WO1995004546A1 - Nouveaux virus de l'immunodeficience humaine et simienne a mutation et vaccins contenant lesdits virus - Google Patents

Nouveaux virus de l'immunodeficience humaine et simienne a mutation et vaccins contenant lesdits virus Download PDF

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WO1995004546A1
WO1995004546A1 PCT/FI1994/000335 FI9400335W WO9504546A1 WO 1995004546 A1 WO1995004546 A1 WO 1995004546A1 FI 9400335 W FI9400335 W FI 9400335W WO 9504546 A1 WO9504546 A1 WO 9504546A1
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rev
hiv
siv
gene
virus
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PCT/FI1994/000335
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Kai Juhani Ernst Krohn
Einari Aavik
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Kai Juhani Ernst Krohn
Einari Aavik
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Priority to AU72639/94A priority Critical patent/AU7263994A/en
Publication of WO1995004546A1 publication Critical patent/WO1995004546A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—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

Definitions

  • Novel mutated human and simian immunodeficiency viruses and vaccines containing said viruses are novel mutated human and simian immunodeficiency viruses and vaccines containing said viruses
  • the present invention relates to novel human and simian immunodeficiency viruses (HIVs and SIVs, respect- ively) having at least one mutation in the rev (the regu ⁇ lator of virion-protein expression) gene of HIV or SIV genome rendering them replication deficient for the rev gene, which can be transcomplemented.
  • HIVs and SIVs novel human and simian immunodeficiency viruses
  • the present invent ⁇ ion further relates to methods for producing said mutated viruses, vaccines containing said live, attenuated virus ⁇ es, and methods of prevention and/or treatment of HIV and/or SIV infections or acquired immunodeficiency syn ⁇ drome (AIDS) and related diseases in primates, including, humans by administering a vaccine containing said mutated virus to confer protection against a virulent wild type HIV and/or SIV to an individual in the need thereof.
  • AIDS acquired immunodeficiency syn ⁇ drome
  • the efficiency of these vaccine preparations can, however, be questioned: the major problem in the develop ⁇ ment of HIV vaccine is the high degree of variability in the virus, especially in the viral envelope protein region and, in particular, in those parts of the envelope protein against which the protective antibody mediated immune re ⁇ sponse (i.e. neutralizing antibodies) is directed.
  • the results of the vaccine studies performed both in humans and in monkeys confirm this.
  • CTLs cytotoxic T lympho ⁇ cytes
  • SIVs (SIV agm and SIV ⁇ groups), which are simian equivalents to HIV, are useful and have been employed as an animal model in the development of a vaccine against AIDS.
  • SIV mac which in e.g. monkeys of Macaca mulata and Macaca fascicularis species causes a disease equal to AIDS in humans, have proved wrong.
  • SIV mac simian immunodeficiency virus
  • Recent results indicate that the protective effect on SIV ⁇ ac in ⁇ fection observed in monkeys vaccinated with a viral envel ⁇ ope protein (Schafferman, A. et al., Proc. Natl. Sci. USA 88: 7126-7130, 1991; Hu, S.-L.
  • the attenuated SIV vaccine preparation used was a cloned SIV mac 239 which contained a deletion in one of the regulatory genes of SIV, namely nef (negative regulatory factor) gene, one function of which apparently being the slowing down of the transcription of the viral genome.
  • nef negative regulatory factor
  • a non-functional NEF can not be readily transcomplemented with genes that do not carry the possibility of recombination.
  • the rev gene based on mutations introduced in another regu ⁇ latory gene, the rev gene, the defective rev function can be transcomplemented with the product of HTLV-I rex gene, and a recombination of the rex gene to the HIV or SIV genome is unlikely due to totally different nucleotide sequences in the rev and rex genes.
  • a major problem involved with the production of attenuated virus vaccines is the difficulty of obtaining viruses from which genes or gene fragments have been re ⁇ moved in the amounts needed for vaccine preparations. It has been reported that, for example, HIV lacking a functional rev gene due to mutations or deletions in any one of the functionally important domains, are unable to produce virions (Sadaie, R.M. et al., Science 239: 910- 913, 1988; Feinberg, M.B. et al., Cell 46: 807-817, 1987).
  • the present invention addresses the above mentioned problems.
  • One object of the present invention is to provide a vaccine comprising a live, attenuated human or simian immunodeficiency virus (HIV or SIV, respectively) into the rev gene of which at least one mutation has been intro- prised.
  • the mutation or mutations introduced cause changes in the amino acid sequence of the part of REV protein that binds to the rev responsive element (RRE).
  • the mutation or mutations cause changes in at least one of the three arginine rich regions of the RRE binding region (RBR) of the REV protein, ren ⁇ dering reduced or deficient replicabilty in vitro, which can be transcomplemented in the presence of the rev gene of HIV or SIV or the rex gene of human T-cell leukemia virus I (HTLV-I) resulting in an infectious but attenuated virus.
  • RBR RRE binding region
  • HTLV-I human T-cell leukemia virus I
  • the present invention provides a live, attenuated vaccine comprising a HIV or SIV into which two or preferably three mutations have been introduced into the part of the rev gene corre- sponding to the RBR of the REV protein and/or optionally having a truncation of the C-terminal part of the rev gene, preferably after the RRE binding region of the REV protein, preventing the expression of the transactivation region of the REV protein.
  • the mutations preferably cause amino acid substitutions in the arginine rich regions of the RBR of the REV protein, whereby two or three arginines are being replaced by glycine, proline or isoleucin.
  • Another object of the present invention is to pro ⁇ vide a process for preparing a live attenuated HIV or SIV vaccine comprising the steps of either transfecting a cell culture constitutively expressing HIV-1 REV or HTLV-I REX, preferably a cell culture of HeLa rev cells or, more pre ⁇ ferably, of HeLa rex cells, with a human or simian immuno ⁇ deficiency provirus into which at least one mutation has been introduced into the rev gene, or cotransfeeting said proviruses together with eucaryotic expression vectors capable of producing HIV REV or HTLV-I REX into a human cell line, subsequently infecting a cell line abortively infected with HTLV-I, such as an MT-2, MT-4 or ATH8 cell line, or another CD4 + human cell line capable of con ⁇ stitutive production of HIV REV or HTLV-I REX, preferably an MT-4 cell line, with the rev deficient mutant virus from the transfected
  • such SIV or HIV mutants are used into which a mutation or mutations have been intro ⁇ quizzed that cause changes in the amino acid sequence of the part of REV protein that binds to the rev responsive element (RRE), more preferably in at least one of the three arginine rich regions of the RBR of the REV protein, rendering reduced or deficient replicabilty in vitro, which can be transcomplemented in the presence of the rev gene of HIV or SIV or the rex gene of human T-cell leu ⁇ kemia virus I (HTLV-I), the result being in an infectious but attenuated virus.
  • RRE rev responsive element
  • HIV or SIV mu ⁇ tants are used that comprise two or preferably three mutations in the part of the rev gene corresponding to the RBR of the REV protein and/or optionally having a trun ⁇ cation of the C-terminal part of the rev gene, preferably after the RRE binding region of the REV protein.
  • the present invention provides a process for preparing a live atte ⁇ nuated HIV or SIV vaccine comprising the steps of trans ⁇ fecting a cell culture of HeLa rex cells with a human or simian immunodeficiency provirus into which two or prefer ⁇ ably three mutations have been introduced into the part of the rev gene corresponding to the RBR of the REV protein and having a truncation of the C-terminal part of the rev gene after the sequence corresponding to the RRE binding region of the REV protein, whereby two or three arginines have been replaced by glycine, proline or isoleucin, sub ⁇ sequently infecting the MT-4 cell line with the rev defi ⁇ cient mutant virus from the transfected cells and recover ⁇ ing the infectious, attenuated virus.
  • a further object of the present invention is to provide human or simian immunodeficiency viruses compris ⁇ ing at least one mutation in the rev gene, preferably in that region of the rev gene which encodes the part of REV protein that binds to the rev responsive element (RRE).
  • More preferred mutants of the invention comprise a muta ⁇ tion or mutations which cause changes in at least one of the three arginine rich regions of the RBR of the REV pro ⁇ tein, rendering reduced or deficient replicabilty in vitro, which can be transcomplemented in the presence of the rev gene of HIV or SIV or the rex gene of HTLV-I re ⁇ sulting in an infectious but attenuated virus.
  • the present invention provides HIV or SIV mutants which contain two or preferably three mutations in the part of the rev encoding the RBR of the REV protein and a truncation of the C-ter ⁇ minal part of the rev gene, preferably after the sequence corresponding to the RRE binding region of the REV pro ⁇ tein, preventing the expression of the transactivation region of the rev gene.
  • the mutations cause amino acid substitutions in the arginine rich regions of the RBR of the REV protein, whereby two or three arginines have been replaced by glycine, proline or isoleucin.
  • Preferred mutants have additionally a truncation of the C-terminal part of the rev gene, preferably after the RBR of the REV protein.
  • the mutants according to the invention are prepared by site directed mutagenesis employing novel primers with the sequence Seq ID NO. 1 to Seq ID NO. 10, which also are an object of the present invention.
  • Still another object of the present invention is to provide a method of prevention and/or treatment of HIV or SIV infection and acquired immunodeficiency syndrome (AIDS) or related diseases, in primates, including humans by administering a vaccine comprising live, attenuated HIV or SIV defective for the rev gene to confer protection against a virulent wild type HIV and/or SIV to an indi ⁇ vidual in the need thereof.
