Note: Descriptions are shown in the official language in which they were submitted.
~9~3S HOE 76/B Q16
The present invention relates to an atoxic, immunogenic
product of tetanus toxin.
The invention provides an atoxic, immunogenic product which
ma~ be obtained from ~etanus toxin by means of a proteinase.
It also provides a tetanus vaccine containing the novel product.
As has already been known, tetanus toxin is highly toxic
towards mammals and also humans and must be detox~cated, in
order to be used as an immunizing agent. It has been known to
detoxicate the tetanus toxin by treating it with formaldehyde.
It has also been known that an atoxic, immunogenic product can
be obtained by a treatment of tetanus toxin ~ith a proteinase.
This atoxic, immunogenic product is labelled as fragment C of
the tetanus toxin in scientific literature.
It was a surprising fact which could not have been ~oreseen
that in the treatment o~ tetanus toxin with a ~roteinase, pre-
ferably with papain, a further substance having especially
advantageous properties is formed which can be characterized
chemically In a definite manner and which may be isolated ac-
cording to known biochemical processes.
The particularly advantageous properties of the product
~res er~,~af~o~ of ~h c
termed fragment B include the absence of toxi~city and the~im-
munogenicity of the same. These properties make the product
suitable as a component of vaccines againSt tetanus~
The present ~nvention therefore provides an atoxic, immuno-
genic product which may be obtained from tetanus tox~n by treat-
ing the same with a proteinase, preferably with papain, which
product IS characterized by the follow~ng parameters:
1. Sedimentation constant S20W 5~66+0~34
,9 2, Immunologic reaction with tetanus antitoxin:
~ 2 -
.
HOE 76/B 016
1(39~63s
.,
parti`~lly identical wi.th.that of tetanus toxi~n,
3, Immunologic reaction with fragment C:
not ident~cal.
4. Molecular weight~ determined by a gel electrophoresi~s in
sodium dodecylsulfate, of 95 000 - 5 000.
5. Cleavable by reduction into a sub-unit having a molecular
weight of 45~0Q + 2 50Q and a sub-unit immunoiogically
,i:dentical with the known derivat~ve of the l~ght chains
of the tetanus toxin, having a molecular weight of 48 0Qn +
2 50Q (each time determined in the sodium dodecyl-sulfate
electrophoresis~.
The variation$ shown in the parameters are due to the
limits of error within the methods of determinati.on~
The sedimentation constant was determined in an aqueous
~5 soluti,on of 0.2 molar NaCl, 0.02 molar Na2HPO4~ Q.03 molar
NaH2PO4 at a pH of 6,8 according to T~ Svedberg and K~O. Peder-
sen, "The Ultra~centri:fuge", The Clarendon Pres$J Oxford, ~940,
in an overlayer cell of an analytical ultracentrifuge, with
re,ference to V~nograd, Proc.Acad.,~ci.. USA, 49, 902 (1963~. The
overlayering action of the test solution containing the product
of the invention was effected on a ~ molar NaCl solution having
a pH value of 7.Q.
The immunologic reactivity was examined while using anti-
sera, which were obtained by the immunisation of rabbits with
tetanus toxoid or the fragment B of the invention, Use was
made of the known double diffusion technique. By means of this
technique it can be shown that the formerly described fragment
C IS clearl~ distin~ui$hed from the fragment B of the invention.
`~9 ~s. could further be shown, fragment B can be dlssociated into
.
1(~9963S
two fragments, one of which shaws the same immunologic behavior
as the derivative of the light chain described in Canadian Patent No.
1,066,621, issued Novenber 20, 1979.
The gel electrophoresis in a 7 % polyacrylamide gel with the
S use of sodium dodecylsulfate as a camponent of the electrophoresis buffer
has been carried out according to K. Weber and M. Osborn, J.Biol.Chem.,
Vol. 244, 4406 - 4412 (1969). Prior to carrying out the gel electro-
phoresis, the samples to be analyzed were mixed in an aqueous solution with
sodium dodecylsulfate up to a concentration of 1 % and were kept for 1
minute in boiling water. m e calculation of the molecular weight is
effected by a ca~parison with standard substances for determining the
molecular weight (Aldolase and Katalase which may be obtained by Messrs.
Boehringer, Mbnnheim, No. of Cat. 15 575 - "Eichproteine Grosse II").
