Method for the inactivation of microbial toxins and attenuation of vaccines

A method for the inactivation of microbial toxins and attenuation of vaccines by treating the toxin or vaccine with a tannin, especially a condensed tannin derived from fruit of the genus Diospyros, particularly the persimmon, without loss of the desired antigenicity.

This invention relates to the inactivation of microbial toxins and the 
attenuation of vaccines and to a new condensed tannin having this utility. 
BACKGROUND OF THE INVENTION 
Toxins produced by microbial agents, for example bacteria, can be used as 
antigens in the process of active immunisation, where the production of 
antibodies is stimulated by the introduction of a specific antigen. 
Frequently, the toxin must be inactivated so that it can be administered 
safely, while at the same time its antigenic effect must be maintained. An 
inactivated toxin, known as toxoid, is commonly prepared from various 
microorganisms by the addition of formaldehyde. Another compound used for 
this purpose is tannic acid, obtained from fermented oak galls. 
The manufacture of toxoids and vaccines involves very close quality control 
and the products require special handling and storage. Legislation has 
been passed in many countries controlling the manufacture, storage and use 
of vaccines in order to ensure the maximum safety. 
Nevertheless, biological preparations of this type exhibit various 
side-effects which are unavoidable even under strict control. For 
instance, pertussis vaccine, which is epidemiologically regarded as a 
necessity, especially in developing areas such as South East Asia and 
Africa, is known to produce side-effects such as swelling at the 
inoculation site, pain, systemic fever, vomitting and also symptoms of 
shock or, occasionally, serious brain damage. There is thus a real need 
for a method which will eradicate the unpleasant side-effects of such a 
vaccine, while at the same time the prophylactic effect is maintained. 
SUMMARY OF THE INVENTION 
We have now found that tannin derived from the persimmon and related 
species can be used to inactivate microbial toxins and to attenuate 
vaccines without affecting their antigenicity. 
The fruit of the persimmon (Diospyros Kaki Thumb.) contains tannin, a 
simple aqueous extraction of which is known as an astringent and as a 
fining agent in the production of sake in Japan. The present invention is 
based on the surprising discovery that tannin from the persimmon fruit and 
the fruit of related species of Diospyros has a wholly unexpected effect 
on the toxins produced by microorganisms and on the side-effects of 
various vaccines. 
According to the present invention there is provided a method for the 
inactivation of a microbial toxin or the attenuation of a vaccine, which 
comprises contacting the toxin or including in the vaccine tannin derived 
from fruit of the genus Diospyros.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
The genus Diospyros is in the family Ebenaceae and includes, for example 
the persimmon itself (Diospyros Kaki Thumb.), the small persimmon 
(Diospyros lotus L.) and ebony (Diospyros ebenum Koenig). 
The tannin may be extracted by any convenient method known and used in the 
extraction of vegetable tannins. 
However, we have found that a particularly useful process for extracting 
the tannin comprises the steps of 
(a) extracting the fruit with water or a water-miscible organic solvent or 
a mixture of water and a water-miscible organic solvent and removing 
insoluble matter; 
(b) except where an alcoholic extraction solvent is used in step (a), 
concentrating the liquid extract, diluting with a lower alcohol and 
removing insoluble matter; 
(c) precipitating tannin by adding to the alcoholic phase a non-solvent for 
tannin; 
(d) dissolving the precipitated tannin in water, sterilising the solution 
and freeze-drying. 
The extraction solvent in step (a) is preferably acetone, but alternatively 
another water-miscible solvent such as methanol or ethanol can be used. 
The non-solvent for the tannin, added to the alcoholic solution to 
precipitate the tannin, is conveniently an organic non-polar "solvent," 
such as an aliphatic ether, e.g., diethyl ether. 
Preferably, the fruit, which may be ripe or unripe is first heated before 
being extracted in step (a). Heat treatment at, for example, 100.degree. 
to 120.degree. C. is advantageous. Step (a) is then effected, preferably 
on the minced fruit, either at ambient temperature or at an elevated 
temperature. 
