JPS6244178A - Recombinant vaccinia virus - Google Patents

Abstract

PURPOSE:To obtain a recombinant vaccinia virus giving weak side reaction after vaccination, by integrating an extraneous DNA to the non-essential DNA region of a specific attenuated Lister temperature-sensitive mutant vaccinia virus strain. CONSTITUTION:The objective recombinant vaccinia virus is produced by integrating an extraneous DNA to the non-essential DNA region of an attenuated Lister temperature-sensitive mutant vaccinia virus strain exhibiting no proliferation property in rabbit renal cell and having small pox size on the chorion of embryonated hen egg. The vaccinia virus to be integrated with the extraneous DNA is an attenutated temperature-sensitive mutant strain of Lister virus which gives smaller pox size (<=3mm) on the chorion of embryonated hen egg than the Lister mother strain. The temperature to inhibit the proliferation of the virus in rabbit renal cell is 41 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a recombinant vaccinia virus, and more specifically, the present invention relates to a recombinant vaccinia virus that does not exhibit high-temperature proliferation in rabbit kidney cells and has a high pock size on the chorioallantoic membrane of cicada eggs. This invention relates to a recombinant vaccinia virus using a small, attenuated Lister temperature-sensitive mutant vaccinia virus strain as a vector.

(Prior art) In recent years, a method for constructing a recombinant vaccinia virus in which foreign DNA is incorporated into a vaccinia virus has been devised, and a recombinant vaccinia virus using infectious disease antigen-encoding DNA as the foreign DNA can be used as a live vaccine. Methods of using
-129971, Special Publication No. 60-500518, etc.).

This method is performed, for example, according to the following steps (
(See Figure 1). That is, first, an appropriate amount of NA is cut out using a restriction enzyme from a DNA region that is not essential for the propagation of vaccinia virus, and this is incorporated into a plasmid to prepare the first hyperply and doblasmid. Then,
Vaccinia virus DNA of this hybrid plasmid
Incorporate exogenous DNA into part A to prepare a second hyperply, doblasmid.

On the other hand, if an animal cell previously infected with vaccinia virus is isolated and a second hybrid plasmid is introduced into the cell, a homologous group between the vaccinia virus and the second hybrid plasmid will be formed in the infected animal cell. Recombination occurs, and a recombinant vaccinia virus that incorporates foreign DNA is formed (for example, the above publication, Cell Engineering Vol.
2. A9, pp. 84-87, 1983).

According to this method, it is possible to incorporate various foreign DNAs depending on the purpose, and it is seen as a promising method for producing a new live vaccine.

However, most of the vaccinia viruses used in conventional methods are the WR strain (see the above publication, Journal of Virology, VOL, 49゜P857-.19
(1984, etc.), these have a strong adverse reaction after smallpox, and the occurrence of post-vaccination encephalitis and intestinal disease is common, so it has been difficult to put them into practical use from the standpoint of safety.

(Problems to be Solved by the Invention) Therefore, as a result of intensive studies to overcome the drawbacks of the prior art, the present inventors found that rabbit kidney cells do not exhibit high-temperature proliferative properties and are grown on the chorioallantoic membrane of chicken eggs. The attenuated non-temperature-sensitive mutant vaccinia virus strain with a small pock size can be used as a vector for recombinant vaccinia viruses, even though it originally shows only extremely weak propagation ability. They discovered that the resulting recombinant strain was equally or even more vulnerable than the parent strain, leading to the completion of the present invention.

(Means for Solving the Problems) Thus, according to the present invention, rabbit kidney cells do not exhibit high-temperature proliferative properties and are grown on the chorioallantoic membrane of hen eggs.

Provided is a recombinant vaccinia virus in which foreign DNA is integrated into the non-essential DNA region of an attenuated Lister temperature-sensitive mutant vaccinia virus strain with a small virus size.

