JPS625132B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a cell-mediated immunostimulant containing an attenuated strain of vaccinia virus as an active ingredient, and to a method for producing the same. The cellular immunostimulant of the present invention is also useful as an antitumor agent. Typical antigens of microbial origin that cause a significant cellular immune response include Mycobacterium tuberculosis in the bacterial domain and vaccinia virus in the viral domain. Regarding the former,
Since it was reported that BCG vaccine or Mycobacterium tuberculosis cell wall substance has immunological anticancer effect,
Although it has recently attracted considerable attention, it has not necessarily been widely applied clinically due to side effects. On the other hand, the anticancer effect of vaccinia virus attracted attention for a time, but humoral immunity is generally established when vaccinated with vaccinia virus. Once this humoral immunity is established, the anticancer effect does not increase, so it has not received much attention recently. The present inventors conducted various studies with the aim of obtaining an attenuated strain of vaccinia virus that suppresses or reduces the humoral immune activity of vaccinia virus but has enhanced cell-mediated immune activity. As a result, after the vaccinia virus was serially cultured in monolayers obtained from tissue culture of mouse kidney cells,
When the virus is transplanted into a monolayer cell layer obtained by tissue culture of chicken fetal cells, and the vaccinia virus is successively cultured in this monolayer cell layer, or the latter, a monolayer cell layer obtained by tissue culture of chicken fetal cells is used. After a significant number of subcultures, the virus becomes attenuated and exhibits little or no virulence in domestic rabbits, and its humoral immune activity is substantially suppressed, preferably However, on the other hand, cell-mediated immune activity was enhanced, and it was recognized that the thus obtained attenuated vaccinia virus strain could be used as a cell-mediated immune stimulant and as an immunological anticancer agent. . Therefore, the gist of the first invention is as follows:
After subculturing the vaccinia virus in a monolayer cell layer obtained from tissue culture of chicken fetal cells or subculturing the vaccinia virus in a monolayer cell layer obtained from tissue culture of mouse kidney cells. An attenuated strain of vaccinia virus obtained by transplanting and subculturing in a monolayer cell layer obtained from tissue culture of fetal chicken cells, which has little or no virulence in domestic rabbits. A cell-mediated immune stimulant characterized by containing as an active ingredient an attenuated strain of vaccinia virus that has been attenuated to such an extent that it does not exhibit the following effects, has substantially suppressed humoral immune activity, but has enhanced cellular immune activity. be. Furthermore, the second aspect of the present invention is that vaccinia virus exhibits little or no pox-inducing power in domestic rabbits in a monolayer cell layer obtained by tissue culture of fetal chicken cells. Vaccinia viruses can be subcultured until they are attenuated to a certain degree and humoral immune activity is substantially suppressed, or the vaccinia virus can be subcultured in monolayers obtained from tissue culture of mouse kidney cells and then cultured in chickens. Transplanted into a monolayer of cells obtained by tissue culture of fetal cells, it is attenuated to the extent that it exhibits little or no pox-inducing power in domestic rabbits, and humoral immune activity is substantially suppressed. The present invention provides a method for producing a cell immunity stimulant made from the attenuated vaccinia virus strain, which comprises subculturing the vaccinia virus strain until subculture, and isolating and purifying the thus obtained attenuated vaccinia virus strain using a conventional method. Examples of vaccinia virus strains to be subcultured to obtain attenuated vaccinia virus strains in the present invention include vaccinia virus Dairen 1 strain kept at the National Institute of Preventive Health, Lister strain, and Ikeda virus strain. stocks, etc. (M. Majer and SAPlotk
−in “Strains of Human Viruses” 250−253
(1972), S. Kargel, Basel, Paris.
(See London and New York). The preparation of a monolayer of mouse kidney cells and a monolayer of chicken fetal cells used as a culture medium for subculturing this vaccinia virus strain is carried out using techniques known in the art for tissue culture of the respective animal cells. This technique is carried out under sterile conditions, and the successive tissue culture of vaccinia viruses in these monolayer cell layers is also carried out in the same way as in ``Introduction to Experimental Virology,'' published by the National Institute of Preventive Health Research Alumni Association, published by Maruzen Shoten ( 1969) and (RCParker
"Methods of Tissue Culture" 3rd edition (1961),
Hoeber, New York and J.Paul “CeII
and Tissue CuIture” 4th edition (1970), Living−
This can be carried out using the known virus culture method described by Stone, Ltd., London). The temperature used for subculture of the virus is preferably in the range of 33 to 37°C. The number of subcultures is repeated until the cultured virus strain exhibits little or no virulence in domestic rabbits. In addition, the presence or absence of smallpox potency is tested using a known method for preparing smallpox vaccines.