In step (d), the aqueous solution of the tannin is preferably dialysed 
before sterilisation, conveniently at an acid pH, e.g., pH 3-5, especially 
pH 4, obtained by addition of an acid such as hydrochloric acid, sulphuric 
acid or acetic acid. 
The tannin is obtained as a pale brown powder. 
We have found that when the extraction process detailed above is used, it 
is possible to obtain a condensed tannin free from other tannin materials, 
which is a new compound and forms part of the present invention. 
Thus, according to the present invention there is further provided a 
condensed tannin derived from fruit of the genus Diospyros, having the 
following properties: 
(a) a pale brown powder in the freeze-dried state; 
(b) soluble in water; sparingly soluble in methanol, ethanol, pyridine, 
acetic acid; insoluble in diethyl ether, ethyl acetate, chloroform; 
(c) aqueous solution gives a dark blue coloration with ferric chloride and 
a brown coloration with lead acetate; 
(d) an infra-red absorption spectrum substantially as shown in FIG. 2 
herein, when measured in liquid paraffin, having main adsorption bands at 
3400, 1615, 1530, 1380, 1200, 1100 and 1040 cm.sup.-1 ; 
(e) an ultra-violet absorption spectrum substantially as shown in FIG. 1 
herein when measured in an aqueous solution, having an absorption maximum 
at about 275 nm; 
(f) a chromatogram substantially as shown in FIG. 3 herein having a single 
peak in a gel filtration using a strongly acidic styrene-divinylbenzene 
copolymer sulphonate resin column and water as a mobile phase; and 
(g) an ultra-centrifuge Schlieren pattern substantially as shown in FIG. 4 
herein at about 46,633 revs/min after 26 minutes. 
This condensed tannin, or the tannin obtained by any of the extraction 
procedures, may be converted into a non-toxic salt such as an alkali metal 
salt, e.g., the sodium or potassium salt, which can also be used in the 
inactivation method of the invention. 
The chemical nature of the condensed tannin was confirmed by the following 
tests. The material was boiled in 20% aqueous hydrochloric acid for 3 
minutes. A major portion of the tannin was obtained as a phlobaphene-like 
precipitate which was filtered off. The reddish purple filtrate contained 
cyanidin and delphinidin. 
The substance was also heated at 100.degree. C. in n-butanol containing 5% 
hydrochloric acid for two hours. The anthocyanidins thus formed were 
isolated and quantitatively determined to show a ratio of cyanidin to 
delphinidin of 1:3. 
A sample of the condensed tannin was subjected to fusion with an alkali at 
185.degree. C. under nitrogen for ten minutes to afford protocatechuic 
acid and gallic acid at a ratio of 1:3, in addition to some 
phloroglucinol. 
These tests, together with the physical characteristics listed above 
indicate that the condensed tannin is indeed a single condensed tannin 
containing as a structural unit gallocatechin and catechin. 
Persimmon tannin, especially the purified condensed tannin, appears to be 
considerably less toxic than tannic acid. Thus, the peritoneal stimulating 
syndrome (e.g., retention of ascites or adhesion of the peritoneum and the 
like) was not observed in Wistar-Imamichi strain male rats of body weight 
200 to 250 g when 0.1 ml of a 0.5% aqueous solution of the tannin was 
injected intraperitoneally. Peritoneal irritation is observed on injection 
of 1-5% aqueous solutions, but no deaths are observed. Injection of 0.1 ml 
of a 1% aqueous solution intravenously at the tail is not lethal, but 
injection of 0.1 ml of a 2% aqueous solution does cause some fatalities. 
No local irritation or swelling is seen when 0.05 ml of a 1.25% aqueous 
solution of persimmon tannin is injected into the plantar region of the 
paw of ddY-strain male mice (5 weeks old). In contrast, a strong 
irritation is observed when only a 0.1% aqueous solution of tannic acid 
(pharmacopoeia grade) is injected. 
A model test for the inactivation of toxins and attenuation of substances 
producing side-effects involves observing the action of the tannin on 
various snake venoms. 
