JPH0349686A - Expression of retrovirus gag protein in eucaryote cell - Google Patents

Abstract

PURPOSE: To ensure HIV to be diagnosed so as to prevent HIV infection by culturing eukaryotic cells transformed by a specific DNA.

CONSTITUTION: First, a recombinant DNA molecule A is obtained by operatively linking to a control element (e.g. lepidopteran cell) setting in function by eukaryotic cells, and including a DNA sequence of the formula which codes for a full-length HIV gag precursor and is deleted from naturally-occurring 5' and 3' franking sequences. Secondly, an insect cell C such as Spodoptera fulgiberda cell is obtained by infection with a recombinant vaculovirus B produced by including the component A. Subsequently, the aimed retrovirus gag precursor protein D is produced by subjecting a cultured product obtained by culturing the cell C to extraction followed by purifying the extract. As necessary, the component D is formulated with a pharmaceutically acceptable carrier to produce a vaccine for protection from HIV infection.

COPYRIGHT: (C)1991,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to the expression of flea in eukaryotic cells. More specifically, the present invention provides immunodeficiency virus gagiU
Concerning the expression of precursor protein.

Oiseishi Sumidan Retrovirus, i.e. Retrovirita (Retro Virita)
Viridae belongs to a large family of icosahedral viruses that have a coiled nucleocapsid in the core structure and an envelope of approximately 150 nm that contains RNA as the genetic material.

Is this family sarcoma and leukemia viruses, immunodeficiency? viruses and lentiviruses']! I! Consists of G viruses.

Human immunodeficiency virus (H IV), the etiologic agent of acquired immunodeficiency syndrome (AIDS) and related disorders, is a member of the retroviridae family.

Human T-lymphotrophic virus type II1 (HTL
There are several isolates including V-Ill), lymph node disease virus (LAV), and AIDS-associated retroviruses (ARV), which have been assigned to the type Il. Related immunodeficiency viruses include HIV type II, which was recently shown to be associated with AIDS in West Africa. Ike's immunodeficiency virus is SIV■. 1 BK
It includes SIV viruses such as 28.

Molecular characterization of the H1v genome indicates that the virus contains all gag-p cells identical to other retroviruses.
It is shown that it exhibits an o l-env organization. In addition, it contains at least five genes not found in more common retroviruses: sorStat3, art/
Contains trs, 3'orf and R. The gag region was stained with 55 kilotaltons of ga by HIV protease.
It encodes three core proteins, p17, l)24 and p16, which are prepared by cleavage of the g precursor protein. The protease is encoded by the pol region.

Recent reports indicate that antibodies to the HIV gag proteins, p17, p24, and p16, are present in human serum from infected individuals in the United States, Europe, and Pakistan, and that antibodies arise early after infection. The presence of these antibodies decreases as the patient progresses towards AIDS.

The gag protein p17 localized in the submembrane is the transmembrane protein g
conveniently located in close contact with p41 and the viral membrane, and with gp24 and possibly gag pl5 viral RNA, thereby playing a central role in the conformational changes involved in viral entry and uncoating processes. is playing. Furthermore, gag
p17 has been found to have a myristic oxidized N-terminus. Myristic oxidation has been implicated in the assembly of virus particles and the transport of virus particles to the plasma membrane. Myristic oxidized proteins are generally localized to the plasma membrane.

Madisen et al. Virology 58:248
(1987) used a DNA sequence consisting of the gag sequence plus 5' untranslated sequences and sequences from the po+ region to isolate Spodoptera frugiverda using the AcMN P V baculovirus.
reported the expression of HIV gagl in cells.

Cochran, European Published Patent Application No. 22
No. 8036 discloses the use of a baculovirus/insect expression system to express certain proteins.

On pages 17a and 26, the AIDS retrovirus protein is mentioned in a list of proteins that can be produced in such a system.

Cochran et al., European Published Patent Application No. 265,785, discloses the expression of HIV envelope proteins in insect cells using a baculovirus expression system.

Cramer et al., Science 23: 1580 (19
86) and Craner et al.
), European Published Patent Application No. 230222, Expression of the gag-pol DNA sequence in yeast and in v
Discloses ivo amputation.

Valenzuela et al.
) is Nature + 298: 347
(1982) report the synthesis of hepatitis B virus surface antigen and its assembly into particles in yeast.

Ellis et al. (European Published Patent Application No. 251460) discloses the aggregation of hepatitis B virus core antigen in yeast and its assembly into particles.

Adams et al.
ELL) 49:11 (1987) reported the assembly of p1 protein and assembly into particles of 'ry-virus-like particles (VLPs) in yeast. For an overview, see p.
We conclude that 1 is the precursor to the major core protein of TyVLPs, and in this respect is functionally similar to the gag precursor protein of retroviruses.

Bishop et al., European Published Patent Application No. 260090, discloses the expression of hepatitis B virus antigens using a baculovirus expression system.

Rusch et al., European Patent Publication No. 11272858, discloses the expression of proteins from HIV GP 160 using a baculovirus expression system.

Despite major research efforts in the AIDS field, there are no diagnostic agents that can be used to monitor the progression of the disease, as well as agents that can prevent primary infection, such as through immune-mediated and cell-to-cell or free viral infection. There is a continuing need for agents that can prevent and suppress secondary infections.

In an aspect of Disclosure 1, the present invention provides a recombinant DNA for the expression of gag precursor protein in eukaryotes comprising a coding sequence operably linked to a regulatory region that functions in a host cell.
Provide molecules.

In a related aspect, the invention provides a host cell comprising the eukaryotic DNA molecule and a culture thereof.

In a further related aspect, the invention provides gag precursor proteins produced by the host cells of the invention, including HIV fa-like particles comprising gag precursor proteins.

In still further related aspects, the invention provides methods for producing recombinant DNA molecules and host cells of the invention,
g precursor proteins and particles, and related materials and methods.

These and other aspects of the invention are described in detail in the disclosure and examples below.

We now demonstrate that the retroviral gag precursor protein can be expressed in recombinant euphemistic cells, and as a result of such expression,
It was found that full-length gag precursor protein could be produced without using the pol DNA sequence and without using the 5° untranslated sequences from the virus. Examples of such cells include insect cells such as Drosophila or Lepidoptera cells; mammalian cell lines; lower eukaryotic cells such as yeast and fungi, including primary mammalian cells, and animal cells. , and cells from insects and transgenic animals.

It was also unexpectedly found that the gag precursor protein differs in size and other physical properties, as well as in antigenicity.
It was also found that particles similar to standard gag particles formed in infected human cells can be formed. During the natural retroviral infection cycle, the gag precursor protein, known in the case of HIV as p55, appears to be morphed into particles consisting predominantly of full-length gag protein.