The number of passages of mouse kidney cells in a monolayer cell layer depends on the culture conditions, but
Preferably, the number of subcultures of chicken fetal cells in a monolayer cell layer is five times or more, although the number of subcultures depends on the culture conditions. The attenuated vaccinia virus strain that has acquired the characteristics of substantially suppressing, preferably substantially eliminating, humoral immune activity but having enhanced cell-mediated immune activity by subculturing in this way is generally It can be isolated and purified using the same methods known in the art for virus vaccine preparation to give the same form as the vaccine preparation. Further, this virus attenuated strain vaccine may be inactivated by known virus inactivation methods such as heating or ultraviolet irradiation. An example of the attenuated strain of vaccinia virus which is an active ingredient of the immunostimulant of the present invention is the vaccinia virus AS strain produced in Example 1 described later, which was developed by the present inventor as an American type strain in the United States.・Culture Your Collection January 22, 1980
Since then, it has been deposited as ATCC No. VR-2010 (see US Pat. No. 4,315,914). Vaccinia virus AS used in the present invention
In summary, the strain is Vaccinia virus Dalian 1, which was described in "Strains of Human Viruses" by M. Majer and SAPlotkin, p. 256 (1972) and used in the production of smallpox vaccines at the National Institute of Preventive Health until 1947. This virus belongs to the Vacciniaceae family of the order Poxvirales and was obtained by subculturing the strain in mouse kidney cells and chicken fetal cells.The subculturing and collection methods are as follows. That is, first, Dalian 1 strain was incubated at 33°C with mouse kidney cells at 115 °C.
The cells were subcultured and tissue cultured, and then subcultured for 5 generations using chicken fetal cells at 33°C. Then, for the purpose of cloning this virus, i.e. purification by blister formation, the virus-containing fetal chicken cells thus obtained and the cell culture were homogenized together and centrifuged (3000p.rm , 15 min) and diluted the supernatant with PBS. The limiting dilution of the virus thus obtained was inoculated into the chorioallantoic membrane of a 12-day-old chicken fetus.
Viral pock 1 produced by culturing
The virus was isolated from the chicken fetus on 12 days after incubation, cultured at 37°C for 48 hours, and the virus was isolated from one virus pox thus obtained.
Furthermore, the virus isolated from the virus pox produced by inoculating chicken fetal chorioallantoic membranes on day 12 of hatching and culturing at 37°C for 48 hours is the AS strain. This AS strain exhibits immunologically neutralizing specificity for the antiserum of the Dalian 1 strain, and although it causes small blisters on the chorioallantoic membrane of chicken fetuses, it exhibits inneutrotropic properties. . Furthermore, the attenuated strain of vaccinia virus used in the present invention is one that the inventor named ASE strain, which is a strain of vaccinia virus Dalian 1 strain that has been passaged for 60 generations using only chicken fetal cells at approximately 33°C. It was obtained by attenuating the Dalian 1 strain in the same manner as the AS strain described above, except that it was subcultured, and it exhibits the same immune responsiveness and antitumor activity as the AS strain. The above-mentioned attenuated strain of vaccinia virus, which constitutes the active ingredient in the immunostimulant of the present invention, does not have the ability to cause pox in humans or domestic rabbits, but its cell-mediated immune activity is far greater than that of previously known attenuated strains of vaccinia virus. It is highly potent and non-neurotropic, and in animals administered with it, not only live vaccinia virus but also
Inactive viruses can also inhibit the growth of malignant tumors. Additionally, it is well known that the immune response of diabetic patients and cancer patients is reduced.
