JP2813066B2 - Test method for neurotoxicity of polio vaccine - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for testing the neurotoxicity of a polio vaccine by inoculating a polio vaccine into the spinal cord of a transgenic non-primate vertebrate into which a gene for a poliovirus receptor has been introduced. .

BACKGROUND ART Polio is a disease of the human central nervous system caused by poliovirus. Poliovirus type 1
It is classified into three serotypes, type 2 and type 3, and is thought to exist in nature in various ways, from those with weak neurotoxicity (attenuated strains) to those with strong neurotoxicity (strongly virulent strains), and shows sensitivity only to primates.

Polio's natural transmission and outbreaks are found exclusively in humans and have existed as an infectious disease among humans since ancient times. Even today, many polio cases occur every year in developing countries, and eliminating them in the near future is a challenge for humankind.
It is a well-known fact that live polio vaccine can be the most effective weapon for that.

Conventionally, an attenuated polio vaccine obtained by attenuating a poliovirus has been manufactured and used. However, the attenuated polio vaccine is rarely pathogenic to a recipient or a contact person (neurotoxicity characterized by paralysis). There is a danger of showing. Therefore, there is a need to produce a safe and effective polio vaccine that does not exhibit the pathogenicity and has a function as a polio vaccine. Tests for

Currently, monkey neurotoxicity tests are conducted worldwide by inoculating a fixed amount of poliovirus into the monkey's spinal cord.
It is performed using a “lesion index” that quantitatively represents the frequency of clinical paralysis and histopathological changes occurring in the central nervous system as an index. Many monkeys such as cynomolgus monkeys are used in the current neurotoxicity test of attenuated polio vaccine.

However, the neurotoxicity test using monkeys has the following problems. 1) The price is remarkably higher than that of general laboratory small animals. 2) There is a risk that monkey handlers will be infected with the pathogenic virus possessed by monkeys. 3) It is becoming difficult to supply monkeys from the standpoint of protecting wildlife. 4) Wild monkeys are not homogenous in genetics, and the results vary greatly from test to test in the same sample test (variation is inevitable).

On the other hand, as described above, poliovirus is not sensitive to animals other than primates, so mice and rats cannot be used for this test.

DISCLOSURE OF THE INVENTION The present invention uses a transgenic primate vertebrate susceptible to poliovirus to correlate with monkey neurotoxicity test results and to perform a simple neurotoxicity test. The aim is to develop a method.

The present inventors have conducted intensive studies based on the above problems, and as a result, poliovirus was inoculated into the spinal cord of a transgenic non-primate vertebrate obtained by introducing a poliovirus receptor gene, whereby poliovirus was inoculated. The present inventors have succeeded in developing a test method which is highly sensitive to neutrophils and can obtain a result which is well correlated with the result of the neurotoxicity test of monkeys, and completed the present invention.

That is, the present invention provides a method for testing the neurotoxicity of a polio vaccine by inoculating the polio vaccine into the spinal cord of a transgenic non-primate vertebrate into which the gene for the poliovirus receptor has been introduced. This is a poison test method. The transgenic non-primate vertebrate includes, for example, a transgenic mouse, a transgenic rat, a transgenic guinea pig, or a transgenic hamster. Examples of the polio vaccine include attenuated poliovirus type 1, type 2 and type 3 strains.

 Hereinafter, the present invention will be described in detail.

The transgenic non-primate vertebrate (hereinafter, referred to as “transgenic animal”) to be used in the neurotoxicity test of the present invention is as described below.

Transgenic mice were produced by Ruibao Ren et al. (Ren,
R., et al., Cell., 63, 353-362, 1990) and Koike et al.
e, S., et al., Proc. Nat'l. Acad. Sci. USA., 88, 951-955,
1991), a mouse of human origin
HeLa cell poliovirus receptor (hereinafter, referred to as "PVR") is a transgenic mouse (hereinafter, referred to as "TgPVR") susceptible to poliovirus, in which the gene of the mouse has been introduced into a mouse gene. TgP used in the present invention
As the VR, any strain of mouse may be used,
The ICR system, C57BL / 10, IQI system, etc., and 4-6 weeks of age are preferred. For example, from an ICR mouse, a homozygous and heterozygous transgenic mouse called ICR-TgPVR21 and a heterozygous transgenic mouse called ICR-TgPVR1 and the like, which are 4 to 6 weeks old, may be mentioned. [Available from the Central Laboratory for Experimental Animals.]
Gender does not matter.

