WO1992006212A1 - Expression amelioree de proteines virales dans des cellules de drosophila - Google Patents

Expression amelioree de proteines virales dans des cellules de drosophila Download PDF

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WO1992006212A1
WO1992006212A1 PCT/US1991/006838 US9106838W WO9206212A1 WO 1992006212 A1 WO1992006212 A1 WO 1992006212A1 US 9106838 W US9106838 W US 9106838W WO 9206212 A1 WO9206212 A1 WO 9206212A1
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protein
drosophila
hiv
gene expression
expression unit
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Mona Ivey-Hoyle
Martin Rosenberg
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Smithkline Beecham Corporation
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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
    • 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
    • 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
    • C12N2740/16122—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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element
    • 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
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • 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
    • C12N2830/75—Vector systems having a special element relevant for transcription from invertebrates

Definitions

  • the present invention relates generally to enhanced expression of viral proteins, and in particular
  • HIV-l Human immunodeficiency virus type 1
  • LTRs long terminal repeats
  • the viral envelope glycoprotein (gpl60) or specific fragments thereof.
  • the env gene encodes the 16.0 kilodalton (kd) precursor glycoprotein of the viral envelope.
  • gpi ⁇ O is cleaved posttranslationally into a 120 kd glycoprotein (gpl20) and a 41 kd glycoprotein (gp41), which are present at the virus surface.
  • gpl20 a 481 amino acid glycoprotein, is derived from the amino terminal two-thirds of the gpl60 glycoprotein. It is exposed on the outside of the virus, and is crucial to the interaction of the virus with its cellular receptor by binding to the CD4 protein present on the surface of helper T.
  • gpl20 thus determines the cellular selectivity of viral infection and contributes to the cytopathogenicity of HIV through its involvement in syncytium formation.
  • gp41 a 345 amino acid protein derived from the carboxyl terminus of gpl60, is an integral membrane protein of HIV-l.
  • gp41 contains a series of hydrophobic amino acids which anchor the protein in the lipid bilayer of the cellular plasma membrane. The carboxyl end of gp41 is believed to protrude into the viral particle.
  • gp41 or a portion thereof is believed to "anchor" gpi20 to HIV and is also responsible for fusion between HIV or HIV-infected cells with uninfected cells displaying surface T. receptors.
  • the portion of gp41 which is believed to be responsible for this fusion is located at the amino terminus. Such fusion is believed to play a role in viral replication. See, e.g., M. Kowalski et al. Science, 237: 1351-55 (1987); D.M. Knight et al, Science, 236: 837-36 ( 1987 ) .
  • viral glycoproteins assume a tertiary structure as viral spikes protruding outwards from the surface of the viral particle. About 70 to 80 spikes are believed to be associated with each newly synthesized viral particle. As the viral particle ages, the spikes disappear, apparently because the association between the gpl20 and gp4l is weak. Thus, for newly synthesized viral particles, this viral glycoprotein spike is believed to be the most immediate target accessible to the immune system following infection.
  • Virus neutralizing antibodies have been reported directed against gpl20 and gp41 epitopes. It has been specifically noted that a target site for type specific neutralizing antibodies is located in the 3' half of the gpl20 glycoprotein molecule.
  • glycosylated gpl60 has previously been obtained in mammalian cells and certain baculovirus insect cells by groups which have also reported the induction of both humoral and cellular immune responses to these antigens.
  • gpl20 has been expressed recombinantly with the use of heterologous promoters in several systems. See, e.g., S. Chakrabarti et al, Nature (London), 320: 535 (1986); S.I. Hu et al. Nature (London), 320: 537 (1986); and M.P. Kieny et al, Biotechnology, 4: 790 (1986).
  • L.A. Lasky et al, Science, 233: 209-212 (1986) constructed a number of plasmids containing mutant env genes for tranfection into mammalian cells, specifically Chinese hamster ovary (CHO) cells.
