WO1990006358A1 - Expression of hiv proteins in drosophila cells - Google Patents

Expression of hiv proteins in drosophila cells Download PDF

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Publication number
WO1990006358A1
WO1990006358A1 PCT/US1989/005155 US8905155W WO9006358A1 WO 1990006358 A1 WO1990006358 A1 WO 1990006358A1 US 8905155 W US8905155 W US 8905155W WO 9006358 A1 WO9006358 A1 WO 9006358A1
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protein
hiv
cells
gpl20
vector
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PCT/US1989/005155
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English (en)
French (fr)
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Hanne Ranch Johansen
Martin Rosenberg
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Smithkline Beecham Corporation
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Priority to JP02500753A priority Critical patent/JP3085704B2/ja
Priority to KR1019900701678A priority patent/KR0152525B1/ko
Publication of WO1990006358A1 publication Critical patent/WO1990006358A1/en
Priority to DK104991A priority patent/DK175461B1/da
Priority to FI912635A priority patent/FI113475B/fi
Priority to NO19912099A priority patent/NO314090B1/no

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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • 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
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    • 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
    • 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 expression of HIV proteins in Drosophila cells and purification of the expressed gene products. More specifically, this invention relates to the production of novel mutant gpl60 and gpl20 gene products by this expression system.
  • HIV-i Human immunodeficiency virus type 1
  • LTRs long terminal repeats
  • gag, pol, and env genes genes that are potential candidates either alone or in concert: as vaccinal agents capable of inducing a protective immune response.
  • the viral envelope glycoprotein (gpl ⁇ O) or specific fragments thereof.
  • the env gene encodes the 160 kilodalton (kd) precursor glycoprotein of the viral Onvelope.
  • gpl ⁇ 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 511 amino acid glycoprotein, is located on the amino terminal two-thirds of the gpl ⁇ O glycoprotein and is exposed on the outside of the virus.
  • gpi20 is crucial to the interaction of the virus with its cellular receptor, the CD4 protein present on the surface of helper T 4 lymphocytes, macrophages, and other cells of the immune system. gpl20 thus determines the tissue 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 gpl ⁇ O, 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 gp4l is believed to protrude into the viral particle.
  • gp41 or a portion thereof is believed to be responsible for fusion between the HIV glycoproteins expressed at the surface of the cell with cells displaying surface T. receptors.
  • the portion of gp41 which is believed to be responsible for this fusion is located at the amino terminal. 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 gp41 is weak. Thus, for newly synthesized v ⁇ ral 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. These researchers secreted a gene product encoded in a plasmid containing the first 50 .amino acids of the glycoprotein D (gD) protein joined in phase to an amino acid sequence (#61-#531) of the env protein, an HBsAg polyA signal, a DHFR gene and the SV40 origin of replication.
  • gD glycoprotein D
  • a recombinant envelope antigen was produced containing 25 amino acids of gD at its amino terminus and lacking 30 residues from the mature processed from of gpl20, and also having a deletion of the gp41 sequence (about 20 amino acids of the carboxyl terminus to the actual 160 kd precursor processing site) .
  • the resulting gene was 520 amino acids in length.
  • the cell-conditioned supernatants contained a 130 kd protein, called gpl30.
  • 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. 0 Fox, Biotechnology, 6 , : 116 (1988) reports the
  • VAXSYN HIV-l vaccine developed by MicroGeneSys. This report does not disclose any details of this vaccine.
  • the recombinant protein was immunoreactive with protein from HIV-infected H9 cells, with antisera to a recombinant fraction of gpi20, with gpl20 itself, with a peptide fragment of gp41, and with human AIDS sera.
  • P.J. Barr et al, Vaccine, 5_: 90 (1987) discloses the expression of HIV proteins in the yeast Saccharomvces cerevisiae. H. Johansen et al, 28th Annual Drosophila
  • the present invention is an HIV env gene expression unit which includes a DNA coding sequence for the desired protein and regulatory sequences necessary for transcription of the protein coding sequence and subsequent translation within a Drosophila cell.