The vaccinia virus used for incorporating foreign DNA in the present invention is a weakly virulent temperature-sensitive mutant strain of Lister strain virus, and has a pock size of 3 plates or less on the chorioallantoic membrane of grown chicken eggs, preferably 3 plates or less. It is less than 1n, which is smaller than the star parent strain, and the temperature at which rabbit kidney cells cannot grow is 41°C, preferably 40.5°C.

Attenuated toxicity here refers to the fact that the central nervous system of rabbits and monkeys is not substantially observed (see Example 4).
．． 5.8 for Japanese white rabbits weighing 0-2.5 kg
Virus t recovered from 10% brain emulsion after 6 days of inoculating (tOgl. TCID 50/dose)
(AOglq TCID 50/d ) is preferably 2 or less, more preferably 1 or less.

Such attenuated temperature-sensitive mutant strains are, for example, those that have extremely poor proliferative properties in Vero cells after subculturing the Lister strain in rabbit kidney cells at 30°C, performing plaque purification, and dividing the strain into cells at 40°C. Furthermore, it can be obtained by allowing the thus obtained strain to form a colony on the chorioallantoic membrane of a hatched chicken egg, and selecting relatively small clones of the colony.

Specific examples of the attenuated temperature-sensitive mutant strain used in the present invention include LA strain and LB strain (CNTM-I-423), which are produced according to the following method.

1) Production of LA strain The original Norister strain was passaged for 36 generations in rabbit kidney cells at 30'r'-, and plaque purification was performed three times to isolate 50 clones. Among these 50 clones, a temperature-sensitive mutant strain with the worst proliferation of Pero cells established from middle kidney cells at 40°C was selected.

When this temperature-sensitive mutant strain was compared with the original strain, it was confirmed that rabbit skin proliferation was better than the original strain, but was comparable to that of the rabbit central haze mutant strain, and significantly weaker than the original strain. A small number of vaccination trials were conducted. As a result, the fever rate was 14 cm and the systemic reaction was mild, but skin formation tended to be delayed, so an attempt was made to isolate Chron, which has poor skin proliferation. The size of the pores on the chorioallantoic membrane of hatched chicken eggs was used as a marker for chron separation. In other words, after 6 passages of the above-mentioned temperature-sensitive mutant strain in rabbit kidney cells, plaque purification was carried out for 2
The pock on the chorioallantoic membrane of the hatched chicken egg was relatively small (2
~3 m) A homogeneous strain was isolated and named LA strain. The growth-inhibiting temperature of this strain was 41°C, and it was much weaker than the parent strain. The strain was isolated and named LB strain. The growth failure temperature of this strain was 40.5°C, and it was more exfoliating than the LA strain.

A comparison of the properties of the star strain and the LB strain used as the parent strain is shown in Table 1.

Table 1 Comparison of Lister strain and LB strain Lister strain LB strain Growth inability temperature (rabbit kidney culture cells) 41°C or higher 40
．． 5℃, large size (rabbit kidney culture cells) (3,5
m) Medium (2.0mm) pock size
Large (4.0 m) Small (0.9 mm) Detailed Proliferative ability in chicken egg chorioallantoic membrane +1+ ÷ Proliferative ability in Pero cells + 10 (Note 1) Central nervous system pathogenic rabbit (Note 2) +l+ +
Cynomolgus monkey (Note 3) Yes (death) No (survival) Invasion of the central nervous system due to peripheral infection Mouse (subcutaneous cortisone acetate inoculation) Yes No mouse (no treatment) Yes No skin growth rabbit + +Human
There is a difference of about 2 tog 1 degree between the two in 10 notes.

Note 2, 10 Based on the amount of virus recovered on day 6 after intracerebral inoculation with TCID5o virus.

Note 3: Intrathalamus inoculation with 10''''TCID5o virus.

These vaccines;
As for the method of incorporating NA, conventional methods may be followed; for example, by using a non-essential DNA region of the vaccinia virus DNA region that, even if deleted, does not have any fundamental effect on the propagation ability of the virus. It can be achieved.