Administration of the immunostimulant of the present invention is expected to restore or enhance such a reduced immune response. The immunostimulant of the present invention is used in an appropriate administration method, and when preparing an injection, the attenuated strain of vaccinia virus is added to the above-mentioned attenuated strain of vaccinia virus with a PH adjuster, a buffer, and a stabilizer. Subcutaneous, intramuscular, or intravenous injections can also be prepared by conventional methods by adding a curing agent, isotonic agent, etc. Vaccine injections can also be lyophilized. When preparing oral solid preparations, attenuated strains of vaccinia virus are combined with conventional excipients, stabilizers,
Furthermore, after adding binders, disintegrants, lubricants, coloring agents, flavoring agents, flavoring agents, etc. as necessary, tablets, coated tablets, granules, powders, capsules, etc. can be made by conventional methods. The dose of the active ingredient of the immunostimulant of the present invention varies depending on the therapeutic purpose and symptoms, but in order to improve cellular immune activity in mice, the dose should be 2 x 10 ⁸ PFU.
(plaque-forming unit)/ml from 0.1ml
It is 0.001 ml and is preferably administered once a day or once every two or three days. The optimum dosage can be readily determined by one skilled in the art by preliminary testing using techniques known in the art. Next, the present invention will be explained with reference to Examples and Test Examples. Example 1 (a) Kidney cells from DDN mice were aseptically removed and added to Hank's BSS (balanced salt salt) supplemented with penicillin and streptomycin.
solution) [i.e. 100 units of penicillin/
After washing with Hanks' balanced salt solubility solution containing 100 μg/ml of streptomycin and 100 μg/ml of streptomycin, the renal cortex was cut into cubes of approximately 3 mm on each side, and the renal tissue of one animal was isolated using Hanks' BSS. Wash twice to remove as much blood as possible, transfer to a 300 ml digestive colven, add 100 ml of balanced salt solution (BSS), shake well, aspirate the liquid with a sterile pipette attached to an aspirator and discard. Add 100 ml of 0.25% trypsin-added BSS that had been warmed to 37°C and incubate magnetically at room temperature.
Stir with a stirrer for 30 minutes with gentle rotation. Thereafter, it is removed from the stirrer, and when the undigested tissue has settled to the bottom of the Kolben, the cell suspension is drawn out and placed in a bottle placed in ice water at 4°C. Separately digest fresh trypsin in BSS solution pre-warmed to 37 °C in a colven.
Add 100 ml to the cell suspension and place on a stirrer for 30 minutes. After that, repeat the same digestion operation several times,
Finally, all the cells were sterilized using a stainless steel wire mesh (80 mesh was used at first, and 120 mesh was used after the second test).
Remove large lumps, transfer to a centrifuge tube, and fill up to about half the volume of the centrifuge tube. Add Hank's BSS solution to half of the cell suspension and centrifuge at 1000 rpm for 3 minutes.
The pellets are resuspended in a small volume of cell culture medium and pooled. The composition of the cell culture medium used for this was YLE liquid (contains yeast extract and lactalbumin hydrolyzate).
100 units/ml of penicillin and 100 μg/ml of streptomycin were added to a mixture of 80 parts (by volume) of Earle BSS) and 20 parts (by volume) of bovine serum. The number of cells in the obtained cell suspension was measured, and the above cell culture medium was newly added to give a 1-3×
After diluting to a concentration of 10 ⁵ cells/ml, dispense 2 ml into test tubes, tightly seal, and culture at 37°C to form a monolayer cell sheet of mouse kidney cells. Once a monolayer cell sheet is formed, refresh the culture medium. (b) The above monolayer cell sheet of mouse kidney cells was inoculated with purified sterile vaccinia virus Dalian 1 strain at an amount of 0.1 moi (MOI), and cultured at approximately 33°C for 4 to 6 days to detect cytopathic effect (CPE). The culture is stopped using the expression of the expression as an indicator, and 1/10th of the culture is used for the second generation. In this way, subculture
It lasted for 115 generations. Thereafter, the skin-muscle part of a 9-day-old chicken fetus was treated in the same manner as the mouse kidney cells described above, and a single cell suspension was cultured as a tissue culture, thereby producing a single cell sheet of chicken fetus cells on a glass test tube. Transfer the virus to this. As in the case of mouse kidney cells, an attenuated strain of vaccinia virus was obtained as a cultured virus that was subcultured at approximately 33°C for five generations. i.e. 5
The second generation chicken fetal cells and the culture solution were homogenized, and the centrifuged supernatant (3000 p.rm 15 minutes) was obtained as a culture virus solution containing an attenuated strain of vaccinia virus. A portion of this cultured virus solution was taken, and 0.1 ml of this was inoculated into the chorioallantoic membrane of five 12-day-old chicken fetuses, and the hatched chicken eggs were further cultured at 37°C for 48 hours. It was found on the chorioallantoic membrane and confirmed to be active as a virus. Furthermore, when we took another part of the cultured virus solution and examined its titer using the chorioallantoic membrane culture method of 12-day-old chicken fetuses, we found that the cultured virus solution from the 5th generation of subculture had a titer of 2.3×10 ⁶ PFU. (plaq−ue−forming
unit)/ml. Furthermore, when 0.1 ml of this cultured virus solution was injected intradermally into a rabbit, no local reactions such as pox were observed at the injection site. The attenuated vaccinia virus strain thus obtained was designated as vaccinia virus AS strain. (c) The above cultured virus solution was prepared using dichlorodifluoroethane using the Epstein method (MAEpstein, “Brit.