  • this invention is a DNA vector which comprises the gene expression unit of the present invention.
  • this invention is a Drosophila cell transfected with the DNA vector of th s invention. In further related aspects, this invention is an
  • HIV env protein or a derivative thereof produced by the transfected insect cells of this invention.
  • the derivative encompasses any HIV env 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 HIV env protein.
  • this invention is a vaccine for stimulating protection against HIV infection, which comprises an immunoprotective and non-toxic -quantity of the HIV env protein produced by this invention.
  • a diagnostic agent useful in detecting presence of HIV infection in a sample of biological fluid which contains a Drosophila cell-produced HIV protein of the invention.
  • the env protein of the present invention may be employed to identify or isolate HIV binding proteins or proteinaceous substances, such as CD4 or derivatives thereof.
  • This invention also relates to a method for production of an HIV env protein, or an immunogenic derivative thereof.
  • the method entails culturing Drosophila cells transfected with an HIV env gene expression unit in a medium suitable for growth of the cells.
  • the transfected cells, cultured in said suitable medium are capable of expressing said protein of interest.
  • the protein may thereafter be collected from the cell or cell culture medium.
  • T e present invention also provides a method for purifying HIV proteins or fragments thereof which bind to a monoclonal antibody reactive with an epitope present on mature gpl20 and also within the gpl60 unprocessed intracellular protein.
  • a monoclonal antibody reactive with an epitope present on mature gpl20 and also within the gpl60 unprocessed intracellular protein is the mouse monoclonal antibody designated 178.1.
  • the method and expression system of the present invention facilitate high-level production of HIV proteins, particularly gpi20, gpl60 and derivatives thereof, in a Drosophila cell structure.
  • the Drosophila cells are transfected by using standard cloning 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 HIV proteins.
  • the method of this invention provides a. continuous, cell expression system for HIV proteins.
  • the protein Upon secretion, the protein is available by purification from the culture medium using conventional techniques. Alternatively, the protein may be produced intracellularly or membrane-bound. The protein may be extracted from the cells using conventional techniques. Alternatively, membrane-bound protein may be employed in a variety of cell-associated assays.
  • a preferred Drosophila cell line for use in the practice of the invention is the D. melanogaster S 2 line.
  • S 2 cells [Schneider, J. Embryol. Exp. Morph. 27 : 353 (1972)] are stable cell cultures of polyploid embryonic Drosophila cells.
  • Introduction of the cDNA coding sequence for gpieo, or its subunits gpl20 or gp4i or derivatives thereof into Drosophila S, cells by DNA transfection techniques produces unexpectedly large amounts of the glycoprotein.
  • Use of the S, Drosoohila 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.
  • Other Drosophila cell culture systems may also useful in the present invention.
  • Some possibly useful cells are, for example, the KC-0 Drosophila Melanogaster cell line which is a serum-free cell line [Schulz et al, Proc. NatA 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.
  • Drosophila 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_ medium.
  • the _ 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, DIS, 58 : 163 (1982).
  • Other conventional media for growth of Drosophila cells may also be used.
  • a recombinant DNA molecule or vector containing an HIV 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 HIV 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 has been recently published.
  • DNA molecules comprising the coding sequence of this invention can be derived from HTLV-Ill 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. 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., gpl20 or gpl60 molecules in which one -or more amino acids have been substituted, added or deleted without significantly 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 typically found upstream from the HIV 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, .
  • 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.
  • An 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) and COPIA LTR promoters.
  • Hsp70 inducible heatshock
  • COPIA LTR promoters COPIA LTR promoters.
  • the SV40 early promoter gives lower levels of expression than the Drosophila metallothionein promoter. Promoters which are commonly employed in the cell expression vectors including, e.g., avian Rous sarcoma virus LTR and simian virus (SV40 early promoter) demonstrate poor function and expression in the Drosoohila system.
  • a desirable gene expression unit or expression- vector for the HIV protein may be constructed by fusing the HIV 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 plas inogen activator (tPA) .
  • Other available signal sequences include, e.g., those derived from Herpes Simplex virus gene HSV-I gD [Lasky et al, Science, supra.].