That is, a first recombinant vector containing part or all of the non-essential DNA region described above is constructed, and a foreign DNA is added to the non-essential DNA region integrated into the first recombinant vector.
By constructing a second recombinant vector into which NA has been inserted, and introducing the second recombinant vector into animal cells that have been previously infected with the temperature-sensitive vaccinia mutant using a conventional method such as the calcium phosphate method, infected animals can be infected. The DN inside the cell
Homologous recombination occurs via the A region, and a recombinant vaccinia virus incorporating foreign DNA is obtained (
For example, JP-A-58-129971, JP-A-60-5
00518 etc.).

The above-mentioned non-essential DNA region used in the present invention may be any region as long as it does not have an essential effect on proliferation, and specific examples thereof include, for example, thymidine kinase (TK).
Examples include a DNA region encoding hemagglutinin (HA), a DNA region encoding hemagglutinin (HA), and an NA region as seen in JP-A-58-129971.

On the other hand, the vector used to construct the first recombinant vector is not particularly limited as long as it can insert non-essential DNA, and specific examples include pBR322, pBR325, pBR327゜pB
Plasmids such as R328, pUC7, pUC8, pUC9, etc., phages such as λ phage, M13 phage, etc., pHC79 (Gene, U, 291.1980
Examples include cosmids such as

Construction of the first recombinant vector may be carried out according to a conventional method. For example, after completely digesting the vaccine virus DNA with an appropriate restriction enzyme, separating and purifying the DNA fragment corresponding to the non-essential region, and It is sufficient to enzymatically link the DNA with a vector that has been cut with a restriction enzyme that can generate sticky ends in DNA that are the same as the enzyme-cleaved ends.

Whether the desired vector was successfully obtained is determined by the DNA containing the recombinant vector that has been enzymatically linked to the cut vector.
Transform (or transduce) bacteria such as E. coli with mixture A, and select from the resulting transformed strains (or transduction method) those that carry the vector into which the desired non-essential region DNA has been inserted. You can check by doing something like

In the present invention, a second recombinant vector is constructed from the thus obtained first recombinant vector and foreign DNA. The foreign DNA used is not particularly limited, but it is preferably DNA encoding an antigen protein of an infectious disease pathogen (e.g., virus, bacteria, parasite, protozoa, etc.) for which the development of a live vaccine is expected. Examples include health virus, varicella stomatitis virus, hepatitis B virus, hepatitis A virus, non-A
Examples include DNA regions encoding antigen tanks such as non-B hepatitis virus, foot-and-mouth disease virus, poliovirus, rabies virus, Japanese encephalitis virus, Vibrio cholerae, Salmonella enterica, Clostridium tetani, Plasmodium, and Schistosoma.

The second recombinant vector may also be constructed according to a conventional method, for example, by cutting the first recombinant vector with a restriction enzyme having a cutting site in the non-essential DNA region of the vaccinia virus, An exogenous DNA fragment having the same adhesive end as the restriction enzyme-cleaved end may be enzymatically linked.

The desired second recombinant vector can be obtained in the same manner as the first recombinant vector.

In the present invention, a recombinant vaccinia virus is then formed by introducing a second recombinant vector into animal cells that have been previously infected with vaccinia virus.

The animal cells used here need only be those in which vaccinia virus can propagate, and specific examples include, for example, T.
K143 (derived from human osteosarcoma), F'L (derived from human amniotic membrane), He1a (derived from human cervical cancer), KB (derived from human nasopharyngeal carcinoma), CJ-1 (derived from monkey kidney), B10-1 (
(derived from monkey kidney), RK-13 (derived from rabbit kidney), L929
(derived from mouse connective tissue).

In addition, the recombinant vector can be introduced into animal cells by conventional methods, such as the calcium phosphate method, liposome method, microinjection method, and the like.

Selection of the constructed recombinant vaccinia virus may be carried out according to conventional methods. For example, when the non-essential DNA region is a DNA region encoding thymidine kinase, a thymidine kinase-deficient strain is used as the infected animal cell, and the homologous ffl Plaques formed by the recombinant vaccinia virus were cultured for 3 days using a cell culture medium containing 25 μI/- of 5-bromodeoxyuricin (BudR) as a plaque-forming medium for the recombinant vaccinia virus. can be obtained by selecting .