These gag particles can be referred to as played core particles or immature core particles. During viral ripening, the precursor is then cleaved into subunit proteins known in the case of HIV as p17, p24 and p16. These gag particles, which now consist predominantly of p17, p24 and p16, can be called core particles or mature fp particles. Also, during virus maturation, apparently during the budding process, a viral membrane is formed around the breaker core or core particle. As shown in the Examples below, HIV gag precursors expressed in recombinant Lepidoptera cells using a baculovirus expression system aggregate to a large extent or form the core of HIV particles that naturally form in infected human cells. packed into particles with similar physical and biological properties and size. The particles of the present invention are overwhelmingly composed of gag precursor protein (99% of the total protein in the particles).
Besides being recognized by anti-gag polyclonal antibodies from the sera of infected patients, otherwise anti-p17 consists of >0% of the full-length gag precursor) and is recognized by anti-gag polyclonal antibodies from the serum of infected patients. Recognized by monoclonal antibodies (MABs).

Since the particles are prepared by recombinant DNA methods disclosed herein, they also lack viral functions necessary for viral ripening and replication, particularly viral RNA and preferably reverse transcriptase and protease functions.

The recombinant eukaryotic cell of the present invention comprises the recombinant DNA of the present invention.
The introduction of the molecule into cells is designed to express the gag precursor protein. The recombinant DNA molecules of the invention consist of the coding region for the gag precursor protein operably linked to regulatory elements that function in the selected host cell. In one aspect of the invention, the other HIV function is ga
It was found that it is not required for the expression of the g precursor protein and for the formation of precore-like particles. DNA sequences encoding other functions, such as amplification functions, selection markers or maintenance functions, are also included in the recombinant DNA molecules of the invention.

The DNA coding region for the gag precursor protein can be prepared from any of several immunodeficiency virus genomic clones or gag-po1 clones reported in the literature. For example, Sham et al.
al. ), Science 226
:1165 (1984); Kramer et al.
real. ), Science 2 3
1:1580 (1986). Alternatively, immunodeficiency virus genome clones can be cloned from standard DNA
It can be prepared from viruses isolated from clinical specimens by cloning techniques. For example, Gallo et al.
atal. ), U.S. Pat. No. 4,520,113; Montagnier et al., U.S. Pat. No. 4,7088ill. Once a genomic fragment consisting of the gag coding region has been cloned, the DNA is restricted to isolate a portion of the DNA coding region and the remaining portion is isolated by the use of synthetic oligonucleotides, as described in the Examples below. By reassembling, regions can be prepared that code only for gag precursors. Alternatively, larger fragments consisting of the gag coding region and additional sequences can be reversely cleaved by use of an exonuclease. In yet another alternative, SIII1! The entire coding region can be prepared using a standard automated DNA synthesizer by synthesizing seven fragments of the coding region and joining them together to form the complete coding region. Although the use of coding sequences lacking naturally occurring 5' and 3' flanking sequences is preferred, fusions of the coding sequences to other immunodeficiency virus sequences, such as envelope protein sequences, are not excluded from preferred embodiments. .

An example of a coding region for the HIV gag precursor protein has the following sequence.

A variety of eukaryotic cells and expression systems can be used to express heterologous proteins. The most widely used of these are yeast, insect, and mammalian cells, but this The invention is not limited to this particular use. Typically, these systems include a hooding sequence for the gene of interest operably linked to regulatory elements, a selection marker, and optionally a replication initiation marker. A recombinant DNA molecule is used that consists of maintenance functions such as dots. Regulatory elements are DNA regions or regions that consist of functions necessary or desirable for transcription and translation. Typically, the regulatory regions are Consists of a promoter for binding and transcription initiation.

Insect cells that can be used in the present invention include Drosophila cells and Lepidoptera (L.
epidoptera) cells. Useful Drosophila cells are S 1, S 2
, S3, KC-0 and D, hyde
i) Contains cells. For example, Schneider et al.
Neider et al. ), Journal of Embryology and Experimental Morphology (J. Embryol. Exp. Mor
ph. ) 2 7, 3 5 3 (1 9 7 2): Schulz et al., Proceedings of the National Academy of Sciences (Proc. Natl.^cad. Sci.
．． ) LISA 83:9428 (1986); Sinclair et al., Molecular and Cellular Biology (Mol.
Cell l. Biol. ), 5: 3 2 0
8 (1985). Drosophila
la) Cells are prepared using calcium phosphate precipitation method, cell fusion method,
Transfection is performed by standard techniques including electroporation and viral transfection methods. Cells are cultured by standard cell culture techniques in a variety of nutrient media, including, for example, M3 medium consisting of balanced salts and essential amino acids. Lindquist, DIS 58:163 (198
2) See.

Promoters known to be useful in Drosophila include mammalian cell promoters as well as Drosophila promoters, with the latter being preferred. Useful Drosophila (
Examples of Drosophila metallothionein bromoproteases are Drosophila metallothionein bromoprotease, 70 kilodalton heat silane protein promoter (HSP70) and COPI.
Includes ALTR. For example, Di Nocera et al.
Nocera et al. ), Brondings
Of National Academy of Sciences (
Proc. Natl. Acad. Sci. ) L
ISA 80:7095 (1983); McGarry et al., Ce
l l) See Sat 2: 903 (1985). Conveniently, for the purpose of DNA propagation, the expression case consisting of the gag coding sequence and regulatory elements is cloned into a bacterial cloning vector prior to transfection of animal cells.

In a preferred embodiment of the invention, the HIV gag precursor is expressed in Lepidoptera cells to produce immunogenic gag particles. Preferably, a baculovirus expression system is used for expression of the gag precursor protein in Lepidoptera cells. In such a system, the expression case consisting of the gag coding sequence and regulatory elements is placed into a standard cloning vector for propagation purposes. The recombinant vector is then co-transfected into Levidoptera cells with DNA from wild-type baculovirus. Recombinant viruses obtained from homologous recombination are then selected, substantially as described by Summers et al., TAES Bull.
．． ) NR 1 5 5 5, 1 9 8 May 7,
Braak purification as described by.

A useful Lepidoptera cell is Trichoplusia ii (Tr
Ichoplusia ni), Spodoptera frugiperda
), Heliothis zea
, Autograca californica (Autogra
phica californica), Rachiplusia ou, Galleria melonella
, Manduca sexta
), or other cells that can be infected with baculoviruses, including nuclear polyhedrosis virus (NPV), single nucleocapsid virus (SNPV) and multinucleocapsid virus (MNPV). Preferred baculoviruses are the NP■ or MNPV baculoviruses. This is because they contain polyhedrin, which is highly expressed in infected cells.
) This is because it contains the gene bromo. In particular, the following examples are Autografica californica (
Autographica california)
(AcMNPV). However, other MNPV and NPV viruses, silkworm viruses, Bombyx mori
i) can also be used. As described above, Lepidoptera cells are co-transfected with DNA comprising an expressed casenote of the invention and infectious baculovirus DNA by standard transfection techniques. for example,
TCIOO supplemented with 10% fetal calf serum (FCS) (
Gibco Europe
: Gardiner et al.
), Journal of Invertebrate Pathology (J. Inverteb Pathol.) 25:3
Cells are cultured according to standard cell culture techniques in a variety of nutrient media, including 63 (1975). Miller et al.
Miller et al. ), Setlow et al.
low et al. ), edited by Genetic Engineerin
g) :Prince Bulls and Methods (Prin
ciples and Methods), vol. 8, New York, Plenum, 1986, p. 27.
See 7-298.

