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Патент USA US3031388

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United States Patent 0 ” 1C6
3,031,378
Patented Apr. 24, 1962
1
2
3,031,378
METHOD OF INACTIVATING VIRUSES
Morizo Ishidate, 608 Koenji 4-chome, Suginamiku,
Tokyo, Japan; Masakatsu Goto, 8-3 Yonbancho, Chi
yodaku, Tokyo, Japan; and Kazuo Ogasawara, 1 Bikin
cho l-chome, Showaku, Nagoya, Japan
No Drawing. Filed May 27, 1960, ?er. No. 32,122
Claims priority, application Japan May 30, 1059
EXAMPLE 2
10'6 ‘dilution of the Japanese B encephalitis virus
(Nakayama strain, i.c., LD50=10-7/0.03 ml.) was pre
pared from infected mouse 1brain, was mixed with pH 7.4
"sodium glucuronate as the same method as previously
. described, and maintained for 1 hour at 22° C., following
in an ice chest for 48 hours. After this procedure the
virus suspensions were inoculated into mice intracerebral
2 Claims. (Cl. 167-78)
In general, formalin, phenol, merthiolate, etc. have been
ly
used as chemical substances for inactivated vaccines of 10
‘
The results for 2 weeks after inoculation are given in '
bacteria and viruses. However, these substances .have
Table 2:‘
been more or less toxic thereby exhibit toxicity to the
host, 50 these chemicals are not desirable.
The present invention is based on a novel knowledge
a
'
Table 2
EXPERIMENTS ON INACTIVA'I‘ION OF THE JAPANESE
B ENOEPHALITIS VIRUS BY SODIUM GLUCURONATE
that, when certain concentration of glucuronolactone, 15
glucuronic acid or its salt will be mixed with viruses in
vitro, the viruses will be inactivated and also the antigenic
properties of the viruses will not be so much reduced
Concentration
of the sodium
glucuronate
Mortality
solution used
as by other known inactivating substances. The glu
curonolactone, glucuronic acid or its salt, especially, so 20
in dilution,
percent
dium salt has hitherto been appreciated as a part of nor
6. 25
12. 5'
25
mal component present in the tissue of animals, so it does
not exhibit any complication when administered to excess.
Control
Accordingly, the product obtained by the present inven
Ratio
Percent
1/10
1/ 10
0/10
10
10
0
9/10
90
‘tion can be advantageously used as improved vaccines.
EXAMPLE 3
The present invention shall now be explained in more
details. A virus suspension is treated at the room tem
The mixture of in?uenza virus (PR 8 strain,
perature (20° C.) or a lower temperature with the addi
EID50=10~7-6/0.2 ml. for 11 old embryonated chicken
tion of glucuronolactone, glucuronic acid or its salt to in
eggs) and sodium glucuronate which harvested from;
activate virus and then pH thereof is adjusted at 6-8. In 30 allantoic ?uid was kept one hour at 22° C. and in an
this case, one must take care that the virus may not be
ice chest for 48 hours. These suspensions were instillated
inactivated by other factors as heat and the like.
into miceintranasally.
,
I
V
The results of experiments in which mice inoculated
The results of the observation for 80 hours are given
‘in Table 3:
with the virus suspensions treated by the application of
the methods for the mouse hepatitis virus, Japanese B en 35
Table 3
cephalitis virus, rabies virus and in?uenza virus are
tabulated in the following.
