+

WO2008151633A2 - Vectors for vaccines against lentivirus infections - Google Patents

Vectors for vaccines against lentivirus infections Download PDF

Info

Publication number
WO2008151633A2
WO2008151633A2 PCT/DK2008/000217 DK2008000217W WO2008151633A2 WO 2008151633 A2 WO2008151633 A2 WO 2008151633A2 DK 2008000217 W DK2008000217 W DK 2008000217W WO 2008151633 A2 WO2008151633 A2 WO 2008151633A2
Authority
WO
WIPO (PCT)
Prior art keywords
vector
hiv
virus
envelope
retroviral
Prior art date
Application number
PCT/DK2008/000217
Other languages
French (fr)
Other versions
WO2008151633A3 (en
Inventor
Martin Tolstrup
Finn Skou Pedersen
Lars ØSTERGAARD
Mogens Ryttergaard Duch
Tea Kirkegaard Nielsen
Original Assignee
Skau Aps
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 Skau Aps filed Critical Skau Aps
Publication of WO2008151633A2 publication Critical patent/WO2008151633A2/en
Publication of WO2008151633A3 publication Critical patent/WO2008151633A3/en

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
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—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
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13045—Special targeting system for 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
    • C12N2740/16011—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
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—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
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6045—RNA rev transcr viruses
    • C12N2810/6054—Retroviridae
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
    • C12N2840/206—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES having multiple IRES

Definitions

  • Retroviruses have the capacity to infect a wide variety of different cells. Moreover, retroviruses transfer their genes from a producer cell to a target cell as a genomic RNA transcript. This RNA is reverse-transcribed after infection and integrated into the DNA genome of the target cell.
  • HIV-1 HIV-1
  • HIV-2 HIV-2 that infect humans. They belong to a group of viruses called Lentiviruses and a virus similar to HIV has been found in African monkeys.
  • the present invention relates to a replication deficient vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition and kit comprising at least one lentiviral envelope polypeptide or at least one nucleic acid sequence encoding a lentiviral envelope polypeptide.
  • the invention is applicable for generation of retroviral particles for specific targeting of host cells.
  • the retroviral particles may be used for integration of trangenes in host cells and induction of an immunogenic response, for example against HIV-1.
  • the invention relates to an RNA of the vector as defined in the present invention.
  • the invention relates to a retroviral provirus produced in a target cell upon reverse transcription of said RNA as defined above or part thereof.
  • the invention pertains to a producer cell comprising any vector according to the present invention.
  • the invention relates to host cell comprising a vector according to the present invention.
  • the present invention relates to a host cell infected with a retroviral particle according to the present invention.
  • the present invention relates to the use of the vector according to the present invention, the producer cell according to according to the present invention, a retroviral particle according to the present invention and/or a host cell according to the present invention for producing a medicament for gene therapy.
  • the present invention relates to the use of the vector according to the present invention, the producer cell according to according to the present invention, a retroviral particle according to the present invention and/or a host cell according to the present invention for producing a medicament for immune therapy.
  • the present invention relates to a pharmaceutical composition containing a therapeutically effective amount of the vector according to the present invention, the producer cell according to according to the present invention, a retroviral particle according to the present invention and/or a host cell according to the present invention.
  • the present invention relates to a method for introducing a nucleotide sequence into target cells, said method comprising infection of target cells with retroviral particle according to the present invention.
  • a vaccine for the prophylaxis or treatment of lentiviral infection comprising any vector according to the present invention, any RNA according to the present invention, any retroviral provirus according to the present invention and/or any retroviral particle according to the present invention.
  • the present invention relates to a vaccine composition
  • a vaccine composition comprising the vaccine according to the present invention for use as a medicament.
  • a further aspect of the present invention relates to a kit comprising a therapeutically effective amount of the vector according to the present invention, the producer cell according to according to the present invention, a retroviral particle according to the present invention and/or a host cell according to the present invention.
  • a further aspect of the present invention relates to vaccine composition
  • vaccine composition comprising a. a lentiviral envelope polypeptide or a fragment, and b. an adjuvant and/or an immunomodulating peptide.
  • Another aspect of the present invention relates to vaccine composition according to the present invention for treatment, amelioration and/or prevention of HIV infection and/or AIDS.
  • a further aspect of the present invention relates to vaccine composition
  • a further aspect of the present invention relates to use of the vector according to the present invention, a producer cell according to the present invention, a retroviral particle according to the present invention, a host cell according to the present invention and/or a vaccine composition according to the present invention for the manufacture of a medicament for gene therapy.
  • a further aspect of the present invention relates to use of the vector according to the present invention, a producer cell according to the present invention, a retroviral particle according to the present invention, a host cell according to the present invention and/or a vaccine composition according to the present invention for the manufacture of a medicament for immune therapy.
  • the present invention relates to use according to the present invention for the manufacture of a medicament for the treatment, amelioration or prevention of HIV and/or AIDS.
  • a final aspect of the present invention relates to use according to the present invention for the manufacture of a medicament for HIV vaccination.
  • Figure 2 Western blot with anti-HIV-1 gp120 of cell lysate from 293T transfected cells.
  • CMV-128 and VSV-G were co-transfected with pLSXN. Forty-eight hours after transfection supematants were filtered through 0.45 ⁇ m and added on D17 CXCR4+/ CD4+ cells. The following day cells were harvested and the percentage of green fluorescent cells were enumerated by flow cytometry. Results shown are the average of at least three independent experiments.
  • FIG. 1 Level of egfp fluorescence evaluated in various cell types.
  • the retroviral vector Egfp-pLXSN was used as comparison to define expression baseline levels of integrated EgfpHIVMo.
  • Pseudotyped particles prepared from 293T cells were used to transduce the indicated cell lines. 48 hr later cells were visualised with fluorescence microscopy.
  • FIG. 10 293T cells transfected with plasmids indicated above lanes.
  • E2R contain a STOP cocon in the Rev exon (hence no Rev production).
  • Anti-Env from HIV+ patient serum used as primary polyclonal antibody.
  • FIG. 1 Western Blot of HIV envelope from transfected HeLa Cells.
  • Figure 12. Western Blot of HIV envelope from transfected NIH3T3 Cells
  • an HIV vaccine is affected by the range of virus subtypes as well as by the wide variety of human populations who need protection and who differ, for example, in their genetic make-up and their routes of exposure to HIV.
  • the occurrence of superinfection indicates that an immune response triggered by a vaccine to prevent infection by one strain of HIV may not protect against all other strains.
  • the effectiveness of a vaccine is likely to vary in different populations unless some innovative method is developed which guards against many virus strains.
  • present invention offers a replication-deficient retroviral vector comprising at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof.
  • the retroviral vector is useful for the production of virus particles displaying a lentivral envelope polypeptide or fragment thereof, which is used as a vaccine to prevent infection with lentiviruses, in particular HIV-1 .
  • the present retroviral vector has improved immunogenic properties compared to known HIV viruses, and in addition displays safety features not available at present as will be described herein.
  • RNA coding for env An example of a spliced and capped RNA transcript coding for a single protein is the RNA coding for env.
  • Other essential retroviral proteins such as e. g. the integrase, reverse transcriptase, protease and capsid protein may be translated as one polypeptide from the capped, full-length RNA transcript. After translation, this polypeptide is proteotytically processed to the different proteins. Hence, this RNA is still monocistronic.
  • Retroviruses utilise for all processes of transcription, RNA processing and translation several host cell mechanisms. Accordingly, various cis-acting sequences, either located in coding or in non-coding regions have been described for different retroviral genomes. These cis-acting sequences interact with various host cell proteins to regulate gene expression, RNA processing, polyadenylation, stability, or nuclear export of viral RNA, which ultimately leads to packaging of the retroviral RNA genome and viral proteins and the formation of viral particles.
  • a retroviral vector is characterised by the ability to harbour a heterologous nucleotide sequence in addition to the vector sequence and to transfer said sequence into a recipient host cell. It has been shown that the disruption of cis-acting elements severely impairs productive generation of infectious viral particles. It is not entirely understood how cis-acting elements influence or control the viral life cycle. Nevertheless, it seems to be clear that disruption of cis-acting elements by randomly inserting a cassette into the genome of recombinant retroviruses results in promoter interference, disturbed splicing balance or lack in packaging efficiency, and finally leads to the loss of viral replication or decreasing viral titers.
  • the vectors of the present invention support the generation of both infectious and non-infectious retroviral particles.
  • Retroviral vectors have been constructed in which a cassette consisting of a translational control element precedes a heterologous gene.
  • translation of the one ORF which is closest to the capping group, starts-as described above-at the first translation-start-codon (ATG or AUG) following the capping group and stops at a stop-codon.
  • AUG first translation-start-codon
  • an additional translational control element e. g. an internal ribosome entry site (IRES) is necessary.
  • IRS internal ribosome entry site
  • a replication-deficient retrovirus devoid of the normally essential genes gag, pol and env relies on packaging cells (producer cells) which harbour these essential genes.
  • the envelope vector can be constructed from any of the complete panel of primary viral isolate envelopes in order to gain a large antibody repertoire.
  • the vector of the present invention may be monocistronic, bicistronic and/or polycistronic
  • the vector enables development of a vaccine against HIV, HIV-1 and/or HIV-2, in particular HIV-1 , that provides the same natural immune protection as the attenuated strains known at present, but with no risk of regenerating an HIV-virus - as only the envelope protein needed for the generation of a specific immune response will be derived from HIV.
  • the retroviral vector lacks all genes for regulatory proteins of HIV and expression.
  • the retroviral vector of the present invention is efficient in relation to enhancing the natural immune response due to for example coexpression of immunostimulatory cytokines/peptides.
  • the vector efficiently transduces cells via CD4 dependent envelopes.
  • the vector is only capable of one infective event using a HIV envelope or any other envelope that is capable of pseudotyping murine leukemi viral vectors, which is packaged in a virion that undertakes "correct" infective events transducing normal HIV-1 target cells.
  • a single-round infectious vector that targets and infects the same cells as HIV the same infectious step as in the live-attenuated virus is mimicked.
  • the vector may in one embodiment coexpress immunostimulatory cytokines/peptides from the same vector, this is expected to generate a superior immune response because there is a dual induction of both humoral and cellular immunity.
  • the great advantage of this approach is in achieving or even surpassing the efficient immunization caused by live-attenuated viruses while improving safety tremendously. Since the single-round infectious vector lacks all of the HIV genes except for one that causes the immune reaction, the risk of virulence reversion is virtually non-existent.
  • the backbone of the vector of the present invention being of non-lentiviral ridge, for example derived from the AKV ⁇ -retrovirus, will be superior in safety issues to other vectors presently being developed and employed.
  • polynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions.
  • percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
  • Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • a "replication-competent retroviral vector” is a vector, which comprises all essential genes for viral propagation, i.e. gag, pol and ENV.
  • Topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water.
  • Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents can be added as necessary.
  • Percutaneous penetration enhancers such as Azone can also be included.
  • compositions for oral administration can form solutions, suspensions, tablets, pills, capsules, sustained release formulations, oral rinses, or powders.
  • the retroviral vector is derived from Avian carcinoma Mill Hill virus 2, Avian leukosis virus, Avian myeloblastosis virus, Avian myelocytomatosis virus 29, Avian sarcoma virus CT10, Fujinami sarcoma virus, Rous sarcoma virus, UR2 sarcoma virus or Y73 sarcoma virus.
  • the alphaviruses are listed in table 1. Each of the alphaviruses specified above is intended to be an individual embodiment.
  • Betaretrovirus Squirrel monkey retrovirus in another embodiment is derived from gammaretroviruses as shown in table 3 below.
  • the vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits comprise at least one lentiviral envelope polypeptide or a fragment thereof, and/or at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof.
  • the lentiviral envelope is selected from the Antiviruses listed in figure 8 and figure 9.
  • the lentiviral envelope is an HIV variant in which the c-terminal tail has been deleted, such as HIV-1 delta-CT, in which the 1 13 c-terminal amino acids has been deleted, as defined in SEQ ID NO.: 2, and/or fragments and/or functional homologous thereof.
  • the said lentiviral envelope polypeptide has a sequence that is for example at least 40%, such as at least 45%, for example at least 50%, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 67%, such as at least 70%, for example at least 72%, such as at least 75%, for example at least 77%, such as at least 80%, for example at least 81 %, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1.
  • a central aspect of the present invention relates to a mammalian expression vector comprising at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof.
  • said vector comprises an intron.
  • the intron is deleted by splicing, thus, facilitating the transport from the nucleus to the cytoplasm of said vector encoded RNA in packaging cells.
  • the vectors of the present invention also comprise a constitutive transport element (CTE).
  • the CTE facilitates the transport of the vector encoded RNA from the nucleus to the cytoplasm of the semipackaging cell.
  • the vector may comprise a Rev responsive element (RRE).
  • the vector of the present invention is a retroviral vector.
  • Retroviral vectors include both replication deficient retroviral vectors, and replication competent retroviral vectors.
  • the present invention relates to a vector, which comprises at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof.
  • the vector may further comprise at least one additional nucleic acid sequence and/or at least one internal ribosomal entry site (IRES).
  • IRES internal ribosomal entry site
  • at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or fragment thereof is preceded by an IRES, and in another embodiment, the at least one additional nucleic acid sequence is preceded by an IRES.
  • IRES is selected from the IRES elements of picornaviridae, retroviridae or retrotransposons, mammalia or combinations thereof.
  • said IRES is selected from the IRES elements of picornavirus.
  • said IRES is selected from the IRES elements of encephalomyocarditis (ECMV).
  • the IRES may be inserted at different locations in the retroviral vector.
  • the IRES is located in a region flanked by the 3'-LTR and the 5'-LTR.
  • the IRES is located in the 3'-l_ong Terminal Repeat (LTR) or the 5'-LTR.
  • the IRES is located in the U3 region of the 3' LTR.
  • the IRES is located in the U3 region between the inverted repeats and the transcription regulatory elements.
  • the present invention relates to a retroviral particle comprising an RNA transcribed from any of the embodiments of the retroviral vectors described herein.
  • the semi-packaging cells are 293T cells comprising Mo-MV GagPol DNA.
  • the retroviral packaging cells or semipackaging cells producing retroviral particles are encapsulated, wherein the capsules have a porous capsule wall which is permeable to said retroviral particles.
  • a carrier may be present independently of an adjuvant.
  • the inclusion of a carrier is relevant in connection with using the viral particles of the present invention are used to boost the immune response due to the ability of the retroviral particles of the present invention to infect, integrate and display the lentiviral envelope or fragments thereof on the surface of a host cell, thereby boosting or enhancing the immune response, in particular the CTL response as discussed elsewhere herein.
  • the function of a carrier can for example be to increase the molecular weight of in particular peptide fragments in order to increase their activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life.
  • the carrier may be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell.
  • a carrier protein could be but is not limited to keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.
  • the carrier must be a physiologically acceptable carrier acceptable to humans and safe.
  • tetanus toxoid and/or diptheria toxoid are suitable carriers in one embodiment of the invention.
  • the carrier may be dextrans for example sepharose.
  • the present invention provides a number of therapeutical applications. It is understood that the vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits may be used for treating a medical condition. Thus, one aspect of the present invention relates to the use of a vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition or/and kit according to the present invention for the manufacture of a medicament for the treatment, prevention and/or amelioration of a clinical condition.
  • Another aspect of the present invention relates to a vector, method, provirus, retroviral particle, composition, vaccine, vaccine composition or/and kit according to the present invention for use as a medicament.
  • the vector, provirus, retroviral particle, use, composition, vaccine, vaccine composition or/and kit when administered to an animal including a human being, is capable of eliciting an immune response against a disease caused by lentivirus, for example HIV, such as HIV-1 or HIV-2, or SIV.
  • Dosis and administration The amount of the immunogenic peptide of the invention in the pharmaceutical composition may vary, depending on the particular application. However, a single dose of the peptide composition is preferably anywhere from about 10 mg to about 5000 mg, more preferably from about 50 mg to about 2500 mg such as about 100 mg to about 1000 mg. Modes of administration include intradermal, subcutaneous and intravenous administration, implantation in the form of a time release formulation, etc. Any and all forms of administration known to the art are encompassed herein.
  • this vector system efficiently couples genotype to phenotype with regard to cellular tropism directed by the HIV-1 envelope protein.
  • This vector construction might be of use in HIV-1 envelope protein targeted vaccine efforts where proper antigen presentation of envelope epitopes may hold promise as well as being a simplified tool in the assessment of HIV-1 entry inhibitors.
  • HIV-1 Env is composed of the two glycoprotein subunits; gp120 and gp41.
  • Gp120 interacts initially with the primary receptor CD4 upon which a second binding site become exposed that binds to a co-receptor (CXCR4/CCR5 and several others) (Huang et al., 2005).
  • CXCR4/CCR5 and several others co-receptor
  • These receptor binding steps activate the fusion machinery located in gp41.
  • the N-terminal fusion peptide is inserted into the target cell membrane upon which the heptad repeat domains (HR1 and HR2) form a coiled-coil structure.
  • Gp41 also comprises the large cytoplasmic tail (CT) of the HIV-1 Env protein.
  • CT cytoplasmic tail
  • retroviruses HIV carry a rather long CT compared to the well studied ⁇ -retroviruses.
  • MMV murine leukemia virus
  • the R-peptide in the CT needs to be cleaved for the Env protein complex to become fusion activated (Januszeski et al., 1997). This mechanism is not present in HIV-1 and the exact function of the CT remains elusive. It has been shown that pseudotyping of MLV particles with HIV-1/2 Env requires the deletion of 1 13 amino acids in the CT (Mammano et al., 1997;Schnierle et al., 1997).
  • Rev/RRE interaction utilises the CRM1 pathways that connects unspliced retroviral RNA to the nuclear pore complex and hence cytoplasmic transport (Emerman et al., 1989;Malim et al., 1989).
  • codon optimisation has been widely utilised in order to obtain an enhanced HIV Env production that is Rev-independent (Kotsopoulou et al., 2000;Vinner et al., 1999).
  • the HIV-1 envelope gene to an egfp gene in the context of the MLV LTR region encompassing all MLV cis-elements needed for replication. Whether the Rev-independence we observe is driven by an unknown MLV CTE element remains to be established. It is also a possibility that the EMCV IRES contributes to the stability and/or nuclear export of the RNA encoding the truncated HIV Env. We can rule out an effect from the coupling of the egfp gene because we have generated an identical vector carrying the neomycin resistance gene in place of egfp that displays similar phenotypic properties.
  • Second round infectivity We intended to produce a conditionally replicational competent vector capable of multiple rounds of infection when Gag and Pol were supplied in trans.
  • Transducing semi-packaging cells i.e. D17 GagPol /Te671 GagPol
  • EgfpHIVMo at MOI ⁇ 1 we were unable to harvest functional pseudotyped viruses.
  • D17 CXCR4+/CD4+ cells we succeeded in directing transduction of D17 CXCR4+/CD4+ cells with pseudotyped viruses when producer cells (semi-packaging cells D17 GagPol /Te671 GagPol) had been infected with EgfpHIVMo at a high MOI (>5) suggesting that the vector production of HIV-1 ⁇ CT Env was low.
  • Table 1 lists the titers obtained from second- round transduction.
  • MLV murine leukemia virus
  • HIV-1 ENV - wild type from reference strain HXB3
  • Vector sequence - EgfpHIVMo Complete nucleotide sequence of the bicistronic vector
  • ACATCTGTAG 3501 AAATTAATTG TACAAGACCC AACAACAATA CAAGAAAAAG
  • TTAACATAAC AAATTGGCTG TGGTATATAA AATTATTCAT AATGATAGTA GGAGGCTTGG
  • GAATTTTTGC TTTCGGTTTT TCGCCGAAAC CGCGCCGCGC 1401 GTCTTGTCTG TCTCAGTATT GTTTTGTCAT TTGTCTGTTC GTTATTGTTT TGGACCGCTT
  • HIV-1 envelope amino acid sequence from reference strain HXB3

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Wood Science & Technology (AREA)
  • Mycology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Plant Pathology (AREA)
  • Communicable Diseases (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention provides a lentiviral envelope polypeptide or a nucleic acid sequence encoding a lentiviral envelope polypeptide, which are capable of inducing an immunogenic response against lentiviral infection, such as HIV infection. Examples of envelopes are HIV-1, HIV-2 or SIV envelope proteins or variants thereof. A specific such envelope variant is devoid of its C-terminal domain. The lentiviral envelope polypeptide or nucleic acid encoding said polypeptide is provided in vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits. Moreover, the lentiviral envelope polypeptide or nucleic acid encoding said polypeptide may be provided in combination with other compounds, such as selective marker genes, adjuvants, and immunomodulating agents. The invention is applicable for generation of retroviral particles for specific targeting of host cells. The retroviral particles may be used for integration of trangenes in host cells and induction of an immunogenic response, for example against HIV-1.

