA. Due to the small amount of serum, some samples with high OD values for the ELISA could not be examined by serum neutralization. A serum sample of SCID-hu mice and vaccinated with pCA-LTR-SHIV KU 2-IN- found negative was also used for the ELISA assay to ensure the reliability of the test.
  • Table 2 Analysis of the neutralizing activity of the sera of the immunized mice. Dilutions (1/10, 1/20, 1/3, 20) were mixed with SHIV KU2 virus (100 TCID 50 ), incubated and then used to inoculate M8166 cells. After 5 days postinoculation, the induced EPCs are enumerated and used to evaluate serum neutralizing activity. Legend: +++ correspond to a strong neutralization, ++ quite high neutralization, + less neutralization, - no neutralization action. For CA-LTR-SHIV K u2, two types of samples are shown, one type of sample having a less neutralizing profile compared to the other two samples.
  • the number of CPEs obtained was higher in M8166 cells infected with serum free SHIV K U2 virus (76 ECP) or with non-immune mouse serum (42 ECP) (negative controls), which allowed us to to set the viral neutralization negativity threshold at 42 ECP (data not shown in the table).
  • the number of ECP becomes almost zero when the virus is incubated with serum diluted 1/10 of the mice immunized with CA-LTR-SHIV DNA K U2-IN- or SHIV K U2- This number increases as serum dilution is increased indicating a dose effect.
  • NOD / SCID-hu mice immunized with the different DNAs.
  • the NOD / SCID-hu mice were immunized by intramuscular injection with a single dose of 50 ⁇ l of CA-LTR-SHIV DNA KU 2-IN-, A4SHIV KU2 or SHIV KU 2, then the splenocytes were used to examine the immune response by ELISPOT to evaluate the proportion of antigen-specific cells producing IFN- ⁇ .
  • FIG. 13 there is a large number of T cells producing human INF- ⁇ in response to stimulation by Gag, Env or Tat + Rev + Nef antigens in the form of SIV or HIV peptides. .
  • Unlabelled lymphocytes from humanized and non-humanized mice were analyzed by flow cytometry to measure basal cell fluorescence (negative control).
  • CD3 + LT is performed with human anti-CD3 antibody coupled to fluorochrome "Pacific Blue”. These cells peak at 10 2 in the profile of isolated cells in SCID-hu mice vaccinated with plasmid pCA-LTR-SHIV KU 2-IN-. This peak is not present in the cells recovered in non-hued SCID mice.
  • a human monoclonal anti-CD8 antibody coupled to APC / Cy7 fluorochrome was used for the detection of CD8 + LTs. It allows the highlighting of a peak located between 10 2 and 3 in the profile of isolated cells in SCID-hu mice vaccinated with pCA-LTR-SHIV K U 2 -IN- and not in non-hued SCID mice.
  • lymphocytes producing the GRA molecules could not be evaluated because no peak difference was observed on the cells from immunized and non-immunized mice labeled with anti-GRA A coupled to Alexa Fluor 647.
  • the cells labeled with the human anti-IFN- ⁇ coupled to the fluorochrome PE-Cy7 showed a sharp peak situated at 10 2 with the SCID-hu mouse cells vaccinated with pCA-LTR-SHIV KU2- IN- which is absent with the cells of non-immunized non-hu SCID mice.
  • CFSE-labeled cells are incubated for 5 days with the peptides (Gag, Env and Tat + Rev + Nef) and then re-stimulated for 6 hours with the cells. same peptides. The cells are then labeled with Ac and examined as above.
  • results of this analysis show the presence of CD3 + and CD8 + cells which produce IFN- ⁇ and which have GRA molecules especially with cells from vaccinated NOD / SCID-hu mice. Indeed, at least 6% of the CD8 + LTs produce IFN- ⁇ and 1.7% produce the GRA A in immunized NOD / SCID-hu mice, compared to only 2.5% and 0.6% respectively in the control mice. These results demonstrate the presence of activated CD3 + CD8 + cells, producing GRA and IFN- ⁇ , which corresponds to an effector cellular immune response induced by our pCA- vaccine.
  • the immune responses are characterized by the presence of a first peak of primary response at 2-4 weeks post-immunization (PI), then later responses from 8-10 weeks PI, and this in the absence of a second immunization. It is very interesting that the intensity of the second peak is often much larger than 1 peak especially for the animal BX80 where the number of cells secreting IFN- ⁇ is multiplied by 3.
  • Table 3 Summary of interferon gamma cytokine secretory T cell (IFN- ⁇ ) ratios in response to stimulation by viral antigens (Gag, Pol, Env and Tat + Rev + Nef) expressed by the vaccine in vaccinated monkeys .
  • the numbers correspond to the number of secretory cells forming one spot per million (10 6 ) of peripheral blood mononuclear cells (PBMC). The weeks of analysis are reported at the top of the table.
  • the results of the multiparametric flow cytometric analyzes confirm those obtained by ELISPOT by revealing that all the animals have developed a response composed of proliferating T cells that are specific for all the antigens expressed by the vaccine vector (Table 4). These responses are heterogeneous between animals and also reveal a first phase of primary response that extends to about 8 weeks PI, followed by a contraction phase (2-4 weeks) and then a re-emergence phase. This longitudinal monitoring of the immune response by multiparametric flow cytometry is continued until the virulent challenge by the test virus SIVmac251 which is carried out at week 52.
  • Table 4 Summary of proliferative CD4 + and CD8 + T cell values in response to antigenic stimulations at the time of the primary expansion, contraction and finally reemergence or secondary expansion phases in vaccinated monkeys. The weeks corresponding to each of the phases are indicated. The numbers correspond to the percentages of T cells specific for each of the antigens that proliferate in response to stimulation relative to the total number of T cells.