In addition, transgenic rats, transgenic hamsters, and transgenic guinea pigs were cloned using the same technique as that used to establish the poliovirus-sensitive transgenic mouse.
Microinjection into the female or male pronucleus of fertilized eggs of various animals, return to the oviduct of the pseudopregnant foster parent, and select the animals in which the human PVR gene has been introduced for the animals that grew and gave birth. Obtainable.

Transgenic rats, transgenic hamsters, and transgenic guinea pigs may be derived from any strain. In the case of transgenic rats, W (Wister) strain, Spraque-Dawky strain, F344 strain, etc. In such cases, Syrian hamster NSJ strains and the like can be used, and in the case of transgenic guinea pigs, Hartley strains and the like can be used.

The virus used in the present invention may be either poliovirus type 1 type 2 or 3, but is preferably a poliovirus type 1 attenuated vaccine strain Sabin type 1 (Sabin, AB, JAMA, 194, 130- 134, 1965) and have the various properties described below, and are expected to have different neurotoxicity (F113, S1A, S1)
B, S1C, S1D, S1D-38 / 2, S1D-38 / 4).

F113 virus is attenuated polio 1 from National Institute of Health
It is a type reference virus and is a standard virus for testing the neurotoxicity of live polio vaccine type 1 in Japan. Also, S1A, S1C,
S1D is a virus having neurotoxicity that can be used as a vaccine virus, and S1B is a highly attenuated virus selected from the Sabin type 1 strain by the plaque cloning method. In addition, S1D-38 / 2, S1D-38 / 4, the vaccine virus S1D, 38 ° C.
Are viruses that have been passaged for 2 or 4 passages, respectively, and have regained virulence to an unacceptable level as a vaccine virus.

The amount of inoculated virus used in the present invention is based on the infectivity of the virus strain. The infectious titer generally refers to the amount of a specimen containing an infectious microorganism that gives an infection rate of 50% when inoculated into cultured cells in equal amounts, and is referred to as “TCID ₅₀ (tissue culture infective). dose) ”.

In the present invention, the infectious titer refers to the amount of virus that gives 50% of the infection rate when the virus is inoculated into cultured cells [GMK2 cells (green monkey-derived kidney cell line; established at the National Institute of Health). Expressed as a unit.

For example, when "10 ⁴ TCID ₅₀ " is described, when a certain amount of virus is inoculated into a GMK2 cell culture tube or a similar cultured cell, a cytopathogenic effect (Cytopathogenic effect; CPE ")
When set to 1 the amounts shown the means having a viral load of ¹⁰⁴ times.

The measurement of TCID ₅₀ can be performed by a roller tube method using GMK2 cells described in Examples described later.

In order to inoculate the transgenic animal with the virus in the spinal cord, the following method is used. That is, the transgenic animal is anesthetized using pentobarbital, ether, or the like, the back of the anesthetized transgenic animal is disinfected with ethanol, and the incision is made along the midline to expose the spine. At this time, on the abdomen of the transgenic animal,
For example, when the intervertebral space is opened by touching the table 1 shown in FIG. 1, the needle is easily inserted.

Next, as shown in FIG. 2, an injection needle is inserted toward the lumbar spinal cord ampulla. When the injection needle is inserted into the lumbar spinal cord, the transgenic animal shows a convulsive reaction on the hind limbs, so that it can be confirmed whether or not the insertion has been made.

Here, the reason why the injection needle is inserted into the lumbar spinal cord ampulla is as follows. That is, in order to conduct a neurotoxicity test, a test system which exhibits a certain degree of sensitivity to poliovirus and is capable of measuring the neurotoxicity of more virulent or less virulent strains is desired. For this purpose, among the organs of transgenic animals, the mRNA of PVR is the most abundant, the PVR is also present in a large amount, and the lumbar spinal cord, which is presumed to be highly sensitive (the most sensitive organ of monkeys to poliovirus) Is preferred).