Description

Vectors for HIV-1 vaccine
All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.
Field of invention
The present invention relates to vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits comprise at least one lentiviral envelope polypeptide or a nucleic acid sequence encoding a lentiviral envelope polypeptide, which are suitable for inducing an immunogenic response againstlentiviral infection, such as HIV infection.
Background of invention Retroviruses have the capacity to infect a wide variety of different cells. Moreover, retroviruses transfer their genes from a producer cell to a target cell as a genomic RNA transcript. This RNA is reverse-transcribed after infection and integrated into the DNA genome of the target cell.
Human immunodeficiency virus (HIV) which is a lentivirus is according to WHO one of the most serious health crisis the world faces today. AIDS has killed more than 25 million people since 1981. In the most severely affected countries, the average life expectancy is now declining to 49 years of age - 13 years less than in the absence of AIDS.
According to L)NAIDS an estimated number of 39.5 million people were living with HIV virus in 2006 and 4.3 million were infected in 2006. In many regions new infections are heavily concentrated in the younger generations (15-24 years of age). Access to treatment and care has greatly increased in recent years. Determining real time trends to HIV incidence and in particular the impact of prevention programmes ideally requires long studies of a large number of people. Given the practical difficulties of conducting such studies focus has been placed on young women and their infants.
Children living with HIV typically acquire infection through a mother-to-child- transmission (MTCT), which occur during pregnancy, delivery or during breastfeeding. The last few years have seen unprecedented political and community mobilisation in response to the HIV pandemic with new funding opportunities and a revitalised public health approach. Renewed efforts are urgently required to increase access to comprehensive and integrated programmes to prevent HIV infection in infants and young children, which will indicate a route to HIV-free generations.
There are two known types of HIV; HIV-1 and HIV-2 that infect humans. They belong to a group of viruses called Lentiviruses and a virus similar to HIV has been found in African monkeys.
The first person with a documented HIV-infection died in 1959. In the early 1980s doctors in the US become aware, that more and more patients suffered from abnormal infections and showed signs of immune failure. The syndrome was named Acquired Immune Deficiency Syndrome -AIDS and it was soon after discovered that HIV was the causative agent for the observed destruction of the immune system.
Initially patients were offered a treatment based solely on pain relief and almost all inevitably died. In mid 90s there were two important breakthroughs in treatment. First of all a new group of antiretroviral agents were discovered and secondly it became possible to measure the amount of HIV virus in blood. These two advances made it possible to treat patients with a combination of different agents and doctors were able to check, whether the treatment actually worked. The result was that the immune system of infected patients gradually became normal and patients lived longer. Today infected people in Western countries are having the same level of quality of life as those not infected and they are able to have children. But this is not the case in developing countries, where more than 95% of those people infected with HIV/AIDS are living. Worldwide more than 25 million people have died from AIDS in the last 25 years.
Globally more than 40 million people are infected by HIV, and approximately 95% of the 140.000 people who get infected every day live in the developing countries, where expensive antiviral drugs are not available. There is an urgent need for an effective vaccine - the only effective solution to the uncontrolled HIV pandemic. During the last few years research has brought up new knowledge on the fundamental biology of HIV- virus which is leading to new antiviral drugs and strategies for vaccine design. In spite of these substantial advances, an effective vaccine does not yet exist. Only attenuated (that is live but weakend) HIV-strains has been able to provide immunity in primate studies even though they will never reach a required safety profile suitable for mass vaccination. Furthermore as several subtypes of HIV exists of which the most prevalent are A, C and D with several CRF's, it has not become completely clear what type of immune response should be induced in order to protect against HIV/AIDS.
There are several examples of vectors expressing HIV envelopes or part thereof in MLV derived retroviral vectors (Mammano et al., Journal of Virology, 1997 Apr;71 (4):3341-45; Neumann et al., Journal of Biotechnology 124 (2006) 615-625). Also a functional bicistronic HIV-1 envelope vector has been reported (Tolstrup at al., AIDS 2007, VoI 21 No 4).
Summary of invention The present invention relates to a replication deficient vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition and kit comprising at least one lentiviral envelope polypeptide or at least one nucleic acid sequence encoding a lentiviral envelope polypeptide. The invention is applicable for generation of retroviral particles for specific targeting of host cells. The retroviral particles may be used for integration of trangenes in host cells and induction of an immunogenic response, for example against HIV-1.
In a primary aspect, the present invention relates to a mammalian expression vector comprising at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof. Embodiments of the mammalian expression vector include retroviral vectors.
In another aspect, the present invention relates to a replication deficient vector comprising at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof.
In another aspect, the invention relates to an RNA of the vector as defined in the present invention. In a further aspect, the invention relates to a retroviral provirus produced in a target cell upon reverse transcription of said RNA as defined above or part thereof.
A further aspect of the invention pertains to a retroviral particle comprising said RNA as defined above, or part thereof.
In a further aspect, the invention relates to a retroviral particle as described herein, wherein said retroviral particle is capable of infecting a CD4 positive cell. In a seventh aspect, the invention relates to said retroviral particle, wherein said retroviral particle is capable of inducing an immunogenic response in a host animal.
In a further aspect, the invention pertains to a producer cell comprising any vector according to the present invention.
In a ninth aspect, the invention relates to host cell comprising a vector according to the present invention.
In a further aspect, the present invention relates to a host cell infected with a retroviral particle according to the present invention.
In a further aspect, the present invention relates to the use of the vector according to the present invention, the producer cell according to according to the present invention, a retroviral particle according to the present invention and/or a host cell according to the present invention for producing a medicament for gene therapy.
In a further aspect, the present invention relates to the use of the vector according to the present invention, the producer cell according to according to the present invention, a retroviral particle according to the present invention and/or a host cell according to the present invention for producing a medicament for immune therapy.
In a further aspect, the present invention relates to a pharmaceutical composition containing a therapeutically effective amount of the vector according to the present invention, the producer cell according to according to the present invention, a retroviral particle according to the present invention and/or a host cell according to the present invention. In a further aspect, the present invention relates to a method for introducing a nucleotide sequence into target cells, said method comprising infection of target cells with retroviral particle according to the present invention.
In a further aspect of the present invention relates to a vaccine for the prophylaxis or treatment of lentiviral infection comprising any vector according to the present invention, any RNA according to the present invention, any retroviral provirus according to the present invention and/or any retroviral particle according to the present invention.
In a further aspect, the present invention relates to a vaccine composition comprising the vaccine according to the present invention for use as a medicament.
A further aspect of the present invention relates to a kit comprising a therapeutically effective amount of the vector according to the present invention, the producer cell according to according to the present invention, a retroviral particle according to the present invention and/or a host cell according to the present invention.
A further aspect of the present invention relates to vaccine composition comprising a. a lentiviral envelope polypeptide or a fragment, and b. an adjuvant and/or an immunomodulating peptide.
Another aspect of the present invention relates to vaccine composition according to the present invention for treatment, amelioration and/or prevention of HIV infection and/or AIDS.
In a further aspect, the present invention relates to use of the vaccine composition according to the present invention for the manufacture of a medicament for the treatment of HIV infection and/or AIDS.
A further aspect of the present invention, relates to vaccine composition comprising the vaccine as defined the present invention for use as a medicament.
A further aspect of the present invention, relates to use of the vector according to the present invention, a producer cell according to the present invention, a retroviral particle according to the present invention, a host cell according to the present invention and/or a vaccine composition according to the present invention for the manufacture of a medicament for gene therapy.
A further aspect of the present invention, relates to use of the vector according to the present invention, a producer cell according to the present invention, a retroviral particle according to the present invention, a host cell according to the present invention and/or a vaccine composition according to the present invention for the manufacture of a medicament for immune therapy.
In yet another aspect, the present invention relates to use according to the present invention for the manufacture of a medicament for the treatment, amelioration or prevention of HIV and/or AIDS.
A final aspect of the present invention, relates to use according to the present invention for the manufacture of a medicament for HIV vaccination.
Description of Drawings
Figure 1 . A) The vector comprises the Akv MLV cis-elements for vector replication (transcription / transport / packaging / reverse transcription and integration). In the backbone is the ampicillin resistance gene for selection in bacterial culture. B) The genomic organization of Akv MLV from were the vector terminal elements are derived and HIV-1 from where the truncated envelope glycoprotein has been used. In addition is the splice donor (SD) and splice acceptor (SA) sites shown to indicate the resulting splicing pattern of the vector EgfpHIVMo. Not drawn to scale.
Figure 2. Western blot with anti-HIV-1 gp120 of cell lysate from 293T transfected cells.
Figure 3. The induction of syncytia by the HIV-1 envelope (ΔCT) was investigated. 293T cells were transfected with EgfpHIVMo and CMV-128 (+/- Rev) in 6-well plate.
Twenty-four hours later 293T cells were co-cultured overnight with HeLa or HeLa CD4+ cells. Syncytia formation was visualized by fluorescence and BF microscopy. A+B) EgfpHIVMo + Rev with HeLa CD4+. C+D) EgfpHIVMo with HeLa CD4+. E+F) EgfpHIVMo with HeLa. G) CMV-128 with HeLa CD4. H) HXB2-env (without CMVRev) with HeLa CD4+. Figure 4. Titer measurement of HIV-1 pseudotyped MLV particles on D17 CXCR4+/ CD4+ was done by transient transfections of 293T cells. CMV-128 and VSV-G were co-transfected with pLSXN. Forty-eight hours after transfection supematants were filtered through 0.45 μm and added on D17 CXCR4+/ CD4+ cells. The following day cells were harvested and the percentage of green fluorescent cells were enumerated by flow cytometry. Results shown are the average of at least three independent experiments.
Figure 5. Level of egfp fluorescence evaluated in various cell types. The retroviral vector Egfp-pLXSN was used as comparison to define expression baseline levels of integrated EgfpHIVMo. Pseudotyped particles prepared from 293T cells were used to transduce the indicated cell lines. 48 hr later cells were visualised with fluorescence microscopy.
Figure 6. Flow cytometric analysis of HIV-1 Env ΔCT cell surface expression was measured by antibody staining. Depicted are the percentage proportions of EGFP +/- and Env +/- cells respectively. Results shown are from one representative experiment.
Figure 7. NIH3T3 cells stable transduced by EgfpHIVMo. From the integrated vector both transgenes are expressed as measured by egfp and syncytium formation upon co-culture with D17 CD4/CXCR4
Figure 8. List of HIV-1 and SIV cpz envelope polypeptide amino acid sequences.
Figure 9. List of HIV-2 and SIV smm envelope polypeptide amino acid sequences.
Figure 10. 293T cells transfected with plasmids indicated above lanes. E2 fulllength 3' genomic sequence with HIV-1 envelope and spliced Tat + Rev expressed from a CMV promoter. E2R contain a STOP cocon in the Rev exon (hence no Rev production). Six- well cell lysate purified protein resuspended in 100 uL PBS. Loaded 25 uL in each lane. Anti-Env from HIV+ patient serum used as primary polyclonal antibody. Secondary goat anti-human IgG labelled with HRP.
Figure 1 1. Western Blot of HIV envelope from transfected HeLa Cells. Figure 12. Western Blot of HIV envelope from transfected NIH3T3 Cells
Figure 13. Western Blot of HIV envelope from MLV pseudotyped particles. Media from transfected 293T cells producing pseudotyped MLV particles was ultracentrifuged for 2 hrs. at 35.000 RPM, 4 QC in Beckham centrifuge. The pettet was lysed in 40 μlTTS and loaded on 8% acrylamide gel. Envelope was detected by blotting with D7 monoclonal antibody.
Figure 14. ELISA endpoint IgG, as described in example 3.
Figure 15. Elispot, as described in example 3.
Detailed description of the invention The development of an HIV vaccine is affected by the range of virus subtypes as well as by the wide variety of human populations who need protection and who differ, for example, in their genetic make-up and their routes of exposure to HIV. In particular, the occurrence of superinfection indicates that an immune response triggered by a vaccine to prevent infection by one strain of HIV may not protect against all other strains. The effectiveness of a vaccine is likely to vary in different populations unless some innovative method is developed which guards against many virus strains.
In one aspect, present invention offers a replication-deficient retroviral vector comprising at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof. The retroviral vector is useful for the production of virus particles displaying a lentivral envelope polypeptide or fragment thereof, which is used as a vaccine to prevent infection with lentiviruses, in particular HIV-1 . The present retroviral vector has improved immunogenic properties compared to known HIV viruses, and in addition displays safety features not available at present as will be described herein.
In a retroviral life cycle the essential genes gag, pol and env are transcribed from the transcription-regulatory cis-elements contained in the U3-region of the 5'Long Terminal Repeat (LTR). Transcription starts at the border of the U3-to the R-region of the 5'LTR and continues towards the polyadenylation site at the end of the R-region in the 3'LTR, where the mature transcript is released. The resulting RNA transcripts comprise full- length as well as spliced retroviral RNA. The 5'-end of the full-length as well as of the spliced retroviral RNA is modified by addition of a capping group. This structure is important for the attachment of ribosomes and thereby for the translation of the RNA. Translation requires besides this binding signal for a ribosome a so-called open- reading-frame ORF, i.e. a DNA or RNA sequence between an ATG/AUG translation start signal and a termination codon. In normal retroviruses or retroviral vectors RNA transcripts comprise only one ORF (monocistronic RNA). This monocistronic RNA is capped and translation of the ORF starts at the first translation-start-codon (e. g. ATG) following the capping group and stops at a stop-codon. Consequently, any coding region downstream of said stop-codon will not be translated into a protein. An example of a spliced and capped RNA transcript coding for a single protein is the RNA coding for env. Other essential retroviral proteins, such as e. g. the integrase, reverse transcriptase, protease and capsid protein may be translated as one polypeptide from the capped, full-length RNA transcript. After translation, this polypeptide is proteotytically processed to the different proteins. Hence, this RNA is still monocistronic.
Retroviruses utilise for all processes of transcription, RNA processing and translation several host cell mechanisms. Accordingly, various cis-acting sequences, either located in coding or in non-coding regions have been described for different retroviral genomes. These cis-acting sequences interact with various host cell proteins to regulate gene expression, RNA processing, polyadenylation, stability, or nuclear export of viral RNA, which ultimately leads to packaging of the retroviral RNA genome and viral proteins and the formation of viral particles.
A retroviral vector is characterised by the ability to harbour a heterologous nucleotide sequence in addition to the vector sequence and to transfer said sequence into a recipient host cell. It has been shown that the disruption of cis-acting elements severely impairs productive generation of infectious viral particles. It is not entirely understood how cis-acting elements influence or control the viral life cycle. Nevertheless, it seems to be clear that disruption of cis-acting elements by randomly inserting a cassette into the genome of recombinant retroviruses results in promoter interference, disturbed splicing balance or lack in packaging efficiency, and finally leads to the loss of viral replication or decreasing viral titers. The vectors of the present invention, however, support the generation of both infectious and non-infectious retroviral particles.
Retroviral vectors have been constructed in which a cassette consisting of a translational control element precedes a heterologous gene. In these cases, translation of the one ORF, which is closest to the capping group, starts-as described above-at the first translation-start-codon (ATG or AUG) following the capping group and stops at a stop-codon. For the translation of any further ORF encoded by such a retroviral RNA transcript an additional translational control element, e. g. an internal ribosome entry site (IRES) is necessary.
For propagation of infectious virus, a replication-deficient retrovirus devoid of the normally essential genes gag, pol and env relies on packaging cells (producer cells) which harbour these essential genes.
The envelope vector can be constructed from any of the complete panel of primary viral isolate envelopes in order to gain a large antibody repertoire.
At the Paul-Ehrlich-lnstitute, Department of Medical Biotechnology based in Langen, Germany a group of researchers have worked on the development of a γ-retroviral vector that express HIV-1 Tat, Rev and Envelope. The clear distinction from the retroviral vector of the presnt invention is that the vector expressing HIV-1 Tat, Rev and Envelope relies on splicing in order to obtain a sufficiently high expression. Furthermore γ-retroviral vector that express HIV-1 Tat, Rev and Envelope results in the co-production of Tat which is undesirable in human trials. This vector facilitates efficient tranduction and subsequent expression of HIV-1 genes in CD4-positive host cells. Induction of both humoral and cellular HIV-1 - specific immune responses in vivo confirmed the potential of this type of γ-retroviral vector as an effective HIV-1 vaccine. Although an ex vivo transduction strategy was employed, the findings demonstrated that re-transplantating Envelope and Rev expressing CD4 positive cells induce a detectable cellular immune response against Rev and Env. Furthermore, a report from the National AIDS Center in Rome, Italy, has tested the use of a self-inactivating lentiviral vector expressing HIV-1 Rev and Envelope to induce an immune response in mice. The present invention encompasses a new rev independent replication deficient retroviral vector expressing the HIV envelope gene together with various immunostimulatory/marker genes. The vector of the present invention may be monocistronic, bicistronic and/or polycistronic The vector enables development of a vaccine against HIV, HIV-1 and/or HIV-2, in particular HIV-1 , that provides the same natural immune protection as the attenuated strains known at present, but with no risk of regenerating an HIV-virus - as only the envelope protein needed for the generation of a specific immune response will be derived from HIV. The retroviral vector lacks all genes for regulatory proteins of HIV and expression. The retroviral vector of the present invention is efficient in relation to enhancing the natural immune response due to for example coexpression of immunostimulatory cytokines/peptides.
The vector efficiently transduces cells via CD4 dependent envelopes. The vector is only capable of one infective event using a HIV envelope or any other envelope that is capable of pseudotyping murine leukemi viral vectors, which is packaged in a virion that undertakes "correct" infective events transducing normal HIV-1 target cells. By utilisation of a single-round infectious vector that targets and infects the same cells as HIV the same infectious step as in the live-attenuated virus is mimicked.
Furthermore the vector may in one embodiment coexpress immunostimulatory cytokines/peptides from the same vector, this is expected to generate a superior immune response because there is a dual induction of both humoral and cellular immunity. The great advantage of this approach is in achieving or even surpassing the efficient immunization caused by live-attenuated viruses while improving safety tremendously. Since the single-round infectious vector lacks all of the HIV genes except for one that causes the immune reaction, the risk of virulence reversion is virtually non-existent.
The vector of the present invention differs from the above used vectors as the expression of the lentiviral envelope polypeptide is Rev-independent. Normally, expression of the immunogenic HIV ENV protein is dependent on existence of the auxiliary gene, Rev, in the HIV virus. Since the vector of the present invention displays expression of a immunogenic lentiviral envelope polypeptide or fragment thereof in the context of a gamma retrovirus, for example the murine Akv-virus, presence of the Rev protein is not necessary. Secondly, the fact that in one embodiment, two independent genes in the bicistronic Akv-based virus exist, enables the incorporation an immunogenic stimulus to boost the response. Finally it is believed that for example the backbone of the vector of the present invention being of non-lentiviral origine, for example derived from the AKV γ-retrovirus, will be superior in safety issues to other vectors presently being developed and employed. (Hum. Gene Ther. 1995
Mar;6(3):289-96. The effect of selection for high-level vector expression on the genetic and functional stability of a single transcript vector derived from a low-leukemogenic murine retrovirus. (Duch M, Paludan K, Lovmand J, Sørensen MS, Jørgensen P, Pedersen FS.)
Terms and definitions
To facilitate understanding of the invention, a number of terms are defined below.
The term "a retroviral vector" comprises a retroviral vector capable of being transcribed into RNA, which can be packaged into a retroviral particle, reverse transcribed into double stranded DNA and inserted into the host genome by the retroviral enzymatic machinery. For translation of the lentiviral envelope polypeptide or fragment thereof envelope, in a particular embodiment, an internal ribosome entry site (IRES) has been inserted upstream of the envelope in the exemplified retroviral expression vector, panel G and H.
The term "heterologous" is used hereinafter for any combination of nucleic acid sequences that is not normally found intimately associated in nature.
The terms "bicistronic" and "polycistronic" as used herein, relates to a transcript encoding a transcript, which comprise two or more open reading frames, respectively. In one embodiment the replication deficient vector of the present invention comprises one open reading frame, two open reading frames, three, four, five or six open reading frames.
The term "polynucleotide" or "nucleic acid sequence" refers to a polymeric form of nucleotides at least 2 bases in length. By "isolated nucleic acid sequence" is meant a polynucleotide that is not immediately contiguous with either of the coding sequences with which it is immediately contiguous (one on the 51 end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA or RNA which is incorporated into a viral vector. The nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double stranded forms of DNA.
The term "polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions. In addition, polynucleotide as used herein can also refer to triple- stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide.
As used herein, the term "polynucleotide" includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein.
It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.
The term "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
A "fragment" is a unique portion of the polynucleotide encoding the chimeric retroviral envelope polypeptide of the present invention which is identical in sequence to but shorter in length than the parent sequence. Similarly the term 'fragment' refers to the the chimeric retroviral envelope polypeptide of the present invention A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide or amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
The term "Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are described and present a detailed consideration of sequence alignment methods and homology calculations, such as VECTOR NTI. The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences will be.
The NCBI Basic Local Alignment Search Tool (BLAST) is available from several sources, including the National Center for Biotechnology Information (NBCI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at http://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www.ncbi. nlm.nih.gov/BLAST/blast_help.html.
Homologs of the disclosed polypeptides are typically characterised by possession of at least 94% sequence identity counted over the full length alignment with the disclosed amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Alternatively, one may manually align the sequences and count the number of identical amino acids. This number divided by the total number of amino acids in your sequence multiplied by 100 results in the percent identity.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
The term "insertion" refers to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double- stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material. The term "operably linked" refers to the situation in which a first nucleic acid sequence, amino acid sequence or ligand is placed in a functional relationship with a second nucleic acid sequence, amino acid sequence or ligand. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences or protein or ligands may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
The term "internal ribosome entry site" (IRES) defines a sequence motif which promotes attachment of ribosomes to that motif on internal mRNA sequences.
Furthermore, all factors needed to efficiently start translation at the AUG-start-codon following said IRES attach to this sequence motive. Consequently, an mRNA containing a sequence motive of a translation control element, e. g. IRES, results in two translational products, one initiating from the 5'end of the mRNA and the other by an internal translation mechanism mediated by IRES. Accordingly, the insertion of a translational control element, such as IRES, operably linked to an ORF into a retroviral genome allows the translation of this additional ORF from a viral RNA transcript. Such RNA transcripts with the capacity to allow translation of two or more ORF are designated bi- or polycistronic RNA transcripts, respectively.
The term "treatment", as used anywhere herein comprises any type of therapy, which aims at terminating, preventing, ameliorating and/or reducing the susceptibility to a clinical condition as described herein. In a preferred embodiment, the term treatment relates to prophylactic treatment, i.e. a therapy to reduce the susceptibility of a clinical condition, a disorder or condition as defined herein.
Thus, "treatment," "treating," and the like, as used herein, refer to obtaining a desired pharmacologic and/or physiologic effect, covering any treatment of a pathological condition or disorder in a mammal, including a human. The effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse affect attributable to the disorder. That is, "treatment" includes (1 ) preventing the disorder from occurring or recurring in a subject who may be predisposed to the disorder but has not yet been diagnosed as having it, (2) inhibiting the disorder, such as arresting its development, (3) stopping or terminating the disorder or at least symptoms associated therewith, so that the host no longer suffers from the disorder or its symptoms, such as causing regression of the disorder or its symptoms, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating, or ameliorating the disorder, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain, and/or immune deficiency.
The terms "prevent," "preventing," and "prevention", as used herein, refer to a decrease in the occurrence of pathological cells in an animal. The prevention may be complete, e.g., the total absence of pathological cells in a subject. The prevention may also be partial, such that the occurrence of pathological cells in a subject is less than that which would have occurred without the present invention. Prevention also refers to reduced susceptibility to a clinical condition.
