JPH0314840B2 - - Google Patents

Description

[Detailed description of the invention]

This invention is unique to viruses of various types of hepatitis, such as certain types of hepatitis seen in patients after blood transfusions, called "non-A" or "non-B", and viral hepatitis B. It relates to preparations with specific immunological properties, in particular antigenic properties (antigenicity). More specifically, the present invention provides highly purified preparations free of human-derived proteins and
Preparations with properties similar to the antigenic properties of HBsAg refer to preparations that do not contain Dein particles. It is known that the specific antigenicity of hepatitis viruses is essentially due to the ``HBs antigen'' or ``HBsAg'' formed by the viral envelope or Dane particles of hepatitis B virus (HBV). ing.
This antigen, which has vaccine activity against viral hepatitis B, is currently obtained from human serum specimens, but other sources of HBsAg are limited due to the special properties of hepatitis B virus (HBV). do not have.
Therefore, the only option seems to be to infect humans, chimpanzees, and perhaps a small number of other primates. Conventionally, it has been impossible to propagate this substance in vitro by cell culture. It is certainly known that malignant liver cancer produces HBsAg. However, there will be strong opposition to using cancerous cells in humans to produce hepatitis vaccines. However, there is no serious risk in using human-derived HBs antigen prophylactically. fact,
HBsAg, which comes from humans with viral hepatitis B, is present in some cases with highly infectious dein particles, and no matter how purified it is, serum-derived HBsAg preparations cannot be used. cannot be removed from Traditionally, highly purified preparations of serum proteins or other antigenic components can cause troublesome immune reactions in the person to whom they are administered, but their content cannot be ignored. That is,
This is because the HBsAg content is originally extremely low compared to proteins in serum (for example, HBsAg is about 50 μg for 80 mg of protein in 1 ml of serum). Although it is difficult to obtain sufficient amounts of virus, it is still difficult to obtain virus in sufficient quantities, but the genome of the hepatitis B virus is made up of partially single-stranded circular DNA, the longest strand of which is approximately 3200 nucleotides. has been revealed (SummersJ.
et al., Proc. Nat. Sci. USA 72 , 4597−4601). Previously, it was only possible to locate the gene encoding the protein portion of the HBsAg antigen, which is involved in the immunological properties of the hepatitis B virus envelope. This gene part is “S
This is particularly seen in the diagrammatic map of the Dane particle genome shown in FIG. 1 below. Figure 1 is a diagrammatic map of the DNA of the Dain particle genome. This DNA contains two strands b ₁ and b ₂ , of which the shorter b ₂
The dashed line portion of the figure is usually missing. this DNA
is known to have only one EcoR site. Arrow f ₁ indicates the direction of the number of nucleotides that make up the longest strand b ₁ , and arrow f ₂ indicates the direction of the number of nucleotides that make up the longest strand b 1.
The direction of transcription of the S gene by the cellular mechanism of cells invaded by hepatitis virus is shown. The EcoR site is therefore numbered 0, or 3182 in the case of hepatitis B virus (Nature, 1979,
vol.281, p646-650). The inner concentric circle e ₁ shows the nucleotide number scale. This circle allows us to identify a certain location of this DNA. 3', 5' and 5', 3' at the bottom of the figure indicate ends having the same number according to the conventional notation of nucleic acid chain ends. At best, the gene encoding the protein portion of the HBsAg antigen has been identified. This gene portion, referred to as the "S gene", is schematically indicated by the arrow S in the figure. The S gene is carried essentially by a fragment of the longest strand b ₁ located between nucleotides 155 and 833 in the direction of transcription of the S gene in FIG. In the genome of Dein particles, the shortest strand _b2 is, for example, T.
A. Landers et al. (J.Viol., 23, 1977, p368-376)
can be "repaired" in vitro by the presence of precursor nucleotides and a polymerase. The hepatitis virus genome repaired in this way (or
Any that can encode the same amino acid sequence
DNA) is hereinafter referred to as DNA HBV for brevity. Therefore, the first object of the present invention is to obtain the entire genomes of viruses involved in various viral hepatitis, especially
The object of the present invention is to provide the above method which can be applied to eukaryotic cell research or expression of all or part of DNA HBV. Another object of the invention is to provide the production of modified vectors capable of carrying out this method. Yet another object of the present invention is to produce reproducible and stable antigens from a system other than human serum, with high purity and immunologically the same specificity as the aforementioned HBsAg or similar antigens (or with constant have some special properties that can be monitored)
The objective is to provide the production of antigen-containing preparations. According to the invention, a method is provided with which it is possible to determine the possibility of converting a particular cell culture with the normally circular or whole genome of the corresponding virus. This method comprises, on the one hand, at least a portion of the viral DNA encoding an immunogenic protein capable of producing in vivo active antibodies against the whole virus in a host which can be infected with the corresponding virus; It involves a procedure consisting in transcribing the cell culture line by means of a vector, especially a plasmid, containing an insertion sequence, usually a viral promoter, under whose control the transcription and translation of the DNA part takes place. According to a first aspect of the invention, the above-mentioned insertion sequence comprises two DNAs oriented in the same direction of transcription.