The dose is preferably 5 to 20 μl as a dose that has little mechanical damage to the spinal cord of the transgenic animal and can be surely inoculated, and 5 μl is the usual dose for mice.

As the injection needle, a two-stage needle as shown in FIG. 3 [the insertion part has a thickness of 33 G (diameter of 0.2 mm) in order to ensure that the needle can be inserted into a target position without penetrating the lumbar spinal cord. ), Length 7 mm].

Although the type of the syringe is not limited, a micro syringe having a size of 25 μl is preferable in consideration of the above-mentioned dose.

After inoculating the virus as described above, the back skin is closed with a commercially available instant adhesive, and the virus inoculating operation is completed.

Clinical observation for a certain period after virus inoculation
Those who show paralysis within 24 hours are excluded from the evaluation as mechanical damage due to vaccination. The normal observation period is 14 days, and PD ₅₀ or LD ₅₀
Alternatively, neurotoxicity is quantified by pathological examination of the central nervous system such as the spinal cord and brain.

PD ₅₀ refers to the amount of virus that causes paralysis in 50% of transgenic animals, and LD ₅₀ refers to the amount of virus that kills 50% of transgenic animals, both of which are clinical figures that are indicators of the neurotoxicity of the virus. It is. In both cases, the lead mince method (Reed, LJ, et al., Am. J.
Hyg. 27, 493-497, 1938) or the Spielman-Kelber method (Lab. Tech. In Rabies, 3rd Ed, Kaplan, MMand Kopro)
wski, H., WHO, 1973).

Items of clinical observation include the presence and degree of paralysis of the limbs, their progression, and changes in body weight. Also,
For a pathological examination, a small animal solid or the brain and spinal cord should be
% Formalin buffer, and embedded in paraffin.
A 9 μm-thick thin section is prepared, a pathological tissue specimen is prepared by galocyanine staining, and the neurotoxicity is observed with an optical microscope using the infiltration of lymphocytes and mononuclear cells and the degeneration and disappearance of nerve cells as indicators. The degree of pathological change in each tissue is determined according to the WHO (World Health Organization) evaluation method for the polio vaccine neurotoxicity test in monkeys (WHO Tech. Rep. Ser., 800, 30-86, 1990). be able to. The outline of the central nervous system of each animal (the medulla (lumbar cord, cervical cord), the brain (midbrain, pons, medulla,
The lesion index for each individual is obtained by dividing the episode of the lesion degree of the cerebrum) by the number of samples, and calculating the average lesion number between animals inoculated with the same viral load.
e; hereinafter referred to as “LS”).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a view in which the back of a mouse is cut.

 FIG. 2 is a diagram showing intraspinal inoculation of a mouse. FIG. 3 is a diagram showing a syringe and a needle.

FIG. 4 shows the results of clinical observation of mice inoculated with the virus.

FIG. 5a shows the mouse with the mouse number 39 (Tg
FIG. 21B is a view showing a histopathological examination result (weak enlargement) of the lumbar spinal cord of 21).

 FIG. 5b is a diagram showing the expansion of FIG. 5a.

FIG. 5c shows the mouse with the mouse number 24 shown in FIG.
FIG. 21 is a diagram showing a histopathological result (weak enlargement) of the cervical spinal cord in 21).

 FIG. 5d is a diagram showing the expansion of FIG. 5c.

FIG. 5e shows the mouse with mouse number 24 (Tg
FIG. 21 is a view showing a result of histopathological examination of the brain of 21), particularly the medullary sublingual nucleus.

FIG. 5f shows the mouse with the mouse number 24 shown in FIG.
FIG. 21 is a view showing a result of histopathological examination of the brain of 21), particularly, medullary olive nuclei.

FIG. 5g is a diagram showing a histopathological examination result (weak enlargement) of the lumbar spinal cord in monkeys.

FIG. 6 is a correlation diagram between the results of the neurotoxicity test of monkeys and the results of the neurotoxicity test of TgPVR.

FIG. 7 is a diagram showing the distribution of viruses in the brain and spinal cord.

DESCRIPTION OF SYMBOLS 1: Platform Best Mode for Carrying Out the Invention Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited to these examples.