A "replication-deficient retroviral vector" according to the present invention, is a vector, which does not comprise all essential genes for viral propagation. The vector may comprise one or more nucleic acid sequences encoding retroviral components, such as an envelope polypeptide. However, the replication-deficient retroviral vector is for example devoid of nucleic acids encoding one or more of the retroviral components gag, pol, or rev, which are normally required for retroviral lifecycle. Generation of retroviral particles derived from a replication-deficient retroviral vector, thus, requires that the remaining components are provided in trans, for example encoded by a nucleic acid sequence comprised in a producer cell.
Moreover, a "replication-competent retroviral vector" according to the present invention, is a vector, which comprises all essential genes for viral propagation, i.e. gag, pol and ENV.
A "pharmaceutically acceptable carrier," "pharmaceutically acceptable diluent," or "pharmaceutically acceptable excipient", or "pharmaceutically acceptable vehicle," used interchangeably herein, refer to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. A pharmaceutically acceptable carrier is essentially non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the carrier for a formulation containing polypeptides would not normally include oxidizing agents and other compounds that are known to be deleterious to polypeptides. Suitable carriers include, but are not limited to, water, dextrose, glycerol, saline, ethanol, and combinations thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the formulation. Adjuvants of the invention include, but are not limited to Freunds's, Montanide ISA Adjuvants [Seppic, Paris, France], Ribi's Adjuvants (Ribi ImmunoChem Research, Inc., Hamilton, MT), I Hunter's TiterMax (CytRx Corp., Norcross, GA), Aluminum Salt Adjuvants (Alhydrogel - Superfos of Denmark/ Accurate Chemical and Scientific Co., Westbury, NY), Nitrocellulose-Adsorbed Protein, Encapsulated Antigens, and Gerbu Adjuvant (Gerbu Biotechnik GmbH, Gaiberg, Germany/C-C Biotech, Poway, CA). Topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents can be added as necessary. Percutaneous penetration enhancers such as Azone can also be included.
"Pharmaceutically acceptable salts" include the acid addition salts (formed with the free amino groups of the polypeptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, mandelic, oxalic, and tartaric. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, and histidine.
Compositions for oral administration can form solutions, suspensions, tablets, pills, capsules, sustained release formulations, oral rinses, or powders.
The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an "effective amount," that is, a dosage sufficient to produce the desired result or effect in association with a pharmaceutically acceptable carrier. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed, the host, and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.
The term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
The term "fusion" according to the present invention comprise cell-cell fusion as well as virus-cell fusion. Cell-cell Fusion or Syncytia formation is a process by which the plasma membranes of two cells merge to form a single continuous double lipid membrane. This process does not happen spontaneously and is often mediate by the surface proteins of enveloped viruses such as the envelope proteins of retroviruses. Virus cell fusion is process by which an enveloped virus mediates merging of its lipid membrane with that of a target cell through interaction of the viral coat protein with a cellular receptor. The result of viral cell fusion process is entry of the viral core into the cytoplasm of a target cell, which is necessary for productive infection.
Vectors
In a primary aspect, the present invention relates to a mammalian expression vector comprising at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof. The vector of the present invention may be any mammalian expression vector. The vector may comprise an intron, which will facilitate the transport from the nucleus to the cytoplasma of the vector encoded RNA in the packaging cells. In another embodiment, the vector is capable of expressing RNA in the cytoplasm by cytoplasmic transcription, which can be translated into envelope polypeptide. The vector is also, in one embodiment, capable of expressing high levels of vector encoded RNA, which is transported to the cytoplasma to be translated into envelope polypeptide as encoded in the vector. The vector of the present invention may be transfected into a packging cell which is capable of producing viral particles comprising said lentiviral envelope polypeptide. In one embodiment, the vector is a retroviral vector. The retroviral vector may be either replication deficient or replication competent. The retroviral vector can be derived from any species of retroviridae. In one embodiment, the retroviral vector is derived from Orthoretrovirinae, comprising Alpharetrovirus, Betaretrovirus, and Gammaretrovirus.
In a specific embodiment, the retroviral vector is derived from Avian carcinoma Mill Hill virus 2, Avian leukosis virus, Avian myeloblastosis virus, Avian myelocytomatosis virus 29, Avian sarcoma virus CT10, Fujinami sarcoma virus, Rous sarcoma virus, UR2 sarcoma virus or Y73 sarcoma virus. The alphaviruses are listed in table 1. Each of the alphaviruses specified above is intended to be an individual embodiment.
Consequently, a retroviral vector according to the present invention derived from each of them are claimed individually.
Table 1. List of alpharetroviruses
Alpharetrovirus Avian carcinoma Mill Hill virus 2
Alpharetrovirus Avian leukosis virus
Alpharetrovirus Avian myeloblastosis virus
Alpharetrovirus Avian myelocytomatosis virus 29
Alpharetrovirus Avian sarcoma virus CTlO
Alpharetrovirus Fujinami sarcoma virus
Alpharetrovirus Rous sarcoma virus
Alpharetrovirus UR2 sarcoma virus
Alpharetrovirus Y73 sarcoma virus
In another specific embodiment, the retroviral vector is derived from Jaagsiekte sheep retrovirus, Langur virus, Mason-Pfizer monkey virus, Mouse mammary tumor virus or Squirrel monkey retrovirus. The betaviruses are listed in table 2. Each of the betaviruses specified herein is intended to be an individual embodiment. Consequently, a retroviral vector according to the present invention derived from each of them may be claimed individually.
Table 2. List of betaretroviruses
Betaretrovirus Jaagsiekte sheep retrovirus
Betaretrovirus Langur virus
Betaretrovirus Mason-Pfizer monkey virus
Betaretrovirus Mouse mammary tumor virus
Betaretrovirus Squirrel monkey retrovirus In another embodiment the retroviral vector according to the present invention is derived from gammaretroviruses as shown in table 3 below.
Table 3. List of gammaretroviruses
o Avian (Reticuloendotheliosis) virus group - Chick syncytial virus o Reticuloendotheliosis virus
- Avian spleen necrosis virus ■ Spleen necrosis virus o Mammalian virus group
■ Murine endogenous retrovirus o Murine leukemia-related retroviruses
■ Epicrionops marmoratus retrovirus ■ lchthyophis kohtaoensis retrovirus
■ Osteolaemus tetraspis retrovirus
■ Sericulus bakeri retrovirus
- Terdus iliacus retrovirus
■ Tomistoma schlegelii retrovirus ■ Viper berus retrovirus
• Xenotropic MuLV-related virus
■ Monodelphis sp. retrovirus o Replication competent viruses
• Feline leukemia virus • Gibbon ape leukemia virus (GALV)
• Murine leukemia virus
• Porcine type-C oncovirus o Replication defective viruses
■ Abelson murine leukemia virus ■ Gardner-Amstein feline sarcoma virus
• Hardy-Zuckerman feline sarcoma virus
■ Harvey murine sarcoma virus
■ Kirsten murine sarcoma virus
• Moloney murine sarcoma virus • Murine osteosarcoma virus
■ Snyder-Theilen feline sarcoma virus
• Spleen focus-forming virus
■ Woolly monkey sarcoma virus o unclassified Gammaretrovirus o Baboon endogenous virus
■ Baboon endogenous virus strain M7 o Feline endogenous virus
■ Feline endogenous virus ECE1
■ Feline endogenous virus RD114 ■ Koala retrovirus o Macaca mulatta type C retrovirus
■ Macaca endogenous retrovirus
■ MLV-related retrovirus
■ Rat leukemia virus - Rat sarcoma virus
■ RD114 retrovirus
- Recombinant M-MuLV/RaLV retrovirus
Mammalian virus group ■ Murine endogenous retrovirus o Murine leukemia-related retroviruses
• Epicrionops marmoratus retrovirus
■ lchthyophis kohtaoensis retrovirus
- Osteolaemus tetraspis retrovirus - Sericulus bakeri retrovirus
■ Terdus iliacus retrovirus
- Tomistoma schlegelii retrovirus
■ Viper berus retrovirus o Xenotropic MuLV-related virus ■ Xenotropic MuLV-related virus VP35
■ Xenotropic MuLV-related virus VP42
■ Xenotropic MuLV-related virus VP62
- Monodelphis sp. retrovirus o Replication competent viruses o Feline leukemia virus
- Feline leukemia provirus (clone CFE-16)
• Feline leukemia provirus (clone CFE-6)
■ Feline leukemia provirus ftt
■ Feline leukemia virus strain A/Glasgow-1 ■ Feline leukemia virus strain B/lambda-B1
■ Feline leukemia virus strain C/FA27
■ Feline leukemia virus strain C/FS246
■ Feline leukemia virus strain C/Sarma
• Feline sarcoma virus ■ Gardner-Arnstein feline leukemia oncovirus B o Gibbon ape leukemia virus (GALV)
■ Simian sarcoma-associated virus o Murine leukemia virus
■ AKR (endogenous) murine leukemia virus • Friend murine leukemia virus
■ Moloney murine leukemia virus
• Murine leukemia virus isolates
• unclassified Murine leukemia virus o Porcine type-C oncovirus • Porcine endogenous retrovirus
• Porcine endogenous type C retrovirus o Replication defective viruses
- Abelson murine leukemia virus
■ Gardner-Arnstein feline sarcoma virus o Hardy-Zuckerman feline sarcoma virus
- Feline sarcoma virus (STRAIN HARDY-ZUCKERMAN 2)
■ Feline sarcoma virus (STRAIN HARDY-ZUCKERMAN 4)
- Harvey murine sarcoma virus
- Kirsten murine sarcoma virus o Moloney murine sarcoma virus ■ Cas-NS-1 murine sarcoma virus
■ FBJ murine osteosarcoma virus
■ Moloney murine sarcoma virus (STRAIN HT-1 )
■ Moloney murine sarcoma virus (STRAIN M1 ) ■ Moloney murine sarcoma virus (strain TS110)
■ Murine sarcoma virus 3611
• Myeloproliferative sarcoma virus
• NS.C58 murine sarcoma virus o Murine osteosarcoma virus - FBR murine osteosarcoma virus
- Snyder-Theilen feline sarcoma virus o Spleen focus-forming virus
• Friend spleen focus-forming virus
■ Rauscher spleen focus-forming virus ■ Woolly monkey sarcoma virus
Thus, the retroviral vector is in one embodiment derived from Chick syncytial virus, Feline leukemia virus, Finkel-Biskis-Jinkins murine sarcoma virus, Gardner-Arnstein feline sarcoma virus, Gibbon ape leukemia virus, Guinea pig type-C oncovirus, Hardy- Zuckerman feline sarcoma virus, Harvey murine sarcoma virus, Kirsten murine sarcoma virus, Moloney murine sarcoma virus, Murine leukemia virus (MLV), Porcine type-C oncovirus, Reticuloendotheliosis virus, Snyder-Theilen feline sarcoma virus, Trager duck spleen necrosis virus, Viper retrovirus or Woolly monkey sarcoma virus. See table 3 for a list of gammaviruses. Each of the gammaviruses specified herein is intended to be an individual embodiment. Consequently, a retroviral vector according to the present invention derived from each of them may be claimed individually. In a particularly preferred embodiment, the retroviral vector is derived from Murine Leukemia Virus (MLV) or Moloney Murine Leukemia Virus (MoMLV) or Akv MLV.
In a specific embodiment, the retroviral vector is derived from Avian (Reticuloendotheliosis) virus group such as Chick syncytial virus, Reticuloendotheliosis virus, Avian spleen necrosis virus , Spleen necrosis virus, Mammalian virus group , Murine endogenous retrovirus , Murine leukemia-related retroviruses , Epicrionops marmoratus retrovirus , lchthyophis kohtaoensis retrovirus , Osteolaemus tetraspis retrovirus , Sericulus bakeri retrovirus , Terdus iliacus retrovirus , Tomistoma schlegelii retrovirus , Viper berus retrovirus , Xenotropic MuLV-related virus , Monodelphis sp. retrovirus, Replication competent viruses , Feline leukemia virus , Gibbon ape leukemia virus (GALV) , Murine leukemia virus , Porcine type-C oncovirus , Replication defective viruses , Abelson murine leukemia virus , Gardner-Arnstein feline sarcoma virus , Hardy-Zuckerman feline sarcoma virus , Harvey murine sarcoma virus , Kirsten murine sarcoma virus , Moloney murine sarcoma virus , Murine osteosarcoma virus , Snyder- Theilen feline sarcoma virus , Spleen focus-forming virus , Woolly monkey sarcoma virus, unclassified Gammaretrovirus , Baboon endogenous virus , Baboon endogenous virus strain M7, Feline endogenous virus , Feline endogenous virus ECE1 , Feline endogenous virus RD1 14, Koala retrovirus , Macaca mulatta type C retrovirus , Macaca endogenous retrovirus, MLV-related retrovirus , Rat leukemia virus , Rat sarcoma virus , RD1 14 retrovirus , Recombinant M-MuLV/RaLV retrovirus, Murine endogenous retrovirus , Murine leukemia-related retroviruses , Epicrionops marmoratus retrovirus , lchthyophis kohtaoensis retrovirus , Osteolaemus tetraspis retrovirus , Sericulus bakeri retrovirus , Terdus iliacus retrovirus , Tomistoma schlegelii retrovirus , Viper berus retrovirus , Xenotropic MuLV-related virus , Xenotropic MuLV- related virus VP35 , Xenotropic MuLV-related virus VP42 , Xenotropic MuLV-related virus VP62, Monodelphis sp. retrovirus, Replication competent viruses , Feline leukemia virus , Feline leukemia provirus (clone CFE-16) , Feline leukemia provirus (clone CFE-6) , Feline leukemia provirus ftt , Feline leukemia virus strain A/Glasgow-1 , Feline leukemia virus strain B/lambda-B1 , Feline leukemia virus strain C/FA27 , Feline leukemia virus strain C/FS246 , Feline leukemia virus strain C/Sarma , Feline sarcoma virus , Gardner-Arnstein feline leukemia oncovirus B, Gibbon ape leukemia virus (GALV) , Simian sarcoma-associated virus, Murine leukemia virus , AKR (endogenous) murine leukemia virus , Friend murine leukemia virus , Moloney murine leukemia virus , Murine leukemia virus isolates , unclassified Murine leukemia virus , Porcine type-C oncovirus , Porcine endogenous retrovirus , Porcine endogenous type C retrovirus, Replication defective viruses , Abelson murine leukemia virus , Gardner-Arnstein feline sarcoma virus , Hardy-Zuckerman feline sarcoma virus , Feline sarcoma virus (STRAIN HARDY-ZUCKERMAN 2) , Feline sarcoma virus (STRAIN HARDY-ZUCKERMAN 4), Harvey murine sarcoma virus , Kirsten murine sarcoma virus , Moloney murine sarcoma virus , Cas-NS-1 murine sarcoma virus , FBJ murine osteosarcoma virus , Moloney murine sarcoma virus (STRAIN HT-1 ) , Moloney murine sarcoma virus (STRAIN M1 ) , Moloney murine sarcoma virus (strain TS1 10) , Murine sarcoma virus 361 1 , Myeloproliferative sarcoma virus , NS.C58 murine sarcoma virus, Murine osteosarcoma virus , FBR murine osteosarcoma virus, Snyder-Theilen feline sarcoma virus , Spleen focus-forming virus , Friend spleen focus-forming virus , Rauscher spleen focus-forming virus or Woolly monkey sarcoma virus. Each of the gammaviruses mentioned above is intended to be an individual embodiment. Consequently, a retroviral vector according to the present invention derived from each of them are claimed individually. In a further embodiment, the retroviral vector is derived from Bovine leukemia virus, Primate T-lymphotropic virus 1 , Primate T-lymphotropic virus 2 or Primate T- lymphotropic virus 3. The deltaviruses are listed in table 4. Each of the deltaviruses mentioned herein is intended to be an individual embodiment. Consequently, a retroviral vector according to the present invention derived from each of them are claimed individually.
Table 4. List of deltaretroviruses
Deltaretrovirus Bovine leukemia virus
Deltaretrovirus Primate T-lymphotropic virus 1
Deltaretrovirus Primate T-lymphotropic virus 2
Deltaretrovirus Primate T-lymphotropic virus 3
In yet a further embodiment, the retroviral vector is derived from Walleye dermal sarcoma virus, Walleye epidermal hyperplasia virus 1 or Walleye epidermal hyperplasia virus 2. The epsilonviruses are listed in table 5. Each of the epsilonviruses mentioned herein is intended to be an individual embodiment. Consequently, a retroviral vector according to the present invention derived from each of them may are individually.
Table 5. List of epsilonretroviruses
Epsilonretrovirus Walleye dermal sarcoma virus Epsilonretrovirus Walleye epidermal hyperplasia virus 1 Epsilonretrovirus Walleye epidermal hyperplasia virus 2
In a primary aspect of the present invention is provided a replication deficient retroviral vector. The vector comprises at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof. In one embodiment, the lentiviral envelope polypeptide or fragment thereof is derived from Bovine immunodeficiency virus, Caprine arthritis encephalitis virus, Equine infectious anemia virus, Feline immunodeficiency virus, Human immunodeficiency virus 1 , Human immunodeficiency virus 2, Puma lentivirus, Simian immunodeficiency virus, Visna/maedi virus or hepatitis C. The lentiviruses, wherefrom the lentiviral envelope or fragment thereof can be derived are listed in table 6. Table 6. List of Antiviruses from which a nucelsic acid sequence encoding an envelope polypeptide or fragment thereof is used according to the present invention.
Lentivirus Bovine immunodeficiency virus
Lentivirus Caprine arthritis encephalitis virus
Lentivirus Equine infectious anemia virus
Lentivirus Feline immunodeficiency virus
Lentivirus Human immunodeficiency virus 1
Lentivirus Human immunodeficiency virus 2
Lentivirus Puma lentivirus
Lentivirus Simian immunodeficiency virus
Lentivirus Visna/maedi virus
Each of the Antiviruses mentioned above is intended to be an individual embodiment. Consequently, a retroviral vector comprising at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof derived from each of them are claimed individually.
Another aspect of the present invention also relates to an RNA, including an mRNA of a vector, provirus, or retroviral particle as defined herein. Another aspect of the present invention relates to a provirus produced in a target cell upon reverse transcription of an RNA according to the present invention. The present invention also relates to a retroviral particle comprising an RNA according to the present invention.
Lentiviral envelope
In one embodiment, the vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits comprise at least one lentiviral envelope polypeptide or a fragment thereof, and/or at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof. In one embodiment, the lentiviral envelope is selected from the Antiviruses listed in figure 8 and figure 9.
The term "lentiviral envelope polypeptide or fragment thereof " in the present context refers to any protein of lentiviral origin as described elsewhere herein which allow a retroviral particle of the present invention to adhere to the membrane of a host cell and/or to enter into the host cell. In the present invention, the envelope polypeptide may also be display on the surface of a viral particle, without making said viral particle infectious or fusogenic, thereby allowing the immune system to recognize different epitopes of said envelope polypeptide.
The vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits of the present invention comprise at least one lentiviral envelope polypeptide or a fragments thereof, and/or a at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof, wherein said lentiviral envelope is derived from Bovine lentivirus group , Bovine immunodeficiency virus , Bovine immunodeficiency virus FL112 , Bovine immunodeficiency virus FL491 , Bovine immunodeficiency virus OK , Bovine immunodeficiency virus R29 , Jembrana disease virus, Equine lentivirus group , Equine infectious anemia virus , Equine infectious anemia virus (CLONE 1369) , Equine infectious anemia virus (clone CL22) , Equine infectious anemia virus (CLONE P3.2-1 ) , Equine infectious anemia virus (CLONE P3.2-2) , Equine infectious anemia virus
(CLONE P3.2-3) , Equine infectious anemia virus (CLONE P3.2-5) , Equine infectious anemia virus (ISOLATE WYOMING) , Equine infectious anemia virus (STRAIN WSU5), Feline lentivirus group , Feline immunodeficiency virus , Feline immunodeficiency virus (isolate Petaluma) , Feline immunodeficiency virus (isolate San Diego) , Feline immunodeficiency virus (isolate TM2) , Feline immunodeficiency virus (isolate wo) , Feline immunodeficiency virus (strain UK2) , Feline immunodeficiency virus (strain UK8) , Feline immunodeficiency virus (strain UT-113) , Lion lentivirus , Panther lentivirus, Puma lentivirus , Puma lentivirus 14 , Puma lentivirus 21 , Ovine/caprine lentivirus group , Caprine arthritis-encephalitis virus , Caprine arthritis encephalitis virus (STRAIN CORK) , Caprine arthritis encephalitis virus (STRAIN G63), Visna/Maedi virus , Small ruminant Lentivirus CA-lreland , Visna/maedi virus 1514 , Visna/maedi virus EV1 , Visna/maedi virus EV1 KV1772 , Visna/maedi virus SA-OMVV , unclassified Ovine/caprine lentivirus , Caprine lentivirus , Ovine progressive pneumonia virus , Small ruminant lentivirus, Primate lentivirus group , Human immunodeficiency virus , Human immunodeficiency virus 1 , HIV-1 group M , HIV-1 group N , HIV-1 group O , Human immunodeficiency virus 3 , Human immunodeficiency virus type 1 (ARV2/SF2 ISOLATE) , Human immunodeficiency virus type 1 (BH10 ISOLATE) , Human immunodeficiency virus type 1 (BH5 ISOLATE) , Human immunodeficiency virus type 1 (BH7 isolate) , Human immunodeficiency virus type 1 (BH8 ISOLATE) , Human immunodeficiency virus type 1 (BRAIN ISOLATE) , Human immunodeficiency virus type 1 (BRU ISOLATE) , Human immunodeficiency virus type 1 (CDC-451 ISOLATE) , Human immunodeficiency virus type 1 (CLONE 12) , Human immunodeficiency virus type 1 (ELI ISOLATE) , Human immunodeficiency virus type 1 (HXB2 ISOLATE) , Human immunodeficiency virus type 1 (HXB3 ISOLATE) , Human immunodeficiency virus type 1 (isolate YU2) , Human immunodeficiency virus type 1 (JH3 ISOLATE) , Human immunodeficiency virus type 1 (JRCSF ISOLATE) , Human immunodeficiency virus type 1 (KB-1 isolate) , Human immunodeficiency virus type 1 (Lai isolate) , Human immunodeficiency virus type 1 (MAL ISOLATE) , Human immunodeficiency virus type 1 (MFA ISOLATE) , Human immunodeficiency virus type 1 (MN ISOLATE) , Human immunodeficiency virus type 1 (NDK ISOLATE) , Human immunodeficiency virus type 1 (NEW YORK-5 ISOLATE) , Human immunodeficiency virus type 1 (NIT-A isolate) , Human immunodeficiency virus type 1 (OYI ISOLATE) , Human immunodeficiency virus type 1 (PV22 ISOLATE) , Human immunodeficiency virus type 1 (RF/HAT ISOLATE) , Human immunodeficiency virus type 1 (SC ISOLATE) , Human immunodeficiency virus type 1 (SF162 ISOLATE) , Human immunodeficiency virus type 1 (SF33 ISOLATE) , Human immunodeficiency virus type 1 (STRAIN UGANDAN / ISOLATE U455) , Human immunodeficiency virus type 1 (WMJ1 isolate) , Human immunodeficiency virus type 1 (WMJ2 ISOLATE) , Human immunodeficiency virus type 1 (Z-84 ISOLATE) , Human immunodeficiency virus type 1 (Z2/CDC-Z34 ISOLATE) , Human immunodeficiency virus type 1 (ZAIRE 3 ISOLATE) , Human immunodeficiency virus type 1 (ZAIRE 6 ISOLATE) , Human immunodeficiency virus type 1 (ZAIRE HZ321 ISOLATE) , Human immunodeficiency virus type 1 Iw12.3 isolate, Human immunodeficiency virus 2 , HIV-2 subtype A , HIV-2 subtype B , Human immunodeficiency virus type 2 (isolate 7312A) , Human immunodeficiency virus type 2 (ISOLATE CAM2) , Human immunodeficiency virus type 2 (ISOLATE D194) , Human immunodeficiency virus type 2 (ISOLATE GHANA-1 ) , Human immunodeficiency virus type 2 (isolate KR) , Human immunodeficiency virus type 2 (ISOLATE NIH-Z) , Human immunodeficiency virus type 2 (ISOLATE SBLISY), Simian immunodeficiency virus , Human T-cell lymphotropic virus type 4 , Simian immunodeficiency virus - agm , Simian immunodeficiency virus - cpz , Simian immunodeficiency virus - mac , Simian immunodeficiency virus - mnd , Simian immunodeficiency virus - mon , Simian immunodeficiency virus - sm , Simian immunodeficiency virus - stm , Simian immunodeficiency virus Qu , Simian immunodeficiency virus SIVsun, Simian-Human immunodeficiency virus , unclassified Lentivirus , Brazilian caprine lentivirus , HIV-like human cancer virus or Ovine lentivirus. Each of the Antiviruses mentioned above is intended to be an individual embodiment. Consequently, a vector, methods, provirus, retroviral particle, use, composition, vaccine, vaccine composition or kit comprising at least one lentiviral envelope polypeptide or a fragment thereof, and/or a at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof, derived from each of the lentiviruses listed above are claimed individually.
In another preferred embodiment, the the vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits of the present invention comprise at least one SIV lentiviral envelope polypeptide and/or a polypeptide encoding an SIV envelope.
In one specific embodiment of the present invention, the vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits of the present invention comprise at least one lentiviral envelope polypeptide derived from HIV-1 , HIV-2 or SIV and/or at least one nucleic acid sequence encoding a lentiviral envelope polypeptide derived from HIV-1 , HIV-2 or SIV. In a preferred embodiment, such heterologous retroviral envelope polypeptide is HIV-1 ENV, such as defined in SEQ ID NO.: 1 , and/or fragments and/or functional homologous thereof. In another preferred embodiment, the lentiviral envelope is an HIV variant in which the c-terminal tail has been deleted, such as HIV-1 delta-CT, in which the 1 13 c-terminal amino acids has been deleted, as defined in SEQ ID NO.: 2, and/or fragments and/or functional homologous thereof.
However, in other embodiments of the present invention, the retroviral envelope polypeptide comprise HIV-1 ENV as defined in SEQ ID NO.: 1 , with a c-terminal deletion, such as a deletion of the 5 c-terminal amino acids, for example the 10 c- terminal amino acids, such as the 15 c-terminal amino acids, for example the 20 c- terminal amino acids, such as the 25 c-terminal amino acids, for example the 30 c- terminal amino acids, such as the 35 c-terminal amino acids, for example the 40 c- terminal amino acids, such as the 45 c-terminal amino acids, for example the 50 c- terminal amino acids, such as the 55 c-terminal amino acids, for example the 60 c- terminal amino acids, such as the 65 c-terminal amino acids, for example the 70 c- terminal amino acids, such as the 75 c-terminal amino acids, for example the 80 c- terminal amino acids, such as the 85 c-terminal amino acids, for example the 90 c- terminal amino acids, such as the 95 c-terminal amino acids, for example the 10O c- terminal amino acids, such as the 104 c-terminal amino acids, for example the 105 c- terminal amino acids, such as the 106 c-terminal amino acids, for example the 107 c- terminal amino acids, such as the 108 c-terminal amino acids, for example the 109 c- terminal amino acids, such as the 110 c-terminal amino acids, for example the 1 1 1 c- terminal amino acids, such as the 112 c-terminal amino acids, for example the 1 14 c- terminal amino acids, such as the 115 c-terminal amino acids, for example the 1 16 c- terminal amino acids, such as the 106 c-terminal amino acids, for example the 107 c- terminal amino acids, such as the 117 c-terminal amino acids, for example the 1 19 c- terminal amino acids, such as the 120 c-terminal amino acids, for example the 121 c- terminal amino acids, such as the 122 c-terminal amino acids, for example the 123 c- terminal amino acids, such as the 124 c-terminal amino acids, for example the 125 c- terminal amino acids, such as the 130 c-terminal amino acids, for example the 135 c- terminal amino acids, such as the 140 c-terminal amino acids, for example the 145 c- terminal amino acids, such as the 150 c-terminal amino acids, for example the 155 c- terminal amino acids, such as the 160 c-terminal amino acids, for example the 165 c- terminal amino acids, such as the 170 c-terminal amino acids, for example the 175 c- terminal amino acids, such as the 180 c-terminal amino acids, for example the 185 c- terminal amino acids, such as the 190 c-terminal amino acids, for example the 195 c- terminal amino acids, such as the 200 c-terminal amino acids, for example the 205 c- terminal amino acids, such as the 210 c-terminal amino acids, for example the 215 c- terminal amino acids, such as the 220 c-terminal amino acids, for example the 225 c- terminal amino acids, such as the 230 c-terminal amino acids, for example the 235 c- terminal amino acids, such as the 240 c-terminal amino acids, for example the 245 c- terminal amino acids, such as the 250 c-terminal amino acids, for example the 255 c- terminal amino acids, such as the 260 c-terminal amino acids, for example the 265 c- terminal amino acids, such as the 270 c-terminal amino acids, for example the 275 c- terminal amino acids, such as the 280 c-terminal amino acids, for example the 285 c- terminal amino acids, such as the 290 c-terminal amino acids, for example the 295 c- terminal amino acids, such as the 300 c-terminal amino acids, for example the 305 c- terminal amino acids, such as the 310 c-terminal amino acids, for example the 320 c- terminal amino acids, such as the 330 c-terminal amino acids, for example the 340 c- terminal amino acids, such as the 350 c-terminal amino acids, for example the 360 c- terminal amino acids, such as the 370 c-terminal amino acids, for example the 380 c- terminal amino acids, such as the 390 c-terminal amino acids, for example the 400 c- terminal amino acids, such as the 410 c-terminal amino acids, for example the 420 c- terminal amino acids.
Further embodiments of the invention include any vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition or kit as described herein, comprising said envelope polypeptides that has a sequence that is at least 36% identical to the amino acid sequence shown in SEQ ID NO.: 5 and/or SEQ ID NO.: 6, or is a fragment of a sequence that is at least 36% identical to the amino acid sequence shown in SEQ ID NO.: 5 and/or SEQ ID NO.: 6.
However, in other embodiments of the present invention the said lentiviral envelope polypeptide has a sequence that is for example at least 40%, such as at least 45%, for example at least 50%, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 67%, such as at least 70%, for example at least 72%, such as at least 75%, for example at least 77%, such as at least 80%, for example at least 81 %, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1. In another embodiment of the present invention said receptor binding domain of said envelope polypeptide is a fragment of a sequence that is for example at least at least 40%, such as at least 45%, for example at least 50%, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 67%, such as at least 70%, for example at least 72%, such as at least 75%, for example at least 77%, such as at least 80%, for example at least 81%, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99% identical to the amino acid sequence shown in SEQ ID NO.: 5 and/or SEQ ID NO.: 6. Reporter gene, suicide gene and selectable markers In one embodiment of the present invention, the vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits comprise a reporter gene and/or a nucleic acid sequence encoding a reporter gene. Thus, in one embodiment, the at least one additional nucleic acid sequence of a vector of the present invention encodes a reporter gene. In the present context the term "reporter gene" refers to any reporter gene that can be used to evaluate whether a host cell harbours the vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition and/or kit according to the present invention. A number of reporter genes and systems for detection exist which will be appreciated by a person skilled in the art. For example the reporter gene of the present invention is selected from the group consisting of the enhanced green fluorescent protein (eGFP), lac Z, dsRed, enhanced yellow fluorescent protein (eYFP), enhanced cyan fluorescent protein (eCFP), enhanced blue fluorescent protein (eBFP) and the human alpha-1 -antitrypsin (hAAT). It is understood that any of the enhanced green fluorescent protein (eGFP), lac Z, dsRed, enhanced yellow fluorescent protein (eYFP), enhanced cyan fluorescent protein (eCFP), enhanced blue fluorescent protein (eBFP) or the human alpha-1 - antitrypsin (hAAT) there are also claimed in separate embodiments. In a preferred embodiment the eGFP gene is used.