It is formed by at least two of these DNAs, such as HBV, with the tail of one attached to the head of the other. In the following description, the term "tandem sequence" is often used expressively, which means, unless otherwise defined, an insertion sequence comprising two DNAs by this method of joining. According to a preferred embodiment of the invention, the above-mentioned insertion sequence comprises at least these two DNAs HBV or
DNA is formed from fragments of HBV,
On each side of the EcoR site oriented in the same direction of transcription, one DNA HBV fragment
Contains the nucleotide sequence of the S gene suitable for encoding a polypeptide involved in the immunological properties of HBs antigen, and has a length such that the other fragment contains the promoter of the S gene . According to a first preferred embodiment of the invention, the oriented DNA of the insertion sequence is constructed by fragments of DNA HBV, the tail of one of these fragments binding to the head of the other fragment at the level of the EcoR site. ing. It will be obvious that non-essential parts of the latter inserted sequence may be deleted. Therefore, such an insertion sequence preferably comprises, on the one hand, a DNA HBV fragment encoding the S gene of the polypeptide responsible for the immunological properties of HBsAg, and, on the other hand, sufficient to contain the promoter of the S gene. DNA HBV fragments of variable length upstream of the former fragment in the direction of transcription, and these two fragments are usually located on either side of the DNA HBV EcoR site. In other words, such an insert array is
In FIG. 1, on the one hand, when the sub-fragment contains the S gene as a whole, it contains the sub-fragment contained between nucleotide positions 0 and 833, and on the other hand, when the sub-fragment contains the S gene as a whole, EcoR cleavage site corresponding to ) and a position at a sufficient distance from this position 3182 (i.e. position 0) (in the opposite direction of transcription), and the above-mentioned promoter is It is now included in the sub-fragments of The latter sub-fragment is usually S
Located upstream of the start of a gene, approximately 235 nucleotides away
Must contain the TATATAA sequence.
More specifically, this insertion sequence is itself at or near nucleotide position 840, and Bgl
Additional DNA located upstream of the site
Let's include the HBV part. According to an additional aspect of the invention, the insertion sequence further comprises a sub-fragment downstream of the S gene in the direction of transcription, which sub-fragment is normally transcribed beyond the DNA HBV Bam H site. It is elongating in the direction of. A preferred insert sequence includes the entire gene encoding the messenger RNA corresponding to the HBs antigen. Referring to FIG. 1, this RNA messenger is indicated by the arrow ARN, its beginning substantially coincident with nucleotide 2800, and it ends between the BamH and Bgl1986 sites. That is, the three sub-fragments mentioned above are the first sub-fragment, that is, the sub-fragment extending from the site upstream of the promoter region to position 3182 [e.g.
No. 2840 (Bal cleavage site) and No. 3182 (i.e. 0
second sub-fragment, i.e. from number 0 to
a fragment containing the S gene sequence up to position 833; and a third sub-fragment, i.e., in the direction indicated by arrow f ₁ , further comprising the terminal nucleotide position of the DNA sequence to be transcribed into RNA from position 833 ( For example, the nucleotide positions shown in Figure 1
This fragment extends to the site corresponding to number 1621). Preferred insertion sequences applicable to the present invention are therefore characterized by having both the S gene and the gene encoding the messenger RNA corresponding to the HBs antigen. As will be clear from the description hereinafter, the present sequence preferably does not have any gene portion encoding an HBc antigen sufficient to be expressed in a protein with immunological properties as an HBc antigen. . Although hereinafter specifically reference is made to DNA HBV, the methods described herein can also be applied to the DNA or corresponding whole genomes of other viruses that cause other types of hepatitis. In order to facilitate the detection of the transformed cells, the present invention preferably simultaneously labels them, e.g. when placed in a special medium, their growth is normal, either naturally or induced for selection purposes. Cells lacking the necessary selectable labeling gene or mutants thereof are used. This cell is able to "compensate" for the deficiency in it (heterogeneous complementary DNA
However, by introducing a fragment of the gene or homologous DNA, it is possible to grow at least in the same medium. This method is carried out by any of the following methods of transforming cells. That is, (1) a single cell that has inserted into its own genome, on the one hand, an insertion sequence specifically derived from DNA HBV, and, on the other hand, complementary DNA (e.g. a mark or marker of selectivity); or (2) two vectors (simultaneous or co-transformation), one vector preferably being a plasmid and containing complementary DNA (e.g. a marker for selectivity). or marker) into its genome, and the other vector is preferably a plasmid;
