CA1063020A - Derivatives of diphtheria toxin, process for preparing them and agents containing them - Google Patents
Derivatives of diphtheria toxin, process for preparing them and agents containing themInfo
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- CA1063020A CA1063020A CA313,332A CA313332A CA1063020A CA 1063020 A CA1063020 A CA 1063020A CA 313332 A CA313332 A CA 313332A CA 1063020 A CA1063020 A CA 1063020A
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- fragment
- diphtheria toxin
- aldehyde
- modified derivative
- diphtheria
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Abstract
ABSTRACT
This invention is directed to a process for the preparation of a modified derivative of fragment B of diphtheria toxin in which fragment B which is free from denaturing agent is combined with an aliphatic mono- or di-aldehyde having a chain length of 1 to 6 carbon atoms at an aldehyde concentration of 0.035 M to 0.35 M and the mixture is allowed to stand for 5 minutes to 50 hours at 1° to 20°C. The aldehyde is removed and the modified derivative of fragment B of diphtheria toxin is recovered. The invention is also directed to this modified derivative. These products are useful in the preparation of diphtheria toxin.
This invention is directed to a process for the preparation of a modified derivative of fragment B of diphtheria toxin in which fragment B which is free from denaturing agent is combined with an aliphatic mono- or di-aldehyde having a chain length of 1 to 6 carbon atoms at an aldehyde concentration of 0.035 M to 0.35 M and the mixture is allowed to stand for 5 minutes to 50 hours at 1° to 20°C. The aldehyde is removed and the modified derivative of fragment B of diphtheria toxin is recovered. The invention is also directed to this modified derivative. These products are useful in the preparation of diphtheria toxin.
Description
The present invention relates to derivatives of the diph-theria toxin, to a process for preparing such derivatives by modification of a partial molecule of the diphtheria toxin and to agents, in particular to diphtheria vaccines, which contain one of such derivatives of the diphtheria toxin.
Conventional diphtheria vaccines for the active immuniz-ation contain almost exclusively the antigen prepared by inact-ivation of the diphtheria toxin with formaldehyde. This substance, also called toxoid, is provided with a great number of determinants, of which only few may play role for the produc-tion of protecting antibodies against diphtheria. Elimination of such determinating groups which are of no importance for the protection is desirable in order to obtain antigenic or immunogenic substances which may have higher specificity and may be better tolerated.
The diphtheria toxin is produced by cells of Coryne-bacterium diphtheriae and set free into the nutrient medium extracellularly as a single polypeptide chain having a molecular we~too`f about 62,000. Mild proteolysis in the presence of compounds which are capable of splitting disulfide bridges in proteins, for example thiol compounds, produce two characteristic fragments of the diphtheria toxin, i.e. the so-called fragment A (molecular weight about 24,000) coming from the NH2-terminal portion of the toxin and the so-called fragment B (molecular weight about 38,000) coming from the carboxy-terminal portion. ~ -Fragment A can be easily isolated by methods of the protein chemistry and is stable in native form in physiological buffer solutions. Fragment B, however, can be stored in solution in isolated state only in the presence of denatured agents such as urea, -- ~
.. . , . . - . : .. .. .
-` 1063~20 guanidine hydrochloride or surface-active detergents. Elimination of the denaturing agent then gives a completely water-insoluble fragment B which impedes handling cons1derably~ The not-denatured - -fragment B, however, is considered to play an important role in the production of protecting antibodies after parenteraf admlni-stration.
Thus, it was a task of the present invention to modify the fragment B of the diphtheria toxin ~n such a manner that it could be kept in solution in a physiologically tolerated medium without loss of its antigenicity. In particular, this fragment B should be able to be used in its modified form as essential immunogenic constituent of diphtheriavaccines and to replace in these ~accines the conventional formaldehyde toxoid.
Now, we have found that this aim can be achie~ed by a process the most important feature of which is to avoid the spontaneous irreversible precipitation of frag~ent B, which can always be ob-served after elimination of the denaturing agents such as urea, by a slight chemical modification oi fragment B either before or after separation of fragment A from the molecular structure of the diphtheria toxin, which is a complex oi fragments A and B.