J. Exp. Path.” vol. 39, p. 436 (1958)).
Next, sucrose density gradient centrifugation (DNPlanterose,
C. Nishimura, NPSalzman, “Virology”18
Vol., p. 294 (1962)) to obtain a titer of 2×.
A virus solution of 10 ⁸ PFU/ml (hereinafter referred to as AS stock stock solution or AS stock 10° solution) was obtained. On the other hand, 15 previously known strains of vaccinia virus (“National Institute of Preventive Health Annual Report” Vol. 30,
(See p. 129 (1976), vol. 31, p. 128 (1977))
and MVA strains (H.Stickls and V.Hochst-ein-
Minzel: “Mu¨nch Med.Wshr.” vol. 113, 1149
- Page 1153 (1971) was similarly inoculated into the chorioallantoic membrane of chicken fetuses on the 12th day of hatching, and the M15 strain was left at 37℃ for 4 days, and the MVA strain was left at 37℃ for 2 days to cause small blisters on the chorioallantoic membrane. The chorioallantoic membranes of those that are secretly infected and alive are taken, homogenized, and then dispersed in PBS (phosphate buffered saline) to prepare a virus solution in the form of an emulsion. Purified and titrated, both at 2×10 ⁸ PFU/
M15 strain stock solution and MVA strain stock solution adjusted to a titer of ml were obtained as a comparative test. Below, the immune response of the AS strain was investigated using the following journal example. Test Example 1 The AS stock solution described above, a 10 ^-1 solution obtained by diluting this 10 times with PBS, or a 10 ^-7 solution obtained by diluting it 10 million times,
A volume of 0.2 ml each was injected into the shaved skin of two domestic rabbits weighing approximately 2 kg, which lacked neutralizing antibodies against vaccinia virus, and observed. Except for the group of rabbits injected with the stock solution, no rabbits showed redness at the injection site, and no induration or swelling characteristic of vaccinia virus was observed. Therefore, the AS strain is recognized to have almost no potency against domestic rabbits. Test Example 2 Detection of IgM antibody productivity using the Cunningham test method SPE (specified-pathogen-free) DDN mice (group of 5 mice) were given 0.2 ml of phosphate buffer solution (PBS solution) containing 10% sheep blood cells in the tail vein. At the same time, ^0.1 ml of each of the AS strain stock solution, M15 stock stock solution, and 0.1 ml of PBS solution (without virus) were injected into the abdomen for the control group. On the 4th day after killing, the PFC of the spleen was injected into the abdomen.
(plaque-forming cell) (Cunnigham AJ, Smith, JB & Mercer, EH
, “J.Exp.Med.” Vol. 124, p. 701 (1966)).
The average measured number of hemolytic plaques was 87.4 for the AS strain inoculated group, the M15 strain inoculated group, and the control group, respectively.
±65.5; 140.8±33.1, 85.0±30.1, and there was a significant increase (p<
0.05), but no significant difference was observed between the AS strain-inoculated group and the AS strain-inoculated group. This allows AS
It was observed that the strain did not substantially increase the number of immune cells in the spleen and therefore did not substantially exhibit humoral immune activity, at least IgM antibody production activity. Test Example 3a Delayed Type Hypersensitivity Reaction
-tivity) sensitization by injecting 0.05 ml of a PBS solution containing 10 ⁸ sheep red blood cells (SRBC)/0.05 ml into the left hind foot pad of SPF ICR mice (10 mice per group). At the same time,
Or, at the time of the second sheep red blood cell injection one week later, each virus stock solution of vaccinia virus AS strain and M15 strain (AS strain 2 × 10 ⁸ PFU/ml) was administered.