The calculation method has been indicated in the above-cited paper by
Weber and Osborn.
Ihe navel atoxic, immunogenic product labelled fragment B
of the tetanus toxm may be obtained according tothe process described
in Canadian Patent No. 1,038,293 issued September 12, 1978, by treating
tetanus tox m with a proteinase. According to a preferred enbodlment,
fragment B is obtained by the treatment of tetanus toxin, which may
be produced fram culture filtrates of clostrium Tetani by way of ion
exchange chramatography, with papain, preferably in the form of a substance
being bound to a carrier, in the presence of a reducing agent.
In this process it has proved to be advantageous to choose
an incubation temperature which is higher than usual, i.e. between 30
and 60 C, preferably between 45 and 55 C. By this measure it is
possible to increase the yield of the product of
HOE 76/B 016
~9963S
the invention. Following the proteolytic action on the tetanus
toxin, the enzyme bound to a carrier is eliminated from the re-
action mixture, suitably by way of centrifuging, and the remain-
ing solution is subjected to a fractionation through a molecular
sieve. In this process the use of dextran cross-linked with
epichlorhydrin has proved to be advantageous, for example
Sephadex(R) G 100 of Firm Pharmacia, Uppsala or Ultrogel(R) of
LKB, Bromma or bio-gel p(R) of Bio-Rad Laboratories, Richmond,
Calif
In case there is used a soluble enzyme preparation, the
separation of the enzyme is effected in the process of frac-
tionation through the molecular sieve.
Once fragment B has been obtained in this manner, an anti-
serum may be prepared with the same by the immunization of test
~5 animals with fragments B, and by means of said antiserum it is
then possible to obtain an immuno-adsorbing agent in common
manner. This agent serves in its turn to extract fragment B in
a particularly pure form from the incubation mixture of said
fermentation solution.
It is also possible to eliminate fragment C or tetanus toxin
from a mixture with fragment B by immuno-adsorptive measures.
For this purpose, an antiserum against the known fragment C is
at first prepared. By means of this antiserum, an immuno-ad-
sorbing agent can be obtained in known manner. Said agent is
suitable in its turn to eliminate the remainder of fragment C
and of tetanus toxin from fragment B, since ragment C as well
as tetanus toxin react with the anti-fragment C-serum, however,
fragment B does not~
~, 29 Other processes of isolation, as they are known to the man
-- 5 --
HOE 76/B 016
1~9963S
skilled in the art for separating proteins having a different
electric charge, are also successful. They include above all
electrophoretic processes as well as processes of ion exchange
chromatography.
The pure fragment B from tetanus toxin which may be obtain-
ed in this manner proves to be non toxic as compared against the
tetanus toxin. Whereas 30 x 106 min.lethal doses are detected
per mg of the tetanus toxin, fragment B has 5 min.lethal doses
per mg. With regard to the toxic symptoms, fragment B is seen
to be completely different from tetanus toxin; it does not show
the symptoms characterized by spastic paralysis. In spite of
the reduction of the toxicity to 1:6x 106 it is advantageous to
treat fragment B with an aldehyde, as has been described in the
following.
For this process the product obtained by means of protei-
nases is treated after its isolation with a protein concentra-
tion of about 1 mg of protein per ml or less in a buffer solu-
tion having a pH value of from 6.0 to 8.5, preferably 7.8, and
~ /~5 ~e~ tre
a molarity in the range of from 0.01 to 0.2 molo with 0.015 to
~o/e~ rer/~r~
0.3 molo of an aliphatic mono- or dialdehyde of ~ to 6 carbon
atoms, preferably formaldehyde, during 14 to 28 days at a tem-
perature in the range of from 20 to 37 C. If desired, the
product may be subjected to a dialysis of 10 to 20 hours, pre-
ferably 15 hours, against a physiologically acceptable solution,
such as 0.15 molar sodium chloride, and/or it may be filtered
under sterile conditions. For preparing the vaccine, the pro-
duct is suitably also mixed with an adjuvant, for example, alu-
minum hydroxide. By dilution with a physiologically acceptable
29 solution, such as 0.1$ molar sodium chloride solution, the con-
' ,' '
- ' ',
.
HOE 76/B 016
1~399635
centration is adjusted to the desired antigen content.