[Example 1] Neurotoxicity test using TgPVR (1) TgPVR A 6-week-old TgPVR derived from a poliovirus-sensitive ICR mouse into which a human-derived HeLa cell PVR gene was introduced.
ICR-TgPVR21 (homozygote or heterozygote; hereinafter referred to as "Tg21") and ICR-TgPVR1 (heterozygote; hereinafter referred to as "Tg1") Are supplied as 5-20 animals per dilution step of virus.

(2) Preparation of virus The virus infection titer (TCID ₅₀ ) of each virus sample to be inoculated was measured in advance.

The TCID ₅₀ was measured by a roller tube method using GMK2 cells.

That is, 0.2m of 10 times serial dilution of each virus material
l for each dilution step 5 or more roller tube cultures of GMK2
Cells were inoculated. After inoculation, the cells were cultured at 36 ° C. for 7 days, and TCID ₅₀ was determined by the appearance of CPE.

Seven attenuated poliovirus 1 strains with different neurotoxicity (F113, S1A, S1B, S1C, S1D, S1D-38 / 2, S1D-38
/ 4; available from the Japan Polio Research Institute)
Medium supplemented with 25% sodium bicarbonate (Medium 19
Using 10), perform 10-fold serial dilution, and use the 3-5 dilution steps (10 ¹
~10 ⁵ TCID ₅₀ / 5μl) was prepared.

(3) Intraspinal Inoculation into TgPVR 0.3 g of 5 mg / ml pentobarbital was injected intraperitoneally with TgPVR and anesthetized. In addition, when additional anesthesia is needed,
Ether inhalation anesthesia was performed.

After the hair on the back of the anesthetized TgPVR was disinfected with ethanol, an incision was made about 2 cm along the midline to expose the spine.

At the time of virus inoculation, first, the abdomen of TgPVR was applied to the abdomen of TgPVR to open the intervertebral space of the thoracolumbar region of the curved spine by applying the platform 1 described in FIG.
The needle was inserted (FIG. 2). TgPVR when needle is inserted into lumbar spinal cord
Shows a convulsive reaction in the hind limbs. After confirming the reaction, 5 μl of the vaccine strain prepared in (2) was added in 5 to 5 per virus dilution.
Twenty TgPVRs were inoculated. The dilution step was set to include a range from a virus amount at which 90% or more of TgPVR develops to a virus amount at which 10% or less of TgPVR develops.

As shown in FIG. 3, a two-stage needle having an insertion portion of 33 G (diameter 0.2 mm, length-mm) was used as an injection needle, and a microsyringe having a size of 25 μl was used as a syringe.

After inoculation, instant adhesive [Aron Alpha (trade name);
The back skin was closed with Toa Gosei Chemical Co., Ltd.].

In addition, TgPVR in which inoculation disorders such as hind limb paralysis were recognized clinically within 24 hours after virus inoculation were excluded from the test.

(4) Neurotoxicity test with TgPVR (i) Clinical observation TgPVR inoculated by the method described in (3) was bred in a cage for each individual, and clinical observation was performed for 14 days.

FIG. 4 shows the results. In FIG. 4, the mark “○” indicates a normal mouse,
"●" indicates paralysis, and "★" indicates moribundity or death.

TgPVR after intraspinal inoculation develops flaccid paralysis on one or both sides of the hind limb and progresses to rarely the forelimb. Most paralysis appeared within 2 to 7 days after inoculation, and after onset, some survived 14 days and died regardless of the amount of inoculated virus. Since the paralyzed mouse cannot drink water at the water supply port at a high position, attention was paid to the position of the water supply port.

Based on such clinical observations, PD ₅₀ and LD ₅₀ were respectively determined by the lead mincing method (Reed, LJ, et al., Am. J. Hyg. 27, 493).
−497, 1938).

As a result, PD ₅₀ and LD ₅₀ for F113 strain of Tg21 is ^{_{10 3.3 TCID 50, 10 3.6 TCID}} 50 , respectively, each of the Tg1 1
The virus load was 0 ^1.6 TCID ₅₀ and 10 ^2.2 TCID ₅₀ .