  • Lasky et al. report secretion of a gene product encoded by a plasmid containing the first 50 amino acids of the glycoprotein D (gD) protein joined in phase to an amino acid sequence comprising (#61-#531) of the HIV env protein.
  • a recombinant envelope antigen was produced containing 25 amino acids of gD at its amino terminus. The resulting gene was 520 amino acids in length.
  • U.S. Patent 4,725,669 also discloses glycoproteins of approximately 160 kd and 120 kd obtained from the human H9/HTLV-III cell line, each having an approximately 90 kd unglycosylated moiety.
  • the HIV-l virus also encodes two regulatory proteins, tat and rev, which govern viral gene expression and which are essential for virus replication.
  • the tat protein increases the expression of both structural and regulatory proteins of HIV while the rev protein selectively increases the synthesis of structural proteins.
  • rev function The precise mechanism of rev function remains unknown. It is known that rev is primarily localized in the nucleolus. This localization is thought to be important for rev function. Hence it is thought that rev regulates gene expression by facilitating export of the nuclear-entrapped mRNA into the cytoplasm. Rev has also been shown to function in a number of different mammalian cell types, e.g., human, monkey and hamster. However, rev regulation has not been demostrated in any non-mammalian system.
  • the present invention is an HIV rev expression unit which includes a DNA coding sequence and regulatory sequences necessary for transcription of the rev protein coding sequence and subsequent translation within a Drosophila cell.
  • this invention is a DNA vector which compri ⁇ as the gene expression unit of the present invention.
  • this invention is a Drosophila cell transfected with the DNA vector of this invention.
  • this invention is an HIV rev protein, or a derivative thereof produced by the transfected cells of this invention.
  • the derivative encompasses any rev protein such as deletions, additions, substitutions or rearrangement of amino acids or chemical modifications thereof which retain the ability to be recognized by antibodies raised to the wild-type rev protein.
  • This invention also relates to a method for enhancing the production of viral proteins in insect cells.
  • the method entails culturing Drosophila cells transfected with a gene expression unit for a viral protein of interest and a rev expression unit in a suitable medium such that the transfected cells are capable of expressing the protein of interest.
  • the protein may thereafter be collected from the cell or cell culture medium.
  • this invention is a whole cell vaccine for stimulating protection against HIV infection, which comprises an immunoprotective and non-toxic quantity of an HIV protein associated with an inactivated Drosophila cell.
  • This invention further relates to a method for protecting a human against disease symptoms associated with HIV infection, which comprises administering to a human a safe and effective amount of the whole cell vaccine of the present invention.
  • the method and expression system of the present invention facilitate high-level production of viral proteins, particularly HIV env proteins and derivatives thereof, in a Drosophila cell culture.
  • the Drosophila cells are transfected by using standard techniques which permit introduction of .foreign DNA into a host cell without adversely affecting the foreign DNA or the host cell.
  • the recombinant Drosophila cells so constructed produce viral proteins.
  • One feature of the present invention is the enhanced expression of viral structural proteins (e.g., env, pol, and gag) when coexpressed with the rev protein in Drosophila.
  • viral structural proteins e.g., env, pol, and gag
  • the rev protein appears to be fully functional when produced by the present invention.
  • the HIV-l env protein, gpl60 is barely expressed in the absence of rev.
  • the levels of gpl60 are enhanced (5 to 10 fold).
  • RNA was dependent on induction of the Drosophila Mt promoter and was independent of Rev.
  • an analysis of fractionated RNA revealed that full-length, unspliced gpl60 mRNA was found in the cytoplasm only in the presence of Rev. In the absence of Rev, this RNA was apparently retained in the nucleus.
  • the method of this invention provides a continuous cell expression system for HIV proteins.
  • the protein of the present invention may be secreted, and purification from the culture medium is by conventional techniques.
  • the protein of the present invention may be produced intracellularly or membrane-bound, and the protein may be extracted from the cells using conventional techniques.
  • membrane-bound protein may be employed in a variety of cell-associated assays, or used as a whole-cell vaccine.
  • a preferred Drosophila cell line for use in the practice of the invention is the D. melanoqaster S 2 line.