  • the HIV DNA coding sequence may also be followed by a polyadenylation (poly A) region, such as an SV40 early 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 HIV protein gene functions.
  • 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 selection of transformed mammalian and bacterial cells, in the Drosophila system, methotrexate can be used to initially high-copy number transfectants.
  • An illustrative plasmid produced, according to the present invention is pgpl60 ⁇ 32, which contains a gpl60 derivative replacing the N-terminal 32 amino acid sequence of gpl ⁇ O with the first amino acid of tPA, serine. This plasmid is further described in Example 1.
  • Another such plasmid vector is pgpl20F ⁇ 32 which contains gpl ⁇ O sequence having the first 32 amino acids replaced with serine and containing a carboxyl deletion of 216 amino acids. This plasmid is also described in Example 1.
  • Still another plasmid which illustrates the derivative proteins of the present invention is pgpl20 ⁇ 32, which contains the entire coding sequence for gpl20 minus the first 32 .amino acids at the N-terminal which are replaced with serine. Additionally, plasmid pgpl20 ⁇ 274 contains a gpl20 protein sequence which has replaced the first 274 amino terminal amino acids with the first amino acid of tPA, ' serine, and containing the remaining amino acids of gpl20 up to the processing site of gpl60. These vector constructions " are described more completely in Example 1.
  • a recombinant DNA molecule or expression vector containing the HIV protein gene expression unit can be transfected into the selected Drosophila cell using standard transfection techniques.
  • Such techniques include, for example, calcium phosphate co-precipitation, cell fusion, electroporation, icroinjection and viral transfection.
  • a two-vector system can be used in the present invention to co-transfect into the Drosophila cell a gene expression unit for the desired HIV protein or derivative and the coding region for the selection system to be used.
  • a preferred illustrative embodiment of this invention is the production of an HIV protein employing a vector containing an HIV protein expression unit, e.g., pgpl20 ⁇ 32, and a vector containing a hygromycin B gene expression unit, e.g., pCOHYGRO.
  • pgpl20 ⁇ 32 contains an expression unit comprising the Drosophila metallothionein promoter, a derivative of the gpl20 gene, and the SV40 poly A site.
  • This gpl20 expression unit in combination with the pCOHYGRO vector system will produce a gpl20 derivative in S- Drosophila cells by maximizing the advantage of hygromycin B resistance for selection.
  • 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 pgpl20 ⁇ 32 and pHGCO, can be used to select methotrexate-resistant cells expression gp!20 or a derivative thereof.
  • the vector pgpl20 ⁇ 32 comprises a gpl20 gene expression unit in which the promoter is the' Drosophila metallothionein promoter.
  • the pHGCO vector comprises a DHFR gene expression unit and is co-transfected with the pgpl20 ⁇ 32 vector, thereby providing the DHFR gene necessary for selection.
  • the two vectors are co-transfected into the S 2 Drosophila cell using the method as described by Wigler et al, Cell, 16: 777
  • the vectors are co-transfected in varying ratios depending upon the particular copy number desired.
  • the transfected cells are then selected, such as in M_ medium containing serum and the appropriate selection agent, e.g. , hygromycin B or methotrexate.
  • the expression of gpl20 " 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 promo ' ter, no inducing agent is required for expression.
  • Transcription and expression of the HIV 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 gpl20 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 [see Example 5].
  • Drosophila S 2 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+ ⁇ °C).
  • Culture medium is M- 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 are cultured in serum-free media.
  • the media is also supplemented with 300 ⁇ g/ml hygromycin B.
  • the S, cells can be grown in suspension cultures, for example, in 250 ml to 2000 ml spinner flasks, with stirring at 50-60 rpm.
  • the cells are grown prior to induction in 1500 ml spinner flasks in media containing 5% serum. — —
  • the HIV 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 HIV protein from the culture media.
  • the use of the cell line S, which secretes the gene product directly into the media is an important feature of the present invention. Direct secretion into the media ° allows utilization of an efficient one-step purification system.
