AU627465B2

AU627465B2 - Expression of retrovirus gag protein in eukaryotic cells

Info

Publication number: AU627465B2
Application number: AU37256/89A
Authority: AU
Inventors: Dirk Gheysen; Eric Jacobs
Original assignee: GlaxoSmithKline Biologicals SA; SmithKline Biological SA
Current assignee: GlaxoSmithKline Biologicals SA
Priority date: 1988-06-03
Filing date: 1989-06-30
Publication date: 1992-08-27
Anticipated expiration: 2009-06-30
Also published as: ZA894137B; AU4320689A; AP8900126A0; AP129A; AU3725689A; WO1991000904A1; IL90381A0; EP0345242A3; PT90731A; NZ229297A; EP0345242A2

Description

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4 1 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 Nest Africa. Other immunodeficiency viruses include the SIV viruses such as SIV -BK 28 Molecular characterization of the HIV genome has demonstrated that the virus exhibits the same overall gag-pol-env organization as other retroviruses. In addition, it contains at least five genes that are not found in more 15 ordinary retroviruses: sor, tat3, art/trs, 3'orf and R. The gag region encodes 3 core proteins, p17, p 24 and p16, which are prepared by cleavage of a 55 kilodalton gag precursor protein by the HIV protease. The protease is encoded by the pol region.

Recent reports have shown that antibodies to the HIV gag proteins, p17, p24 and p16, are present in human sera from infected individuals in the United States and Europe and that antibodies arise early after infection. The presence of these antibodies declines as the individual proceeds towards AIDS.

The gag protein p17 with its submembrane localization is 25 well positioned to be in close contact with the transmembrane protein gp41 and the viral membrane and with gag p24 and possibly gag p15 viral RNA thereby playing a central role in the conformational changes involved in the viral entry and uncoating process. Furthermore, gag p17 has been found to have a myristylated N-terminus. Myristylation has been implicated in virion assembly and transport of viral components to the plasma membrane. Myristylated proteins are generally localized in the plasma membrane.

Madisen et al., Virology 158:248 (1987), report expression of the HIV gag protein in Spodoptera frugiperda cell using the S* 4 2404K 050388 2ri i ii Si ii i id,_iill 4 44 4 4 4 4 *e 4 1 14 I 4 4 44,4 1 AcMNPV Baculovirus using a DNA sequence comprising 51 untranslated sequences and sequences from the pol region in addition to the gag sequence.

Cochran, EP-A-228,036, disclose use of a Baculovirus/insect 5 expression system to express certain proteins. At pages 17a and 26, AIDS retrovirus core proteins are mentioned among a list of proteins which may be produced in such system.

Cochran et al., EP-A-265,785, disclose expression of HIV envelope proteins in insect cells using a Baculovirus expression system.

Kramer et al., Science 231:1580 (1986) and Kramer et al., EP-A-230,222, report expression of a gag-pol DNA sequence in yeast and in vivo cleavage of the precursor by the HIV protease expressed from this region.

Valenzuela et al., Nature 298:347 (1982), report synthesis the Hepatitis B Virus surface antigen in yeast and its assembly into particles.

Ellis et al., EP-A-251,460, disclose synthesis of the Hepatitis B Virus core antigen in yeast and its assembly into 20 particles.

Adams et al., Cell 49:111 (1987), report synthesis of the pl protein of Ty-virus-like particles (VLP's) in yeast and assembly into particles. The outlines conclude that pl is a precursor to the major core proteins of Ty-VLP's and, in this respect, is functionally similar to the gag precursor protein of retroviruses.

Bishop et al., EP-A-260,090, disclose expression of Hepatitis B Virus antigens using a baculovirus expression system.

30 Rusche et al., EP-A-272,858, disclose expression of proteins derived from HIV GP 160 using a baculovirus expression system.

#0* 0 4 14444 2404K 050388 3 r^ 1! eL;iwt~ 9* .9 9 9 999 9 4i q 99 9 9i *9*9I 9 990 9 9 49 99 9 .9 9 *9 99l 4 0C* 1 In spite of major research efforts in the area of AIDS, there continues to be a need for diagnostic reagents which can be used to monitor disease progression and for agents which can prevent primary infection, such as via immunization, and for agents which can prevent or inhibit secondary infection, such as by cell-to-cell transmission or by free virus infection.

Summary of the Invention In one aspect, this invention is a recombinant DNA molecule for expression of gag precursor protein in eukaryotic cells which comprises a coding sequence there for operatively linked to a regulatory region which functions in the host cell.

In related aspects, this invention is host cells comprising the recombinant DNA molecule and cultures thereof.

15 In further related aspects, the invention is the gag precursor protein produced by the host cells of the invention, including a HIV core-like particle comprising the gag precursor protein.

In yet further related aspects, the invention is a process 20 for producing the recombinant DNA molecule and the host cell of the invention, a process for producing the gag precursor protein and particles of the invention, and related compositions and methods.

These and other aspects of the invention are fully 25 described in the disclsoure and Examples which follow.

Detailed Description of the Invention It has now been found that retroviral gag precursor protein can be expressed in recombinant eukaryotic cells and that such 30 expression can result in production of full-length gag precursor protein without use of pol DNA sequences and without use of 5' untranslated sequences from the virus. Exemplary of such cells are cells from lower eukaryotes such as yeast and fungi and animal cells including insect cells such as Drosophila or Lepidoptera cells; mammalian cell lines; mammalian primary cells, and insects and transgenic animals.

2404K 050388 4 9 9 9 s4 f rr o i" 1 It has also been found, unexpectedly, that the gag precursor protein can form particles which resemble authentic gag particles formed in infected human cells in size and other physical properties and in antigenicity. During a natural retrovirus infection cycle, it appears that gag precursor protein, known in the case of HIV as p55, is formed largely into particles comprising predominantly full-length gag protein. These gag particles can be referred to as pre-core particles or immature core particles. Then, during viral maturation, the precursor is cleaved into the subunit proteins known in the case of HIV as p17, p 24 and p16. These gag particles, now comprised predominantly of p17, p24 and p16, can be referred to as core particles or as mature core particles.

Also during viral maturation, apparently during the budding 15 process, the viral membrane is formed around the pre-core or core particles. As shown in the Examples below, HIV gag precursor expressed in recombinant Lepidoptera cells using a Baculovirus expression system are largely aggregated or packaged in particles which have physical and biological properties and dimensions similar to those of the core of HIV particles formed naturally in infected human cells. The particles of the invention comprise predominantly gag precursor protein (greater than 90% of all protein in the particles is full length gag precursor) but nevertheless are recognized 25 after brief treatment with Triton X100 by anti-pl7 monoclonal antibodies (MABs), anti-p24 MABs and anti-pl6 MABs in addition to being recognized by anti-gag polyclonal antibodies from sera of infected patients. The particles, because they are prepared by recombinant DNA techniques as disclosed herein, lack viral 30 functions required for viral maturation and replication especially viral RNA and also, preferably, reverse transciptase and protease functions.

The recombinant eukaryotic cells of the invention are engineered to express the gag precursor protein by introduction into the cells of the recombinant DNA molecule of the invention. The recombinant DNA molecule of the invention 2404K

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050388 5 2 ,i Ir~l-e 3.

4400 4h 4' 04 4 4t.4 4 O 44 o 4 49 9O 4o 4 0 4 44 4 1 comprises a coding region for the gag precursor protein operatively linked to a regulatory element which functions in the selected host cells. As an aspect of this invention, it has been found that other HIV functions are not required for expression of the gag precursor protein and for pre-core-like particle formation. DNA sequences coding for other functions, for amplification functions, selection markers or maintenance functions, can also be comprised within the recombinant DNA molecule of the invention.