The above-mentioned insertion sequence is inserted into the genome and modified in advance. After culturing in the above medium, colonies transformed with this inserted sequence are collected. As long as the DNA HBV can be expressed by the cells in the culture, tandem sequences (or those having more than one DNA HBV unit) or insertion sequences as generally described above can be used without the presence of a vector. It must behave normally like the corresponding circular DNA introduced into cells. The use of such vectors, especially plasmids, instead of the circular DNA itself has the advantage that large quantities of this vector can be obtained after amplification by cloning bacteria of the preconstructed vector in vitro. brought about. According to the invention, therefore, circular DNA or whole viruses, genomes,
In particular, DNA of hepatitis viruses other than HBV can be tested without regard to the exact location of the promoter under whose control transcription takes place. The above insertion sequences, especially those derived from DNA HBV, can be used to induce expression of the insertion sequences in eukaryotic cell cultures, particularly those of mouse or human origin. Clear vectors for labeling transformed cells,
Especially when using plasmids containing markers,
It is preferred to use a plasmid containing "complementary DNA" such as the thymidine kinase gene of the Herpes simplex HSV-1 virus which is excised from the viral genome by specific enzymatic restriction such as BamH. The thymidine kinase gene from this virus corresponds to a homologous gene (phosphorylating enzyme of exogenous thymidine supplied from the culture medium) that has been adapted to direct the synthesis of thymidine kinase in a variety of eukaryotic cells. The possibility of overcoming genetic defects in certain eukaryotic cells (particularly in mice) has already been published by M. Wigler et al. for the endogenous thymidine kinase gene by induced or stimulated mutations (Cell et al. vol., 223-232, 1977), the defective cells, called TK-, do not contain thymidine in a medium known as ``HAT medium (containing hypolyxanthine and aminopterin in addition to thymidine). -Selected because it does not have the ability to synthesize kinases. It is known that when using this medium, thymidine phosphate can be synthesized only through a metabolic route called the "rescue circuit," and this circuit is responsible for the TK of cells that can grow within it. This suggests that genetic integrity was preserved. However, as soon as TK ^- cells are modified by incorporating the herpesvirus TK gene, they can grow in the same medium and this organism can be genetically transmitted to progeny by subsequent cell divisions. is possible. What was previously "TK ^- " is repaired and becomes "TK ⁺ " in the cells after uptake.
The plant acquires the ability to phosphorylate thymidine in the above medium, and growth is observed. Needless to say, the DNA fragment containing the thymidine kinase gene can be combined with other suitable complementary DNA.
It is possible to replace it with . Other examples of complementary genes that can be used in the construction of vectors according to the invention include those containing dihydroforate reductase (DHFR), and more generally any of the numerous examples of naturally occurring complementary genes. be able to. Complementary DNA can, of course, include natural genes.
It can also be synthesized, in particular enzymatically, by copying the corresponding ARN messenger. As for the co-transformation technique itself, it is advantageous to use vectors into which complementary genes have been inserted and inducible DNA HBV fragments in a high ratio of more than 100, for example on the order of 1000. The higher the ratio, the greater the number of cells co-transformed simultaneously by the two vectors. It is advantageous to rely on the same basic vector to form the two modified vectors used for co-transformation. A particularly preferred basic vector is constructed with plasmid pBR322. To form one of these modified cotransformation vectors (pCP10), it would be possible to insert a "DNA HBV tandem" into its EcoR site. Pvu site or the same pBR322 plasmid.
A second co-transformation vector (pAGO) can be obtained by inserting the tkHSV gene into the BamH site. Regarding culture conditions, a well-cultured medium should be replaced at the time of conversion or some time later with a medium in which non-complementary cells cannot grow (e.g., a selected complementary
If the DNA is the thymidine kinase gene
It is obvious that we must change to HAT medium). Once the co-transformation has taken place and in particular the above-mentioned preferred modified plasmid becomes active in mouse fibroblast cells, the co-transformed cells produce particles in the culture medium with immunological properties characteristic of the hepatitis B virus envelope. Significant results constituted by the excretion of HBsAg, especially natural HBsAg isolated from human serum.
Bringing about its agglutination by antibodies capable of agglutinating the antigen. The presence of excretory particles that can be agglutinated by anti-HBgAg antibodies may be caused by, for example, fluorescent anti-HBsAg and anti-
It can be detected by conventional radioimmunoassay methods such as indirect immunofluorescence using IgG rabbit serum. The amount produced is then measured by a direct passive hemagglutination test method used for natural HBsAg and known per se. What is particularly striking is that cells excreted during mouse cell culture contain human antibodies anti-HBc and anti-HBc.