TEST A 
In a test tube, 0.1 ml of a solution of 40.gamma. of the venom of the Erabu 
sea snake (Lacticauda semifasciata) in distilled water was neutralised 
with an equal amount of an aqueous solution of the tannin and the 
resulting solution is injected intraperitoneally into 5 week old 
ddY-strain male mice. The venom is found to be inactivated at a tannin 
concentration of at most 0.039%, while the purified condensed tannin is 
effective at a concentration of 0.025%. In contrast, the venom is not 
inactivated by tannic acid J.P. concentrations of up to 1.25%. 
TEST B 
In an in vitro test in which 0.1 ml of a solution of 20.gamma. of the venom 
of the Habu snake (Trimeresurus flavoviridis) in distilled water was 
admixed with 0.1 ml of an aqueous solution of persimmon tannin, 
subcutaneous bleeding in rabbits was inhibited at a concentration of 
0.078% or above. A concentration of at least 5% is required for similar 
inhibition by tannic acid J.P. 
TEST C 
Snake venom bound with persimmon tannin cannot be separated with a 
centrifuge and no precipitation line is observed in a precipitation 
reaction with antiserum using the gel-difusion method. In contrast, tannic 
acid J.P. bound to snake venom can be separated on a centrifuge whereupon 
the venom again becomes virulent. Also, a precipitation line is observed 
in a precipitation reaction by the gel difusion method. 
Persimmon tannin, especially the condensed tannin defined above can thus be 
used for the inactivation of toxins, particularly microbial toxins. 
Particular toxins of interest are those from bacteria such as Bordetella 
pertussis, Corynebacterium diphtheriae, Clostridium tetani, Clostridium 
botulinum, Staphylococcus; and Streptococcus. Attenuation of substances 
producing side-effects e.g., pyrogens in vaccines such as typhoid, 
paratyphoid, cholera, pertussis, equine strangles and anthrax can also be 
attenuated. 
While tannin from the fruit of the persimmon itself is preferred, the 
fruits of other species of Diospyros can equally be used. 
The amount of tannin to be added to the toxin or vaccine will, of course, 
vary depending on the nature of the toxin or vaccine and the strength. In 
general it will be satisfactory to add a minor amount of an aqueous 
solution having a persimmon tannin concentration of not more than 0.1%. 
The use of persimmon tannin, particularly the condensed tannin has a number 
of advantages, which may be summarised as follows: 
(1) pronounced inactivation of a toxic action can be achieved using a 
relatively small amount of the tannin so that little consideration need be 
paid to any toxicity of the tannin itself; 
(2) aqueous solutions of the tannin are thermally stable; 
(3) an irreversible bond with toxic substances is formed; 
(4) the desired antigenicity is maintained when a toxin is inactivated or a 
substance producing side-effects is attenuated; and 
(5) the material may be easily obtained by a simple method in good yield 
from a readily available starting material. 
The following Examples illustrate the invention further. 
EXAMPLE 1 
EXTRACTION AND ISOLATION OF CONDENSED TANNIN 
Persimmon fruits (Diospyros Kaki Thumb.) having seeds and calyces removed 
(10 kg) and a small amount of distilled water were heated to 120.degree. 
C. for about 1 hour. The fruit was then minced and 15 liters of acetone 
were added and the mixture left to soak at ambient temperature for 24 
hours. The mixture was then filtered and the filtrate concentrated under 
reduced pressure to about 2 liters. To this concentrate 10 liters of 
methanol were added and the mixture again filtered. To the filtrate were 
added 6-10 liters of diethyl ether and the precipitate which separated was 
recovered. The precipitate was dissolved in 7.5 liters of distilled water, 
adjusted to pH 4 with dilute hydrochloric acid, placed in a cellulose tube 
and then dialysed against running water. The contents of the tube were 
then filtered and sterilised under high pressure at 120.degree. C. for 20 
minutes. The resulting aqueous solution was freeze-dried to give 50 g of 
condensed tannin as a pale-brown fine powder. 