A DNA coding region for gag precursor protein can be prepared from any of the several immunodeficiency virus genomic clones or gag-pol clones reported in the literature. See, for example, Shaw et al., Science 226:1165(1984); Kramer et al., Science 231:1580(1986). Alternatively, an immunodeficiency 15 virus genomic clone can be prepared from virus isolated from clinical specimens by standard DNA cloning techniques. See, for example, Gallo et al., U.S. Patent 4,520,113; Montagnier et al., U.S. Patent 4,708,818. Having cloned a fragment of the genome which comprises the gag coding region, a region which codes only for the gag precursor can be prepared by restricting the DNA so as to isolate a portion of the DNA coding region and reconstructing the remaining portions through use of synthetic oligonucleotides, such as described in the Examples, below.

Alternatively, a larger fragment comprising the gag coding 25 region and additional sequences can be cut back through use of exonucleases. In yet another alternative procedure, the entire coding region can be synthesized using standard automated DNA synthesizers by synthesizing fragments of the coding region and ligating these together to form a complete coding region.

While use of a coding sequence which lacks the naturally occurring 5' and 3' flanking sequences is preferred, fusion of the coding sequence to other immunodeficiency virus sequences, envelope protein sequences, is not precluded from the preferred embodiments.

4444 4 4 004 0 0 a) 44 4 4 4 2404K 050388 -6i L j I 4r An exemplary coding region for the HIV gag precursor protein has the following sequence.

1 ATG GGT GC AGA 000 TCA 4 44 4 444, 4$ 44 444, 44 4 4 4, 4 4,4 4 4 4,44 4 44I 4,, 44 44, 4 4I 44 4, 44 4 4, .4,44 4,b 44404O 4,4 44 4,4 4 14 4 4, Met 37 TTA Leu 73 GGA Gly 109 GCA Ala 145 GGC Gly 181 CTO Leu 217 GAA lu 253 CTC Leu 289 ACC Thr 325 AAC Aa 361 GAC Asp 397 CCT Pro 433 CAG Gin Gly

GAT

Asp

MG

Lys

AGC

Ser

CTG

Leu

OGA

Gly

GA

Glu

TAT

Tyr

AAG

Lys

AAA

Lys

ACA

Thr

ATA

Ile

GCC

Ala Ala

CGA

Arg

AAA

Lys

AGG

Arg

TTA

Leu

CAG

Gin

CTT

Leu

TGT

CyR

GAA

GIu

AGT

Ser

GGA

Gly

GTG

Val

ATA

lie rg

TGG

Trp

AAA

Lys

GAG

Glu

GAA

Glu

CTA

Leu

AGA

Arg

GTG

Val

GCT

Ala

AAG

Lya

CAC

His

CAG

Gin

TCA

Ser Ala

GMA

Gilu

TAT

Tyr

CTA

Leu

ACA

Thr

CAA

Gln

TCA

Ser

CAT

His

TTA

Leu

AAA

Lys

AGC

Ser

AAC

Asn

CCT

Pro Ser

AAA

Lys

AAA

Lya

GAA

Glu

TCA

Ser

CCA

Pro

TTA

Leu

CAA

Gin

GAC

Asp

AAA

Lys

AG?

Ser

ATC

Ile

AGA

Arg

GTA

val

ATT

Ile

TTA

Leu

CGA

Arg

GAA

Glu

TCC

Ser

TAT

Tyr

AGG

Arg

AAG

Lya

GCA

Ala

CAG

G. n

CAG

Gin

ACT

Thr

OCT

Ala 7-

TTA

Lou

CGG

Arg

AAA

Lys

TTC

Phe

GGC

Gly

CTT

LQu

AAT

As n

ATA

Ile

ATA

Ile

CAG

Gin

GTC

Val

GGG

Gly

TTA

Leu

TTC

Phe

ACC

Ser

TTA

Lou

CAT

His

GCA

Ala

TGT

CYn

CAG

Gin

ACA

Thr

GAG

Glu

GAG

Giu

CAA

Gin

AGO

Ser

CAA

Gin

AAT

Asn

AGC

Ser OCC GGA Gly Gly AGG CCA Arg Pro ATA GTA le Val OTT AAT Val Asn AGA CAA Arg Gln ACA GGA Thr Gly GTA GCA Val Ala ATA AAA Ile Lys GAA GAG Glu Glu GCA OCA Ala Ala CAA AAT Gin Asn ATG GTA Met Val GCA TGG Ala Trp CCA GAA Pro Glu

GAA

alu

GGG

Gly

TGG

Trp

CCT

Pro

ATA

Ile

TCA

Scr

ACC

Thr

GAC

Asp

CAA

Gin

GCT

Ala

TAC

Tyr

CAT

His

OTA

Val

GTA

Val 36 72 108 144 180 216 252 288 324 360 396 432 468 469 AAA GTA GTA GAA GAG AAG Lys Val Val Giu OlU Lys 2404K 050388 505 ATA CCC ATG TTT TCA GCA TTA TCA GAA GGA GCC ACC 540 tie Pro Met Phe Ser Ala Leu See' Giu Gly Ala Thr 541 CCA CAA GAT TTA AAC ACC ATG CTA AAC ACA GTG GGG 576 Pro Gin Asp Leu Aan Thr Met Leu Asn Thr Val Gly 577 GGA CAT CAA GCA GCC ATG CAZ ATG TTA AAA GAG ACC 612 Glv His Gin Ala Ala Met Gin Met Leu Lys Glu Thr

I

613 ATC AAT GAG GAA GOT GCA GAA TGG GAT AGA GTA CAT 648 Ile Asn Glu Glu Ala Ala Giu Trp Asp Arg Val His 649 CCA GTG CAT GCA GGG CCT ATT GOA CCA GGC CAG ATG 684' Pro Val His Ala Gly Pro Ile Ala Pro Gly Gin Met 685 AGA GAA OCA AGG GGA' AGT GAO ATA OCA OGA ACT ACT 720 Arg Giu Pro Arg Gly Ser A~p Ile Ala Gly Thr Thr 721 AGT ACC CTT CAG GAA CAA ATA GGA TGG ATG ACA AAT 756 Ser Thr Leu Gin Giu Gin Ile Gly Trp Met Thr An 757 AAT CCA COT ATO CCA GTA GGA GAA ATT TAT AAA AGA 792 Asn Pro Pro Ile Pro Val Gly Giu Ile Tyr Lys Arg 793 TGG ATA ATC CTG GGA TTA AAT AAA ATA GTA AGA ATG 828 Trp Ile Ile Leu Gly Leu Asn Lye Ile Val Arg Met 829 TAT AGO CCT ACC AGC ATT CTG GAC ATA AGA CAA GGA 864 Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gin Gly *4I 865 CCA AAA GAA CCT TTT AGA GAO TAT GTA GAO CGG TTO 900 Pro Lys Giu Pro Phe Arg Asp Tyr Val Asp Arg Phe 901 TAT AAA ACT-CTA AGA GCC GAG CAA GOT TCA CAG GAG 936 Tyr Lys Thr Leu Arg Ala Giu Gin Ala Ser Gin Glu 04 4 937 OTA AAA AAT TGG ATG ACA GAA ACO TTG TTG GTO CAA 972 Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gin 973 AAT GOG AAC OCA GAT TGT AAG ACT ATT TTA AAA GCA 1008 Asn Ala As Pro Asp Cys Lys Thr Ile Leu Lys Ala 1009 TTG GGA COA GCG GOT ACA CTA GAA GAA ATG ATG ACA 1044 k Leu Gly Pro Ala Ala Thr Leu Giu Giu Met Met Thr 2404K 050388 -8- -Impw

I-

1045 GCA TOT CAG GGA GTA GGA GGA CCC. GGC CAT AAG GCA Ala Cys Gin Gly Val Gly Gly Pro Gly Hlis Lys Ala 1081 AGA GTT TTG GCT GAA GCA ATG AGO CAA GTA ACA AAT Arg Val Leu Ala Giu Ala Met Ser Gin Val Thr Asn 1117 ACA GCT ACC ATA ATG ATG Thr Ala Thr lie Met Met

CAG

Gin AGA GCC AAT TTT AGO Arg Gly Asn Phe Arg 4, 8~ 4 4 t, 4 44.