It contains no traces of HBcAg and HBeAg antigen detectable by direct immunofluorescence using HBe/1,2,3. By contacting the cotransformed cells themselves with anti-HBsAg serum, indirect immunofluorescence reveals that individualized cytoplasmic particles predominate in the cotransformed cells. After cell lysis,
These particles can be spotted. In many cases,
Excretory particles account for the majority of particles that can be agglutinated by anti-HBsAg antibodies that can be produced. The particles formed, whether excreted into the culture medium or retained in the cytoplasm of co-transformed cells, still have the properties described in the Examples below. However, when human HeLa cells were transformed with circularly cloned DNA of hepatitis B virus, it was found that not only HBs but also cells with complete virus particles containing HBc antigen were excreted into the culture medium. (Reference 9 below). According to an additional improvement of the present invention, in the insertion sequence as described above derived from DNA HBV, a certain portion other than the S gene of DNA HBV is excised in advance, thereby encoding the entire dein particle. It is advantageous to use only valid genomes for which it is no longer possible. In particular, insertion sequences lacking sufficient gene portions to encode HBc antigen can be used, thereby preventing production of HBc antigen by infected cells. These insertion sequences can therefore be used to modify vectors, which can be used to transform eukaryotic cells, whether mouse fibroblasts or human cells.
Suitable to perform without the possibility of HBc antigen production. In particular, this DNA insertion part (insert)
At nucleotide positions 1986 and 2425, Bgl
B lacking the fragment linked at the end of the site
It is advantageous to construct with hepatitis virus DNA. Referring to the diagram in FIG. 1, arrow C indicates inside the C gene its relative position with respect to the total DNA. In particular, it is also possible to use the original DNA HBV insert sequence with deletion of larger fragments, as described above. Of course, this inserted DNA may further contain additional deletions as long as they do not affect the expression ability or translation phase of the inserted sequence. Similarly,
In the present invention, viruses belonging to subtypes other than those contemplated in the specification and examples
It also extends to the use of equivalent inserted sequences with respect to the DNA-equivalent sequence, or modified sequences in certain parts without altering the ability of the expression product to immunoreact with antibodies to the HBs antigen. Thus, cell cultures of mouse fibroblasts transformed with vectors modified with insertion sequences such as those described above can be obtained without human-derived components, especially without serum proteins.
It is possible to obtain antigen preparations with the immunological properties of HBsAg antigens free of Dein particles and the characteristic antigens contained therein within the limits of detection. Furthermore, it has been established that proteins excreted into the culture medium have in fact vaccine immunological activity, which is currently being used to demonstrate the immunogenicity of native HBsAg extracted from human serum. Experimentally, when this substance was administered in vivo to mice or rabbits, hepatitis virus,
A human anti-inflammatory drug that is particularly active against hepatitis B virus.
Demonstrated by its ability to induce in vivo production of antibodies similar to HBsAg antibodies. The present invention has a size in the range of about 18-25 nm,
DNA polymerase free, Dane particles, HBe
Contains no antigen, HBc antigen, and is found in human serum.
It is aggregated by antibodies that cause an agglutination reaction with HBs antigen, but is not detected by anti-HBc antibodies and anti-HBe antibodies, and is immunogenic and has properties that can be used as a vaccine against hepatitis B virus. Type B, which does not cause infectious hepatitis and is long enough to encompass the S gene, a promoter region containing the TATATAA sequence that is upstream of the S gene in terms of the direction of transcription and controls the transcription of the S gene. A vector containing an insert sequence consisting of a DNA fragment of the hepatitis B virus that includes a region of the DNA of the hepatitis virus and, on the other hand, a region downstream of the S gene and sufficiently long to allow expression of the S gene, where the fragment is This gene contains an EcoR site between the promoter region and the S gene, and the entire gene encodes messenger RNA corresponding to the HBs antigen). The present invention relates to particles that do not contain any human-derived components and vaccine compositions comprising the particles and a pharmaceutically acceptable carrier. The present invention also provides human-derived Dein particles, HBc
It also relates to novel preparations constituting vaccines consisting of proteins having the immunogenic and immunological properties of HBsAg antigens with a degree of purity free of antigen and serum components. The invention more particularly describes the use of this antibody (which can be recovered from the culture medium or cell lysate of the co-transformed cells) and optionally a suitable pharmaceutical carrier to constitute an active vaccine against viral hepatitis. The present invention relates to a vaccine composition that can be administered orally or parenterally. The present invention may furthermore be used as a standard or control with respect to the purity of the HBsAg antigen (whether naturally occurring or not), in particular its relative content of serum proteins or other antigens such as HBcAg or HBeAg. It also relates to experimental reagents containing a certain amount of these antigens. The invention is further illustrated by examples relating to the application of modified plasmids (which themselves form part of the invention) for the production of antigens with the immunological activity of HBsAg. Example 1 Construction of a recombinant containing B virus hepatitis virus DNA 200 ng of pBR322 was injected with endonuclease EcoR.