Production in insect cells can also be disrupted by infecting insect larvae. For example, the gag precursor can be obtained from Triehoprusia ni by growing the recombinant paculovirus of the invention with a very small amount of wild-type baculovirus and then extracting the gag precursor from the hemolymph about 2 days later. ) can be produced in caterpillars.

Bromote promoters for use in Lepidoptera cells include promoters from the baculovirus genome. A promoter of the polyhedrin gene is preferred. Because polyhedrin (poly-hedr)
in) protein is inherently more expressed than other baculovirus proteins. The polyhedrin gene promoter from the AcMNPV virus is preferred. Sussers et al.
et at. )TAES Bul1.) TAES Bul1. NR 1 5 5 5
, May 1987; Sgiith et a
l. ), European Published Patent Application No. 127839; Smith et al.
So+ith et al. ), Proceedings of the National Academy of Sciences (Proc. Natl. AcadSci.) L]S
A82:8404 (1985): and Cochrane (C
och-ran), see European Published Patent Application No. 228036.

For expression in mammalian cells, the expression case is similarly cloned into a cloning vector and then used to transfect mammalian cells. Preferably, the vector further comprises a DNA function for gene amplification, e.g. a DHFRp expression cassette, and may also further consist of a function for selection and/or amplification, e.g. resistant cassette. Other functions can also be used, such as those for transcriptional promotion. If desired, further functions may be included in the vector for stable episomal maintenance, such as maintenance functions of bovine papilloma virus. The mammalian cell vector may also be a recombinant virus, such as recombinant vaccinia or other smallpox virus.

For example, Paoletti et a
l. ), U.S. Patent No. 4,603,112; Paoletti et al., Proceedings of the National Academy of Sciences (Proc. Natl., Acad. Dxi.)
See US 81:193 (1984).

Useful mammalian cells include Chinese hamster ovary (
CHO), NI H3T3, COS-7, CVI, mouse or rat myeloma, }IAK, Vero, He
human diploid cells such as la, MRC-5 and w138, or chicken lymphoma cell lines. Perform transfection and cell culture by standard techniques. Furthermore, production in mammalian cells is also disrupted by expression in transgenic animals. For example, g
The ag precursor is expressed using a casein promoter and can be purified from milk.

Promoters useful in mammalian cell lines and mammalian primary cells include the SV40 early and late gene promoters, metallothionein promoters, viral LTRs such as the Rous sarcoma LTR, Moloney sarcoma virus (MSV) LTR or mouse mammary tumor virus (MMTV) LTR. , or hybrid promoters such as the adenovirus major late promoter and the hybrid BK virus and adenovirus major late promoters.

Regulatory regions may also consist of downstream functions, such as regions for polyadenylation, or other functions, such as transcriptional enhancer sequences.

Yeasts that can be used in the practice of the present invention include the genera Hanensula, Pichi
a), Kluveroayces,
Schizosaccharo+
syces), Candida and Saccharomyces. Saccharomyces cerevisiae
omyces cerevisiae) is a preferred yeast host. A useful promoter is copper-inducible (cuP1
) fluorophores, glycolytic gene promoters, e.g. TDH3
, PGK and AD}{, and PH05 and AR
Contains the G3 promoter. For example, Myanohara et al.
iyanohara et al. ), Proceedings of the National Academy of Sciences (Proc. 1iatl.Acad.sci.)
tlsA80:1 (1983): Mellor et al., Gene
), 24:l (1983); Hitzemann et al.
itzeman et al. ), Sci
ence), 219:620 (1983); Cabezon et al., Proceedings of the National Academy of Sciences (Proc. Natl. Acsd. Sci.)
See USA 8 1: 6 5 94 (1984).

In the case of gag precursor protein particles produced according to the present invention, particles consisting of gag precursor are preferred, but natural gag
It should be understood that particles consisting of derivatives of g precursors can also be prepared. For example, one or more nucleotides or amino acids shown in the above sequence can be deleted, substituted, or added without substantially adversely affecting immunogenic cross-reactivity with authentic gag epitopes. In other words, such derivatives that are immunologically similar to authentic gag particles in that they are recognized by antibodies include p17, p24 and p16.
Occurs for at least one of the following. Such derivatives, which include antigenic regions of the HIV genome and may include amino acids from other regions, do not encode other HIV functions, such as the protease function or reverse transcriptase function of the pot region.

Additionally, such derivatives retain the ability to form particles in insect cell culture as disclosed herein.

In this case, gag consists of a hybrid protein having l or more evitopes in addition to gag evitopes.
It is within the skill of those skilled in the art to prepare the particles. Such further shrimp may be of HIV origin or may be derived from other pathogenic microorganisms, such as hepatitis B virus or herpesvirus.

The gag precursor protein is expressed in secreted or membrane-bound form. It is isolated from the conditioned medium by standard techniques of protein isolation and purification. Detergents can be added to separate proteins from cell membrane material. Following treatment with a detergent, such as Triton xlOO, Tween or sodium dodecyl sulfate (SDS), the proteins or particles are subjected to a series of ultrafiltration steps, ultracentrifugation steps, selective precipitation with eg ammonium sulfate or PEG. , density gradient centrifugation and/or affinity chromatography on CsCQ or sucrose gradients, immunoaffinity chromatography HPLC, reversed phase H
Purification can be achieved by chromatographic steps such as PLC, cation and anion exchange, molecular sieve chromatography and preparative isoelectric focusing. During or following purification, proteins or particles can be treated with, for example, formaldehyde, glutaraldehyde or NAE to enhance stability and immunogenicity. In view of the discovery disclosed herein that gag precursors can form immunogenic particles in the absence of other viral functions, when gag precursors are expressed in nonparticulate form, yeast It is conceivable that expression of the hepatitis B surface antigen could allow the particles to be shaped collectively, as was shown in the case of the hepatitis B surface antigen. See, for example, European Published Patent Application No. 1 35435. Such gag precursor protein particles are within the scope of the present invention.

Hl'l/gag precursor proteins or particles produced according to the present invention are useful as diagnostic agents for the detection of exposure to HIV. The proteins and particles are also useful in vaccines to prevent infection or inhibit or prevent the progression of disease.

Example The present invention will be explained in more detail with reference to Examples below.

Restriction enzymes and other agents were used essentially as directed by the manufacturer.