In another embodiment of the present invention, the vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits comprise a suicide gene and/or a selection gene encoding a selective marker. The selection gene of the present invention may be any gene suitable for for example selecting cells harbouring the vector constructs of the present invention. Typically the selection gene is a gene that confers resistance to antibiotics or drugs. Examples of such selection genes is the puromycin resistance gene (Puro), the tetracycline resistance gene, the streptomycin resistance gene, the hygromycin B resistance gene (Hygro), the zeocin resistance gene (zeo), the neomycin resistance gene (neo),and the blasticidin resistance gene (Bst). Therefore, the selection gene of the present invention is selected from the group consisting of puromycin resistance gene (Puro), the tetracycline resistance gene, the streptomycin resistance gene, the hygromycin B resistance gene (Hygro), the zeocin resistance gene (zeo), the neomycin resistance gene (neo) and the blasticidin resistance gene (Bst). In a preferred embodiment the selection gene is selected from the group consisting of puromycin resistance gene (Puro), the hygromycin B resistance gene (Hygro), the zeocin resistance gene (zeo), the neomycin resistance gene (neo) and the blasticidin resistance gene (Bst). It is appreciated that the resistance gene is selected from any of puromycin resistance gene (Puro), the tetracycline resistance gene, the streptomycin resistance gene, the hygromycin B resistance gene (Hygro), the zeocin resistance gene (zeo), the neomycin resistance gene (neo) or the blasticidin resistance gene (Bst). In a preferred embodiment the resistance gene is the neomycin resistance gene. In another preferred embodiment, the selective marker is neomycin phosphotransferase II.
The suicide gene and/or a selection gene encoding a selective marker according to the present invention can also be Herpes simplex virus thymidine kinase (HSV-TK).
Adjuvants lmmunostimulatory adjuvants augment antigen-specific immune responses by physical localization and improved presentation of antigen, and by provocation of inflammatory or innate immune responses (Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Immunol Cell Biol 2004;82(5):488-96). A key feature in the innate immune system is its capability to detect foreign organisms using a set of cell receptors termed pattern-recognition receptors (PRR). One family of PRRs are Toll-Like Receptors, such as Toll-Like Receptor 9 (TLR9). TLR9 detects unmethylated CpG dinucleotides, which are relative common in the genomes of most bacteria and DNA viruses.
Use of CpG oligodeoxynucleotides (ODNs) as adjuvants has been tested in several vaccine trials. Cooper (2005) used CpG 7909 as an adjuvant to a hepatitis B vaccination schedule in HIV patients and after 12 months seroprotective titres were found in 100% of subjects in the CpG group compared to 63% in the control group (p=0.008). In a recent study immunotherapy with a ragweed-toll-like receptor 9 agonist vaccine for allergic rhinitis appeared to offer long-term clinical efficacy in the treatment of ragweed allergic rhinitis (Creticos PS, Schroeder JT and Hamilton RG, et al.
Immunotherapy with a ragweed-toll-like receptor 9 agonist vaccine for allergic rhinitis. N Engl J Med 2006;355(14): 1445-55). TLR9-receptor agonists are also currently being evaluated as adjuvant to novel malaria vaccine candidates but they are also being used in a number of cancer trials. Some of the shortcomings of regular vaccination are: need for several boosts to achieve protection delay in rise of protective antibodies
• prevalence of vaccine non-responders (as outlined above - this is particularly a problem for immune-compromised individuals) cost of antigen and vaccine production which is a very significant limitation in the development of new conjugated pneumococcal vaccines, poorly protective antibodies with low affinity - this has been observed in a number of trials with pneumococcal vaccines in HIV-infected individuals. • Fall in antibody titre over time.
These shortcomings can be overcome by the effects of TLR9-agonists:
Reduce number of vaccinations required to achieve seroprotection (this was demonstrated in the Engerix and CpG7909 trial (Cooper CL, Davis HL and Angel JB, et al. CPG 7909 adjuvant improves hepatitis B virus vaccine seroprotection in antiretroviral-treated HIV-infected adults. AIDS 2005;19(14):1473-9))
Accelerate seroconversion, possibly permitting post-exposure vaccination Reduce non-responders rate • Reduce amount of antigen required
• Increase antibody avidity and protective activity
• More sustained antibody levels
Thus in one embodiment, the vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits according to the present invention comprise an adjuvant and/or a nucleic acid sequence encoding an adjuvant.
In one embodiment, the adjuvant of the present invention is an immunostimulatory adjuvant. Examples of such adjuvants are toll-like receptor agonists, such as agonists for TLR9, including CpG ODNs.
TLR agonist may in one embodiment be utilized as a mean of attracting and activating antigen presenting cells (APC; primarily Monocyte/macrophages and dendritic cells). This allows to selectively targeting the infection of pseudotyped MLV particles to the activated APC and thus promoting immunological cross-talk. In yet another embodiment, the vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits according to the present invention comprises at least one immunomodulating peptide and/or at least one nucleic acid sequence encoding an immunomodulating peptide. Specifically, said immunomodulating peptide may be an immunostimulating polypeptide, an immunodominant polypeptide and/or a genetic adjuvant. Such embodiments of the present invention include vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits comprising at least one cytokine and/or hormone, and/or at least one nucleic acid sequence encoding a cytokine and/or a hormone. Examples of cytokines include without restriction lnterleukin-2 (IL-2), lnterleukin-4 (IL-4) , lnterleukin-10 (IL-10) , Granulocyte-macrophage colony stimulating factor (GM-CSF), Vascular endothelial growth factor (VEGF), lnterleukin-12 (IL-12) , Fibroblast growth factor (FGF), lnterleukin-7 (IL-7) , lnterleukin-6 (IL-6) , Tumor Necrosis Factor-alpha (TNF-a) , Tumor Necrosis Factor-beta (TNF-b), Lymphotactin, Interferon-alpha (IFN-a), Interferon-beta (IFN-b), Interferon-gamma (IFN-g), Tumor Necrosis Factor (TNF), lnterleukin-15 (IL-15) , lnterleukin-5 (IL-5) , lnterleukin-13 (IL- 13) , lnterleukin-1 a (IL-1 alfa) , lnterleukin-1 b (IL-1 beta) , lnterleukin-18 (IL-18), MCP-1 , MIP-I a, MIP-I b, RANTES, TCA-3, CD80, CD86, CD40L, CCL3, CCL4, CCL5, Lymphocyte Chemotactic Factor (LCF), Erythropoietin (EPO), Prothymosin-alpha,
Thymopoietin, Thymosin-alpha-1. Each of the cytokines mentioned herein is intended to be an individual embodiment. Consequently, a vector, method, provirus, use, composition, vaccine, vaccine composition and/or kit comprising at least one heterologous nucleic acid sequence encoding a cytokine polypeptide or a fragment thereof derived from each of them are claimed individually.
Immunomodulating polypeptides may also be expressed by the vector by including at least one nucleic acid sequence encoding an immunomodulating polypeptide. A number of immunomodulating peptides are known to the person skilled in the art.The immunomodulating polypeptides may be immunostimulatory adjuvants, immunostimulatory cytokines or immunostimulant polypeptides other than cytokines. Non-limiting examples of immunomodulating polypeptides according to the present invention are shown in table 6a, table 6b and table 6c. Table 6a. List of preferred immunostimulatory adjuvants which can be used as immunostimulatory adjuvants according to the present invention.
Figure imgf000038_0001
Table 6b. List of immunostimulatory cytokines, which can be used as immunostimulatory adjuvants according to the present invention. Genetic adjuvants other than cytokines and conventional adjuvants.
Figure imgf000039_0001
(From Methods. 2003 Nov;31(3):243-54. Adjuvant formulations and delivery systems for DNA vaccines. Sasaki S, Takeshita F, Xin KQ, lshii N, Okuda K.)
Table 6c. List of immunostimulatory cytokines, which can be used as immunostimulatory adjuvants according to the present invention. (Vaccine. 2001 Mar 21 ;19(17-19):2647-56. Genetic adjuvants for DNA vaccines. Scheerlinck JY.)
Figure imgf000039_0002
Constitutive transport element (CTE)
The vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits of the present invention, in one embodiment, further comprise a constitutive transport element (CTE) that is characterised in that it serves as a signal of nuclear export of unspliced viral RNAs. The CTE of the present invention may be selected from the CTEs listed below in table 6D.
Table 6D CTE elements
Figure imgf000040_0001
In a particular embodiment the CTE is derived from for Mason-Pfizer monkey virus, in another preferred embodiment the CTE is derived from the Woodchuck Hepatitis virus, for example the Woodchuck Hepatitis virus posttranscriptional regulatory element (WPRE).
Internal ribosomal entry site (IRES)
The present invention also relates to a vector comprising at least one additional nucleic acid sequence and at least one internal ribosomal entry site (IRES). Thus, the vector according to the present invention comprises, in one embodiment, two additional nucleic acid sequences, three, four, five or six additional nucleic acid sequences and at least two IRES elements, three, four, five or six IRES elements. In one embodment the at least one IRES are of different origin. Specifically, the heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or fragment thereof may be preceded by an IRES. Similarly, any of the at least one additional nucleic acid sequences as mentioned herein may be preceded by an IRES. In one embodiment of the present invention the lentiviral envelope polypeptide or fragment thereof is not translated under the control of IRES. An IRES preceding a nucleic acid sequence results in the translation of said sequence under the control of the IRES. The IRES elements according to the present invention may be derived from picornaviridae, retroviridae or retrotransposons, mammalia or combinations thereof. In a specific embodiment, the IRES is selected from the IRES elements of encephalomyocarditis (ECMV) or another picornavirus. Specifically, the IRES according to the present invention can be derived from the IRES element of elF4G as defined in SEQ ID NO.: 7. In further embodiments of the present invention, the vector comprises an IRES element, which is selected from any af the IRES elements listed in the tables below.
Table 7. IRES elements which can be used according to the present invention. RNA 2006 Oct;12(10):1755-85
Figure imgf000042_0001
Table 7. continued
Figure imgf000043_0001
Figure imgf000044_0001
Table 8. Reported viral IRES elements which can be used according to the present invention. RNA. 2006 Oct;12(10):1755-85.
Figure imgf000045_0001
Figure imgf000046_0001
Table 9. Reported minimal IRES modules. RNA. 2006 Oct;12(10):1755-85
Figure imgf000047_0001
Table 10. list of viral IRES elements, which can be used according to the present invention. Curr Gene Ther. 2004 Mar;4(1):15-31.
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
In a specific embodiment of the present invention, the vector, RNA, provirus, mRNA or retroviral particle comprises an IRES, said IRES located in the 3'-LTR or the 5'-LTR, or in a region flanked by the 3'-LTR and the 5'-LTR. In particular, said IRES may be located in the R region of both the 5'-LTR and/or 3'-LTR. The U3 region of the 3'-Long Terminal Repeat or the U5 region of the 5'-LTR. In yet another specific embodiment of the present invention, said IRES is located in the U3 region between the inverted repeats and the transcription regulatory elements.
Preferred vectors A central aspect of the present invention relates to a mammalian expression vector comprising at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof. In one embodiment, said vector comprises an intron. The intron is deleted by splicing, thus, facilitating the transport from the nucleus to the cytoplasm of said vector encoded RNA in packaging cells. The vectors of the present invention also comprise a constitutive transport element (CTE). The CTE facilitates the transport of the vector encoded RNA from the nucleus to the cytoplasm of the semipackaging cell. Moreover, the vector may comprise a Rev responsive element (RRE). The presence of an RRE in the RNA transcript facilitates its transport from the nucleus to the cytoplasma of the semipackaging cell, when REV/REX is coecpressed. In one specific embodiment, the vector is transcribed in the cytoplasm, thereby producing high levels of transcript, which can be translated into envelope polypeptide. This envelope polypeptide may then be incorporated into viral particles in a packaging/producer cell.
In a specific embodiment, the vector of the present invention is a retroviral vector. Retroviral vectors include both replication deficient retroviral vectors, and replication competent retroviral vectors. In one embodiment, the present invention relates to a vector, which comprises at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof. The vector may further comprise at least one additional nucleic acid sequence and/or at least one internal ribosomal entry site (IRES). In one embodiment, at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or fragment thereof is preceded by an IRES, and in another embodiment, the at least one additional nucleic acid sequence is preceded by an IRES. The vectors of the present invention are preferably derived from gamma-retroviruses, for example Murine Leukemia Virus (MLV), Moloney Murine Leukemia Virus (MoMLV), or Akv MLV. Preferably, the at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof is selected from the nucleic acid sequences encoding envelope polypeptides derived from HIV-1 , HIV-2, or SIV. In a specific embodiment, the at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof encodes HIV-1 envelope as defined in SEQ ID NO.: 1 , or a fragment thereof. In another specific embodiment, the at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof encodes a C- terminally truncated HIV-1 envelope, such as defined in SEQ ID NO.: 2, or a fragment thereof. In yet another specific embodiment, the at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof encodes an SIV envelope.
The vectors of the present invention, also encompass vectors as defined above, wherein said at least one additional nucleic acid sequence is a reporter gene. Different embodiments of reporter genes according to the present invention include reporter genes encoding enhanced green fluorescent protein (eGFP), lac Z, dsRed, enhanced yellow fluorescent protein (eYFP), enhanced cyan fluorescent protein (eCFP), enhanced blue fluorescent protein (eBFP) and the human alpha- 1 -antitrypsin (hAAT). It is understood that any of the enhanced green fluorescent protein (eGFP), lac Z, dsRed, enhanced yellow fluorescent protein (eYFP), enhanced cyan fluorescent protein (eCFP), enhanced blue fluorescent protein (eBFP) or the human alpha-1 -antitrypsin (hAAT). However, the at least one additional nucleic acid sequence may also encode a selective marker, such as neomycin phosphotransferase II, and/or a suicide gene. Moreover, the at least one additional nucleic acid sequence may encode an immunomodulating polypeptide or peptide, such as an immunostimulating polypeptide, a genetic adjuvant, cytokines and/or hormones.
Important embodiments of the present invention include vectors, wherein said IRES is selected from the IRES elements of picornaviridae, retroviridae or retrotransposons, mammalia or combinations thereof. In a specific embodiment, said IRES is selected from the IRES elements of picornavirus. In another specific embodiment, said IRES is selected from the IRES elements of encephalomyocarditis (ECMV). The IRES may be inserted at different locations in the retroviral vector. In one embodiment, the IRES is located in a region flanked by the 3'-LTR and the 5'-LTR. In another embodiment, the IRES is located in the 3'-l_ong Terminal Repeat (LTR) or the 5'-LTR. In yet another embodiment, the IRES is located in the U3 region of the 3' LTR. In a further embodiment, the IRES is located in the U3 region between the inverted repeats and the transcription regulatory elements.
The design of the vectors of the present invention allows the vectors to be used for vaccination purposes. Thus, in one embodiment, the lentiviral envelope encoded by the vectors as defined by the present invention is capable of inducing an immunogenic response in a host animal. For example, said immunogenic response is an antibody response and/or cytotoxic T Lymphocyte (CTL) response.
In one embodiment, the immunogenic response of a vector according to the present invention is directed against a retroviral particle expressing said lentiviral envelope. In a specific embodiment, the lentiviral envelope is incorporated in said retroviral particle. The immunogenic response may thus be directed against the retroviral particle in the host animal. Thus, the retroviral particle is not required to be infectious and/or fusogenic. In a preferred embodiment, the envelope polypeptide as described herein is expressed and displayed on the surface of said retroviral particle. The present invention, thus relates to a vector, wherein said envelope is incorporated in said retroviral particle.
In another embodiment, the immunogenic response is a CTL response, wherein said vector is integrated into the genome of a host cell.
In a preferred embodiment, the retroviral vector of the present invention is derived from Akv MLV, and comprises a first nucleic acid sequence encoding eGFP and an additional nucleic acid sequence encoding the truncated HIV-1 ENV delta-CT (SEQ ID NO.: 2) preceded by an ECMV IRES element. This vector is defined in SEQ ID NO.: 3. In another preferred embodiment, the retroviral vector of the present invention is derived from Akv MLV, and comprises a first nucleic acid sequence encoding neomycin phosphotransferase Il and an additional nucleic acid sequence encoding the truncated HIV-1 ENV delta-CT as defined in SEQ ID NO.: 2, preceded by an ECMV IRES element. This vector is defined in SEQ ID NO.: 4. Retroviral particles and producer cells
One aspect of the present invention relates to a retroviral particle comprising a lentiviral envelope peptide as defined elsewhere herein, or a retroviral particle comprising an RNA according to the present invention. In one specific embodiment of the present invention, the lentiviral envelope polypeptide is derived from HIV-1 In a preferred embodiment, the retroviral envelope polypeptide is HIV-1 ENV, such as defined in SEQ ID NO.: 1 , and/or fragments and/or functional homologous thereof. In another preferred embodiment, the lentiviral envelope is an HIV variant in which the c-terminal tail has been deleted, such as HIV-1 delta-CT, in which the 1 13 c-terminal amino acids has been deleted, as shown in SEQ ID NO.: 2, and/or fragments and/or functional homologous thereof.
The retroviral particle of the present invention is derived from any of the retroviruses, wherefrom the vectors of the present invention may be derived, as describes above. In a preferred embodiment, the retroviral particle is a gamma-retroviral particle.
Further embodiments of the invention include any retroviral particle comprising an envelope polypeptide that has a sequence that is at least 36% identical to the amino acid sequence shown in SEQ ID NO.: 5 and/or SEQ ID NO.: 6, or is a fragment of a sequence that is at least 36% identical to the amino acid sequence shown in SEQ ID NO.: 5 and/or SEQ ID NO.: 6.
In another aspect, the present invention relates to a retroviral particle comprising an RNA transcribed from any of the embodiments of the retroviral vectors described herein.
In a preferred embodiment, the lentiviral envelope polypeptide as described herein is expressed on the surface of the retroviral particle. Cell surface expression of envelope polypeptides may be detected by persons skilled in the art. One method of detecting surface expression of ENV is provided in example 5 below. Moreover, incorporation of ENV into retroviral particles may in one example be detected by the method shown in example 6
In one embodiment, the retroviral particles of the present invention are fusogenic and/or infectious. However, in another embodiment, the retroviral particles of the present invention are non-fusogenic and/or non-infectious. Specifically, in one embodiment, the retroviral particle of the present invention is not capable of mediating fusion of said retroviral particle and cells expressing receptors for HIV. However, in another embodiment, the retroviral particle of the present invention is capable of mediating fusion of said retroviral particle and cells expressing receptors for HIV
A number of different assays are known for the skilled practitioner for detecting fusogenicity and/or infectiousness of a retroviral particle. Example 7 provides one such methodology for detecting fusogenicity of retroviral particles.
The retroviral vector of the present invention is constructed as a replication-defective vector based on any of the retroviruses mentioned elsewhere herein. A replication- defective retroviral vector is characterised in that, one or more genes essential for virus replication, packaging of viral RNA and/or formation of infective particle, have been deleted from the retroviral vector. Thus, to reconstitute the viral life cycle and generate viral particles comprising such replication defective vectors a specialised producer cell providing the deleted genes is needed. Such producer cell are constructed by transducing a cell with DNA constructs encoding the genetic information of the retroviral proteins, which are essential for packaging a retroviral vector genome and generating viral particles.
In the presence of a retroviral vector genome (the process known as transduction) a producer cell will generate infectious viral particles which comprise the retroviral genome derived from the vector. The viral particles produced in this manner will be released and can infect another producer cell. Such producer cells are also designated as packaging cells. In one embodiment, the retroviral particle of the present invention is obtained by transfection of a producer cell with a retroviral vector or part thereof, or an RNA or part thereof according to the present invention. In another embodiment, the retroviral particle of the present invention is obtained by transfection of a semipackaging cell with a vector or part thereof, or an RNA or part thereof according to the present invention.
In one aspect, the present invention relates to a producer cell comprising a vector as defined elsewhere herein. In one example, a producer cell of the present invention does not comprise lentiviral tat or rev and/or rex originating from HTLV. Packaging/producer cells are often produced by transfecting cells with genetic information and/or genes essential for retroviral particle formation. The culture of packaging cells is subsequently supertransfected with the vector DNA. Supertransfection here describes another or a second transfection event, namely the transfection of the packaging cell with the vector. The resulting supertransfected packaging cell will subsequently produce infectious viral particles comprising the vector RNA genome. Said particles, which will be released from the packaging cell, can be isolated. It should be noted that only supertransfected packaging cells produce infectious viral particles. Accordingly, the transduction efficiency directly correlates with the amount of infectious viral particles produced.
However, the problem of low transduction efficiency is overcome by the present invention. A replication-defective retroviral vector is provided, said vector comprising a gene encoding a lentiviral envelope polypeptide or fragment thereof. The lentiviral envelope polypeptide or part thereof may be selected among the lentiviral envelopes as listed elsewhere herein. According to one embodiment of the present invention, a nucleic sequence encoding a lentiviral envelope polypeptide or fragment thereof is under translational control of a heterologous IRES. Alternatively, a nucleic sequence encoding a lentiviral envelope polypeptide or fragment thereof may be present as in the vector backbone and is expressed in the transcript of the viral RNA and translated by the host cell machinery. Since the vector according to the present invention encodes itself an envelope, a packaging cell needs only to provide the proteins encoded by gag and/or pol. Such packaging cells comprising a gag and/or pol encoding DNA construct, but no env encoding DNA construct are known as semi-packaging cell.
Advantageously, this semi-packaging cell is not resistant to superinfection since these cells do not express envelope protein prior to transfection with the retroviral particle. Consequently, no envelope protein binds to the cellular receptor and thus, no resistance is mediated in said cell. Consequently, viral particles are only generated and released after transfection of the semi-packaging cell with the vector. The released retroviral particles comprising a gene of a functional lentiviral envelope polypeptide or fragment thereof are able to infect further semi-packaging cells in culture. Thus, the vector of the present invention is in one embodiment, replication-competent in the semi-packaging cells and thus allows for an easy and highly efficient production of the retroviral particles in high titers. Infection of target cells (also known as host cells) lacking gag and/or pol, by infectious particles produced in these semi-packaging cells result in the transfer of the genetic information of the vector only. Since this vector according to this embodiment does not comprise the gag and/or pol, no further replication of the vector in the target cell is observed. Accordingly, said replication- defective vector is a safe vector e. g. for use in gene therapy or for use as a vaccine as described herein. However, the present invention also encompass semi-packaging cells, which are capable of producing non-infectious retroviral particles.
In a particular embodiment of the invention, the producer cell does not comprise lentiviral tat or rev for example HIV-1 tat or rev. Also the rex gene originating from HTLV is not present in the producer cell.
The semi-packaging cells of the present invention are selected from all types of eukaryotic cells e.g. mammalian cells, yeast cells. In a further embodiment cells from old world momkeys and humans for production of infectious viral particles. For viral particle production capable of activating the the innate immune system, all eukaryotic cells except cells derived from old world monkeys and humans may be used in the as semi-packaging cells. Examples of cells used are Human embryonic Kidney cells HEK 293, HEK 293T, Human rhabdomysarcoma cells TE671 , Canine osteosarcome cells D17, murine fibroblast cells NIH3T3.
In a particular embodiment the semi-packaging cells are 293T cells comprising Mo-MV GagPol DNA.
In another embodiment, the retroviral particle according to the present invention comprises vesicular stomatitis virus envelope (VSV-G), the amphotropic murine leukemia virus envelope, Mutated SL3-2 envelope, Xenotropic murine leukaemia virus envelope, 10A1 virus envelope, Hepatitis virus C envelope, gibbon ape leukaemia virus. Human T-cell lymphotropic virus, or envelopes of endogenous human retroviruses. In one embodiment, the retroviral particle comprises the G glycoprotein of the vesicular stomatitis virus envelope (VSV-G).
The retroviral particles are preferably capable of infecting animal cells, such as mammalian cells, preferably human cells. In a specific embodiment, the retroviral particles are capable of infecting stem cells. In another specific embodiment, the retroviral particles are capable of infecting a CD4 positive cell. Moreover, the retroviral particles of the present invention are capable of inducing an immunogenic response in a host animal, preferably a human being. In a preferred embodiment, the immunogenic response is directed towards a retroviral particle of the present invention in said host animal. Thus, it is not required that the retroviral particle is infectious and/or fusogenic. The immunogenic response may directed against the retroviral particle it self. In one embodiment, the host animal is a human being.
In a specific embodiment, the retroviral packaging cells or semipackaging cells producing retroviral particles are encapsulated, wherein the capsules have a porous capsule wall which is permeable to said retroviral particles.
In a specific embodiment, the retroviral particles of the present invention comprise a lentiviral emvelope polypeptide as defined elsewhere herein, wherein said envelope polypeptide is glycosylated. In a preferred embodiment, said envelope polypeptide comprises alpha-gal. In a specific embodiment, the retroviral particles of the present invention comprise s lentiviral envelope polypeptide or fragment thereof, which comprise gal-alfa1 -3Galbeta1 -4GIcNAc-R epitopes on the envelope protein. Thus, in a preferred embodiment, the packaging/producer cells of the present invention comprise alpha-galactosidase enzyme, which make them capable of producing retroviral particles comprising alpha-gal labeled envelope polypeptides.
Target cells (Host cells)
A primary aspect of the present invention relates to a retroviral particle, as defined herein, capable of infecting specific target cell. Infection of a target cell can occur by receptor mediated endocytosis and/or membrane fusion, as described elsewhere.
One aspect of the present invention relates to a host cell comprising a vector or part thereof of the present invention. Another aspect of the present invention relates to a host cell comprising a retroviral particle of the present invention.
One embodiment of the present invention relates to a replication deficient retrovirus or any of the vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits described herein comprising a retroviral envelope polypeptide, capable of mediating infection of a cell, by use of the CD4- CXCR4/CCR5 receptor pathway. Thus, embodiments of the present invention comprise all retroviral particles or any of the vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits described herein comprising a retroviral envelope polypeptide, capable of mediating infection of a CD4-positive cell, and/or a CXCR4/CCR5-positive cell.