and then 2.6 units of alkaline phosphatase in Tris-HCl buffer (100 nM, PH 8).
and treated at 60°C for 60 minutes. After extraction twice with phenol and then three times with ether, the DNA was precipitated with ethanol. Dissolve the solid residue in water and add to this solution 100 ng of EcoHBV DNA [the cloned double-stranded entire genome of hepatitis B virus cut into a linear form at its unique EcoR site]. ] (PNAS, vol.76, P.2222~
2226.1979) was added. The ligation described in reference 1 above was then performed. 100 μg/ml diaminopimelic acid and 20 μg/ml
E. coli DP50 was cultured in a medium composed of medium L containing thymidine. This bacterium was then transformed according to the above method by mixing recombinants selected for resistance to 100 μg/ml ampicillin and 15 μg/ml tetracycline, yielding 900 colonies. . This is all done in situ hybridization (Eco HBV as a probe).
DNA was tested for the presence of HBV DNA, of which 800 colonies were positive. Of these, 16 colonies with high hybridization activity were collected, the plasmid was extracted, and its structure was determined using EcoR.
Analyzed in the presence of Xho, Hind and Xba. The structure of the obtained recombinant is schematized in Figures 2a and 2b, one for Eco HBV
One shows the DNA gene and the other shows the structure of the modified PCP10 plasmid. Here, at the level of the EcoR site, a pBR 322 DNA fragment constructed by two concatenated Eco HBV DNA fragments with one head attached to the other tail has been inserted into the plasmid (tandem head −tail insertion). Of the 16 colonies collected above, 14 were
It has a pCP10 type plasmid, and the inserted Eco
HBV DNA showed a head-to-tail tandem sequence. Hind and Xba DNA fragments were able to determine the insertion direction of Eco HBV DNA.
This is due to endonuclease digestion. Two Eco HBV DNAs in pCP10 plasmid
Integration of the fragment was demonstrated by digestion in the presence of Xho endonuclease, which excised a DNA fragment similar in size to the Eco HBV DNA from the hybrid plasmid pCP10 with 10726 base pairs. was produced. In Figures 2a and 2b, Eco HBV DNA
Each position of the S gene in the hybrid plasmid pCP10 is indicated by a thick arrow S, respectively. Small side arrows indicate the relative positions of certain restriction enzyme sites in the corresponding DNA strands. The positions of ampicillin resistance factor (Ap ^R ) and tetracycline resistance factor (Tc ^R ) are also shown schematically, respectively. Similarly, the herpes simplex virus HSV was added to the Pvu site of the pBR322 plasmid.
-1 thymidine kinase gene (HSV-tk)
The pAG0 plasmid obtained by inserting (Reference 2 below) is shown in Figure 2c. Culture and transformation of the above thymidine kinase-deficient mouse cells with the plasmid (Ltk ^- ) Thymidine kinase-deficient LM mouse cells mutant (Ltk ^- )
was cultured. Then Graham and Van Der Eb
For transformation, the DNA corresponding to the plasmid was added to the cells grown in a single phase (see below) in a Falcon flask according to the Stow and Wilkie modification (see reference 4 below) of the Stow and Wilkie method (see reference 3 below). 25cm ²
2×10 ⁶ cells per cell were inoculated. In performing this transformation, the pAGO plasmid (linearized with Hind endotaclease) and the pCP10 plasmid, linearized with or without the same enzyme, were used simultaneously. In all tests, the pAGO/pCP10 molecular weight ratio was on the order of 1/1000. DNA concentration at least 10μ
DNA salmon sperm was used as a carrier to adjust to g/ml. After conversion, the cell culture was
ml hypoxanthine, 0.1 μg/ml aminopterin, and 5 μg/ml thymidine. 24 hours after conversion, 100-fold concentrated in the medium
HAT solution was added and the concentrated HAT solution was replaced after one week and every three days thereafter. When cultured for 15 days in the presence of the two plasmids (co-transformation) in selective HAT medium, colony formation was observed. Twenty days after co-transformation, HBsAg production in the medium was detected by radioimmunoassay. Single plasmid as control
No production of HBsAg was observed from cultures transformed with pAGO and formed under the same conditions. tk ⁺ resistant to HAT medium 20 days after cotransformation
Colonies were collected with a Pasteur pipette and transferred to tissue culture on microplates. Colonies were passaged every 5 days and kept continuously and under selection pressure in HAT medium. 5 obtained by cotransformation in the presence of linear pCP10
and 10 colonies obtained by cotransforming mouse cells with circular pCP10.