Accordingly, the subject of the present invention is a process for preparing a derivati~es of fragment B of the diphtheria toxin, which comprises treating an aqueous solution of the diph- -theria toxin with an aliphatic mono- or di-aldehyde having a chain length of 1 to 6 carbon atoms, preferably formaldehyde, with an aldehyde concentration of 0.0015 M to 0.035 ~S, preferably 0.002 to 0.02 M, at 1 - 20 C, preferably 1 - 6 C, for 5 minutes to 50 hours, reicting the toxin, before or after the treatment ,- . "
- -- . . ~. ,, ;. ; , , ' ~
with aldehyde, în the presence oi a compound splittlng o~f disul~ide bridges, with a proteolytic enzyme, recovering the derivative of fragment B from the solution obtained in the pre-sence of a denaturing agent by protein-chemical isolation pro-C~SS~S ~JU S~p~l ~L~IIg iL 1~ Lh~ ~r~a~u~ a~Gr.t.
Aiter isolation of the derivative of fragment B, the de-naturing agent is eliminated by dialysis or other comparable methods which permit the separation of low molecular substances, for example by gel filtration. The aldehyde used ior the reaction and which has not been completely consumed can be eliminated from the batches by similar methods.
The derivative prepared according to the method of the in-vention, when diluted in physiologically tolerated, isotonically aqueous media, does no longer show the denaturation and precipitation phenomena known from the native fragment B.
In the same manner as ~ragment B, the derivative does not have the toxicity of the diphtheria toxin. It has the same behaviour as the antigen and is capable of ~nduc_ing protecting antibodies against diphtheria in the human or animal organism.
The derivative prepared according to the invention can be purified by the conventional proteln-chemical methods. How-ever, in view oi' the high toxicity oi' the native diphtheria toxin and the fact that protein fractionation methods lead only very seldom to a 100% separation oi accompanying substances, especially oi those protein bodies having a similar structure, it is oi advantage to submit the product prepared according to the invention to another treat~ent with aldehyde, which treatment comprises combining the derivative oi iragment B prepared according to the ~-.
...... . . . . .
':
.. . .
. ' . I ' , . . . .
, . :' ", 1063~Z0 invention with an aliphatic mono- or dl-aldehyde having a chain length of 1 to 6 carbon atoms, preferably formaldehyde, with an aldehyde concentration of up to 0.035 to 0.35 ~5 and allowing the whole to stand for 14 to 28 days at 20 - 37C, removing the aldehyde? for example by dialysis. and isolatin~ the modified derivatives of said fragment B.
This additional treatment by aldehyde gives the fragment B
an increased stability in aqueous media. Thus, it was assumed that this stable derivative of the fragment B could also be obtained by reacting the diphtheria toxin, as known in the pre-paration oi the fragment B, with a proteolytic enzyme in the pre-sence of substances splitting off di-sulfide bridges, for example thiol compounds, combining the solution ~ith a denaturing agent, racovering the fragment B advantageously ~y chromatography, in particular by gel iiltration, remov~ng the denaturing agent by gel eiltration-and subsequently allowing the fragment B so ob-tain-d, immedi&tely thereafter, to stay together with an ali-phatic mono- or dialdehyde having a chain length of 1 to 6 carbon atoms, preferably formaldehyde, at a concnntration of 0.0015 Ml to 0.03~ M~ for 5 minutes to 50 hours at 1 to 20 C. If desired, a second treatment with aldehyde for the modification of the deri-vative may follow. However, the fragment B may also be reacted directly after its isolation with the increased quantity of al-dehyde.
Isolation of a modified fragment B from th~ conventional diphtheria toxoid is not possible, because the use o~ essentially higher quantities of formaldehyde produces a co-valent cross-linkage between the fragments A and B. However, there is no impeding reason not to react the solution containlng the frag-. . .
.
. ' . ' ' ' - ' : ' ' . ' . :.
' ' ~ ' ' ' ~ ' :' ., : , , ., ment B with aldehydes in order to obtain derivatives having the advantageous properties regarding stability and antigenicity.