solution), 10 ⁷ PFU/ml virus solution diluted 20 times, and 10 ⁶ PFU/ml virus solution diluted 200 times,
In addition, a virus solution inactivated by heating the AS stock solution at 60℃ for 30 minutes (inactivated AS strain 2
Inoculate mice by subcutaneously injecting 0.1 ml of 10 ⁸ PFU/ml solution. Control mice were injected with PBS solution without sheep red blood cells. One week after the first sheep red blood cell injection, the thickness of the right hind footpad of all mice was measured with a caliper, and then ¹⁰⁸ sheep red blood cells/
Inject 0.05 ml of PBS solution containing 0.05 ml into the footpad (i.e., second sheep red blood cell injection), measure the thickness of the footpad again 24 hours later, and measure the degree of swelling as a delayed-type hypersensitivity reaction (DTH reaction). ) as a guideline.
The results are shown in Table 1 below.

[Table] As is clear from the results in Table 1, the virus was injected one week before the second sheep red blood cell injection, that is, at the time of the first sheep red blood cell injection, that is, the group inoculated with the virus at the time of the first sheep red blood cell injection. Among them, only the group inoculated with the AS strain showed significantly strong swelling. As a result, it was confirmed that the cell-mediated immune activity of the AS strain was significantly stronger than that of the previously known virus strain M15. It is known that such a DTH reaction test is used as a measure of activity to activate cell-mediated immunity (“Journal of the National Cancer
51, 5, pp. 1669-1675 (1973) and “Journal of Experimental Medicine”, 139
See Vol. 528-524 (1974). Test Example 3(b) MVA strain stock solution (titer 2×10 ⁸ PFU/ml) previously prepared as a comparative example sample, 20-fold diluted 10 ⁷ PFU/ml virus solution, and 200-fold diluted 10 ⁶ PFU/ml Using the virus solution, the ability to enhance the DTH reaction was investigated in the same manner as in Test Example 3a. The results are shown in Table 2.
In contrast, 2-fold dilutions, 20-fold dilutions, and 200-fold dilutions of the AS stock solution were also tested.

[Table] As is clear from the results in Table 2, the vaccinia virus AS strain has a stronger ability to enhance the DTH reaction than the MVA strain. In other words, although the virus titer against chicken fetal chorioallantoic membrane was the same, the AS strain, which had less virulence or affinity for domestic rabbits, tended to have a stronger ability to enhance the DTH response and, therefore, a stronger cellular immune activity. Next, we investigated whether the AS strain has antitumor activity in the following test example. Test Example 4 Sarcoma 180 cells were intraperitoneally injected into SPF ICR mice (5 weeks old, female) at 10 ² cells/0.1 ml, and 24 hours later, AS strain suspension (10 ⁶ PFU/
0.1 ml) was injected subcutaneously, and the same amount was injected every third day thereafter. In this treatment group, after virus inoculation
At day 21, all mice survived, survival rate 100
%, but in the untreated control group the survival rate was 20%.
It stopped. Test Example 5 SPF ICR mice (5 weeks old) were intraperitoneally injected with 0.1 ml of a PBS solution containing ¹⁰⁷ sarcoma 180 cells/ml heated at 60°C for 30 minutes, and then administered for 1 week. After that, PBS solution containing ¹⁰⁶ cells/ml of the same sarcoma 180 cells/ml was added.
0.1ml each was inoculated subcutaneously into the right limb. Immediately after this AS
Inject 0.1 ml of 10 ⁷ PFU/ml of AS strain or 0.1 ml of 10 ⁷ PFU/ml of AS strain inactivated by heating at 60°C for 30 minutes, followed by injection of 0.1 ml of the same virus every 3 days. ml
The animals were killed 20 days after the sarcoma inoculation. When the tumor weight was compared with the control group, it was 5.47±
1.89g compared to 2.65 in the group treated with live AS strain virus.