When cultured in HAT medium, HBsAg was produced in all the media. Although the amount of HBsAg produced varied depending on the culture and was within the range of 1:30, the amount produced was stable and remained stable even after several generations of subculture. This HBsAg was recovered from the supernatant of the medium by centrifugation, purified by density gradient centrifugation using cesium chloride, and collected from the 1.20 g/ml density band. After incubation at 37° C. for 1 hour, the HBsAg thus obtained was completely neutralized with an anti-HBsAg serum solution (10/1 ratio) to an extent detectable by radioimmunoassay. According to electron micrographs, these particles were spherical particles with a size of 18 nm to 25 nm (average 22 nm). And this one is isolated from human serum.
Although the morphology was reminiscent of a 22 nm spherical particle antigen,
At least under the experimental conditions, there were no fibrous structures observed in antigens extracted from human serum, and no Dane particles were detected. The following table shows the production ecology of mouse cells tk ^- cotransformed with the plasmids used. Here, the ratio P/
N is the rate of disintegrations per minute (dpm) of supernatant in control medium when the HBsAg dose was determined by immunological testing 20 days after cell co-transformation.
The ratio of dpm is shown, and when the P/N ratio is 2.1 or more, it is considered that there is a significant difference. Here, N is the supernatant of control (comparison) cells.
Corresponding to the activity of the background noise when contacted with radiolabeled antibodies against HBsAg, P is definitely transduced by sequences derived from HBV DNA as evidenced by greater activity. Corresponds to the dpm number determined for the supernatant of the transformed cell line. Ratio P/N
exceeds 2.1, the corresponding cell line (active P
) is also effectively transformed,
It was well concluded that this is a cell line capable of converting HBsAg particles.

[Table] Using the probe Eco HBV DNA prepared by the method of Weinstok et al. (reference 7 below), pre-washed cells were prepared by the method of Wahl et al. After lysing the protein, digesting the protein with proteinase, extracting and purifying the cellular DNA, and estimating its number, the amount of intracellular HBsAg synthesized was equivalent to approximately one-third of the HBsAg excreted into the medium. It turns out that it does. Under our experimental conditions, the most active clones produced up to 150 ng/ml of HBsAg. As mentioned above, this HBsAg does not contain Dein particles, but in addition to this, human-derived anti-HBc and anti-
At least detectable by direct immunofluorescence using HBe/1,2,3 fluorescent antibodies (Reference 8 below)
HBcAg and HBeAg were not observed. Furthermore, the production of DNA polymerase by DNA polymerase activity assay by the method of Kaplan et al.
It was not found in the centrifugation retentate of the supernatant, nor in the lysates of transformed cells. High molecular weight DNA of certain cloned products was extracted, digested in the presence of EcoR, Hind and Xho, and the resulting fragments were fractionated by electrophoresis on an agarose gel, transferred to a nitrocellulose filter, and fragment p ³²
Hybridization with an Eco HBV DNA probe labeled with pCP10 demonstrated that several copies of plasmid pCP10 can be integrated into cellular DNA. Also, plasmid copies and possibly cellular DNA contained in transformed cells
HBV on both sides between the fragments incorporated into
The presence of DNA dimers was observed. According to kinetic studies, under the experimental conditions described above,
Assuming that each particle contains approximately 100 molecules of polypeptide, it can be excreted by the cell into the medium
HBsAg antigen particles are 2×10 ⁴ to 4×10 ⁴ particles/
cells/24 hours, i.e. 2,000,000 to 4,000,000
Polypeptide molecules/cells/24 hours were determined. Example 2 DNA from which most of the C gene has been excised in advance
Expression of vectors in eukaryotic cells modified by insert sequences derived from HBV (1) Linear DNA at the EcoR site of HBV
Cloning of pCP9 by insertion of pBR322 plasmid pBR322 hydrolyzed by EcoR
The plasmid was treated with alkaline phosphatase and then an equimolar amount of EcoR-cleaved
DNA was ligated in the presence of HBV. (2) A PAC series containing a DNA HBV fragment cut with Bgl and a DNA HBV fragment cut with BamH.
Clone construction with PANC The pASO plasmid was hydrolyzed with BamH and then treated with alkaline phosphatase.
After cleavage with Hind and Pst (sites not present in DNA HBV), the pCP10 plasmid described above,
More specifically, the dimeric (i.e., tandem array) was purified by electroelution on an agarose gel.
From the DNA HBV-containing fragment, HBV
I got the fragment. These fragments were partially hydrolyzed with restriction enzymes Bgl or BamH under conditions that resulted in only two-site cleavage, and the hydrolysates were ligated with equimolar amounts of pAGO plasmid previously cleaved with BamH. (This ligation method offers the advantage that the BamH and Bgl sites are characterized by a common cohesive end).