However, when using a Lister temperature-sensitive mutant vaccinia virus strain with a growth incapacity temperature of 40.5°C, it is extremely difficult to obtain a recombinant vaccinia virus, and a medium with a low concentration of Bu dR is used. Plaques of recombinant vaccinia virus can be selected only after culturing for 5 days or more.

(Effects of the Present Invention) Thus, according to the present invention, the non-essential DNA region of the Lister temperature-sensitive mutant vaccinia virus strain that does not exhibit high-temperature proliferation in rabbit kidney cells and has a small pock size on the Wf chicken egg chorioallantoic membrane. By incorporating foreign DNA,
It is possible to obtain a recombinant vaccinia virus that is not invasive to the central nervous system due to peripheral infection of mice and has weak side effects after inoculation.

In other words, as will be explained in detail in Example 4, the recombinant virus was judged to be equivalent to the parent strain in animal tests, or to be less neurovirulent than the parent strain based on the results of the rabbit central nervous system pathogenicity test. This suggests that it is physically safe.

(Example) The present invention will be described in more detail with reference to Examples below.

Example 1 (1) Production of the first recombinant evector (Grasmid PCZO2) containing the vaccine virus TK gene (second
(See figure) 5tJ pBR328 was digested with Ee oRI, extracted with phenol/chloroform (1:1), cleaved pBR328 was recovered by ethanol precipitation, and then both ends were made blunt by treating with S1 enzyme. did. This DNA fragment was ligated to obtain a plasmid (pcZOl) that does not have an EcoRI cleavage site.

Selection of pcZOl was performed by transforming competent E. coli strain C600 with the ligated DNA and selecting chloramphenicol-sensitive transformants.

Next, 15 μm of plasmid pcZO was treated with HlndDI, extracted with phenol or chloroform, precipitated with ethanol, and then the DNA was recovered. The 5'-terminal phosphoric acid was removed by alkaline phosphatase treatment, and the DNA was recovered again by phenol-chloroform extraction and jetanol precipitation. The cleaved 0.5 μy pcZOI DNA was
0.1 μg of purified vaccinia virus (WR strain) H containing the vaccinia virus thymidine kinase (TK) gene
The hybrid plasmid obtained by ligation to the lndI[[J fragment was named pcZO2.

The presence of the vaccinia virus TK gene in pcZO2 was confirmed by the following flat head analysis.

That is, the ligated DNA allows for competent E. coli H
B101 strain was transformed, ampicillin-resistant and tetracycline-sensitive bacteria were selected, and then Birungheim (
Blrnboim) and Doly's method [
Nucleitsk Acid Research, 7.1513~p
(1979)] and extracted the plasmid with HlndI [
This was confirmed by electrophoresis after digestion with I and examining whether a fragment having the same length as the original vaccine virus HtndlJ fragment was present.

(2) Creation of a second recombinant vector (Grasmid pcZO3) containing the HBsAg gene Grasmid pBRHBadr72 (nucleitsu 7'/y) containing the gene encoding the hepatitis B surface antigen (HBmAg)" Research, VOL 11
, A 13, 4601-4610.1983)
After digesting 10 μN with BamHI and XhoI, approximately 1.27 k was obtained by low melting point electrophoresis (40°C, 16 hours).
The DNA fragment of b was isolated. Next, after confirming the DNA fragments by ethidium bromide staining, the dal was excised, extracted with phenol, and then precipitated with ethyl-sinol.
A 27 kb DNA fragment was recovered. The recovered DNA fragment was mixed with 10mM Trla-HCt (PH8,0), 1mM
The DNA was dissolved in 10 μt of a buffer containing EDT, treated with DNA polymerase to make the ends blunt, extracted with phenol/chloroform, and recovered by precipitation with jetanol.