Example 1 Vector Construction pRIT12982 (DT 12-16)
1305 base pairs for the N-terminal region of the precursor protein (b
This is a vector consisting of the coding sequence of p). It is an HIV genome clone (Shaw et al.
tal. ), Science, E. 2: 1 165 (1984)) The Clal-Bgl fragment of the gag precursor protein coding region derived from l- was fused with the N-terminal coding sequence of the gag precursor protein. prepared by ligating to oligonucleotides. The oligonucleotide has the following sequence: 5°C ATG GGT GCT AGA GCT T
CC GTG TTG TCC GGT GGT CA
A TTG (.AT 3 CCA CGA TCT CGA AGG CAC A
AC AGG CCA CCA CTT AAC CT
A GCNcol
ClaalpRIT12983 encodes the C-terminal portion of gag precursor protein (7)25
This vector consists of a gi region of 0 bp. That is H
The BglIt-Mae[l fragment of the gag precursor coding region derived from the IV genome clone was
It was prepared by linking to a synthetic oligonucleotide with the C-terminal coding sequence of the ag precursor protein. The oligonucleotide has the following sequence: Tokuyo S' G TCA CM TM AcATAG (.
AT CC 3'TT ATT TCT AT
C CTA GGA GCTMaell
Xhol. 250bp Bgln-TAA-Bam}II-X from pRIT12983 in ptlc previously cut with BamHI and Salt
130 from pRIT12982 to the hol fragment
5 base pairs (bp) BamHI (Nco I)-Bg l
I tied the II7 ragment. The resulting plasmid, identified as pRIT13001, is therefore B a m
H](Nco I) - gag on BamH l casenote
Contains the entire coding region for the precursor protein.

The baculovirus expression vector combines the BamHI7 fragment from pRIT]3001 with BamH of pAc373.
prepared by inserting into one site. Smith:)
(Smith et al.), Proceedings.
Of Na, /-1 Nall Academy of Sciences (Proc. Natl. Acad. Sci.)
, tlsA 82:8404 (1985). pA
c373 allows foreign genes to be cloned into its Bam81 site, and the strong polyhedrin
) is a baculovirus transfer vector containing a modified polyhedrin gene expressed under the control of bromovirus. Summers et al. (S
Ummers et al. ), Texas Agricultural Experiment Station Bulletin (Texas Agricultural Exp/
Station Bulletin) NR 1555
(May 1987). A derivative of plasmid pAc373 with a small deletion and large upstream strong polyhedrin (pQlyhedrin) promoter was also used as an expression vector. Minor modifications are made to the recombinant virus il v
It did not appear to affect itro expression or proliferation. Insertion of the gag coding sequence into the baculovirus vector resulted in plasmid pRIT13003.

The mammalian cell expression vector combines the BamHI fragment from pRIT13001 into SV40 in pSV529.
prepared by ligating downstream of the late promoter (Gheysen et al., Journal of Molecular and Genetic Genetics (J. Mol.^ppt. Genet.)
1:385 (1982)). This vector is pR
Identical to 1T13002.

A yeast expression vector was prepared as follows.

Nco I-Bg l II fragment l) RIT
12982 and inserted into the yeast plasmid downstream of the ARG3 promoter and in its decoding phase (Cabezon et al., Proceedings of the National Academy of Sciences (Proc. Na. Acad.Sci.)U
SA 81:6594 (1984)), vector pRIT12984 (DT14-20)
get. The C-terminal part of the gag precursor protein is converted into Bg II
pR as I-BamH I fragment]T1298
3 and inserted into the BglI1 site of pRIT12984 to create yeast vector pRIT12985 (DT16-
26) was obtained. Thus, pRIT12985 is ARA
It consists of the coding sequence for the full-length gag precursor, operably linked to the 3-bromo sequence and lacking other HHIV sequences.

In addition, it consists of a replication function from a yeast 2 micron vector and a URA3 gene selection marker.

Example 2 Expression in insect cells Substantially as described by Summers et al.
, TAES Bu
ll. ) NR 1555, May 1987, supra, recombinant baculovirus transfected with pRI713003 was prepared.

Spodobutella frugiberda (S.f.) cells were cotransfected with wild type (wt) AcMNPV baculovirus DNA and plasmid pRIT13003 at 1 μg and 50 μg, respectively. The obtained virus particles were obtained by collecting the supernatant. Using the medium containing the virus, S. f. infected cells. wt AcN
Using virus particles containing both PV DNA and DNA recombined with DNA encoding the p55gag precursor protein. f. Subsequent infection of the cells resulted in cells expressing gag protein instead of polyhedrin protein.

"Clear plaque" (0.1
−0.01% frequency) were further screened by filter hybridization using a gag-specific probe. Gag probes were hybridized to enumerate Nakoblak, followed by further plaque purification (2-3 times) before virus stocks were generated; the virus stocks were also tested by immunoenzymatic assay (ELISA). Next, S. f, Cells were infected with these recombinant gag virus strains at a multiplicity of infection (Mol) of 1-10 and after 24 h, 48 h, 3 days and 5 days conditioned medium (supernatant)
and/or a portion of the cells were treated with Triton xlOO to a final ag degree of 1% and anodized.

p55 polyclonal antibodies or AIDS patients (Zylan (Z)
airan)) using antisera from a pool of
The gag precursor protein assembled in infected insect cells was observed by Western brosometry. A prominent band at a molecular weight (Mr) of 54 kilodaltons (k d) was observed in all tested sera and p55 polyclonal antisera.

A band at 54 kd was also detected when conditioned medium was tested after 48 hours, 3 days and 5 days. When cell extracts were analyzed, bands at M r 49 k d and M r 47 kd (minor) as well as a band at M 30 kd could also be detected. This latter band with obvious Mr3Qkd was only detected with the p55 polyclonal antibody, but not with the serum of AIDS-infected individuals. HIV (Molt/H
TLV-m) infected Mol t cells at least 10 times more. p expressed in f cells
55 "epitope" and approximately 80 times more p55 "epitope" in S. cerevisiae infected with gag recombinant virus than in the conditioned medium of Molt/HTLV-III cells.
I. It was observed to be present in the conditioned medium of II cells.

In the second assay experiment, 48 hours, 3 days and 5
conditioned medium (2 ml to 200 ml ultracentrifuged (1 ml))
00000xg for 1 hour) resulting in a small beleft, which was analyzed on SDS-gel and also by immunopronoting method. One band at M r 5 5 k d was detected with specific antibodies against p17, p24 and p55. was recognized. Only a small amount of decomposition products in Mr49-46 could be detected. A band at 55 kd can be observed on the Comassie stained gel,
It was 20-80% pure. This band corresponded to the immunoplot and was recognized by antibodies against p17, p24 and p55 vorivebutide.

In the third assay experiment, 4 in a microfuge at 1200 Orpm.
Centrifugation of 8-hour, 3-day, and 5-day conditioned media for 5 to 20 minutes resulted in bands on the SDS-gel at Mr55kd, as revealed by antibodies against p17, p24, and p55 polypeptides. and HIV cell lysate (Molt/HTLV-
nl) As compared to the 55kd band, HIV-1
It was specific for the p55gag precursor.

In the fourth assay experiment, 48-hour, 3-day, and 5-day conditioned medium (150 mR to 1,000 mg, containing aprotinin, added 24 hours postinfection and also at each time of harvest) was first introduced. Treatment was performed by adding Tween 20 to a final concentration of 0.01%. Then,
Polyethylene Glyfur, Mr6kd (P
E G 6 0 0 0 ) ( 2 M Na
A solution of 40% w/v) in CQ was added to give a final concentration of 10% or 5%. After 4 hours at 4°C, or preferably overnight at 4°C, the precipitate was centrifuged at 4°C, 5000 rpm for 10 minutes. Then P.E.
HBS containing 0.1% Tween 20
-Buffer (Hank's Balanced Salt, Flow Laboratories (
Flow Laboratories), 18-102
-54) 200μ+! In a Beckman rotor TLAI0 at 4°C.
0 (Beckman Instruments)
nstruments), Fullert
on), California), approximately 35 at 5000 rpm.
minutes or at 4°C.
) SW 4 1 c+ - 25000r at Yuichi
sucrose gradient (4°C) IBS buffer containing 10 μQ/mQ of abrotinin,
20%-60% in 0.1% Noeen 20, Sigma Chemical Company (Sign+a Chemical
Co. ), St. Louis, Missouri).