  • AIDS acquired immunodeficiency syndrome
  • a vac ⁇ cine comprising a live, attenuated HIV or SIV virus in which the mutations have been introduced in that region of the rev gene which encodes the part of REV protein that binds to the rev responsive element (RRE), more preferably a mutation or mutations have been introduced which cause changes in at least one of the three arginine rich regions of the RRE binding region of the REV protein, rendering reduced or deficient replicabilty in vitro, which can be transcomplemented in the presence of the rev gene of HIV or SIV or the rex gene of human T-cell leukemia virus I
  • HTLV-I HTLV-I
  • the present invention provides a method of prevention and/or treatment of HIV and/or SIV infections or acquired immunodeficiency syndrome (AIDS) and related diseases in primates including humans by administering a vaccine comprising comprising a live, vaccine HIV or SIV into which two or preferably three mutations have been introduced into the part of the rev gene corresponding to the RBR of the REV protein and optionally having a truncation of the C-terminal part of the rev gene, preferably after the RRE binding region of the REV protein, preventing the expression of the transac- tivation region of the REV protein.
  • a vaccine comprising comprising a live, vaccine HIV or SIV into which two or preferably three mutations have been introduced into the part of the rev gene corresponding to the RBR of the REV protein and optionally having a truncation of the C-terminal part of the rev gene, preferably after the RRE binding region of the REV protein, preventing the expression of the transac- tivation region of the REV protein.
  • the mutations prefer ⁇ ably cause amino acid substitutions in the arginine rich regions of the RBR of the REV protein, whereby two or three arginines are being replaced by glycine, proline or isoleucin. More preferably the HIV or SIV mutants comprise a truncation of the C-terminal part of the rev gene after the RRE binding region of the REV protein, preventing the expression of the transactivation region of the REV pro ⁇ tein.
  • Figure 1 depicts the Northern blotting analysis of RNA samples isolated from HeLa, HeLa rev and HeLa rex cells transfected with the rev-defective SIV clone PBj- 1.5.
  • Lane 1 HeLa/pBR322 (negative control); Lane 2: HeLa/PBj-1.5; Lane 3: HeLa rev/PBj-1.5; Lane 4: HeLa rex/PBj-1.5.
  • Figure 2 shows the SIV p27 core antigen content in supernatants of HeLa, HeLa rev or HeLa rex cell lines three days after they were transfected with the rev-de ⁇ fective SIV clone PBj-1.5 or with the control plasmid pBR322.
  • Figure 3 depicts the SIV p27 core antigen product ⁇ ion resulting from the infection of human T-cell lines H9 and MT-4 with supernatants from HeLa, HeLa rev or HeLa rex cells transfected with the rev-defective SIV clone PBj- 1.5 or with the control plasmid pBR322. Supernatants were harvested at day 7 post infection.
  • Figure 4 is a schematic presentation of the mutagenesis procedure SIV. The draw- ings are not in the right scale. The shaded-in areas rep- resent SIV mac 251 DNA, the thick lines represent vector se ⁇ quences and the thin lines represent single-stranded DNA and oligomeric primers.
  • Figure 5 shows the primer pair used in the mutage ⁇ nesis. The newly introduced changes are marked by aste ⁇ risks.
  • the pBKl wild type sequence corresponds to SIV mac 251 provirus nucteotides 8810 to 8877 (GenBank accession M19499)
  • Figure 6 shows the mutations introduced in the part of the SIV rev gene coding for the RBR together with the corresponding amino acid substitutions (A).
  • the corre ⁇ sponding amino acid changes in SIV TAT and SIV ENV of clone pBKl of SIVmac isolate 251 (B).
  • the unchanged resi ⁇ dues appear as dots.
  • the pBKl sequence presented corre ⁇ sponds to SIV mac 251 nucleotides 8816 to 8864.
  • Figure 7 depicts SIV proviral DNA detected by PCR amplification in the peripheral blood mononuclear cells of two monkeys inoculated with the rev defective SIV clone PBK1M15 and of two non-infected control monkeys as assayd by 1.5% agarose/TAE gel electrophoresis.
  • Lanes 1 to 5 samples from monkey 1 taken at day 0, 5, 9, 35 and 65 post inoculation with 12,500 TCIU 50 of the pBKlM15 virus
  • Lanes 6 to 9 samples from monkey 2 taken at day 0, 5, 9 and 35 post inoculation with 12,500 TCIU 50 of the pBKlM15 virus
  • Lanes 10 and 12 samples from the two control monkeys
  • Lane 11 molecular weight markers
  • Lane 13 to 18 samples from the infected/noninfected MT-4 cell mixtures contain- ing 10 "1 to 10 "6 infected cells
  • Lane 19 reagent control.
  • Figure 8 illustrates the antibody activity measured with an ELISA assay towards synthetic NEF peptides in the serum of the two monkeys inoculated with the pBKlMl ⁇ virus before and ten days after inoculation.
  • Figure 9 is an experimental scheme of the setup concerning the replication of rev defective HIV in various cell types.
  • Figure 10 is a schematic presentation of the muta ⁇ genesis procedure of HIV.
  • the drawings are not in the right scale.
  • the shaded-in areas represent the clone pro ⁇ virus of the HIVSF2 isolate, the thick lines represent vector sequences and the thin lines represent single- stranded DNA and oligomeric primers.
  • GAG i.e. components of the viral particles.
  • HIV and SIV mutants which are rendered defect- ive but can still express at least some part of the REV molecule but in a nonfunctional form.
  • HIV or SIV mutants are prepared by site-directed mutagenesis of the rev gene and, in partic ⁇ ular, of the region of the rev gene which encodes the rev responsive element (RRE) binding region of the REV protein of HIV or SIV.
  • the mutations allow the viruses to produce regulatory proteins but make them incapable of producing any structural viral proteins or at least large amounts of them thereby rendering the viruses substantially replica- tive deficient.
  • This deficiency can then be transcomple ⁇ mented with a corresponding regulatory gene, HIV-1 rev; or with the rex gene of a related human retrovirus, the human T-cell leukemia virus I (HTLV-I), resulting in an infect ⁇ ious but attenuated virus.
  • the present invention further deals with the diffi ⁇ culty of producing sufficient amounts of the virus needed for vaccine production.
  • the yield of the virus is further increased if the supernatants containing infectious rev deficient virus are used to infect cells from a cell line abortively infected with HTLV-I or other CD4 + human cell line capable of con- stitutive production of HIV REV or HTLV-I REX.
  • the genomes of the human and simian immunodeficiency viruses (HIV-1 and HIV-2 and SIV, respectively encode nonstructural or regu ⁇ latory proteins.
  • the REV protein enables the virus to produce selectively either regulatory proteins or virion components (Haseltine, W.A., N. Engl. J. Med. 320: 1487-1489, 1989).
  • REV is necessary for the transport of mRNA transcripts from nucleus to cytoplasm (Knight, D. et al., Science 236: 837-840, 1987) and for the translation (D'Agostino, D.M. et al., Mol. Cell. Biol.
  • mRNAs messenger RNAs
  • rev genes have been shown to exist in the HIV-1, HIV-2 and SIV mac (Malim, M. et al., Proc. Natl. Acad. Sci. USA 8_6: 8222-8226, 1989).
  • HTLV human T-cell leukemia viruses
  • rex Hidaka, M. et al., The EMBO Journal 2 : 519-523, 1988.
  • the phylogenic relationship between diffe ⁇ rent related immunodeficiency viruses appears in the following scheme. Retroviridae
  • RRE rev responsive elements
  • H histidine
  • N asparagine
  • D aspartic acid
  • E glutamic acid
  • Q glutamine
  • K lysine
  • R arginine
  • S serine
  • T threonine
  • A alanine
  • W tryptophane
  • this sequence can form an amphi- pathic alpha-helix (Giniger, E. and Ptashne, M., Nature 330: 670-672, 1987), which could bind to a B-form of double-stranded RNA within the stem/bulge structures in RRE.
  • the arginines contained in the three arginine rich stretches of the RRE region are located on one side of the helix.
  • Mutated HIV or SIV constructs in which the argi ⁇ nines are changed to any other amino acid which straight ⁇ ens the computer-predicted alpha-helical structure in the RRE-binding region of REV in such a way that a full-length protein molecule with significantly lower or missing affi- nity towards RRE is created and, provided that the defect ⁇ ive REV function can be transcomplemented with HIV-1 REV or with HTLV-I REX, enables the production of HIV or SIV mutants noncompetent for replication but infectious in amounts needed for vaccines.
  • this con ⁇ cept of mutating the rev gene especially the regions of the rev gene coding for the arginine rich region of REV, is used.
  • a mutation or mutations are thus introduced into rev gene by site di- rected mutagenesis, especially into that region of the rev gene which encodes the part of REV protein that binds to the rev responsive element (RRE) (REV RBR), and in parti ⁇ cular at least one of the three arginine rich regions of the RRE binding region of the REV protein of HIV or SIV.
  • RRE rev responsive element
  • arginine(s) in the arginine rich region of HIV REV or SIV REV is (are) replaced by another amino acid (or other amino acids) which is (are) capable of breaking the -helical structure of RBR to form a rigid linear struc ⁇ ture yielding to a REV protein with significantly lower or missing affinity for RRE.
  • Any amino acid can be used as the substituting amino acid, with the provision that it is capable of dis ⁇ integrating the ⁇ -helix structure which appears to be essential as regards the activity of REV.
  • Suitable amino acids include, but are not limited to, glycine, proline and isoleucine. Due to its small size, glycine is pre ⁇ ferred.