Similar material can be obtained using fruits of Diospyros lotus and 
Diospyros ebenum. 
EXAMPLE 2 
INACTIVATION OF ALPHA STREPTOCOCCUS TOXIN 
(a) The toxin employed had a minimum lethal dose (M.L.D.) of 0.55 mcg in 
R.F.V.L./strain male mice having a body weight of 25 g.+-.2 g. 
To 0.1 ml samples of toxin solutions containing 1.1 mcg (2 MLD) were 
admixed equal volumes of aqueous persimmon tannin solutions prepared by 
multiple dilution of 0.5% up to 0.0019%. The mixtures were left at ambient 
temperature for 10 minutes and groups of 3 animals were each 
intraveneously injected with 0.2 ml of the mixture per animal. The state 
of the animals was then observed fo 48 hours. In a controlled experiment 
samples of the toxin were mixed with equal volumes of tannic acid 
solutions containing up to 0.1%. 
At persimmon tannin concentrations of more than 0.0039% at least some 
animals survived and at a concentration of 0.0078% or higher all animals 
survived. In contrast, all animals died in the 24 hours following 
administration when tannic acid was used, even at a concentration of 0.1%. 
(b) The above test used a relatively crude tannin extract which had not 
been dialysed. Using the purified condensed tannin, this time in 
ddY-strain male mice having a body weight of 25=2 g, at concentrations of 
1.5, 2.0 and 2.5 MLD per 0.1 ml, the following results were obtained. 
Protection against fatalities was observed at a concentration of 0.00125% 
when 1.5 MLD was injected and at 0.0025% when 2.5 MLD was injected. 
EXAMPLE 3 
INACTIVATION OF PERTUSSIS GUNDEL TOXIN 
The toxin used was a supernatant prepared from 200 mcg/ml of living 
microorganisms by ultrasonic disintegration and subsequent refrigerated 
centrifugation at 10,000 g for 30 minutes. This toxic solution produce 
local bleeding in a guinea pig on subcutaneous injection of 0.05 ml of a 
1,000-fold diluted solution. 
For the test, the toxic solution was diluted 100 times and then mixed with 
an equal volume of an aqueous persimmon tannin solution. Different 
mixtures were made containing different concentrations of tannin. The 
mixtures were left for 10 minutes and a 0.1 ml portion was then 
subcutaneously injected into guinea pigs. After 24 hours inactivation was 
estimated by observation of induced bleeding. 
Results: at an aqueous tannin concentration of 0.125%, no bleeding was 
observed. Slight bleeding was observed in the case of a 0.031% solution, 
although this bleeding was less than that produced by a 100-fold dilution 
of the toxin used as a control. 
EXAMPLE 4 
REDUCTION IN LEUCOCYTE INCREASE BY PERTUSSIS VACCINE 
Reduction in leucocyte increase, considered to be a side-effect of 
pertussis vaccine when injected, was examined using aqueous persimmon 
tannin samples, including the purified condensed tannin. 
Method: the pertussis vaccine used was a suspension in phosphate buffer 
which contained 40 billion/ml of Bordetella pertussis and thimerosal at a 
1/10,000 concentration. Equal volumes of the pertussis suspension and 
persimmon tannin solutions having various concentrations were mixed and 
left at ambient temperature for 10 minutes. The mixtures (0.5 ml portions) 
were then intraperitoneally injected into ddY-strain male mice (4 weeks 
old) and the number of peripheral leucocytes in each animal was determined 
after 72 hours. 
As controls, there were used two groups of animals, one of which was 
injected with a twice diluted sample of the pertussis suspension in 
phosphate buffer and the other injected only with the phosphate buffer. 
The results are shown in the following Table. In the Table, column A 
refers to the use of a relatively crude persimmon tannin extract while 
column B refers to the use of a purified condensed tannin. 
