AP129A - Expression of retrovirus gag protein eukaryotic cells - Google Patents

Field of the Invention

This invention relates to expression of proteins in eukaryotic cells. More particularly it relates to the expression of immunodeficiency virus gag precursor protein.

Background of the Invention

Retroviruses, that is, viruses within the family, Retroviridae, are a large family of enveloped, icosohedral viruses of about 150 nm having a coiled nucleocapsid within the core structure and having RNA as the genetic

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material. The family comprises the oncoviruses such as the sarcoma and leukemia viruses, the immunodeficiency viruses 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.

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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 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 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, pl7, p24 and pl6, 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 pl7 with its submembrane localization is well positioned to be in close contact with the transmembrane protein gp41 and the viral membrane and with gag p24 and possibly gag pl5 viral RNA thereby playing a central role in the conformational changes involved in the viral entry and uncoating process. Furthermore, gag pl7 has been found to have a myristylated N-terminus.

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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.

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.

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 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 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

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and that such 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.

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 pl7, p24 and pl6. These gag particles, now comprised predominantly of pl7, p24 and pl6, can be referred to as core particles or as mature core particles. Also during viral maturation, apparently during the budding 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 after brief treatment with Triton X100 by anti-pl7 monoclonal antibodies (MABs), anti-p24 MABs and anti-pl6 MABs in

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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 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 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, e.g., 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 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 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, e.g., envelope protein sequences, is not precluded from the preferred embodiments.

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An exemplary coding region for the HIV gag precursor protein has the following sequence.

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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 include Drosophila cells and Lepidoptera cells. Useful Drosophila cells include SI, 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 media, including, e.g., 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, 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

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- 10 BAD ORIGINAL Q 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 Bui1. NR 1555, May, 1987.

Useful Lepidoptera cells include cells from Trichoplusia ni, Spodoptera fruqiperda, Heliothis zea, Autographica californica, Rachiplusia ou. Galleria melonella, Manduca sexta or other cells which can be • -- - · · v 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 californica (AcMNPV). However, other MNPV and NPV viruses can also be employed the silkworm virus, Bombyx mori. Lepidoptera cells are co-transfected with DNA comprising the 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 variety of nutrient media, including, for example, TC100 (Gibco Europe; Gardiner et al., J. Inverteb. Pathol.

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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 et al. Proc. Natl. Acad. Sci. USA 82:8404(1985); and Cochran, EP-A-228,036.

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 amplification, e.g., a DHFR expression cassette, and may also comprise additional functions for selection and/or amplification, e.g., a neomycin resistance cassette for G418 selection. Other functions, such as for transcription enhancement can also be employed. Yet other functions can be comprised within the vector for stable episomal 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 vaccinia 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

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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 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, 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., Science 219:620 (1983); Cabezon et al., Proc. Natl. Acad. Sci. USA 81:6594 (1984).

In the case of the gag precursor protein particles produced in accordance with this invention, it is to be understood that although particles comprising the gag precursor are preferred, particles comprising derivatives of the native gag precursor can also be prepared. For example, one or more nucleotides or amino acids shown in the sequence above can be deleted, substituted or added

BAD ORIGINAL 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 pl7, p24 and pl6. 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, e.g., Hepatitis B Virus or Herpes Virus.

The 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, e.g., 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, e.g., ammonium sulfate or PEG, density gradient centrifugation in CsCl or sucrose gradients and/or chromatographic steps, such as affinity chromatography, immunoaffinity chromatography, HPLC, reversed phase HPLC, cation and anion exchange, size exclusion chromatography and preparative isoelectric focusing. During or following purification, the protein or particles can be treated with, e.g., formaldehyde, glutaraldehyde or NAE to enhance stability or immunogenicity. In view of the discovery herein disclosed that the gag precursor can form

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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, e.g., 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.

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 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 synthetic oligonucleotide having the N-terminal coding sequence of the gag precursor protein. The oligonucleotide has the sequence:

5' C ATG GGT GCT AGA GCT TCC GTG TTG TCC GGT GGT GAA TTG GAT 3 CCA CGA TCT CGA AGG CAC AAC AGG CCA CCA CTT AAC CTA GC

Ncol Clal pRIT12983 is a vector which comprises a 250 bp region which codes for the C-terminal portion of gag precursor

BAD ORIGINAL protein. It was prepared by ligating a Bglll-Maelll 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

5' G TCA CAA TAA AGA TAG GAT CC 3'

TT ATT TCT ATC CTA GGA GCT

Maelll XhoI.