  • the spent culture media can be added directly to the column and the protein eluted using 1.5 M KSCN in phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • a preferred purification technique enabling large-scale efficient production of the HIV proteins of the invention employs an immunoaffinity column containing a monoclonal antibody directed against an epitope present in gpl60 and present in mature secreted gpl20 proteins . 0 Such a monoclonal is advantageous because of its capacity to recognize the protein sequence in more than one configuration.
  • An antibody having these characteristics and useful in immunoaffinity columns for various HIV proteins, derivatives or fragments thereof is designated 5 178.1. This monoclonal antibody is described in greater detail in Example 3.
  • Such a column of the invention may be made by coupling an antibody with the characteristics of 178.1 to a conventional absorbant carrier, such as Sephadex, under appropriate conventional conditions of pK, 0 temperature and the like. Such a purification column and procedure may be utilized to separate the HIV proteins and fragments of the present invention.
  • monoclonal antibodies may be used in this purification procedure.
  • Other new monoclonal antibodies useful in this invention may be developed by now-conventional techniques.
  • the glycoproteins produced by Drosophila cells are completely free of contaminating materials, e.g., mammalian, yeast, bacterial and more importantly, other HIV viral materials. Drosophila-produced HIV proteins have also been demonstrated to possess different pattern of gly ⁇ osylation 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 wit a pharmaceutically acceptable carrier.
  • the composition can be syste ically 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. Additionally, the recombinant proteins of the
  • present invention may be used as components of vaccines to innoculate mammalian subjects against HIV infection. These proteins may be used alone or with other recombinant proteins or therapeutic vaccinal agents. Components of such a vaccine would be determined , by one of skill in the art.
  • proteins of the present invention may be useful as diagnostic agents for the detection of ° the presence of HIV infection or antibodies to an HIV infective agent in biological fluids, such as blood, serum, saliva and the like.
  • proteins of the invention may also be employed in methods to identify - and/or isolate HIV-binding proteins or other HIV-binding 5 substances in biological fluids and tissues, e.g., sCD4 or derivatives thereof.
  • the proteins may thus be components in kits to perform such methods .
  • a protein is employed to contact the substance or an 0 impure mixture containing the substance under conditions to promote binding between the protein and the HIV-binding substance.
  • a conventional assay to detect the occurrence of binding e.g., detection of radioactive labels or the like, is also part of the method. The presence of binding 5 between the protein and the binding substance is, therefore, indicative of HIV binding.
  • the binding event could be followed by a conventional procedure to purify the bound 0 entity formed by the protein of the present invention and the HIV-binding substance from the mixture.
  • Other components of such diagnostic systems and kits may be conventional components of diagnostic kits and may be selected by those of skill in the art. 5
  • the following examples illustrate the construction of exemplary vectors and transformants of the invention, the preferred purification system and assays for determination of the production level of the glycoproteins gpl20 and gpl ⁇ O. These examples are not to be considered as limiting the scope of this invention.
  • 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 deleted the poison sequences [Mellon et al, Ceil, 27: 297 (1982)]. These sequences are inhibitory to amplification of the vector.
  • This vector was digested with Sail and Aat2 which removes a small piece of pBR322, and the digested ends were filled in. The missing piece of pBR322 is then replaced with a cassette containing the Drosophila metallothionein promoter on an end-filled
  • a HindllI-Xbal fragment containing the entire env gene was isolated from an HIV-isolate clone BH10 [L. Ratner et al, cited above; GenBank] .
  • the entire gpl ⁇ O 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 gpl ⁇ O gene.
  • the digestion with Ndel cut the gpl ⁇ O sequence at amino acid #32.
  • the Sacl digestion cuts 3" of the gpl60 gene, including in the sequence 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 Bglll, end-filled, and subsequently cut with Sacl, which deletes the mature tPA sequence. The Bglll site is positioned at the first amino acid of tPA. Consequently, the resulting vector pgpi60 ⁇ 32 codes for a modified gpl ⁇ O protein which has replaced the N-terminal 32 amino acids of gpl60 w.ith serine.