As starting materials, there are used diphtheria-contain-ing aqueous media such as those which are also used for the isolation of fragment B, for example culture filtrates which are more or less pre-purified and enriched with regard to diphtheria toxin, highly purified toxin preparations or solutions of frag-ment B.
Proteolytic enzymes used in the preparation of fragment B are pronase, papain, subtilisin, preferably, however, trypsin (3.4.21.4), in soluble form or in a form bound to a solid car-rier. It has been found advantageous to stop the proteolytic reaction with the aid of an inhibitor, for example trypsin (3.4.21.4) with the aid of a trypsin inhibitor from animal or-gans or plants. If proteolytic enzymes bound to a carrier are used, the application of inhibitors is not necessary.
As compounds which split off di-sulfide bridges, thiol-compounds are preferred. In this respect, there may be used, for example cysteine, mercapto-ethanol, di-thioerythritol, or ~pref,era~l~ di-thiothreitol.
Denaturing agents in the sense of the present invention are chemical compounds with the aid of which a dissociation of hydrogen linkages in protein molecules is made possible.
Known agents which dissociate hydrogen linkages are, for example urea or guanidine hydrochloride.
Urea is known for the fact that, with its aid fragment B can be kept in solution at concentrations of ~0.6 M. Advan-tageously, about 4 to 6 M of urea or about 2 to 4 M of guanidine hydrochloride are used.
': :
- .
^ 1063~20 In addition to the methods described herein ln the present invention, the present invention also relates to derivati~es of the diphtheria toxin, which derivatives are characterized by the parameters found during their preparation.
Eventually, the invention also relates to agents which contain the diphtheria toxin derivative o~ the invention, in particular to diphtheriavaccines which are to be used as prophylactic drugs against diphtheria or for the manufacture of diphtheria antisera or also of dia~n~stic preparations. For increasing the stability in solution of the product of the invention, it may be suitable to add amino-acids or carbohydrates to the physiologically tole-rated aqueous medium in which it i8 dissolved.
; -The potency of the products as vaccines is proved by the following tests which show the good tolerance and the potency of the products of the invention .
Tolerance test: ~
!
Since care must be taken with the conventional formaldehyde toxo~d for allergic, delayed reactions, the product of the in-vention was compared with the conventional toxoid in the following test:
Groups oi 10 Guinea pigs each were given:
A. 0.5 ml of the product accordi~g to Example 1 ~ith 0.1 mg of protein/ml, adsorbed on 0.02% Al(OH)3-Gel and 3 weeks later 0.5 ml of the same antigen with 0.04 mg/ml without Al(OH)3.
B. 0.5 ml of a convc.-tional formaldehyde toxoid with 0.1 mg protein/ml, adsorbed OD 0.02% of Al(OH)3 and 3 weeks later 0.5 ml of the same antigen with 0.04 mg/ml without Al(OH)3.
~ 7 ~
. ..... . . . . ..
12 Days later, all animals were given intracutaneously 0.1 ml of the two antigens in three dilutions (10 ~g/ml, 1 ~g/ml and 0.1 ~g/ml). The thickness of the skin at the injection site was measured after 24 and 48 hours. The average increase of the thickness of the skin as a measure of the aller-gic reaction was higher with the conventional toxoid than ~ith the product of the invention.
Average increase of the thickness of the skin in 1/10 nm after the injection, measured with a cutimeter (apparatus for measuring the thickness of the skin manufactured by Messrs.
Hauptner) A Animals immunized with the product of the invention Reaction 24 hours 48 hours , . . .
Challenge with the product of the invention 5.3 0.16 Challenge with conventional toxoid 7.0 1.8 B. Animals immunized with the conventional toxoid Challenge with the product of the invention 5.7 3.0 Challenge with conventional toxoid 9.1 4.9 Potency test Groups of 5 Guinea pigs each were given subcutaneously 0.5 ml (20 ug) of a suspension of the product prepared according to Example 1, adsorbed on 0.2% Al(OH)3 gel. 4 Weeks later the animals were poisoned with 0.3 mg of diphtheria toxin, correspond-ing to 10 minimum letal doses (= dlm - dosis letalis minima).