  • S, cells [Schneider, J. Embryol. Exp. Morph. 27: 353 (1972)] are stable cell cultures of polyploid embryonic Drosophila cells.
  • Introduction of the DNA coding sequence for gpl20, or derivatives thereof, into Drosophila S 2 cells by DNA transfection techniques produces unexpectedly large amounts of the glycoprotein.
  • Use of the S, Drosophila cell has many advantages, including, but not limited to, its ability to grow to a high density at room temperature. Stable integration of the selection system has produced up to 1000 copies of the transfected gene expression unit into the cell chromosomes.
  • Drosophila cell culture systems may also be useful in the present invention.
  • Some possibly useful cells are, for example, the KC-O Drosophila Welanoqaster cell line which is a serum-free cell line [Schulz et al, Proc. Nat'l Acad. Sci. USA, 83: 9428 (1986)]. Preliminary studies using the KC-0 line have suggested that transfection is more difficult than with S 2 cells.
  • Another cell line which may be useful is a cell line from Drosophila hydei. Protein expression can be obtained using the hydei cell line; however, transfection into this cell line can result in the transfected DNA being expressed with very low efficiency [Sinclair et al, Mol. Cell. Biol., 5. 3208 (1985)].
  • Other available Drosophila cell lines which may be used in this invention include S- and S 3 .
  • the Drosophila cells selected for use in the present invention can be cultured in a variety of suitable culture media, including, e.g., M 3 medium.
  • M 3 medium consists of a formulation of balanced salts and essential amino acids at a pH of 6.6. Preparation of the media is substantially as described by Lindquist, PIS, 58: 163 (1982). Other conventional media for growth of Drosophila cells may also be used.
  • a recombinant DNA molecule or vector containing a viral protein gene expression unit can be used to transfect the selected Drosophila cells, according to the invention.
  • the gene expression unit contains a DNA coding sequence for a selected viral protein or for a derivative thereof.
  • Such derivatives may be obtained by manipulation of the gene sequence using traditional genetic engineering techniques, e.g., mutagenesis, restriction endonuclease treatment, ligation of other gene sequences including synthetic sequences and the like. See, e.g., T. Maniatis et al. Molecular Cloning, A Laboratory Manual., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
  • the HIV DNA coding sequence which includes rev, has been published. See, Ratner et al, Nature 313:277-284 (1985) or Wain-Hobson et al, .Cell 40:9-17 (1985).
  • the nucleotide sequence is also available from GenB.ank (clone BH10, Ratner et al, supra) ⁇
  • DNA molecules comprising the coding sequence of this invention can be derived from HTLV-III infected cells using known techniques (see, Hahn et al. Nature 312:166-169 (1984)), or, in the alternative, can be synthesized by standard oligonucleotide techniques, or via PCR. Moreover, there are numerous recombinant host cells containing the cloned DNA coding sequences, which are widely available.
  • Derivatives can then be prepared by standard techniques, including DNA synthesis. Such derivatives may include, e.g., rev, gpl20 or gpl60 molecules in which one or more amino acids have been substituted, added or deleted without signi icantly adversely affecting the binding capacity or biological characteristics of the protein. Derivatives of these proteins may also be prepared by standard chemical modification techniques, e.g., acylation, methylation.
  • the regulatory region typically contains a promoter region which functions in the binding of RNA polymerase and in the initiation of RNA transcription.
  • the promoter region is found upstream from the protein coding sequence.
  • Preferred promoters are of Drosophila origin, e.g., the Drosophila metallothionein promoter [Lastowski-Perry et al, J. Biol. Chem., 260: 1527 (1985)].
  • This inducible promoter directs high-level transcription of the gene in the presence of metals, e.g., CuSO..
  • metals e.g., CuSO.
  • Use of the Drosophila metallothionein promoter results in the expression system of the invention retaining full regulation even at very high copy number. This is in direct contrast to the use of the mammalian metallothionein promoter in mammalian cells in which the regulatory effect of the metal is diminished as copy number increases. In the Drosophila expression system, this retained inducibility effect increases expression of the gene product in the Drosophila cell at high copy number.