  • mutations are introduced to positions 1 and/or 2, more preferably to positions 1, 2 and 3 of the RRE-binding region corresponding to amino acids 38, 39 and 40 of the SIV REV protein.
  • site directed mutageneses To create the HIV or SIV mutants of the present invention, standard methods for site directed mutageneses are employed. Thus, to introduce the specific mutations in the part of the rev gene which encodes the RRE-binding domain, site directed mutageneses using a pair of specific oligomeric primers and PCR (polymerase chain reaction) amplification or oligonucleotide-mediated mutagenesis (Hutchison, CA. et al., J. Biol. Chem. 253: 6651, 1978; for review, see Smith, M. , Annu. Rev. Genet. 1 ⁇ : 43, 1985) can be used.
  • PCR polymerase chain reaction
  • any HIV-1 or SIV iso ⁇ late cloned in a suitable plasmid can be used.
  • useful HIV-1 clones are HIV SF2 and HIV MN isolates, and for useful SIV X clones, SIV mac 251 and SIV mac 239 isolates.
  • a preferred source for the rev gene is SIV mac 251 wild type cloned proviral DNA pBKl comprising a 10277 bp insert in pS72 vector.
  • cleavage performed e.g. with Spel and PstI results in fragments of suitable size.
  • the 0,9 kb DNA fragment is separated electro- phoretically with agarose gel, such as Seaplaque low gelling agarose (FMC BioProducts, Rockland, ME, USA), purified by suitable means, e.g. by the Sephaglas BandPrep kit (Pharmacia P-L Biochemicals, Wisconsin, USA), and then ligated to a competent vector, such as pUC19 (Yanisch- Perron, C.
  • any other suitable cloning vector synthetized or commercial, e.g. pBlueScript products (Sratagene, La Jolla, Ca, USA), pGEM and pSP series (Promega Corporation, Madison, Wiscon- sin, USA) just to mention some, which is first cleaved with the suitable restriction enzymes, e.g. Xbal and PstI, to create compatible ends.
  • suitable restriction enzymes e.g. Xbal and PstI
  • the ligation product obtained is then propagated by transforming into suitable host cells. Transformation can be performed by standard methods, e.g. the electroporation method described by Dower et al., 1988 (Nucl. Acids Res. 16: 6127, 1988) or the method decribed by Hanahan (J. Mol. Biol. 166: 557, 1983). For the pUC19, the latter method is preferred.
  • the use of E. coli TGI cells which have been made competent cells by the method also described by Hanahan (supra) is preferred.
  • Other E. coli cells, such as E. coli JM 109 can also be used.
  • the transformation can, for example, be performed by thawing the frozen competent host cells in wet ice, adding the cells to the ligation mixture prepared above and after a further incubation in ice for 20 to 30 mi ⁇ nutes, subjecting the mixture to short (e.g. 1 min) heat- shock in water bath (e.g. 42°C), and after about 5 minutes at the room temperature, adding the LB-medium or any other culture medium competent to the host cells used. The transformation cultures are then further incubated for 30 to 60 minutes at the optimum temperature of the bacterial cells (usually 37°C) with gentle rocking.
  • short e.g. 1 min
  • heat- shock in water bath e.g. 42°C
  • the transformation cultures are then further incubated for 30 to 60 minutes at the optimum temperature of the bacterial cells (usually 37°C) with gentle rocking.
  • the selection for transformants can be made on agar plates in the culture medium used in the transformation supplemented with a suitable antibiotic, such as ampicil- lin, kanamycin, tetracyclin and chloramphenicol.
  • a suitable antibiotic such as ampicil- lin, kanamycin, tetracyclin and chloramphenicol.
  • LB-medium plus 1.5% agar plus 100 ⁇ g/ml ampicillin is suitable.
  • the true recombinants are then detected by using, for example, the blue/white color selection method with X-Gal and IPTG (Ullmann, A. et al., J. Mol. Biol. 24: 339, 1967; Messing, J., Methods Enzymol.
  • the plasmids are extracted e.g. by the alkaline procedure described by Birnboim and Doly (Nucleic Acids Res. 7: 1513, 1979), analyzed by agarose gel electrophoresis, and one of the mutants is arbitrarily picked for further processing by the PCR.
  • the oligomeric primers used in the site directed mutagenesis procedure with the PCR can be commercially available primers or, alternatively, desired primers can be prepared by using e.g. Cyclone DNA synthesizer (Milli- pore, MA, USA).
  • the specific mutations in the regions of the rev gene encoding the arginine rich stretches are first introduced by PCR in two steps.
  • the first reaction mixture includes a universal primer specific for the vector used, e.g.
  • 17mer USP M13 universal sequencing primer, New England Biolabs, MA, USA
  • a pair of primers for SIV mu- tants, see Figure 5
  • the plasmid prepared above MgCl 2 and dNTPs (deoxynucleotides) in a suitable buffer (for example, the standard lxPCR buffer containing 50 mM KC1, 20 mM TRIS-HC1, pH8.8)
  • a suitable buffer for example, the standard lxPCR buffer containing 50 mM KC1, 20 mM TRIS-HC1, pH8.8.
  • the actual pH, the concentration of dNTPs, the primers and MgCl 2 depend on the reactants (input DNA and primers).
  • the PCT protocol used is also dependent on the nature of input DNA and primers.
  • another specific mutagenesis primer for SIV mutants, see Figure 5
  • another universal primer e.g. 16mer RSP (M13 Reverse Sequencing Primer, New England Bio- labs, MA, USA
  • the PCR is preferably carried out with 1 cycle comprising 5 minutes at 94°C, 2 minutes at 37°C and 1 minute at 72°C, and 23 cycles comprising 1 minute at 94°C, one minute at 50°C and one minute at +72°C after the addition of Taq polymerase (Boehringer-Mannheim, Darmstadt, Germany).
  • Taq polymerase Boehringer-Mannheim, Darmstadt, Germany.
  • the reaction mixture from PCR is then analyzed electrophoreti- cally on agarose and staining with ethidium bromide to verify the amplification reaction products.
  • samples from the two previous reaction mixtures are combined with USP and RSP primers in buffer, and annealed by cooling from 72"C to the room temperature for about 30 to 60 minutes.
  • the DNAs are subjected to sec ⁇ ond amplification as described above. After completion, the DNA is recovered by precipitation and the precipitates are collected by centrifugation, washed and dissolved in water.
  • the DNAs are then cleaved with suitable restriction endonucleases, in the case of SIV mutants preferably with
  • the mutations are analyzed by standard methods; the dideoxy sequencing by a Sequenase version 2.0 kit (USB, Ohio, USA) is preferred.
  • the plasmid DNAs are first isolated from over night cultures by the alkaline method (Molecular cloning: a laboratory manual, 2nd edition, Eds. Sambrook. J. et al., Cold Spring Harbor Laboratory Press, 1989), phenol extracted, EtOH precipi ⁇ tated, RNase treated, PEG (polyethylene glycol) precipi ⁇ tated and dissolved in buffer, e.g. in TE buffer contain- ing 10 mM TRIS-HC1 pH8.0 and 0.1 mM EDTANa 2 .
  • mutant plasmid DNAs that contained the desired mutations are first cleaved with appropriate restriction enzymes, such as Nhel and PstI, and separated by electro- phoresis.
  • appropriate restriction enzymes such as Nhel and PstI
  • the Nhel/PstI fragment (0.2 kb) the mutated RBR is isolated and purified e.g. with the Sephaglas BandPrep kit (Pharmacia P-L Biochemicals, Wisconsin, USA).
  • pMACl which contains a 3,5 kb Hindlll/EcoRI fragment of SIV Bac 251 DNA from pBKl, cloned by one of the inventors, and includes the second coding exon of the rev gene.
  • any other suitable vector is cleaved with the restriction enzymes Nhel and PstI, purified and ligated to the Nhel/PstI fragment obtained above by using T4 DNA ligase and transformed into E. coli TGI cells or other suitable cells as described above. Sample colonies are picked and analyzed by dideoxy sequencing. Instead of RSP, another oligomeric primer complementary to the 3' end of SIV tat is used.
  • pMACl-derivatives containing the verified mutations and the parental pBKl plasmid are cleaved with the restriction enzymes Nhel and EcoRI.
  • the Nhel/EcoRI fragment from pBKl is ligated to different Nhel/EcoRI fragments from pMACl-derivatives and transformed into E. coli JM109 cells (Boehringer-Mannheim, Darmstadt, Germany) or to other suitable (recombination deficient) cells, pre ⁇ ferably recA " mutants. After large scale purification of mutant proviral DNA, the mutations are verified once again by sequencing.
  • the mutations can also be introduced by the oligo- nucleotide-directed in vitro mutagenesis process, which is shortly described in terms of the preparation of HIV mu ⁇ tants of the invention starting from the proviral DNA from HIV SF2 proviral DNA for illustrating purposes only, since for the skilled artisan it is immidiately evident that the process can be applied, with appropriate variations, to any other suitable HIV or SIV DNA source.
  • HIVSF2 pro ⁇ viral DNA is cleaved with Xhol and Sad, and inserted into pBluescript S/K (+) vector or another suitable vector which has been digested with Xhol and Sad.
  • the recom ⁇ binant plasmid thus obtained is grown in a culture medium, e.g. in LB-medium, supplemented with ampicillin and when necessary the culture can be coinfected with a helper virus, such as M13K07.
  • the recombinant plasmid will be packed into virus-like particles and after purification of the single-stranded recombinant plasmid DNA, the RRE re ⁇ sponsive region encoding part of the rev gene is altered by using specific primers and the Oligonucleotide-directed in vitro mutagenesis system version 2.1 kit (Amersham, Buckinghamshire, England).