0.2 to 0.5m each from about 40-50% sugar
The C fraction was collected, frozen at -20''C, and -24/Ig
AIDS biotinylated antiserum or AIDS antiserum/I
g Core POD (HIV-anti-core EIA1 Abbott
Abbott Laboratories
es) was tested using a specific antigen capture immunoenzymatic method. OD immunoenzymatic method pick of 1 was detected,
This indicates that p55 protein migrated as particles or "aggregated structures" on the C-I sugar gradient. The bipolar fractions and surrounding fractions were immunoblotted with p17, p24, or p55 antibodies. Mr in SDS-reducing gel
1 major band at 55 Kd was determined by Mo I t/H I Lv- prepared essentially as described above.
(2) Compared to cell extracts, p55 gag precursor protein was detected.

In the fifth anomaly experiment, S, f. From 150 rM of culture to 5% PEG6000, essentially as described for the fourth assay experiment.
J:. The lees were prepared.

200μ HBS buffer containing 0.1% Tween 20
12 medium (:PE06000 pellets were collected.

1 After centrifugation at 5000xg for 1 minute, the supernatant was
5M CsCQ solution (0.3 volumes HBS-buffer, 10mM) Lys-HCg (pH 8.0), lm
Mx ethylenediaminetetraacetic acid (EDTA), O. 1% Twee720 and Abrotinin lOμg/m(1)1
1.5m12. This suspension was heated at 18°, 44°
Centrifugation was performed for approximately 72 hours at 000 rpm in a Becman Rotor 50 Ti. Fractions of 300 μQ each were collected, frozen at −20° C., and tested by specific antigen capture immunoenzymatic assay (HIV anti-core EIA). Density approximately 1.28 and 1.20
A band was recognized in g/cm', and the core-like particles had an apparent 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 clearly budding onto the cell surface. Immuno Gold Transmission (im
(aunogold transmission) electron microscopy revealed particles recognized by p24 and p55 antibodies. The p17, p24 and p55 epitopes were also recognized by immunogold labeling in electron microscopy preparations after brief treatment of purified particles with Triton XIOO. The particles were approximately spherical and approximately 100-150 nm in diameter. The particle has an electron lucent center surrounded by a dark ring and an outer shell.
luscent center) and apparently have many p17, p24, p16, and p55 epitopes on the inner surface of the particle.

Therefore, this example demonstrates the necessity of expression and distribution of HIV brecor-like (and core-like) particles consisting of the immunodeficiency virus gag precursor protein. The particles are
Overwhelmingly (over 90% of the total protein) consists of the full-length gag precursor protein, in the absence of DNA sequences of viral origin other than the gag precursor sequence, and thus, a reservoir such as retroviral protease and reverse transcriptase. formed in the absence of viral functions.

S. f. To demonstrate that the HIV gag precursor protein produced in cells is effectively myristically oxidized, we infected 3 x 10 cells in F25 cm' flasks with recombinant p55 gag baculovirus at MOT 5. After treatment, 500 μCi myristic acid NET
-830 (DuPont, Wilmington)
ington), Delav-are)
48 hours p. i. Labeled with. Subsequently, conditioned media and cells were prepared separately for Western blotting and SDS-gel radioautography. The conditioned medium contained l at 55kd, which was also recognized as a gag precursor in Western blots, as revealed by antibodies directed against p17, p24, p55+.
showed the major bands of Two other labeled secondary bands were detected for Mr49-46-47kd and the same antibody senotes (p17, p24,
p55). 1% triton x
Cell lysates made in lOO and frozen at -20°C were analyzed by radioautography and Western blotting on 12.5% laemli gels.
bands at 55 kd each (as well as apparently ga
g) showing a minor band at 58 kd corresponding to a translational frameshift as described above for the retroviral HTV-1 viral genome and a more prominent band at Mr49-47-46) and a degraded biobottom at 30-27 kd, the latter band was not radioactive (contained no myristic acid).

Example 31'i@Expression in milk cells Calcium phosphate precipitation method (Wigieret a1.
), Cell, Vol. 6:777 (1979))
Blasmid pRIT13002 was introduced into CosI and CVl cells by. Transfusion's 48
At this time and 110 hours later, cells and media were annotated using a specific immunoenzymatic method for gag antigen expression. Cell extracts (lO@cells) were adjusted to 1% Triton xlOO or 0.5% DQC-NP40. p17, p
using immunoenzymatic capture antigen tests involving polyclonal and monoclonal antibodies against p55 or p55;
The p55 antigen was detected using the DuPont RIA test (NEW-040), which involves competition with purified p24 bebutide. The expression levels obtained were determined by ])24RIA DuPont test, 4 and 1
It was between 0 ng/m+2.

Example 4 Expression in Yeast Cells Expression of plasmid pRIT12985 into S. cerevisiae (S.
．． cerevisiae) strain 02276b (ura
3-durQ'roci). Yeast extract (using glass beads or zimolase (zy
p55 antigen was detected in mid-log phase cells disrupted by spheroblastization with molase). The p55 antigen was detected using immunoenzymatic tests involving polyclonal and monoclonal antibodies against the p24 peptide, or using DuPont Radioimmune assay (RIA) involving incubation with purified p24 peptide. Detected.

The p55 protein synthesized in S. cerevisiae was observed in Western blotting using a p17 or 1)24-specific monoclonal antibody, which was determined by the molecular weight of the p55 antigen obtained from infected cells. It has a similar molecular weight.

When cell extracts were obtained in the absence of detergents, a significant fraction of the antigen was retained in the membrane cells.

This antigen fraction was collected using Triton xlOO.

The use of detergent either before or after isolation enhanced antigenicity as determined by RIA. g produced in yeast
The ag precursor was shown to be myristic oxidized by labeling with tritiated myristic acid and was clearly associated with the cell plasma membrane as shown by electron microscopy.

The above examples demonstrate the expression of gag precursor protein in animal cell culture and the expression of immunodeficiency virus Brecoa-like particles in Lepidoplera cells using a baculovirus expression system. The proteins and/or particles thus prepared are purified and formulated into a vaccine for parenteral administration to humans at risk of exposure to HIV, in order to protect the vaccinated person from the onset of symptoms of illness associated with HIV infection. . Each vaccine dose consists of an amount of protein or particles that is safe, ie, effective to induce an immune response while not causing significant adverse side effects.