  • the Drosophila actin 5C gene promoter [B.J. Bond et al, Mol. Cell. Biol., 6 : 2080 (1986)] is also a desirable promoter sequence.
  • the actin 5C promoter is a constitutive promoter and does not require addition of metal. Therefore, it is better-suited for use in a large scale production system, like a perfusion system, than is the Drosophila metallothionein promoter.
  • a additional advantage is that the absence of a high concentration of copper in the media maintains the cells in a healthier state for longer periods of time.
  • Drosophila promoters examples include, e.g., the inducible heatshock (Hsp70), the COPIA LTR, and the ⁇ -tubulin promoters.
  • Hsp70 inducible heatshock
  • COPIA LTR the COPIA LTR
  • ⁇ -tubulin promoters examples include, e.g., the inducible heatshock (Hsp70), the COPIA LTR, and the ⁇ -tubulin promoters.
  • the SV40 early promoter gives lower levels of expression than the Drosophila metallothionein promoter.
  • Promoters which are commonly employed in the mammalian cell expression vectors including, e.g., avian Rous sarcoma virus LTR and simian virus (SV40 early promoter) demonstrate poor function and expression in the Drosophila system.
  • a gene expression unit or expression vector for the viral protein of interest may also be constructed by fusing the viral protein coding sequence to a desirable signal sequence.
  • the signal sequence functions to direct secretion of the protein from the host cell.
  • a signal sequence may be derived from the sequence of tissue plasminogen activator (tPA) .
  • tPA tissue plasminogen activator
  • Other available signal sequences include, e.g., those derived from Herpes Simplex virus gene HSV-I gD [Lasky et al. Science, supra.].
  • the DNA coding sequence for the protein of interest may also be followed by a polyadenylation (poly A) region, such as an SV40 early, or SV40 late, or metallothionein poly A region.
  • poly A region which functions in the polyadenylation of RNA transcripts appears to play a role in stabilizing transcription.
  • a similar poly A region can be derived from a variety of genes in which it is naturally present. This region can also be modified to alter its sequence provided that polyadenylation and transcript stabilization functions are not significantly adversely affected.
  • the recombinant DNA molecule may also carry a genetic selection marker, as well as the viral protein gene.
  • the selection marker can be any gene or genes which cause a readily detectable phenotypic change in a transfected host cell. Such phenotypic change can be, for example, drug resistance, such as the gene for hygromycin B resistance.
  • a selection system using the drug methotrexate, and prokaryotic dihydrofolate reductase (DHFR) gene can be used with Drosophila cells.
  • the endogenous eukaryotic DHFR of the cells is inhibited by methotrexate. Therefore, by transfecting the cells with a plasmid containing the prokaryotic DHFR which is insensitive to methotrexate and selecting with methotrexate, only cells transfected with and expressing the prokaryotic DHFR will survive.
  • methotrexate can be used to achieve initially high-copy number transfectants. Only cells which have incorporated the protective prokaryotic DHFR gene will survive. Concomitantly, these cells have the gene expression unit of interest.
  • a recombinant DNA molecule or expression vector containing the viral protein gene expression unit and the rev gene expression unit can be transfected into the Drosophila cell using standard transfection techniques.
  • transfection techniques include, for example, calcium phosphate co-precipitation, cell fusion, electroporation, microinjection and viral transfection.
  • a one, two, or three vector system can be used in the present invention to transfect a Drosophila host cell.
  • the gene expression unit for the desired protein e.g., an HIV env protein or derivative
  • the rev expression unit and the coding region for a selectable marker are all located on different vectors. It is noted that all three elements, the desired protein expression unit, the rev expression unit, and the selectable marker can also be found on one or two vectors.
  • a preferred illustrative embodiment of this invention is the production of an HIV env protein employing a vector containing an HIV protein expression unit, e.g., pgpl60 ⁇ 32, a vector containing the rev expression unit, e.g., pMtRev, and a vector containing the hygromycin B gene expression unit, e.g., pCOHYGRO.