Collect clones sensitive to tetracycline,
Each possible insertion was analyzed to isolate it. Thus, clones PAC12, PAC14, PAC16
and PANC34 were obtained. In Figure 1, the arrows accompanying the corresponding symbols correspond to the DNA HBV sequences contained in the resulting recombinant plasmid.
Linearized in BamH or Bgl sites
The insertion position of pBR322 is shown. Each of the above clones further has the following characteristics. PAC12: Bgl fragment 1986-2840 deleted; PAC14: Bgl fragment 1986-2425 deleted; PAC16: pBR322 genome inserted into DNA HBV 1986 site; PANC34: DNA inserted into HBV BamH1400 site
Contains pBR322 genome. per 2 x ¹⁰⁶ cells using these clones
LTK ⁻ cells were transformed at a rate of 2 μg of plasmid. After 4 weeks, the TK ⁺ colonies were fully grown.
By the radio immunoassay method described above
The presence of HBs was tested. The "+" and "-" symbols in Figure 1 indicate the amount of blood excreted into the medium.
Expressing the characteristics of the S gene in the form of HBs antigen,
The capabilities of various recombinant plasmids with or without were shown respectively. The expression of the S gene in clones PAC14 and PAC16 and the absence of expression in clone PAC12
It is shown that transcription of the S gene is initiated in the viral DNA, more specifically in the restriction fragment Bgl2425-2840. From the beginning of the "pre-S" region, which appears to control transcription of the S gene,
TATATAA, which is located at the 72nd nucleotide in the upstream direction and is called the "TATATAA box"
An array exists. The fact that some PANC34 clones did not express under these experimental conditions is probably related to the fact that the DNA encoding the mRNA stops after the BamH site. That is, under this specific condition, HBV
Inserts inserted into PANC34 clones that are derived from DNA no longer look in the direction of transcription.
derived from HBV DNA, which encodes the terminal part of RNA.
Does not contain DNA fragments and HBV DNA
It is thought that expression did not occur because the DNA was excised at the DNA binding site of the corresponding plasmid. Especially important is the production of HBs.
The fact that the PAC14 clone lacks the viral C gene eliminates the risk of production of transformed cell virus particles, regardless of its nature, and therefore it cannot be used for vaccine production. can. Therefore, the present invention can provide a product of purity not previously available. The present invention more particularly relates to a preparation of protein particles having the immunological properties of HBsAg that are essentially free of serum proteins, which can be prepared by conventional radioimmunoassay techniques. Contains no detectable Dane particles. Furthermore, it is completely free of proteins, especially serum proteins of human origin, and DNA polymerase. The invention also relates to the above-mentioned novel insertion sequence itself and to vectors, in particular plasmids, containing this sequence, which have the characteristic that they themselves contain the promoter for the transcription of the S gene. These vectors can, if necessary, contain specific vectors corresponding to the proteins desired for expression in eukaryotic cells.
It is particularly advantageous in that it can be modified by insertion of DNA sequences. In fact, this vector is particularly advantageous in that it is not toxic to the cells in question. The promoter is not repressed because it causes the cell to express the S gene. In addition, this vector allows excretion of the synthesized protein directly into the culture medium. One of the above-mentioned newly inserted sequences contains, in addition to the S gene, DNA in the "pre-S" region (this is located in the DNA of the viral hepatitis virus, immediately upstream of the S gene and in the opposite direction of transcription). This pre-S region contains 489 nucleotides and 163
encodes a polypeptide having 10 amino acids. Tiollais et al., Science, 1981.vol.213, P.406
See −411. ), the messenger RNA gene of the S gene of hepatitis B virus, and the promoter of hepatitis B virus DNA, especially the TATAA sequence (T stands for thymidine and A stands for adenine). This substance also specifically contains the DNA of the hepatitis B virus.
It consists of a DNA sequence corresponding to a sequence localized between tacleotides 2425 and 2840. It is clear that within the scope of the invention are arrays comprising several units of the type described above, especially "tandem" arrays. The ability of the vector modified according to the present invention to express expression in eukaryotic cells is due to the fact that transcription of the HBsAg gene occurs under the control of the viral promoter contained in the fragment derived from HBV DNA. This is considered to be substantiated. These vectors, such as plasmids pCP10, PAC14 or PAC16, can themselves be used as vectors for the expression of foreign DNA previously inserted into these vectors into eukaryotic cells. A great advantage of the modified vector according to the invention also lies in the fact that it does not induce lysis of the host cell. Furthermore, the S gene has a sequence signal, and under its control, it is thought that the hybrid protein is excreted into the culture medium based on the expression of the foreign DNA inserted into the above vector into the host cell. . Recovery of this hybrid protein from the culture medium would clearly be greatly facilitated. Although the method of the present invention has been primarily described by its application to DNA HBV, it also extends to any circular DNA or whole genome, particularly for "non-A" and "non-type" viral hepatitis. Type B” virus circular shape
It can be applied to DNA research. Below, the state of the art related to the present invention and the references mentioned in the Examples of the specification are described. (1) Tiollais, p., Perricaudet, M.,
Petterson, U. and Phillipson, L. (1976).