All of the animals survived the poisoning. When the same pro-duct was administered in the form of a vaccin free of adsorbate and in a dose of 100 ~g/animals, a survival rate of 20~ was obtained.
In order to be able to make a comparison with the condition , . .. ,, . . . , . , , :
1063~20 of humans who already have a d~phtheria-antitoxin tlter, groups of 5 Guinea pigs each were pre-im~unized with the conventional toxoid. The dose of conventionalvaccine was so selected that the antitoxin titer after 28 days was always below 0.125 IU/ml of serum. ~n the ~ day, antigens which haa been prepared according to the Examples of the present invention, were administered in different doses as adsorDate-free vaccins and the antitoxin titer was determined 10 days later. Animals which h~d been given 0.2 to 0.4 ~g/animal of the product according to Example 2, showed a 3~ to 60-fold increase of the antitoxin titer.
The following Examples illus~rate the invention:
EXAI~LE 1:
-Diphtheria toxin (100,000 floculation units e Lf-units , corresponding to about 270 mg of protein) was combined, as a 0.1%-protein solution in a 0.1 M phosphate buffer, at pH 7.8, with iormaldehyde up to a final concentration of 0 015 M of aldehyde and allowed to stand for 16 hours at 4 C. The solution was filled into a dialysis hose and dialyzed against the 10-fold quantity of 0.1 M trishydroxymethylaminome~hane-HCl buf~er (TRIS) havin~
a pH of 8.0, ~ ina~ted with t~in(3.4.21.4) (2 u~) in the presence of dithiothreitol (DTT) (1.5 mg/ml) for 60 minutes at 37 C and evenb~ly oxbined with a t~ypsin (3.4.21.4) inh~bitorfrom bovine lun~s (3 ~g/ml). After concentration of the solution to a final con-centration of 10 mg of protein per ml on an ultrafilter, urea was added to a concentration of 6 ~Vl und the solution was intro-duced into a column (4.5 x 100 c.~) of Sephadex(~) G-150 (Trade-mark of ~less rs. Pharmacia, Uppsala), equilibrated with 6 ~ of urea in 0.1 ~ of Tris buffer, at p~ 8.0, and DTT 0.001 ~. Elu- -tion was effected with the equilibration mixture. ~pon chrom~-_ 9 _ ' ,, , '.. ' '' ' ' ' ', " ' ', ' ', ' ' '' " ~
.. . . . . . . .
.. , . , , , . ~ - -tography, three protein peaks were found by measuring the UV-absorption at 280 nm. The first, smallest peak represented high molecular material which was rejected. The second peak represented the derivative of fragment B. It could be subjected to further purification by a repeated chromatography. Peak 3 represented fragment A.
The solution of the isolated derivative of fragment B was diluted with 0.1 M phosphate buffer, at pH 7.8, to 500 ml and then dialyzed againt the same buffer~until a qualitative urea analysis became negative. The formaldehyde concentration of the urea-free solution of the derivative of fragment B was then adjusted to 0.07 M and incubated for 21 days at 37 C. After a finaly dialysis against 0.15 M of NaCl to remove any free form-aldehyde, the modified derivative of fragment B was isolated.
It was concentrated by ultra-filtration and processed into a . . .
vaccin in known manner. As stabilizer, glycin (0.1 M) or lysin (0.05 M) could be used.
Instead of formaldehyde, there could also be used, re-~ferred to the molar quantity of aldehyde, glutardialdehyde, pro-pionaldehyde or butyraldehyde, either in both steps or in onei step only in order to bbtain a product with the same properties of easy solubility and good antigenicity.