______________________________________ 
Table for Example 4 
Tannin Concen- 
Number of leucocytes 
tration (Average of 5 animals) 
% A B 
______________________________________ 
Treated 0.1 4860 4750 
animals 0.02 6200 5200 
0.004 13320 11300 
0.0008 26620 24500 
B. pertussis 22820 
22820 
Control only 
Buffer only 4310 4310 
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It can be seen from the results that a complete inhibition of leucocyte 
increase can be achieved at a persimmon tannin concentration of 0.1%, 
while a moderate effect on leucocyte increase is exerted at lower 
concentrations, a concentration of 0.004% being sufficient to reduce the 
leucocyte increase to about half its normal value. 
EXAMPLE 5 
INFLUENCE UPON ANTIGENICITY OF PERTUSSIS VACCINE 
Using the same microorganism suspension as in Example 4 above and a 0.04% 
aqueous solution of persimmon tannin, equal volumes of the two liquids 
were mixed. As a control the twice diluted microorganisms suspension in 
phosphate buffer was used. 
These vaccines were diluted 5, 25 and 125 times and then intraperitoneally 
injected into ddY-strain male mice (4 weeks old) in 0.5 ml portions. 
Ten days after the injection, the animals were intracerebrally challenged 
with the live microorganism and thereafter the survival of the animals was 
observed over 14 days. No significant difference was noted in the survival 
of the animals. Accordingly, it was seen that the immunogenic effect in 
every case was the same and the persimmon tannin had no adverse effect on 
antigenicity. 
EXAMPLE 6 
INACTIVATION OF DIPHTHERIA TOXIN 
Purified diphtheria toxin was diluted from 200 to 3,200 times and an equal 
volume of a 0.2% aqueous solution of persimmon tannin was mixed with each 
dilute liquid. Controlled samples were obtained by mixing physiological 
saline solution instead of persimmon tannin. 
Hartley strain male guinea pigs having an average body weight of 300.+-.10 
g were divided into groups of three animals. Each animal was 
subcutaneously injected with 0.2 ml of the test liquid in the dorsal 
region and the survival was observed for 7 days. 
Results: the controlled group of animals was observed to die within 24 
hours of injection with the 200 and 400-fold dilutions of toxin, and 
within 48 hours in the case of injection with 800-fold dilutions of toxin. 
Animals treated with toxin solutions diluted at least 1600 time survived. 
All animals in the group treated with toxin plus tannin survived. 
Autopsy of the guinea pigs which had died indicated noticable bleeding in 
the adrenal glands in every case. 
EXAMPLE 7 
ATTENUATION OF TETANUS TOXIN 
Equal volumes of tetanus toxin solution and aqueous persimmon tannin 
solution were mixed and left at room temperature for 10 minutes. 
R.F.V.L/strain female mice having an average body weight of 23.+-.2 g were 
divided into groups of 3 and each animal was subcutaneously injected with 
0.4 ml of the test liquid prepared as above. A series of experiments was 
conducted, using successive samples in which the tannin was diluted while 
the quantity of toxin was maintained constant; 
Results: when tetanus toxin was applied at 150 MLD all the animals died 
within 24 hours when only the toxin was injected. 
(i) All the animals survived with a tannin solution of 0.75%, but all died 
with a tannin solution of 0.375%. 
(ii) All animals survived with a condensed tannin solution of 0.5%, but all 
animals died with a condensed tannin concentration of 0.25% the average 
time of death was 72 hours. 
From these results it will be seen that tetanus toxin is effectively 
attenuated by persimmon tannin. 
EXAMPLE 8 
EVALUATION OF PERSIMMON TANNIN USING SNAKE VENOM 
Method: the venom of the Erabu sea snake (Lacticauda semifasciata) 
(40.gamma.) in 0.1 ml was mixed with an equal volume of solutions of the 
purified condensed tannin at various dilutions. The resulting mixture was 
injected intramuscularly into ddY-strain female mice (5 weeks old; 
40.gamma. of venom equals 2 MLD). 
Results: protection against fatality was observed using 0.025% solutions of 
the condensed tannin. Protection against 4 MLD required 0.05% and against 
6 MLD required 0.1% tannin solutions.