  • pgpl60 ⁇ 32 contains an expression unit comprising the Drosophila metallothionein promoter, a derivative of the gpl60 gene, and the SV40 poly A site. This gpl60 expression unit in combination with rev and the pCOHYGRO vector system will produce a gpl60 derivative in S 2 Drosophila cells.
  • the antibiotic hygromycin B can be used to select for those cells containing the transfected vectors.
  • a more complete description of this embodiment is described in Example 2.
  • an expression system employing the DHFR gene/methotrexate selection system consisting of the vectors pgpl60 ⁇ 32, pMtRev and pHGCO, can be used to select methotrexate-resistant cells expressing gpl60 or a derivative thereof.
  • the pHGCO vector comprises a DHFR gene expression unit and is co-transfected with pgpl60 ⁇ 32 and pMtRev, thereby providing the DHFR gene necessary for selection.
  • the vectors are ..transfected into Drosophila S 2 cells using conventional techniques.
  • Vectors containing the protein expression unit of interest (e.g., HIV gpl60) and the rev expression unit are preferably present in the same molar ratios.
  • the vector having the coding sequence for the selectable marker may be added in varying ratios depending upon the particular copy number of the gene of interest desired.
  • transfected cells are then selected, such as in 3 medium containing serum and the appropriate selection agent, e.g., hygromycin B or methotrexate.
  • the appropriate selection agent e.g., hygromycin B or methotrexate.
  • the expression of gpl60 is induced by the addition of an appropriate inducing agent for the inducible promoter.
  • an appropriate inducing agent for the inducible promoter For example, cadmium or copper are inducing agents for the metallothionein promoter. Heat is the inducing agent for the Hsp70 promoter.
  • constitutive promoters such as the actin 5C promoter, no inducing agent is required for expression.
  • Transcription and expression of the viral protein coding sequence in the above-described systems can be monitored. For example. Southern blot analysis can be used to determine copy number of the gpl60 gene. Northern blot analysis provides information regarding the size of the transcribed gene sequence [see, e.g., Maniatis et al, cited above] . The level of transcription can also be quantitated. Expression of the selected HIV protein in the recombinant cells can be further verified through Western blot analysis and activity tests on the resulting glycoprotein.
  • Drosophila S_ cells are especially suited to high-yield production of protein in the method of the present invention.
  • the cells can be maintained in suspension cultures at room temperature (24+l°C) .
  • Culture medium is M 3 supplemented with between 5 and 10% (v/v) heat-inactivated fetal bovine serum (FBS) .
  • FBS heat-inactivated fetal bovine serum
  • the culture medium contains 5% FBS.
  • the cells may be cultured in serum-free media.
  • the media is also supplemented with 300 ⁇ g/ml hygromycin B. In this media, the S 2 cells can be grown in suspension cultures, for example, in 250 ml to
  • the cells are grown prior to induction in 1500 ml spinner flasks in media containing 5% serum.
  • the viral protein can be isolated from the spent media, e.g., by use of a monoclonal antibody affinity column.
  • Other known protein purification steps e.g., metal chelates, various affinity chromatography steps or absorption chromatography, can be used to purify the viral protein from the culture media.
  • glycoproteins produced by Drosophila cells are completely free of contaminating materials, e.g., mammalian, yeast, bacterial and more importantly, other (HIV) viral materials.
  • Drosophi1a-produced HIV proteins have also been demonstrated to possess different pattern of glycosylation than that reported by other systems, e.g., mammalian systems.
  • the HIV proteins and derivatives produced, according to the present invention may be useful in a variety of products.
  • these recombinant proteins may be used in pharmaceutical compositions for the treatment of HIV-infected subjects.
  • Such a pharmaceutical composition comprises a therapeutically effective amount of the HIV protein or derivative of the invention in admixture with a pharmaceutically acceptable carrier.