These vaccines may be used by themselves, but also in com-
bination with other vaccines. ~ For combination there are sui-
table, above all, diphtheria toxoid, pertussis immunogenic
agents, poliomyelitis viruses, or measles viruses. By way of
adsorption tests it can be shown that about one half of all
protecting antibodies directed against tetanus toxin are direct-
ed against fragment B.
The following Examples serve to further illustrate the
present invention.
E X A M P L E 1:
~f~
t~ Clostrium-tetani are fermented in a latham medium. The
toxin formed in this process is adsorbed at diethylaminoethyl
cellulose ion exchangers and is elu_ted by a gradiently rising
molarity of a phosphate buffer having a pH value of 7 from 0.01
mole to 0.4 mole. The fractions containing the toxin are again
separated by chromatography in a column which is filled with
Sephadex(R) G 100, and the fractions containing the toxin are
extracted and combined.
2.5 Grams of tetanus toxin in a solution having a final
concentration of 15 mg of tetanus toxin/ml of a 0~1 molar phos-
phate buffer of a pH of 6.5 containing 0.01 mole of Na2EDTA
and 0.001 mole of cystein-HCl are mixed with 40 mg of papain.
The papain contains 30 units of enzymatic activity/mg of sub-
stance. At first, the mixture is maintained for 1 hour at
45 C, and thereafter for another 2 hours at 55 C.
For the separation of the tetanus toxin there may be used
preferably also papain bound to a carrier.
SO ~9/~/
29 For th;s purpose, a solution of 500 ml' of papain,dialyzed
- 7 ~
HOE 76/B 016
1~9963S
against ~r0.1 molar Na2CO3 buffer of a pH of 10.0 is combined
with 50 mg/m~ of an agarose gel activated with cyanobromide
and the mixture is maintained for 24 hours at 4 C. The reac-
tion product washed several times with a solution containing
4 moles of urea and 0.5 mole of NaCl per liter is used in a
manner analogous to that of the soluble enzyme for the proteo-
lytic separation of the tetanus toxin.
Upon cooling of the mixture, the volume is concentrated by
means of an ultrafilter, and the mixture is then introduced
into a column of a length of 10 x 100 cm filled with Sephadex(R)
G 100. The elution is effected with a 0.1 molar trishydroxy-
methylaminomethane-hydrochloric acid buffer having a pH value
of 8.0 and containing 1 mole of NaCl.
During the elution, the adsorption in the range of the
wave length of 280 nm is measured continuously. The fractions
showing an adsorption in this range are collected separately.
There are formed 4 fractions, the first of which representing
a double peak.
In the course of a repeated chromatography carried out
under the same conditions, the two peaks are separated, and
finally the second peak is isolated. This latter peak con-
tains the atoxic, immunogenic product of the invention, the
fragment B from tetanus toxin.
The fragment B of the invention can be obtained in a
particularly simple manner, if an antiserum directed against
the known fragment C (10 ml with 1 000 IU/ml~ is bound in
common manner to agarose activated by BrCN, and a preparation
of fragment B ~hich has been partially purified by gel chroma-
29 tography is subsequently mixed with such an immuno-adsorbing
- 8 -
HOE 76/B 016
~099635
agent. After 60 minutes of stirring, the gel is separated to-
gether with the impurities bound to it, and fragment B is ob-
tained by another gel chromatography.
It is also possible to neutralize the impurities by adding
anti-fragment C-antiserum (IgG fraction) and to separate the
f ~
_Lu~ *L}~ complex compounds from fragment B by way of gel
chromatography.
Finally, fragment B of the invention may also be obtained
if an immuno-adsorbing agent for fragment B is prepared via an
antiserum, with the aid of a fragment B once prepared. For this
purpose, known processes may be applied. By means of the immu-
no-adsorbing agent, fragment B is adsorbed selectively from
the mixture with other reaction products from the proteinase
treatment of tetanus toxin, whereupon it may be eluted selecti-
vely from the immuno-adsorbing agent.
E X A M P L E 2:
Fragment B is diluted with 0.~ molar phosphate buffer
solution of a pH of 6.5 to 200 ~g of protein per ml, mixed with
0.06 % formaldehyde and allowed to stand at 37 C for 21 days.
Thereafter, the solution is dialyzed for 16 hours against
several t~mes its volume of 0.15 molar sodium chloride solu-
tion and is then processed in common manner into a vaccine.
_ g .~
.