  • the primers used can be com ⁇ suddenly available primers or can be synthetized e.g. by using the Cyclone DNA synthesizer (Millipore, MA, USA).
  • the primer containing a specific mutation or mutations is annealed to the tem- plate single-stranded plasmid and the complementary strand is synthetized in an appropriate buffer in the presence of dNTPs, one of which is chemically modified to make the nascent strand resistant to the cleavage with Neil endo- nuclease by using Klenow enzyme.
  • the template DNA will then be nicked by Neil or any other suitable restriction enzyme capable of introducing single-stranded nick, e.g. Pvul, digested with exonuclease, and after nuclease in- activation, the new strand will be synthesized with Klenow enzyme.
  • the DNA is then transformed into E. coli TGI cells and randomly picked colonies are sequenced to specify mu ⁇ tations introduced.
  • the mutant plasmid will then be digested with Aval and the 0,5 fragment is reinserted to the parent plasmid cleaved with Aval. Then the resulting plasmid will be di- gested with Drain and Xhol restriction endonucleases and the 2.3 kb insert will be again reinserted into the par ⁇ ent plasmid to obtain the desired mutant.
  • the SIV obtained contained mutations in the parts of the rev gene encoding the RRE binding region. These mutants were de ⁇ noted as pBKlM12, pBKlM15, pBKlM16, pBKlM2, pBKlM3, pBKlM4, and pBKlM5.
  • the mutated sequences are depicted in Figure 5.
  • the sequences of the altered arginine rich RRE binding regions and corresponding amino acid changes in SIV TAT and SIV ENV are shown (Fig. 6A and 6B).
  • the strategy employed in the construction of the mutated SIV or HIV viruses that can be used as live atte ⁇ nuated vaccines or in the form of a DNA vaccine is based on the fact that the rev gene product, the REV protein has three important functionally active regions; the oligomeration region (OR), located in the N-terminal part of the molecule, is necessary for assembly, by oligo ⁇ meration, of 3 to 4 REV proteins that bind as a complex to the REV responsive elements (RRE) on non-spliced and singly-spliced mRNAs.
  • the RRE binding region (RBR) is another functionally active region and the activity of REV is in fact based on a proper binding process to RRE.
  • transactivation region (TR) located at the C-terminus of the molecule is required for the actual ac- tivity of REV.
  • TR transactivation region located at the C-terminus of the molecule.
  • Our object was to generate mutations that would lead to expression of at least part of the REV pro ⁇ tein but that would still be functionally inactive.
  • stop-codons or deletions in the rev gene, leading to dele ⁇ tion of the C-terminal transactivation region would make a functionally inactive REV protein. Constructs carrying these mutations may be important if the mutated virus is given to a vaccinee in DNA form.
  • mutants such as pBKlM5, pBKlM15 and pBKlM16 will be totally non-replicative and thus, in terms of safety, superior to the others.
  • the viral proteins expressed by these mutants are con ⁇ fined to the regulatory proteins coded for by the doubly- spliced mRNAs, the immune response caused by such an atte- nuated virus is restricted.
  • mutants such as pBKlM12 or pBKlM3, as well as the corresponding HIV mutants which are still replicative but where the rep ⁇ lication is decreased as compared to the wild type virus would be less safe but cause a broader and stronger immune response.
  • a totally replicative defective virus such as the one represented in the pBKlM15 construct
  • a boost- er immunization with a replicative but still attenuated virus such as the one exemplified in the pBKlM12 and PBK1M3 constructs.
  • HIV-1 rev can com ⁇ pensate in trans for a defective rev gene in both HIV-1 and HIV-2 viruses.
  • HIV-2 rev in contrast, compensates only for a defective rev gene in HIV-2 (Malim, M. et al., Proc. Natl. Acad. Sci. USA 6: 8222-8226, 1989; Sakai, H. et al., J. Virol. 64: 2202-2207, 1990).
  • HIV-2 REV may in fact even inhibit the function of HIV-1 REV, possibly due to ineffective/aberrant multimerization of the two REV molecules on the RRE of HIV-1 (Garrett, E.D. and B. Cullen, J. Virol. j36: 4288-4294, 1992).
  • HTLV-I REX human T cell leukemia virus type I
  • HTLV-I rex can also compen ⁇ sate for a defective rev gene.
  • Pre ⁇ ferably HeLa cells constitutively expressing HTLV-I rex are transfected with HIV or SIV mutants of the present invention. Other suitable cells can also be used.
  • Trans ⁇ fection can be performed by any of the methods known in the art.
  • Cotransfection of the HIV or SIV mutant viruses of the present invention together with eucaryotic expression vectors capable of producing HIV REV or HTLV-I REX into a suitable human cell line can also be used.
  • the cells are harvested by trypsinization and re-plated in a culture medium, such as RPMI-1640, at 37°C.
  • a culture medium such as RPMI-1640
  • the following day mutant DNA are mixed with CaCl 2 in a suitable buffer. After 10 to 20 minutes at the room tem ⁇ perature, the mixture is added dropwise to the cells and the incubator's C0 2 content is adjusted. After 16 to 24 hours the cells are washed with a fresh medium containing fetal calf serum (FCS), and incubated until harvested.
  • FCS fetal calf serum
  • the supernatants con- taining infectious rev deficient virus are then used to infect cells from a cell line abortively infected with HTLV-1 and expressing low amounts of the REX protein, such as an MT-2, MT-4 or ATH8 cell line, or some other CD4 + human cell line capable of constitutive production of HIV REV or HTLV-I REX.
  • a cell line abortively infected with HTLV-1 is preferred.
  • the latter infection step increases the amount of the virus up to 1,000-fold, normally 300 - 800-fold.
  • the attenuated but infectious SIV or HIV virus pre ⁇ pared according to the invention is then purified by ultrafiltration and ultracentrifugation or by any other suitable conventional means.
  • the virus is stored in frozen form e.g. in liquid nitrogen.
  • the mutated SIV and HIV viruses of the present in ⁇ vention can be used either as live attenuated vaccines where the viruses are given to the vaccinee in the form of a life virus, or as a DNA vaccine where the vaccinees are injected with purified plasmid DNA containing the mutated HIV/SIV genome in a suitable expression vector.
  • the purified virus can be dissolved in a physiologically acceptable buffer, such as PBS, and stored in frozen state.
  • a physiologically acceptable buffer such as PBS
  • the virus can be freeze dried and stored at the room temperature whereupon the vaccine is reconstituted before vaccination by adding a given amount, for example 0.5 - 1.0 ml, of the buffer.
  • the virus dose to be used naturally varies but should contain approximately the amount of virus corresponding to the amount obtainable from a few milliliters of the cell culture.
  • the vaccinees should receive 10 4 to 10 5 infectious units of the virus to ensure the infection of a sufficient number of target cells.
  • the purified DNA is pre- cipitated by ethanol, dried and stored either in frozen state or, for short periods, at the room temperature.
  • the vaccine is reconsti ⁇ tuted by the addition of about 0.5 to 1.0 ml of an appro- priate physiological buffer, such as PBS.
  • the vaccines of the present invention can be admin ⁇ istered through several routes.
  • the live atte ⁇ nuated virus of the invention is given either intravenous ⁇ ly, intramuscularly or through gastrointestinal route on the mucosa of the intestine.
  • the vaccine preparation is packed e.g. in enteral capsules that resist the proteolytic activity of the gastric juice in the stomach but are degraded in the intestine.
  • intramuscular admin ⁇ istration is preferred, but intravenous and subcutaneous routes can also be chosen.
  • the vaccines of the present invention are preferab ⁇ ly used prophylactically to protect individuals, preferab- ly humans, in a high risk of infection of HIV, but may also be advantageous in immunotherapy of patients already infected by HIV or SIV.
  • the vaccine administered either as a life attenuated vaccine or as a DNA vaccine, can be given in association with any suitable agent or drug pro- moting the effect of the vaccine or the clinical state of the patient.
  • drugs such as azidothymidine (Zidovudin) can be combined with the vaccination protocol.
  • Immunologic adjuvants and immunomodulators, such as cyto- kines may also be used in conjunction with immunization to promote cell-mediated responses.
  • PBj-14 is an acutely lethal variant of simian immunodeficiency virus from sooty mangabeys (SIV ⁇ mm ) and always causes fatal disease in pig-tailed maca ⁇ ques (Fultz, P. N. et al., AIDS Res. Hum. Retroviruses 5 : 397-409, 1989).
  • SIV ⁇ mm sooty mangabeys
  • One of the several clones obtained from this isolate, PBj-1.5 contains a transition at the ini ⁇ tiation codon of rev and shows no reverse transcriptase activity upon CEMxl74 cells (Dewhurst, S. et al., Nature 345: 636-640, 1989).
  • HeLa cells (NIH AIDS Research and Reference Reagent Program Catalog #153) and HeLa cells constitutively ex ⁇ pressing HIV-1 rev (HeLa rev) or HTLV-I rex (HeLa rex) (both generous gifts from Dr Georg Pavlovski, NIH, Bethes- da, MI, USA) were transfected with PBj-1.5 or with a con ⁇ trol plasmid pBR322 DNA.
  • the transfeetions of HeLa, HeLa rev and HeLa rex cells were performed essentially as described by Chen and Okayama (Biotechniques 6: 632-638, 1988).
  • the cells were harvested by trypsinization and replated in amounts of 1 to 2 x 10 5 per a 25 cm 2 flask con- taining an RPMI 1640 medium.