  • the composition can be systemically administered either parenterally, intravenously or subcutaneously.
  • the therapeutic composition for use in this invention is in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • the preparation of such a parenterally acceptable protein solution having due regard to pH, isotonicity, stability and the like, is within the skill of the art.
  • the dosage regimen will be determined by the attending physician, considering various factors which modify the action of drugs, e.g., the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors.
  • the pharmaceutical carrier and other components of a pharmaceutical formulation would be selected by one of skill in the art.
  • the recombinant proteins of the present invention may be used as whole cell vaccines to innoculate mammalian subjects against HIV infection.
  • the cells may be inactivated by physical (e.g., heat) or chemical means (e.g. addition of glutaraldehyde) .
  • physical e.g., heat
  • chemical means e.g. addition of glutaraldehyde
  • the preparation of vaccines is generally described in Voller et al. (eds.), New Trends and Developments in Vaccines, University Park Press, Baltimore, Maryland (1978).
  • the tPA expression vector pMTtPA (also called pDMtPA) was used.
  • This vector is a derivative of vector pMLl, a small pBR322 vector containing the beta-lactamase gene which has the poison sequences [Mellon et al. Cell, 27: 297 (1982)] deleted from it. These sequences are inhibitory to amplification of the vector.
  • This vector was digested with Sail and Aatll which removes a small piece of pBR322, and the digested ends were filled in.
  • Hindlll-Xbal fragment containing the entire env gene was isolated from an HIV-isolate clone BH10 [L. Ratner et al. Nature, 3_13:277-84 (1985); see also GenBank].
  • the entire gpl60 sequence was then inserted into a Ncol-Xbal digested vector pDSPl.
  • the resulting vector, SU2 was digested with Ndel, followed by treatment with mung bean nuclease and subsequently digested with Sacl, thus isolating the gpl60 gene.
  • the digestion with Ndel cut the gpl60 sequence at amino acid #32.
  • the Sacl digestion cuts 3' of the gpl60 gene, including part of the original pDSPl vector containing a polylinker. This fragment was inserted into the above-described expression vector pMTtPA which had been digested with Bqlll, end-filled, and subsequently cut with Sacl, which deletes the mature tPA sequence. This creates a coding sequence for the first 36 amino acids of tPA (i.e., signal sequence) fused to 795 amino acids of gpl60 beginning with amino acid number 32 (asp) of the mature viral molecule and ending at the natural gpl60 stop codon.
  • signal sequence i.e., signal sequence
  • Another vector containing a modified gene sequence was constructed by digesting pgpl60 ⁇ 32 with HindiII and Sacl, thereby removing the carboxyl terminus of gpl60. Approximately two-thirds of the sequence coding for gp41 is removed by this digestion. Thus, this gpl60 sequence is missing the first 31 amino acids and the last 216 amino acids of the natural gpl60 sequence. The deleted sequence at the carboxy terminus was replaced by a short synthetic DNA linker encoding a stop codon on an Hindlll-Sacl fragment. The 6-amino-acid linker sequence is as follows: 5'AGCTTTGACTGACTGAGCT 3' .
  • Yet another vector containing a mutant gpl60 gene was constructed by digesting pgpl60 ⁇ 32 with Styl and Xbal, thereby deleting all of the sequence for gp41 and about 30 amino acids at the carboxyl terminus of the gpl20 glycoprotein sequence.
  • This fragment was replaced by a synthetic Styl-Xbal linker sequence coding for the correct carboxyl terminus of gpl20 from the Styl site to the processing site of gpl20-gp41.
  • This sequence was followed by a stop codon.
  • This sequence thereby contained all of the coding sequence for gpl20 minus the first 31 amino acids and none of the gp41 coding sequence.
  • Still another exemplary vector containing a mutant gpl20 gene was constructed as follows: a 720-base pair carboxyl terminal fragment of gpl20 was isolated by a partial digestion of pgpl20 ⁇ 32 with Bglll followed by a Xbal digestion. This fragment was now inserted in place of the tPA gene into the Bglll-Xbal cut pMTtPA expression vector.