  • Another vector containing a modified gene sequence coding for HIV-l surface glycoprotein gpl ⁇ O was constructed by digesting pgpl60 ⁇ 32 with HindiII and Sacl, thereby removing the carboxyl terminal of gpl60. Approximately two-thirds of the sequence coding for gp41 is removed by this digestion. Thus, this gpl ⁇ O sequence is missing the first 32 amino acids and the last 216 amino acids of the natural gpl60 sequence. The deleted sequence was replaced by a short synthetic linker sequence coding " for a stop codon on an Hindlll-Sacl fragment.
  • the 6-amino-acid linker sequence is as follows:
  • Yet another vector containing a mutant gpl ⁇ O gene was constructed by digesting pgpl60 ⁇ 32 with Styl and Xbal, thereby deleting all of the sequence of protein 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 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 32 ° amino acids and none of the gp4l 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 partial digestion of pgpi20 ⁇ 32 with Bglll followed by 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 codes for a gpl20 protein which has replaced the first 274 amino terminal amino acids with the first amino acid of tPA, serine.
  • 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 ⁇ _ ⁇ coli gene coding for hygromycin B phosphotransferase (hygromycin B cassette) was cloned into pUCOPIA using standard cloning techniques.
  • the hygrymycin 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.
  • pCOHYGRO was co-transfected into S 2 Drosophila cells together with one of the vectors carrying a gpl60 mutant gene under the control of the Drosophila metallothionein promoter as described above.
  • the vector employed is pgpl20 ⁇ 32.
  • the transfected cells were selected in M 3 medium containing 5% serum and 300 ⁇ g/ml of hygromycin B. After 2 . to 3 days under identical conditions, the untransfected cells stop dividing and begin to die. The time of selection in order to obtain stable, growing hygromycin B-resistant cells in the transfected cultures is approximately two to three weeks.
  • the ratios of the two vectors were varied.
  • the ratio in this example was 20:1. Similar ratios have been employed for other gpl ⁇ O mutant vectors of this invention. This ratio is the same when any of the gpl60 mutant vectors are used.
  • Cells were maintained as suspension cultures in 250 ml to 2000 ml spinner flasks.
  • Culture medium was _ supplemented with 300 ⁇ g/ml hygromycin B. Cultures were incubated at 24+l°C and stirred at 50-60 rpm. Cell densities were typically maintained between 10 and 10 cells per ml in 3 medium supplemented with hygromycin B. CuSO. was added to a final concentration of 500 ⁇ , and the cultures were allowed to grow for 3 to 4 days- in serum-free media prior to harvesting the modified gpl20 glycoprotein.
  • the proteins, according to this method produced were approximately 100 MW, and the level of expression was higher than any ' other reported gpl20/gpl60 expression in any eukaryotic cell system.
  • the purified modified gpi20 expressed as described above, is capable of inhibiting virus infection in tissue culture, binds T 4 and reacts to antibodies to gpl20.
  • An affinity purification column employing a novel monoclonal antibody was used in the purification scheme applied to the above-described mutant gpl60/gpl20 proteins.
  • This monoclonal antibody may be characterized as being capable of reacting with non-denatured HIV glycoprotein products present in cell lysate and with mature gpl20 as secreted into the supernatant of a yeast culture.
  • One such monoclonal antibody specific for the epitope which is contained both in the unprocessed gpl ⁇ O recombinant molecule and in the full-size processed gpl20 protein is a mouse monoclonal antibody 178.1.
  • mice Eight-week-old Balb/c mice were injected three times subcutaneously and intraperitonally with the partially purified (1.5 - 3% purity) yeast-recombinant gpl60 in Freund's adjuvant at 4-week intervals. After a resting period of 3 months, one mouse was sacrificed, and its spleen cells were fuse with myeloma cells [see, e.g., R.P. Siraganian et al, Meth. Enz. , 9_2: 17 (1983); EMBO Course on Hybridoma Production, Basel Inst. for Immunol. (1980)].