For example, each dose may contain from 1 to 1000 μg, preferably from 1 O to 500 μg, of gag precursor protein or particles in a pharmaceutically acceptable carrier, such as an aqueous solution buffered to about pH 5 to 9, preferably pH 6 to 8. Become. The vaccine may also contain adjuvants, such as aluminum hydroxide, muramyl dipeptide or Quil A
Contains saponins such as. Useful buffers include sodium or ammonium cations and acetic acid, citric acid,
Includes phosphate or glutamate anions.

Other pharmaceutically acceptable carriers or excipients can be used to render the formulation isotonic or stabilized, such as sodium chloride, glucose, mannitol, albumin or polyethylene glycols.

Vaccines can be lyophilized for convenient storage and handling. Such vaccines are reconstituted prior to administration. Alternatively, gag proteins or particles can be formulated into liposomes or ISCOMS by known techniques. An example of a vaccine dose is 100 gag particles adsorbed on aluminum hydroxide in water buffered to pH 7 with sodium acetate.
Consists of μg.

In another embodiment of the invention, the gag protein or particles are mixed with one or more other antigens by co-expression or co-formulation in the same cell culture. Such other antigens include other HIV antigens, such as envelope proteins,
can be an antigen derived from gp160 or gp120;
or from one or more other pathogenic organisms, cells or viruses, such as hepatitis B surface antigen to confer protection against hepatitis B virus or herpesvirus glycoproteins to confer protection along with herpesviruses. can be an antigen.

Although other routes of administration are also useful in inducing a protective response,
Preferably, the vaccine is administered parenterally, for example intramuscularly (i.
m) or subcutaneously (s c).

The vaccine may be administered in a single dose or in multiple doses, eg 2 to 4. Immune protection can be confirmed by assaying serum anti-gag antibody levels. Thereafter, revaccination can be performed as needed, eg, annually.

As a diagnostic agent, gag proteins or particles can be used in any standard diagnostic assay, such as immunoenzymatic or RIA methods, to detect the presence of anti-1{TV antibodies in clinical specimens. Such diagnostic agents can be used in combination with other HIV antigens to monitor disease progression. Use of gag proteins or particles as diagnostic agents generally involves contacting human or other animal serum or other body samples with proteins or particles that are preferably bound or otherwise attached or captured. , and then anassying the binding of anti-gag antibodies from serum or other samples to the gag protein or particles. Such assays can be accomplished by standard techniques including substantially quantifying the binding of added labeled anti-gag antibody.

Example 5 Construction and Expression of a Mutant p55 Gene To investigate the possible role of N-myristoylation in the assembly and formation of extracellular gag particles, we constructed a glycine deletion mutant. Therefore, pR
The BamHI-Claf fragment (see Example 1) in IT12982 was replaced with a synthetic oligonucleotide linker-syn3. syn3 encodes the true N-terminal amino acids of the gag protein, except that the second glycine codon is deleted. This mutant BamHIp55 expression cassette was subcloned into the BamH1 site of the Bakuguchi expression vector pACYMI (Matsuura et al.
), Journal of General Pyrology (J
．． Gen. Virol. ) 68: l 233
(1987)), recombinant Braak was obtained and selected essentially as in Example 1. AcGag31, a recombinant virus with a glycine deletion mutation in the gag gene
-18 using S. f. infected. gag precursor protein, as determined by immunoenzymatic assay,
effectively synthesized. By metabolic labeling with sH2listic acid, substantially as described in Example 2,
It was revealed that there was no uptake of myristic acid, and N
It was determined that deletion of one terminal glycine was sufficient to prevent myristoylation of the gag precursor protein. Analysis of cell extracts in Western broth (see Example 1) showed a prominent band of 55 kd and low molecular weight degradation products. The obtained protein band pattern is S.
f. I. It was similar to that of the wild type (wt) p55 protein expressed in Ill cells. In contrast to the wt p55 recombinant, the glycine mutant was p.p. i. No gag protein was detected. Thus, mutated p55 protein was detected only within infected cells. Thus, the myristoylation process appears to be required for extracellular release of p55 products. Scanning electron microscopy (SEM) revealed that the cell surface was fairly smooth and showed no particles.

Ac gag3 1-18 (My i) recombinant virus for 24, 48 and 66 hours p, i. Thin secLion transmission performed on infected cells
n) Electron microscopy and immunogold
Gold) labeling revealed that the non-myristoylated aga protein was effectively expressed and was either dispersed in the cytoplasm or associated with gray amorphous structures within the cytoplasm and the nucleus. These intracellular particles or particulate structures are morphologically similar to myristoylated gag recombinants (AcGag7), since they exhibit a double electron-dense ring structure and do not contain a lipid bilayer derived from the Mibuda membrane. This is different from the extracellular particles obtained in . Neither gag protein nor budding structure was observed at the cell membrane.

These results indicate that myristoylation of the gag precursor is likely required for its plasma membrane positioning, budding, and extracellular particle release. However, myristoylation
It does not appear to be necessary for multimer assembly of five molecules. The accumulation of non-myristoylated p55 products in the nucleus (and kernel) is a surprising phenomenon.

Example 6 Assembly and expression of truncated p55 precursor protein HIV precursor gag protein 17)! )16 (COOH-
In order to investigate the role of gag p
A gag deletion mutant encoding only the 17-p24 precursor portion was created.

BamH l -C f r 1 of pRIT13003
The gag fragment was purified and the synthetic nucleotide sequence S' GGC CAT AAG GCA AGA GT
T TTA GTT AGT TAG 3'3' T
A TTC CG7 TCT CAA AAT CAA
TCA ATC CTA GS' was ligated, gel purified, and cloned into the BamHI site of pAcYMl.

This linker sequence contains the true amino acids COOH of the HIV p24 core protein at one end and two additional amino acids, valine and serine. Using this recombinant plasmid, the procedure was performed substantially as described above (see Example 1).
) with AcMNPV DNA. f. Cells were co-transfected. Recombinant Braaks were screened as described in Example 1.

Using the selected recombinant virus, AcCfrl, S. f
infected cells. Truncated gag-polypeptide (p1
7-24) was detected at the expected molecular weight position of 41 kd, which was found to be associated with p17 and p17 by Western blot analysis.
It reacted with 24 single clones. Although the p17-24 product was expressed overwhelmingly inside the cells, analysis of the conditioned medium by Western blotting revealed that a small amount of extracellular p17-24 was expressed.
17-24 products could be detected. PEG precipitation and ultracentrifugation of conditioned medium of the Ctrl mutant gagW white yielded no detectable pl''7-24 product. Electron microscopic analysis revealed that budding or extracellular gag particles No evidence of the presence of . shape gag
Proteins (p17-p24) are localized at the cell membrane and around these tubular extensions, but in electron-dense "ring" structures.
No typical p55 particle structure could be detected. Presumably, this indicates that the p17-p24 products are unable to assemble into multimeric structures, ie cap-shaped bodies, at the cell membrane. These results suggest that at least a portion of the p16 polybebutide of the gag precursor polybebutide is required for particle formation.

pAcGag31-18 The 669 bp EcoRV-Ps t 17 fragment of the non-myristoylated p55 gene was converted into pAC Cfrl EcoRV-Ps11±940
By exchanging with a 0 bp long fragment, C f
A glycine deletion mutant of the r I cassette (non-myristoylated p17-24) was created. This mutant did not exhibit membrane protrusions as described above, but did display p17 and p24 imsunogold decorations scattered in the cytoplasm and nucleus.