EXAMPLE 2:
Diphtheria toxin (100,000 floculation units = Lf-units, corresponding to about 270 mg of protein) was dialyzed, as 0.1%
solution in 0.1 M trishydroxymethylaminomethane-HCl buffer, having a pH of 8.0, then incubated with trypsin (2 ug/ml) in the pres-ence of dithioerythritol (DTE) 1.5 mg/ml) for 60 minutes at 37C
and eventually combined with a trypsin (3.4.21.4) inhibitor of bovine lungs :
:
-. . . : . :: . . : . , 1063~Z0 (3 ~g/ml). Formaldehyde was theD added up to a concentration of 0.015 M oi aldehyde and the whole was allowed to stand ior -16 ho~rs at 4 C. Urea was added to this solution to a concen-tration of 6 M/l and the derivat~v;e of fragment B was isolated as described in Example 1. If desired, the following reaction with 0.07 N of iormaldehyde could be carried out as described in Example 1.
EXA~LE 3:
Diphtheria toxin (100,000 Iloculation units ~ Lf-units, corresponding to about 270 mg of protein) was treated, as a 0.1~ protein solution, without the pre-treatment with 0.015 M of aldehyde, was treated with ~qpsm (3.4.21.4) in the:pre ~ oe of di-thiothreitol as described in ExaD~le 1 and then further treated with 0.07 M oi aldehyde as described for the reaction of the deri.vative of fragment B;eventual~y, the modified derivative of the iragment B was isolated. .
.
This application is a division of Serial No. 237,446 filed October 10, 1975.
~ ' , , , . . , , , ~ . . . . . .. .. .
- , ~ ;, - , :,:, ~,:
,- .. - , . .
Conventional diphtheria vaccines for the active immuniz-ation contain almost exclusively the antigen prepared by inact-ivation of the diphtheria toxin with formaldehyde. This substance, also called toxoid, is provided with a great number of determinants, of which only few may play role for the produc-tion of protecting antibodies against diphtheria. Elimination of such determinating groups which are of no importance for the protection is desirable in order to obtain antigenic or immunogenic substances which may have higher specificity and may be better tolerated.
The diphtheria toxin is produced by cells of Coryne-bacterium diphtheriae and set free into the nutrient medium extracellularly as a single polypeptide chain having a molecular we~too`f about 62,000. Mild proteolysis in the presence of compounds which are capable of splitting disulfide bridges in proteins, for example thiol compounds, produce two characteristic fragments of the diphtheria toxin, i.e. the so-called fragment A (molecular weight about 24,000) coming from the NH2-terminal portion of the toxin and the so-called fragment B (molecular weight about 38,000) coming from the carboxy-terminal portion. ~ -Fragment A can be easily isolated by methods of the protein chemistry and is stable in native form in physiological buffer solutions. Fragment B, however, can be stored in solution in isolated state only in the presence of denatured agents such as urea, -- ~
.. . , . . - . : .. .. .
-` 1063~20 guanidine hydrochloride or surface-active detergents. Elimination of the denaturing agent then gives a completely water-insoluble fragment B which impedes handling cons1derably~ The not-denatured - -fragment B, however, is considered to play an important role in the production of protecting antibodies after parenteraf admlni-stration.
Thus, it was a task of the present invention to modify the fragment B of the diphtheria toxin ~n such a manner that it could be kept in solution in a physiologically tolerated medium without loss of its antigenicity. In particular, this fragment B should be able to be used in its modified form as essential immunogenic constituent of diphtheriavaccines and to replace in these ~accines the conventional formaldehyde toxoid.
Now, we have found that this aim can be achie~ed by a process the most important feature of which is to avoid the spontaneous irreversible precipitation of frag~ent B, which can always be ob-served after elimination of the denaturing agents such as urea, by a slight chemical modification oi fragment B either before or after separation of fragment A from the molecular structure of the diphtheria toxin, which is a complex oi fragments A and B.
Accordingly, the subject of the present invention is a process for preparing a derivati~es of fragment B of the diphtheria toxin, which comprises treating an aqueous solution of the diph- -theria toxin with an aliphatic mono- or di-aldehyde having a chain length of 1 to 6 carbon atoms, preferably formaldehyde, with an aldehyde concentration of 0.0015 M to 0.035 ~S, preferably 0.002 to 0.02 M, at 1 - 20 C, preferably 1 - 6 C, for 5 minutes to 50 hours, reicting the toxin, before or after the treatment ,- . "
- -- . . ~. ,, ;. ; , , ' ~
with aldehyde, în the presence oi a compound splittlng o~f disul~ide bridges, with a proteolytic enzyme, recovering the derivative of fragment B from the solution obtained in the pre-sence of a denaturing agent by protein-chemical isolation pro-C~SS~S ~JU S~p~l ~L~IIg iL 1~ Lh~ ~r~a~u~ a~Gr.t.