On the other hand, 1 μg of EcoRI linker was phosphorylated at the 5/terminus with 10 units of kinase in Kainesi/Bacfur (37°C, 30 minutes), and this
A DNA fragment was added and subjected to lidarization (12°C, 16 hours). The reaction product was treated with phenol and chloroform, and DNA was recovered by ethanol precipitation. After digestion by adding 20 units of EcoRI, the reaction product was treated with phenol and chloroform, followed by ethanol precipitation.

Meanwhile, plasmid pC2O2 was added to 2 units of Ec in buffer solution.
Linearization was performed by treatment with oRI/μll DNA at 37°C for 2 hours. 50rnM of linearized plasmid
TrisHCt (d49.0), 1 mM Mg (,
t2. The 5' end of the plasmid was dephosphorylated by incubating at 37°C for 30 minutes with 0.1 units of bovine small intestine alkaline foster enzyme in a buffer containing 0.1 mM ZnC22 and 1 mM Sunominomiminone. DNA was recovered by extraction with equal volumes of phenol and chloroform and ethanol precipitation. 0.1 pg of this linear plasmid was added to 66 mM
Trim-HCt (pH 7,5), 6, 6 mM Mg
After treatment at 12°C for 15 hours in Cl2.10 mM DTT and 0.5 mM ATP, a 1.27 kb (D EcoRI fragment 0.2 μi
combined with. This Grasmid was used to transform E. coli HB 101 strain, and the transformed E. coli was transformed to 1.5
% agar, 37°C in LB medium supplemented with 50 μg/l ampicillin.
, and cultured for 15 hours. Transformed E. coli grown on agar was grown in LB liquid medium supplemented with 50 μl/d ampicillin at 37°C.
After culturing for 15 hours, the Birnboim & Doly
Purified using the method. Ten DNA samples each were digested in buffer with 5 units of E (!ORI) for 1 hour at 37°C.
1.2 of plasmid DNA by agarose electrophoresis
A 7 kb Ee oRI fragment was screened.

1.27 kb EeoRI hepatitis B virus DNA
Since fragments can be inserted in the forward or reverse orientation within their respective plasmids, the orientation of the inserted genes was screened. Screening was performed by digesting the plasmid derived from plasmid pczO2 with Xhol in a buffer solution for 1 hour, analyzing the generated DNA fragments by 7-ga-Sumi electrophoresis, and comparing their lengths.
A plasmid in which the Ag gene is inserted in the forward direction of the vaccine virus TK gene promoter is called pcZO.
3. pcZO4 plasmid inserted in the opposite direction
It was named.

(3) Creation of recombinant vaccinia virus A virus strain (6α) cultured in Petri dishes and attenuated to RK-13 cells (
Attenuated strain LA, temperature incapable of growth in rabbit kidney cells: 41°C, pock size on aged chicken egg chorioallantoic membrane: 2-3 m.
The acquisition method from the parent stock is as described above F)) o, IP
45 minutes after inoculation with FU, Grasmid pcZO with the HBsAg-gene inserted into the vaccine virus TK gene.
Dissolve 312 μg in 2.21 nl of sterile water and pour
A DNA-calcium phosphate coprecipitate was prepared by the method of Proteins, Nucleic Acids, Enzymes, 27, 340, 1985), and 0.5 m7 of it was dropped onto infected RK-13 cells. 3
Leave it in an incubator at 37°C for 0 minutes and leave it in a 5-inch CO□ incubator.
Eagle ME containing 0% tallow serum
M 4.5 go was added.

Three hours later, the culture medium was exchanged, and after culturing for 48 hours, the cultured cells were frozen and thawed three times and treated with ultrasound (1 minute).

To confirm the insertion of the HBmAg gene in the recombinant, 63
The above plaque-forming virus was inoculated into TK-negative (TK-) 143 cells cultured in a vetri dish, and 45 minutes later, L-chi agar, 12% beef tallow serum, and Eagl@MEM containing 25μji/jllBUdR were layered. 0 infected cells after culture for 1 day
．． Stained with 01% neutral red. Black formation rate is approximately 0.0
It was 0.05%. Remove the agar medium from the Petri dish, store it at 4°C, and transfer the virus to the cell surface remaining at the bottom of the Petri dish by pressing a sterilized nylon membrane with 0.5N.
After repeating the treatment for 10 min with NaOH and 5 min with 1 M Tris-HCl buffer three times, 1.5 M NaCl 0.5 M
)') Treated with hydrochloric acid buffer for 5 minutes.