  • anti-SHIV antigen antibodies The detection of anti-SHIV antigen antibodies has been performed by a commercial ELISA kit which makes it possible to detect anti-HIV-1 Env antibodies. Longitudinal examination of the sera collected at each blood collection point showed the presence of anti-Env antibodies from week 20 PI in animal BX80 and from week 8 for animal BX73. The presence of antibodies in the positive sera was confirmed by Western blotting against SHIV proteins showing a strong signal against Gag-p27 protein as well as a signal against glycoproteins gp160 / gp120.
  • T cell responses are directed against all viral antigens expressed by the vaccine vector. They are persistent and pursue a classic pattern of expansion, contraction and memorization. The presence of central memory and memory effector T cells was confirmed by phenotyping.
  • the SIV gag, pol, vif, vpx and vpr genes were deleted following double digestion with the Nar1 and Sph1 enzymes, and the SIV nef gene. was deleted following partial digestion with the enzyme Nru1 and total digestion with the enzyme Not 1. The remaining fragment was then purified. This fragment, carrying the tat, rev and env genes of the HIV boxed by the LTRs of CAEV and transported by the plasmid pET, was used to introduce the 5kb fragment carrying the gag, pol, vif, vpx and vpr genes of HIV.
  • the vector CAL-HIV-IN-, deleted coding sequences of the integrase consists of the sequence SEQ ID NO: 18.
  • the DNA of this vector was introduced into GHOST-CXCR4 and HEK-293 T cells by transfection using ExGen and the protocol recommended by the manufacturer.
  • the transfected GHOST-CXR4 cells then became fluorescent attesting to the expression of the viral proteins of HIV-1 by the vaccine vector and more particularly the Tat protein which transactivates the expression of the GFP (Green Fluorescent Protein) gene under the control of the HIV LTR.
  • GFP Green Fluorescent Protein
  • the supernatant of HEK-293T cells transfected with the vaccine vector CAL-HIV-IN- was used to inoculate M8166 indicator CD4 + T cells that developed characteristic cytopathic effects of HIV infection. These results provide evidence that the proteins expressed by the vaccine vector have assembled into viral particles allowing the infection of M8166 indicator cells. These cells with cytopathic effects did not produce a virus capable of re-infecting M8166 indicator cells and inducing cytopathic effects. This result indicates the CAL-HIV-IN- is associated with a single cycle of replication in the absence of integration.
  • the supernatants of HEK-293T cells transfected with the vaccine vector were harvested 24h, 48h and 72h post transfection and then examined for the presence of Gag p24 antigens by ELISA. Measurements of the amounts of this protein are reported in Table 5. demonstrate an increasing accumulation of this protein ranging from 100 ng / ml to 24h up to 135 ng / ml at 72 hours post transfection.
  • Table 5 Evaluation of secreted H IV-1 Gag p24 protein in the supernatant of HEK 293T cells transfected with the vaccine vector CAL-HIV-IN-. Quantification of the p24 protein accumulated in the supernatant of the HEK 293T cells after 24, 48 and 72 h post-transfection with the CAL-HIV-IN-vector DNA.
  • mice Three groups of BALB / C Mice (6 per group) of 6 weeks were used: 2 groups were used for immunization and the third group was a control group. The 2 groups of immunized mice were injected with 100 ⁇ g / mouse of CAL-HIV-IN-vector DNA by the intramuscular route. Animals in one group were sacrificed at 2 weeks and the other at 3 weeks post immunization (PI). The control mice were sacrificed at 2 weeks Pl. The spleens of each of the mice were removed, the splenocytes were isolated and then used either for the ELISPOT assay or for the multiparametric flow cytometry analysis as described above.
  • Table 6 Summary of the results of the ELISPOT analysis on splenocytes of BABL / c mice immunized with the vaccine vector CAL-HIV-IN- at 2 and 3 weeks post-immunization.
  • the splenocytes isolated from the spleens of mice immunized with 100 ⁇ g per intramuscular mouse were examined by the IFN- ⁇ ELISPOT test to evaluate the number of T cells secreting this cytokine in response to the stimulations with the peptide pools (Gag, Env and Tat + Rev + Nef, TRN). Spot number averages for each antigen and for the 6 mice examined after 2 and 3 weeks post-immunization are reported.
  • the results of the flow cytometric analysis demonstrate immune responses in CD4 + and CD8 + T cells specific for all the antigens expressed by the vaccine vector CAL-HIV-IN-.
  • Table 7 Summary of the results of the multiparametric flow cytometry analysis. The figures correspond to the percentages of T cells specific for each of the antigens that proliferate in response to the antigenic stimulation relative to the total number of T cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Virology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • AIDS & HIV (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
EP12756752.7A 2011-09-12 2012-09-12 Genomes lentiviraux chimeriques non-integratifs comme vaccins contre hiv-1 Withdrawn EP2756087A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR1158096A FR2979919B1 (fr) 2011-09-12 2011-09-12 Genomes lentiviraux chimeriques non-integratifs comme vaccins innovants contre vih-1
PCT/EP2012/067863 WO2013037841A2 (fr) 2011-09-12 2012-09-12 Génomes lentiviraux chimériques non-intégratifs comme vaccins innovants contre le hiv-1