'4 I I 4' It 4 4~ I~ 4 I 1153 AAC CAA AGA MAG ATGOGTT MAG TOT TTC A.T TOT GGC Asn. Gin Arg Lys Met Val. Lys Cy5 Phe Aari Cys Giy 1189 AAA GAA GGG CAC ACA GCC AGA AAT TOO AGO C CCT Lys Giu Gly His Thr Ala Arg Asn Cys Arg Ala Pro 1.225 AGO AAA AAG CCC TGT TGG AAA TGT GGA AAG GAA Gc-A Axg Lys Lys Gly Cys Trp Lys Cys Gly Lys (Zlu Gly 1261 CAC CAA ATO AAA GAT TGT ACT GAG AGA CAG GCT M-T His Gin Met Lys Asp Cya Thr Oiu Ary Gin Ala Asn 1297 TTT TTA GGG MAG ATC TGG COT TOC TAC AAG GGA AGO Phe Leu Gly Lys Ilie Trp Pro Ser Tyr Lys Gly Arg 1.333 CCA GCC AAT TTT CTT GAG AGC AGA CCA GAG CCA ACA Pro Gly Asn Phe Leu Gin Ser Arg Pro Giu Pro Thr 1369 GCC CCA CCA TTT CTT GAG AGC AGA OCA GAG CCA ACA Ala Pro Pro Phe Leu Gin. Ser Arg Pro Glu Pro Thr 1405 GCC CCA OCA GAA GAG AGO TTO AGG TOT COG GTA GAG Ala Pro Pro Giu Giu Ser Phe Arg Ser Gly Val Giu 1441 ACA ACA ACT CCC COT CAG AAG GAG GAG COG ATA GAO Thr Thr Thr Pro Pro Gin Lys Gin Glu Pro Ilie Asp 1477 AG, CAA OTG TAT COT TTA ACT TOO OTO AGA TOA OTO Lys Glu Leu Tyr Pro Leu Thr Ser Leu Arg Ser Leu 1080 1116 1152 1188 1224 1260 1296 13312 1368 1404 1440 1476 1512 8 4 8, I 4

I

4 1 4884 44 88

A

4 8 4 4 8 '4 1513 TTT 000 MAC GAC COO TOO TCA CAA TMA Phe Gly Asn. Asp Pro Ser Ser Gin End 1539 2 404K 050388 9- L~i_ -7 Z_ o ac o 4 D a 4g 40 4 o O a t 4 4 0 V) I 44U C £4 81 a 40* 1 A variety of eukaryotic cells and expression systems are available for expression of heterologous proteins.

The most widely used among these are yeast, insect and mammalian systems, although the invention is not limited to use of these. Typically, these systems employ a recombinant DNA molecule comprising a coding sequence for the gene of interest operatively linked to a regulatory element, a selection marker and, in some cases, maintenance functions such as an origin of replication. A regulatory element is a DNA region or regions which comprise functions necessary or desirable for transcription and translation.

Typically, the regulatory region comprises a promoter for RNA polymerase binding and initiation of transcription.

Insect cells which can be used in the invention 15 include Drosophila cells and Lepidoptera cells. Useful Drosophila cells include S1, S2, S3, KC-0 and D. hydei cells. 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 by standard techniques, including calcium phosphate precipitation, cell fusion, electroporation and viral transfection. Cells are cultured in accordance with standard cell culture procedures in a variety of nutrient 25 media, including, M3 media which consists of balanced salts and essential amino acids. See, Lindquist, DIS 58:163 (1982).

Promoters known to be useful in Drosophila include mammalian cell promoters as well as Drosophila promoters, 30 the latter being preferred. Examples of useful Drosophila promoters include the Drosophila metallothionein promoter, the 70 kilodalton heatshock protein promoter (HSP70) and the COPIA LTR. See, for example, DiNocera et al., Proc.

Natl. Acad. Sci. USA 80:7095 (1983); McGarry et al., Cell 42:903 (1985). Conveniently, an expression cassette comprising the gag coding sequence and regulatory element 0 4 0 0 2404K 050388 10 1 can be cloned within a bacterial cloning vector for purposes of propagating the DNA prior to transfection of the animal cells.

In the preferred embodiments of this invention, the HIV gag precursor is expressed in Lepidoptera cells to produce immunogenic gag particles. For expression of the gag precursor protein in Lepidoptera cells, use of a Baculovirus expression system is preferred. In such system, an expression cassette comprising the gag coding sequence and regulatory element is placed into a standard cloning vector for purposes of propagation. The recombinant vector is then co-transfected into Lepidoptera cells with DNA from a wild type Baculovirus. Recombinant viruses resulting from honologous recombination are then selected and plaque purified substantially as described by Summers et al., TAES Bull. NR 1555, May, 1987.

Useful Lepidoptera cells include cells from Trichoplusia ni, Spodoptera frugiperda, Heliothis zea, Autographica californica, Rachiplusia ou, Galleria o 20 melonella, Manduca sexta or other cells which can be infected with Baculoviruses, including nuclear polyhedrosis viruses (NPV), single nucleocapsid viruses (SNPV) and multiple nucleocapsid viruses (MNPV). The preferred Baculoviruses are NPV or MNPV Baculoviruses because these contain the polyhedrin gene promoter which is highly expressed in infected cells. Particularly exemplified hereinbelow is the MNPV virus from Autographica 4o californica (AcMNPV). However, other MNPV and NPV viruses SO can also be employed the silkworm virus, Bombyx mori.

30 Lepidoptera cells are co-transfected with DNA comprising Sthe expression cassette of the invention and with the DNA of an infectious Baculovirus by standard transfection techniques, as discussed above. Cells are cultured in accordance with standard cell culture techniques in a S 35 variety of nutrient media, including, for example, TC100 (Gibco Europe; Gardiner et al., J. Inverteb. Pathol.

2404K 050388 11

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2 1 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 by infecting insect larvae. For example, the gag precursor can be produced in Trichoplusia ni caterpillars by feeding the recombinant Baculovirus of the invention along with traces of wild type Baculovirus and then extracting the gag precursor from the hemolymph after about two days.

Promoters for use in Lepidoptera cells include promoters from a Baculovirus genome. The promoter of the polyhedrin gene is preferred because the polyhedrin protein is naturally over expressed relative to other Baculovirus proteins. The polyhedrin gene promoter from the AcMNPV virus is preferred. See, Summers et al., TAES Bull. NR 1555, May 1987; Smith et al., EP-A-127,839; Smith S. et al. Proc. Natl. Acad. Sci. USA 82:8404(1985); and i Cochran, EP-A-228,036.

20 For expression in mammalian cells, the expression cassette is likewise cloned within a cloning vector and is then used to transfect the mammalian cells. The vector preferably comprises additional DNA functions for gene I amplification, a DHFR expression cassette, and may also comprise additional functions for selection and/or amplification, a neomycin resistance cassette for G418 selection. Other functions, such as for transcription S. enhancement can also be employed. Yet other functions can "be comprised within the vector for stable episomal oo, 30 maintenance, if desired, such as maintenance functions of Bovine Papilloma Virus. The mammalian cell vector can also be a recombinant virus, such as a recombinant o* vaccinia or other pox virus. See, Paoletti, et al., 0: U.S. Patent 4,603,112; Paoletti, et al., Proc. Natl. Acad.

S 35 Sci. U.S. 81:193 (1984).

Useful mammalian cells include cells from Chinese 2404K 050388 12 1 hamster ovary (CHO), NIH3T3, COS-7, CVI, mouse or rat myeloma, HAK, Vero, HeLa, human diploid cells such as and N138, or chicken lymphoma cell lines.

Transfection and cell culture are carried out by standard techniques. Production in mammalian cells can also be accomplished by expression in transgenic animals. For example, the gag precursor can be expressed using a casein promoter and purified from milk.