CD4 (cluster of differentiation 4) is a glycoprotein that is found primarily on the surface of helper T cells, as well as regulatory T cells and dendritic cells. CD4 is a member of the immunoglobulin superfamily. It has four immunoglobulin domains (D1 to D4) that are exposed on the extracellular surface of the cell: D1 and D3 resemble immunoglobulin variable (IgV) domains, while D2 and D4 resemble immunoglobulin constant (IgC) domains. On T cells, CD4 is the co-receptor for the T cell receptor (TCR) and recruits the tyrosine kinase lck.
CD4 is also a primary receptor used by HIV-1 to gain entry into host T cells. The HIV-1 envelope gp120 protein attaches to CD4, creating a shift in the conformation of the viral gp120 protein, which allows HIV-1 to bind to two other cell surface receptors on the host cell (the chemokine receptors CCR5 and CXCR4). Following another change in shape of a different HIV-1 envelope protein (gp41 ), HIV inserts a fusion peptide into the host T cell that allows the outer membrane of the virus to fuse with the T-cell membrane. HIV infection leads to a progressive reduction in the number of T cells possessing CD4 receptors and, therefore, the CD4 count is used as an indicator to help physicians decide when to begin treatment in HIV-infected patients.
The terms "CD4-positive cell" or "CXCR4/CCR5-positive cell" as used herein, relates to any cells expressing CD4 receptor or CXCR4/CCR5 receptor, respectively. The CD4 receptor and/or CXCR/CCR5 receptor may be accessible from the extracellular space at the cellular membrane.
In one embodiment, the retroviral particle comprises the G glycoprotein of the vesicular stomatitis virus envelope (VSV-G), the amphotropic murine leukemia virus envelope, Mutated SL3-2 envelope, Xenotropic murine leukaemia virus envelope, 10A1 virus envelope, Hepatitis virus C envelope, gibbon ape leukaemia virus, human T-cell lymphotropic virus or envelopes of endogenous human retroviruses In a particular embodiment of the present invention, the producer cell does not comprise lentiviral tat or rev, for example HIV-1 tat or rev. Also the rex gene originating from HTLV is not present in the host cell.
CTL response
The vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits according to the present invention is capable of inducing an immunogenic response in a host animal, for example in a human. The immunogenic response may be divided into to two types of responses, the antibody response and the cytotoxic T lymphocyte (CTL) response.
In the antibody response, specific antibodies are important in and may protect against viral infections. The most effective type of antiviral antibody is "neutralizing" antibody - this is antibody which binds to the virus, usually to the viral envelope of the virus particle or capsid proteins, and which blocks the virus from binding and gaining entry to the host cell. Virus specific antibodies may also act as opsonins in enhancing phagocytosis of virus particles - this effect may be further enhanced by complement activation by antibody-coated virus particles e.g. through production of the viral particles in eukaryotic cells e.g. mouse cells that ads gal-alfa1-3Galbeta1 -4GIcNAc-R epitopes on the envelope protein. In addition, in the case of some viral infections, viral proteins are expressed on the surface of the infected cell. These may act as targets for virus-specific antibodies, and may lead to complement-mediated lysis of the infected cell, or may direct a subset of natural killer cells to lyse the infected cell through a process known as antibody-directed cellular cytotoxicity (ADCC). At mucosal surfaces (such as the respiratory and gastrointestinal tracts), virus infection may induce the production of specific antibodies of the IgA isotype, which may be protective against infection at these surfaces. Not all antibodies to viruses are protective, however, and in certain cases an antibody to the virus may facilitate its entry into a cell through Fc receptor-mediated uptake of the antibody coated particle. Such antibodies are called enhancing antibodies.
During the course of a viral infection, antibody is most effective at an early stage, before the virus has gained entry to its target cell. In this respect, antibody is relatively ineffective in primary viral infections, due mainly to the lag phase in antibody production. Preformed antibody, particularly neutralising antibody, however, is an effective form of protective immunity against viral infections, as witnessed by the success of many viral vaccines, which work by stimulating virus-neutralising antibody responses.
The principal effector cells which are involved in clearing established viral infections are the virus specific cytotoxic T lymphocytes (CTL), for example the CD8+ cytotoxic T lymphocytes. These cells recognise (viral) antigens which have been synthesised within cell's nucleus or cytosol, and which have been degraded. They are presented at the cell's surface as short peptides associated with self class I MHC molecules. The recognition of antigen by CD8+ T cells is, therefore, distinct from that of CD4+ T cells in several respects. It requires synthesis of the target antigen within the cell (and is therefore restricted largely to virally infected or tumour cells); it is "restricted" by class I MHC molecules (as opposed to MHC class Il restriction for CD4+ T cells); MHC class I molecules are expressed on almost all somatic cells, so virtually any cell, on infection with virus, can act as a "target" cell for antigen specific CTL (contrasts with the limited tissue distribution of class Il MHC); recognition of an antigen presenting cell (APC) by an antigen-specific CTL usually results in the destruction of the APC.
In the context of the present invention the immune response is produced against the lentiviral envelope polypeptide or fragment thereof which is displayed on the host cells or the virus particles carrying a lentiviral envelope. The virus particles carrying a lentiviral envelope may be given to an animal, including a human, as a vaccine. The vaccine is produced as described herein using a vector of the present invention and/or a retroviral particle and given to an animal for example a human being. The retroviral particle produced according to the present invention may infect a host cell and upon integration of the vector of the present invention into the genome of the host cell, transcription and translation by the host cell, the lentiviral envelope polypeptide is presented on the surface of the host cell. The host cell targeted in this manner will be subject to a CTL response.
In one embodiment of the present invention the vector described herein is able to induce an immune response. The response may be an antibody response following vaccination with retroviral particles using the vector of the present invention. However, in yet another embodiment the immune response is a CTL response. In a specific embodiment, the immunogenic response is a CTL response, wherein said vector, RNA, mRNA of the present invention is integrated into the genome of a host cell. The ability of the vector or retroviral particle to infect, integrate and display the lentiviral envelope or fragments thereof on the surface of a host cell the lentiviral envelope polypeptide provides a means of boosting or enhancing the immune response as compared to viral vaccines that are not able to infect human cells. Viral vaccines that cannot infect human cells and subsequently display the viral envelope polypeptide on the surface of the host cell is not able to elicit a CTL response in comparable levels as the vaccine according to the present invention. In a specific embodiment of the present invention, however, is provided non-infectious retroviral particles, which express a lentiviral envelope as described herein and display said envelope on its surface, thereby allowing the immune system to recognize said envelope or fragment thereof.
The vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits according to the present invention can thus be used alone or in combination with other vaccines directed against Antiviruses as defined elsewhere herein, for example HIV.
During the course of a viral infection with for example HIV, antibody is most effective at an early stage, before the virus has gained entry to its target cell. In this respect, antibody is relatively ineffective in primary viral infections, due mainly to the lag phase in antibody production. Preformed antibody, particularly neutralising antibody, however, is an effective form of protective immunity against viral infections, as witnessed by the success of many viral vaccines, which work by stimulating virus-neutralising antibody responses. In contrast to existing vaccines against Antiviruses, the present invention thus provides an additional feature which renders the vaccine capable for eliciting a CTL response.
Non-infectious retroviral particles...
Vaccine In one aspect, the present invention relates to a vaccine for the prophylaxis or treatment of lentiviral infection comprising a vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition and/or kit of the present invention, the RNA of the lentiviral vector, the retroviral provirus, and/or a retroviral particle according to the invention as described herein. In a particular embodiment the vaccine is to be given against infection with HIV, in particular HIV-1. The present invention therefore also pertains to a vaccine composition which is administered to an animal including a human being, in which the vaccine is capable of eliciting an immune response against a disease caused by a lentivirus, in particular HIV-1.
A primary aspect of the present invention, thus, relates to a vaccine composition comprising a) lentiviral envelope polypeptide or a fragment thereof; and b) an adjuvant and/or an immunomodulating peptide. Examples of adjuvants and immunomodulating peptides are described elsewhere herein.
One embodiment of the present invention combines the vaccine compositions and/or the lentiviral envelope polypeptides or fragments thereof of the present invention with various materials such as adjuvants, to produce vaccines, immunogenic compositions, etc. Adjuvants, broadly defined, are substances which promote immune responses. Frequently, the adjuvant of choice is Freund's complete or incomplete adjuvant, or killed B. pertussis organisms, used e.g. in combination with alum precipitated antigen. A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) at pages 61 -63. Goding notes, however, that when the antigen of interest is of low molecular weight, or is poorly immunogenic, coupling to an immunogenic carrier is recommended. Examples of such carrier molecules include keyhole limpet haemocyanin, bovine serum albumin, ovalbumin and fowl immunoglobulin. Various saponin extracts have also been suggested to be useful as adjuvants in immunogenic compositions. Recently, it has been proposed to use granulocyte-macrophage colony stimulating factor (GM-CSF), a well known cytokine, as an adjuvant (WO 97/28816).
The vaccine compositions according to the invention preferably comprise an adjuvant and/or a carrier and/or immunomodulating peptides. Examples of useful adjuvants and carriers are given herein below. Thus, the viral particles present in the composition can be associated with a carrier such as e.g. a protein or an antigen-presenting cell to a T celll.
In particular the use of adjuvants is desired when the viral particles of the present invention are used to boost the immune response due to the ability of the retroviral particles of the present invention to infect, integrate and display the lentiviral envelope or fragments thereof on the surface of a host cell, thereby boosting or enhancing the immune response, in particular the CTL response as discussed elsewhere herein.
Adjuvants are any substance whose admixture into the vaccine composition increases or otherwise modifies the immune response of the retroviral particle coated with the lentiviral envelope polypeptide or fragment thereof as defined elsewhere herein. Carriers are scaffold structures, for example a polypeptide or a polysaccharide, to which the retroviral particle coated with the lentiviral envelope polypeptide or fragment thereof is capable of being associated.
Adjuvants could for example be selected from the group consisting of AIK(SO4J2, AINa(SO4J2, AINH4 (SO4), silica, alum, AI(OH)3, Ca3 (PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 1 1687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1 '2'-dipalmitoyl-sn - glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP- PE), RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80.RTM. emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes or other lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, lnterleukin 1 , lnterleukin 2, Montanide ISA-51 and QS-21 . Preferred adjuvants to be used with the invention include oil/surfactant based adjuvants such as Montanide adjuvants (available from Seppic, Belgium), preferably Montanide ISA-51. The most preferred adjuvants are adjuvants suitable for human use.
Montanide adjuvants (all available from Seppic, Belgium), may be selected from the group consisting of Montanide ISA-51 , Montanide ISA-50, Montanide ISA-70, Montanide ISA-206, Montanide ISA-25, Montanide ISA-720, Montanide ISA-708, Montanide ISA-763A, Montanide ISA-207, Montanide ISA-264, Montanide ISA-27, Montanide ISA-35, Montanide ISA 51 F, Montanide ISA 016D and Montanide IMS, preferably from the group consisting of Montanide ISA-51 , Montanide IMS and Montanide ISA-720, more preferably from the group consisting of Montanide ISA-51. Montanide ISA-51 (Seppic, Inc.) is oil/surfactant based adjuvants in which different surfactants are combined with either a non-metabolizable mineral oil, a metabolizable oil, or a mixture of the two. They are prepared for use as an emulsion with an aqueous solution comprising the protein belonging to the Bcl-2 protein family or peptide fragment thereof. The surfactant is mannide oleate. QS-21 (Antigenics; Aquila Biopharmaceuticals, Framingham, MA) is a highly purified, water-soluble saponin that handles as an aqueous solution. QS-21 and Montanide ISA-51 adjuvants can be provided in sterile, single-use vials.
A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) at pages 61 -63. Goding notes, however, that when the antigen of interest is of low molecular weight, or is poorly immunogenic, coupling to an immunogenic carrier is recommended. Examples of such carrier molecules include keyhole limpet haemocyanin, bovine serum albumin, ovalbumin and fowl immunoglobulin. Various saponin extracts have also been suggested to be useful as adjuvants in immunogenic compositions. Recently, it has been proposed to use granulocyte-macrophage colony stimulating factor (GM-CSF), a well known cytokine, as an adjuvant (WO 97/28816).
Desirable functionalities of adjuvants capable of being used in accordance with the present invention are listed in the below table.
Table 1 1. Modes of adjuvant action Action Adjuvant type Benefit
1. Generally small molecules or Upregulation of immune lmmunomodula proteins which modify the response. Selection of Th1 or tion cytokine network Th2
2. Presentation Generally amphipathic molecules Increased neutralizing antibody or complexes which interact with response. Greater duration of immunogen in its native response conformation
3. CTL • Particles which can bind or Cytosolic processing of protein induction enclose immunogen and yielding correct class 1 restricted peptides which can fuse with or disrupt cell membranes Simple process if promiscuous
• w/o emulsions for direct peptide(s) known attachment of peptide to cell surface MHC-1
4. Targeting • Particulate adjuvants which Efficient use of adjuvant and bind immunogen. Adjuvants immunogen which saturate Kupffer cells
• Carbohydrate adjuvants which As above. May also determine target lectin receptors on type of response if targeting macrophages and DCs selective
5. Depot • w/o emulsion for short term Efficiency Generation Microspheres or nanospheres for Potential for single-dose long term vaccine
Source: Cox, J.C., and Coulter, A.R. (1997). Vaccine 15, 248-56.
A vaccine composition according to the present invention may comprise more than one different adjuvant. Furthermore, the invention encompasses a therapeutic composition further comprising any adjuvant substance including any of the above or combinations thereof. It is also contemplated that the retroviral particle coated with the lentiviral envelope polypeptide or fragment thereof, and the adjuvant can be administered separately in any appropriate sequence.
A carrier may be present independently of an adjuvant. In particular, the inclusion of a carrier is relevant in connection with using the viral particles of the present invention are used to boost the immune response due to the ability of the retroviral particles of the present invention to infect, integrate and display the lentiviral envelope or fragments thereof on the surface of a host cell, thereby boosting or enhancing the immune response, in particular the CTL response as discussed elsewhere herein.
The function of a carrier can for example be to increase the molecular weight of in particular peptide fragments in order to increase their activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life. The carrier may be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell. A carrier protein could be but is not limited to keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid. For immunization of humans, the carrier must be a physiologically acceptable carrier acceptable to humans and safe. However, tetanus toxoid and/or diptheria toxoid are suitable carriers in one embodiment of the invention. Alternatively, the carrier may be dextrans for example sepharose.
Accordingly, the invention encompasses a therapeutic composition further comprising an adjuvant substance including any of the above or combinations thereof. It is also contemplated that the antigen, i.e. retroviral particle of the invention and the adjuvant can be administered simultaneously or separately in any appropriate sequence.
The pharmaceutical compositions may be prepared and administered using any conventional protocol known by a person skilled in the art. In example 5 a non-limiting example of preparation of a vaccine composition according to the invention is given as well as a non-limiting example of administration of such as a vaccine. It will be appreciated by the person skilled in the art that the protocol may be easily adapted to any of the vaccine compositions described herein.
In on embodiment of the invention, the pharmaceutical compositions, vaccines and vaccine compositions of the invention are useful for the prophylaxis of HIV infection or for treatment of HIV infection in a human being, where the human being is receiving treatment for the infection. In a further embodiment, the pharmaceutical compositions, vaccines and vaccine compositions of the invention are suitable for the treatment, amelioration and/or prevention of a lentiviral infection, such as HIV infection, including HIV-1 infection, and AIDS.
In preferred embodiments, the pharmaceutical composition of the invention is a vaccine composition. The pharmaceutical composition may thus be an immunogenic composition or vaccine capable of eliciting an immune response to lentiviral infection. As used herein, the expression "immunogenic composition or vaccine" refers to a composition eliciting at least one type of immune response directed against lentiviral envelope polypeptides or fragments thereof. Thus, such an immune response may be any of the types mentioned above: A CTL response where CTLs are generated that are capable of recognising the HLA/polypeptide complex presented on cell surfaces resulting in cell lysis, i.e. the vaccine elicits the production in the vaccinated subject of effector T-cells having a cytotoxic effect against the host cells harbouring the retroviral vector or RNA thereof; an antibody response giving rise to the production of anti-HIV antibodies.
Kits
One aspect of the present invention relates to a kit comprising a therapeutically effective amount of a vector, provirus, retroviral particle, composition, vaccine, and/or vaccine composition of the present invention.
Medical applications
The present invention provides a number of therapeutical applications. It is understood that the vectors, methods, proviruses, retroviral particles, uses, compositions, vaccines, vaccine compositions and kits may be used for treating a medical condition. Thus, one aspect of the present invention relates to the use of a vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition or/and kit according to the present invention for the manufacture of a medicament for the treatment, prevention and/or amelioration of a clinical condition.
Another aspect relates to a vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition or kit of the present invention for treating, ameliorating and/or preventing a clinical condition.
In a preferred embodiment, said clinical condition is lentiviral infection, such as HIV infection, such as HIV-1 infection and/or AIDS. In another preferred embodiment, said treatment is prophylactic treatment, for example by reducing the susceptibility of lentiviral infection, such as HIV infection or AIDS.
Thus, one aspect of the present invention relates to the use of a vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition or/and kit according to the present invention for the manufacture of a medicament for the treatment, prevention and/or amelioration of lentiviral infection, such as HIV infection and/or AIDS. Also the present invention relates to a vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition or kit of the present invention for treating, ameliorating and/or preventing lentiviral infection, incuding HIV infection and/or AIDS. The present invention also relates to use of a vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition or/and kit according to the present invention for the manufacture of a medicament for lentiviral vaccination, such as HIV vaccination.
Another aspect of the present invention relates to a vector, method, provirus, retroviral particle, composition, vaccine, vaccine composition or/and kit according to the present invention for use as a medicament. In one embodment, the vector, provirus, retroviral particle, use, composition, vaccine, vaccine composition or/and kit, when administered to an animal including a human being, is capable of eliciting an immune response against a disease caused by lentivirus, for example HIV, such as HIV-1 or HIV-2, or SIV.
One aspect of the present invention relates to the use of a vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition or/and kit according to the present invention for the manufacture of a medicament. In one aspect, the present invention relates to the use of a vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition or/and kit according to the present invention for the manufacture of a medicament for gene therapy. Another aspect relates to the use of a vector, method, provirus, retroviral particle, use, composition, vaccine, vaccine composition or/and kit according to the present invention for the manufacture of a medicament for immune therapy.
Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the vector, the producer cell, a retroviral particle and/or a host cell according to the present invention.
The invention also relates to a method for introducing a nucleotide sequence into target cells, said method comprising infection of target cells with a retroviral particle as defined elsewhere herein. This method may for example be used for the production of transgenic animals, said method comprising infection or transduction of embryonic stem cells with a retroviral particles or a vector of the present invention.
Dosis and administration The amount of the immunogenic peptide of the invention in the pharmaceutical composition may vary, depending on the particular application. However, a single dose of the peptide composition is preferably anywhere from about 10 mg to about 5000 mg, more preferably from about 50 mg to about 2500 mg such as about 100 mg to about 1000 mg. Modes of administration include intradermal, subcutaneous and intravenous administration, implantation in the form of a time release formulation, etc. Any and all forms of administration known to the art are encompassed herein. Also any and all conventional dosage forms that are known in the art to be appropriate for formulating injectable immunogenic peptide composition are encompassed, such as lyophilized forms and solutions, suspensions or emulsion forms containing, if required, conventional pharmaceutically acceptable carriers, diluents, preservatives, adjuvants, buffer components, etc.
The pharmaceutical compositions and/or vaccine compositions may be prepared and administered using any conventional protocol known by a person skilled in the art. In examples 3-5 non-limiting examples of preparation of a vaccine composition according to the invention is given as well as a non-limiting example of administration of such as a vaccine. It will be appreciated by the person skilled in the art that the protocol may be easily adapted to any of the vaccine compositions described herein. In a further embodiment of the invention, the pharmaceutical composition of the invention is useful for treating an individual suffering from a clinical condition characterized by expression of IDO, such as cancer and infections.
The immunoprotective effect of the composition of the invention can be determined using several approaches known to those skilled in the art. A successful immune response may also be determined by the occurrence of DTH reactions after immunization and/or the detection of antibodies specifically recognizing the polypeptide(s) of the vaccine composition.
Vaccine compositions according to the invention may be administered to an individual in therapeutically effective amounts. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
The vaccine compositions and/or pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular. Administration of pharmaceutical compositions is accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial (directly to the tissue), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the methods of prophylaxis and treatment with the vaccine composition.
For example, the vaccine compositions can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the vaccine, comprising any of the herein described compounds can be employed as a prophylactic or therapeutic agent. Also any and all conventional dosage forms that are known in the art to be appropriate for formulating injectable immunogenic peptide composition are encompassed, such as lyophilized forms and solutions, suspensions or emulsion forms containing, if required, conventional pharmaceutically acceptable carriers, diluents, preservatives, adjuvants, buffer components, etc.
Preferred modes of administration of the vaccine composition according to the invention include, but are not limited to systemic administration, such as intravenous or subcutaneous administration, intradermal administration, intramuscular administration, intranasal administration, oral administration, rectal administration, vaginal administration, pulmonary administration and generally any form of mucosal administration. Furthermore, it is within the scope of the present invention that the means for any of the administration forms mentioned in the herein are included in the present invention.
A vaccine according to the present invention can be administered once, or any number of times such as two, three, four or five times. Administering the vaccine more than once has the effect of boosting the resulting immune response. The vaccine can further be boosted by administering the vaccine in a form or body part different from the previous administration. The booster shot is either a homologous or a heterologous booster shot. A homologous booster shot is a where the first and subsequent vaccinations comprise the same constructs and more specifically the same delivery vehicle especially the same viral vector. A heterologous booster shot is where identical constructs are comprised within different viral vectors.
Examples
Example 1
Example 1 describes the generation of two retroviral bicistronic vectors derived from the Akv murine leukemia virus with the egfp or the neomycin resistance gene, an IRES element, and a truncated version of the HIV-1 envelope gene. The vector transgenes are efficiently expressed upon stable integration. In addition the vector transduces CXCR4+/CD4+ cells via the encoded truncated HIV-1 envelope. The production of the HIV-1 envelope protein is Rev-independent and without being bound by theory one explanation is that inherent cis-elements for RNA transport is present in the Akv-MLV genome, or alternatively that the ECMV IRES element is responsible for the Rev- independent production of the truncated HIV-1 envelope.
By allowing single round of infection this vector system efficiently couples genotype to phenotype with regard to cellular tropism directed by the HIV-1 envelope protein. This vector construction might be of use in HIV-1 envelope protein targeted vaccine efforts where proper antigen presentation of envelope epitopes may hold promise as well as being a simplified tool in the assessment of HIV-1 entry inhibitors.
Construction of EgfpHIVMo
Cloning of EgfpMo carrying the HIV-1 (HXB2) envelope gene was performed by PCR amplification of the HIV-1 envelope gene from pgTat-CMV kindly provided by Prof. Jørgen Kjems. A premature stop codon was introduced by mutating codon 713 (primers 5'CGTTCTTAAGAACGATAATGGTTAATATCCCTGCC and δ'AGTCACACCTGCTGGCCATGAGAGTGAAGGAGAAATAT) this leads to a truncated HIV-1 Envelope which has been shown to be necessary for HIV-1 /2 pseudotyping of Mo-MLV particles (Mammano et al., 1997;Schnierle et al., 1997). Restriction enzyme linkers are indicated in bold 3' AfIII and 5' Aarl. Aarl produces a 3' overhang that is compatible with Ncol. This digested fragment was ligated in either the EgfpMo or NeoMo vector in 5' Ncol and 3' AfIII sites. The EgfpMo and NeoMo are retroviral bicistronic vectors with Akv MLV background having the GagPol reading frame exchanged with the genes encoding either egfp or Neo and an ECMV IRES element (Bahrami et al., 2003).
Western Blot Expression of HIV-1 Env from the EgfpHIVMo and NeoHIVMo were analysed by western blotting of cell lysate from 293T transiently transfected. Five million pelleted cells were lysed in 2x SDS sample buffer. The cell lysate was briefly centrifuged to remove cell debris. Five μL sample was loaded on a denaturing polyacrylamide gel. The proteins were transferred to a Hybond nitrocellulose membrane (Amersham Biosciences) and HRP-conjugated anti-gp120 (Europa Bioproducts, UK) was incubated overnight. Visualisation was performed with ECL luminescence (Cell Signalling, USA). The positive control CMV-128 is a CMV promoter driven HIV-1 envelope with an identical C-terminal truncation In addition the vector encodes rev (Schnierle et al., 1997), kindly provided by Dr. B. Schnierle.
Syncytium Assay
