AP129A - Expression of retrovirus gag protein eukaryotic cells - Google PatentsExpression of retrovirus gag protein eukaryotic cells Download PDF
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- AP129A AP129A AP8900126A AP8900126A AP129A AP 129 A AP129 A AP 129A AP 8900126 A AP8900126 A AP 8900126A AP 8900126 A AP8900126 A AP 8900126A AP 129 A AP129 A AP 129A
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- precursor protein
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- C07—ORGANIC CHEMISTRY
- 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
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- 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
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C12N2740/00—Reverse Transcribing RNA Viruses
- C12N2740/00011—Reverse Transcribing RNA Viruses
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
- C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Expression of Retrovirus gag Proteinin Eukaryotic CellsAbstract of the Disclosure
Retrovirus gag precursor protein is expressed in eukaryoticcells by recombinant DNA techniques and is used to induceimmunoprotection in humans at risk of exposure to HIV and todiagnose exposure.
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Expression of Retrovirus gag Proteinin Eukaryotic Cells
Field of the Invention
This invention relates to expression of proteins ineukaryotic cells. More particularly it relates to theexpression of immunodeficiency virus gag precursor protein.
Background of the Invention
Retroviruses, that is, viruses within the family,Retroviridae, are a large family of enveloped, icosohedralviruses of about 150 nm having a coiled nucleocapsidwithin the core structure and having RNA as the genetic
BAD ORIGINAL 1 10 15 20 25 30 material. The family comprises the oncoviruses such asthe sarcoma and leukemia viruses, the immunodeficiencyviruses and the lentiviruses.
Human Immunodeficiency Virus (HIV), the etiologic agent of acquired immune deficiency syndrome (AIDS) and related disorders, is a member of the Retroviridae family. There exist several isolates of HIV including human T-lymphotropic virus type-III (HTLV-III), the lymphadenopathy virus (LAV) and the AIDS-associated retrovirus (ARV) which have been grouped in type 1.
Related immunodeficiency viruses, include HIV type 2, which was shown recently to be associated with AIDS in
West Africa. Other immunodeficiency viruses include the SIV viruses such as SIV „ -BK28. mac
Molecular characterization of the HIV genome hasdemonstrated that the virus exhibits the same overallgag-pol-env organization as other retroviruses. Inaddition, it contains at least five genes that are notfound in more ordinary retroviruses: sor, tat3, art/trs, 31orf and R. The gag region encodes 3 core proteins, pi7,p24 and pl6, which are prepared by cleavage of a 55kilodalton gag precursor protein by the HIV protease. Theprotease is encoded by the pol region.
Recent reports have shown that antibodies to the HIVgag proteins, pl7, p24 and pl6, are present in human serafrom infected individuals in the United States and Europeand that antibodies arise early after infection. Thepresence of these antibodies declines as the individualproceeds towards AIDS.
The gag protein pl7 with its submembrane localizationis well positioned to be in close contact with thetransmembrane protein gp41 and the viral membrane and withgag p24 and possibly gag pl5 viral RNA thereby playing acentral role in the conformational changes involved in theviral entry and uncoating process. Furthermore, gag pl7has been found to have a myristylated N-terminus. APO00129
BAD ORIGINAL ft 35
Myristylation has been implicated in virion assembly andtransport of viral components to the plasma membrane.Myristylated proteins are generally localized in theplasma membrane.
In spite of major research efforts in the area ofAIDS, there continues to be a need for diagnostic reagentswhich can be used to monitor disease progression and foragents which can prevent primary infection, such as viaimmunization, and for agents which can prevent or inhibitsecondary infection, such as by cell-to-cell transmissionor by free virus infection.
Summary of the Invention
In one aspect, this invention is a recombinant DNAmolecule for expression of gag precursor protein ineukaryotic cells which comprises a coding sequence therefor operatively linked to a regulatory region whichfunctions in the host cell.
In related aspects, this invention is host cellscomprising the recombinant DNA molecule and culturesthereof.
In further related aspects, the invention is the gagprecursor protein produced by the host cells of theinvention, including a HIV core-like particle comprisingthe gag precursor protein.
In yet further related aspects, the invention is aprocess for producing the recombinant DNA molecule and _thehost cell of the invention, a process for producing thegag precursor protein and particles of the invention, andrelated compositions and methods.
These and other aspects of the invention are fullydescribed in the disclsoure and Examples which follow.
Detailed Description of the InventionIt has now been found that retroviral gag precursor protein can be expressed in recombinant eukaryotic cells
35 1 10 15 20 25 30 and that such expression can result in production offull-length gag precursor protein without use of pol DNAsequences and without use of 5' untranslated sequencesfrom the virus. Exemplary of such cells are cells fromlower eukaryotes such as yeast and fungi and animal cellsincluding insect cells such as Drosophila or Lepidopteracells; mammalian cell lines; mammalian primary cells, andinsects and transgenic animals.
It has also been found, unexpectedly, that the gagprecursor protein can form particles which resembleauthentic gag particles formed in infected human cells insize and other physical properties and in antigenicity.During a natural retrovirus infection cycle, it appearsthat gag precursor protein, known in the case of HIV asp55, is formed largely into particles comprisingpredominantly full-length gag protein. These gagparticles can be referred to as pre-core particles orimmature core particles. Then, during viral maturation,’the precursor is cleaved into the subunit proteins knownin the case of HIV as pl7, p24 and pl6. These gagparticles, now comprised predominantly of pl7, p24 andpl6, can be referred to as core particles or as maturecore particles. Also during viral maturation, apparentlyduring the budding process, the viral membrane is formedaround the pre-core or core particles. As shown in theExamples below, HIV gag precursor expressed in recombinantLepidoptera cells using a Baculovirus expression systemare largely aggregated or packaged in particles which havephysical and biological properties and dimensions similarto those of the core of HIV particles formed naturally ininfected human cells. The particles of the inventioncomprise predominantly gag precursor protein (greater than90% of all protein in the particles is full length gagprecursor) but nevertheless are recognized after brieftreatment with Triton X100 by anti-pl7 monoclonalantibodies (MABs), anti-p24 MABs and anti-pl6 MABs in AP000129 BAD ORIGINAL & 35 4 1 10 15 20 25 30 addition to being recognized by anti-gag polyclonalantibodies from sera of infected patients. The particles,because they are prepared by recombinant DNA techniques asdisclosed herein, lack viral functions required for viralmaturation and replication especially viral RNA and also,preferably, reverse transciptase and protease functions.
The recombinant eukaryotic cells of the invention areengineered to express the gag precursor protein byintroduction into the cells of the recombinant DNAmolecule of the invention. The recombinant DNA moleculeof the invention comprises a coding region for the gagprecursor protein operatively linked to a regulatoryelement which functions in the selected host cells. As anaspect of this invention, it has been found that other HIVfunctions are not required for expression of the gagprecursor protein and for pre-core-like particleformation. DNA sequences coding for other functions,e.g., for amplification functions, selection markers ormaintenance functions, can also be comprised within therecombinant DNA molecule of the invention. - ... A DNA coding region for gag precursor protein can beprepared from any of the several immunodeficiency virusgenomic clones or gag-pol clones reported in theliterature. See, for example, Shaw et al., Science226:1165(1984); Kramer et al., Science 231:1580(1986)Alternatively, an immunodeficiency virus genomic clone canbe prepared from virus isolated from clinical specimens bystandard DNA cloning techniques. See, for example, Galloet al., U.S. Patent 4,520,113; Montagnier et al., U.S.Patent 4,708,818. Having cloned a fragment of the genomewhich comprises the gag coding region, a region whichcodes only for the gag precursor can be prepared byrestricting the DNA so as to isolate a portion of the DNAcoding region and reconstructing the remaining portionsthrough use of synthetic oligonucleotides, such asdescribed in the Examples, below. Alternatively, a larger
35 1 fragment comprising the gag coding region and additionalsequences can be cut back through use of exonucleases. Inyet another alternative procedure, the entire codingregion can be synthesized using standard automated DNAsynthesizers by synthesizing fragments of the codingregion and ligating these together to form a completecoding region. While use of a coding sequence which lacksthe naturally occurring 5' and 3' flanking sequences ispreferred, fusion of the coding sequence to otherimmunodeficiency virus sequences, e.g., envelope proteinsequences, is not precluded from the preferred embodiments.
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An exemplary coding region for the HIV gag precursorprotein has the following sequence. 1 ATG Met GGT Gly GCG Ala AGA Arg GCG Ala TCA Ser GTA Val TTA Leu AGC Ser GCG Gly GGA Gly GAA Glu 37 TTA GAT CGA TGG GAA AAA ATT CGG TTA AGG CCA GGG Leu Asp Arg Trp Glu Ly« lie Arg Leu Arg Pro Gly 73 GGA AAG AAA AAA TAT AAA TTA AAA CAT ATA GTA TGG Gly Lys Lys Lys Tyr Lys Leu Lys His He Val Trp 109 GCA AGC AGG GAG CTA GAA CGA TTC GCA GTT AAT CCT Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 145 GGC CTG TTA GAA ACA TCA GAA GGC TGT AGA CAA ATA Gly Leu Leu Glu Thr ser Glu Gly Cys Arg Gin lie 181 CTG GGA CAG CTA CAA CCA TCC CTT CAG ACA GGA TCA Leu Gly Gin Leu Gin Pro Ser Leu Gin Thr Gly Ser 217 GAA GAA CTT AGA TCA TTA TAT AAT ACA GTA GCA ACC Glu Glu Leu Arg Ser Leu Tyr Asn Thr Val Ala Thr 253 CTC TAT TGT GTG CAT CAA AGG ATA GAG ATA AAA GAC Leu Tyr Cy· Val His Gin Arg lie Glu He Lys Asp 289 ACC AAG GAA GCT TTA GAC AAG ATA GAG GAA GAG CAA Thr Lys Glu Ala Leu Asp Lys He Glu Glu Glu Gin 325 AAC AAA AGT AAG AAA AAA GCA CAG CAA GCA GCA GCT Asn Lys Ser Ly· Lys Lys Ala Gin Gin Ala Ala Ala 361 GAC ACA GGA CAC AGC AGT CAG GTC AGC CAA AAT TAC Asp Thr Gly His Ser Ser Gin Val Ser Gin Asn Tyr 397 CCT ATA GTG CAG AAC ATC CAG GGG CAA ATG GTA CAT Pro lie Val Gin Asn He Gin Gly Gin Met Val His 433 CAG GCC ATA TCA CCT AGA ACT TTA AAT GCA TGG GTA Gin Ala lie Ser Pro Arg Thr Leu Asn Ala Trp Val 469 AAA GTA GTA GAA GAG AAG GCT TTC AGC CCA GAA GTA Ly· Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val
BAD ORIGINAL 10 15 20 25 30 505 ATA He CCC Pro ATG Met TTT Phe TCA Ser GCA Ala TTA Leu TCA Ser GAA Glu GGA Gly GCC Ala ACC Thr 540 541 CCA CAA GAT TTA AAC ACC ATG CTA AAC ACA GTG GGG 576 Pro Gin Asp Leu Asn Thr Met Leu Asn Thr Val Gly 577 GGA CAT CAA GCA GCC ATG CAA ATG TTA AAA GAG ACC 612 Glv His Gin Ala Ala Met Gin Met Leu Lys Glu Thr 613 ATC AAT GAG GAA GCT GCA GAA TGG GAT AGA GTA CAT 648 He Asn Glu Glu Ala Ala Glu Trp Asp Arg Val His 649 CCA GTG CAT GCA GGG CCT ATT GCA CCA GGC CAG ATG 684 Pro Val His Ala Gly Pro lie Ala Pro Gly Gin Met 685 AGA GAA CCA AGG GGA AGT GAC ATA GCA GGA ACT ACT 720 Arg Glu Pro Arg Gly Ser Asp He Ala Gly Thr Thr 721 AGT ACC CTT CAG GAA CAA ATA GGA TGG ATG ACA AAT 756 Ser Thr Leu Gin GlU Gin He Gly Trp Met Thr Asn 757 AAT CCA CCT ATC CCA GTA GGA GAA ATT TAT AAA AGA 792 Aan Pro Pro lie Pro Val Gly Glu lie Tyr Lys Arg 793 TGG ATA ATC CTG GGA TTA AAT AAA ATA GTA AGA ATG 828 Trp lie He Leu Gly Leu Asn Lys lie Val Arg Met 829 TAT AGC CCT ACC AGC ATT CTG GAC ATA AGA CAA GGA 864 Tyr Ser Pro Thr Ser lie Leu Aep He Arg Gin Gly 865 CCA AAA GAA CCT TTT AGA GAC TAT GTA GAC CGG TTC 900 Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe 901 TAT AAA ACT CTA AGA GCC GAG CAA GCT TCA CAG GAG 936 Tyr Lys Thr Leu Arg Ala Glu Gin Ala Ser Gin Glu 937 GTA AAA AAT TGG ATG ACA GAA ACC TTG TTG GTC CAA 972 Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gin 973 AAT GCG AAC CCA GAT TGT AAG ACT ATT TTA AAA GCA 1008 Asn Ala Asn Pro Asp Cys Lys Thr lie Leu Lys Ala 1009 TTG GGA CCA GCG GCT ACA CTA GAA GAA ATG ATG ACA 1044 Leu Gly Pro Ala Ala Thr Leu Glu Glu Met Met Thr AP 0 0 0 1 2 9 35 -8 - BAD ORIGINAL jg 1045 GCA Ala TGT CAG GGA GTA GGA GGA CCC GGC CAT AAG GCA 1080 Cys Gin Gly Val Gly Gly Pro Gly His Lys Ala 1081 AGA GTT TTG GCT GAA GCA ATG AGC CAA GTA ACA AAT 1116 Arg Val Leu Ala GlU Ala Met Ser Gin Val Thr Asn 1117 ACA GCT ACC ATA ATG ATG CAG AGA GGC AAT TTT AGG 1152 Thr Ala Thr He Met Met Gin Arg Gly Asn Phe Arg 1153 AAC CAA AGA AAG ATG GTT AAG TGT TTC AAT TGT GGC 1188 Asn Gin Arg Lya Met Val Lys Cya Phe Asn Cys Gly 1189 AAA GAA GGG CAC ACA GCC AGA AAT TGC AGG GCC CCT 1224 Lys Glu Gly His Thr Ala Arg Asn Cys Arg Ala Pro 1225 AGG AAA AAG GGC TGT TGG AAA TGT GGA AAG GAA GGA 1260 Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly 1251 CAC CAA ATO AAA GAT TGT ACT GAG AGA CAO GCT AAT 1296 Hia Gin Met Lya Asp Cya Thr Glu Arg Gin Ala Asn 1297 τττ TTA GGG AAG ATC TGG CCT TCC TAC AAG GGA AGG 1332 Phe Leu Gly Lys lie Trp Pro Ser Tyr Lys Gly Arg 1333 CCA GGG AAT TTT CTT CAG AGC AGA CCA GAG CCA ACA 1368 Pro Gly Asn Phe Leu Gin Ser Arg Pro Glu Pro Thr 1369 GCC CCA CCA TTT CTT CAG AGC AGA CCA GAG CCA ACA 1404 Ala Pro Pro Phe Leu Gin Ser Arg Pro Glu Pro Thr 1405 GCC CCA CCA GAA GAG AGC TTC AGG TCT GGG GTA GAG 1440 Ala Pro Pro Glu Glu Ser Phe Arg Ser Gly Val Glu 1441 ACA ACA ACT CCC CCT CAG AAG CAG GAG CCG ATA GAC 1476 Thr Thr Thr Pro Pro Gin Lys Gin Glu Pro lie Asp 1477 AAG GAA CTG TAT CCT TTA ACT TCC CTC AGA TCA CTC 1512 Lys Glu Leu Tyr Pro Leu Thr Ser Leu Arg Ser Leu 1513 TTT GGC AAC GAC CCC TCG TCA CAA TAA 1539 Phe Gly Asn Asp Pro Ser Ser Gin End
1 10 15 20 25 30 A variety of eukaryotic cells and expression systemsare available for expression of heterologous proteins.
The most widely used among these are yeast, insect andmammalian systems, although the invention is not limitedto use of these. Typically, these systems employ arecombinant DNA molecule comprising a coding sequence forthe gene of interest operatively linked to a regulatoryelement, a selection marker and, in some cases, maintenancefunctions such as an origin of replication. A regulatoryelement is a DNA region or regions which comprise functionsnecessary or desirable for transcription and translation.Typically, the regulatory region comprises a promoter forRNA polymerase binding and initiation of transcription.
Insect cells which can be used in the inventioninclude Drosophila cells and Lepidoptera cells. UsefulDrosophila cells include SI, S2, S3, KC-0 and D. hydeicells. See, for example, Schneider et al., J. Embryol.
Exp. Morph. 27:353 (1972); Schulz et al., Proc. Natl.
Acad. Sci. USA 83:9428 (1986); Sinclair et al., Mol. Cell.Biol. 5:3208 (1985). Drosophila cells are transfected bystandard techniques, including calcium phosphateprecipitation, cell fusion, electroporation and viraltransfection. Cells are cultured in accordance withstandard cell culture procedures in a variety of nutrientmedia, including, e.g., M3 media which consists ofbalanced salts and essential amino acids. See, Lindquist,DIS 58:163 (1982).
Promoters known to be useful in Drosophila includemammalian cell promoters as well as Drosophila promoters,the latter being preferred. Examples of useful Drosophilapromoters include the Drosophila metallothionein promoter,the 70 kilodalton heatshock protein promoter (HSP70) andthe COPIA LTR. See, for example, DiNocera et al., Proc.Natl. Acad. Sci. USA 80:7095 (1983); McGarry et al., Cell42:903 (1985). Conveniently, an expression cassettecomprising the gag coding sequence and regulatory element AP 0 0 0 1 2 9 35 - 10 -
BAD ORIGINAL Q can be cloned within a bacterial cloning vector forpurposes of propagating the DNA prior to transfection ofthe animal cells.
In the preferred embodiments of this invention, theHIV gag precursor is expressed in Lepidoptera cells toproduce immunogenic gag particles. For expression of thegag precursor protein in Lepidoptera cells, use of aBaculovirus expression system is preferred. In suchsystem, an expression cassette comprising the gag codingsequence and regulatory element is placed into a standardcloning vector for purposes of propagation. Therecombinant vector is then co-transfected into Lepidopteracells with DNA from a wild type Baculovirus. Recombinantviruses resulting from honologous recombination are thenselected and plaque purified substantially as described bySummers et al., TAES Bui1. NR 1555, May, 1987.
Useful Lepidoptera cells include cells fromTrichoplusia ni, Spodoptera fruqiperda, Heliothis zea,Autographica californica, Rachiplusia ou. Galleriamelonella, Manduca sexta or other cells which can beinfected with Baculoviruses, including nuclearpolyhedrosis viruses (NPV), single nucleocapsid viruses(SNPV) and multiple nucleocapsid viruses (MNPV). Thepreferred Baculoviruses are NPV or MNPV Baculovirusesbecause these contain the polyhedrin gene promoter whichis highly expressed in infected cells. Particularlyexemplified hereinbelow is the MNPV virus from Autographicacalifornica (AcMNPV). However, other MNPV and NPV virusescan also be employed the silkworm virus, Bombyx mori.Lepidoptera cells are co-transfected with DNA comprisingthe expression cassette of the invention and with the DNAof an infectious Baculovirus by standard transfectiontechniques, as discussed above. Cells are cultured inaccordance with standard cell culture techniques in avariety of nutrient media, including, for example, TC100(Gibco Europe; Gardiner et al., J. Inverteb. Pathol.
BAD ORIGINAL 1 10 15 20 25 30 25:363 (1975)) supplemented with 10 % fetal Calf serum(FCS). See, Miller et al., in Setlow et al., eds.,
Genetic Engineering: Principles and Methods, Volume 8,
New York, Plenum, 1986, pages 277-298.
Production in insect cells can also be accomplished byinfecting insect larvae. For example, the gag precursorcan be produced in Trichoplusia ni caterpillars by feedingthe recombinant Baculovirus of the invention along withtraces of wild type Baculovirus and then extracting thegag precursor from the hemolymph after about two days.
Promoters for use in Lepidoptera cells includepromoters from a Baculovirus genome. The promoter of thepolyhedrin gene is preferred because the polyhedrinprotein is naturally over expressed relative to otherBaculovirus proteins. The polyhedrin gene promoter fromthe AcMNPV virus is preferred. See, Summers et al., TAESBull. NR 1555, May 1987; Smith et al. , EP-A-127,839; Smithet al. Proc. Natl. Acad. Sci. USA 82:8404(1985); andCochran, EP-A-228,036.
For expression in mammalian cells, the expressioncassette is likewise cloned within a cloning vector and isthen used to transfect the mammalian cells. The vectorpreferably comprises additional DNA functions for geneamplification, e.g., a DHFR expression cassette, and mayalso comprise additional functions for selection and/oramplification, e.g., a neomycin resistance cassette forG418 selection. Other functions, such as for transcriptionenhancement can also be employed. Yet other functions canbe comprised within the vector for stable episomalmaintenance, if desired, such as maintenance functions ofBovine Papilloma Virus. The mammalian cell vector canalso be a recombinant virus, such as a recombinantvaccinia or other pox virus. See, e.g., Paoletti, et al.,U.S. Patent 4,603,112; Paoletti, et al. , Proc. Natl. Acad.Sci. U.S. 81:193 (1984).
Useful mammalian cells include cells from Chinese AP000129 35 12 BAD ORIGINAL 0 1 10 15 20 25 30 hamster ovary (CHO), NIH3T3, COS-7, CVI, mouse or ratmyeloma, HAK, Vero, HeLa, human diploid cells such asMRC-5 and WI38, or chicken lymphoma cell lines.
Transfection and cell culture are carried out by standardtechniques. Production in mammalian cells can also beaccomplished by expression in transgenic animals. Forexample, the gag precursor can be expressed using a caseinpromoter and purified from milk.
Promoters useful in mammalian cell lines or mammalianprimary cells include the SV 40 early and late genepromoters, the metallothionein promoter, viral LTR's suchas the Rous sarcoma LTR, the Moloney sarcoma virus (MSV)LTR or the mouse mammary tumor virus (MMTV) LTR, or theadenovirus major late promoter and hybrid promoters suchas a hybrid BK virus and adenovirus major late promoter.The regulatory region can also comprise downstreamfunctions, such as regions for polyadenylation, or otherfunctions, such as transcription enhancer sequences.
Yeasts which can be used in the practice of theinvention include those of the genera Hanensula, Pichia,Kluveromyces, Schizosaccharomyces, Candida and
Saccharomyces. Saccharomyces cerevisiae is the preferredyeast host. Useful promoters include the copper inducible(CUP1) promoter, glycolytic gene promoters, e.g., TDH3, PGK and ADH, and the PHO5 and ARG3 promoters. See, e.g.,Miyanohara et al., Proc. Natl. Acad. Sci. USA 80:1 (1983);Mellor et al. , Gene 24; 1 (1983); Hitzeman et al., Science219:620 (1983); Cabezon et al., Proc. Natl. Acad. Sci. USA81:6594 (1984).
In the case of the gag precursor protein particlesproduced in accordance with this invention, it is to beunderstood that although particles comprising the gagprecursor are preferred, particles comprising derivativesof the native gag precursor can also be prepared. Forexample, one or more nucleotides or amino acids shown inthe sequence above can be deleted, substituted or added 35
BAD ORIGINAL 1 10 15 20 25 30 without substantially adversely affecting the immunogeniccross-reactivity with authentic gag epitopes. In otherwords, such derivatives immunologically similar toauthentic gag particles in theat they are recognized byantibodies raised against at least one of pl7, p24 andpl6. Such derivatives, while they may include amino acidsfrom other regions, including antigenic regions of the HIVgenome, do not encode other HIV functions, such as theprotease function of the pol region or the reversetranscriptase function. In addition, such derivativesretain the ability to form particles in insect cellculture as disclosed herein. In this case, it is withinthe skill of the art to prepare gagd particles comprisinghybrid proteins having one or more epitopes additional tothe gag epitopes. Such additional epitopes can be of HIVorigin or can be derived from other pathogenic organisms,e.g., Hepatitis B Virus or Herpes Virus.
The gag precursor protein is expressed in secretedform and in membrane bound form. It is isolated fromconditioned medium by standard techniques of proteinisolation and purification. Detergents can be added inorder to free the protein from cell membrane material.Following treatment with detergent, e.g., Triton X100, aTween or sodium dodecyl sulfate (SDS), the protein orparticles can be purified by a series of ultrafiltrationsteps, ultracentrifugation steps, selective precipitationswith, e.g., ammonium sulfate or PEG, density gradientcentrifugation in CsCl or sucrose gradients and/orchromatographic steps, such as affinity chromatography,immunoaffinity chromatography, HPLC, reversed phase HPLC,cation and anion exchange, size exclusion chromatographyand preparative isoelectric focusing. During or followingpurification, the protein or particles can be treatedwith, e.g., formaldehyde, glutaraldehyde or NAE to enhancestability or immunogenicity. In view of the discoveryherein disclosed that the gag precursor can form AP 0 0 0 1 2 9
35 immunogenic particles in the absence of other viralfunctions, it is believed that when gag precursor isexpressed in non-particulate form, it can be caused toform particles synthetically, as has been shown to be thecase for the hepatitis B surface antigen followingexpression in yeast. See, e.g., EP-A-135,435. Such gagprecursor protein particles are encompassed within thescope of this invention.
The HIV gag precursor protein and particles producedin accordance with this invention are useful as diagnosticagents for detection of exposure to HIV. The protein andparticles are also useful in vaccines for the preventionof infection or for the inhibition or prevention ofdisease progression.
The Examples which follow are illustrative but notlimiting of the invention. Restriction enymes and otherreagents were used substantially in accordance with thevendors' instructions.
Example 1. Vector Construction pRIT12982 (DT 12-16) is a vector which comprises a 1305 base pair (bp) coding sequence for the N-terminal region of gag precursor protein. It was prepared by ligating a Clal-Bglll fragment of the gag precursor protein coding region derived from an HIV genomic clone(Shaw et al., Science 226:1165 (1984)) to a syntheticoligonucleotide having the N-terminal coding sequence ofthe gag precursor protein. The oligonucleotide has thesequence:
5' C ATG GGT GCT AGA GCT TCC GTG TTG TCC GGT GGT GAA TTG GAT 3CCA CGA TCT CGA AGG CAC AAC AGG CCA CCA CTT AAC CTA GC
Ncol Clal pRIT12983 is a vector which comprises a 250 bp regionwhich codes for the C-terminal portion of gag precursor 15
BAD ORIGINAL 10 15 20 25 30 35 protein. It was prepared by ligating a Bglll-Maelllfragment of the gag precursor coding region derived froman HIV genomic clone to a synthetic oligonucleotide havingthe C-terminal coding sequence of the gag precursorprotein. The oligonucleotide has the sequence:
STOP 5' G TCA CAA TAA AGA TAG GAT CC 3'
TT ATT TCT ATC CTA GGA GCT
The 1305 base pair (bp) BamHI(NcoI)-BglII fragmentfrom pRIT12982 was ligated to the 250 bpBglH-TAA-BamHI-XhoI fragment from pRIT12983 in pUC 12which had been previously cut with BamHI and Sail. Theresulting plasmid, identified as pRIT13001, thereforecontains the entire coding region for the gag precursorprotein on a BamHI(NeoI)-BamHI cassette. A baculovirus expression vector was prepared byinserting the BamHI fragment from pRIT13001 into the BamHIsite in pAc373. See, Smith, et al., Proc. Natl. Acad.
Sci. USA 82:8404(1985). pAc373 is a baculovirus transfervector containing a modified polyhedrin gene into which aforeign gene can be cloned into a BamHI site and expressedunder the control of the strong polyhedrin promoter. SeeSummers, et al., Texas Agricultural Exp. Station BulletinNR 1555 (May 1987). A derivative of plasmid pAc373 havinga small deletion present far upstream the strong polyhedrin promoter was also used as an expressionvector. The slight modification did not appear to affectin vitro expression or growth of the recombinant virus.Insertion of the gag coding sequence into the Baculovirusvector resulted in plasmid pRIT13003. A mammalian cell expression vector was prepared byligating the BamHI fragment from pRIT13001 downstream of APO 0 0 12 9 16 bad original the SV40 late promoter in pSV529 (Gheysen et al., J. Mol.Appl. Genet. 1:385 (1982)). This vector is identified aspRIT13002. A yeast expression vector was prepared as follows. AnNcol-Bglll fragment was isolated from pRIT12982 andinserted into a yeast plasmid downstream of and in-framewith the ARG3 promoter (see, Cabezon et al. , Proc Natl.Acad. Sci. USA 81:6594 (1984)) giving rise to the vector,pRIT12984 (DT14-20). The C-terminal protion of the gagprecursor protein was isolated from pRIT12983 as aBglll-BamHI fragment and was inserted into the Bglll siteof pRIT 12984, giving rise to the yeast vector, pRIT12985(DT16-26). pRIT12985 thus comprises a coding sequence for the full gag precursor, devoid of other HIV sequences,operatively linked to the ARG3 promoter. In addition, itcomprises replication functions from the yeast 2 micronvector and a URA3 gene selection marker.
Example 2. Expression in Insect Cells
Recombinant Baculovirus transfected with pRIT13003were prepared substantially as described by Summers, etal., TAES Bull. NR 1555, May 1987, cited above.
Spodoptera frugiperda (S.f.) cells were cotransfectedwith wild type (wt) AcMNPV Baculovirus DNA and plasmidpRITl3003 at 1 pg and 50 pg, respectively. Resultingvirus particles were obtained by collecting thesupernatants. The virus-containing media were used toinfect S.f. cells in a plaque assay. Subsequent infectionof S.f. cells using the viral particles which include bothwt AcNPV DNA and DNA recombined with the DNA encoding thep55 gag precursor protein resulted in cells expressing thegag protein instead of the polyhedrin protein.
The "clear plaques" (0.1 - 0.01% frequency) obtainedin the plaque assay were further screened by filterhybridization with a gag specific probe. Plaques whichhybridized to the gag probe were scored and subsequently
BAD ORIGINAL 1 10 15 20 25 30 35 further plaque purified (2-3 times) before a virus stockwas generated; the virus stock was also tested by ELISA. t S.f. cells were then infected with these recombinant gagvirus stocks at a multiplicity of infection (MOI) of 1-10and after 24 hr, 48 hr, 3 days and 5 days, aliquots of theconditioned medium (Supernatant) and/or cells were treatedwith Triton X100 to a final concentration of 1% andassayed.
The gag precursor protein synthesized in infectedinsect cells was observed in Western blots using p55polyclonal antibodies or antiserum from a pool of AIDSpatients (Zairan). A pre-dominant band at molecularweight (Mr) of 54 kilodaltons (kd) was observed with alltested sera and with p55 polyclonal antisera. A band atMr 54 kd was also detected when testing conditioned mediumafter 48 hr, 3 days and 5 days. Bands at Mr 49 kd and Mr47 kd (minor) and a band at Mr 30 kd could also be seenwhen cell extracts were analyzed. This latter band withapparent Mr 30 Kd is only detected with p55 polyclonalantibodies and not with serum of AIDS infected persons.
It was observed that at least 10 times more p55 "epitopes"expressed in S.f. cells than in Molt cells infected withHIV (Molt/HTLV-III) and about 80 times more p55 "epitopes"were present in the conditioned medium of S.f. cellsinfected with a gag recombinant virus than in theconditioned medium of Molt/HTLV-III cells.
In a second assay experiment, ultrafiltration (100,000x g, 1 hr.) of the 48 hr, 3 day and 5 day conditionedmedia (2 ml to 200 ml) resulted in a small pellet whichwas analysed on SDS-gels and which was also analysed byimmunoblotting. One band at Mr 55 kd was recognized withspecific antibodies against pl7, p24 and p55,. Only verysmall amounts of degraded products at Mr 49-46 could bedetected. On Coomassie-stained gels, a band at 55 kdcould be seen which was 20-80% pure. This bandcorresponded with the immunoblot and was recognized byantibodies against pl7, p24 and p55 polypeptides.
BAD ORIGINAL AP 0 0 0 1 2 9 8
In a third assay experiment, centrifugation (1 ml) ofthe 48 hr, 3 day and 5 day conditioned media in amicrofuge at 12000 rpm for 5 to 20 minutes produced a bandon SDS-gels at Mr 55 kd which was specific for HIV-I p55gag precursor as revealed by antibodies against pl7, p24and p55 polypeptides and as compared to the HIV celllysate (Molt/HTLV-III) 55 kd band.
In a fourth assay experiment, 48 hr, 3 day and 5 dayconditioned media (150 ml to 1 liter, containing 1 pg/mlof aprotinin which was added at 24 hrs. post-infection andalso at the times of harvest) was treated first byaddition of Tween 20 to 0.01% final concentration. Then,a solution of polyethylene glycol, Mr 6 kd, (PEG6000) (40% w/v in 2M NaCl) was added to 10 % or 5 % finalconcentration. After 4 hours at 4°C or preferentiallyovernight at 4°C this precipitate was centrifuged at5000 rpm for 10 min at 4°C. The PEG pellet was thentaken up in 200 μΐ to 1 ml HBS-buffer (Hanks balancedsalt, Flow Laboratories, 18-102-54) containing 0.1 % Tween20 and centrifuged in sucrose gradients (20 % - 60 % inHBS-buffer, 0.1 % Tween 20 at 4°C containing 10 μΐ/mlaprotinin, Sigma Chemical Co., St. Louis, Missouri) forabout 35 min at 50,000 rpm in a Beckman rotor TLA100(Beckman Instruments, Fullerton, California) at 4°C, orfor about 18 hr at 25,000 rpm on a Beckman SW41 rotor at4°C. Fractions of 0.2 to 0.5 ml, respectively, fromapproximately 40-50% sucrose, were collected, frozen at-20°C and tested either with a specific antigen captureElisa assay such as -24/Ig AIDS antiserum biotinylated orAIDS antiserum/Ig core POD (HIV-1 anticore EIA, AbbottLaboratories). One OD Elisa pick was detected,demonstrating that on surcrose gradients the p55 gagprotein migrated as particles or "aggregated structures".The pick fractions and the surrounding fractions wereimmunoblotted with pl7, p24 or p55 antibodies. One majorband at Mr 55 kd in the SDS-reducing gels was detected
BAD ORIGINAL $ 1 10 15 20 25 30 35 corresponding to p55 gag precursor protein as compared toan extract of Molt/HILV-IlI cells prepared substantiallyas described above.
In a fifth assay experiment, a 5 % PEG6000 precipitatewas prepared substantially as described for the fourthassay experiment from 150 ml of a S.f. culture which hadbeen co-infected with the gag precursor recombinantBaculovirus and with a recombinant Baculovirus whichexpressed the HIV envelope protein at a MOI of 3 to 5.
The PEG6000 pellet was taken up in 200 ul of HBS-buffercontaining 0.1 % Tween 20. After centrifugation at 15000x g for 1 min, the supernatant was mixed with 11.5 ml of a 1.5 M CsCl solution (0.3 volumes HBS-buffer, 10 mMTris-HCL (pH 8.0), 1 mM ethylendiamine tetraacetate(EDTA), 0.1 % Tween 20 and 10 ug/ml of aprotinin). Thissuspension was centrifuged in a Beckman Rotor 50Ti forabout 72 hr at 44000 rpm at 18°. Fractions of 300 uleach were collected, frozen at -20°C and tested with aspecific antigen capture Elisa assay (HIV anticore EIA). 3
Bands at densities of about 1.28 and 1.20 g/cm wererecognized, the core-like particle apparently having the 3 density of 1.28 g/cm .
Electron microscopy confirmed the presence of pre-core(and core) -like particles in the conditioned medium.Scanning electron microscopy revealed particles whichapparently were budding onto the cell surfaces.
Immunogold transmission electron microscopy revealedparticles which were recognized by p24 and and p55antibodies. Also, pl7, p24 and p55 epitopes wererecognized by immunogold labelling after brief treatmentwith Triton X100 of purified particles in electronmicroscopic preparations. The particles wereapproximately spherical and of about 100 - 150 nm indiameter. The particles display electron luscent centerssurrounded by a dark staining ring and an outer shell andappear to have the majority of the pl7, p24, pl6 and p55epitopes on the inside surface of the particle, -20- AP 0 0 0 1 2 9 BAD ORIGINAL ft 1 10 15 20 25 30
This Example, therefore, demonstrates expression andsecretion of HIV pre-core-like (and core-like) particlescomprising immunodeficiency virus gag precursor protein.The particles comprise predominantly (greater than 90% oftotal protein) full length gag precursor protein and areformed in the absence of DNA sequences of viral originother than the gag precursor sequence and, hence in theabsence of other viral functions such as the retrovirusprotease and reverse transcriptase.
To demonstrate that the HIV gag precursor protein made in S.f. cells is efficiently myristylated, 3 X 106 cells2 in F25 cm flask, were labelled at 48 hr p.i. with 500pCi myristic acid NET-830 (Dupont, Wilmington, Delaware)for 18 hr after they had been infected with recombinantp55 gag baculoviruses at MOI of 5. Subsequently, theconditioned medium and the cells were processed separatelyfor western blotting and SDS-gel radioautography.Conditioned medium displayed one major band at 55 kd whichwas also recognized as gag precursor in western blots asrevealed by antibodies against pl7, p24, p55. Two otherlabelled minor bands were detected at Mr 49-46-47 kd andwere recognized specifically by the same set of antibodies(pl7, p24, p55) in the western blot. Cell lysates made in1 % triton x 100 and frozen at -20°C displayed onradioautography of the 12.5 % Laemli gel and western blotrespectively band at 55 kd (and a minor band at 58 kdwhich apparently corresponded to the translation frameshift as described for the gag retroviral HIV-l virusgenome and more prominent bands at Mr 49-47-46 anddegradation products at Mr 30-27 kd the latter bands werenot radioactive (containing no myristic acid).
Example 3. Expression in Mammalian Cells:
The plasmid pRITl3002 was introduced via the
Ca-phosphate coprecipitation technique (Wigler, et al.,Cell 16:777(1979)) in CosI and CV1 cells. At 48 hr and
1 10 15 20 25 30 35 110 hr post-transfection, the cells and culture mediumwere assayed using an ELISA specific for gag antigenexpression. Cell extracts (106 cells) were adjusted to1 % Triton X100 or 0.5 % DOC-NP40. The p55 antigen wasdetected using ELISA capture antigen tests involvingpolyclonal and monoclonal antibodies to pl7, p24 or p55 orusing the Dupont RIA test (NEK-040), involving acompetition with purified p24 peptide. The expressionlevels obtained were between 4 and 10 ng/ml as measured bythe p24 RIA Dupont test.
Example 4. Expression in yeast cells
The plasmid pRIT12985 was introduced into theS. cerevisiae strain 02276b (ura3~ durO*1 rod-).
The p55 antigens were detected in yeast extracts (cells inmid-log phase, broken using glass beads or spheroplastingwith zymolase). The p55 was detected using ELISA tests,involving polyclonal and monoclonal antibodies to the p24peptide, or using the Dupont radioimmune assay (RIA)involving competition with the purified p24 peptide.
The p55 protein synthesized in S. cerevisiae wasobserved in Western blots, using pl7 or p24 specificmonoclonal antibodies, and has a molecular weight similarto that of the p55 antigen obtained from infected cells.
When cellular extracts were obtained in the absence ofdetergents, an important fraction of the antigen wasretained in the "membrane pellet". This fraction ofantigen was recovered using Triton X100. Use ofdetergents either prior to or after isolation enhancedantigenicity as measured in the RIA. The gag precursorproduced in yeast was shown to be myristilated bylabelling with tritiated myristic acid and was apparentlyassociated with cell plasma membrane as shown by electronmicroscopy.
The above Examples demonstrate expression of gagprecursor protein in animal cell culture and expression ofimmunodeficiency virus pre-core-like particles in AP000129 BAD ORIGINAL ft
Lepidoptera cells using a Baculovirus expression system.The protein and/or particles thus prepared are purifiedand formulated into a vaccine for parenteral administration to humans in danger of exposure to HIV, inorder to protect the vaccinees from onset of diseasesymptoms associated with HIV infection. Each vaccine dosecomprises an amount of the protein or particle which issafe, i.e., does not cause significant adverse sideeffects, but which is effective in inducing an immune A. response. For example, each dose comprises 1 to 1000 ug,preferably 10 to 500 ug, of gag precursor protein orparticle in a pharmaceutically acceptable carrier, e.g.,an aqueous solution buffered to about pH 5 to 9,preferably pH 6 to 8. The vaccine can also comprise anadjuvant, e.g., aluminum hydroxide, muramyl dipeptide or asaponin such as Quil A. Useful buffers include buffersderived from sodium or ammonium cations and acetate,citrate, phosphate or glutamate anions. Otherpharmaceutically acceptable carriers or diluents can beused to adjust isotonicity or to stabilize theformulation, e.g., sodium chloride, glucose, mannitol,albumin or polyethylene glycol. The vaccine can belyophilized for convenience of storage and handling. Suchvaccine is reconstituted prior to administration.Alternatively, the gag protein or particle can beformulated in liposomes or ISCOMS by known techniques. Anexemplary vaccine dose comprises 100 ug of gag particlesadsorbed on aluminum hydroxide in water buffered to pH 7with sodium acetate.
In an alternative embodiment of the invention, the gagprotein or particle is mixed with one or more otherantigens by coexpression in the same cell culture or byco-formulation. Such other antigens can be other HIVantigens, e.g., antigens derived form the envelopeprotein, gpl60 or gpl20, or can be antigens derived fromone or more other pathogenic organisms, cells or viruses, BAD ORIGINAL ft 1 10 15 20 25 30 such as hepatitis B surface antigen for conferringprotection against Hepatitis B Virus or antigens derivedfrom the Herpes Virus glycoprotein for conferringprotection against Herpes Virus.
The vaccine is preferably administered parenterally,e.g., intramuscularly (im) or subcutaneously (sc),although other routes of administration may be useful inelicitng a protective response. The vaccine isadministered in a one-dose or multiple-dose, e.g., 2 to 4,course. Immunoprotection can be ascertained by assayingserum anti-gag antibody levels. Thereafter, vaccinees canbe revaccinated as needed, e.g., annually.
As a diagnostic reagent, the gag protein or particlescan be used in any of the standard diagnostic assays, suchas an ELISA or RIA, to detect the presence of anti-HIVantibodies in clinical specimens. Such diagnostic can beused in conjunction with other HIV antigens to monitordisease progression. Use of the gag protein or particleas a diagnostic reagent will generally involve contactinga sample of human or other animal serum or other bodyfluid with the protein or particle, preferably bound orotherwise affixed or entrapped, and then assaying forbinding of anti-gag antibodies from the serum or othersample to the gag protein or particles. Such assay can beaccomplished by standard techniques, including byquantitating binding of subsequently added labelledanti-gag antibodies.
The above description and examples fully disclose theinvention and the preferred embodiments thereof. Theinvention, however, is not limited to the embodimentsspecifically disclosed herein but, rather, encompasses allimprovements, variations and modifications thereof whichcome within the scope of the following claims. ΑΡ000129 35 24 BAD ORIGINAL fl EXAMPLE 5,
CONSTRUCTION AND EXPRESSION OF A MUTANT Pr559a*3GENE
In order to examine the potential role of theN-myr s itoylation in the assembly and formation of extracellulargag particles we have constructed a glycine deletion mutant.Therefore a synthetic oligonucleotide linker syn3 was for the BamHI-Clal fragment in pRIT12982 (seeSyn3 encodes the genuine N-terminal amino acids ofprotein except that the second glycine codon isThis mutant BamHI Pr559a9 expression cassette wassubcloned into the BamHI site of the baculo expression vectorpAcYMl (Matsuura et al., J. Gen. Virol. 68.:1233 (1987)) andrecombinant plaques were obtained and selected essentially asdescribed in Example 1. The recombinant virus, AcGag 31-18,harbouring the glycine deletion mutation of the gag gene wasused to infect S.f. cells. The gag precursor protein wasefficiently synthesized as determined by an ELISA assay.Metabolic labelling with 3H-myristic acid essentially as in Example 2 revealed no myristic acid incorporationthat deletion of the N-terminal glycine wasto prevent myristoylation of the GAG precursorprotein. Analysis of the cell extracts in Western blots (seeExample 1) showed a prominent band of 55 kd and lower M.W.degradation products. The obtained pattern of protein bands wassimilar to the wild type (wt) Pr559a9 protein expressed inS.f. cells. In contrast with the wt Pr559a9 recombinant, nogag protein could be detected with the glycine mutant 2 daysp.i. using PEG or ultracentrifugation of the conditionedmedium. Thus the mutated Pr559a9 protein was only detectedwithin the infected cells. The myristoylation process thusseems to be required for the extracellular release of thePi559a9 product. Scanning electron microscopy (SEM) revealed rather smooth, showing no particles,electron microscopy and immunogold substituted,example 1).the gagdeleted. describedconf i rmingsufficient 48 and 66 hrsvirus revealed that the cell surface wasThin section transmission labelling performed on cells infected 24 hrs,p.i. with the Ac gag 31-18 (Myr-) recombinanttliat the non-myristoylated GAG protein was efficiently expressed, scattered in the cytoplasm or associated to greyamorphous structures within the cytoplasm and the nucleus.These i,ntracellu 1 ar particles or particulate structures aremorphologically different from the extracellular particlesobtained with the myristoylated gag recombinant (AcGag7) asthey display a double electron dense ring structure andcontain a lipid bilayer derived from the cell membrane. GAG protein nor budding structures were observed at themembrane.
These gag precuror do not
Neither cell results demonstrate that the myristoylation of theappears to be required for its plasma membranelocation, budding and extracellular particle release.Myristoylation does not seem however to be required for thenmltimeiic assembly of the Pr559a9 molecules. Accumulation ofthe non-myristoy 1 ated Pr559a9 products within the nucleus(and nucleoli) is a surprising phenomenon. 24(a)
BAD ORIGINAL EXAMPLE_£x. construction and expression of a truncated
Pr55^a9 PRECURSOR PROTEIN
In order to examine the role of the pl6 (COOH-end) ofthe HIV precursor GAG protein we made a GAG deletion mutantwhich encodes only the pl7-p24 precursor part of GAG.
The BamHI-CfrI GAG fragment of pRIT13003 was purifiedand ligated with a synthetic oligonucleotide sequence 5' GGC CAT AAG GCA AGA GTT TTA GTT AGT TAG 3’ TCT CAA AAT CAA TCA ATC CTA G 5'in the BamHI-site of pAcYMI. Thisgenuine amino acid COOH-end of the two additional amino acids, Valineplasmid was used to co-transfect S, 3' TA TTC CGTpurified and clonedsequence contains thecore protein andThis recombinant
AcMNPV DNA essentially asRecombinant plaques were described before (seescreened as described in andf . and gellinkerHIV p24Serine,cells with
Example 1. A selected recombinant virus, Ac CfrI, was used toinfect S.f. cells. A truncated gag-polypeptide (pl7-24) wasdetected at the expected M.W. of 41 Kd and which reacted inWestern blot analysis with pl7 and p24 monoclonals. The pl7-24product was predominantly expressed inside the cells but asmall amount of extracellular pl7-24 product could be detectedwhen analyzing the conditioned medium by Western blotting. PEGprecipitation and ultracentrifugation of the conditioned mediumof the CfrI mutant gag protein did not result in detectablepl7-p24 product. Electron microscopy analysis showed noevidence of budding or extracellular gag particles. Largeprofusions 1-4 μιη long in the form of tubular structures whichare longitudinally connected to the cell membrane surface couldbe detected early in infection. Immunogold labelling showedthat the truncated GAG protein (pl7-p24) was localized at thecell membrane and at the periphery of these tubular extensions,but no electron dense "ring" structures - typical of thePr559a9 particle structures - could be detected. Thisprobably indicates that the pl7-p24 product is not able toassemble in multimeric structures, i.e., cap formations, at thecell membrane. These results suggest that at least a part ofthe pl6 polypeptide of the GAG precursor polypeptide isnecess'a ry for particle formation. A glycine deletion mutant of the CfrI cassette(non-myristoy1 a ted pl7-24) was made by exchanging theEcoRV-Pstl fragment of 669 bp of the pAcGag 31-18non-myristoyla ted Ργ55^Α0 gene with the pAC CfrI EcoRV-Pstl +9400 bp long fragment. This mutant displayed no protrusions ofmembranes as described above but showed pl7 and p24 immunogolddecoration scattered in the cytoplasm and nucleus. APO 0012 9 24(b) -
EXAMPUE_7 x CONSTRUCTION_ANQ_EXPR£SSION OF A GAG-POL PROTEIN
To express the gag-pol products, we have included most(about 80 %) of the pol gene into the baculovirus transfervector carrying the Pr559a9 expression cassette (pRIT13003).
The pol gene UNA fragment is a BglH (2093) - EcoRI (4681)restriction fragment from BH10 (Shaw et al., Science 2.26:1165(1984). A poly-stop synthetic DNA fragment 5* AAT TCC TAA CTAACT AAG 3* 31GGA TTG AT TGA TTC CTA G 5'was added at the Eco RI site. The resulting baculovirusexpression plasmid, LE-8-4, was used in a co-transfectionexperiment to generate recombinant plaques essentially asdescribed in Example 1.
In this recombinant construct, the myristoylatedPr559a9 as well as a gag-pol product resulting from theHIV-specific translational frame-shift in S.f. cells, areexpected to be produced, and subsequently processed by theprotease. Recombinant baculovirus harbouring the gag-pol genewas screened and selected essentially as described in Example1. In S.f. cells infected with such a gag-pol recombinantvirus, VAC 8-5, no gag or gag-pol products were detected whenthe conditioned medium was analysed by Western Blot orpLecipitated with PEG.
Cell extracts however, did show a strong doublet bandat 24 Kd and a band at 17 Kd which reacted with p24 and pl7monoclonal antibodies in Western blots. Very small amounts ofthe precursor Pr559a9 band and intermediate 41 Kd (46 Kd)bands could also be detected in Western blots. This indicatesthat the protease is active in the gag-pol fusion protein,expressed by translational frame-shift in S.f. cells. Thisresults in pl7, p24 polypeptides and intermediates (41 Kd, 46-49 Kd, 55 Kd). The large precursor gag-pol product was notdetected with our p55, pl7 or p24 antibodies.
Electron microscopy showed on rare occasions a fewparticles budding at the cell membrane. These particles seem tobe morphologially similar to the above described Pr559a9particles. Co-infection experiments with recombinant virusesharbouring the Pr559a9 and the gag-pol gene did not result indetectable particles displaying a morphological difference such
EXAMPLE 8CONSTRUCTION AND EXPRESSION OF THE SIV Pr579a9GENE IN S.F, CELLS
The gag gene of Simian Immunodeficiency virus (SIV)was subcloned from the molecularly cloned SIVrnac-BK28 (giftof J. Mullins; see Hirsh et a 1. , (J.eJLJL 4.2.:307 ( 1987) and Kestleret al., Nature 3JL1:619 (1988)). A 3504 bp Kpnl fragment of thepBK28 genome (nucleotides 1212 to 4716) was subcloned intopUC8. Two internal fragments of the gag gene, the 5' fragmentFnur>n( 1201) - Pst ( 1959) and the 3’ fragment Pstl ( 1959) -Hphl (2803) were purified and synthetic oligonucleotidelinkers, linker 1:
GAT CC ACC ATG GGCG TGG TAC CCG and linker 3: TGCTGCACCTCAATTCTCTCTTTGGAGGAGACCAGTAGAGATCTGGTACAACGACGTGGAGTTAAGAGAGAAACCTCCTCTGGTCATCTCTAGACwere ligated to adequate gag fragments to reconstituteentire the
G was used at thesecond codon,constructionsby sequencing.
precursor gag gene. In a separate experiment a linker 2:GATCC ACC ATG GCCTGG TAC CGG 5* fragment, to introduce a mutation in thenamely, GGC (Gly) to GCC (Ala). The different were cloned into blue scribe vectors and verified. The N-terminal fragment (BamHI-Pstl) and thecarboxy-termina1 fragment (PsI-Bglll) were isolated and clonedinto the BamHI digested, alkaline phosphatase treated pAcYMIbaculovirus expression vector. The pAC gag Myr+ plasmidcontains the native SIV gag gene and the pAc gag Myr-contains the mutated (Gly to Ala) gene. S.f. cells weretransfected with a mixture of purified AC MNPV viral DNA (1respective recombinant transfer plasmids (50 pg)as described in Example 1. The recombinant plaqueswere screened and selected as described in Example 1.
The SIV gag Pr579a9 precursor polypeptide wasefficiently synthesized in infected insect cells as observed inWestern blots using the rabbit antiserum to SIV (metrizamide -gradient purified SIV-BK28 virus) or a monoclonal directed the COOH-end of the HIV p24 polypeptide, which appearrecognize the SIV core protein.
In a second assay experiment it was demonstrated thatthe SIV native gag precurser gene expressed in S.f. cells wasefficiently myristoy1 a ted in contrast to the culture infectedwith the glycine to alanine mutant in which no myristoylationof the precursor Pr57ya9 protein could be detected whenanalysed on SDS-PAGE and radioautography.
As in the case of HIV-gag precursor protein we alsoobserved gag particle formation and release of particles in theconditioned medium when the infected cultures were analysed byultracentrifugation, sucrose gradients and electron microscopy py) and theessentia 1 ly againstalso to ΑΡ00 0 1 2 9 - 24(d) -
BAD ORIGINAL £ (TEM and SEM). Similar SIV-gag Pr579a9 particles as thoseobtained when expressing HIV-Pr559a9 precursor gene in S.f.cells were observed. The extracellular gag particles formcrescent structures at the cell membrane which assemble intotypical buds that closely resemble immature virus buddingparticles. The SIV Pr579a9 as the HIV Pr559a9 particleswere about 100-120 nm in diameter and showed a light greytransluscent center surrounded by a tick dark electron densering and an outer lipid bilayer. Experiments with the SIVnon-myristoylated (Gly to Ala) mutant confirmed theobservations made with the HIV-non myristoylated Pr559a9mutant that N-myristoylation is essential for budding andextracellular particle formation.
The difference between the gag precursor protein of HIV and SIVis that the latter forms also intracellular particles andparticulate structure when the native SIV gag protein(myristoylated) is expressed. This could be explained asfollows : the expression level is about 3 times higher than theHIV gag expression level and maybe not all the SIV Pr579a9molecules are myristoylated. Also more degradation products,especially a myristoylated p27 protein band could be detectedin WB of cultures infected with the SIV Pr579a9 nativeconstruct.
It is possible that the cellular structures of about40 nm in diameter and sometimes up to 1 pm. long - which areobserved at late stage of infections - are composed at least inpart of these degradation gag products. This could resemble thep24 core assembly into tubular structures observed in some rarecases of retroviral core maturation. Also when the p24 coreprotein of HIV-1 is expressed in E. coli, tubular structurescontaining p24 protein have been observed. Part of theintracellular particles observed with the native SIV gagprecursor recombinant take form near the cell membrane, wherethey appear to differentiate into virus-like particles buddingas described above. This process is reminiscent of the viralmaturation of type D retrovirus which involves intermediateintracellular type A particles. - 24(e)
X BAD ORIGINAL &
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