2x SSC (1x SSC, 0,15M NaC1,0,
015Mc, H4(OH)(COONa), ) and baked at 80°C for 2 hours. 4x SET (
0,6M NaC2,0,08MTrls HCL, 4
mMEDTAp)17.8) -10x Dsnhar
It was treated with dt-0.1% SDS at 68°C for 2 hours. 4x SET -10x @nhardt -0.1% SD
S-0,1% Na4P20. - s o μg/'
Add denatured salmon sperm DNA and hepatitis B DNA labeled with P by nick translage and incubate at 68°C for 14 days.
Time was high. After washing, a nylon membrane plate was overlaid with an X-ray film, and autoradiography was performed to confirm the presence of the spot.The X-ray film was then placed on agar stored at 4°C, and plaques that matched the spots were found to contain the HBaAg gene. A recombinant (strain rLA) containing the virus was identified and isolated using a sterile Pasteur pipette.

Example 2 Lister temperature-sensitive mutant vaccinia virus, attenuated LB strain (temperature at which growth is ineffective in rabbit kidney cells: 40.5°C, pock size on aged chicken egg chorioallantoic membrane, approximately 0.5°C).
99-1CNC-1-423) and the selection method of the recombinant vaccinia virus was changed as follows. ) was obtained.

Recombinant selection method: 6-plate) TK negative (TK-) 1 cultured in IJ dish
43 cells were inoculated with the above plaque-forming virus, and 45 minutes later, the cells were inoculated with 1% agar, 12% tallow serum, and 15μVmjBudR.
Eagle MEM with additives was layered and cultured for 3 days, then Evacuation MEM of the same composition was layered and cultured for an additional 3 days.

Then, the infected cells were stained with 0.01% neutral scarlet, and the following
It was treated in the same manner as in Example 1.

Comparative Example 1 The same procedure as in Example 1 was carried out, except that Lister original strain (LO strain, temperature incapable of proliferation in rabbit kidney cells > 41°C, pock size 4 on the chorioallantoic membrane of grown chicken eggs) was used as the vaccinia virus. An experiment was conducted to obtain a recombinant vaccinia virus (rLo strain).

Comparative Example 2 Example 1 except that the WR strain (temperature incapable of growth in rabbit kidney cells > 41°C) was used as the vaccinia virus.
Experiments were carried out in the same manner as above, and recombinant vaccinia virus (
rWR stock) wo profit*.

Example 3 Expression of recombinant cells in 9RK-13 cells cultured in a 25 cm culture bottle.
, I PFUO recombinant and parent strain were inoculated, and after virus adsorption for 1 hour, Eagle (Ea
gle) MEM medium was added and cultured at 37°C for 14 days. As a control, uninfected RK-13 cells were seeded. After culturing, freeze-thaw three times, sonicate, and centrifuge (3,
000 rpm, 10 minutes) to remove cell debris, and the amount of HBsAg in the supernatant was determined by enzyme-linked immunosorbent assay (EIA).

Abbott Lab). The beads treated with anti-)tBaAg antibody were allowed to stand at room temperature for 15 hours with the centrifuged supernatant for 0.2 d, and the beads were incubated with peroxidase-labeled anti-HBs.
0.21 d of Ag goat antiserum was added and reacted at 40°C for 1 hour. After washing the beads three times with 5 g of distilled water, transfer them to a test tube for enzyme reaction and add 0.31!7! of hydrogen peroxide solution containing orthophenysaldiamine dihydrochloride. was added to react for 30 minutes, and IN sulfate IWLt was added to stop the enzyme reaction.