Aiter isolation of the derivative of fragment B, the de-naturing agent is eliminated by dialysis or other comparable methods which permit the separation of low molecular substances, for example by gel filtration. The aldehyde used ior the reaction and which has not been completely consumed can be eliminated from the batches by similar methods.
The derivative prepared according to the method of the in-vention, when diluted in physiologically tolerated, isotonically aqueous media, does no longer show the denaturation and precipitation phenomena known from the native fragment B.
In the same manner as ~ragment B, the derivative does not have the toxicity of the diphtheria toxin. It has the same behaviour as the antigen and is capable of ~nduc_ing protecting antibodies against diphtheria in the human or animal organism.
The derivative prepared according to the invention can be purified by the conventional proteln-chemical methods. How-ever, in view oi' the high toxicity oi' the native diphtheria toxin and the fact that protein fractionation methods lead only very seldom to a 100% separation oi accompanying substances, especially oi those protein bodies having a similar structure, it is oi advantage to submit the product prepared according to the invention to another treat~ent with aldehyde, which treatment comprises combining the derivative oi iragment B prepared according to the ~-.
...... . . . . .
':
.. . .
. ' . I ' , . . . .
, . :' ", 1063~Z0 invention with an aliphatic mono- or dl-aldehyde having a chain length of 1 to 6 carbon atoms, preferably formaldehyde, with an aldehyde concentration of up to 0.035 to 0.35 ~5 and allowing the whole to stand for 14 to 28 days at 20 - 37C, removing the aldehyde? for example by dialysis. and isolatin~ the modified derivatives of said fragment B.
This additional treatment by aldehyde gives the fragment B
an increased stability in aqueous media. Thus, it was assumed that this stable derivative of the fragment B could also be obtained by reacting the diphtheria toxin, as known in the pre-paration oi the fragment B, with a proteolytic enzyme in the pre-sence of substances splitting off di-sulfide bridges, for example thiol compounds, combining the solution ~ith a denaturing agent, racovering the fragment B advantageously ~y chromatography, in particular by gel iiltration, remov~ng the denaturing agent by gel eiltration-and subsequently allowing the fragment B so ob-tain-d, immedi&tely thereafter, to stay together with an ali-phatic mono- or dialdehyde having a chain length of 1 to 6 carbon atoms, preferably formaldehyde, at a concnntration of 0.0015 Ml to 0.03~ M~ for 5 minutes to 50 hours at 1 to 20 C. If desired, a second treatment with aldehyde for the modification of the deri-vative may follow. However, the fragment B may also be reacted directly after its isolation with the increased quantity of al-dehyde.
Isolation of a modified fragment B from th~ conventional diphtheria toxoid is not possible, because the use o~ essentially higher quantities of formaldehyde produces a co-valent cross-linkage between the fragments A and B. However, there is no impeding reason not to react the solution containlng the frag-. . .
.
. ' . ' ' ' - ' : ' ' . ' . :.
' ' ~ ' ' ' ~ ' :' ., : , , ., ment B with aldehydes in order to obtain derivatives having the advantageous properties regarding stability and antigenicity.
As starting materials, there are used diphtheria-contain-ing aqueous media such as those which are also used for the isolation of fragment B, for example culture filtrates which are more or less pre-purified and enriched with regard to diphtheria toxin, highly purified toxin preparations or solutions of frag-ment B.
Proteolytic enzymes used in the preparation of fragment B are pronase, papain, subtilisin, preferably, however, trypsin (3.4.21.4), in soluble form or in a form bound to a solid car-rier. It has been found advantageous to stop the proteolytic reaction with the aid of an inhibitor, for example trypsin (3.4.21.4) with the aid of a trypsin inhibitor from animal or-gans or plants. If proteolytic enzymes bound to a carrier are used, the application of inhibitors is not necessary.