  • the following day 5 to 10 ⁇ g of PBj-1.5 or pBR322 plasmid DNA was mixed with 0,5 ml of 0,25 M CaCl 2 to which 0,5 ml 2xBES buffer (50 mM N,N-bis- 2-hydroxyethyl-2-aminoethanesulfonic acid, 280 mM NaCl, 1,5 mM Na 2 HP0 4 .2H 2 0) was added.
  • 0,5 ml 2xBES buffer 50 mM N,N-bis- 2-hydroxyethyl-2-aminoethanesulfonic acid, 280 mM NaCl, 1,5 mM Na 2 HP0 4 .2H 2 0
  • each transfected cell line was quantitated by measuring the amount of a viral core antigen p27 released to the culture supernatant by using a commercially available ELISA kit (Abbott, Abbott Park, II 60064, USA, or Coulter, Hialeah, FI, USA).
  • the super- natants were filtered trough 0,22 ⁇ m cellulose acetate filters and the absorbances were compared to those ob ⁇ tained with known amounts of a standard p27 antigen in ⁇ cluded in the kit.
  • absorbance optical den ⁇ sity
  • RNA A cytoplasmic fraction of RNA was extracted from HeLa, HeLa rev and HeLa rex cells transfected with PBj- 1.5 and pBR32248 post transfection. Extraction of RNA and subsequent analysis by formaldehyde gel electrophoresis and by Northern blotting were performed by standard meth ⁇ ods (Molecular cloning: a laboratory manual, eds. Sambrook, J. et al, 2nd edition, Cold Spring Harbor Lab ⁇ oratory Press, USA, 1989).
  • PBS phosphate buffered saline
  • RNA extraction buffer [0.14 M NaCl, 1.5 mM MgCl 2 , 10 mM TRIS-HC1, pH 8.6, 0.5% Nonidet P-40, 1 mM dithiotreitol (DTT) and 1000 units/ml RNasin ribonuclease inhibitor (Promega Corporation, WI, USA)].
  • the cells were vortexed, centrifuged at 12,000 g for 90 seconds, and an equal volume of Proteinase K digestion buffer [0.2 M TRIS-HC1 pH 8.0, 25 mM EDTANa 2 pH 8.0, 0.3 M NaCl and 2% sodium dodecyl sulphate (SDS)] containing 50 mg/ml of Proteinase K (Boehringer-Mannheim, Darmstadt, Germany) was added to the supernatant. After incubation for 30 minutes at 37°C, the RNA was extracted once with 400 ⁇ l of phenol:chloroform (1:1) and precipitated by adding 400 ⁇ l of isopropanol.
  • Proteinase K digestion buffer [0.2 M TRIS-HC1 pH 8.0, 25 mM EDTANa 2 pH 8.0, 0.3 M NaCl and 2% sodium dodecyl sulphate (SDS)] containing 50 mg/ml of Proteinase K (Boehringer-Mannheim, Darm
  • cyto- plasmic RNA from each sample was analyzed by electrophor ⁇ esis on 1.5% agarose gel (Seakem GTG agarose; FMC Bioprod- ucts, ME, USA) containing 2.2 M formaldehyde.
  • the running buffer (10 mM Na-phosphate, pH 7.0) was recirculated by pumbing it from one reservoir to the other, and the elec ⁇ trophoresis was performed at +4°C at 25 volts for 12 to 14 hours.
  • the gels were stained with a fluorescent dye ethidium bromide (EtBr; Sigma Chem- ical Company, MO, USA) at a concentration of 0,5 ⁇ g/ml in water, destained in water and photographed under UV-illu ⁇ mination (wavelength 302 nm). Subsequently, the gels were treated with 50 mM NaOH for 30 minutes by soaking them in 50 mM NaOH in 20xSSC solution (3M NaCl, 0,3 M Na-citrate pH 7.0) for 30 minutes and transferred by capillary elution to Hybond-N nylon membranes (Amersham, Buckingham ⁇ shire, England).
  • EtBr ethidium bromide
  • the filter was wrapped with plastic film and the RNA side of the membrane was exposed to UV-light (254 nm ⁇ for 4 minutes to covalently bind the RNA to the mem ⁇ brane.
  • the filter was sealed into a plastic bag and pre- hybridized for 1 hour at 65°C in 20 ml of a solution con ⁇ taining lOxSSC (0.1 M NaCl, 0.15 M Na-citrate, pH7,0), 5xDenhardt's solution [0.5% Ficoll 400, 0.5% bovine serum albumin (BSA), 0.5% polyvinylpyrrolidone], 0.1% SDS and 100 ⁇ g/ml denaturated salmon sperm DNA.
  • a solution con ⁇ taining lOxSSC 0.1 M NaCl, 0.15 M Na-citrate, pH7,0
  • 5xDenhardt's solution [0.5% Ficoll 400, 0.5% bovine serum albumin (BSA), 0.5% polyvinylpyrrolidone
  • the hybridization was performed at 65°C overnight in the same solution con ⁇ taining approximately 50 ng of a SIV LTR probe (a 0.6 kb StuI fragment purified from PBj-1.5 proviral DNA) labelled with a 3z P-dCTP (Amersham, Buckinghamshire, England) by the random-priming method described by Hodgson, C.P. and Fisk, R.Z. (Nucleic Acids Res. 15: 6295, 1987) to specific acti ⁇ vity of 5xl0 8 cpm/ ⁇ g (counts per minute per microgram of input DNA).
  • the hybridized filters were washed with 2xSSC twice at the room temperature for 15 to 30 minutes, twice at 37°C for 15 to 30 minutes, and at 65°C for 5 minutes and exposed to an X-ray film (X-HRG, Fuji) with an inten ⁇ sifying screen (Enlightening Plus, Du Pont) at -70°C for several days.
  • X-HRG X-ray film
  • Enlightening Plus Du Pont
  • HeLa cells transfected with the rev defective SIV clone PBj-1.5 are shown in Figure 1.
  • HeLa rex/PBj-1.5 cells ex ⁇ pressed three classes of mRNAs (1.8 - 2.2 kb, 4.5 kb and 9.4 kb, corresponding to double spliced, singly-spliced and unspliced mRNA, respectively) (Lane 4), whereas the HeLa rev/PBj-1.5 cells expressed mainly mRNAs of 1.8 to 2.2 kb and minor amounts of 4.5 and 9.4 kb mRNA (Lane 3).
  • the HeLa/PBj-1.5 cells failed to show any large unspliced genomic or singly-spliced env mRNA (Lane 2). Control transfections with pBR322 of any of the cell lines showed no SIV mRNA in the Northern blotting analysis.
  • the results of the SIV p27 core antigen assay demonstrate the production of large amounts of the virus by HeLa rex/PBj-1.5 cells and produc- tion of minor but significant amounts of the virus by HeLa rev/PBj-1.5 cells.
  • HeLa/PBj-1.5 cells and control trans ⁇ fections of any of the cell lines with pBR322 were nega ⁇ tive in the p27 core antigen assay.
  • the supernatants from the different HeLa cultures transfected as described above were used to infect H9 and MT-4 cell lines.
  • the H9 cell line is a CD4 positive lymphoid T-cell line (NIH AIDS Research and Reference Reagent Program Catalog #87).
  • An MT-4 cell line (NIH AIDS Research and Reference Reagent Program Catalog #120) is a CD4 positive lymphoid T-cell line abortively infected with HTLV-I (one integrated copy/cell) and it has low or un- detectable HTLV-I virus production (Harada, S. et al., Science 229: 563-566, 1985).
  • Culture supernatants were collected from each of the transfection experiments, cleared by centrifugation (900g for 10 minutes), filtered through a 0.22 ⁇ m dis- posable filter, and 3 ⁇ l of the supernatant were used to infect 10 7 MT-4 or H9 cells grown in RPMI 1640 supplemented with 10% FCS at 37°C. The cell cultures were then main ⁇ tained at 37°C and fed every three days with a fresh medium. Samples for a p27 antigen assay were taken at day 7, 14 and 21 post infection.
  • HIV-1 clone pHXB2107 contains a defective rev gene caused by a stop codon at position 6 (Sadaie R. et al., Science 239: 910-913, 1988).
  • Another HIV-1 clone, pHXB2 f ⁇ (Bam P3), contains a four bp insertion at the BamHI site at nucleotide position 8053 leading to frame shift at amino acid position 59 of the rev gene (Feinberg M. et al., Cell 46: 807-817, 1986).
  • HeLa, HeLa rev and HeLa rex cell lines were transfected with the two pHXB2 mutants and with a control plasmid pBR322, and the determinations of viral mRNA by the Northern blotting were performed as de ⁇ scribed in Example 1.
  • each transfected cell line was quantitated by measuring the amount of viral core antigen p24 released to the culture supernatant by using a commercially available ELISA kit (Abbott, Abbott Park, II 60064, USA). The supernatants were filtered trough 0,22 ⁇ m cellulose acetate filters and the absorbances were com ⁇ pared with those obtained with known amounts of a standard • p24 antigen included in the kit. Results are shown in Table 2. In the assay, absorbance of 0.080 was considered positive according to the manufacturer's recommendation.
  • HeLa pBR322 0,060 negative HeLa pHXB2 107 0,051 negative HeLa pHXB2 f ⁇ 0,049 negative
  • HeLa REV pBR322 0,053 negative HeLa REV pHXB2 107 ⁇ 2,000 positive HeLa REV pHXB2 f ⁇ 0,233 positive
  • HeLa REX pBR322 0,049 negative HeLa REX pHXB2 107 >2,000 positive HeLa REX pHXB2 f ⁇ 0,488 positive
  • Results given in Table 2 demonstrate abundant prod ⁇ uction of a p24 antigen in the HeLa rev and HeLa rex cell lines transfected with the pHXB2 107 clone while only a low p24 production was observed with the pHXB2 f ⁇ clone.