To express the gag-po+ product, we included most of the pol gene (approximately 80%) into a baculovirus transfer vector carrying the p55 expression cassette (pRIT13003). . pol
Genetic DNA fragment Bg 11 from BHIO
1 (2093)-EcoR1 (4681) restriction fragment (Sham et al.
．． ), Science 22nd: 116
5 (1 984). Poly-stopped synthetic DNA fragment 5゜ AAT TCC TAA CTA ACT AA
G 3゜3゜ GGA TTG AT TGA TT
C CTA G 5' was added to the EcoR] site. The resulting baculovirus expression plasmid, LE-8-4, was used in co-transfectin experiments to obtain recombinant plaques essentially as described in Example 1.

In this recombinant assembly, myristoylated p55 and S. It was expected that a gag-pol product derived from an HIV-specific translational frameshift in f cells would be produced and subsequently proscenosed by proteases. Recombinant baculoviruses carrying the gag-po1 gene were screened and selected substantially as described in Example 1. S. cerevisiae infected with such gag-pol recombinant virus, VAC8-5. f. In cells, gag or gag-po
No product was detected.

However, cell extracts showed p
24 and p17 monoclonal antibodies showed a strong double band at 24 kd and a band at 17 kd. Very small amounts of the precursor p55 band and the intermediate 4 1kd (46Kd) band were also detected in the Western blot.

This is because protease D. f. It is shown to be active in gagpot fusion proteins expressed by translational frameshifting in cells.

As a result, p17, p24 polybebutide and intermediate (
4 1Kd, 46-49Kd, 55Kd). The large precursor gag-pol product is our p55,
It was not detected with p17 or p24 antibodies.

Electron microscopy showed only occasional budding of some particles at the cell membrane. These particles appear to be morphologically similar to the p55 particles described above. Co-infection experiments with recombinant viruses harboring p55 and gag-pot genes revealed that a denser (cone-shaped) core (p2
4) No detectable particles showing morphological differences such as structure were obtained.

The gag gene of immunodeficiency virus (SIV) is molecularly cloned into SIV. ．． cBK28 (gifted by J. Mullins; Hirun:) (formerly rsh et al.), Ce
ll) 49: 307 (1987) and Kesler et al., Fuichi +-
(Nature) 3 3 1619 (1988)).

The 3504 bp Kpnt fragment (nucleotides 1212 to 4716) of the pBK28 genome was pL]
It was subcultured into c8. Two internal fragments of the gag gene, the 5' fragment FnuDII (12
01)-Psi (1959) and 3° fragment Ps t 1 (1959)-Hph1 (28
03) and synthesized oligonucleotide linkers, Linker 1: GAT CC ACC ATG GGCG TGGTA
C CCG and linker 3. TGCTGCACCTCAATDCTCTTTTGG
AGGAGACCAGTAGAGATCTGGTACM
CCACGTGGAGTTAAGACAGAMCC
The gag fragment was adapted using TCCTC TGG TCA TCTCTAGAC to restore the entire precursor gag gene. In another experiment, relinker 2. GATCC ACC ATG GCC G TGG TAC CGG is used in the 5° fragment to mutate the second coton, i.e. GGC (G + y) or GCc (Al
a). Different silk solids were cloned into Bluescribe vectors and confirmed by sequencing. The N-terminal aqueous fragment (BamHI-Pstl) and the carboxy-terminal fragment (Pstl-Bgln) were isolated and cloned into the BamH1-digested alkaline phosphatase-treated pAcYMI baculovirus expression vector.

The pAc gag Myy' plasmid contains a mutated (G I y to Ala) gene.

S. f. Cells were incubated with purified AC MNPV viral DNA (1 μg) substantially as described in Example 1.
) and each recombinant transfer plasmid (50 μg
) was transfected with a mixture of

Recombinant Braaks were screened and selected as described in Example 1.

Rabbit antiserum against SVI (metrizamide (met)
rizaa + ide) one gradient purified SIV-BK
28 virus) ltH IV p 2 4 F7
>COOH The SIVgag Pr57'' precursor polypeptide is efficiently synthesized in infected insect cells, as observed in Western bronotations using monoclonal polypeptides oriented toward one end, which also It appears to recognize core protein.

In a second assay experiment, cultures infected with glycine or alanine mutants in which myristoylation of the precursor p,57 mat protein was not detected when analyzed by SDS-PAGE and radioautography. In contrast, S. f, S expressed in cells
It was shown that the IV native gag precursor gene was effectively myristoylated.

As in the case of the HIV-gag precursor protein, we found that when infected cultures were analyzed by ultracentrifugation, sucrose gradients and electron microscopy (TEM and SEM), gag particle shapes and particles were also observed in conditioned media. The release of was observed. The HIV-p55 precursor gene was transferred to S. f, SIV-gag Pr57 eag particles similar to those obtained when expressed in cells were observed. Extracellular gag particles form half-moon-shaped structures at the cell membrane that assemble into typical buds, much like mature virus budding particles.

Like HIVp55 particles, SIVPr57t'''C are approximately 100-120 nm in diameter and have a tick
It showed a pale gray translucent center surrounded by a dark electron-dense ring and an external lipid bilayer. SIV non-myristoylated (Gly
or A l a) Through experiments on mutants,
The observation made with the HIV-non-myristoylated p55 mutant that N-myristoylation is essential for budding and extracellular particle formation was confirmed.

The difference between the gag precursor proteins of HIV and SIV is that when the native SIV gag protein (myristoylated) is expressed, the latter also forms intracellular particles and particulate structures. This can be explained as follows. The expression level is about three times higher than the H IV g ag expression level, probably not all sIV Pr57'' molecules are myristoylated. Also, more degradation products, especially myristoylated A p27 protein band could be detected in the WB of cultures infected with the SIVPr57"' native construct.

Cellular structures approximately 4 Q nm in diameter and sometimes up to 1 μm in length - observed at later stages of infection - are responsible for these degraded gag
There is a possibility that it is produced in at least a part of the raw wood. This can be said to be similar to the assembly of the p24 core into a tubular structure that is rarely observed in the case of retrovirus core ripening.

Furthermore, the p24 core protein of HIV-1 is expressed by E.
When expressed in E. coli, tubular structures containing the p24 protein have been observed. Some of the intracellular particles observed in natural SIV gag precursor recombinants adopt a morphology close to the cell membrane, where they appear to differentiate into virus-like particle budding as described above. This process is reminiscent of viral maturation of type D retroviruses, which contain intermediate intracellular type 8 particles.

Although preferred specific examples of the present invention have been described above, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the present invention, and these are also within the scope of the present invention. It is.