EXAMPLE 1
10"“ dilution of mouse hepatitis virus (Busher strain,
i.p., LD50=10-7-6/0.2 ml.) was prepared with a Bacto
Brain Heart Infusion Broth of Difco Laboratories. Three
10"’ dilutions are prepared with 25%, 12.5% and 6.25%
EXPERIMENTS ON INACTIVATION OF THE INFLUENZA
VIRUS \VITH SODIULI GLUCURONATE
Concentration
of the sodium
glucuronate
40'
,
Morbidity
.
solution used
in dilution,
percent
Ratio
Percent
6. 25
12.5
25
Control
0/8
0/8
0/7
3/7
0
0
43
sodium glucuronate adjusted pH 7.04 with phosphate
buffer solution, and were maintained at the room tempera
ture for one hour and then in an ice chest for 12 and 45
48 hours. 0.2 ml. of the solution was intraperitoneally
0
inoculated in each mouse ( d‘ dd strain 10 to 12 g.) . Same
procedure was performed with 10-6 and 10-5 dilutions
EXAMPLE 4
of the mouse hepatitis virus. For controls, 10*5, 10-6
and 10-7 virus suspensions were prepared with only phos 50
‘phate butter solution. ,
104, 10-5 and 10-6 dilutions of the rabies virus (CVS
strain, i.c., LD50=10‘*7~4/0.2 ml.) were prepared in the
same manner as in Example 1, were kept 1 hour at 22°
C. and in an ice chest for 48 hours and were inoculated
The results for 10 days after inoculation of mixture
virus and sodium glucuronate are given in Table 1.
Table 1
EXPERIMENTS ON INACTIVATION OF‘ THE MOUSE 55
HEPATI‘I‘IS VIRUS‘ WITH SODIUM GLUCURONATE
respectively into mice intracerebrally (0” dd strain 10 to
12 g.).
The results are given in Table 4:
Table 4
Concen- Concentration of the mouse hepatitis virus dilution
tration of
Time of the sodi
leaviug um gluin the ice curonate
c
est
> (4° 0.)
10'"
10'‘5
solution
used in
EXPERIMENTS ON INACTIVATION O'F RABIES VIRUS
WITH SODIUM GLUCURONATE
1
,
Mortality
10*7
_
60
Mortality
Mortality
dilution,
percent
sodium
12 hours
48 hours
85
60
50
100
10
0
0
95
4/20
2/20
0/20
17/20
1/20
0/20
0/20
12/20
20
10
0
85
5
0
0
60
1/10
0/10
0/10
5/10
0/10
0/10
0/10
4/10
10
0
0
50
0
0
0
40
10'!
10-8
Mortality
Mortality
Mortality
solution used
65
17/20
12/20
10/20
20/20
2/20
0/20
0/20
19/20
10“4
glucuronate
_
Ratio Percent Ratio Percent Ratio Percent
6. 25
12. 5
25
Control
6. 25
12. 5
25
Controls
Concentration of the rabies virus dilution
Concentra
tion of the
.
70
in dilution,
percent
6. 25
12. 5
25
Control
Ratio
Percent
8/10
0/10
0/10
10/10
80
0
0
100
Ratio . Percent
0/10
0/10
0/10
10/10
0
0
0
100
Ratio
Percent
0/10
0/10
0/10
7/10
0
0
0
70
3,031,378
bodies in each group were tested by intracerebral inocula
From these experiments it is obvious that sodium glu
curonate inactivates viruses in vitro. Likewise, glucuronic
tion by the conventional method.
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R1340 0
Norm-Antigen 1: Sodium glucuronate inactivated virus. 11: Formalinized virus. III: M/SOO N-benzyl
(bis-?-ehloroethyD-amine N-oxide hydrochloride inactivated virus.
IV: Heat-inactivated virus.
sisting of glucuronolactone, glucuronic acid and alkali
(2) Comparison of neutralizing antibodies production
metal glucuronate and maintaining the pH at between 5-8.
of inactivated rabies by various methods.
Testing method.-—As in Example 5 (2) the mouse was 65
References Cited in the ?le of this patent
inoculated with the above treated virus. 7 days after the
“Glucuronolactone
(1952),” Corn Products Sales Co.
?nal immunization, the mice were sucri?ed and the sera
Chem. Div., 17 Battery PL, NY. 4, N.Y., 20 pp.
were obtained respectively. Titer of neutralizing anti
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