Promoters useful in mammalian cell lines or mammalian primary cells include the SV 40 early and late gene promoters, the metallothionein promoter, viral LTR's such as the Rous sarcoma LTR, the Moloney sarcoma virus (MSV) LTR or the mouse mammary tumor virus (MMTV) LTR, or the adenovirus major late promoter and hybrid promoters such as a hybrid BK virus and adenovirus major late promoter.

The regulatory region can also comprise downstream functions, such as regions for polyadenylation, or other functions, such as transcription enhancer sequences.

Yeasts which can be used in the practice of the o o" 20 invention include those of the genera Hanensula, Pichia, Kluveromyces, Schizosaccharomyces, Candida and Saccharomyces. Saccharomyces cerevisiae is the preferred yeast host. Useful promoters include the copper inducible (CUP1) promoter, glycolytic gene promoters, TDH3, PGK and ADH, and the PH05 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., Science 219:620 (1983); Cabezon et al., Proc. Natl. Acad. Sci. USA S81:6594 (1984).

o 30 In the case of the gag precursor protein particles tote produced in accordance with this invention, it is to be understood that although particles comprising the gag oo** precursor are preferred, particles comprising derivatives 00 of the native gag precursor can also be prepared. For 35 example, one or more nucleotides or amino acids shown in the sequence above can be deleted, substituted or added 2404K 050388 13 1 without substantially adversely affecting the immunogenic cross-reactivity with authentic gag epitopes. In other words, such derivatives immunologically similar to authentic gag particles in theat they are recognized by antibodies raised against at least one of p1 7 p24 and p16. Such derivatives, while they may include amino acids from other regions, including antigenic regions of the HIV genome, do not encode other HIV functions, such as the protease function of the pol region or the reverse transcriptase function. In addition, such derivatives retain the ability to form particles in insect cell culture as disclosed herein. In this case, it is within the skill of the art to prepare gagd particles comprising hybrid proteins having one or more epitopes additional to the gag epitopes. Such additional epitopes can be of HIV origin or can be derived from other pathogenic organisms, Hepatitis B Virus or Herpes Virus.

SThe gag precursor protein is expressed in secreted form and in membrane bound form. It is isolated from conditioned medium by standard techniques of protein isolation and purification. Detergents can be added in order to free the protein from cell membrane material.

Following treatment with detergent, Triton X100, a Tween or sodium dodecyl sulfate (SDS), the protein or particles can be purified by a series of ultrafiltration steps, ultracentrifugation steps, selective precipitations with, ammonium sulfate or PEG, density gradient centrifugation in CsCl or sucrose gradients and/or "chromatographic steps, such as affinity chromatography, 30 immunoaffinity chromatography, HPLC, reversed phase HPLC, cation and anion exchange, size exclusion chromatography and preparative isoelectric focusing. During or following .*co purification, the protein or particles can be treated with, formaldehyde, glutaraldehyde or NAE to enhance S 35 stability or immunogenicity. In view of the discovery herein disclosed that the gag precursor can form i 2404K 050388 14 -7 i i 1 immunogenic particles in the absence of other viral functions, it is believed that when gag precursor is expressed in non-particulate form, it can be caused to form particles synthetically, as has been shown to be the case for the hepatitis B surface antigen following expression in yeast. See, EP-A-135,435. Such gag precursor protein particles are encompassed within the scope of this invention.

The HIV gag precursor protein and particles produced in accordance with this invention are useful as diagnostic agents for detection of exposure to HIV. The protein and particles are also useful in vaccines for the prevention of infection or for the inhibition or prevention of disease progression.

The Examples which follow are illustrative but not limiting of the invention. Restriction enymes and other reagents were used substantially in accordance with the vendors' instructions.

20 Examples 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 ClaI-BglII fragment of the gag precursor protein coding region derived from an HIV genomic clone (Shaw et al., Science 226:1165 (1984)) to a synthetic oligonucleotide having the N-terminal coding sequence of the gag precursor protein. The oligonucleotide has the 30 sequence: 9 9* *r 9 9,99 94 9 o 99 9e0 99 999 999.

9a 9 99 9 99 oo 99 9 9e 0 9 9 C ATG GGT GCT AGA GCT TCC GTG TTG TCC GGT GGT GAA TTG CCA CGA TCT CGA AGG CAC AAC AGG CCA CCA CTT AAC Ncol GAT 3' CTA GC Clal pRIT12983 is a vector which comprises a 250 bp region which codes for the C-terminal portion of gag precursor 2404K 050388 15 i 1 ii I f I 10 protein. It was prepared by ligating a BglII-MaeIII fragment of the gag precursor coding region derived from an HIV genomic clone to a synthetic oligonucleotide having the C-terminal coding sequence of the gag precursor protein. The oligonucleotide has the sequence:

STOP

G TCA CAA TAA AGA TAG GAT CC 3' TT ATT TCT ATC CTA GGA GCT MaeIII Xhol.

i4 i The 1305 base pair (bp) BamHI(Ncol)-BglII fragment from pRIT12982 was ligated to the 250 bp BglII-TAA-BamHI-XhoI fragment from pRIT12983 in pUC 12 which had been previously cut with BamHI and Sall. The resulting plasmid, identified as pRIT13001, therefore contains the entire coding region for the gag precursor S. protein on a BamHI(Ncol)-BamHI cassette.

A baculovirus expression vector was prepared by 20 inserting the BamHI fragment from pRIT13001 into the BamHI 0, site in pAc373. See, Smith, et al., Proc. Natl. Acad.

t° Sci. USA 82:8404(1985). pAc373 is a baculovirus transfer vector containing a modified polyhedrin gene into which a foreign gene can be cloned into a BamHI site and expressed under the control of the strong polyhedrin promoter. See Summers, et al., Texas Agricultural Exp. Station Bulletin NR 1555 (May 1987). A derivative of plasmid pAc373 having 0 a small deletion present far upstream the strong polyhedrin promoter was also used as an expression So 30 vector. The slight modification did not appear to affect in vitro expression or growth of the recombinant virus.

Insertion of the gag coding sequence into the Baculovirus vector resulted in plasmid pRIT13003.

A mammalian cell expression vector was prepared by ligating the BamHI fragment from pRIT13001 downstream of

L

2404K 050388 16 lp

V

-i i cl L~Yli 1 the SV40 late promoter in pSV529 (Gheysen et al., J. Mol.

Appl. Genet. 1:385 (1982)). This vector is identified as pRIT13002.

A yeast expression vector was prepared as follows. An NcoI-BglII fragment was isolated from pRIT12982 and inserted into a yeast plasmid downstream of and in-frame with 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 gag precursor protein was isolated from pRIT12983 as a BglII-BamHI fragment and was inserted into the BglII site of 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, it comprises replication functions from the yeast 2 micron vector and a URA3 gene selection marker.

Example 2. Expression in Insect Cells 20 Recombinant Baculovirus transfected with pRIT13003 E. were prepared substantially as described by Summers, et al., TAES Bull. NR 1555, May 1987, cited above.

Spodoptera frugiperda cells were cotransfected with wild type (wt) AcMNPV Baculovirus DNA and plasmid pRIT13003 at 1 pg and 50 pg, respectively. Resulting virus particles were obtained by collecting the supernatants. The virus-containing media were used to infect S.f. cells in a plaque assay. Subsequent infection of S.f. cells using the viral particles which include both 30 wt AcNPV DNA and DNA recombined with the DNA encoding the L p55 gag precursor protein resulted in cells expressing the gag protein instead of the polyhedrin protein.

9 The "clear plaques" (0.1 0.01% frequency) obtained .o in the plaque assay were further screened by filter hybridization with a gag specific probe. Plaques which hybridized to the gag probe were scored and subsequently 2404K A 050388 17 i f 1 further plaque purified (2-3 times) before a virus stock was generated; the virus stock was also tested by ELISA.

S.f. cells were then infected with these recombinant gag virus stocks at a multiplicity of infection (MOI) of 1-10 and after 24 hr, 48 hr, 3 days and 5 days, aliquots of the conditioned medium (Supernatant) and/or cells were treated with Triton X100 to a final concentration of 1% and assayed.