  • the myeloma cells used are a subclone of the Sp 2 /O-Agl4 line previously selected for optimal growth in agar medium and high fusion efficiency [J.D. Franssen et al, Proc. XXIX Colloq. Protids Biol. Fluids, 29 : 645-649 (1981)]. After about ten days, supernatants were withdrawn for screening in a capture ⁇ LISA, using a commercial monospecific anti-gpl20 reagent [Biochorm, Seromed Ref. D7324] as capture antibody.
  • Nunc Immunoplate I (nr 4-39454) were coated overnight at 4 ⁇ C with 50 ⁇ l of a solution of 5 ⁇ g/ml of sheep anti-gpl20 IgGs in PBS.
  • the plates 1 were washed with washing buffer (PBS, Tween 20 0.1%) and saturated with 100 ⁇ l of saturation buffer [PBS, Newborn Calf Serum 4%, bovine serum albumen (BSA) 1%, Tween 20 0.1%] for 1 hour at 37°C.
  • PBS washing buffer
  • BSA bovine serum albumen
  • the cloned hybridomas were then grown in vivo by injecting 2 to 5 x 10 hybridoma cells in the peritoneal cavity of Balb/c mice pretreated by intraperitoneal injection of 5 pristane (2, 6, 10, 14-tetramethyl pentadecane) .
  • the monoclonal antibodies selected from the above procedure were characterized by Western blot analysis (WB), radioimmuno precipitation assay (RIPA) , purification, biotin-labeling and competition assays.
  • I 5 the s-ame assay.
  • a Western blot (WB) analysis was performed according to conventional techniques to demonstrate that 178.1 is capable of binding HIV virus isolated from human cells infected with HTLV-III B [Moltg/HT V-HIg] .
  • Radio immuno precipitation assays were performed, as described in P.J. Kanki et al, Science, 228: 1199 (1985) to demonstrate that 178.1 could immunoprecipitate the human cells infected with HTLV-Ill virus strains.
  • This monoclonal recognizes an epitope that is apparently conserved between gpl ⁇ O and gpl20 and thus, when used in the purification technique described in Example 4 below, provides an added ° advantage for the production of gpl60/gpl20 glycoproteins in various constructs.
  • CM Drosophi1a-conditioned media
  • PMSF phenylmethylsulfonyl fluoride
  • EDTA ethylenedi mine tetraacetic acid
  • CM was filtered through a .45 ⁇ m Durapore membrane using a pellicon (Millipore) device.
  • CM was applied to S-sepharose fast flow (Pharmacia) (5 liters; 25.2 cm x 11 cm) at a linear flow rate (LFR) of 37 ml/cm h 'equilibrated in Buffer A, containing 20 mM 2-[N-morpholino]ethanesulfonic acid (MES), pH 6.0.
  • Buffer A containing 20 mM 2-[N-morpholino]ethanesulfonic acid (MES), pH 6.0.
  • Buffer B containing 20 mM MES, pH- 6.0, 0.4M NaCL.
  • the S-sepharose-eluted gpl20 ⁇ 32 was applied to an anti-gpl20 mouse monoclonal-sepharose 4B column (60 ml;
  • 178.1 was produced according to Example 3 above. This hybridoma was seeded at 2 x 10 cells/ml and cultured for four days in Dulbecco's Modified Eagle Medium [Hazelton Research Products] supplemented with ° 4.5 grams/liter glucose, 2 ⁇ M gluta ine and 10% serum. CM containing 178.1 antibody was filtered (0.2 ⁇ m membrane) and applied to a protein A-sepharose (Pharmacia) (17 ml; 1.5 cm x 10 cm) equilibrated in 0.1M Tris, pH 8.2. Antibody was eluted with 0.1M sodium citrate, pH 3.5 and
  • Purified anti-gpl20 monoclonal antibody was coupled to CNBr-activated sepharose 4B (Pharmacia) , according to manufacturer's instructions at a density of 2 mg antibody/ml resin and with a coupling efficiency of
  • This affinity resin will specifically bind gpl20 protein through the interaction of the antibody with a unique structural epitope on gpl20.
  • 25 according to this purification technique is 80-90% with an estimated yield of 8.5 mg/30 liters conditioned media. Recovery is estimated at between 25-50%.