±1.26g, and 2.57±1.38g in the group treated with the inactivated AS strain virus, indicating that the live or inactivated AS strain significantly suppressed the growth of tumor cells. Test Example 6 Sugimura et al.’s method (“Experimental Stomach
Cancer;Methods in Cancer Research”, 7
Vol. 245-308 (1973) Academic Press, New York), the carcinogen MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) was
160μg/ml and surfactant Tween
SPF Wistar rats (group of 8 males, 6 weeks old) were given tap water containing 3.36 mg/ml of 60 as drinking water ad libitum while shielded from light. Changed the water. AS strain 10 ⁷ PFU/ml
A total of 62 injections were carried out by subcutaneously injecting 1 ml/dose of AS strain or 10 ⁶ PFU/ml of AS strain every 3 days at the start of the experiment. Stomachs were examined 218 days later and compared with a control group given only MNNG. When examining polyps that had developed in the forestomach, five out of eight animals in the AS strain 10 ⁷ PFU/ml treated group had no polyps at all. The remaining three animals had only one or five suspected polyps. The overall average for this treatment group was an incidence of 0.6 polyps per animal. In contrast, in the control group, the number of polyps occurring per animal was remarkable at an average of 17. Also
In the group treated with AS strain 10 ⁶ PFU/ml, the average number of polyps per animal was 9.8. Regarding the number of polyps that occurred in the glandular stomach area, the AS strain was ¹⁰⁶ PFU/ml.
In the treatment group, the number of polyps was on average less than 2.2 per animal and 1.5 in the AS strain 10 ⁷ PFU/ml treatment group.
In contrast, in the control group, there were an average of 3.8 pieces per animal. In addition, histopathological findings also indicate that AS
In the strain ¹⁰⁷ PFU/ml treatment group, the resulting polyps were classified according to the WHO classification (K. Oota and LHSobin “Histo
−logical typing of gastric and oesophageal
Tumors, International histological classif−
ieation of Tumors“No.8, WHOGeneva
(1977), there is nothing that should be recognized as malignant. In contrast, in the control group, there were no benign polyps, indicating that administration of the AS strain clearly had an anticancer effect. Test Example 7 Using SPF CDF mice (8 weeks old, female), each group of 10 mice received IMC ascites tumor cells ( ¹⁰⁶ cells/0.1 ml) at 0.1
Inject subcutaneously in ml portions and inactivate with UV rays after 24 hours.
0.1 ml of AS strain (10 ⁷ PFU/ml) was subcutaneously injected every 3 days. The tumor cells were sacrificed 30 days after injection and the weight of the tumor was measured and was 9.96±6.47 g. In contrast, in the untreated control group, the tumor weight was
13.91±4.80g, clearly (p<0.05) AS
The results showed that administration of the strain suppressed tumor development. Test Example 8 DDN of a group of 10 mice was given 0.025 ml/mouse of AS stock stock solution or PBS solution containing 1 strain of Dalian (titer 10 ⁸ PFU/ml).
Brain specimens were prepared using Coons' method by injecting the mouse into the brain and killing one mouse every day.
H., Leduc, E.H. & Kaplan, M.H., “J.Exp.
Med, vol. 93, p. 173 (1951)). When examined using the indirect fluorescent antibody method and the antiserum labeling method using fluorescein isothiocyanate, it was found that the virus was always present in the brain specimens after 72 hours in the Dalian 1 strain inoculation group. However, no virus could be detected in the AS-inoculated group up to 8 days after vaccination.Thus, it was confirmed that the AS strain had no neurotropism.Example 2. The skin and muscle of a 9-day-old chicken fetus was treated in the same manner as the mouse kidney cells in Example 1 (a) above, and tissue cultured as a single cell suspension, whereby a single cell sheet of chicken fetus cells was tested. 0.1 moi (M.
OI), cultured at about 33°C for 4 to 6 days, and stopping the culture using the expression of cytopathic effect (CPE) as an indicator, and transplanting one-tenth of the culture to the second generation. In this way, subculturing was carried out for 60 generations using only chicken fetal cells, and an attenuated strain of vaccinia virus was obtained as the cultured virus for the 60th generation. i.e.