Publications (1)

Publication Number Publication Date
EP2756087A2 true EP2756087A2 (fr) 2014-07-23

Family

ID=46829786

Family Applications (1)

Application Number Title Priority Date Filing Date
EP12756752.7A Withdrawn EP2756087A2 (fr) 2011-09-12 2012-09-12 Genomes lentiviraux chimeriques non-integratifs comme vaccins contre hiv-1

Country Status (10)

Country Link
US (1) US9879230B2 (es)
EP (1) EP2756087A2 (es)
JP (1) JP6324892B2 (es)
CN (1) CN103946385B (es)
CA (1) CA2848484C (es)
FR (1) FR2979919B1 (es)
IL (1) IL231494B (es)
MX (1) MX354571B (es)
WO (1) WO2013037841A2 (es)
ZA (1) ZA201402676B (es)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180179256A1 (en) * 2015-05-04 2018-06-28 Epivax, Inc. Modified H7 Hemagglutinin Glycoprotein of the Influenza A/Shanghai/2/2013 H7 Sequence
US10286063B2 (en) 2015-05-04 2019-05-14 Epivax, Inc. Modified H7 hemagglutinin glycoprotein of the Influenza A/Shanghai/2/2013 H7 sequence
RU2752498C2 (ru) * 2015-11-24 2021-07-28 Глэксосмитклайн Интеллекчуал Проперти Дивелопмент Лимитед Стабильные клеточные линии для продуцирования ретровирусов

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999056774A1 (en) * 1998-04-30 1999-11-11 University Of Southern California Viral chimeras comprised of caev and hiv-1 genetic elements

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5354844A (en) 1989-03-16 1994-10-11 Boehringer Ingelheim International Gmbh Protein-polycation conjugates
EP0752000A1 (en) * 1994-03-24 1997-01-08 Syngenix Limited Packaging-deficient lentiviruses
US7488485B2 (en) * 2003-09-16 2009-02-10 University Of Kansas Medical Center DNA vaccine compositions and methods of use
DE102005003207A1 (de) * 2005-01-17 2006-07-27 Geneering Gmbh Lentivirales Gentransfersystem für Ribozym-vermittelte RNA-Reparatur