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

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

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 a small deletion present far upstream the strong polyhedrin promoter was also used as an expression 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

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BAD ORIGINAL 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 Ncol-Bglll 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 Bglll-BamHI fragment and was inserted into the Bglll 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

Recombinant Baculovirus transfected with pRIT13003 were prepared substantially as described by Summers, et al., TAES Bull. NR 1555, May 1987, cited above.

Spodoptera frugiperda (S.f.) cells were cotransfected with wild type (wt) AcMNPV Baculovirus DNA and plasmid pRITl3003 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 wt AcNPV DNA and DNA recombined with the DNA encoding the p55 gag precursor protein resulted in cells expressing the gag protein instead of the polyhedrin protein.

The clear plaques (0.1 - 0.01% frequency) obtained 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

BAD ORIGINAL ft further plaque purified (2-3 times) before a virus stock was generated; the virus stock was also tested by ELISA.

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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 p55 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 antibodies 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 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 was analysed on SDS-gels and which was also analysed by immunoblotting. One band at Mr 55 kd was recognized with specific antibodies against pl7, p24 and p55,. Only very small amounts of degraded products at Mr 49-46 could be detected. On Coonassie-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 pl7, p24 and p55 polypeptides.

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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 p55 gag precursor as revealed by antibodies against pl7, 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) (40 % w/v in 2M NaCl) 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 μΐ to 1 ml HBS-buffer (Hanks balanced salt, Flow Laboratories, 18-102-54) containing 0.1 % Tween 20 and centrifuged in sucrose gradients (20 % - 60 % in HBS-buffer, 0.1 % Tween 20 at 4°C containing 10 μΐ/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 4°C. Fractions of 0.2 to 0.5 ml, respectively, from approximately 40-50% sucrose, were collected, frozen at -20°C and tested either with a specific antigen capture Elisa assay such as -24/Ig AIDS antiserum biotinylated or AIDS antiserum/Ig core POD (HIV-1 anticore EIA, Abbott Laboratories). One OD Elisa pick was detected, demonstrating that on surcrose gradients the p55 gag protein migrated as particles or aggregated structures. The pick fractions and the surrounding fractions were immunoblotted with pl7, p24 or p55 antibodies. One major band at Mr 55 kd in the SDS-reducing gels was detected

BAD ORIGINAL $ corresponding to p55 gag precursor protein as compared to an extract of Molt/HILV-IH 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 5.

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

1.5 M CsCl solution (0.3 volumes HBS-buffer, 10 mM Tris-HCL (pH 8.0), 1 mM ethylendiamine tetraacetate (EDTA), 0.1 % Tween 20 and 10 ug/ml of aprotinin). This suspension was centrifuged in a Beckman Rotor 50Ti for about 72 hr at 44000 rpm at 18°. Fractions of 300 ul each were collected, frozen at -20°C and tested with a specific antigen capture Elisa assay (HIV anticore EIA).

Bands at densities of about 1.28 and 1.20 g/cm were recognized, the core-like particle apparently having the 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 which apparently were budding onto the cell surfaces.

Immunogold transmission electron microscopy revealed particles which were recognized by p24 and and p55 antibodies. Also, pl7, p24 and p55 epitopes were recognized by immunogold labelling after brief treatment 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 pl7, p24, pl6 and p55 epitopes on the inside surface of the particle,

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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 10⁶ cells 2 in F25 cm flask, were labelled at 48 hr p.i. with 500 pCi myristic acid NET-830 (Dupont, Wilmington, Delaware) for 18 hr after they had been infected with recombinant p55 gag baculoviruses 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 revealed by antibodies against pl7, 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 (pl7, p24, p55) in the western blot. Cell lysates made in 1 % triton x 100 and frozen at -20°C displayed on 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-l 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).

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

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 pl7, 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~ durO*¹ rod^-).

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) involving competition with the purified p24 peptide.

The p55 protein synthesized in S. cerevisiae was observed in Western blots, using pl7 or p24 specific monoclonal antibodies, and has a molecular weight similar to that of the p55 antigen obtained from infected cells.

When 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 X100. Use of detergents either prior to or after isolation enhanced 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

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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, i.e., does not cause significant adverse side effects, but which is effective in inducing an immune

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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 adjuvant, e.g., 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, e.g., 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. 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.

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, e.g., antigens derived form the envelope protein, gpl60 or gpl20, or can be antigens derived from one or more other pathogenic organisms, cells or viruses,

BAD ORIGINAL ft 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, e.g., 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, e.g., 2 to 4, course. Immunoprotection can be ascertained by assaying serum anti-gag antibody levels. Thereafter, vaccinees can be revaccinated as needed, e.g., annually.

As a diagnostic reagent, the gag protein or particles can be used in any of the standard diagnostic assays, such as an ELISA or RIA, to detect the presence of anti-HIV 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 as a diagnostic reagent will generally involve contacting 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 accomplished by standard techniques, including by quantitating binding of subsequently added labelled anti-gag antibodies.

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.

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EXAMPLE 5,

CONSTRUCTION AND EXPRESSION OF A MUTANT Pr559^a*3 GENE

In order to examine the potential role of the N-myr s itoylation in the assembly and formation of extracellular gag particles we have constructed a glycine deletion mutant. Therefore a synthetic oligonucleotide linker syn3 was for the BamHI-Clal fragment in pRIT12982 (see Syn3 encodes the genuine N-terminal amino acids of protein except that the second glycine codon is This mutant BamHI Pr559^a9 expression cassette was subcloned into the BamHI site of the baculo expression vector pAcYMl (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 in Example 2 revealed no myristic acid incorporation that deletion of the N-terminal glycine was to prevent myristoylation of the GAG precursor protein. Analysis of the cell extracts in Western blots (see Example 1) showed a prominent band of 55 kd and lower M.W. degradation products. The obtained pattern of protein bands was similar to the wild type (wt) Ργ55^³^ protein expressed in S.f. cells. In contrast with the wt Pr559^a9 recombinant, no gag protein could be detected with the glyc.ine mutant 2 days p.i. using PEG or ultracentrifugation of the conditioned medium. Thus the mutated Pr55^^a^ protein was only detected within the infected cells. The myristoylation process thus seems to be required for the extracellular release of the Pi559^a9 product. Scanning electron microscopy (SEM) revealed rather smooth, showing no particles, electron microscopy and immunogold substituted, example 1). the gag deleted.

described conf i rming sufficient and 66 hrs virus revealed that the cell surface was Thin section transmission labelling performed on cells infected 24 hrs, p.i. with the Ac gag 31-18 (Myr^-) recombinant tliafc 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 i,ntracellu 1 ar particles or particulate structures are morphologically different from the extracellular particles obtained with the myristoylated gag recombinant (AcGag7) as they display a double electron dense ring structure and contain a lipid bilayer derived from the cell membrane.

GAG protein nor budding structures were observed at the membrane.

These gag precuror do not

Neither cell results demonstrate that the myristoylation of the appears to be required for its plasma membrane location, budding and extracellular particle release. Myristoylation does not seem however to be required for the nmltimeiic assembly of the Pr55^^a<3 molecules. Accumulation of the non-myristoy 1 ated Pr559^a9 products within the nucleus (and nucleoli) is a surprising phenomenon.

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EXAMPLE--fL· CONSTRUCTION AND EXPRESSION OF A TRUNCATED Pr559^a9 PRECURSOR PROTEIN

In order to examine the role of the pl6 (COOH-end) of the HIV precursor GAG protein we made a GAG deletion mutant which encodes only the pl7-p24 precursor part of GAG.

The BamHI-CfrI GAG fragment of pRIT13003 was purified and 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. This genuine amino acid COOH-end of the two additional amino acids, Valine plasmid was used to co-transfect S,

3' TA TTC CGT purified and cloned sequence contains the core protein and This recombinant

AcMNPV DNA essentially as Recombinant plaques were described before (see screened as described in and f .

and gel linker HIV p24 Serine, cells with

Example 1)

Example 1.

A selected recombinant virus, Ac CfrI, was used to infect S.f. cells. A truncated gag-polypeptide (pl7-24) was detected at the expected M.W. of 41 Kd and which reacted in Western blot analysis with pl7 and p24 monoclonals. The pl7-24 product was predominantly expressed inside the cells but a small amount of extracellular pl7-24 product could be detected when analyzing the conditioned medium by Western blotting. PEG precipitation and ultracentrifugation of the conditioned medium of the CfrI mutant gag protein did not result in detectable pl7-p24 product. Electron microscopy analysis showed no evidence of budding or extracellular gag particles. Large profusions 1-4 μιη 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 (pl7-p24) was localized at the cell membrane and at the periphery of these tubular extensions, but no electron dense ring structures - typical of the Fr559^a9 particle structures - could be detected. This probably indicates that the pl7-p24 product is not able to assemble in inultimeric structures, i.e., cap formations, at the cell membrane. These results suggest that at least a part of the pl6 polypeptide of the GAG precursor polypeptide is necess'a ry for particle formation.

A glycine deletion mutant of the CfrI cassette (non-myristoy1 a ted pl7-24) was made by exchanging the EcoRV-Pstl fragment of 669 bp of the pAcGag 31-18 non-myristoyla ted Ργ55^Α0 g_ene with the pAC CfrI EcoRV-Pstl + 9400 bp long fragment. This mutant displayed no protrusions of membranes as described above but showed pl7 and p24 immunogold decoration scattered in the cytoplasm and nucleus.

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EXAMPLE_7,. CONSTRUCTION _AND_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 transfer vector carrying the Pr559^a9 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 CTA ACT AAG 3*

3¹GGA TTG AT TGA TTC CTA G 5' 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 Example 1.

In this recombinant construct, the myristoylated Pr559^a9 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 pLecipitated with PEG.

Cell extracts however, did show a strong doublet band at 24 Kd and a band at 17 Kd which reacted with p24 and pl7 monoclonal antibodies in Western blots. Very small amounts of the precursor Pr559^a9 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 pl7, p24 polypeptides and intermediates (41 Kd,

46-49 Kd, 55 Kd). The large precursor gag-pol product was not detected with our p55, pl7 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 Pr559^a9 particles. Co-infection experiments with recombinant viruses harbouring the Pr559^a9 and the gag-pol gene did not result in detectable particles displaying a morphological difference such

EXAMPLE 8CONSTRUCTION AND EXPRESSION OF THE SIV Pr579^a9 GENE IN S.F, CELLS

The gag gene of Simian Immunodeficiency virus (SIV) was subcloned from the molecularly cloned SIV_rnac-BK28 (gift of J. Mullins; see Hirsh et al., C.eJLJL 4.2.:307 ( 1987) and Kestler et al., Nature 3J_L:619 (1988)). A 3504 bp Kpnl fragment of the pBK28 genome (nucleotides 1212 to 4716) was subcloned into pUC8. Two internal fragments of the gag gene, the 5' fragment Fnur>n( 1201) - Pst ( 1959) and the 3’ fragment Pstl ( 1959) Hphl (2803) were purified and synthetic oligonucleotide linkers, linker 1:

GAT CC ACC ATG GGC G TGG TAC CCG and linker 3:

TGCTGCACCTCAATTCTCTCTTTGGAGGAGACCAGTAGAGATCTGGTAC AACGACGTGGAGTTAAGAGAGAAACCTCCTCTGGTCATCTCTAGAC were ligated to adequate gag fragments to reconstitute entire the

G was used at the second codon, constructions by sequencing.

precursor gag gene. In a separate experiment a linker 2: GATCC ACC ATG GCC TGG TAC CGG

5* fragment, to introduce a mutation in the namely, GGC (Gly) to GCC (Ala). The different were cloned into blue scribe vectors and verified . The N-terminal fragment (BamHI-Pstl) and the carboxy-termina1 fragment (PsI-Bglll) 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 mutated (Gly to Ala) gene. S.f. cells were transfected with a mixture of purified AC MNPV viral DNA (1 respective recombinant transfer plasmids (50 pg) as described in Example 1. The recombinant plaques were screened and selected as described in Example 1.

The SIV gag Pr575^a9 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 the COOH-end of the HIV p24 polypeptide, which appear 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 myristoy1 a ted in contrast to the culture infected with the glycine to alanine mutant in which no myristoylation of the precursor Pr57y^a9 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 in the conditioned medium when the infected cultures were analysed by ultracentrifugation, sucrose gradients and electron microscopy py) and the essentia 1 ly against also to

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(TEM and SEM). Similar SIV-gag Pr579^a9 particles as those obtained when expressing HIV-Pr559^a9 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 Pr579^a9 as the HIV Pr559^a9 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 Pr559^a9 mutant that N-myristoylation is essential for budding and extracellular particle formation.

The difference between the gag precursor protein of HIV and SIV is that the latter forms also intracellular 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 Pr579^a9 molecules are myristoylated. Also more degradation products, especially a myristoylated p27 protein band could be detected in WB of cultures infected with the SIV Pr579^a9 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 appear to differentiate into virus-like particles budding as described above. This process is reminiscent of the viral maturation of type D retrovirus which involves intermediate intracellular type A particles.

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