The fusion capability of the NeoHIVMo / EgfpHIVMo expressed HIV-1 Env ΔCT was tested by a cell-cell fusion assay. Briefly 293T cells were seeded at 3*104 /cm2 in a 6- well plate and transfected the following day by 1 μg DNA using the CaP04-method. Twenty-four hours later cells were rinsed and co-cultured with 3*104 /cm2 HeLa CD4+ or D17 CXCR4+/CD4+. After 24 hrs incubation syncytia formation was visualized by bright field and fluorescent microscopy. HXB2-env is a full length HIV-1 envelope plasmid not encoding Rev (Page et al., 1990).
Stably integrated EgfpHIVMo surface expression of HIV-1 envelope was assayed by 1 :1 overnight co-culture of cells in question with HeLa CD4+ or D17 CXCR4+/CD4+. Cells and Titer Determination
Human kidney epithelial 293 cells and TE671 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % foetal bovine serum (FBS) and 1 % penicillin/streptomycin. Canine D17 cells were maintained in modified Eagle's medium α (MEM- α) with 10 % FBS and 1 % penVstrep. Mouse NIH 3T3 cells were grown in DMEM with 10 % Newborn Calf Serum (NCS) and 1 % pen./strep. Human T lymphocytes Jurkat (E6-1 ) were maintained in RPMI with 10 % FBS and 1 % pen./strep.
D17 CXCR4+/CD4+ cells were created by transduction of the retroviral vector pBabe.Fusin and selection of 10 μg/mL puromycin. Subsequently the cells were transfected by pT4b (Maddon et al., 1985). The cells were double stained with mouse anti-CD4 and goat anti mouse IgG-PE (Abeam, Cambridge, UK) and FACS sorted for high CD4 expression to a purity of 98 %.
The capability of pseudotyping MLV particles with HIV-1 Envelope has previously been achieved when deleting the cytoplasmic tail. The verification of pseudotyping capabilities was performed by co-transfection of 293T cells with EgfpHIVMo and Mo-
MLV GagPol expression construct (Morita et al., 2000). Briefly, 293T cells were seeded at 7*104 cells/cm2 in an 80 cm2 bottle. Transfection of 5 μg EgfpHIVMo DNA and 3 μg Mo-MLV GagPol DNA was performed by CaPO4 precipitation. Forty-eight hours later virus containing supernatant was assayed for transduction capability on D17 CXCR4+/CD4+ cells by 3 fold dilutions. D17 CXCR4+/CD4+ cells were seeded in 12- well at 5*104 /cm2 the viral supernatant was added and left on overnight. Twenty-four hours after transduction cells were harvested and the titer was determined by flow cytometric determination of the number of EGFP expressing cells. The CMV-128 and VSV-G was co-transfected with the MLV derived pLXSN that contains an egfp gene.
Vector Rescue
EgfpHIVMo was stable integrated in D17 or Te671 cells and expressed both transgenes as measured by EGFP visualisation and syncytia formation when co- cultured with CXCR4+/CD4+ cells (results presented in Table 1 ). The cells were FACS sorted based on the EGFP expression and a purity of 97 % was obtained. The cells were seeded 5*104 cells/cm2 in a 6-well plate. The following day Lipofectamine 2000 transfection of 0.5 μg MLV GagPol DNA and 0.5 μg VSV-G DNA was performed as described by the manufacturer (Invitrogen). After 24 hrs medium was replenished. Forty-eight hours after transfection viral supernatant was harvested and filtered through 0.45 μm on D17 CXCR4+/CD4+. The vector rescue was measured by fluorescence microscopy.
Multiple round infection Semi-packaging cells either D17 or Te671 stably transfected and selected to express MLV GagPol were used for second round infection experiment. EgfpHIVMo viruses were produced with VSV-G and utilised for transduction of the semi-packaging cells. The titer of the virus stock was measured by serial dilution on D17 cells and flow cytometry analysis as described. We transduced the semi-packaging cells at MOI=I and at MOI>5.
Flow cytometry
HIV-1 Env expression was assayed by flow cytometry. 293T cells were transfected by EgfpHIVMo and CMV-128. Forty-eight hours later 106 cells were harvested washed with PBS and double stained first with F105 anti-HIV-1 Env and second with goat anti- human IgG conjugated to phycoerythrin (Abeam, Cambridge, UK). Env expression was measured by flow cytometry. Quantification of Env and EGFP signals was done in FlowJo version 6.3.
Results
The human immunodeficiency virus type 1 (HIV-1) is one of the major health concerns globally with merely 38 million people infected and more than 2.8 million deaths annually as of 2005 (UNAIDS www.unaids.org). Continued efforts to develop novel treatment opportunities and progress in vaccine research are of primary importance in the future control of HIV-1.
Retroviruses are lipid-enveloped viruses with their envelope (Env) proteins arranged in trimeric spikes on the virion surface. HIV-1 Env has been studied in great detail and much knowledge regarding structure and function has been gathered (Chen et al., 2005;Kwong et al., 1998;Zhu et al., 2006). During entry the Env protein undergoes large conformational changes resulting in the mixing of the viral and cellular membrane. This leads to the internalisation of the viral core, reverse transcription and integration of the viral genome. The entry process of HIV-1 has recently been targeted in the treatment of HIV-1 infected patients by the licensing of Enfuvirtide in 2003 (Lazzarin et al., 2003). With the introduction of Enfuvirtide in the antiretroviral treatment of HIV-1 infected patients new challenges in the detection and testing of fusion inhibitor resistant strains have emerged. The entry step in the viral replication cycle holds promise with regard to further development of entry inhibitors because of the plethora of protein-protein interactions that occur during fusion.
HIV-1 Env is composed of the two glycoprotein subunits; gp120 and gp41. Gp120 interacts initially with the primary receptor CD4 upon which a second binding site become exposed that binds to a co-receptor (CXCR4/CCR5 and several others) (Huang et al., 2005). These receptor binding steps activate the fusion machinery located in gp41. The N-terminal fusion peptide is inserted into the target cell membrane upon which the heptad repeat domains (HR1 and HR2) form a coiled-coil structure. These complex rearrangements bring the lipid membranes into proximity and membrane fusion takes place (Eckert & Kim, 2001 ).
Gp41 also comprises the large cytoplasmic tail (CT) of the HIV-1 Env protein. Among retroviruses HIV carry a rather long CT compared to the well studied γ-retroviruses. In the γ-retrovirus murine leukemia virus (MLV) the R-peptide in the CT needs to be cleaved for the Env protein complex to become fusion activated (Januszeski et al., 1997). This mechanism is not present in HIV-1 and the exact function of the CT remains elusive. It has been shown that pseudotyping of MLV particles with HIV-1/2 Env requires the deletion of 1 13 amino acids in the CT (Mammano et al., 1997;Schnierle et al., 1997).
Retroviral mRNA splicing patterns differ between the simple γ-retroviruses and the complex Antiviruses. The HIV-1 genome contains 9 genes which are translated from more than 40 different single and multiple spliced mRNA species (reviewed in (Stoltzfus & Madsen, 2006)). The HIV-1 envelope mRNA contains an important regulatory element for viral gene expression; the Rev Responsive Element (RRE). The RRE is bound by the Rev protein connecting the unspliced mRNA species to the nuclear export pathway thereby facilitating cytoplasmic localisation of genomic RNA (Cullen, 2003). The Rev/RRE interaction utilises the CRM1 pathways that connects unspliced retroviral RNA to the nuclear pore complex and hence cytoplasmic transport (Emerman et al., 1989;Malim et al., 1989). For vaccine design purposes codon optimisation has been widely utilised in order to obtain an enhanced HIV Env production that is Rev-independent (Kotsopoulou et al., 2000;Vinner et al., 1999).
In the present study we sought to develop an Akv MLV retroviral vector expressing the HIV-1 envelope gene as to utilise the pseudotyping capabilities between the MLV species and HIV. By constructing a bicistronic vector we coupled the expression of the HIV-1 envelope and the egfp or the neo gene in a Rev-independent manner. This vector design may be used in vaccination purposes as well as being a tool for the testing and development of HIV-1 fusion inhibitors, where the use of HIV-1 pseudotyped MLV particles would have advantages in the ease of use for testing in clinical settings (Coakley et al., 2005;Siegert et al., 2005).
Results and Discussion Vector design
The two vectors EgfpHIVMo and NeoHIVMo were constructed with the cis regulatory elements from Akv MLV. In that backbone we inserted a bicistronic cassette containing the egfp gene or the neomycin resistance gene, an ECMV IRES element and a truncated HIV-1 envelope gene. Figure 1 depicts the entire vector map and the vector design with regard to parental MLV and HIV-1 splice patterns. We designed the vector as to omit the canonical multiple splice sites 3' in HIV-1 envelope. A similar vector construction albeit with the normal MLV envelope gene and the functional use thereof has previously been described from our group (Bahrami et al., 2003;Bahrami et al., 2004). The integrity of EgfpHIVMo and NeoHIVMo were verified by restriction analysis and sequencing. The entire vector sequences can be found as additional material.
Transgene expression
The transgene expression from the vector was assayed at several levels. The correct translation from the IRES element of HIV-1 Env was investigated by western blotting of cell lysates of transiently transfected 293T cells. Figure 2 depicts the gp140 Env precursor (normally gp160 but this version is C-terminal truncated by 1 13 amino acids) and the gp120. Both vectors (NeoHIVMo and EgfpHIVMo) appear to sustain production of HIV-1 Env that is cleaved correct. The vector CMV-128 encodes both an identical truncated version of HIV-1 Env and the rev gene (Schnierle et al., 1997). The primary translational product from the bicistronic transcripts is Neo/EGFP; the latter is visualized by fluorescence microscopy and/or flow cytometry (see Figure 3). To further elucidate the functionality of the expressed HIV-1 Env ΔCT we tested the ability of transfected cells to induce syncytia via envelope-receptor interaction. Figure 3,A+B clearly identifies syncytia formation localised to the EGFP signal. In addition there was a dependence on the CD4 receptor. HeLa (CD4 negative) did not induce syncytia upon co-culture (figure 3,E+F). The vector NeoHIVMo was equally efficient in the formation of syncytia.
Lastly the cell surface expression (CSE) of HIV-1 Env ΔCT was measured by flow cytometry. Figure 6 depicts the proportions of EGFP pos/neg and Env pos/neg respectively. It is evident that the proportions of double positive cells (EGFP+/Env+) are only present in the EgfpHIVMo transfected cells whereas CMV-128 display a single positive phenotype (EGFP-/Env+). Moreover in the panel with EgfpHIVMo the two single positive populations EGFP-/Env+ and EGFP+/Env- are very low suggesting that the expression of the two transgenes is strictly coupled as expected from the bicistronic vector design.
Transduction of CXCR4+/CD4+ cells By co-transfecting 293T cells with Mo-MLV GagPol and the vector EgfpHIVMo pseudotyped particles were produced that showed transduction capability of
CXCR4+/CD4+ cells. Figure 4 displays the viral titers on D17 CXCR4+/CD4+ cells. We achieved a titer of 2*105 IU/mL supernatant from transient transfections.
Several papers utilising HIV envelope pseudotyping of MLV particles have reported titers in the range 5*104 - 1 *105 IU/mL with a further improvement by Thaler et al. 2001 who constructed a packaging cell line reaching 5*105 IU/mL (Mammano et al.,
1997;Schnierle et al., 1997;Thaler & Schnierle, 2001 ).
Rev-independent HIV-1 ΔCT envelope expression As can be seen in Figure 4 there is no effect of Rev in this system on EgfpHIVMo transduction capability. These findings were substantiated in the syncytium assay. No effect on size and number of syncytia was observed with or without Rev in experiment with EgfpHIVMo. This is depicted in Figure 3 A+B (with CMV-Rev) vs. C+D (without CMV-Rev). In contrast Figure 3 panel G and H clearly shows the Rev-dependence of the HIV-1 Env. Panel G is CMV-128 that carries an internal Rev production whereas pane H is full-length HXB2 Env that is Rev-dependent. This suggests that inherent features of the Akv MLV genome may sustain Rev-independent transport of vector RNA. We and others have recently identified regions in the MLV packaging signal that are important for viral RNA transport (Basyuk et al., 2005;Smagulova et al., 2005). The observations are in line with a report from Nack and Schnierle 2003 where a truncated HIV-1 envelope was inserted in a replication competent MLV (containing Gag and Pol) (Nack & Schnierle, 2003). In that construct there appears to be Rev independent viral RNA processing, which the authors suggest may be due to the naturally occurring intron being spliced out upstream of envelope. This is not the explanation in this vector system where the normal MLV splice donor located 5' is not utilised because 1 ) All downstream splice acceptors are deleted 2) The egfp gene would be spliced out but all transfected/tranduced cells are green fluorescent (see Figure 1 B). Furthermore as indicated in Table 1 it was possible to rescue the vector from stably transduced cells by transfecting with MLV GagPol and VSV-G and transferring the vector as subsequently measured by EGFP and HIV-1 Env expression.
Recently Graf et al. 2006 have made an elegant study converting an egfp gene into a Rev-dependent phenotype by codon exchange as to resemble the HIV-1 gag gene in nucleotide composition (Graf et al., 2006). It was established that the modified egfp gene utilized CRM-1 mediated export as opposed to the normal egfp that remained
LMB insensitive. In addition they inserted the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV) which rescued the modified egfp and mediated export via an alternative pathway (Graf et al., 2006).
Here we have coupled the HIV-1 envelope gene to an egfp gene in the context of the MLV LTR region encompassing all MLV cis-elements needed for replication. Whether the Rev-independence we observe is driven by an unknown MLV CTE element remains to be established. It is also a possibility that the EMCV IRES contributes to the stability and/or nuclear export of the RNA encoding the truncated HIV Env. We can rule out an effect from the coupling of the egfp gene because we have generated an identical vector carrying the neomycin resistance gene in place of egfp that displays similar phenotypic properties.
Second round infectivity We intended to produce a conditionally replicational competent vector capable of multiple rounds of infection when Gag and Pol were supplied in trans. Transducing semi-packaging cells (i.e. D17 GagPol /Te671 GagPol) with the vector EgfpHIVMo at MOI < 1 we were unable to harvest functional pseudotyped viruses. In contrast we succeeded in directing transduction of D17 CXCR4+/CD4+ cells with pseudotyped viruses when producer cells (semi-packaging cells D17 GagPol /Te671 GagPol) had been infected with EgfpHIVMo at a high MOI (>5) suggesting that the vector production of HIV-1 ΔCT Env was low. Table 1 lists the titers obtained from second- round transduction.
Again this confirms the results from Nack and Schnierle 2003 who observed significantly reduced second round mobilisation of the HIV-1 Env into MLV particles despite plentiful viral RNA transport (Nack & Schnierle, 2003). In contrast we could induce syncytia formation between cells stably integrated with one EgfpHIVMo provirus even at a low MOI which shows that the HIV-1 envelope is properly translated and transported to the plasma membrane from the stable provirus (data not shown).
The generation of a bicistronic MLV vector expressing a truncated version of the HIV-1 envelope presents a novel approach in the development of retroviral vectors. We envision the utilisation of the vector in resistance testing of HIV-1 fusion inhibitors. In addition the vector can be used in forced in vitro evolutionary studies on functional domains of the HIV-1 envelope and the effects on fusion competence, because the vector contains the possibility to sustain multiple rounds of infection when supplying GagPol in trans.
Lastly the HIV-1 envelope protein in such a bicistronic vector context may be of promise in HIV vaccine development. The advantage of this vector construction is the capability to perform one infection event of natural HIV-1 target cells (via CD4 and CXCR4) and to process the HIV-1 Env correctly from the integrated provirus.
Example 2
Western blot of HIV envelope from transfected HeLa CeIIe, NIH3T3 cells and from MLV pseudotypes cells. Western Blot Protocol
Materials and reagents 5X SDS Loading Dye for 50ml
25OmM Tris HCI pH6.8
10% SDS
0.5 % Bromophenol blue 0.25g
50% Glycerol 25ml Weigh out SDS and bromophenol blue.
Add Tris and ddH2O to 25 ml and vortex to mix, and then add glycerol. Store at room temperature. Heat to 37O before use to ensure SDS is in solution.
NOTE- Bromophenol blue is very difficult to weigh out -"static" .
Reducing agent is to be added to the loading dye before use. DTT is routinely used but alternatively βmercaptoethanol can be used.
DTT - Add DDT to final concentration of 10OmM in sample to be loaded. 1 M
DDT stocks are stored at -2O0C in single use aliquots βmercaptoethanol -Add 25μl of βmercaptoethanol to 1.5ml of x5 loading dye fresh on the day of use.
SDS Running buffer (x10 stock- 0.025M Tris, 0.19M glycine, pH8.3, 1 %
SDS)
121.2g Tris base
576g glycine 40g SDS
Dissolve in 3.5L of dH2O in a large container with stirring. Make up to 4L with dH2O. DO NOT TITRATE. Store at room temperature. Dilute to 1 X on day of use
Lower Gel Buffer - pH 8.8, 1.5M Tris, .4% SDS Tris base 182 g SDS 4.0 g ddH2O 900ml
Stir until dissolved, then bring to pH 8.8 with concentrated HCI. Make up to 1 L with ddH2O and store at 4°C. Upper Gel Buffer- pH 6.8, .5M Tris, .4% SDS
Tris base 30 g
SDS 2.O g ddH2O 400ml
Stir until dissolved , then bring to pH 6.8 with concentrated HCI. Make up to 500ml with ddH20 and store at 40C.
Acrylamide stock 40% 29:1 Acryl /Bis Amresco cat# 0311 -500 / BIORAD cat# 161 -0146 10% APS- 10% ammonium persulphate in ddH2O Solution is made fresh daily and stored at 4°C. Ammonium persulphate powder (Merk cat# 443073E) is hydroscopic and the lid must be sealed with parafilm. Invitrogen SEE BLUE molecular weight markers cat#LC5625
Semi dry transfer buffers Anode 1 buffer- .3M Tris pH 10.4, 20% methanol I M Tris pH 10.4 150ml 100% methanol 100ml dH2O 250ml
Anode 2buffer- .025M Tris pH 10.4, 20% methanol I M Tris pH 10.4 12.5ml 100% methanol 100ml dH2O 387.5ml
Cathode buffer- .025M Tris pH 9.4, 4OmM aminocaproic acid, 20% methanol
OPTIONAL 0.1 % SDS (better transfer of high MW proteins) IM Tris pH 9.4 25ml
Aminocaproic acid 5.248 g
100% methanol 200ml dH2O 775ml
CHECK Ph OF SOLUTIONS AND ADJUST ACCORDINGLY
Whatman 3mmm Chromatography paper Cat# 3030 917
PDVF membrane NEN Life Sciences Cat #NEF1002
Buffers for detection by ECL PBSTT 0.3% Tween 20, 0.05% Triton-x-100 in 1 X PBS
PBST 0.3% Tween 20 in 1 X PBS
PBSTTE 0.3% Tween 20, 0.05% Triton-x-100, 1 OmM EDTA in 1 X
PBS
PBSTB 0.3% Tween 20, 3% BSA in 1 X PBS Tween 20 (Amresco cat# 9005-64-5)
Triton-x-100 (Merk cat# 30632.4N)
PBS supplied at 10X from Media Prep Unit University of Melbourne
Microbiology Department
ECL substrate - Supersignal, Pierce Cat # 3408 Hyperfilm ECL (Amersham cat#RPN3103K
Developer and fixer
Stripping buffer (homemade)
2% SDS v/v 62.5mM Tris pH6.8
NB. Must add 2-β Mercaptoethanol to a final concentration of 1% v/v before use
OR
Commerical Stripping buffer
Pierce Restore western blot stripping buffer (catalogue number 21059)
12. Protocol Procedure for running SDS PAGE gel
12.1 Clean plates, stand, 1.5mm spacers etc. with 100% ethanol. Assemble plates in casting stand and mark plates at twice the depth of the comb from the top of the plates.
12.2 Mix ingredients in 50ml tube and pour to 5mm above mark on plates. Gently overlay with 5mm of hydrated butanol and allow to polymerize. (approximately 1 hour). NB. The volumes given for stacking and lower gel mixes are sufficient for one 18x15.5cm large Hoeffer gel.
10% SDS separating gel
Lower Gel Buffer 8.0ml ddH2O 16ml
40% Acralamide 8.0ml
10% APS 50μl
TEMED 25μl
12.3 Wash butanol from top of gel and drain off water with paper towel. Mix stacking gel in 50ml tube, fill to top of plates and insert comb, taking care to eliminate all bubbles. Allow the gel to polymerize (approximately 30 minutes)
5% stacking gel
Upper gel buffer 3.75ml ddH2O 9.5ml
40% Acrylamide 1.8ml 10% APS 50μl
TEMED 30μl
12.4 Thaw lysates from -800C and store on ice. At this point samples may be standardized at this point for transfection efficiency (or another marker such as p24 concentration) Samples should be made up to equal volume with 1 x TTS buffer. Blank samples should be prepared for empty lanes to minimize the smile effect using lysis buffer and 5x dye with the appropriate reducing agent. Invitrogen SEE BLUE markers are included as a size comparison in all gels. 15μl of markers are used and treated in exactly the same way as the samples (loading dye, reducing agent, denatured)
Standardizing for Transfection Efficiency using EGFP
Using a cell lysate containing high amounts of EGFP (ie. BRIGHT green visually) make a 1 :2 dilution series in cell lysis buffer. Add a set volume into a black costar cluster plate (cat #3631 ) including same volume of unknown samples. Read the plate on the fluro stage @473/520nm on
FugiFLA-3000 phosphoimager. Obtain a equation for a standard curve using linear regression of the dilution series. Dilute the unknown samples according to the equation obtained. CHECK the fluorescence intensity of the diluted samples in equal volume to confirm correct dilutions have been made. 12.5 Combine sample and x5 loading dye with the appropriate reducing agent. From this point do not put the samples on ice or the SDS in the loading dye will precipitate.
12.6 Flick tubes to mix samples and pulse in microfuge. Denature samples by heating to 950C for 5 minutes in a heating block. Pulse tubes again to ensure all of sample is loaded.
12.7 When stacking gel is set, remove comb and wash out the wells twice with ddH2O removing water from the wells with a blunt canula. Load samples into wells quickly, and then carefully overlay with x1 SDS running buffer using a transfer pipette.
12.8 Assemble gel in running tank taking care to remove bubbles from the bottom of the gel plate. Run at 8mAmps PER GEL overnight on a Biorad
3000 powerpac. (16-18 hours) or alternatively at 20mAmps for approximately 6 hours.
Procedure for transfer by semi dry method
12.9 Pre soak gel in cathode buffer for 15 minutes Cut 9 sheets of Whatman 3mmm Chromatography paper Cat# 3030 917 and 1 sheet of PDVF membrane NEN Life Sciences Cat #NEF1002 to the size of the gel (15cm x15cm). Soak 3 sheets of paper in each of the three buffers. Soak the membrane in 100% methanol for 5 minutes, then for 10 minutes in PFW
(ensure that the membrane wets) and finally for 5 minutes in anode 2 buffer. All solutions can be reused with the exception of the cathode buffer used to pre-soak the gel. 12.10 Stack 3 sheets of paper from anode 1 on the base(+ve) and remove air bubbles by rolling with a pipette. Mop up any excess buffer with paper towel. Stack 3 sheets of paper from anode 2 followed by the membrane, gel and lastly the cathode soaked papers. Take care not to handle the membrane but to exclude air bubbles. Place lid on apparatus (-ve) and carefully attach leads. Transfer proteins at.45 Amps for 1 .5 -2 hours for a single gel ( .65 Amps for 2 hours for 2 gels) on the high resistance powerpac (Biorad 200). This equates to approximately 5mA/cm2.
12.11 Block the membrane in PBS 5% skim milk powder (Diploma) overnight at 4°C for optimal results. Clean excess acrylamide from the surface of the membrane by gently wiping the surface with gloved fingertips. Occasionally the membrane may dry out slightly during the transfer process and will appear to be slightly streaky. To re-hydrate the membrane wash for 5 minutes in PBST, then rinse well in PBS prior to blocking.
12.12 Western blot detection by ECL
All incubations and washes are performed on a rocking platform. • After blocking, wash the membrane 3X 10 minutes in PBSTT • Rinse 1 X in PBS
• Incubate with primary antibody @1 :1000 to 1 :10000 diluted in PBSTB overnight at 4 0C for optimal sensitivity ( or 3 hours at room temperature)
• Wash 3X 5min with PBSTT at RT. • Wash 3X 5 min with PBST at RT.
• Incubate with secondary antibody (conjugated to horseradish- peroxidase , @ 1 :1000 to 1 :50000 in PBSTB for 1 hour at room temperature (or 4°C overnight)
• Wash blot 4X 10 min in PBSTTE (OMIT IF USING SUPERSIGNAL) • Wash 4X 10min PBST
• Rinse with PBS
• Remove filter to a clean tray, draining off excess PBS on paper towel.
• Mix ECL reagents (Supersignal, Pierce Cat # 3408) in 1 :1 ratio , add to filter (protein face up) and incubate for EXACTLY 1 min with manual rocking.
• Remove filter from tray , draining excess liquid on paper towel and wrap in gladwrap.
• Immediately expose to Hyperfilm ECL (Amersham cat#RPN3103K) Adjust exposure according to strength of signal.
12.13 As a guide to exposure times - If you can see a signal try a touch exposure, if no signal is visible try a 5 min exposure. Use a fluorescent ruler to align markers (Fluorescence seen after a 5min exposure) 12.14 Then films are dry transfer the marks corresponding to the molecular weight markers onto the films.
HeLa Cells were seeded at 2,5*105 in T25 bottle. Transfected using Lipofectamine according to protocol. Two days later cells and supernatant were harvested in TTS buffer. Total protein concentration were determined by BCA protein assay kit as described by manufacturer.
Westernblot as descripted in "Western blot protocol"
Block membrane with 5% milk in PBS 0,1% tween 20 at least 2 hrs Add 3 μl primary antibody (D7 Goat anti-Env) to 10 ml milk incubate at least 2 hrs or o/n in refrigerator Wash for 5 * 5 min's with PBST
Add secondary Ab (1 -3 μL) to 10 ml milk incubate at least 2 hrs or o/n in refrigerator Wash membrane 5 * 5 min's with PBST Dye with supersignal west pico chemilumnescent substrat and picture at Kodak chemiluminescent station
The results of the western blots are shown in figure 11 , figure 12, and figure 13.
Example 3
IFN ELISpot data
ELISA protokol:
Coat plates o/n: - Env-anti Ab binds to well bottom when exposed to high salt cone, (turning all ways)
- Use re-use lid, plates from drawer and built-in rags.
- For 100 wells make up 11 ml: 9,9 ml ddH2O 1,1 ml 10x COAT ELISA15
2,2 μl D7 (1 mg/μl)
- Add 100 μl/well and store o/n in 4°c
Finish up: - Prepare 50 ml 5% milk (PBS 0,1% Tween + 2,5 g milk powder) - Wash plate; 2* PBST bath, 2* PBST bath, 2*( Ix)PBS bath, hit out on tissue
- Add 200 μl/well milk
- Incubate 1 hr RT, defreeze samples 10 min's before time runs out
- Empty in sink, and add env protein, 100 μl/well: 9 ml 5% milk
1 ml NL4-3 (ELISA)
- Incubate RT for at least 2 hrs (up to four)
- Wash plate; 2* PBST bath, 2* PBST bath, 2*( Ix)PBS bath, hit out on tissue
- Add samples: 100 μl/well 5% milk, add 1 μl/well sample serum remember to write on lid and do neg and pos control
- Make up secondary Ab mix (goat + sheep serum) and incubate at least 40 min's. 10,2 ml 5% milk 1 : 1000 ab:
10,2 μl Goat anti-mice HRP 102 μl 1% norm, sheep serum
- Incubate plate 1 hr RT
- Wash plate; 2* PBST bath, 2* PBST bath, 2*( Ix)PBS bath, hit out on tissue
- Add secondary Ab mix lOOμ I/well.
- Make up POD substrate 30 min's before use to reach RT, 10 ml: 1 tablet 3,3'5,5-tetramethyl- benzidinemid fridge
1 ml DMSO, pour in hood to keep bottle sterile -kept across from hood, glass bottle
-> shake teen min's on vortexer in 50 ml falcon
- Add: 9 ml Phosphor-citrate ph 5mid fridge 50 ml falcon, red rack
2 μl Hydroperoxideblack cap alufoi1' red rack
- Incubate 1 hr.
- Wash plate; 2* PBST bath, 2* PBST bath, 4*(Tx)PBS bath, hit out on tissue
- Add POD sub, lOOμl/well, shake, incubate 10 min's
- Develop
Results of ELISA experiments are shownin figure 14 and figure 15.
Cross-reaction of mice ab to D7 (sheep ab) could give a false pos result. To avoid this
Ab mix is made up from Goat-anti-mouse and norm, sheep sera, so that mice ab that would have bound to D7 now binds to the free sheep ab.
Example 4 lmmunogenicity of retroviral particles
The development of a broad and neutralising antibody response is vital for a protective HIV-1 vaccine (Mascola et al 2000, Nat Medicine. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies). In addition, the induction of specific and effective cytotoxic T lymphocytes has been shown to be required for infection control (Schmitz et al 1999, Science. Control of Viremia in Simian Immunodeficiency Virus Infection by CD8+ Lymphocytes). Thus, the development of vaccine strategies that encompass both arms of the immune sysem are thus important. Differential MHC I and Il antigen presentation is a key factor for initiation of a potent immune response. There appear to be short-commings in antigen presentation when antigens are administered solely as peptides or DNA. In contrast cross-talk between antigen presenting cells and T helper lymphocytes are promoted in vaccine strategies based on either infectious or non-infectious viral particles. A potent induction of cell mediated immunity can be achieved even with whole-killed viral particles (McBurney et al 2007, Virology. Membrane embedded HIV-1 envelope on the surface of a virus-like particle elicits broader immune responses than soluble envelopes). This is likely a consequence of improved antigen uptake and systemic immunestimulation in macrophages and dendritic cells (Buonaguro et al 2006, J Virol. Baculovirus-derived human immunodeficiency virus type i virus-like particle activate dendritic eels and induce ex vivo t-cell responses). To achieve potent immunogenic retroviral particles, γ-retroviral particles may be produced with functonal HIV-1 envelope trimers on the surface. These particles can function as a superior immunogenic particle avoiding any risk associated with viral inactivation procedures
Example 5
Detection of cell surface expression of ENV by Flow cytometry:
The cells are labeled with anti HIV-ENV antibodies through incubation of the cells with the Ab for 45 min on ice. Followed by washing of the unbound Ab with PBS. The cell- anti envelope Ab complex is subsequently incubated with a fluorescent labled Ab against the primary ENV-binding Ab for 45 min. on ice followed by a second PBS wash. A flow cytometer will be used to detect fluorescence associated with the cells, which is indicative of ENV expression.
Example 6
Detection of incorporation of ENV into retroviral particles by Flow cytometry: Supernatant containing retroviral particles is incubated with cells expressing the CD4 receptor for 45 min. on ice followed by PBS wash. The cells are subsequently labeled with anti HIV-ENV antibodies through incubation of the cells with the Ab for 45 min on ice. followed by washing of the unbound Ab with PBS. The cell-anti envelope Ab complex is subsequently incubated with a fluorescent labled Ab against the primary ENV-binding Ab for 45 min. on ice followed by a second PBS wash. A flow cytometer will be used to detect fluorescence associated with the cells, which is indicative of ENV expression.
Example 7
Detection of fusogenicity of the ENV by syncytia assay:
An Env-expressing plasmid is transfected into 293T cells. Two days later, the ENV- expressing cells are co-cultivated with D17 cells expressing CD4 (10,000 cells pr. square centimeter) and one or more of the HIV co-receptors (ie. CXCR4, CCR5 etc. 10,000 cells pr. square centimeter). Fusogenicity of the ENV protein can be detected by examination of the level of cell-cell fusion in a microscope.
References
Bahrami, S., Duch, M., Pedersen, F. S., 2004. Change of tropism of SL3-2 murine leukemia virus, using random mutational libraries. J.Virol. 78, 9343-9351. Bahrami, S., Jespersen, T., Pedersen, F. S., Duch, M., 2003. Mutational library analysis of selected amino acids in the receptor binding domain of envelope of Akv murine leukemia virus by conditionally replication competent bicistronic vectors. Gene 315, 51-61.
Basyuk, E., Boulon, S., Skou Pedersen, F., Bertrand, E., Vestergaard Rasmussen, S., 2005. The packaging signal of MLV is an integrated module that mediates intracellular transport of genomic RNAs. J.Mol.Biol. 354, 330-339.
Chen, B., Vogan, E. M., Gong, H., Skehel, J. J., Wiley, D. C, Harrison, S. C, 2005. Structure of an unliganded simian immunodeficiency virus gpl20 core. Nature 433, 834-841.
Coakley, E., Petropoulos, C. J., Whitcomb, J. M., 2005. Assessing chemokine co-receptor usage in HIV. Curr.Opin.Infect.Dis. 18, 9-15.
Cullen, B. R., 2003. Nuclear mRNA export: insights from virology. Trends Biochem.Sci. 28, 419-424.
Eckert, D. M., Kim, P. S., 2001. Mechanisms of viral membrane fusion and its inhibition. Annu.Rev.Biochem. 70, 777-810.
Graf, M., Ludwig, C, Kehlenbeck, S., Jungert, K., Wagner, R., 2006. A quasi-lentiviral green fluorescent protein reporter exhibits nuclear export features of late human immunodeficiency virus type 1 transcripts. Virology.
Halene S, Wang L, Cooper RM, Bockstoce DC, Robbins PB, Kohn DB. Improved expression in hematopoietic and lymphoid cells in mice after transplantation of bone marrow transduced with a modified retroviral vector. Blood 1999; 94(10):3349-3357.
Huang, C. C, Tang, M., Zhang, M. Y., Majeed, S., Montabana, E., Stanfield, R. L., Dimitrov, D. S., Korber, B., Sodroski, J., Wilson, I. A., Wyatt, R., Kwong, P. D., 2005. Structure of a V3-containing HIV-I gpl20 core. Science 310, 1025-1028.
Januszeski, M. M., Cannon, P. M., Chen, D., Rozenberg, Y., Anderson, W. F., 1997. Functional analysis of the cytoplasmic tail of Moloney murine leukemia virus envelope protein. J.Virol. 71, 3613-3619.
Kotsopoulou, E., Kim, V. N., Kingsman, A. J., Kingsman, S. M., Mitrophanous, K. A., 2000. A Rev-independent human immunodeficiency virus type 1 (HFV-l)-based vector that exploits a codon-optimized HIV-I gag-pol gene. J.Virol. 74, 4839-4852.
Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., Hendrickson, W. A., 1998. Structure of an HIV gpl20 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393, 648- 659.
Lazzarin, A., Clotet, B., Cooper, D., Reynes, J., Arasteh, K., Nelson, M., Katlama, C, Stellbrink, H. J., Delfraissy, J. F., Lange, J., Huson, L., Demasi, R., Wat, C, Delehanty, J., Drobnes, C, Salgo, M., 2003. Efficacy of enfuvirtide in patients infected with drug- resistant HIV-I in Europe and Australia. N.Engl. J.Med. 348, 2186-2195.
Lund AH, Duch M, Lovmand J, Jorgensen P, Pedersen FS. Complementation of a primer binding site-impaired murine leukemia virus-derived retroviral vector by a genetically engineered tRNA-like primer. J Virol 1997; 71(2):1191-1195.
Maddon, P. J., Littman, D. R., Godfrey, M., Maddon, D. E., Chess, L., Axel, R., 1985. The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family. Cell 42, 93-104.
Mammano, F., Salvatori, F., Indraccolo, S., De, R. A., Chieco-Bianchi, L., Gottlinger, H. G., 1997. Truncation of the human immunodeficiency virus type 1 envelope glycoprotein allows efficient pseudotyping of Moloney murine leukemia virus particles and gene transfer into CD4+ cells. J.Virol. 71, 3341-3345.
Morita, S., Kojima, T., Kitamura, T., 2000. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7, 1063-1066.
Nack, U., Schnierle, B. S., 2003. Replacement of the murine leukemia virus (MLV) envelope gene with a truncated HIV envelope gene in MLV generates a virus with impaired replication capacity. Virology 315, 209-216.
Page, K. A., Landau, N. R., Littman, D. R., 1990. Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity. J.Virol. 64, 5270-5276.
Reddy, T. R., Xu, W., Mau, J. K., Goodwin, C. D., Suhasini, M., Tang, H., Frimpong, K., Rose, D. W., Wong-Staal, F., 1999. Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-I Rev. Nat.Med. 5, 635-642.
Reddy, T. R., Xu, W. D., Wong-Staal, F., 2000. General effect of Sam68 on Rev/Rex regulated expression of complex retroviruses. Oncogene 19, 4071-4074. Schnierle, B. S., Stitz, J., Bosch, V., Nocken, F., Merget-Millitzer, H., Engelstadter, M., Kurth, R., Groner, B., Cichutek, K., 1997. Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells. Proc.Natl.Acad.Sci.U.S.A 94, 8640-8645.
Siegert, S., Thaler, S., Wagner, R., Schnierle, B. S., 2005. Assessment of HIV-I entry inhibitors by MLV/HIV-1 pseudotyped vectors. AIDS Res.Ther. 2, 7. Smagulova, F., Maurel, S., Morichaud, Z., Devaux, C, Mougel, M., Houzet, L., 2005. The highly structured encapsidation signal of MuLV RNA is involved in the nuclear export of its unspliced RNA. J.Mol.Biol. 354, 1118-1128.
Stoltzfus, C. M., Madsen, J. M., 2006. Role of viral splicing elements and cellular RNA binding proteins in regulation of HIV-I alternative RNA splicing. Curr.HIV.Res. 4, 43- 55.
Thaler, S., Schnierle, B. S., 2001. A packaging cell line generating CD4- specific retroviral vectors for efficient gene transfer into primary human T-helper lymphocytes. Mol.Ther. 4, 273-279.
Vinner, L., Nielsen, H. V., Bryder, K., Corbet, S., Nielsen, C, Fomsgaard, A., 1999. Gene gun DNA vaccination with Rev-independent synthetic HIV-I gpl60 envelope gene using mammalian codons. Vaccine 17, 2166-2175.
Zhu, P., Liu, J., Bess, J., Jr., Chertova, E., Lifson, J. D., Grise, H., Ofek, G. A., Taylor, K. A., Roux, K. H., 2006. Distribution and three-dimensional structure of AIDS virus envelope spikes. Nature 441, 847-852. Tolstrup M, Selzer-Plon J, Laursen AL, Bertelsen L, Gerstoft J, Duch M, Pedersen FS, Ostergaard L., Full fusion competence rescue of the enfuvirtide resistant HIV-I gp41 genotype (43D) by a prevalent polymorphism (137K).AIDS. 2007 Feb 19;21(4):519-21.
Sequences
SEQ ID NO.: 1
HIV-1 ENV - wild type (from reference strain HXB3)
Nucleotide: atgagagtgaaggagaaatatcagcacttgtggagatgggggtggagatggggcaccatgctccttgggatgttgatga tctgtagtgctacagaaaaattgtgggtcacagtctattatggggtacctgtgtggaaggaagcaaccaccactctattttgt gcatcagatgctaaagcatatgatacagaggtacataatgtttgggccacacatgccggtgtacccacagaccccaacc cacaagaagtagtattggtaaatgtgacagaaaattttaacatgtggaaaaatgacatggtagaacagatgcatgagga tataatcagtttatgggatcaaagcctaaagccatgtgtaaaattaaccccactctgtgttagtttaaagtgcactgatttgaa gaatgatactaataccaatagtagtagcgggagaatgataatggagaaaggagagataaaaaactgctctttcaatatc agcacaagcataagaggtaaggtgcagaaagaatatgcatttttttataaacttgatataataccaatagataatgatact accagctatacgttgacaagttgtaacacctcagtcattacacaggcctgtccaaaggtatcctttgagccaattcccatac attattgtgccccggctggttttgcgattctaaaatgtaataataagacgttcaatggaacaggaccatgtacaaatgtcagc acagtacaatgtacacatggaattaggccagtagtatcaactcaactgctgttaaatggcagtctagcagaagaagagg tagtaattagatctgtcaatttcacggacaatgctaaaaccataatagtacagctgaacacatctgtagaaattaattgtac aagacccaacaacaatacaagaaaaaaaatccgtatccagaggggaccagggagagcatttgttacaataggaaa aataggaaatatgagacaagcacattgtaacattagtagagcaaaatggaatgccactttaaaacagatagctagcaa attaagagaacaatttggaaataataaaacaataatctttaagcaatcctcaggaggggacccagaaattgtaacgcac agttttaattgtggaggggaatttttctactgtaattcaacacaactgtttaatagtacttggtttaatagtacttggagtactgaa gggtcaaataacactgaaggaagtgacacaatcacactcccatgcagaataaaacaatttataaacatgtggcaggaa gtaggaaaagcaatgtatgcccctcccatcagcggacaaattagatgttcatcaaatattacagggctgctattaacaag agatggtggtaataacaacaatgggtccgagatcttcagacctggaggaggagatatgagggacaattggagaagtg aattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcaga gagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgt caatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgag gcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatac ctaaaggatcaacagctcctggggatttggggttgctctggaaaactactttgcaccactgctgtgccttggaatgctagttg gagtaataaatctctggaacagatttggaatcacacgacgtggatggagtgggacagagaaattaacaattacacaag cttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgg gcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtt taagaatagtttttgctgtactttctgtagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaat cccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgat tagtgaacggatccttagcacttatctgggacgatctgcggagcctgtgcctcttcagctaccaccgcttgagagacttact cttgattgtaacgaggattgtggaacttctgggacgcagggggtgggaagccctcaaatattggtggaatctcctacaata ttggagtcaggagctaaagaatagtgctgttagcttgctcaatgccacagctatagcagtagctgaggggacagatagg gttatagaagtagtacaagaagcttatagagctattcgccacatacctagaagaataagacagggcttggaaaggatttt gctataa
SEQ ID NO.: 2
C-terminally truncated HIV-1 envelope (HIV-1 ENV delta-CT) (from reference strain
HXB3)
atgagagtgaaggagaaatatcagcacttgtggagatgggggtggagatggggcaccatgctccttgggatgttgatga tctgtagtgctacagaaaaattgtgggtcacagtctattatggggtacctgtgtggaaggaagcaaccaccactctattttgt gcatcagatgctaaagcatatgatacagaggtacataatgtttgggccacacatgcctgtgtacccacagaccccaacc cacaagaagtagtattggtaaatgtgacagaaaattttaacatgtggaaaaatgacatggtagaacagatgcatgagga tataatcagtttatgggatcaaagcctaaagccatgtgtaaaattaaccccactctgtgttagtttaaagtgcactgatttgaa gaatgatactaataccaatagtagtagcgggagaatgataatggagaaaggagagataaaaaactgctctttcaatatc agcacaagcataagaggtaaggtgcagaaagaatatgcatttttttataaacttgatataataccaatagataatgatact accagctataagttgacaagttgtaacacctcagtcattacacaggcctgtccaaaggtatcctttgagccaattcccatac attattgtgccccggctggttttgcgattctaaaatgtaataataagacgttcaatggaacaggaccatgtacaaatgtcagc acagtacaatgtacacatggaattaggccagtagtatcaactcaactgctgttaaatggcagtctagcagaagaagagg tagtaattagatctgtcaatttcacggacaatgctaaaaccataatagtacagctgaacacatctgtagaaattaattgtac aagacccaacaacaatacaagaaaaagaatccgtatccagagaggaccagggagagcatttgttacaataggaaa aataggaaatatgagacaagcacattgtaacattagtagagcaaaatggaataacactttaaaacagatagctagcaa attaagagaacaatttggaaataataaaacaataatctttaagcaatcctcaggaggggacccagaaattgtaacgcac agttttaattgtggaggggaatttttctactgtaattcaacacaactgtttaatagtacttggtttaatagtacttggagtactgaa gggtcaaataacactgaaggaagtgacacaatcaccctcccatgcagaataaaacaaattataaacatgtggcagaa agtaggaaaagcaatgtatgcccctcccatcagtggacaaattagatgttcatcaaatattacagggctgctattaacaag agatggtggtaatagcaacaatgagtccgagatcttcagacctggaggaggagatatgagggacaattggagaagtg aattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcaga gagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcct caatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgag gcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatac ctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttg gagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaag cttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgg gcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtt taagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattaa
SEQ ID NO.: 3
Vector sequence - EgfpHIVMo: Complete nucleotide sequence of the bicistronic vector
EgfpHIVMo.
1 TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG
101 TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG
CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA 201 CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC
ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC
TCTTCGCTAT
301 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT TTTCCCAGTC ACGACGTTGT AAAACGACGG
CCAGTGAATT
401 CTACCTTACG TTTCCCCGAC CAGAGCTGAT GTTCTCAGAA
AAACAAGAAC AAGGAAGTAC AGAGAGGCTG GAAAGTACCG GGACTAGGGC
CAAACAGGAT 501 ATCTGTGGTC AAGCACTAGG GCCCCGGCCC AGGGCCAAGA
ACAGATGGTC CCCAGAAACA GAGAGGCTGG AAAGTACCGG GACTAGGGCC
AAACAGGATA
601 TCTGTGGTCA AGCACTAGGG CCCCGGCCCA GGGCCAAGAA
CAGATGGTCC CCAGAAATAG CTAAAACAAC AACAGTTTCA AGAGACCCAG AAACTGTCTC
701 AAGGTTCCCC AGATGACCGG GGATCAACCC CAAGCCTCAT
TTAAACTAAC CAATCAGCTC GCTTCTCGCT TCTGTACCCG CGCTTATTGC
TGCCCAGCTC
801 TATAAAAAGG GTAAGAACCC CACACTCGGC GCGCCAGTCC TCCGATAGAC TGAGTCGCCC GGGTACCCGT GTATCCAATA AAGCCTTTTG
CTGTTGCATC
901 CGAATCGTGG TCTCGCTGAT CCTTGGGAGG GTCTCCTCAG
AGTGATTGAC TGCCCAGCCT GGGGGTCTTT CATTTGGGGG CTCGTCCGGG
ATTTGGAGAC 1001 CCCCGCCCAG GGACCACCGA CCCACCGTCG GGAGGTAAGC
TGGCCAGCGA TCGTTTTGTC TCCGTCTCTG TCTTTGTGCG TGTGTGTGTG
TGTGCCGGCA
1101 TCTAC I I I I I GCGCCTGCGT CTGATTCTGT ACTAGTTAGC
TAACTAGATC TGTATCTGGC GGCTCCGTGG AAGAACTGAC GAGTTCGTAT TCCCGACCGC
1201 AGCCCTGGGA GACGTCTCAG AGGCATCGGG GGGGGGATCC
AGAGCTCGAG ATGGTGAGCA AGGGCGAGGA GCTGTTCACC GGGGTGGTGC
CCATCCTGGT 1301 CGAGCTGGAC GGCGACGTAA ACGGCCACAA GTTCAGCGTG
TCCGGCGAGG GCGAGGGCGA TGCCACCTAC GGCAAGCTGA CCCTGAAGTT
CATCTGCACC
1401 ACCGGCAAGC TGCCCGTGCC CTGGCCCACC CTCGTGACCA CCCTGACCTA CGGCGTGCAG TGCTTCAGCC GCTACCCCGA CCACATGAAG
CAGCACGACT
1501 TCTTCAAGTC CGCCATGCCC GAAGGCTACG TCCAGGAGCG
CACCATCTTC TTCAAGGACG ACGGCAACTA CAAGACCCGC GCCGAGGTGA
AGTTCGAGGG 1601 CGACACCCTG GTGAACCGCA TCGAGCTGAA GGGCATCGAC
TTCAAGGAGG ACGGCAACAT CCTGGGGCAC AAGCTGGAGT ACAACTACAA
CAGCCACAAC
1701 GTCTATATCA TGGCCGACAA GCAGAAGAAC GGCATCAAGG
TGAACTTCAA GATCCGCCAC AACATCGAGG ACGGCAGCGT GCAGCTCGCC GACCACTACC
1801 AGCAGAACAC CCCCATCGGC GACGGCCCCG TGCTGCTGCC
CGACAACCAC TACCTGAGCA CCCAGTCCGC CCTGAGCAAA GACCCCAACG
AGAAGCGCGA
1901 TCACATGGTC CTGCTGGAGT TCGTGACCGC CGCCGGGATC ACTCTCGGCA TGGACGAGCT GTACAAGTAA AGCGGCCGTA CGCGTTGATC
AGTTAACGAA
2001 TTCGAAGGGT CCCAGGCCTC GGAGATCTGG GCCCATGCGG
CCGCCCCCTA ACGTTACTGG CCGAAGCCGC TTGGAATAAG GCCGGTGTGC
GTTTGTCTAT 2101 ATGTTATTTT CCACCATATT GCCGTCTTTT GGCAATGTGA
GGGCCCGGAA ACCTGGCCCT GTCTTCTTGA CGAGCATTCC TAGGGGTCTT
TCCCCTCTCG
2201 CCAAAGGAAT GCAAGGTCTG TTGAATGTCG TGAAGGAAGC
AGTTCCTCTG GAAGCTTCTT GAAGACAAAC AACGTCTGTA GCGACCCTTT GCAGGCAGCG
2301 GAACCCCCCA CCTGGCGACA GGTGCCTCTG CGGCCAAAAG
CCACGTGTAT AAGATACACC TGCAAAGGCG GCACAACCCC AGTGCCACGT
TGTGAGTTGG 2401 ATAGTTGTGG AAAGAGTCAA ATGGCTCTCC TCAAGCGTAT
TCAACAAGGG GCTGAAGGAT GCCCAGAAGG TACCCCATTG TATGGGATCT
GATCTGGGGC
2501 CTCGGTGCAC ATGCTTTACA TGTGTTTAGT CGAGGTTAAA AAACGTCTAG GCCCCCCGAA CCACGGGGAC GTGGTTTTCC TTTGAAAAAC
ACGATAATAC
2601 CATGGGAGCA GAAGACAGTG GCAATGAGAG TGAAGGAGAA
ATATCAGCAC TTGTGGAGAT GGGGGTGGAG ATGGGGCACC ATGCTCCTTG
GGATGTTGAT 2701 GATCTGTAGT GCTACAGAAA AATTGTGGGT CACAGTCTAT
TATGGGGTAC CTGTGTGGAA GGAAGCAACC ACCACTCTAT TTTGTGCATC
AGATGCTAAA
2801 GCATATGATA CAGAGGTACA TAATGTTTGG GCCACACATG
CCTGTGTACC CACAGACCCC AACCCACAAG AAGTAGTATT GGTAAATGTG ACAGAAAATT
2901 TTAACATGTG GAAAAATGAC ATGGTAGAAC AGATGCATGA
GGATATAATC AGTTTATGGG ATCAAAGCCT AAAGCCATGT GTAAAATTAA
CCCCACTCTG
3001 TGTTAGTTTA AAGTGCACTG ATTTGAAGAA TGATACTAAT ACCAATAGTA GTAGCGGGAG AATGATAATG GAGAAAGGAG AGATAAAAAA
CTGCTCTTTC
3101 AATATCAGCA CAAGCATAAG AGGTAAGGTG CAGAAAGAAT
ATGCA I I I I I TTATAAACTT GATATAATAC CAATAGATAA TGATACTACC
AGCTATAAGT 3201 TGACAAGTTG TAACACCTCA GTCATTACAC AGGCCTGTCC
AAAGGTATCC TTTGAGCCAA TTCCCATACA TTATTGTGCC CCGGCTGGTT
TTGCGATTCT
3301 AAAATGTAAT AATAAGACGT TCAATGGAAC AGGACCATGT
ACAAATGTCA GCACAGTACA ATGTACACAT GGAATTAGGC CAGTAGTATC AACTCAACTG
3401 CTGTTAAATG GCAGTCTAGC AGAAGAAGAG GTAGTAATTA
GATCTGTCAA TTTCACGGAC AATGCTAAAA CCATAATAGT ACAGCTGAAC
ACATCTGTAG 3501 AAATTAATTG TACAAGACCC AACAACAATA CAAGAAAAAG
AATCCGTATC CAGAGAGGAC CAGGGAGAGC ATTTGTTACA ATAGGAAAAA
TAGGAAATAT
3601 GAGACAAGCA CATTGTAACA TTAGTAGAGC AAAATGGAAT AACACTTTAA AACAGATAGC TAGCAAATTA AGAGAACAAT TTGGAAATAA
TAAAACAATA
3701 ATCTTTAAGC AATCCTCAGG AGGGGACCCA GAAATTGTAA
CGCACAGTTT TAATTGTGGA GGGGAATTTT TCTACTGTAA TTCAACACAA
CTGTTTAATA 3801 GTACTTGGTT TAATAGTACT TGGAGTACTG AAGGGTCAAA
TAACACTGAA GGAAGTGACA CAATCACCCT CCCATGCAGA ATAAAACAAA
TTATAAACAT
3901 GTGGCAGAAA GTAGGAAAAG CAATGTATGC CCCTCCCATC
AGTGGACAAA TTAGATGTTC ATCAAATATT ACAGGGCTGC TATTAACAAG AGATGGTGGT
4001 AATAGCAACA ATGAGTCCGA GATCTTCAGA CCTGGAGGAG
GAGATATGAG GGACAATTGG AGAAGTGAAT TATATAAATA TAAAGTAGTA
AAAATTGAAC
4101 CATTAGGAGT AGCACCCACC AAGGCAAAGA GAAGAGTGGT GCAGAGAGAA AAAAGAGCAG TGGGAATAGG AGCTTTGTTC CTTGGGTTCT
TGGGAGCAGC
4201 AGGAAGCACT ATGGGCGCAG CCTCAATGAC GCTGACGGTA
CAGGCCAGAC AATTATTGTC TGGTATAGTG CAGCAGCAGA ACAATTTGCT
GAGGGCTATT 4301 GAGGCGCAAC AGCATCTGTT GCAACTCACA GTCTGGGGCA
TCAAGCAGCT CCAGGCAAGA ATCCTGGCTG TGGAAAGATA CCTAAAGGAT
CAACAGCTCC
4401 TGGGGATTTG GGGTTGCTCT GGAAAACTCA TTTGCACCAC
TGCTGTGCCT TGGAATGCTA GTTGGAGTAA TAAATCTCTG GAACAGATTT GGAATCACAC
4501 GACCTGGATG GAGTGGGACA GAGAAATTAA CAATTACACA
AGCTTAATAC ACTCCTTAAT TGAAGAATCG CAAAACCAGC AAGAAAAGAA
TGAACAAGAA 4601 TTATTGGAAT TAGATAAATG GGCAAGTTTG TGGAATTGGT
TTAACATAAC AAATTGGCTG TGGTATATAA AATTATTCAT AATGATAGTA GGAGGCTTGG
4701 TAGGTTTAAG AATAGTTTTT GCTGTACTTT CTATAGTGAA TAGAGTTAGG CAGGGATATT AACCATTATC GTTCTTAAGA CAATAGAAGA TTGTAAATCA
4801 CGTGAATAAA AGATTTTATT CAGTTTACAG AAAGAGGGGG
GAATGAAAGA CCCCTTCATA AGGCTTAGCC AGCTAACTGC AGTAACGCCA
TΠTGCAAGG 4901 CATGGGAAAA TACCAGAGCT GATGTTCTCA GAAAAACAAG
AACAAGGAAG TACAGAGAGG CTGGAAAGTA CCGGGACTAG GGCCAAACAG
GATATCTGTG
5001 GTCAAGCACT AGGGCCCCGG CCCAGGGCCA AGAACAGATG
GTCCCCAGAA ACAGAGAGGC TGGAAAGTAC CGGGACTAGG GCCAAACAGG ATATCTGTGG
5101 TCAAGCACTA GGGCCCCGGC CCAGGGCCAA GAACAGATGG
TCCCCAGAAA TAGCTAAAAC AACAACAGTT TCAAGAGACC CAGAAACTGT
CTCAAGGTTC
5201 CCCAGATGAC CGGGGATCAA CCCCAAGCCT CATTTAAACT AACCAATCAG CTCGCTTCTC GCTTCTGTAC CCGCGCTTAT TGCTGCCCAG
CTCTATAAAA
5301 AGGGTAAGAA CCCCACACTC GGCGCGCCAG TCCTCCGATA
GACTGAGTCG CCCGGGTACC CGTGTATCCA ATAAAGCCTT TTGCTGTTGC
ATCCGAATCG 5401 TGGTCTCGCT GATCCTTGGG AGGGTCTCCT CCTCTGTCGG
TCGACCTGCA GGCATGCAAG CTTGGCGTAA TCATGGTCAT AGCTGTTTCC
TGTGTGAAAT
5501 TGTTATCCGC TCACAATTCC ACACAACATA CGAGCCGGAA
GCATAAAGTG TAAAGCCTGG GGTGCCTAAT GAGTGAGCTA ACTCACATTA ATTGCGTTGC
5601 GCTCACTGCC CGCTTTCCAG TCGGGAAACC TGTCGTGCCA
GCTGCATTAA TGAATCGGCC AACGCGCGGG GAGAGGCGGT TTGCGTATTG
GGCGCTCTTC 5701 CGCTTCCTCG CTCACTGACT CGCTGCGCTC GGTCGTTCGG
CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC
AGAATCAGGG
5801 GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCG I I I I I CCATAGGCTC
CGCCCCCCTG
5901 ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG
AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC
TCGTGCGCTC 6001 TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC
TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CAATGCTCAC GCTGTAGGTA
TCTCAGTTCG
6101 GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC
CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG
6201 TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA
CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT
GGTGGCCTAA
6301 CTACGGCTAC ACTAGAAGGA CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG
CAAACAAACC
6401 ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA
TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTΠTCTACG
GGGTCTGACG 6501 CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT
GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA
GTTTTAAATC
6601 AATCTAAAGT ATATATGAGT AAACTTGGTC TGACAGTTAC
CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT
6701 GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG
GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA
CCGGCTCCAG 6801 ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG
CAGAAGTGGT CCTGCAACTT TATCCGCCTC CATCCAGTCT ATTAATTGTT
GCCGGGAAGC
6901 TAGAGTAAGT AGTTCGCCAG TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC
TTCATTCAGC
7001 TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA
TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA
AGTAAGTTGG 7101 CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA
TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG ACTGGTGAGT
ACTCAACCAA
7201 GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT
TGCCCGGCGT CAATACGGGA TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC
7301 ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT
TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA
TCTTCAGCAT
7401 CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG AAATGTTGAA
TACTCATACT
7501 CTTCC I I I I I CAAT ATT ATT G AAGCATTTA TCAGGGTTAT
TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA ATAAACAAAT
AGGGGTTCCG 7601 CGCACATTTC CCCGAAAAGT GCCACCTGAC GTCTAAGAAA
CCATTATTAT CATGACATTA ACCTATAAAA ATAGGCGTAT CACGAGGCCC
TTTCGTC
SEQ ID NO.: 4
Vector sequence - NeoHIVMo: Complete nucleotide sequence of the bicistronic vector
NeoHIVMo.
1 TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG 101 TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA
GCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA
201 CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT
301 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT
TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT 401 CTACCTTACG TTTCCCCGAC CAGAGCTGAT GTTCTCAGAA AAACAAGAAC AAGGAAGTAC
AGAGAGGCTG GAAAGTACCG GGACTAGGGC CAAACAGGAT 501 ATCTGTGGTC AAGCACTAGG GCCCCGGCCC AGGGCCAAGA ACAGATGGTC CCCAGAAACA
GAGAGGCTGG AAAGTACCGG GACTAGGGCC AAACAGGATA 601 TCTGTGGTCA AGCACTAGGG CCCCGGCCCA GGGCCAAGAA CAGATGGTCC CCAGAAATAG
CTAAAACAAC AACAGTTTCA AGAGACCCAG AAACTGTCTC
701 AAGGTTCCCC AGATGACCGG GGATCAACCC CAAGCCTCAT TTAAACTAAC CAATCAGCTC GCTTCTCGCT TCTGTACCCG CGCTTATTGC TGCCCAGCTC
801 TATAAAAAGG GTAAGAACCC CACACTCGGC GCGCCAGTCC TCCGATAGAC TGAGTCGCCC
GGGTACCCGT GTATCCAATA AAGCCTTTTG CTGTTGCATC 901 CGAATCGTGG TCTCGCTGAT CCTTGGGAGG GTCTCCTCAG AGTGATTGAC TGCCCAGCCT
GGGGGTCTTT CATTTGGGGG CTCGTCCGGG ATTTGGAGAC 1001 CCCCGCCCAG GGACCACCGA CCCACCGTCG GGAGGTAAGC TGGCCAGCGA TCGTTTTGTC
TCCGTCTCTG TCTTTGTGCG TGTGTGTGTG TGTGCCGGCA 1101 TCTACTTTTT GCGCCTGCGT CTGATTCTGT ACTAGTTAGC TAACTAGATC TGTATCTGGC
GGCTCCGTGG AAGAACTGAC GAGTTCGTAT TCCCGACCGC
1201 AGCCCTGGGA GACGTCTCAG AGGCATCGGG GGCCCGCTGG GTGGCCCAAT CAGTAAGTCC GAGTCCTGAC CGATTCGGAC TATTTGGGGC CCCTCCTTTG
1301 TCGGAGGGGT ACGTGGTTCT TTTAGGAGAC GAGAGGTCCA AGCCCTCGCC GCCTCCATCT
GAATTTTTGC TTTCGGTTTT TCGCCGAAAC CGCGCCGCGC 1401 GTCTTGTCTG TCTCAGTATT GTTTTGTCAT TTGTCTGTTC GTTATTGTTT TGGACCGCTT
CTAAAAACGG ATCCGTCGAC CTGCAGCCAA GCTTCACGCT 1501 GCCGCAAGCA CTCAGGGCGC AAGGGCTGCT AAAGGAAGCG GAACACGTAG AAAGCCAGTC
CGCAGAAACG GTGCTGACCC CGGATGAATG TCAGCTACTG
1601 GGCTATCTGG ACAAGGGAAA ACGCAAGCGC AAAGAGAAAG CAGGTAGCTT GCAGTGGGCT TACATGGCGA TAGCTAGACT GGGCGGTTTT ATGGACAGCA 1701 AGCGAACCGG AATTGCCAGC TGGGGCGCCC TCTGGTAAGG TTGGGAAGCC CTGCAAAGTA
AACTGGATGG CTTTCTTGCC GCCAAGGATC TGATGGCGCA 1801 GGGGATCTGA TCAAGAGACA GGATGAGGAT CGTTTCGCAT GATTGAACAA GATGGATTGC
ACGCAGGTTC TCCGGCCGCT TGGGTGGAGA GGCTATTCGG 1901 CTATGACTGG GCACAACAGA CAATCGGCTG CTCTGATGCC GCCGTGTTCC GGCTGTCAGC
GCAGGGGCGC CCGGTTCTTT TTGTCAAGAC CGACCTGTCC 2001 GGTGCCCTGA ATGAACTGCA GGACGAGGCA GCGCGGCTAT CGTGGCTGGC CACGACGGGC
GTTCCTTGCG CAGCTGTGCT CGACGTTGTC ACTGAAGCGG
2101 GAAGGGACTG GCTGCTATTG GGCGAAGTGC CGGGGCAGGA TCTCCTGTCA TCTCACCTTG CTCCTGCCGA GAAAGTATCC ATCATGGCTG ATGCAATGCG
2201 GCGGCTGCAT ACGCTTGATC CGGCTACCTG CCCATTCGAC CACCAAGCGA AACATCGCAT
CGAGCGAGCA CGTACTCGGA TGGAAGCCGG TCTTGTCGAT 2301 CAGGATGATC TGGACGAAGA GCATCAGGGG CTCGCGCCAG CCGAACTGTT CGCCAGGCTC
AAGGCGCGCA TGCCCGACGG CGAGGATCTC GTCGTGACCC 2401 ATGGCGATGC CTGCTTGCCG AATATCATGG TGGAAAATGG CCGCTTTTCT GGATTCATCG
ACTGTGGCCG GCTGGGTGTG GCGGACCGCT ATCAGGACAT 2501 AGCGTTGGCT ACCCGTGATA TTGCTGAAGA GCTTGGCGGC GAATGGGCTG ACCGCTTCCT
CGTGCTTTAC GGTATCGCCG CTCCCGATTC GCAGCGCATC
2601 GCCTTCTATC GCCTTCTTGA CGAGTTCTTC TGAGCGGGAC TCTGGGGTTC GAAGGCCAAT TGGGCCACCG GTGCTAGCCC CCTAACGTTA CTGGCCGAAG
2701 CCGCTTGGAA TAAGGCCGGT GTGCGTTTGT CTATATGTTA TTTTCCACCA TATTGCCGTC
TTTTGGCAAT GTGAGGGCCC GGAAACCTGG CCCTGTCTTC 2801 TTGACGAGCA TTCCTAGGGG TCTTTCCCCT CTCGCCAAAG GAATGCAAGG TCTGTTGAAT
GTCGTGAAGG AAGCAGTTCC TCTGGAAGCT TCTTGAAGAC 2901 AAACAACGTC TGTAGCGACC CTTTGCAGGC AGCGGAACCC CCCACCTGGC GACAGGTGCC
TCTGCGGCCA AAAGCCACGT GTATAAGATA CACCTGCAAA 3001 GGCGGCACAA CCCCAGTGCC ACGTTGTGAG TTGGATAGTT GTGGAAAGAG TCAAATGGCT
CTCCTCAAGC GTATTCAACA AGGGGCTGAA GGATGCCCAG
3101 AAGGTACCCC ATTGTATGGG ATCTGATCTG GGGCCTCGGT GCACATGCTT TACATGTGTT TAGTCGAGGT TAAAAAACGT CTAGGCCCCC CGAACCACGG
3201 GGACGTGGTT TTCCTTTGAA AAACACGATA ATACCATGAG AGTGAAGGAG AAATATCAGC
ACTTGTGGAG ATGGGGGTGG AGATGGGGCA CCATGCTCCT 3301 TGGGATGTTG ATGATCTGTA GTGCTACAGA AAAATTGTGG GTCACAGTCT ATTATGGGGT
ACCTGTGTGG AAGGAAGCAA CCACCACTCT ATTTTGTGCA 3401 TCAGATGCTA AAGCATATGA TACAGAGGTA CATAATGTTT GGGCCACACA TGCCTGTGTA
CCCACAGACC CCAACCCACA AGAAGTAGTA TTGGTAAATG 3501 TGACAGAAAA TTTTAACATG TGGAAAAATG ACATGGTAGA ACAGATGCAT GAGGATATAA
TCAGTTTATG GGATCAAAGC CTAAAGCCAT GTGTAAAATT 3601 AACCCCACTC TGTGTTAGTT TAAAGTGCAC TGATTTGAAG AATGATACTA ATACCAATAG
TAGTAGCGGG AGAATGATAA TGGAGAAAGG AGAGATAAAA 3701 AACTGCTCTT TCAATATCAG CACAAGCATA AGAGGTAAGG TGCAGAAAGA ATATGCATTT
TTTTATAAAC TTGATATAAT ACCAATAGAT AATGATACTA 3801 CCAGCTATAA GTTGACAAGT TGTAACACCT CAGTCATTAC ACAGGCCTGT CCAAAGGTAT
CCTTTGAGCC AATTCCCATA CATTATTGTG CCCCGGCTGG 3901 TTTTGCGATT CTAAAATGTA ATAATAAGAC GTTCAATGGA ACAGGACCAT GTACAAATGT
CAGCACAGTA CAATGTACAC ATGGAATTAG GCCAGTAGTA
4001 TCAACTCAAC TGCTGTTAAA TGGCAGTCTA GCAGAAGAAG AGGTAGTAAT TAGATCTGTC AATTTCACGG ACAATGCTAA AACCATAATA GTACAGCTGA
4101 ACACATCTGT AGAAATTAAT TGTACAAGAC CCAACAACAA TACAAGAAAA AGAATCCGTA
TCCAGAGAGG ACCAGGGAGA GCATTTGTTA CAATAGGAAA 4201 AATAGGAAAT ATGAGACAAG CACATTGTAA CATTAGTAGA GCAAAATGGA ATAACACTTT
AAAACAGATA GCTAGCAAAT TAAGAGAACA ATTTGGAAAT 4301 AATAAAACAA TAATCTTTAA GCAATCCTCA GGAGGGGACC CAGAAATTGT AACGCACAGT
TTTAATTGTG GAGGGGAATT TTTCTACTGT AATTCAACAC 4401 AACTGTTTAA TAGTACTTGG TTTAATAGTA CTTGGAGTAC TGAAGGGTCA AATAACACTG
AAGGAAGTGA CACAATCACC CTCCCATGCA GAATAAAACA
4501 AATTATAAAC ATGTGGCAGA AAGTAGGAAA AGCAATGTAT GCCCCTCCCA TCAGTGGACA AATTAGATGT TCATCAAATA TTACAGGGCT GCTATTAACA
4601 AGAGATGGTG GTAATAGCAA CAATGAGTCC GAGATCTTCA GACCTGGAGG AGGAGATATG
AGGGACAATT GGAGAAGTGA ATTATATAAA TATAAAGTAG 4701 TAAAAATTGA ACCATTAGGA GTAGCACCCA CCAAGGCAAA GAGAAGAGTG GTGCAGAGAG
AAAAAAGAGC AGTGGGAATA GGAGCTTTGT TCCTTGGGTT 4801 CTTGGGAGCA GCAGGAAGCA CTATGGGCGC AGCCTCAATG ACGCTGACGG TACAGGCCAG
ACAATTATTG TCTGGTATAG TGCAGCAGCA GAACAATTTG 4901 CTGAGGGCTA TTGAGGCGCA ACAGCATCTG TTGCAACTCA CAGTCTGGGG CATCAAGCAG
CTCCAGGCAA GAATCCTGGC TGTGGAAAGA TACCTAAAGG
5001 ATCAACAGCT CCTGGGGATT TGGGGTTGCT CTGGAAAACT CATTTGCACC ACTGCTGTGC CTTGGAATGC TAGTTGGAGT AATAAATCTC TGGAACAGAT
5101 TTGGAATCAC ACGACCTGGA TGGAGTGGGA CAGAGAAATT AACAATTACA CAAGCTTAAT
ACACTCCTTA ATTGAAGAAT CGCAAAACCA GCAAGAAAAG 5201 AATGAACAAG AATTATTGGA ATTAGATAAA TGGGCAAGTT TGTGGAATTG GTTTAACATA
ACAAATTGGC TGTGGTATAT AAAATTATTC ATAATGATAG 5301 TAGGAGGCTT GGTAGGTTTA AGAATAGTTT TTGCTGTACT TTCTATAGTG AATAGAGTTA
GGCAGGGATA TTAACCATTA TCGTTCTTAA GACAATAGAA
5401 GATTGTAAAT CACGTGAATA AAAGATTTTA TTCAGTTTAC AGAAAGAGGG GGGAATGAAA GACCCCTTCA TAAGGCTTAG CCAGCTAACT GCAGTAACGC 5501 CATTTTGCAA GGCATGGGAA AATACCAGAG CTGATGTTCT CAGAAAAACA AGAACAAGGA
AGTACAGAGA GGCTGGAAAG TACCGGGACT AGGGCCAAAC 5601 AGGATATCTG TGGTCAAGCA CTAGGGCCCC GGCCCAGGGC CAAGAACAGA TGGTCCCCAG
AAACAGAGAG GCTGGAAAGT ACCGGGACTA GGGCCAAACA 5701 GGATATCTGT GGTCAAGCAC TAGGGCCCCG GCCCAGGGCC AAGAACAGAT GGTCCCCAGA
AATAGCTAAA ACAACAACAG TTTCAAGAGA CCCAGAAACT 5801 GTCTCAAGGT TCCCCAGATG ACCGGGGATC AACCCCAAGC CTCATTTAAA CTAACCAATC
AGCTCGCTTC TCGCTTCTGT ACCCGCGCTT ATTGCTGCCC
5901 AGCTCTATAA AAAGGGTAAG AACCCCACAC TCGGCGCGCC AGTCCTCCGA TAGACTGAGT CGCCCGGGTA CCCGTGTATC CAATAAAGCC TTTTGCTGTT
6001 GCATCCGAAT CGTGGTCTCG CTGATCCTTG GGAGGGTCTC CTCCTCTGTC GGTCGACCTG
CAGGCATGCA AGCTTGGCGT AATCATGGTC ATAGCTGTTT 6101 CCTGTGTGAA ATTGTTATCC GCTCACAATT CCACACAACA TACGAGCCGG AAGCATAAAG
TGTAAAGCCT GGGGTGCCTA ATGAGTGAGC TAACTCACAT 6201 TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC CAGCTGCATT
AATGAATCGG CCAACGCGCG GGGAGAGGCG GTTTGCGTAT 6301 TGGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG
AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC
6401 ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC
6501 TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA
CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC 6601 CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC
TTCGGGAAGC GTGGCGCTTT CTCAATGCTC ACGCTGTAGG 6701 TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT
CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG 6801 AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA
GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA
6901 GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC
7001 GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC
AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA 7101 CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT
CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG 7201 AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT
AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT 7301 TCATCCATAG TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA
TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT 7401 CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG
GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG 7501 TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT
TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG 7601 GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC
AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA 7701 GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT AATTCTCTTA
CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA
7801 GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA
7901 AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG
TTGAGATCCA GTTCGATGTA ACCCACTCGT GCACCCAACT 8001 GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA GGAAGGCAAA
ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG 8101 AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT
GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA 8201 ATAGGGGTTC CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT
ATCATGACAT TAACCTATAA AAATAGGCGT ATCACGAGGC 8301 CCTTTCGTC
SEQ ID NO.: 5
HIV-1 envelope amino acid sequence (from reference strain HXB3)
mrvkekyqhlwrwgwrwgtmllgmlmicsateklwvtvyygvpvwkeatttlfcasdakaydtevhnvwathagvpt dpnpqewlvnvtenfnmwkndmveqmhediislwdqslkpcvkltplcvslkctdlkndtntnsssgrmimekgeik ncsfnistsirgkvqkeyaffykldiipidndttsytltscntsvitqacpkvsfepipihycapagfailkcnnktfngtgpctnv stvqcthgirpwstqlllngslaeeewirsvnfldnaktiivqlntsveinctrpnnntrkkiriqrgpgrafvtigkignmrqah cnisrakwnatlkqiasklreqfgnnktiifkqssggdpeivthsfncggeffycnstqlfnstwfnstwstegsnntegsdtitl pcrikqfinmwqevgkamyappisgqircssnitgllltrdggnnnngseifrpgggdmrdnwrselykykwkieplgv aptkakrrwqrekravgigalflgflgaagstmgaasmtltvqarqllsgivqqqnnllraieaqqhllqltvwgikqlqarila verylkdqqllgiwgcsgkllcttavpwnaswsnksleqiwnhttwmewdreinnytslihslieesqnqqekneqellel dkwaslwnwfnitnwlwyiklfimivgglvglrivfavlswnrvrqgysplsfqthlpiprgpdrpegieeeggerdrdrsirlv ngslaliwddlrslclfsyhrlrdlllivtrivellgrrgwealkywwnllqywsqelknsavsllnataiavaegtdrviewqeay rairhiprrirqgleriH*
SEQ ID NO.: 6
C-terminally truncated HIV-1 envelope (HIV-1 delta-CT) amino acid sequence (from reference strain HXB3) Mrvkekyqhlwn/vgwrwgtmllgmlmicsateklwvtvyygvpvwkeatttlfcasdakaydtevhnvwathagvpt dpnpqevvlvnvtenfnmwkndrnveqmhediislwdqslkpcvkltplcvslkctdlkndtntnssgrmimekgeikn csfnistsirgkvqkeyaffykldiipidndttsytltscntsvitqacpkvsfepipihycapagfailkcnnktfngtgpctnvst vqcthgirpwstqlllngslaeeewirsvnftdnaktiivqlntsveinctrpnnntrkkiriqrgpgrafvtigkignmrqahc nisrakwnatlkqiasklreqfgnnktiifkqssggdpeivthsfncggeffycnstqlfnstwfnstwstegsnntegsdtitl pcrikqfinmwqevgkamyappisgqircssnitgllltrdggnnnngseifrpgggdmrdnwrselykykwkieplgv aptkakrrwqrekravgigalflgflgaagstmgaasmtltvqarqllsgivqqqnnllraieaqqhllqltvwgikqlqarila verylkdqqllgiwgcsgkllcttavpwnaswsnksleqiwnhttwmewdreinnytslihslieesqnqqekneqellel dkwaslwnwfnitnwlwyiklfimivgglvglrivfavlswnrvrqgy
SEQ ID NO.: 7
IRES sequence from elF4G. Sequence 1-357 of the mRNA 5' UTR.
tctagatggg ggtcctgggc cccagggtgt gcagccactg acttggggac tgctggtggg gtagggatga gggagggagg ggcattgtga tgtacagggc tgctctgtga gatcaagggt ctcttaaggg tgggagctgg ggcagggact acgagagcag ccagatgggc tgaaagtgga actcaagggg tttctggcac ctacctacct gcttcccgct ggggggtggg gagttggccc agagtcttaa gattggggca gggtggagag gtgggctctt cctgcttccc actcatctta tagctttctt tccccagatc cgaattcgag atccaaacca aggaggaaag gatatcac
Complete mRNA sequence GenBank ace nr . D12686