Gene 1 , 49-63 (2) Colbere-Garapin, F., Chousterman, S.,
Horodniceanu, F., Kourilsky, P. and
Garapin, AC (1979), Proc, Natl.Acad.Sci,
USA, 76 , 3755−3759 (3) Graham, FL and Van der Eb, AJ.
(1973), Virology 52 , 456-458 (4) Stow, N.D., and Wilkie, N.N.
(1976), J.Gen.Virol. 33 , 447-458 (5) Southern.EM (1975), J.Mol.Biol. 98 , 503
−517 (6) Wahl, G.M., Stern, M. and Stark, G.
R. (1979), Proc, Natl, Acad, Sci, USA,
76, 3683−3687 (7) Weinstock, R., Sweet, R., Weiss, M.,
Cedar, H. and Axel, R. (1978), Proc. Natl.
Acad, Sci, USA, 75 , 1299−1303 (8) Trepo, C., Hantz, L., Vitvitski, L.,
Chevallier, P., Williams, A., Lemaire,
J, M. and Stepjian, M. (1978) in Viral
Hepatitis, eds. Vyas, GN, Cohen, SN and Schmid, R. (The Franklin Institute
Press) pp.203−209 (9) Shalomz. Hirschman et al., Proc. Natl, Acad,
Sci, USA, 77 , (9), pp5507-5511 (1980),
“Expression of Cloned hepatitis B
virus DNA in human cell cultures”

[Brief explanation of the drawing]

Figures 1, 2a, 2b and 2c show schematic maps of suitable plasmids for use in the present invention. In the figure, the numbers in brackets indicate the numbers of the aforementioned references.

Claims (1)

[Claims] 1. Having a size in the range of about 18 to 25 nm, containing no DNA polymerase, containing Dane particles, HBe antigen,
It contains no HBc antigen at all, and is aggregated by antibodies that cause an agglutination reaction with HBs antigen in human serum, but is not detected by anti-HBc antibodies or anti-HBe antibodies, and is immunogenic. It exhibits properties as a vaccine against hepatitis viruses, does not cause infectious hepatitis, and on the other hand, contains the S gene, a TATATAA sequence that is located upstream of the S gene in the direction of transcription and controls the transcription of the S gene. of hepatitis B virus that is long enough to encompass the promoter region.
A vector containing an insert sequence consisting of a DNA fragment of the hepatitis B virus that includes a region of the DNA and, on the other hand, a region downstream of the S gene and long enough to allow expression of the S gene, where the fragment comprises the promoter region and Between the S gene
Contains an EcoR site and the entire gene is HBs
Particles free of human-derived components that can be obtained by genetic engineering from eukaryotic cells transformed with Messenger RNA (encoding the antigen). 2. The particles of claim 1 having a density of about 1.20 g/ml in a CsCl gradient. 3. Particles according to claim 1, which do not induce an autoimmune response in treated patients. 4 Dein particles, having a size in the range of approximately 18 to 25 nm, containing no DNA polymerase, in an amount sufficient to confer immunity against infection with hepatitis B virus;
It does not contain any HBe antigen or HBc antigen, and is aggregated by antibodies that cause an agglutination reaction with HBs antigen in human serum, but is not detected by anti-HBc antibodies or anti-HBe antibodies, and is immunogenic. , TATATAA, which exhibits vaccine properties against hepatitis B virus, does not cause infectious hepatitis, and is located upstream of the S gene in terms of the direction of transcription and controls the transcription of the S gene. consisting of a region of hepatitis B virus DNA long enough to include the promoter region containing the sequence and, on the other hand, a region downstream of the S gene and long enough to allow expression of the S gene. Transformation using a vector containing an insertion sequence (wherein the fragment contains an EcoR site between the promoter region and the S gene, and the entire gene encodes messenger RNA corresponding to HBs antigen) A vaccine composition comprising particles free of human-derived components that can be obtained by genetic engineering from eukaryotic cells and a pharmaceutically acceptable carrier. 5. The vaccine composition of claim 4 having a density of about 1.20 g/ml in a CsCl gradient. 6. The vaccine composition according to claim 4, which does not induce an autoimmune response in treated patients.