Patent Applicant: SmithKline Biologicals Société Anonymous

Claims (1)

Claims: (1) operably linked to a regulatory element that functions in a eukaryotic cell, encoding the full-length immunodeficiency virus gag precursor protein, and with naturally occurring 5' and 3'flankings; A recombinant DNA molecule characterized in that it consists of a DNA sequence lacking sequence. (2) the regulatory element functions in yeast, insect or mammalian cells, and the ga8 precursor protein functions in HIVg
The recombinant DN according to claim (1), which is an ag precursor protein.
A molecule. (3) The regulatory element is Lepidoptera (Lepidoptera).
ra) The recombinant DNA molecule according to claim 2, which functions in cells. (4) The regulatory element is polyhedrin (polyhedrin).
The recombinant DNA molecule according to claim 3, which comprises n) a gene promoter. (5) Any one of claim No. (2), (3) or (4)
A recombinant baculovirus comprising the recombinant DNA molecule described in . (6) An insect cell characterized by being infected with the recombinant baculovirus according to claim (5). (7) The insect cell according to claim (6), which is a Lepidoptera cell. (8) Spodoptera frugiperda
Claim No. (6), which is a cell of A. ra frugiperda.
Insect cells described. (9) The regulatory element is Drosophila (Drosophila).
a) The recombinant DNA molecule according to claim (2), which functions in cells. (10) A Drosophila (Drosop) characterized by being transformed with the recombinant DNA molecule according to claim (9).
hila) cells. (11) The recombinant DNA molecule according to claim (2), wherein the regulatory region functions in mammalian cells. (12) A recombinant vaccinia virus comprising the recombinant DNA molecule according to claim (11). (13) A mammalian cell comprising the recombinant DNA molecule according to claim (11). (14) A mammalian cell infected with the recombinant vaccinia virus according to claim (12). (15) CHO cells, COS-7 cells, NIH-3T3
cells, CVI cells, mouse or rat myeloma cells,
The mammalian cell according to claim 13, which is selected from the group consisting of HAK cells, vero cells, HeLa cells, WI38 cells, MRC-5 cells, or chicken lymphoma cells. (16) The recombinant DNA molecule according to claim (2), wherein the regulatory region functions in yeast. (17) The regulatory element is CUP1, TDH3, PGK, A
The recombinant DNA molecule according to claim 16, which comprises a DH, PHO5 or ARG3 promoter. (18) A recombinant yeast cell comprising the recombinant DNA molecule according to claim (16). (19) A recombinant S. cerevisiae (S. cerevisiae) comprising the recombinant DNA molecule according to claim (17).
visiae) cells. (20) Particles immunologically similar to standard immunodeficiency virus gag particles were collected from Lepidoptera (Lepidoptera).
a) a recombinant DNA molecule expressed in a cell, operably linked to a regulatory element functional in a Lepidoptera cell, for all or part of the immunodeficiency virus gag precursor protein, or for immunodeficiency virus gag precursor protein; A recombinant DNA molecule, characterized in that it consists of a DNA sequence encoding a hybrid protein having all or part of the viral gag precursor protein. (21) encodes a full-length HIV gag precursor protein that lacks other HIV functions and is mostly a full-length H1Vga precursor protein.
Claim No. (20), which expresses particles consisting of g precursor protein.
Recombinant DNA molecules described. (22) A recombinant DNA molecule operably linked to a regulatory region that functions in Lepidoptera cells and comprising a coding sequence for the immunodeficiency virus gag precursor protein. (23) The recombinant DNA molecule according to claim 22, wherein said coding sequence is for a full-length HIV gag precursor protein lacking other HIV functions. (24) The regulatory element is polyhedrin (polyhedrin).
in) claim (20) consisting of a gene promoter, (
21), (22) or (23). (25) A recombinant baculovirus comprising the recombinant DNA molecule according to claim (20) or (22). (26) A recombinant baculovirus comprising the recombinant DNA molecule according to claim (24). (27) Lepidoptera (Lepi) characterized by being infected with the recombinant baculovirus according to claim (25)
doptera) cells. (28) Spodoptera frugiperda, which is infected with the recombinant baculovirus according to claim (26).
cell. (29) A gag precursor protein produced by culturing the cell according to claim (6). (30) A ga produced by culturing the cell according to claim No. (13) or (18).
g precursor protein. (31) A gag precursor protein produced by culturing the cell according to claim (27). (32) Gag precursor protein particles, characterized in that they are isolated from a conditioned medium from the culture of the cells according to claim (6). (33) A gag precursor protein particle produced by culturing the cell according to claim (27). (34) An immunogenic particle which is immunologically similar to a standard immunodeficiency virus gag particle and is characterized by being composed of a gag precursor protein produced by a recombinant eukaryotic cell. (35) Anti-p16, anti-p24 and anti-
Mostly full-length HIV, recognized by the p17 antibody and lacking the viral functions necessary for viral maturation and replication.
The immunogenic particle according to claim 34, comprising a gag precursor protein. (36) gag produced by recombinant eukaryotic cells
A vaccine characterized by comprising a precursor protein. (37) gag produced by recombinant eukaryotic cells
A vaccine characterized by comprising precursor protein particles. (38) A sample of serum or other body fluid from an animal is brought into contact with the gag precursor protein according to any one of claims (29), (30), (31), or (32). A method for collecting data useful for diagnosing exposure of an animal to an immunodeficiency virus. (39) Diagnosis of exposure of an animal to an immunodeficiency virus, characterized by contacting a sample of serum or other body fluid from the animal with the immunogenic particles according to claim (34) or (35). How to collect useful data. (40) Claim No. (29) for use as a vaccine agent;
The gag precursor protein according to any one of (30), (31), or (32). (41) Claim No. (29) for use as a vaccine agent for providing protection in humans against infection by HIV.
, (30), (31) or (32). (42) Claim No.
Any one of 29), (30), (31) or (32)
gag precursor protein described in. (43) The immunogenic particle according to claim (34) or (35), which is used as a vaccine agent. (44) Claim No. (34) for use as a vaccine for providing protection in humans against infection by HIV.
Or the immunogenic particle according to (35). (45) Claim No.
34) or the immunogenic particle described in (35). (46) characterized by linking a DNA sequence encoding a full-length immunodeficiency virus gag precursor protein and lacking naturally occurring 5' and 3' flanking sequences to regulatory elements that function in eukaryotic cells. A method for producing a recombinant DNA molecule expressing the immunodeficiency virus gag protein. (47) the regulatory element is functional in yeast, insect, or mammalian cells, and the gag precursor protein is
The method according to claim 46, wherein the gag precursor protein is a gag precursor protein. (48) The regulatory element is Lepidoptera (Lepidoptera).
Claim No. (47), which functions in tera) cells.
Manufacturing method described. (49) The regulatory element is polyhedrin (polyhedrin).
The method according to claim (48), comprising: in) a gene promoter. (50) from a DNA sequence operably linked to a regulatory element that functions in a eukaryotic cell, encoding a full-length immunodeficiency virus gag precursor protein, and lacking naturally occurring 5' and 3' flanking sequences. recombinant DNA
culturing recombinant eukaryotic cells transformed with the molecule; isolating the gag precursor protein particles produced thereby; and then combining the isolated gag precursor protein particles with a pharmaceutically acceptable carrier. A method for producing a vaccine for protecting humans against diseases caused by infection with HIV, characterized by: