JP3001610B2 - Method for producing retrovirus core structure and particles produced thereby - Google Patents

Description

The present invention relates to a cleaved form of a virus belonging to the family Retroviridae.
The present invention relates to a method for producing particles comprising a gag protein, particles produced by the production method, and vaccines containing the particles as an active ingredient.More specifically, at least the gag gene and pol not containing the env gene of a virus belonging to the family Retroviridae. Env of a virus that has been infected with a viral vector incorporating the gene or belongs to the retroviridae family
A virus vector containing at least the gag gene without the gene and a virus belonging to the retroviridae family
Cultivating and expressing animal culture cells infected with a virus vector that does not contain the env gene but has at least the pol gene incorporated therein, and produces particles composed of the cleaved gag protein obtained by cleaving the gag protein precursor The invention relates to a method, particles produced in such a way, and a vaccine comprising the particles as an active ingredient.

(Prior Art) Recently, the onset and spread of acquired immunodeficiency syndrome (AIDS) has become a worldwide concern, and prevention and treatment measures have become an urgent issue.

Several years ago, various retroviruses considered to be causative viruses such as AIDS and adult T-cell leukemia were isolated and identified, and their nucleotide sequences were determined. as a result,
They began to be reported as similar or identical. In particular, HILV-III and LA, which are pathogenic viruses of AIDS
V and ARV are the same virus, and HIV (human immunodefic
iency virus).

Retroviral particles are composed of many proteins, and are known as the envelope glycoprotein (env protein, HIV
In the case of -I, the precursor env-gp160 is cleaved to gp12
0 and gp41), reverse transcriptase (in the case of HIV-I, there are p65 and p51), nuclear protein (called gag protein, and in the case of HIV-I, it is a precursor. gag-p55 is cleaved to produce three gags, p17, p24, and p15
Become protein. In the amino acid sequence of gag-p55, an amino acid sequence equivalent to p17, an amino acid sequence equivalent to p24,
The amino acid sequence corresponding to p15 is continuous in this order). Regulatory gene products such as tat, vif, rev, and nef, which are said to be unique to HIV, are known, but the vif product does not exist in HIV particles.

gp120, which is an env protein, has a function of immobilizing virus particles on cell membranes during infection, and gp41 has a function of allowing viral genes to enter cells. The gag protein, p24, is thought to be the main component of the core protein, and p17 is the matrix protein. Reverse transcriptase is encoded by the pol gene. The pol gene also encodes p10 (protease protein) and p31 (endonuclease, also referred to as having integrase activity), and a group of viral enzymes encoded by these pol is called pol protein. vif encodes p23, which is thought to be involved in virion assembly.

In order to obtain an effective AIDS vaccine, it is necessary to include an appropriate antigen to induce antibodies capable of neutralizing the virus. Env as such antigen
Proteins, particularly gp120 implicated in infectivity
(Cell, 53 , 483, (1988)) have been selected as the most promising candidates, and research has been carried out to produce a gene encoding this antigen protein by a genetic engineering technique for recombination into Escherichia coli (for example, Science, 228 , 93-96,
(1985)) and so on.

However, the analysis of the genome sequences of HIV isolated from various patients and infected persons revealed that the HIV env gene had extremely severe mutations (Scientific American, 253 , 84- 93,
(1985)), the technique using this site may not be able to respond to all HIV.

On the other hand, the gene encoding the gag protein or the pol gene encoding the reverse transcriptase that forms the framework of the virus particles may have a low probability of mutation,
Vaccines that use these gene products as antigens are considered to be advantageous, and studies that produce them by genetic engineering to convert reverse transcriptase into E. coli (for example, Science (Scienc)
e), 236 , 305-308, (1987)), studies on the production of gag-p55, p24, and the like in a recombinant polynuclear virus system (for example, Virology, 158 , 248-250, (198)
7)), Studies on the synthesis of a polypeptide that binds to an antibody that specifically binds to the antigenic determinant site of p17 (JP-A-63-27498)
No.), report that p17 monoclonal antibody has AIDS neutralizing activity (Journal of Virology (J.Vi.
rol.), 63 , 267-272, (1989)). In December 1987, the inventors also presented a vaccinia virus incorporating the gag gene and the pol gene at an academic meeting of the AIDS Research Association.

However, the expression of non-cleavable gag proteins such as gag-p55 was confirmed by recombination of the entire gag gene of the retrovirus.In that case, regardless of the presence or absence of recombination of the pol gene, expression of the cleaved gag protein was performed. Was not confirmed.

In addition, HIV-free non-infectious retroviral particles that do not contain gp160 and gp120 have been known (WO88 / 09670).
However, it was obtained by treating a retrovirus, and it was not known that a retrovirus core structure containing no env protein without producing a retrovirus using genetic recombination could be produced.

(Problems to be Solved by the Invention) As a result of intensive studies, the present inventors have expressed gag protein and pol protein without expressing retrovirus env protein in cells infected with a viral vector. By doing so, a cleaved gag protein is expressed and constituted by the cleaved gag protein.
The present inventors have found that non-infectious retrovirus core structure particles having the same antigenicity as a part of the structure of the retrovirus particle can be obtained, and have completed the present invention.

(Means for Solving the Problems) In the present invention, the gene to be incorporated into the virus vector is derived from a virus belonging to the genus Retrovirus. The virus belonging to the genus Retrovirus is not particularly limited, and specific examples include a virus belonging to the subfamily of Oncoviridae, a virus belonging to the subfamily of Lentivirus, and more specifically, HT belonging to the subfamily of Oncoviridae.
HI belonging to the subfamily Lentivirus, such as LV-I and HTLV-II
VI, HIV-II and the like.

In the present invention, the term "env protein" refers to an env protein that controls retrovirus infection and pathogenicity and a mutant or modified product thereof, and does not mean an env protein that has no influence on infection or a modified env protein that has lost pathogenicity. In the present invention, env
The absence of the gene means that the gene encoding the env protein is not contained in a form capable of expressing the gene, and a gene derived from the env gene not related to retrovirus infection or pathogenicity (part of the env gene). Or a modification of the env gene). Gp160 disclosed in WO88 / 09670
And since retrovirus particles that do not contain gp120 do not have gp120, it is unlikely that the genes contained therein will be taken into cells.However, if the genes contained in the particles are taken into cells, all the retrovirus genes will be deleted. However, since the particles of the present invention do not contain the env gene, there is no such risk. However, in the present invention, en
When a modified product of the v gene is contained, it is preferable that the modified product of the env gene is not also contained, since the pathogenicity may be restored by mutation or the like.

In the present invention, the retroviral cDNA to be incorporated into the viral vector may be any one that can express the gag protein and / or the pol protein and does not contain the env gene in cultured animal cells infected with the viral vector. Not limited. For example, gag of HIV-I
Encodes the gene and pol gene and does not contain the env gene
cDNA is pNL43 (Journal of virology (JV
irol.), 59 , 2, 284, (1986)) and HIV integrated into the chromosome of AIDS patients in the form of a provirus.
-I is once cloned using a λ phage or the like and used to construct a plasmid containing the entire cDNA of HIV-I, or the RNA genome of HIV-I isolated from a patient is reverse-transcribed and converted to cDNA. And then transfer to the plasmid
It can be obtained by constructing a plasmid containing the entire cDNA of HIV-I, cutting the obtained plasmid with an appropriate restriction enzyme, and preparing a DNA fragment containing both coding regions of the gag protein and the pol gene.

The virus vector used in the present invention is not particularly limited as long as it can infect animal cells and express the gag gene and the pol gene. For example, Vaccinia virus, Avipox virus, Baculo virus and the like can be mentioned.

The method for constructing a virus vector incorporating the gag gene and / or pol gene used in the present invention is not particularly limited. For example, when a vaccinia virus is used as the virus vector, the gene to be integrated is changed from the gene encoding the surface antigen protein of Japanese encephalitis virus to the gag gene and / or pol gene of the retrovirus, and the vaccinia virus used is always preferred. Except that it is not necessary to satisfy the conditions,
Can be constructed by the method described in the above item.

Next, a virus vector incorporating the constructed cDNA fragment encoding the gag gene and the pol gene, or a virus vector incorporating the cDNA fragment encoding the gag gene and the cDNA fragment encoding the pol gene are incorporated. The virus vector thus infected is used to infect cultured animal cells, and the cleaved gag protein and pol protein are expressed in the culture supernatant.

The cultured animal cell used in the present invention may be any one in which a virus vector can be propagated, and as a specific example, when the virus vector is a vaccinia virus,
For example, TK ^- 143 (derived from human osteosarcoma), FL (derived from human amniotic membrane), Hela (derived from human cervical cancer), KB (derived from human nasopharyngeal carcinoma), RK13 (derived from rabbit kidney), CV-1 (derived from monkey kidney) ), BSC-1 (derived from monkey kidney), L929 (derived from mouse connective tissue), CE (chick embryo) cells, DEF (chick fetal fibroblast), SW480 (derived from human colon cancer), and the like. When the virus vector is an avipox virus, the animal cultured cells used in the present invention include chicken-derived cells such as fibroblasts for chicken embryos, and also include embryonated chicken egg chorioallantoic membrane.

However, under general culture conditions, the expression level of the cleaved gag protein is low depending on the cells used.
When Vaccinia virus in which the HIV-1 gene was incorporated into V-1 was infected, almost no expression of the cleavage type gag protein was observed. A combination of a virus vector having a high expression level of the cleavage type gag protein and cultured animal cells is preferable, and such an example is a case where a vaccinia virus incorporating the HIV-I gene is infected into SW480.

The cleavage of the gag protein expressed here is considered to be due to pol protein having protease activity such as p10.

Purification of the cleaved gag protein from the culture supernatant is not particularly limited. For example, common law (Science, 2
24 , 497-500, (1984)), the supernatant may be subjected to sucrose or potassium tartrate density gradient centrifugation, followed by cesium chloride sedimentation equilibrium centrifugation. Cleavable gag
The protein forms particles and is recovered.

Particles composed of the cleaved gag protein of the present invention, for example, when the retrovirus is HIV-I and is fractionated by specific gravity, a specific gravity of about 1.15, which is the same as that of HIV-I particles, is observed.

The method of confirming in which fraction the particles are present in each stage of the purification is not particularly limited. For example, ga
There are methods that use antibodies against the g protein and methods that measure reverse transcriptase activity. Particles composed of the cleaved gag protein of the present invention may or may not contain reverse transcriptase, but usually at least partly contain reverse transcriptase. Therefore, the fraction containing the particles of the present invention can be determined by measuring the reverse transcriptase activity.

The measurement of the reverse transcriptase activity may be performed according to a conventional method. For example,
The recombinant virus-infected cell supernatant was poly-A according to the description in the Practical Microbiology Training Program (ed.
Is used as a template and oligo-dT as a primer to measure the incorporation of ³² P-dTTP into the acid-insoluble fraction.

The particles of the present invention are associated with an envelope-like substance which is considered to be a lipid bilayer derived from the biological membrane of a cultured animal cell infected with a viral vector. These particles can function as antigens while having a lipid bilayer, but when it is not preferable to have a lipid bilayer, treatment with an organic solvent such as ether under conditions that do not inactivate the protein, or freezing It can be crushed and removed by melting.

The particles do not contain the env protein and its genes that are responsible for infecting cells, and therefore do not show pathogenicity. Other genes may or may not be included. Similarly, as long as the structure is formed by the cleavage type gag protein, it may or may not contain proteins other than the env protein. Also, the presence or absence of an envelope-like lipid bilayer is not important. The particles have antigenicity, and the antigenicity is derived from the gag protein, which is considered to have less mutation than the env protein, or from the pol protein and the gag protein when included. Furthermore, since it has the same or similar structure as a part of the virus particles in the natural world, it can be expected that the antigenicity is higher than that of the gag protein, the pol protein or a mere mixture thereof.

Retroviruses have been identified as pathogenic viruses for many intractable diseases such as AIDS, adult T-cell leukemia, cancer, and rheumatism.
Although it has been estimated that vaccines are not effective for many diseases caused by retroviruses, particles having the same or similar structure to the core of the retrovirus of the present invention are excellent. The effect can be expected.

(Effect of the Invention) Thus, according to the present invention, gag derived from retrovirus
Culturing cells infected with a viral vector incorporating the cDNA encoding the protein and the cDNA encoding the pol protein into a genomic region that is non-essential for the growth of the virus, comprising the cleaved gag protein, env protein and env A gene-free retroviral core structure is obtained. This structure has the same structure as that of a part of the structure of the retrovirus particle, but has no infectivity. Therefore, it can be expected that an effective retrovirus vaccine can be obtained by using this structure as an active ingredient. .

(Example) Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 (1) Preparation of recombinant vector pUHK (see FIG. 1) pUC9 (manufactured by Pharmacia) was digested with EcoR I and Pst I, treated with polymerase, and the cut end was blunt-ended.
Ligation was performed to create pUC9 lacking the EcoRI site. EcoR I
Vaccinia virus after cutting site-defective pUC9 with HindIII
It was ligated to the HindIII J fragment of the vaccinia virus WR strain containing the TK gene to obtain a recombinant vector pUHK.

(2) Construction of recombinant vector pNZ68K2 (see FIG. 2) The 7.5K promoter of vaccinia virus WR strain (Moss et.al, Cell, 125 , 805-813, (198
1)) was inserted into the Hinc II site of pUC19 (manufactured by Pharmacia), and subsequently cut with Hind III and EcoRI in order to obtain a DNA fragment (about 350 bp) having a polylinker site before and after the 7.5K promoter. .

The DNA fragment treated with S1 nuclease and the plasmid pUHK obtained in (1) digested with EcoRI digested with polymerase were ligated, and the resulting recombinant plasmid was named pNZ68K2.

(3) Preparation of recombinant plasmid W-gag (see FIG. 3) After digesting pNL43 containing the entire cDNA of HIV-I with BglII and EcoRI, a fragment of about 5.1 kbp was separated by low melting point agarose electrophoresis. The fragment was recovered by phenol extraction.

In this fragment, as shown in FIG. 3, the gag gene,
And the pol gene region.

Next, the pNZ68K2 obtained in (2) was digested with BamHI and EcoRI, and the DNA containing the gag gene and the pol gene prepared above was digested.
Ligation with the fragment and recombinant plasmid W-gag
I got

(4) Production of recombinant vaccinia virus (see Fig. 3) 0.1 pfu / cell of vaccinia virus WR strain was inoculated into CV-1 cells cultured in a 25 cm ² culture bottle, and 45 minutes later, the recombinant plasmid W- Dissolve gag in 2.2 ml of sterilized water, and use Hitaka et al. (Protein, nucleic acid, enzyme, 27 , 340, (198
A DNA-calcium phosphate coprecipitate was prepared by the method 5)), and 0.5 ml of the coprecipitate was dropped on the infected CV-1 cells. 30
For 5 minutes at 37 ° C in a 5% CO ₂ incubator for 5%
4.5 ml of Eagle MEM medium containing fetal calf serum was added. Part 3
After a lapse of time, the culture medium was exchanged, and after 48 hours, the cultured cells were freeze-thawed three times together and sonicated for 30 seconds.

To confirm the揮入of the gag gene, and pol genes of the recombinant, TK-negative cultured in a Petri dish of 6cm (TK ^-) 14
3 cells were inoculated with the plaque forming virus, and after 45 minutes,
1% agar, 12% fetal calf serum, 25μg / ml BUdtR added Eagle ME
After the M medium was layered and cultured for 3 days, the infected cells were stained with 0.01% neutral red. The plaques formed are isolated with a Pasteur pipette and diluted in Eagle MEM medium. The same operation was repeated once again for the isolated plaque to purify the plaque. The virus obtained from the purified plaque was propagated, DNA was prepared from the proliferating virus, and the resulting recombinant vaccinia virus was subjected to HIV-dot blot hybridization using a cDNA encoding the gag gene and the pol gene as a probe. It was confirmed that the virus was a recombinant virus having in its genome a DNA encoding the gag gene and pol gene derived from I.
20-1-8.

(6) Expression of recombinants in cell supernatants SW480 cells cultured in a 9 cm dish for tissue culture
i. Inoculated with 10-20 V-20-1-8, infected at 37 ° C. for 1 hour, withdrawn from the virus solution, washed with Egale MEM,
Eagle MEM supplemented with 5% fetal calf serum was added. 16 hours later, the cell supernatant was collected and 10 mM Tris-hydrochloric acid (pH 7.4) -0.1 M NaCl
-1 Overlaid on a density gradient of 20 to 60% potassium tartrate prepared with 1 mM EDTA solution, and rotor SW47 (manufactured by Beckman)
Centrifugation was performed at 35,000 rpm for 16 hours. After centrifugation, each fraction was divided.