Claims

Claims
1. A mammalian expression vector comprising at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof.
2. The mammalian expression vector according to claim 1 , wherein said vector comprise an intron.
3. The vector according to any of the preceding claims, which is transcribed in the cytoplasm, thereby producing high levels of transcript, which can be translated into envelope polypeptide.
4. The vector according to any of the preceding claims, wherein said vector comprises a constitutive transport element (CTE).
5. The vector according to any of the preceding claims, wherein said vector comprises a Rev responsive element (RRE).
6. The vector according to claim 1 , wherein said vector is a retroviral vector
7. The vector according to claim 5, wherein said vector is a replication deficient retroviral vector.
8. The vector according to claim 5, wherein said vector is a replication competent retroviral vector.
9. The vector according to any of the preceding claims, further comprising at least one additional nucleic acid sequence and at least one internal ribosomal entry site (IRES).
10. The vector according to any of the preceding claims, wherein said at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or fragment thereof is preceded by an IRES.
11. The vector according to any of the preceding claims, wherein said at least one additional nucleic acid sequence is preceded by an IRES.
12. The vector according to any of the preceding claims , wherein the vector is derived from gamma-retroviruses.
13. The vector according to claim 12, wherein the vector is derived from Murine Leukemia Virus (MLV).
14. The vector according to claim 13, wherein the vector is derived from Moloney Murine Leukemia Virus (MoMLV).
15. The vector according to claim 13, wherein the vector is derived from Akv MLV.
16. The vector according to any of the preceding claims, wherein the at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof is selected from the nucleic acid sequences encoding envelope polypeptides derived from HIV-1 , HIV-2, or SIV.
17. The vector according to any of the preceding claims, wherein the at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof encodes HIV-1 envelope as defined in SEQ ID NO.: 1 , or a fragment thereof.
18. The vector according to any of the preceding claims, wherein the at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof encodes a C-terminally truncated HIV-1 envelope as defined in SEQ ID NO.: 2, or a fragment thereof.
19. The vector according to any of the preceding claims, wherein the at least one heterologous nucleic acid sequence encoding a lentiviral envelope polypeptide or a fragment thereof encodes an SIV envelope.
20. The vector according to any of the preceding claims, wherein said at least one additional nucleic acid sequence is a reporter gene.
21. The vector according to claim 20, wherein said reporter gene encodes enhanced green fluorescent protein (eGFP), lac Z, dsRed, enhanced yellow fluorescent protein (eYFP), enhanced cyan fluorescent protein (eCFP), enhanced blue fluorescent protein (eBFP) and the human alpha-1 -antitrypsin (hAAT). It is understood that any of the enhanced green fluorescent protein
(eGFP), lac Z, dsRed, enhanced yellow fluorescent protein (eYFP), enhanced cyan fluorescent protein (eCFP), enhanced blue fluorescent protein (eBFP) or the human alpha-1 -antitrypsin (hAAT).
22. The vector according to any of the preceding claims, wherein said at least one additional nucleic acid sequence encodes a selective marker.
23. The vector according to claim 22, wherein said selective marker is neomycin phosphotransferase II.
24. The vector according to any of the preceding claims, wherein said at least one additional nucleic acid sequence encodes a suicide gene.
25. The vector according to any of the preceding claims, wherein said at least one additional nucleic acid sequence encodes an immunomodulating polypeptide or peptide.
26. The vector according to claim 25, wherein said immunomodulating polypeptide is an immunostimulating polypeptide.
27. The vector according to claim 25, wherein said immunomodulating polypeptide is an genetic adjuvant.
28. The vector according to claim 26, wherein said immunostimulating polypeptide is selected from cytokines or hormones.
29. The vector according to any of the preceding claims, wherein said IRES is selected from the IRES elements of picornaviridae, retroviridae or retrotransposons, mammalia or combinations thereof.
30. The vector according to claim 29, wherein said IRES is selected from the IRES elements of picornavirus
31. The vector according to claim 29, wherein said IRES is selected from the IRES elements of encephalomyocarditis (ECMV).
32. The vector according to any of the preceding claims 5, wherein said IRES is located in a region flanked by the 3'-LTR and the 5'-LTR.
33. The vector according to any of the preceding claims, wherein said IRES is located in the 3'-Long Terminal Repeat (LTR) or the 5'-LTR.
34. The vector according to claim 33, wherein said IRES is located in the U3 region of the 3' LTR.
35. The vector according to claim 33, wherein said IRES is located in the LJ3 region between the inverted repeats and the transcription regulatory elements.
36. The vector according to any of the preceding claims, wherein said encoded lentiviral envelope is capable of inducing an immunogenic response in a host animal.
37. The vector according to claim 36, wherein said immunogenic response is an antibody response and/or cytotoxic T Lymphocyte (CTL) response.
38. The vector according to claim 36 or 37, wherein said immunogenic response is directed against a retroviral particle expressing said lentiviral envelope.
39. The vector according to any of the preceding claims, wherein said lentiviral envelope is incorporated in said retroviral particle.
40. The vector according to claim 36, wherein said immunogenic response is a CTL response, and wherein said vector is integrated into the genome of a host cell.
41. An RNA of the vector according to any of the preceding claims 1 to 40.
42. A retroviral provirus produced in a target cell upon reverse transcription of the RNA according to claim 41.
43. An RNA of the retroviral provirus according to claim 42.
44. A retroviral particle comprising an RNA as defined in claim 41 , or part thereof.
45. The retroviral particle according to claim 44 obtained by transfection of a producer cell with the vector according to claims 1 to 40 or the RNA according to claim 41 , or part thereof.
46. The retroviral particle according to claim 44 obtained by transfection of a semipackaging cell with the vector according to claims 1 to 40 or the RNA according to claim 41 , or part thereof.
47. A retroviral particle comprising a lentiviral envelope or fragment thereof as defined in any of claims 16 to 19.
48. The retroviral particle according to any of claims 44 to 47, wherein said lentiviral envelope polypeptide or fragment thereof is expressed on the surface of the retroviral particle.
49. The retroviral particle according to any of claims 44 to 48, wherein said lentiviral envelope polypeptide or fragment thereof comprises gal-alfa1-3Galbeta1- 4GIcNAc-R epitopes on the envelope protein.
50. The retroviral particle according to any of claims 44 to 49, wherein said retroviral particle comprises vesicular stomatitis virus envelope (VSV-G), the amphotropic murine leukemia virus envelope, Mutated SL3-2 envelope, Xenotropic murine leukaemia virus envelope, 10A1 virus envelope, Hepatitis virus C envelope, gibbon ape leukaemia virus, Human T-cell lymphotropic virus, or envelopes of endogenous human retroviruses.
51. The retroviral particle according to any of claims 44 to 50, which is not capable of mediating fusion of said retroviral particle and cells expressing receptors for HIV.
52. The retroviral particle according to any of claims 44 to 50, which is capable of mediating fusion of said retroviral particle and cells expressing receptors for
HIV.
53. The retroviral particle according to any of claims 44 to 52, wherein said retroviral particle is capable of infecting human cells.
54. The retroviral particle according to any of claims 44 to 53, wherein said retroviral particle is capable of infecting stem cells.
55. The retroviral particle according to any of claims 44 to 54, wherein said retroviral particle is capable of infecting a CD4 positive cell.
56. The retroviral particle according to any of claims 44 to 55, wherein said retroviral particle is capable of inducing an immunogenic response in a host animal.
57. The retroviral particle according to any of claims 44 to 56, wherein said immunogenic response is directed towards said retroviral particle in said host animal.
58. The retroviral particle according to any of claims 44 to 57, wherein said host animal is a human being.
59. A producer cell comprising a vector according to any one of preceding claims 1 to 40.
60. The producer cell according to claim 59, wherein said producer cell does not comprise lentiviral tat and/or rev and/or rex originating from HTLV.
61. A host cell comprising a vector according to any one of preceding claims 1 to 40.
62. A host cell infected with a particle according to claims 44 to 58.
63. A pharmaceutical composition comprising a therapeutically effective amount of the vector according to any of the preceding claims 1 to 40, the producer cell according to claim 59 or 60, a retroviral particle according to any of preceding claims 44 to 58 and/or a host cell according to any of preceding claims 61 to 62.
64. A method for introducing a nucleotide sequence into target cells, said method comprising infection of target cells with a retroviral particle according to any of claims 44 to 58.
65. The method according to claim 64 for the production of transgenic animals, said method comprising infection or transduction of embryonic stem cells with said retroviral particles or the vector according to any of the preceding claims 1 to 40.
66. A vaccine for the prophylaxis or treatment of lentiviral infection comprising the vector according to any of the preceding claims 1 to 40, the RNA according to claim 41 or 43, the retroviral provirus according to claim 42 and/or a retroviral particle according to any of preceding claims 44 to 58.
67. The vaccine according to claim 66, wherein said lentiviral infection is HIV infection.
68. A vaccine composition comprising a. a lentiviral envelope polypeptide or a fragment, and b. an adjuvant and/or an immunomodulating peptide.
69. The vaccine composition according to claim 68, wherein said lentiviral envelope polypeptide is as defined in any of claims 16 to 19.
70. The vaccine composition according to claim 68, wherein said envelope polypeptide is comprised in a retroviral particle according to any of claims 44 to 58.
71. The vaccine composition according to claim 68, wherein said adjuvant is selected from the group consisting of AIK(SO4)2, AINa(SO4)2, AINH4 (SO4), silica, alum, AI(OH)3, Ca3 (PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl- nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-sn - glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80.RTM. emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes or other lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, lnterleukin 1 , lnterleukin 2, Montanide ISA-51 and QS-21. Preferred adjuvants to be used with the invention include oil/surfactant based adjuvants such as Montanide adjuvants (available from Seppic, Belgium), preferably Montanide ISA-51.
72. A vaccine composition according to claim 68 for treatment, amelioration and/or prevention of HIV infection and/or AIDS.
73. Use of the vaccine composition according to any of claims 68 to 71 for the manufacture of a medicament for the treatment of HIV infection and/or AIDS.
74. A vaccine composition comprising the vaccine as defined in claim 66 for use as a medicament.
75. The composition of claim 68, wherein the vaccine composition when administered to an animal including a human being, is capable of eliciting an immune response against a disease caused by lentivirus.
76. A kit comprising a therapeutically effective amount of the vector according to any of the preceding claims 1 to 40, the producer cell according to claim 59, a retroviral particle according to any of preceding claims 42 and 44 to 58 and/or a host cell according to any of preceding claims 61 to 62.
77. Use of the vector according to any of the preceding claims 1 to 40, a producer cell according to claim 59 or 60, a retroviral particle according to any of preceding claims 44 to 58, a host cell according to claim 61 or 62 and/or a vaccine composition according to claim 74 or 75 for the manufacture of a medicament for gene therapy.
78. Use of the vector according to any of the preceding claims 1 to 40, a producer cell according to claim 59 or 60, a retroviral particle according to any of preceding claims 44 to 58, a host cell according to claim 61 or 62 and/or a vaccine composition according to claim 74 or 75 for the manufacture of a medicament for immune therapy.
79. The use according to claim 77 or 78 for the manufacture of a medicament for the treatment, amelioration or prevention of HIV and/or AIDS.
80. The use according to any of claims 77 to 78 for the manufacture of a medicament for HIV vaccination.
PCT/DK2008/000217 2007-06-15 2008-06-13 Vectors for vaccines against lentivirus infections WO2008151633A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DKPA200700861 2007-06-15
DKPA200700861 2007-06-15

Publications (2)

Publication Number Publication Date
WO2008151633A2 true WO2008151633A2 (en) 2008-12-18
WO2008151633A3 WO2008151633A3 (en) 2009-04-23

Family

ID=39816797

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2008/000217 WO2008151633A2 (en) 2007-06-15 2008-06-13 Vectors for vaccines against lentivirus infections

Country Status (1)

Country Link
WO (1) WO2008151633A2 (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103520717A (en) * 2013-09-02 2014-01-22 中山大学 Application of alanine serving as vaccine adjuvant
US8652460B2 (en) 1998-10-01 2014-02-18 University Of Southern California Gene delivery system and method of use
US8829173B2 (en) 2008-09-26 2014-09-09 Tocagen Inc. Recombinant vectors
US9642921B2 (en) 2012-12-20 2017-05-09 Tocagen Inc. Cancer combination therapy and recombinant vectors
US9669049B2 (en) 2010-10-31 2017-06-06 Tocagen Inc. Compositions comprising gamma retrovirus vectors and methods of treating proliferative disorders
US10035983B2 (en) 2008-09-26 2018-07-31 Tocagen Inc. Gene therapy vectors and cytosine deaminases
CN111263816A (en) * 2017-10-23 2020-06-09 吴云涛 Lentiviral-dependent vaccine particles reduce viral rebound and viral stocks in vivo
US11065311B2 (en) 2012-10-25 2021-07-20 Denovo Biopharma Llc Retroviral vector with mini-promoter cassette
US11279949B2 (en) 2015-09-04 2022-03-22 Denovo Biopharma Llc Recombinant vectors comprising 2A peptide

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999020742A2 (en) * 1997-10-20 1999-04-29 Universita' Degli Studi Di Padova A packaging cell line producing siv-pseudotyped mlv

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BABIUK L A ET AL: "Induction of immune responses by DNA vaccines in large animals" VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 21, no. 7-8, 30 January 2003 (2003-01-30), pages 649-658, XP004401599 ISSN: 0264-410X *
CAVER T E ET AL: "A novel vaccine regimen utilizing DNA, vaccinia virus and protein immunizations for HIV-1 envelope presentation" VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 17, no. 11-12, 1 March 1999 (1999-03-01), pages 1567-1572, XP004158286 ISSN: 0264-410X *
HAMMARSKJOELD MARIE-LOUISE ET AL: "Human immunodeficiency virus env expression becomes rev-independent if the env region is not defined as an intron" JOURNAL OF VIROLOGY, vol. 68, no. 2, 1994, pages 951-958, XP002500726 ISSN: 0022-538X *
JIAN-MING X ET AL: "Immunogenic potential of rgp120 from African HIV-1 subtype A" VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 14, no. 10, 1 July 1996 (1996-07-01), pages 993-1000, XP004057631 ISSN: 0264-410X *
WANG ET AL: "Relative contributions of codon usage, promoter efficiency and leader sequence to the antigen expression and immunogenicity of HIV-1 Env DNA vaccine" VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 24, no. 21, 22 May 2006 (2006-05-22), pages 4531-4540, XP005463444 ISSN: 0264-410X *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8652460B2 (en) 1998-10-01 2014-02-18 University Of Southern California Gene delivery system and method of use
US8741279B2 (en) 1998-10-01 2014-06-03 University Of Southern California Gene delivery system and methods of use
US10035983B2 (en) 2008-09-26 2018-07-31 Tocagen Inc. Gene therapy vectors and cytosine deaminases
US8829173B2 (en) 2008-09-26 2014-09-09 Tocagen Inc. Recombinant vectors
US10407666B2 (en) 2008-09-26 2019-09-10 Tocagen Inc. Recombinant vectors
US9669049B2 (en) 2010-10-31 2017-06-06 Tocagen Inc. Compositions comprising gamma retrovirus vectors and methods of treating proliferative disorders
US11065311B2 (en) 2012-10-25 2021-07-20 Denovo Biopharma Llc Retroviral vector with mini-promoter cassette
US9642921B2 (en) 2012-12-20 2017-05-09 Tocagen Inc. Cancer combination therapy and recombinant vectors
CN103520717A (en) * 2013-09-02 2014-01-22 中山大学 Application of alanine serving as vaccine adjuvant
CN103520717B (en) * 2013-09-02 2015-11-18 中山大学 Alanine is as the application of vaccine adjuvant
US11279949B2 (en) 2015-09-04 2022-03-22 Denovo Biopharma Llc Recombinant vectors comprising 2A peptide
CN111263816A (en) * 2017-10-23 2020-06-09 吴云涛 Lentiviral-dependent vaccine particles reduce viral rebound and viral stocks in vivo
US11850281B2 (en) 2017-10-23 2023-12-26 George Mason Research Foundation, Inc. Rev-dependent lentiviral vaccine particles for reducing viral rebound and viral reservoirs in vivo

Also Published As

Publication number Publication date
WO2008151633A3 (en) 2009-04-23

Similar Documents

Publication Publication Date Title
CN111593073B (en) Double-reporter gene framework vector, four-plasmid pseudovirus packaging system and new packaging corolla pneumonia pseudovirus
WO2008151633A2 (en) Vectors for vaccines against lentivirus infections
ES2388527T3 (en) HIV vaccines based on multiple HIV clade Env
DK2346995T3 (en) GENETERIVE VECTORS AND CYTOSINDEAMINASES
DK175857B1 (en) Recombinant retrovirus
US9598469B2 (en) HIV-1 south african subtype C env proteins
US7618642B2 (en) Expression cassettes encoding modified human immunodeficiency virus type 1 subtype C envelope glycoproteins
KR20220125332A (en) Compositions and methods for targeting PCSK9
JP2004508064A (en) Enhanced first generation adenovirus vaccine expressing codon-optimized HIV1-GAG, POL, NEF and modifications
US20020168346A1 (en) Novel lentiviral packaging cells
JP2003523721A (en) Polynucleotides encoding antigenic HIVC-type polypeptides, polypeptides, and uses thereof
US20080095833A1 (en) Polynucleotides encoding antigenic hiv type b polypeptides, polypeptides, and uses thereof
JP2009082136A (en) Polynucleotides encoding antigenic type B HIV polypeptides and / or antigenic type C HIV polypeptides, polypeptides thereof and uses thereof
JP2004535190A (en) Molecular clones having mutated HIV gag / pol, SIV gag and SIV env genes
WO2021236325A1 (en) Covid-19 vaccine using non-integrating lentiviral vectors
AU2014365777A1 (en) Cancer immunotherapy by delivering class II MHC antigens using a VLP-replicon
US20030194800A1 (en) Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof
JP4627950B2 (en) Molecular clone with mutant HIV gag / pol, SIV gag and SIV env genes
CN111065408A (en) immunogenic composition
US20060165664A1 (en) Method of inducing an enhanced immune response against hiv
JP2006503800A (en) Methods for inducing an enhanced immune response against HIV
EP4132590A1 (en) Modified vectors for production of retrovirus
Baranick Characterization and utilization of simple replication-competent retroviral vectors
ZA200400793B (en) Polynucleotides encodin antigenic HIV type C polypeptides and uses thereof.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08758226

Country of ref document: EP

Kind code of ref document: A2

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08758226

Country of ref document: EP

Kind code of ref document: A2

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载