The gag precursor protein synthesized in infected insect cells was observed in Western blots using polyclonal antibodies or antiserum from a pool of AIDS patients (Zairan). A pre-dominant band at molecular weight (Mr) of 54 kilodaltons (kd) was observed with all tested sera and with p55 polyclonal antisera. A band at Mr 54 kd was also detected when testing conditioned medium after 48 hr, 3 days and 5 days. Bands at Mr 49 kd and Mr 47 kd (minor) and a band at Mr 30 kd could also be seen when cell extracts were analyzed. This latter band with apparent Mr 30 Kd is only detected with p55 polyclonal S" 20 antibodies and not with serum of AIDS infected persons.

It was observed that at least 10 times more p55 "epitopes" I l expressed in S.f. cells than in Molt cells infected with HIV (Molt/HTLV-III) and about 80 times more p55 "epitopes" were present in the conditioned medium of S.f. cells infected with a gag recombinant virus than in the conditioned medium of Molt/HTLV-III cells.

In a second assay experiment, ultrafiltration (100,000 x g, 1 hr.) of the 48 hr, 3 day and 5 day conditioned media (2 ml to 200 ml) resulted in a small pellet which 30 was analysed on SDS-gels and which was also analysed by immunoblotting. One band at Mr 55 kd was recognized with specific antibodies against p17, p 24 and p55,. Only very small amounts of degraded products at Mr 49-46 could be detected. On Coomassie-stained gels, a band at 55 kd could be seen which was 20-80% pure. This band corresponded with the immunoblot and was recognized by antibodies against p17, p24 and p55 polypeptides.

2404K 050388 18 1 In a third assay experiment, centrifugation (1 ml) of the 48 hr, 3 day and 5 day conditioned media in a microfuge at 12000 rpm for 5 to 20 minutes produced a band on SDS-gels at Mr 55 kd which was specific for HIV-I gag precursor as revealed by antibodies against p17, p24 and p55 polypeptides and as compared to the HIV cell lysate (Molt/HTLV-III) 55 kd band.

In a fourth assay experiment, 48 hr, 3 day and 5 day conditioned media (150 ml to 1 liter, containing 1 pg/ml of aprotinin which was added at 24 hrs. post-infection and also at the times of harvest) was treated first by addition of Tween 20 to 0.01% final concentration. Then, a solution of polyethylene glycol, Mr 6 kd, (PEG6000) w/v in 2M NaC1) was added to 10 or 5 final concentration. After 4 hours at 4°C or preferentially overnight at 4°C this precipitate was centrifuged at 5000 rpm for 10 min at 4°C. The PEG pellet was then taken up in 200 pl to 1 ml HBS-buffer (Hanks balanced salt, Flow Laboratories, 18-102-54) containing 0.1 Tween 20 20 and centrifuged in sucrose gradients (20 60 in 4 4 HBS-buffer, 0.1 Tween 20 at 4°C containing 10 pl/ml aprotinin, Sigma Chemical Co., St. Louis, Missouri) for about 35 min at 50,000 rpm in a Beckman rotor TLA100 (Beckman Instruments, Fullerton, California) at 4°C, or for about 18 hr at 25,000 rpm on a Beckman SW41 rotor at Fractions of 0.2 to 0.5 ml, respectively, from I' approximately 40-50% sucrose, were collected, frozen at -200C and tested either with a specific antigen capture Elisa assay such as -24/Ig AIDS antiserum biotinylated or 9 30 AIDS antiserum/Ig core POD (HIV-1 anticore EIA, Abbott Laboratories). One OD Elisa pick was detected, Sdemonstrating that on surcrose gradients the p55 gag protein migrated as particles or "aggregated structures".

The pick fractions and the surrounding fractions were immunoblotted wth p17, p24 or p55 antibodies. One major band at Mr 55 kd in the SDS-reducing gels was detected 2404K 050388 19

I

1 corresponding to p55 gag precursor protein as compared to an extract of Molt/HILV-III cells prepared substantially as described above.

In a fifth assay experiment, a 5 PEG6000 precipitate was prepared substantially as described for the fourth assay experiment from 150 ml of a S.f. culture which had been co-infected with the gag precursor recombinant Baculovirus and with a recombinant Baculovirus which expressed the HIV envelope protein at a MOI of 3 to The PEG6000 pellet was taken up in 200 ul of HBS-buffer containing 0.1 Tween 20. After centrifugation at 15000 x g for 1 min, the supernatant was mixed with 11.5 ml of a M CsC1 solution (0.3 volumes HBS-buffer, 10 mM Tris-HCL (pH 1 mM ethylendiamine tetraacetate 15 (EDTA), 0.1 Tween 20 and 10 ug/ml of aprotinin). This r suspension was centrifuged in a Beckman Rotor 50Ti for about 72 hr at 44000 rpm at 18 Fractions of 300 ul S' 2 each were collected, frozen at -20 0 C and tested with a specific antigen capture Elisa assay (HIV anticore EIA).

20 Bands at densities of about 1.28 and 1.20 g/cm 3 were recognized, the core-like particle apparently having the density of 1.28 g/cm 3 Electron microscopy confirmed the presence of pre-core (and core) -like particles in the conditioned medium.

Scanning electron microscopy revealed particles which apparently were budding onto the cell surfaces.

Immunogold transmission electron microscopy revealed particles which were recognized by p24 and and antibodies. Also, p17, p 24 and p55 epitopes were 30 recognized by immunogold labelling after brief treatment *04# S, with Triton X100 of purified particles in electron microscopic preparations. The particles were approximately spherical and of about 100 150 nm in diameter. The particles display electron luscent centers surrounded by a dark staining ring and an outer shell and appear to have the majority of the p17, p24, p16 and epitopes on the inside surface of the particle.

2404K 050388 20 Ir i ]*B

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1 This Example, therefore, demonstrates expression and secretion of HIV pre-core-like (and core-like) particles comprising Immunodeficiency virus gag precursor protein.

The particles comprise predominantly (greater than 90% of total protein) full length gag precursor protein and are formed in the absence of DNA sequences of viral origin other than the gag precursor sequence and, hence in the absence of other viral functions such as the retrovirus protease and reverse transcriptase.

To demonstrate that the HIV gag precursor protein made in S.f. cells is efficiently myristylated, 3 X 10 6 cells in F25 cm 2 flask, were labelled at 48 hr p.1. with 500 pCi myristic acid NET-830 (Dupont, Wilmington, Delaware) for 18 hr after they had been infected with recombinant 15 p55 gag baculovlruses at MOI of 5. Subsequently, the conditioned medium'and the cells were processed separately for western blotting and SDS-gel radioautography.

Conditioned medium displayed one major band at 55 kd which was also recognized as gag precursor in western blots as 20 revealed by antibodies against p17, p24, p55. Two other labelled minor bands were detected at Mr 49-46-47 kd and were recognized specifically by the same set of antibodies (p17, p24, p55) in the western blot. Cell lysates made in 1 triton x 100 and frozen at -20°C displayed on 25 radioautography of the 12.5 Laemli gel and western blot respectively band at 55 kd (and a minor band at 58 kd which apparently corresponded to the translation frame shift as described for the gag retroviral HIV-1 virus genome and more prominent bands at Mr 49-47-46 and degradation products at Mr 30-27 kd the latter bands were not radioactive (containing no myristic acid).

Ir

II

0$* Example 3. Expression in Mammalian Cells: The plasmid pRIT13002 was introduced via the Ca-phosphate coprecipitation technique (Wigler, et al., Cell 16:777(1979)) in CosI and CV1 cells. At 48 hr and 2404K 050388 21 L j_ <2 S It t t i t t 44 .4o= 0 I t t0 a* 4 4 4 4 4 4 4 4 04 0 4 4 4 4 1 110 hr post-transfection, the cells and culture medium were assayed using an ELISA specific for gag antigen expression. Cell extracts (106 cells) were adjusted to 1 Triton X100 or 0.5 DOC-NP40. The p55 antigen was detected using ELISA capture antigen tests involving polyclonal and monoclonal antibodies to p17, p24 or p55 or using the Dupont RIA test (NEK-040), involving a competition with purified p24 peptide. The expression levels obtained were between 4 and 10 ng/ml as measured by the p24 RIA Dupont test.