The 60th generation chicken fetal cells and the culture solution were homogenized, and the centrifuged supernatant (3000 p.r.m for 15 minutes) was obtained as a culture virus solution containing an attenuated strain of vaccinia virus. Take a part of this cultured virus solution and add 5
When 0.1 ml was inoculated into the chorioallantoic membrane of a 12-day-old chicken fetus and the embryonated chicken eggs were cultured at 37°C for an additional 48 hours, small smallpox appeared as in the case of the AS strain obtained in Example 1. It was found on the chorioallantoic membrane and confirmed to be active as a virus. In addition, another part of the cultured virus solution was examined for titer using the chorioallantoic membrane culture method of 12-day-old chicken fetuses, and the titer of this cultured virus solution was 1.9×10 ⁶ PFU/ml. . Furthermore, when 0.2 ml of this cultured virus solution was injected intradermally into a rabbit, no local reactions such as pox were observed at the injection site. The attenuated vaccinia virus strain thus obtained was designated as the vaccinia virus ASE strain. Below, the immune response of the ASE strain was investigated in the following test example. Test Example 9 A stock solution of 1.9×10 ⁶ PFU/ml of the ASE strain of Example 2, and a 10 ^-1 solution obtained by diluting this 10 times with PBS,
Alternatively, inject 0.2 ml each of the ^10-6 solution diluted 1 million times into the shaved skin of two healthy domestic rabbits weighing approximately 1.5 kg without neutralizing antibodies against vaccinia virus. Observed. ASE stock stock solution (10
A slight redness was observed in the group of rabbits injected with ゜liquid), but
There were no other rabbits that showed redness at the injection site, and no induration, swelling, bumps, or swelling characteristic of vaccinia virus.
No local reactions such as pox were observed. Therefore, the ASE strain is recognized to have almost no potency against domestic rabbits. Next, the antitumor activity of the ASE strain was investigated in the following test example. Test Example 10 The ASE strain obtained in Example 2 was further inoculated into the chorioallantoic membrane of chicken eggs hatched for 12 days, cultured at 36.5°C for 48 hours, and then subcultured for 3 generations.
A virus solution of ASE strain with a titer of 10 ⁸ PFU/ml was obtained. On the other hand, sarcoma sarcoma 180 cells were added to 30 SPF ICR mice (5 weeks old, male) at 5×10 ⁵ cells/
The amount of mice was injected intraperitoneally. Thirty ICR mice inoculated with mouse sarcoma sarcoma 180 strain were divided into three groups of 10 mice each, one group was a control group (untreated), and the other groups were treated with ASE strain and AS strain, respectively. Immediately after the sarcoma cell injection, 10 ⁷ PFU/0.1 ml of the ASE strain or AS strain virus was injected into the ASE strain-treated group, the AS strain-treated group, and the local area of the sarcoma cell injection.
When the diameters of the tumors were measured 3 weeks after inoculation, they were 18.4 ± 1.8 mm, 6.8 ± 6.5 mm, and 5.1 ± 6.9 mm in the control group, ASE strain-treated group, and AS strain-treated group, respectively, and the tumor weights were 6.96±1.97g, 2.01
They were ±1.83g and 1.5g ±1.5g. obviously
The ASE strain was shown to have similar tumor suppressive effects as the AS strain.

Claims (1)

[Scope of Claims] 1. Vaccinia virus is subcultured in a monolayer cell layer obtained by tissue culture of chicken fetal cells, or vaccinia virus is subcultured in a monolayer cell layer obtained by tissue culture of mouse kidney cells. After subculturing the virus, it was transplanted into a monolayer of cells obtained from tissue culture of fetal chicken cells.
Vaccinia obtained by subculturing
A vaccinia virus that is an attenuated strain of the virus and has been attenuated to the extent that it exhibits little or no virulence in domestic rabbits, and has substantially suppressed humoral immune activity but enhanced cell-mediated immune activity.・A cell-mediated immune stimulant characterized by containing an attenuated virus strain as an active ingredient. 2. Vaccinia virus is attenuated to the extent that it exhibits little or no virulence in domestic rabbits in a monolayer cell layer obtained by tissue culture of fetal chicken cells, and it substantially suppresses humoral immune activity. or subculturing vaccinia virus in a monolayer cell layer obtained from tissue culture of mouse kidney cells until inhibition is achieved, or subculture the vaccinia virus in a monolayer cell layer obtained from tissue culture of fetal chicken cells. The vaccinia obtained in this way are transplanted into a rabbit and subcultured until it is attenuated to the extent that it exhibits little or no pox-inducing power and that humoral immune activity is substantially suppressed. - A method for producing a cell-mediated immunostimulant made from the above-mentioned attenuated vaccinia virus strain, which comprises isolating and purifying the attenuated virus strain by a conventional method.