  • the f ⁇ clone behaves as a dominantly negative mutant where the missing rev func- tion can not be readily transcomplemented with rev nor with rex gene products.
  • the dominant negative effect of the HXB2 f ⁇ mutation is due to the fact that the defective REV protein, lacking the C-terminal transactivation re ⁇ gion, still binds through the arginine-rich region (amino acids 36 to 50) to the RRE of the nonspliced and singly- spliced mRNAs and thus inhibits the binding to the RRE of the functionally active normal REV or REX proteins.
  • SIV_. ac 251 wild type cloned proviral DNA pBKl (10277 bp insert in pP72 vector) was received from prof. James Mullins (Stanford University, CA USA).
  • pBKl is a deriva ⁇ tive (5' flank of chromosomal DNA has been removed) of pBK28 (available from NIH AIDS Research and Reference Reagent Program Catalog #133) which is identical with pBKl as regards the provirus and thus can also be used.
  • the specific mutations of the parts of the SIV rev encoding the RRE-binding domain were introduced by using a pair of oligomeric primers and PCR (polymerase chain reaction) amplification (Saika, R.K.
  • the pBKl plasmid DNA was cleaved with the restriction enzymes Spel and PstI and the 0.9 kb fragment (SIV Bao 251 nucleotides 8017-8947, GenBank locus name SIV ⁇ Sl, accession n:o M19499, Y00269) was cut from 1% Seaplaque low gelling agarose (FMC BioProducts, Rock- land, ME, USA), purified by using a Sephaglas BandPrep kit (Pharmacia P-L Biochemicals, Wisconsin, USA) and ligated to pUC19 (Yanisch-Perron, C.
  • the ligation reaction mixture consisted typically of a lxligase buffer [50 mM TRIS-HC1 pH8.0, 10 mM MgCl 2 , 1000 ⁇ g/ml BSA, 0,25 mM adenosine triphosphate (ATP), 1 mM DTT] , 5 ⁇ g/ml of a vector, insert DNA in an amount twice as large as that of the vector in moles, and 1 Weiss unit of T4 DNA ligase (Boehringer-Mannheim, Darmstadt, Ger ⁇ many).
  • the ligation reaction mixtures were incubated for two hours at 15°C and then transformed into E. coli TGI competent cells produced by the method of Hanahan (Hana- han, D., J. Mol. Biol. 166: 557, 1983)
  • the transformation was performed as follows. The frozen competent E. coli TGI bacteria were thawed in wet ice, aliqouts of 100 ⁇ l were added to the ligation mix ⁇ tures and the resulting mixtures were, after brief vortex- ing, incubated in ice for further 20 to 30 minutes. After this the mixtures were subjected to heat-shock in a water bath at 42 "C for 1 minute and were then allowed to stand for 5 minutes at the room temperature.
  • LB- medium 1% Bactotryptone (Difco Laboratories, Detroit, Michigan, USA), 0,5% yeast extract (Difco Laboratories, Detroit, Michigan, USA), 1% NaCl] were added and the tubes were incubated for 30 to 60 minutes at 37°C with gentle rocking on a laboratory shaker.
  • the selection for transformants was performed on LB-plates (LB-medium plus 1.5% agar) supplemented with 100 mg/ml of ampicillin.
  • the true recombinants were detected by blue/white color selection by adding 10 ⁇ l of 50 mg/ml of X-Gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) in dimethylformamide (DMFA) and 20 ⁇ l of 0.1 M isopropyl- thiogalactoside (IPTG) per plate.
  • X-Gal 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside
  • IPTG isopropyl- thiogalactoside
  • the specific mutations in the rev gTV arginine-rich region were first introduced by PCR in two steps.
  • the first reaction mixture contained 10 pico- moles of 17mer USP (M13 universal sequencing primer, New England Biolabs, MA, USA), 10 picomoles of a specific mutagenesis primer (MIL, M2L, M3L, M4L or M5L), 2 ng of pMll, and 250 ⁇ M dNTP (deoxynucleotide triphoshates dATP, dGTP, dCTP and dTTP) in a lxPCR buffer (50 mM KC1, 1.5 mM MgCl 2 , 20 mM TRIS-HC1 pH 8.8).
  • the second PCR reaction was performed parallelly, and the reaction mixture (mix B) was identical to mix A but contained 10 picomoles of a speci ⁇ fic mutagenesis primer (M1U, M2U, M3U, M4U or M5U) and 10 picomoles of 16mer RSP (M13 Reverse Sequencing Primer, New England Biolabs, MA, USA) instead of USP and MIL primers.
  • Mix B was identical to mix A but contained 10 picomoles of a speci ⁇ fic mutagenesis primer (M1U, M2U, M3U, M4U or M5U) and 10 picomoles of 16mer RSP (M13 Reverse Sequencing Primer, New England Biolabs, MA, USA) instead of USP and MIL primers.
  • the PCR was carried out with 1 cycle comprising 5 minutes at 94°C, 2 minutes at 37°C and 1 minute at 72°C, and 23 cycles comprising 1 minute at 94°C, 1 minute at 50°C and 1 minute at +72°C after 2.5 U of Taq polymerase (Boehringer-Mannheim, Darmstadt, Germany) was added.
  • the total volumes of the PCR samples were 50 ⁇ l.
  • aliquots of 5 ⁇ l were analyzed by electrophoresis by using 2% agarose in 40 mM TRIS-acetate, 1 mM EDTANa 2 .
  • the gel was stained with EtBr at a concentration of 0.5 mg/ml in water for 30 minutes at the room temperature to verify the amplification.
  • the second cycle of the PCR was performed as follows: aliquots of 0.1 ⁇ l from the two previous reaction mixtures were combined and supplemented with 5 ⁇ l of a lOxPCR buffer, 10 ⁇ l of 1.25 mM dNTPs and 10 picomoles of USP and RSP primers, and up to 50 ⁇ l of water was added, and the annealing was conducted by cooling from 72°C to the room temperature in 30 minutes. After the addition of 2.5 U of Taq polymerase per reaction mixture the DNAs were amplified as described above. After completion, the DNA was precipitated by adding 1/10 volumes of 5M NaCl and 2 volumes of EtOH at -20°C for 1 hour.
  • Precipitates were collected by centrifugation in a microfuge for 10 minutes at 12,000 g at the room temperature, washed once with 70% of aqueous ETOH and dissolved in 20 ⁇ l of water.
  • the DNAs were cleaved with the restriction endonucleases EcoRI and Hindlll, fragments were separated and transformed into E. coli TGI.
  • the mutations were analyzed by dideoxy sequencing by using a Sequenase version 2.0 kit (USB, Ohio, USA).
  • the plasmid DNAs were isolated from 1.5 ml of over night cultures of the cells in the LB-medium containing 100 ⁇ g/ml ambicillin by the alkaline method described in Molecular cloning: a laboratory manual, 2nd edition, eds. Sambrook. J. et al. , Cold Spring Harbor Laboratory Press, 1989, phenol extracted, EtOH precipitated, RNase treated, PEG (polyethylene glycol) precipitated and dissolved in 50 ⁇ l of TE (10 mM TRIS-HCl pH ⁇ .O, 0.1 mM EDTANa 2 ). 18 ⁇ l of plasmid DNA (approx. 3-5 ⁇ g of DNA) were and sequenced by using the RSP primer.
  • mutant plasmid DNAs that contained the desired mutations were cleaved with the restriction enzymes Nhel and PstI and electrophoresed by using a 1,5% agarose/TAE gel.
  • the 0.2 kb fragment was isolated and purified by a Sephaglas Band- Prep kit (Pharmacia P-L Biochemicals, Wisconsin, USA).
  • pMACl pUC19 containing 3.7 kb Hindlll/- EcoRI DNA fragment from pBKl provirus 3' end containing rev siv 2nd coding exon
  • Nhel and PstI restriction enzymes Nhel and PstI
  • the two DNAs were ligated by using T4 DNA ligase and transformed into ___• coli TGI cells as described above. Sample colonies were picked and analyzed by dideoxy sequencing. Instead of RSP, an oligomeric primer complementary to the 3' end of tat g ⁇ v was used.
  • E. coli JM109-J5 cells kindly donated by Dr. V. Hirsch. National Institute of Allergy and Infectious Diseases, Rockville, Maryland, USA.
  • the parent strain of the E. coli JM109-J5 cells E. coli JM109 cells (Boeh ⁇ ringer-Mannheim, Darmstadt, Germany), can also be used. After large scale purification of mutant proviral DNA, the mutations were verified once again by sequencing.
  • SIV mutants obtained were denoted as pBKlM12, PBK1M15, pBKlM16, pBKlM2, pBKlM3, pBKlM4, and pBKlM5.
  • the mutated sequences are depicted in Figure 6A.
  • the sequences of the altered arginine-rich RRE binding regions and corresponding amino acid changes in SIV TAT and SIV ENV are shown in Figure 6B.
  • the HeLa, HeLa rev and HeLa rex cells were trans ⁇ fected with the SIV mutants pBKlM2, pBKlM3, pBKlM4, PBK1M12, pBKlMl ⁇ ja pBKlM16 prepared in Example 3 and with pBR322 and pBKl, which serve as a negative control and a positive control, respectively, essentially as described in Example 1 by using 5 mg of DNA per near confluent 25 cm 2 flask.