  • the resulting vector, pl20 ⁇ 274 contains a coding sequence for the. first 36 amino acids of tPA (i.e., the signal sequence) fused to amino acid number 275 of the mature gpl20 molecule.
  • An ApaLI-SacI fragment was isolated from plasmid pgpl60 ⁇ 32 containing the majority of the gpl60 coding sequence.
  • a Bglll-ApaLI fragment encoding the N-terminus of the mature gpl60 coding sequence was generated by the PCR technique using the natural gpl60 coding sequence from the BH10 clone (see (b)) as the template. (The Bglll site was introduced at the first codon of mature gpl60).
  • This Bglll-ApaLI fragment and the ApaLI-SacI fragment were used to replace the pgpl60 ⁇ 32 coding sequence which was removed by digestion with Bglll-Sacl.
  • the resulting vector encodes the entire mature gpl60 coding sequence and contains all of the regulatory elements as found in gpl60 ⁇ 32.
  • tPA coding sequence i.e., for the signal sequence and mature protein
  • pMtpolyA This plasmid is herein referred to as pMtpolyA.
  • pMtRev is then contructed by inserting an Xbal-Xhol fragment encompassing rev cDNA isolated from plasmid pH3art (Rosen et al., Proc Nat'l Acad Sci USA, 85:2071-6 (1988)) into the Xbal-Xhol sites of the polylinker region of pMtpolyA.
  • the resulting vector encodes the Drosophila metallothionein promoter, the rev protein, and the SV40 polyA region.
  • pUCOPIA A commercially available plasmid, pUC18 [BRL] containing a BamHI and Smal site was used.
  • the 5' LTR from an integrated COPIA element (357 base pairs) was cloned into the BamHI site of vector pUCl ⁇ , resulting in the vector designated pUCOPIA.
  • COPIA is a representative member of the disperse middle repetition sequences found scattered through the Drosophila genome [Rubin et al, in Cold Spring Harbor Symp. Quant. Biol., 45: 619 (1980)].
  • the vector pUCOPIA was cut at the Smal site and the E.
  • coli gene coding for hygromycin B phosphotransferase (hygromycin B cassette) was cloned into pUCOPIA using standard cloning techniques.
  • the hygromycin B cassette was isolated on a Hindlll-BamHI fragment of 1481 base pairs from the vector DSP-hygro [Gertz et al, Gene, 25: 179 (1983)].
  • the hygromycin B cassette contains the sequence coding for the hygromycin B phosphotransferase gene and the SV40 early poly A region.
  • the Hindi11 and BamHI sites were filled in using T. DNA polymerase, and the hygromycin B cassette was ligated into the Smal site of the vector pUCOPIA producing vector pCOHYGRO.
  • pCOHYGRO was transfected into S 2 Drosophila cells together with a vector carrying a gpl60 mutant gene (e.g., pgpl60 ⁇ 32) and the rev gene, both of which were under the control of the Drosophila metallothionein promoter as described above.
  • a total of 20 ⁇ g of plasmid DNA was used in each transfection which consisted of 10 ⁇ g of the hygromycin B selection plasmic pCOHYGRO and 10 ⁇ g total of pMtl60 ⁇ 32 and pMtRev.
  • the transfected cells were selected in M 3 medium containing
  • the protein encoded by gpl60 ⁇ 32 appears to be cleaved to produce a gpl20-sized molecule.
  • This gpl20-sized molecule rapidly dissociates from the cell and is found in the culture medium.
  • This gpl20 protein also recognizes and binds to a soluble form of the human CD4 protein and thus retains at least its receptor recognition properties.
  • the dissociation of the Drosophila expressed gpl20 molecule appears to be due to the fact that it is lacking the N-terminal 31 amino acids of the mature viral protein. Expression from an otherwise identical gpl60 construct (i.e., pgpi60 ⁇ 0) in which these 31 amino acids have been restored produces gpl20 which remains associated with the cells.