The absorbance at a wavelength of 492 nm was measured, and HBiAg
The antigen amount was calculated with reference to HBsAg positive and negative controls that were performed at the same time. The results are shown in Table 2.

Table 2 HB Hatake Ag Production from Recombinant Example 4 In Vivo Marker Confirmation Test In order to confirm the difference between the in vivo marker of the parent strain and the in vlvo properties of the recombinant into which the HBaAg gene has been inserted, rabbit central A nervous system pathogenicity test and a central nervous system invasiveness test by peripheral infection of mice of the genus were conducted.

b) Rabbit central nervous system pathogenicity test 2.0 to 2.5 kII healthy Japanese white-like rabbits.
10 various vaccinia viruses #10 TCI
Inoculate 0.25 d of D5o no'filus solution into the brain, and
After clinical observation for one day, the surviving rabbits were sacrificed, virus was recovered from the brain, and histopathological examination was performed. The results are shown in Table 3. In addition, + in Table 3 means mild, ox means moderate, +
indicates that the song is moderate to advanced, and the song is advanced.

The recombinant strain of the present invention has a smaller amount of virus recovered from the brain and is significantly less pathogenic to the central nervous system than the recombinant strain WR strain or the original Star strain. Furthermore, in comparison with the parent strain, the recombinant strain showed a tendency to have weaker meningeal, choroid plexus, and parenchymal lesions than the parent strain. This tendency is particularly noticeable in the case of LeB.

Mouth) Invasiveness test on the central nervous system by peripheral infection of mice.
5O from 3TC was inoculated intraperitoneally and 2~ subcutaneously with cortezone acetate at the same time to examine whether the virus invaded the central nervous system from the periphery and multiplied by Willemy. After vaccination 3
, 4th, 5th, 64th, and 7th, blood and brain samples were collected, and the amount of virus in the blood and brain was measured.

The results are as follows.

No virus was recovered from the blood or brain of the recombinant vaccinia virus of the present invention or its parent strain. on the other hand,
Virus was recovered from the blood and brain of the recombinant WR strain, and a large difference was observed in the ability of the virus to invade the central nervous system and the ability of the virus to multiply there in both methods.

The mechanism of occurrence of post-vaccination encephalitis and encephalopathy has been explained by the fact that vaccinia virus reaches the meninges and choroid plexus as a breeding ground by Willemy and causes inflammatory changes. The above animal test is considered to be a model system for analyzing the developmental mechanism of post-vaccination encephalitis and encephalopathy, and the recombinant virus has low central nervous system pathogenicity and does not show central nervous system invasiveness, and is a highly safe virus that can be used as a live vaccine. It may also be possible to use it as

[Brief explanation of the drawing]

FIG. 1 is a drawing showing an outline of the operating procedure of the present invention, and FIG. 2 is a drawing showing the operating procedure in Embodiment 1.

Claims (1)

[Scope of Claims] 1. Foreign DNA is present in the non-essential DNA region of an attenuated Lister temperature-sensitive mutant vaccinia virus strain that does not show high-temperature growth in rabbit kidney cells and has a small pox size on the chorioallantoic membrane of hatched chicken eggs. Integrated recombinant vaccinia virus. 2. The recombinant vaccinia virus according to claim 1, wherein the temperature at which the Lister temperature-sensitive mutant vaccinia virus strain cannot grow in rabbit kidney cells is 41°C. 3. The recombinant vaccinia virus according to claim 2, wherein the pock size of the Lister temperature-sensitive mutant vaccinia virus on the chorioallantoic membrane of hatched chicken eggs is 3 mm or less. 4. The non-essential DNA region of Lister temperature-sensitive mutant vaccinia virus strain is DNA encoding thymidine kinase
The recombinant vaccinia virus according to claim 1, which is a region of the invention. 5. The recombinant vaccinia virus according to claim 1, wherein the foreign DNA is DNA encoding a protein of an infectious disease pathogen. 6. The recombinant vaccinia virus according to claim 5, wherein the protein is a hepatitis B surface antigen.