50 mM Tris-HCl (pH 8.0), 10 mM dithiothreitol, 75 mM KCl, 5 mM MgCl ₂ , 10 μg / ml poly-A, 5 μg /
ml oligo-dT, 5% reaction mixture containing 0.05% NP40 [α-
³² P] -dTTP (400 Ci / mmol, 10 mCi / ml) was added in an amount of 5 to 10 µ. This reaction solution is dispensed into a 96-well U-shaped plate in a number symmetric to the fraction, and each fraction or 10 mM Tris-hydrochloric acid (pH 7.4) -0.1 M NaCl
10 ml of a 1 mM DTA solution was added, and the mixture was reacted at 37 ° C. for 1 hour. During the reaction, a grid of 2 cm was drawn on the nitrocellulose filter with a pencil at intervals of 2 cm. After the reaction was completed, 10 μ of each fraction and the reaction solution of the control was spotted on each section on the nitrocellulose filter. After the filter was completely dried by air drying, it was washed in a 5% trichloroacetic acid-5% sodium pyrophosphate solution with shaking for 15 minutes to remove unreacted radioactive precursor. This was followed by a 5 minute wash in 5% trichloroacetic acid and a 5 minute wash in 50% ethanol.
After drying, each compartment was cut open along the base of the substrate, placed in a scintillation vial, and the uptake was measured with a liquid scintillation counter in a toluene scintillator. The incorporation of 3 times or more of the control was regarded as positive, and it was judged that there was reverse transcriptase activity.

Next, the fraction in which reverse transcriptase activity was observed was collected.
Fractionated by SDS-polyacrylamide gel electrophoresis and blotted on a nitrocellulose filter (Analytical Biochem., Anal. Biochem. 11
2 , 195-203, 1981)). As a detection method, ¹²⁵ I-labeled protein A, anti-p24 monoclonal antibody (Japanese Journa
l of Cancer Research, 78 , 235-241, (1987)) by the so-called Western blotting method.
Expression of the cleaved gag protein was confirmed, and V-20-1-
It was found that 8 infected SW480 cells produced a cleaved gag protein p24 having a molecular weight of 24,000 daltons in the supernatant. Considering the structure of gag-p55, since p24 corresponding to the amino acid sequence of the central part of gag-p55 has been produced, p17 and p15 corresponding to the amino acid sequences at both ends of gag-p55 are produced.
Is also considered to be produced.

The fraction with a specific gravity of about 1.15 where reverse transcriptase activity was detected was further purified by equilibrium centrifugation with cesium chloride sedimentation, and p24 was confirmed in the fraction with a specific gravity of about 1.15 where reverse transcriptase activity was detected.

When the above-mentioned fraction in which p24 was confirmed was observed by an electron microscope by a so-called negative staining method, spherical particles having a diameter of about 110 to 150 nm different from those of vaccinia virus were observed.

[Brief description of the drawings]

FIG. 1 shows plasmid pUHK, and FIG. 2 shows plasmid pNZ68K.
2 and 3 show the procedure for constructing the plasmid W-gag.

Continuation of front page (56) References JP-A-2-124951 (JP, A) Journal of Virology, Vol. 63, No. 3, p. 1451 -1454 (MAR1989)

Claims (3)

(57) [Claims] 1. A vaccinia bacterium which does not contain at least the env gene of a virus belonging to the retrovirus but incorporates the gag gene and pol gene of the virus belonging to the retrovirus.
A virus-infected animal cell is cultured, and isolated from the culture supernatant. (1) Specific gravity of 1.15; (2) env protein-free; (3) Retro with reverse transcriptase activity A method for producing virus core structure particles. 2. The virus belonging to the retrovirus is HIV-
The method for producing retrovirus core structure particles according to claim 1, which is I, HIV-II, HTLV-I, or HTLV-II. 3. The retrovirus according to claim 1 or 2,
Retrovirus core structure particles produced by the method for producing core structure particles.