Example 4. Expression in yeast cells The plasmid pRIT12985 was introduced into the S. cerevisiae strain 02276b (ura3- dur0h rocl-).

15 The p55 antigens were detected in yeast extracts (cells in mid-log phase, broken using glass beads or spheroplasting with zymolase). The p55 was detected using ELISA tests, involving polyclonal and monoclonal antibodies to the p24 peptide, or using the Dupont radioimmune assay (RIA) 20 involving competition with the purified p24 peptide.

The p55 protein synthesized in S. cerevisiae was observed in Western blots, using p17 or p24 specific monoclonal antibodies, and has a molecular weight similar to that of the p55 antigen obtained from infected cells.

25 Nhen cellular extracts were obtained in the absence of detergents, an important fraction of the antigen was retained in the "membrane pellet". This fraction of antigen was recovered using Triton XIOO. Use of detergents either prior to or after isolation enhanced 30 antigenicity as measured in the RIA. The gag precursor produced in yeast was shown to be myristilated by labelling with tritiated myristic acid and was apparently associated with cell plasma membrane as shown by electron microscopy.

The above Examples demonstrate expression of gag precursor protein in animal cell culture and expression of immunodeficiency virus pre-core-like particles in 2404K 050388 22 le 4, *I 4 0; 4.

4 4.

44.44

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404$ 1 Lepidoptera cells using a Baculovirus expression system.

The protein and/or particles thus prepared are purified and formulated into a vaccine for parenteral administration to humans in danger of exposure to HIV, in order to protect the vaccinees from onset of disease symptoms associated with HIV infection. Each vaccine dose comprises an amount of the protein or particle which is safe, does not cause significant adverse side effects, but which is effective in inducing an immune response. For example, each dose comprises 1 to 1000 ug, preferably 10 to 500 ug, of gag precursor protein or particle 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 an 15 adjuvant, aluminum hydroxide, muramyl dipeptide or a saponin such as Quil A. Useful buffers include buffers derived from sodium or ammonium cations and acetate, citrate, phosphate or glutamate anions. Other pharmaceutically acceptable carriers or diluents can be used to adjust isotonicity or to stabilize the formulation, sodium chloride, glucose, mannitol, albumin or polyethylene glycol. The vaccine can be lyophilized for convenience of storage and handling. Such vaccine is reconstituted prior to administration.

25 Alternatively, the gag protein or particle can be formulated in liposomes or ISCOMS by known techniques. An exemplary vaccine dose comprises 100 ug of gag particles adsorbed on aluminum hydroxide in water buffered to pH 7 with sodium acetate.

30 In an alternative embodiment of the invention, the gag protein or particle is mixed with one or more other antigens by coexpression in the same cell culture or by co-formulation. Such other antigens can be other HIV antigens, antigens derived form the envelope protein, gp160 or gp120, or can be antigens derived from one or more other pathogenic organisms, cells or viruses, 444, 0 2404K 050388 23 -3 1 1 such as hepatitis B surface antigen for conferring protection against Hepatitis B Virus or antigens derived from the Herpes Virus glycoprotein for conferring protection against Herpes Virus.

The vaccine is preferably administered parenterally, intramuscularly (im) or subcutaneously (sc), although other routes of administration may be useful in elicitng a protective response. The vaccine is administered in a one-dose or multiple-dose, 2 to 4, course. Immunoprotection can be ascertained by assaying serum anti-gag antibody levels. Thereafter, vaccinees can be revaccinated as needed, annually.

As a diagnostic reagent, the gag protein or particles can be used in any of the standard diagnostic assays, such 15 as an ELISA or RIA, to detect the presence of anti-HIV w antibodies in clinical specimens. Such diagnostic can be used in conjunction with other HIV antigens to monitor disease progression. Use of the gag protein or particle 0 as a diagnostic reagent will generally involve contacting 20 a sample of human or other animal serum or other body fluid with the protein or particle, preferably bound or otherwise affixed or entrapped, and then assaying for binding of anti-gag antibodies from the serum or other sample to the gag protein or particles. Such assay can be 25 accomplished by standard techniques, including by quantitating binding of subsequently added labelled Sm anti-gag antibodies.

Example 5. Construction and Expression of a Mutant 'i 30 Gene In order to examine the potential role of the N-myrsitoylation in the assembly and formation of extracellular gag particles we have constructed a glycine deletion mutant. Therefore a synthetic oligonucleotide linker syn3 was substituted, for the BamHI-ClaI fragment in pRIT12982 (see example Syn3 encodes the genuine F 2404K 050388 -24- LL~j 4 i 4 t 4 4O I 001

I

(4 t 4 o4 44 1 N-terminal amino acids of the gag protein except that the second glycine codon is deleted. This mutant BamHI expression cassette was subcloned into the BamHI site of the baculo expression vector pAcYMI (Matsuura et al., J.

Gen. Virol. 68:1233 (1987)) and recombinant plaques were obtained and selected essentially as described in Example 1. The recombinant virus, AcGag 31-18, harbouring the glycine deletion mutation of the gag gene was used to infect S.f. cells. The gag precursor protein was efficiently synthesized as determined by an ELISA assay.

Metabolic labelling with 3H-myristic acid essentially as described in Example 2 revealed no myristic acid incorporation confirming that deletion of the N-terminal glycine was sufficient to prevent myristoylation of the gag precursor protein. Analysis of the cell extracts in Western blots (see Example 1) showed a prominent band of kd and lower M.W. degradation products. The obtained pattern of protein bands was similar to the wild type (wt) protein expressed in S.f. cells. In contrast with the 20 wt p55 recombinant, no gag protein could be detected with the glycine mutant 2 days p.i. using PEG or ultracentrifugation of the conditioned medium. Thus the mutated p55 protein was only detected within the infected cells. The myristoylation process thus seems to be 25 required for the extracellular release of the p55 product.

Scanning electron microscopy (SEM) revealed that the cell surface was rather smooth, showing no particles. Thin section transmission electron microscopy and immunogold labelling performed on cells infected 24 hrs, 48 and 66 30 hrs p.i. with the Ac gag 31-18 (Myr-) recombinant virus revealed that the non-myristoylated gag protein was efficiently expressed, scattered in the cytoplasm or associated to grey amorphous structures within the cytoplasm and the nucleus. These intracellular particles or particulate structures are morphologically different 2404K 050388 25 *a 4 4 4 o 1 1 I.~UX~3~I~U~ 0 4a 4 14 44 11 *9 4 0 4.4.) #4 4 o 44 o 4 0 1 frrm the extracellular particles obtained with the myristoylated gag recombinant (AcGag7) as they display a double electron dense ring structure and do not contain a lipid bilayer derived from the cell membrane. Neither gag protein nor budding structures were observed at the cell membrane.

These results demonstrate that the myristoylation of the gag precuror appears to be required for its plasma membrane location, budding and extracellular particle release. Myristoylation does not seem however to be required for the multimeric assembly of the p55 molecules.

Accumulation of the non-myristoylated p55 products within the nucleus (and nucleoli) is a surprising phenomenon.

Example 6. Construction and Expression of a Truncated Precursor Protein In order to examine the role of the p16 (COOH-end) of the HIV precursor gag protein we made a gag deletion mutant which encodes only the p17-p24 precursor part of gag.

The BamHI-CfrI gag fragment of pRIT13003 was purified and ligated with a synthetic oligonuc.leotide sequence 5' GGC CAT AAG GCA AGA GTT TTA GTT AGT TAG 3' 3' TA TTC CGT TCT CAA AAT CAA TCA ATC CTA G and gel purified and cloned in the BamHI-site of pAcYMI.

This linker sequence contains the genuine amino acid COOH-end of the HIV p24 core protein and two additional amino acids, Valine and Serine. This recombinant plasmid was used to co-transfect S.f. cells with AcMNPV DNA 30 essentially as described before (see Example 1).

Recombinant plaques were screened as described in Example 1.

A selected recombinant virus, Ac CfrI, was used to infect S.f. cells. A truncated gag-polypeptide (p17-24) 35 was detected at the expected M.W. of 41 Kd and which 2404K 050388 26 4 444*l 44 44 4o 1 reacted in Western blot analysis with p17 and p24 monoclonals. The p17-24 product was predominantly expressed inside the cells but a small amount of extracellular p17-24 product could be detected when analyzing the conditioned medium by Western blotting. PEG precipitation and ultracentrifugation of the conditioned ii medium of the Cfrl mutant gag protein did not result in detectable p17-p24 product. Electron microscopy analysis showed no evidence of budding or extracellular gag particles. Large protusions 1-4 pm long in the form of tubular structures which are longitudinally connected to the cell membrane surface could be detected early in infection. Immunogold labelling showed that the truncated gag protein (p17-p24) was localized at the cell membrane and at the periphery of these tubular extensions, but no electron dense "ring" structures typical of the particle structures could be detected. This probably indicates that the p17-p24 product is not able to assemble S in multimeric structures, cap formations, at the 20 cell membrane. These results suggest that at least a part of the p16 polypeptide of the gag precursor polypeptide is 4| 4necessary for particle formation.

A glycine deletion mutant of the Cfrl cassette (non-myristoylated p17-24) was made by exchanging the EcoRV-PstI fragment of 669 bp of the pAcGag 31-18 non-myristoylated p55 gene with the pAC Cfrl EcoRV-PstI 9400 bp long fragment. This mutant displayed no protrusions of membranes as described above but showed p17 and p24 immunogold decoration scattered in the cytoplasm 30 and nucleus.

o a e Example 7. Construction and Expression of a gaq-pol Protein and the Processed Polypeptides To express the gag-pol products, we have included most (about 80 of the pol gene into the baculovirus transfer o 4 vector carrying the p55 expression cassette 2404K 050388 27 1 (pRIT13003). The pol gene DNA fragment is a BglII (2093) EcoRI (4681) restriction fragment from BH10 (Shaw et al., Science 226:1165 (1984). A poly-stop synthetic DNA fragment 5' AAT TCC TAA CTA ACT AAG 3' 3'GGA TTG AT TGA TTC CTA G was added at the Eco RI site. The resulting baculovirus expression plasmid, LE-8-4, was used in a co-transfection experiment to generate recombinant plaques essentially as described in imple i.

In this recombinant construct, the myristoylated as well as a gag-pol product resulting from the HIV-specific translational frame-shift in S.f. cells, are expected to be produced, and subsequently processed by the protease. Recombinant baculovirus harbouring the gag-pol gene was screened and selected essentially as described in Example 1. In S.f. cells infected with such a gag-pol recombinant virus, VAC 8-5, no gag or gag-pol products were detected when the conditioned medium was analysed by Western Blot or precipitated with PEG.

20 Cell extracts however, did show a strong doublet band at 24 Kd and a band at 17 Kd which reacted with p24 and p17 monoclonal antibodies in Western blots. Very small amounts of the precursor p55 band and intermediate 41 Kd (46 Kd) bands could also be detected in Western blots.

This indicates that the protease is active in the gag-pol fusion protein, expressed by translational frame-shift in S.f. cells. This results in p17, p 2 4 polypeptides and intermediates (41 Kd, 46-49 Kd, 55 Kd). The large precursor gag-pol product was not detected with our 30 p17 or p24 antibodies.

Electron microscopy showed on rare occasions a few particles budding at the cell membrane. These particles seem to be morphologially similar to the above described particles. Co-infection experiments with recombinant 35 viruses harbouring the p55 and the gag-pol gene did not result in detectable particles 9 i 9

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94 99 4 4 999 9 99 9 9 9, 9 94 9 9 0 9 #9 2404K 050388 28 r LI:

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I I% i 1 displaying a morphological difference such as a more condensed (cone-shaped) core (p24) structure, typical of a mature retroviral (HIV) particle.

Example 8. Construction and Expression of the SIV Pr 57 gag Gene in S.F. Cells The gag gene of Simian Immunodeficiency virus (SIV) was subcloned from the molecularly cloned SIVmac-BK 28 (gift of 3. Mullins; see Hirsh et al., Cell 49:307 (1987) and Kestler et al., Nature 331:619 (1988)). A 3504 bp KpnI fragment of the pBK28 genome (nucleotides 1212 to 4716) was subcloned into pUC8. Two internal fragments of the gag gene, the 5' fragment FnuDII( 1201) Pst (1959) and the 3' fragment PstI (1959) HphI (2803) were purified and synthetic oligonucleotide linkers, linker 1: GAT CC ACC ATG GGC G TGG TAC CCG and linker 3:

TGCTGCACCTCAATTCTCTCTTTGGAGGAGACCAGTAGAGATCTGGTAC

20 AACGACGTGGAGTTAAGAGAGAAACCTCCTCTGGTCATCTCTAGAC were ligated to adequate gag fragments to reconstitute the entire precursor gag gene. In a separate experiment a linker 2: GATCC ACC ATG GCC G TGG TAC CGG was used at the 5' fragment, to introduce a mutation in the second codon, namely, GGC (Gly) to GCC (Ala). The different constructions were cloned into blue scribe vectors and verified by sequencing. The N-terminal 30 fragment (BamHI-PstI) and the carboxy-terminal fragment (PsI-BglII) were isolated and cloned into the BamHI digested, alkaline phosphatase treated pAcYMI baculovirus expression vector. The pAC gag Myr plasmid contains the native SIV gag gene and the pAc gag Myr- contains the 35 mutated (Gly to Ala) gene. S.f. cells were transfected with a mixture of purified AC MNPV viral DNA (1 pg) and *r l1*

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2404K 050388 29 j r i ~+C 1 the respective recombinant transfer plasmids (50 pg) essentially as described in Example 1. The recombinant plaques were screened and selected as described in Example 1.

The SIV gag Pr 57 gag precursor polypeptide was efficiently synthesized in infected insect cells as observed in Western blots using the rabbit antiserum to SIV (metrizamide gradient purified SIV-BK28 virus) or a monoclonal directed against the COOH-end of the HIV p24 polypeptide, which appear also to recognize the SIV core protein.

In a second assay experiment it was demonstrated that the SIV native gag precurser gene expressed in S.f. cells was efficiently myristoylated in contrast to the culture infected with the glycine to alanine mutant in which no myristoylation of the precursor Pr 57 gag protein could be detected when analysed on SDS-PAGE and radioautography.

As in the case of HIV-gag precursor protein we also observed gag particle formation and release of particles 20 in the conditioned medium when the infected cultures were analysed by ultracentrifugation, sucrose gradients and Selectron microscopy (TEM and SEM). Similar SIV-gag Pr 57 gag particles as those obtained when expressing precursor gene in S.f. cells were observed. The extracellular gag particles form crescent structures at the cell membrane which assemble into typical buds that closely resemble immature virus budding particles. The SIV Pr 57 gag as the HIV p55 particles were about 100-120 nm .in diameter and showed a light grey transluscent center surrounded by a tick dark electron dense ring and an outer lipid bilayer. Experiments with the SIV non-myristoylated (Gly to Ala) mutant confirmed the observations made with the HIV-non myristoylated p55 mutant that N-myristoylation ,to is essential for budding and extracellular particle 35 formation.

The difference between the gag precursor protein of HIV and SIV is that the latter forms also intracellular 2404K 050388 30 L 1 particles and particulate structure when the native SIV gag protein (myristoylated) is expressed. This could be explained as follows the expression level is about 3 times higher than the HIV gag expression level and maybe not all the SIV Pr 57 gag molecules are myristoylated.

Also more degradation products, especially a myristoylated p27 protein band could be detected in WB of cultures infected with the SIV Pr 57 ga g native construct.

It is possible that the cellular structures of about 40 nm in diameter and sometimes up to 1 pm long which are observed at late stage of infections are composed at least in part of these degradation gag products. This could resemble the p24 core assembly into tubular structures observed in some rare cases of retroviral core maturation. Also when the p24 core protein of HIV-1 is expressed in E. coli, tubular structures containing p24 protein have been observed. Part of the intracellular particles observed with the native SIV gag precursor recombinant take form near the cell membrane, where they 20 appear to differentiate into virus-like particles budding as described above. This process is reminiscent of the ft viral maturation of type D retrovirus which involves intermediate intracellular type A particles.

The above description and examples fully disclose the invention and the preferred embodiments thereof. The invention, however, is not limited to the embodiments specifically disclosed herein but, rather, encompasses all improvements, variations and modifications thereof which *come within the scope of the following claims.

I

2404K 050388 31 i

Claims

1. A recombinant DNA molecule comprising a DNA sequence which codes for a full length immunodeficiency virus gag precursor protein and which is devoid of the naturally occurring 5' and 3' flanking sequences, operatively linked to a regulatory element as hereinbefore defined which functions in eukaryotic cells.

2. The recombinant DNA molecule of claim 1 in which the regulatory element is one which functions in yeast, insect or mammalian cells and the gag precursor protein is the HIV gag precursor protein. I Is t* t~

3. The recombinant DNA molecule of claim 2 in which the I I regulatory element is one which functions in Lepidoptera cells.

4. The recombinant DNA molecule of claim 3 in which the regulatory element comprises the polyhedrin gene promoter. A recombinant Baculovirus comprising the recombinant DNA molecule of claim 2, 3 or 4. tI

6. An insect cell infected with the recombinant Ac Baculovirus of claim

7. The insect cell of claim 6 which is a Lepidoptera cell.

8. The insect cell of claim 6 which is a Spodoptera frugiperda cell.

9. The recombinant DNA molecule of claim 2 in which the regulatory element is one which functions in Drosophila cells. 91 1114,ejhaclc.00 1,37256.res,32 33 A Drosophila cell transformed with the recombinant DNA molecule of claim 9.

11. The recombinant DNA molecule of claim 2 in which the regulatory element is one which functions in mammalian cells.

12. A recombinant vaccinia virus comprising the recombinant DNA molecule of claim 11.

13. A mammalian cell comprising the recombinant DNA molecule of claim 11. 9 9 .o 14. A mammalian cell infected with the recombinant :vaccinia virus of claim 12. O 0 e1 S* 15. The mammalian cell of claim 13 which is selected from the group consisting of CHO cells, COS-7 cells, NIH- 3T3 cells, CV1 cells, mouse or rat myeloma cells, HAK cells, vero cells, HeLa cells, W138 cells, MRC-5 cells or chicken lymphoma cells. 4 9* i

16. The recombinant DNA molecule of claim 2 in which the regulatory element is one which functions in yeast.

17. The recombinant DNA molecule of claim 16 in which the regulatory element comprises the CUP1, TDH3, PGK, ADH, PHO5 or ARG3 promoter.

18. A recombinant yeast cell comprising the recombinant DNA molecule of claim 16.

19. A recombinant S. cerevisiae cell comprising the recombinant DNA molecule of claim 17. A recombinant DNA molecule for expressing in Lepidoptera cells a particle which is immunologically 911119,ejhcmc.001,37256.res,33 :OX y -34- similar to authentic immunodeficiency virus gag particles which molecule comprises a DNA sequence which codes for all or a portion of an immunodeficiency virus gag precursor protein or for a hybrid protein having all or a portion of an immunodeficiency virus gag precursor protein, operatively linked to a regulatory element as hereinbefore defined which functions in Lepidoptera cells.

21. The recombinant DNA molecule of claim 20 for expressing a particle comprising predominantly full S, length HIV gag precursor protein which codes for full length HIV gag precursor protein devoid of other HIV oo functions.

22. A recombinant DNA molecule comprising a coding 0e 00 sequence for an immunodeficiency virus gag precursor protein operatively linked to a regulatory element which functions in Lepidoptera cells.

23. The recombinant DNA molecule of claim 22 in which the coding sequence is for a full length HIV gag precursor protein devoid of other HIV functions. 0o0 S 0a

24. The recombinant DNA molecule of claim 20, 21, 22 or 23 in which the regulatory element comprises the polyhedrin gene promoter. o A recombinant Baculovirus comprising the recombinant DNA molecule of claim 20 or 22.

26. A recombinant Baculovirus comprising the recombinant DNA molecule of claim 24.

27. A Lepidoptera cell infected with the recombinant Baculovirus of claim 7 911119,ejhcmc.001,37256.res,34

28. A Spodoptera frugiperda cell infected with the recombinant Baculovirus of claim 26.

29. A gag precursor protein produced by culturing cells of claim 6. A gag precursor protein produced by culturing cells of claim 13.

31. A gag precursor protein produced by culturing cells

33. A gag precursor protein particle produced by culturing cells of claim 27.

34. An immunogenic particle comprising gag precursor protein produced by recombinant eukaryotic cells which i ,particle is immunologically similar to authentic immunodeficiency virus gag particles. The immunogenic particle of claim 34 which comprises 1 predominantly full length HIV gag precursor protein, which is recognized by anti-pl6, anti-p24 and anti-pl7 antibodies and which lacks viral functions required for viral maturation and replication.

36. A vaccine comprising gag precursor protein produced by recombinant eukaryotic cells.

37. A vaccine comprising gag precursor protein particles produced by recombinant eukaryotic cells.

38. A method for collecting data useful in the diagnosis of exposure of an animal to an immunodeficiency virus 911119,ejhcmc.001,37256.res,35 36 which comprises contacting a sample of serum or other bodily fluid from the animal with a gag precursor protein of claims 29, 30, 31 or 32.

39. A method for collecting data useful in the diagnosis of exposure of an animal to an immunodeficiency virus which comprises contacting a sample of serum or other bodily fluid from the animal with the immunogenic particle of claim 34 or A process for preparing a recombinant DNA molecule for expressing an immunodeficiency virus gag protein r which comprises ligating a DNA sequence which codes for a full length immunodeficiency virus gag precursor protein and which is devoid of the naturally occurring 5' and 3' flanking sequences to a regulatory element as t, hereinbefore defined which functions in eukaryotic cells.

41. The process for a claim 40 in which the regulatory element is one which functions in yeast, insect or mammalian cells and the gag precursor protein is the HIV gag precursor protein.

42. The process of claim 41 in which the regulatory element is one which functions in Lepidoptera cells.

43. The process for claim 42 in which the regulatory element comprises the polyhedrin gene promoter.

44. A method of preparing a vaccine for protecting a human against disease caused by infection by HIV which comprises culturing recombinant eukaryotic cells which have been transformed with a recombinant DNA molecule comprising a DNA sequence which codes for a full length HIV gag precursor protein and which is devoid of naturally occurring 5' and 3' flanking sequences, operatively linked to a regulatory element as C 911119,ejhcmc.001,37256.res,36 7<W 7 37 hereinbefore defined which functions in the eukaryotic cells; isolating the gag precursor protein particles produced thereby; and combining the isolated gag precursor protein particles with a pharmaceutically acceptable carrier. *r r 9, D *9* 19 4 d .9 .4 It 4) 4 A recombinant DNA molecule of claim 1, a cell or virus comprising a said molecule, or a product expressed thereby, substantially as hereinbefore described with reference to the examples. Dated this 19th day of November, 1991. SMITHKLINE BIOLOGICALS By Its Patent Attorneys DAVIES COLLISON CAVE 404#94 r 0 .41 I '4494It 444wi .41 i 14( 4.9*4 I 4 911119,ejhanc001,37256.res,37