Therefore, these results indicate that both Tg21 and Tg1 show clear dose dependence, and can be used as an assay system. However, Tg1 was paralyzed in 50% or more at a viral load of 10 ² TCID ₅₀ , and tended to be too sensitive, and Tg21 had moderate sensitivity.

Clinical observation records of Tg21 were performed three times in three months to confirm the reproducibility of the test. In each case 4-5 TgPVRs per virus concentration were used. Then, PD ₅₀ of each experiment showed the very close and ^{_{10 3.25 TCID 50 ~10 3.33 TCID 50}} , was shown to be highly reproducible experimental system. Moreover, in TgPVR, as described later, the occurrence of paralysis and the presence of pathological changes were in good agreement, so that clinical symptoms using PD ₅₀ or LD ₅₀ as an index were sufficient indicators of neurotoxicity.

(Ii) Histopathological examination In conducting histopathological examination of TgPVR, TgPVR that died or died during the clinical observation period on the 14th day for TgPVR that survived for 14 days (TgPVR that died On the day, the brain and spinal cord were fixed with 10% formalin.

For the brain, the cortex, midbrain, thalamus, pons, cerebellum, medulla,
With respect to the spinal cord, 3 to 6 pieces of the cervical cord, thoracic cord, and lumbar cord were embedded in paraffin by a conventional method, and 6 to 9 μm thin sections were prepared as samples for histopathological examination.
The thin sections were stained with gallocyanine and then examined microscopically. The degree of pathological change in each tissue was in accordance with the evaluation method of the polio vaccine neurotoxicity test in monkeys of WHO.

 Table 1 shows the results of pathological changes in the central nervous system of TgPVR.

In the column of “mouse number” in Table 1, each number corresponds to the number of Tg21 described in (i) in FIG. In the column of “clinical observation”, “P (−)” means that no paralysis was observed during the period of 14 days,
“P (+)” means that paralysis was observed. P
In the case of (+), "4-9 (S)" means that paralysis occurred on the fourth day after virus inoculation and died on the ninth day. The same applies to “6-9 (S)” and “4-7 (S)”.

In the column of “Histopathological observation”, the observation site of the brain is 1 in the cortex, 2 is the thalamus, and 3 to 6 indicate the midbrain, cerebellum, pons, and medulla, respectively. The observation site of the spinal cord
"CC" indicates cervical spinal cord, "TC" indicates thoracic spinal cord, and "LC" indicates lumbar spinal cord. Further, in the observation column, "-" indicates that the tissue was not observed.

As is clear from Table 1, the strongest lesion was found in the lumbar spinal cord, which was the site of inoculation, and the presence or absence of a lesion in the lumbar spinal cord coincided well with the onset of paralysis. Lumbar spinal cord lesions mainly consisted of degeneration and loss of motor neurons in the anterior horn of gray matter, and mild infiltration of mononuclear cells such as microglia and lymphocytes as an inflammatory response was observed (Figure 5).
a, b). Lesions similar to the lumbar spinal cord were found in the cervical cord and thoracic cord of TgPVR, which had lesions in the lumbar spinal cord, although the degree of the lesion was light, but the lesion was similar to the lumbar spinal cord (Fig. 5c, d). In the brain, mild lesions were frequently observed, especially in the medulla and pons (FIGS. 5e and 5f).

Thus, TgPV with lesions in the lumbar spinal cord at the inoculation site
In almost all cases of R, the lesions spread to the medulla and pons via the thoracic and cervical spinal cords (Table 1), indicating that virus transmission through the central nervous system of Tg21 is easy.
It shows that PVR can be used as an animal system to examine the neurotoxicity of poliovirus in place of monkeys.

In the central nervous system of TgPVR inoculated intraspinally, lesions similar to those of monkeys (FIG. 5g) were observed. The most common sites of lesions are similar to monkeys, but the characteristic feature is that mononuclear cells such as microglia and lymphocytes have less proliferation and infiltration than monkeys (Fig. 5a- g). This pathological image is not only TgPVR which died suddenly due to paralysis,
It is also observed in TgPVR who has been paralyzed for a long time. In the case of PVRTg, which died suddenly, degeneration and disappearance of nerve cells were mainly observed.