Abstract

La présente invention se rapporte à un nouveau procédé destiné à l'expression améliorée de protéines virales, et en particulier des glycoprotéines de VIH dans des cellules de Drosophila.
PCT/US1991/006838 1990-09-28 1991-09-20 Expression amelioree de proteines virales dans des cellules de drosophila WO1992006212A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994003620A2 (fr) * 1992-07-29 1994-02-17 Chiron Corporation Procede permettant d'accroitre l'expression de proteines virales
WO1995011307A1 (fr) * 1993-10-22 1995-04-27 Institut Pasteur Vecteur nucleotidique, composition le contenant et vaccin pour l'immunisation a l'encontre d'une hepatite
US5654398A (en) * 1993-06-03 1997-08-05 The Regents Of The University Of California Compositions and methods for inhibiting replication of human immunodeficiency virus-1
US6133244A (en) * 1993-10-22 2000-10-17 Institut Pasteur Method for immunization against hepatitis B
US7732423B2 (en) 1994-04-27 2010-06-08 Institut Pasteur Nucleotide vector, composition containing such vector, and vaccine for immunization against hepatitis

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US5008373A (en) * 1986-11-01 1991-04-16 Oxford Gene Systems Limited Fusion proteins and particles

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Bio/Technology, Vol. 9, issued February 1991, CULP et al., "Regulated Expression Allows High Level Production and Secretion of HIV-1 gp 120 Envelope Glycoprotein in Drosophila Schneider Cells", pages 173-177, see entire document. *
First International Conference on Gene Regulation, Oncogenesis, AIDS, issued 15-21 September 1989, ARTHOS et al., "Interaction of the HIV Envelope with human CD4 Receptor", see Abstract. *
International Conferences AIDS, Vol. 5, issued 4-9 June 1989, JOHANSEN et al., "Stable expression and secretion of recombinant HIV-1 envelope protein in Drosophila Schneider Cells", page 584, see meeting Abstract. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994003620A2 (fr) * 1992-07-29 1994-02-17 Chiron Corporation Procede permettant d'accroitre l'expression de proteines virales
WO1994003620A3 (fr) * 1992-07-29 1994-05-11 Procede permettant d'accroitre l'expression de proteines virales
EP1298215A3 (fr) * 1992-07-29 2007-09-26 Aventis Pasteur, S.A. Production de glycoprotéine H de CMV recombinante immunologiquement réactive et tronquée
EP1298215A2 (fr) * 1992-07-29 2003-04-02 Aventis Pasteur, S.A. Production de glycoprotéine H de CMV recombinante immunologiquement réactive et tronquée
US5767250A (en) * 1992-07-29 1998-06-16 Chiron Corporation Complexes comprising truncated CMV gH polypeptides and escort proteins
US5654398A (en) * 1993-06-03 1997-08-05 The Regents Of The University Of California Compositions and methods for inhibiting replication of human immunodeficiency virus-1
US6133244A (en) * 1993-10-22 2000-10-17 Institut Pasteur Method for immunization against hepatitis B
US6429201B1 (en) 1993-10-22 2002-08-06 Institut Pasteur Method for immunization against hepatitis B
FR2711670A1 (fr) * 1993-10-22 1995-05-05 Pasteur Institut Vecteur nucléotidique, composition le contenant et vaccin pour l'immunisation à l'encontre d'une hépatite.
US6635624B1 (en) 1993-10-22 2003-10-21 Institut Pasteur Nucleotide vector composition containing such vector and vaccine for immunization against hepatitis
WO1995011307A1 (fr) * 1993-10-22 1995-04-27 Institut Pasteur Vecteur nucleotidique, composition le contenant et vaccin pour l'immunisation a l'encontre d'une hepatite
US7825097B2 (en) 1993-10-22 2010-11-02 Institut Pasteur Nucleotide vector vaccine for immunization against hepatitis
US7732423B2 (en) 1994-04-27 2010-06-08 Institut Pasteur Nucleotide vector, composition containing such vector, and vaccine for immunization against hepatitis

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