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999056774A1 (en) * 1998-04-30 1999-11-11 University Of Southern California Viral chimeras comprised of caev and hiv-1 genetic elements

Also Published As

Publication number Publication date
FR2979919B1 (fr) 2015-12-11
JP2014527812A (ja) 2014-10-23
JP6324892B2 (ja) 2018-05-16
CA2848484A1 (fr) 2013-03-21
FR2979919A1 (fr) 2013-03-15
MX354571B (es) 2018-03-12
MX2014002953A (es) 2014-07-10
US9879230B2 (en) 2018-01-30
CN103946385A (zh) 2014-07-23
ZA201402676B (en) 2015-05-27
US20140370051A1 (en) 2014-12-18
WO2013037841A2 (fr) 2013-03-21
CA2848484C (fr) 2021-03-23
IL231494A0 (en) 2014-04-30
CN103946385B (zh) 2016-03-16
IL231494B (en) 2018-05-31
WO2013037841A3 (fr) 2013-05-23

Similar Documents

Publication Publication Date Title
JP6480028B2 (ja) レンチウイルス遺伝子導入ベクター及びそれらの医薬品への適用
JP6625716B2 (ja) ヒト免疫不全ウイルス感染に対する防御免疫を誘導するための方法および組成物
EP2676676B1 (en) Recombinant viral vectors
US20130189754A1 (en) Immunoselection of recombinant vesicular stomatitis virus expressing hiv-1 proteins by broadly neutralizing antibodies
Schneider et al. Simian lentiviruses—the SIV group
EP0245136B1 (fr) Vecteur viral, codant pour une glycoprotéine du virus responsable du SIDA, vaccin et anticorps
EP2398496B1 (en) Vesicular stomatitis virus vectors encoding hiv env epitopes
US20110110892A1 (en) Vectors for delivering disease neutralizing agents
Xiao et al. Parainfluenza virus 5 priming followed by SIV/HIV virus-like-particle boosting induces potent and durable immune responses in nonhuman primates
KR20180127402A (ko) 사이클릭 디뉴클레오타이드를 포함하는 바이러스 입자의 제조 방법 및 암 치료를 위한 상기 입자의 용도
CA2848484C (fr) Genomes lentiviraux chimeriques non integratifs comme vaccins innovants contre le hiv-1
Baier et al. Complete nucleotide sequence of a simian immunodeficiency virus from African green monkeys: a novel type of intragroup divergence
FRANCHINI et al. Phylogenesis and genetic complexity of the nonhuman primate retroviridae
Li et al. Mucosal IL-4R antagonist HIV vaccination with SOSIP-gp140 booster can induce high-quality cytotoxic CD4+/CD8+ T cells and humoral responses in macaques
Moussa et al. A novel non-integrative single-cycle chimeric HIV lentivector DNA vaccine
WO1987007642A1 (fr) Vaccin, contenant la proteine f du virus du sida
Pahar et al. Single epitope mucosal vaccine delivered via immuno-stimulating complexes induces low level of immunity against simian-HIV
WAKRIM et al. Superinfection of HIV-2-preinfected macaques after rectal exposure to a primary isolate of SIVmac251
WO2004039945A2 (en) Preventive and therapeutic aids vaccines
Villinger et al. Induction of long-term protective effects against heterologous challenge in SIVhu-infected macaques
Mahmoudi Development and characterization of novel dendritic cell (DC)-targeting vaccine against human immunodeficiency virus (HIV)-1 envelope conserved elements (CEs)
Amarasena et al. Z. Li1, M. Khanna1, 2, SL Grimley3, P. Ellenberg3, CA Gonelli3, Wen Shi Lee3
Bhakta A Functional Analysis Of The Human Immunodeficiency Virus
EP1423418A1 (fr) Gene env mute, glycoproteine env mutee et utilisations
Mostafa Characteristics and Relevance of the Interaction between Staufen-1 and Rev-related retroviral Proteins

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20140411

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20161027

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20230718

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: UNIVERSITE GRENOBLE ALPES

Owner name: INSTITUT NATIONAL DE RECHERCHE POUR L'AGRICULTURE, L'ALIMENTATION ET L'ENVIRONNEMENT

Owner name: CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (C.N.R.S.)

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20231129