[Example 2] Sensitivity of TgPVR by intraspinal inoculation method Four lines (Tg1, Tg5, Tg8 and Tg21) established by Koike et al.
Of TgPVR, the Tg1, if the F113 of 10 ⁶ TCID ₅₀ type 1 Sabin virus neurotoxin strength test standard virus was inoculated into the brain, any not show symptoms, cause finally paralysis 10 ^6.5 TCID ₅₀ Was. This very high virus concentration indicates that Tg1 inoculation into the brain is not suitable for testing the neurotoxicity of conventional vaccines.

On the other hand, as a result of intraspinal inoculation of T113 with F113 virus, 8 out of 10 mice (80.0%) even when F113 had 10 ² TCID ₅₀
Paralysis was observed (FIG. 4).

As a control, normal non-transgenic mice
ICR mice were intraspinally inoculated with 10 ⁶ TCID _{50 of} F113, but showed no symptoms.

Thus, the intraspinal inoculation of F113 against Tg1 was more than 10,000 times more sensitive than the intracerebral inoculation. There was no gender difference in this sensitivity.

Based on the results of FIG. 4, the results of calculating the PD ₅₀ of the F113 strain for Tg1, 10 cause paralysis in a small amount of viral load of ^1.6 TCID _50, showed very high sensitivity.

In addition, even when 10 ³ TCID _{50 of} F113 was intraspinally inoculated against Tg21, paralysis appeared in 4 out of 14 animals (28.6%) (FIG. 4).

From the above, it was found that intraspinal inoculation of virus against TgPVR was effective for neurotoxicity test.

[Comparative Example 1] Neurotoxicity test using monkeys The neurotoxicity test using monkeys was carried out according to the WHO method.

The samples used were the seven viruses used in the TgPVR test, and 0.1 ml (10 ⁶ TCID ₅₀ ) of each sample was inoculated into the spinal cord of 12 cynomolgus monkeys. However, S1D-38 / 2 and S1D-
For 38/4, we performed with 5 cynomolgus monkeys. Nineteen days after the inoculation, the animals were dissected and the central nervous system was examined histopathologically.

In the histopathological examination of the central nervous system, the pathological changes in the brain and spinal cord were defined as 1 for mononuclear cell proliferation / infiltration only, and 2 or more for neuronal degeneration. Of the cases involving neuronal degeneration, 2 cases where there is cell infiltration with local neuronal degeneration, 3 cases where there is cell infiltration with widespread neuronal degeneration, 3 The most severe case was designated as the maximum of 4. 7 brains (cortex, midbrain, thalamus,
Left and right lesions were recorded for the pons, cerebellum and upper and lower medulla, 12 cervical cords, and 18 lumbar spinal cords. Then, the average value of each of the brain, cervical spine, and lumbar spinal cord was synthesized, and the number divided by 3 was used as the number of lesions in one monkey, and the average value of the number of lesions in monkeys used in the experiment was used as the LS of the test virus strain. .

For 7 viruses derived from one strain of Sabin including F113, neurotoxicity test using monkeys (indicating LS) and TgPV
Table 2 shows the results of a comparison with a neurotoxicity test using Tg21 of R (PD ₅₀ as an index).

LS of monkeys of four viruses, F113, S1A, S1C and S1D
Is 0.77 to 0.85, and the PD ₅₀ by TgPVR is 10 ^3.7 to
10 ^3.3 TCID ₅₀ .

For the S1B virus, LS is 0.33, which is very attenuated compared to other viruses, and PD ₅₀ is 10 ^4.3 TCID ₅₀ and F113.
Was also more attenuated than the other four viruses, including

For the S1D-38 / 2 and S1D-38 / 4 viruses, the LS is 1.32 and 2.07, respectively, and the PD ₅₀ is 10 ^2.9 TCID ₅₀ and 1 respectively.
0 was ^2.4 TCID ₅₀ .

From the above, in neurotoxicity tests using monkeys, the degree of central nervous system lesion is used as an indicator of neurotoxicity, but PV
In RTg, since the onset of paralysis and the presence of lesions were in good agreement, it was found that the clinical symptoms of PD ₅₀ and LD ₅₀ could be sufficient indicators of neurotoxicity.