  • the assay described below is a non-isotopic 35 assay utilizing an enzyme and a substrate for the detection of gpl20 or fragments thereof, which was employed in detecting the gpl20 proteins produced by the methods and compositions of the present invention.
  • the criteria for detecting gpl20 is dependent on antibody specificity.
  • a colorless substrate (1 mg/ml of OPD in citrate buffer with 4 ⁇ l of 35% hydrogen peroxide per 10 ml of buffer) was added.
  • the hydrogen peroxide was added just prior to adding substrate to the wells.
  • These plates were incubated for 8 minutes on a shaker and the reaction stopped by adding 100 ⁇ l of 0.1 M sodium fluoride to each well.
  • the substrate turns deep yellow.
  • Optical density, or intensity of the color which is proportional to the amount of gpl20 captured, was read on a plate reader at 450 nanometers, and a standard curve was constructed with concentrations of unknowns calculated.

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WO2013006688A3 (en) * 2011-07-05 2013-04-04 Duke University N-terminal deleted gp120 immunogens
US10092638B2 (en) 2011-10-03 2018-10-09 Duke University GP120 immunogens and methods inducing neutralizing antibodies to human immunodeficiency virus

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Gene (Amsterdam, The Netherlands), Volume 25, Issued 1983, GRITZ et al "Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae", pages 179-188, see the entire document. *
Journal of Biological Chemistry (Baltimore, USA), Volume 260, Issued February 1985, LASTOWSKI-PERRY et al., "Nucleotide sequence and expression of a Drosophila metallothionein", pages 1527-1530. see the entire document. *
Journal of Embryology and Experimental Morphology (Oxford, UK), Volume 27, Issued 1972, SCHNEIDER, "Cell lines derived from late embryonic stages of Drosophila Melanogaster", pages 353-365, see the entire document. *
Journal of Virological Methods (Amsterdam, The Netherlands), Volume 18, Issued 1987, PALKER et al., "Purification of envelope Glycoproteins of human T cells lymphotropic virus type I (HTLV-I) by affinity chromatography", pages 243-256, see the entire document. *
Journal of Virology, (Washington, USA) Volume 63, Issued September 1989, SUN et al., "Generation and characterization of monoclonal antibodies to the putative CD4- binding domain of human immunodeficiency virus type 1 gp120", pages 3579-3585, see the entire document. *
Molecular and Cellular Biology (Washington, USA), Volume 5, Issued February 1985, SUGDEN et al., "A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus", pages 410-413, see the entire document. *
Molecular and Cellular Biology (Washington, USA), Volume 6, Issued June 1986, BOND et al., "The Drosophila malanogaster actin 5C gene uses two transcription initiation sites and three polyadenylation sites to express multiple mRNA species", pages 2080-2088, see the entire document. *
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WO2013006688A3 (en) * 2011-07-05 2013-04-04 Duke University N-terminal deleted gp120 immunogens
EP2739300A4 (en) * 2011-07-05 2015-06-03 Univ Duke N-TERMINATED REMOTE GP120 IMMUNOGENESE
AU2012279018B2 (en) * 2011-07-05 2017-06-08 Children's Medical Center Corporation N-terminal deleted GP120 immunogens
US10040826B2 (en) 2011-07-05 2018-08-07 Duke University Human immunodeficiency virus type 1 (HIV-1) N-terminal deleted GP120 immunogens
AU2017225144B2 (en) * 2011-07-05 2019-04-18 Children's Medical Center Corporation N-Terminal Deleted GP120 Immunogens
US11053285B2 (en) 2011-07-05 2021-07-06 Duke University Nucleic acids encoding human immunodeficiency virus type 1 (HIV-1) N-terminal deleted gp120 immunogens and methods of use
US10092638B2 (en) 2011-10-03 2018-10-09 Duke University GP120 immunogens and methods inducing neutralizing antibodies to human immunodeficiency virus
US10835599B2 (en) 2011-10-03 2020-11-17 Duke University Methods to identify prime and boost immunogens for use in a B cell lineage-based vaccination protocol

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