  • the production of virus was analyzed by the p27 core antigen assay as in Example 1.
  • pBR322 - (neg. ( neg. (neg. pBKl 1.078 (pos. 1,081 ( pos . 0, 811 (pos.
  • PBK1M3 0.960 (pos. 1,050 ( pos . 1 , 076 (pos.
  • PBK1M4 0.610 (pos. 1,022 ( pos . 1 , 056 (pos.
  • PBK1M15 0.049 (neg. 0,294 ( pos . 0, 873 (pos.
  • PBK1M16 0.051 (neg. 0,070 ( neg. 0, 261 (pos.
  • mutant pBKlM3 replicated almost to the same extent as the wild type, as assessed by the p27 antigen production.
  • the production of a p27 antigen by pBKlM4 was about 60% of the wild type, while the p27 anti ⁇ gen production by mutant pBKlM12 was only 30% of that of the wild type.
  • Mutants pBKlM15 and pBKlM16 produced no antigen in the HeLa cell cultures. When HeLa rev cells were transfected with the negative and positive controls or with the mutants, mutants pBKlM12, pBKlM13 and pBKlM4 produced the same amount of a p27 antigen as the wild type pBKl.
  • mutant pBKlM15 produced about 30% of the antigen as compared with the wild type, while the p27 antigen production by the pBKlM16 mutant was negative.
  • the infectious virus could, however, still be rescued from the HeLa rev/pBKlM16 cultures by infecting the MT-4 cells (see Table 4 below).
  • all proviruses produced a virus with a comparable efficiency, but again, mutant pBKlM16 was less effective and produced 3 to 4 times less antigen than the original pBKl. These experiments were repeated three times.
  • the absolute value of p27 core antigen pro- duction varied, but the proportions in the level of repli ⁇ cation (p27 antigen production) between different mutants and the wild type pBKl provirus remained consistent.
  • the crucial difference in the pBKlM15 and pBKlM16 mutants compared with the wild type pBKl provirus and with the other mutants is the stop codon at position 50. As this stop codon prevents the translation of the C-terminal part of the REV protein and thus produces only truncated proteins without the transactivation region, these mutants should theoretically behave as dominantly negative and inhibit the action of wild type REV and REX. This is, how ⁇ ever, not the case with pBKlM15 and pBKlM16, and this is due to the fact that the RRE binding region (RBR) of the REV protein has also been changed.
  • RBR RRE binding region
  • the cells were washed twice with phosphate buffered saline (PBS, 0,02 M K/Na phosphate, 0.13 M NaCl, pH 7.4) to re- move the unbound virus and supplemented with an RPMI-1640 medium containing 10% FCS, 100 U of penicilline and 100 ⁇ g/ml of streptomycin.
  • PBS phosphate buffered saline
  • FCS phosphate buffered saline
  • the cells were fed every third day by adding a fresh culture medium, and aliquots for a SIV p27 core antigen assay were collected first at 12 hours after infection and thereafter weekly.
  • the results of the infection experiments are presented in Table 4.
  • pBKlM12 mutant behave as attenuated viruses. Lymphocytes can be infected with these viruses, but upon infection they produce either no viral structural proteins (pBKlM15 and pBKlM16) or only low amounts of viral structural proteins (pBKlM12). These mutants can therefore be used as live attenuated vaccines.
  • Example 5
  • the SIV rev defective pBKlM15 was selected for the in vivo vaccine experiments with cynomolgus monkeys (Macaca fascicularis). As shown above, this clone is totally rev deficient, but can readily be transcomple ⁇ mented with REV and REX; and thus a high amount of an in ⁇ fectious virus can be obtained from the MT-4 cultures in ⁇ fected with the supernatant from HeLa rex transfections.
  • the vaccine virus was consequently produced as follows:
  • HeLa rex cells were transfected with 5 ⁇ g of plasmid DNA/semiconfluent 25 cm 2 flask, as described in detail in Example 4A. The supernatants were harvested at 24 hours post transfection, centrifuged at 2000 rpm and filtrated through 0.2 ⁇ m disposable filter. The superna- tants were used to infect 10 7 MT-4 cells which were then cultured for 20 days. At days 4, 8, 12, and 16 post in ⁇ fection, fresh noninfected MT-4 cells were added, together with a fresh tissue culture medium (RPMI-1640/10% FCS).
  • RPMI-1640/10% FCS fresh tissue culture medium
  • Blood samples were taken from the monkeys at day 5 and 9, and thereafter every two weeks. They were tested for virus isolation and for the presence of a p27 core antigen in plasma.
  • peripheral blood mononuclear cells (10 6 /ml) were cultured with PHA (10 ⁇ g/ml) up to four weeks, and the supernatant was analyzed for virus production by the p27 antigen assay.
  • DNA PCR using SIV gag gene specific primers was used to demonstrate viral DNA in the peripheral blood lympho ⁇ cytes. The PCR was performed essentially as described in Example 1.
  • venous blood was drawn from the monkeys at days 5, 9, 24 and 35 post inoculation.
  • the mononuclear cells were isolated by Hypaque-isopague (Phar- macia) centrifugation.
  • MT-4 cells infected with the same clone and producing large amounts of the virus were used.
  • the infected MT-4 cells were mixed with non-infected MT-4 cells in different proportions.
  • 10 5 cells were used for the PCR assay by using primers spe- cific for the SIV, ⁇ gag gene.
  • the samples were electrophoresized in agarose gel electropho ⁇ resis and viewed under UV light. The results are shown in figure 7.
  • Antibody response to SIV structural proteins was determined by the Western blotting method.
  • Antibody acti ⁇ vity towards overlapping synthetic peptides, representing the SIV NEF protein was also performed by the ELISA method described by Ovod et al. (AIDS 6: 25 - 34, 1992).
  • synthetic 16-mer peptides overlapping by 8 amino acids were synthesized by the Zinzer peptide synthesizer and the instructions given by the manufacturer.
  • the pep ⁇ tides were dissolved in 0.15 M carbonate buffer, pH 9.3, at a concentration of 10 ⁇ g/ml and the ELISA plates (NUNC, Roskilde, Denmark) were incubated with the peptide mix ⁇ tures for 24 hours (100 ⁇ l per well).
  • the plates were then washed with PBS, and PBS containing 5 % skimmed milk powder was added to block the nonspecific binding. After an additional washing with PBS, the monkey sera diluted 1:100 in PBS containing 2 % normal goat serum were added and the plates were incubated at 4"C overnight. The plates were rewashed, peroxidase conjugated rabbit anti-monkey IgG (Cappel Laboratories, West Chester, PA, USA), was added and the plates were incubated for 1 hour. The plates were rewashed, peroxidase conjugated goat anti-rabbit IgG was added at a 1:1000, dilution and the plated were incu ⁇ bated for 1 hour at room temperature.
  • peroxidase conjugated rabbit anti-monkey IgG Cappel Laboratories, West Chester, PA, USA
  • the rev deficient SIV mutant, pBKlM15 thus behaves as an ideal live attenuated vaccine. It causes a transient infection which is abolished by the immune response raised in the vaccinated animal.
  • a booster injection was given.
  • the booster virus was given in the form of infective autologous T cells.
  • 100 ml of M15-infected culture of MT-4 cells (pas ⁇ sage 8, showed strong syncytia formation) was centrifuged lOOOg for 10 min. The supernatant was filtered through a 450 nm filter and concentrated 10 fold with Centriprep-30 R (Amicon Inc. Beverly, MA, USA ) cartridges.
  • the concen ⁇ trated supernatant was purified by ultracentrifugation for 150 min at 100,000g through 15% (w/v) sucrose onto 60% sucrose cushion. Fractions were collected, assayed for p27 antigen and after titration the viral stock was stored in aliquots at -70°C. This M15 viral stock was used to in ⁇ fect PBMC from the two vaccinated monkeys on the 3rd day after stimulation with PHA by adding 3 ml of Ml5 virus stock to 9xl0 6 PHA-blasts. Samples of 5xl0 5 cells were taken prior to infection, immediately after infection as well as 24h and 48h post infection for later PCR analysis.
  • the PHA blasts (5xl0 6 ) were centrifuged, washed once with sterile PBS (phosphate- -saline), resuspended in 5 ml of sterile PBS and the in ⁇ fected cells were introduced to monkeys intra venously. At this occasion, the monkeys received an estimated amount of 10 6 infected cells.
  • sterile PBS phosphate- -saline
  • the two vaccinated and two control monkeys were challenged with 100 monkey infectious doses of titrated SIVmac251 isolate, grown in monkey peripheral blood leuko- cytes (a generous gift from Dr. Ronald Desrosiers, New England Primate Center, Harvard School of Medicine, USA).
  • the outcome of the challenge experiment illustrated in table 5 shows classical viremia followed by seroconversion in the nonvaccinated animals. Wild-type SIV could regular- ly be isolated from their peripheral blood lymphocytes.
  • p27 antigen assay was constantly negative, but virus isolation was positive at three weeks. There ⁇ after, virus isolation has been constantly negative in the vaccinees.
  • Peripheral blood mononuclear cells were separated with NycoPrep TM 1.077Animal (Nycomed Pharma ASD, Oslo
  • SI virus was isolated by co-culturing 10 6 blood mononuclear cells with 5xl0 6 174xCEM with 10 ⁇ g/ml ConA and lOU/ml recombinant human IL-2. Fresh medium (RPMI 1640 supplemented with 10% fetal calf serum) was added weekly. Supernatant was sampled and tested for SIV p27 antigen. Repeatedly positive samples with increasing p27 concentration were considered posi ⁇ tive. Faint PCR reactivity was seen in one of the vac ⁇ cinees still at 6 weeks, while in the other the PCR test became negative.