6 is a correlation diagram between the LS value and PD ₅₀ summarizes these results. Both are very well correlated, and the correlation coefficient γ =
It was -0.96.

Therefore, from these results, TgPVR can be used to test the neurotoxicity of a virus with different neurotoxicity even with the same Sabin type 1 virus with the same sensitivity as monkeys. It turns out that it is possible.

[Reference Example 1] Investigation of virus distribution in the central nervous system Tg21 was inoculated intracellularly with 10 ⁴ TCID ₅₀ of F113 virus.
The virus was recovered from the brain, spinal cord, and blood, and the distribution of the virus in the central nervous system was examined for three days immediately after the inoculation, with the aim of examining which part of the central nervous system the inoculated virus spread and its spread. The method is as follows.

To TG21, a 10 ⁴ TCID ₅₀ of F113 is a viral load of developing almost 100% paralysis, TG21 inoculated into spinal cord (1 group 9 animals), the two animals respectively after 1, 2 and 3 days of TgPVR The brain, spinal cord and blood were collected and virus was isolated. The brain and the spinal cord were each thickened by 2 heads, and the brain and the spinal cord 1 were added with 9 virus culture medium (Medium 199), and cooled with a homogenizer to form an emulsion at 2000 rpm and 20 rpm.
After centrifugation for 0.4 minutes, the supernatant was filtered with a pore size of 0.45 μm to obtain a separation material. As the blood, heparinized blood collected from two animals was used as a separation material as it was.

 FIG. 7 shows the results.

The inoculated F113 strain started to grow immediately in the spinal cord, peaked on the second day, and grew in the brain a few days after inoculation. No virus was recovered from the blood. A similar experiment was performed with normal non-transgenic ICR mice, but no results were obtained in which the virus was propagated in the brain, spinal cord and blood.

INDUSTRIAL APPLICABILITY The present invention can provide a more simplified test method for attenuated polio vaccine neurotoxicity test, which is highly sensitive to poliovirus.

According to the present invention, the results of clinical observation with TgPVR and the results of the neurotoxicity test in monkeys are well correlated. In addition, a large number of substantially homogeneous solids can be handled relatively easily, and stable data on the neurotoxicity of polio vaccine virus can be obtained. Furthermore, a very wide range of neurotoxicity tests can be performed, from virulent strains of poliovirus to attenuated viruses.

Therefore, it is possible to carry out a number of genetic characterization tests, in particular, a test on the neurotoxicity of a vaccine virus against monkeys, which has a function as a polio vaccine without showing pathogenicity, thereby producing a safe and effective polio vaccine. can do.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Satoshi Koike 3-18-22 Honkomagome, Bunkyo-ku, Tokyo Inside the Tokyo Metropolitan Institute of Clinical Medicine (72) Inventor Akio Nomoto 7-11-11 Ikegami, Ota-ku, Tokyo (72) Inventor Hiromichi Yonekawa 46-6, Marugasaki-cho, Omiya City, Saitama Prefecture (72) Inventor Choji Taya 1-13-16 Tabata, Kita-ku, Tokyo (72) Inventor Tatsuji Nomura Shibuya-ku, Tokyo Jingumae 6-chome 35-3-3-308 (56) References Special Table Hei 4-504200 (JP, A) Tokyo Medical University Magazine Vol. 45 No. 1 (1987) p23-p25 (58) Fields investigated (Int. Cl. ⁶ , DB name) G01N 33/15 A61K 39/13 C12N 15/09 Biosys Previews jois

Claims (4)

(57) [Claims] 1. A method for testing the polio vaccine neurotoxicity by inoculating a polio vaccine into the spinal cord of a transgenic non-primate vertebrate into which a poliovirus receptor gene has been introduced. Force test method. 2. The method according to claim 1, wherein the spinal cord is a lumbar spinal cord ampulla. 3. The method of claim 1, wherein the transgenic non-primate vertebrate is a transgenic mouse, a transgenic rat,
The neurotoxicity test method according to claim 1 or 2, which is a transgenic guinea pig or a transgenic hamster. 4. A polio vaccine comprising poliovirus type 1, 2
The neurotoxicity test method according to claim 1 or 2, which is a type 3 or type 3 attenuated strain.