  • PCR detection of SIV pro ⁇ viral DNA was used: Synthetic oligos 5'TTA GGC TAC GAC CCG GCG GAA AGA3' and 5'ATA GGG GGT GCA GCC TTC TGA CAG3' were applied as gag primers. As a gag-specific hybridization probe 5'ACT GTC TGC GTC ATC TGG TG3' (Stahl-Hennig et al. 1992) was used. The oligomers were synthesized using com ⁇ dismissal DNA syntehesis kit and MilliGen DNA synthesizer (Millipore Corporation, USA).
  • the DNA from 100,000 monkey peripheral blood mononuclear cells was extracted as described (Albert and Feny ⁇ 1990) and was supplemented with 10 pmol both of gag primers, 5 ⁇ l of lOxTaq buffer (Promega Corporation, USA), dNTPs up to 125 ⁇ M (Pharmacia Biotech, Sweden), water to 50 ⁇ l, 2 units of Taq polymerase (Boehringer-Mannheim, Germany) and the amp ⁇ lification reaction was carried out for 35 cycles.
  • the gel was blotted onto HybondN membrane (Amersham Ltd., UK), hybridized with 32 P- labelled gag probe at 40°C in lOxSSC, 0.1% SDS, 100 ⁇ g/ ml herring sperm DNA, 5xDenhardt's and washed twice in 2xSSC, 0.1%SDS at 40°C and twice at 55°C.
  • the filters were ex ⁇ posed to X-ray film overnight.
  • CD4 lymphocytes Percentage of CD4- and CD8- positive lymphocytes were stained from whole blood using 0KT4-phycoerytrin (Ortho) and anti-leu2a-FITC (Becton- Dickinson), erythrocytes lysed with FACS Lysing Solution, and subsets analyzed with FACScan flow cytometer, as in ⁇ structed by the manufacturer.
  • CD4 positive helper T-lymphocytes fell in the non-vaccinated control animals to about half of the normal value (from 45% to 20.4% in monkey #3, from 43% to 25% in monkey #4), while no change could be seen in the vaccinees (from 36.1% to 33.2% in monkey #1 and from 50% to 48.2% in monkey #2).
  • Table 5 Plasma antigenemia, virus isolation (p27 produc- tion by cultured PBMC) and PCR data from 100,000 PBMC of two M15 immunized Macaca fascicularis monkeys and from two control monkeys after challenge with monkey-grown SIV
  • an analogous attenuated live HIV-1 vaccine can be constructed.
  • Such a construct is prepared by the same principles as with the described SIV mutants.
  • the transactivation region of the HIV REV is abolished by deletions and/or by stop codons before the transactivation region but after the RRE bind- ing region (RBR).
  • RBR RRE bind- ing region
  • the cons ⁇ gagt will synthesize a truncated HIV-1 REV molecule which is nonactive and which does not inhibit the transacti- vating effect of HTLV-I REX.
  • Figure 9 One possible way of producing such an HIV-1 mutant is depicted in Figure 9.
  • MT-4 cells are grown in large containers to produce high amounts of the vaccine virus.
  • the vaccine virus is purified from the supernatant by concentration with ultrafiltration, followed by ultracentrifugation in a sucrose gradient against a 60% sucrose cushion.
  • the su ⁇ pernatant can be used as such for vaccination purposes, after filtration through a 0.2 ⁇ m filter.
  • HIV-1 SF2 which is a stan ⁇ dardized challenge virus stock readily available
  • the HIV-1 SF2 proviral DNA is cleaved with the restriction endonucleases Xhol and Sad, and the 2.9 kb fragment is inserted into pBluescript S/K (+) vector digested with Xhol and Sad.
  • the recombi- nant plasmid pSF29X/S thus obtained is grown in the LB- medium supplemented with ampicillin and the culture is coinfected with a helper virus such as M13K07.
  • the pSF29 will be packed into virus-like particles and after purifi- cation of the single-stranded pSF29 DNA, the part of the rev gene encoding RRE responsive region is altered by using specific primers and the Oligonucleotide-directed in vitro mutagenesis system version 2.1 kit (Amersham, Buckinghamshire, England).
  • the primers used can be commer- cially available primers or can be synthetized e.g. by using the Cyclone DNA synthesizer (Millipore, MA, USA).
  • the primers used in the production of HIV mutants are syn ⁇ thetized by using the Cyclone DNA synthesizer (Millipore, MA, USA).
  • the primer containing a specific mutation (or mutations) is (are) annealed to the template single-stranded pSF29 DNA and the complement ⁇ ary strand is synthetized in an appropriate buffer in the presence of dNTPs, one of which is chemically modified to make the nascent strand resistant to the cleavage with Neil endonuclease, by using a Klenow enzyme.
  • the template DNA will then be nicked by Neil digested with exonuclease, and after nuclease inactivation, the new strand will be synthetized with a Klenow enzyme.
  • the DNA is then trans- formed into E. coli TGI cells, and randomly picked colo ⁇ nies are sequenced to specify mutations introduced.
  • the mutant plasmid is then be digested with Aval and the 0,5 fragment is reinserted to the parent plasmid cleaved with Aval. Then the resulting plasmid, called e.g. pMUTANT 1.1, is digested with Drain and Xhol restriction endonucleases and the 2.3 kb insert is again reinserted into the parent proviral plasmid pHIVSF2 to obtain pHIVSF- 2M1.
  • This and the other mutants are analyzed by transfecting HeLa, Hela rev anf HeLa rex cell lines. The supernatants are transferred to cultures of MT-4, H9 and human PBMC cells to monitor the infectivity of the mutant viral progeny.
  • the viral p24 antigen content in culture supernatants is determined as a measure of the rate of replication of specific mutants in given cell cultures. Additionally, the effect of each altered RRR bind ⁇ ing domain on viral replication is tested also in cotrans- fection experiments. After the in vitro experiments, the virus is purified by ultracentrifugation and tested in chimpanzees in which it is administered either intrave- nously and/or intravaginally.
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO: 2: TGACGGTTGG TCTATCC
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO: 3: GCCAGGAGGG AGACGGTGGA 20
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 7: GAGAGGGCCG TGGCAACAGC T
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 8: CTCTGCCACC TCTCCCGGC 19
  • MOLECULE TYPE DNA (genomic)

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Abstract

La présente invention se rapporte à de nouveaux virus de l'immunodéficience humaine et simienne (respectivement HIV et SIV), présentant au moins une mutation dans le gène r^_e^_v^_ (le régulateur de l'expression protéine-virion) du génome d'HIV ou de SIV produisant une déficience de réplication pour le gène r^_e^_v^_, et qui peuvent subir une complémentation en trans. La présente invention se rapporte également à des procédés de production desdits virus à mutation à des vaccins contenant ces virus vivants et atténués, ainsi qu'à des procédés de prévention et/ou de traitement d'infections par HIV et/ou SIV ou du syndrome de l'immunodéficience acquise (SIDA) et de maladies apparentées chez les primates, y compris les humains, par l'administration, à toute personne nécessitant un tel traitement, d'un vaccin contenant lesdits virus à mutation afin de protégér ladite personne contre le virus HIV et/ou SIV virulent de phénotype sauvage.
PCT/FI1994/000335 1993-08-06 1994-08-03 Nouveaux virus de l'immunodeficience humaine et simienne a mutation et vaccins contenant lesdits virus WO1995004546A1 (fr)

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WO1996027389A1 (fr) * 1995-03-08 1996-09-12 Neovacs Immunogenes denues de toxicite derivant d'une proteine de regulation retrovirale, anticorps, procede de preparation et compositions pharmaceutiques les renfermant
FR2731355A1 (fr) * 1995-03-08 1996-09-13 Neovacs Nouveaux immunogenes, nouveaux anticorps, procede de preparation et compositions pharmaceutiques les renfermant
WO1997032983A1 (fr) * 1996-03-05 1997-09-12 The Regents Of The University Of California Virus de l'immunodeficience feline vivants recombines et vaccins d'adn proviraux
US6200575B1 (en) 1996-03-07 2001-03-13 Neovacs Non-toxic immunogens derived from a retroviral regulatory protein antibodies preparation process and pharmaceutical compositions comprising them

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EP0406557A2 (fr) * 1989-05-25 1991-01-09 Sandoz Ltd. Répresseur multivalent de gènes
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996027389A1 (fr) * 1995-03-08 1996-09-12 Neovacs Immunogenes denues de toxicite derivant d'une proteine de regulation retrovirale, anticorps, procede de preparation et compositions pharmaceutiques les renfermant
FR2731355A1 (fr) * 1995-03-08 1996-09-13 Neovacs Nouveaux immunogenes, nouveaux anticorps, procede de preparation et compositions pharmaceutiques les renfermant
US6132721A (en) * 1995-03-08 2000-10-17 Neovacs Non-Toxic immunogens derived from a retroviral regulatory protein, antibodies, preparation method therefor, and pharmaceutical compositions containing same
WO1997032983A1 (fr) * 1996-03-05 1997-09-12 The Regents Of The University Of California Virus de l'immunodeficience feline vivants recombines et vaccins d'adn proviraux
US6004799A (en) * 1996-03-05 1999-12-21 The Regents Of The University Of California Recombinant live feline immunodeficiency virus and proviral DNA vaccines
US6200575B1 (en) 1996-03-07 2001-03-13 Neovacs Non-toxic immunogens derived from a retroviral regulatory protein antibodies preparation process and pharmaceutical compositions comprising them

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