As compounds which split off di-sulfide bridges, thiol-compounds are preferred. In this respect, there may be used, for example cysteine, mercapto-ethanol, di-thioerythritol, or ~pref,era~l~ di-thiothreitol.
Denaturing agents in the sense of the present invention are chemical compounds with the aid of which a dissociation of hydrogen linkages in protein molecules is made possible.
Known agents which dissociate hydrogen linkages are, for example urea or guanidine hydrochloride.
Urea is known for the fact that, with its aid fragment B can be kept in solution at concentrations of ~0.6 M. Advan-tageously, about 4 to 6 M of urea or about 2 to 4 M of guanidine hydrochloride are used.
': :
- .
^ 1063~20 In addition to the methods described herein ln the present invention, the present invention also relates to derivati~es of the diphtheria toxin, which derivatives are characterized by the parameters found during their preparation.
Eventually, the invention also relates to agents which contain the diphtheria toxin derivative o~ the invention, in particular to diphtheriavaccines which are to be used as prophylactic drugs against diphtheria or for the manufacture of diphtheria antisera or also of dia~n~stic preparations. For increasing the stability in solution of the product of the invention, it may be suitable to add amino-acids or carbohydrates to the physiologically tole-rated aqueous medium in which it i8 dissolved.
; -The potency of the products as vaccines is proved by the following tests which show the good tolerance and the potency of the products of the invention .
Tolerance test: ~
!
Since care must be taken with the conventional formaldehyde toxo~d for allergic, delayed reactions, the product of the in-vention was compared with the conventional toxoid in the following test:
Groups oi 10 Guinea pigs each were given:
A. 0.5 ml of the product accordi~g to Example 1 ~ith 0.1 mg of protein/ml, adsorbed on 0.02% Al(OH)3-Gel and 3 weeks later 0.5 ml of the same antigen with 0.04 mg/ml without Al(OH)3.
B. 0.5 ml of a convc.-tional formaldehyde toxoid with 0.1 mg protein/ml, adsorbed OD 0.02% of Al(OH)3 and 3 weeks later 0.5 ml of the same antigen with 0.04 mg/ml without Al(OH)3.
~ 7 ~
. ..... . . . . ..
12 Days later, all animals were given intracutaneously 0.1 ml of the two antigens in three dilutions (10 ~g/ml, 1 ~g/ml and 0.1 ~g/ml). The thickness of the skin at the injection site was measured after 24 and 48 hours. The average increase of the thickness of the skin as a measure of the aller-gic reaction was higher with the conventional toxoid than ~ith the product of the invention.
Average increase of the thickness of the skin in 1/10 nm after the injection, measured with a cutimeter (apparatus for measuring the thickness of the skin manufactured by Messrs.
Hauptner) A Animals immunized with the product of the invention Reaction 24 hours 48 hours , . . .
Challenge with the product of the invention 5.3 0.16 Challenge with conventional toxoid 7.0 1.8 B. Animals immunized with the conventional toxoid Challenge with the product of the invention 5.7 3.0 Challenge with conventional toxoid 9.1 4.9 Potency test Groups of 5 Guinea pigs each were given subcutaneously 0.5 ml (20 ug) of a suspension of the product prepared according to Example 1, adsorbed on 0.2% Al(OH)3 gel. 4 Weeks later the animals were poisoned with 0.3 mg of diphtheria toxin, correspond-ing to 10 minimum letal doses (= dlm - dosis letalis minima).
All of the animals survived the poisoning. When the same pro-duct was administered in the form of a vaccin free of adsorbate and in a dose of 100 ~g/animals, a survival rate of 20~ was obtained.
In order to be able to make a comparison with the condition , . .. ,, . . . , . , , :
1063~20 of humans who already have a d~phtheria-antitoxin tlter, groups of 5 Guinea pigs each were pre-im~unized with the conventional toxoid. The dose of conventionalvaccine was so selected that the antitoxin titer after 28 days was always below 0.125 IU/ml of serum. ~n the ~ day, antigens which haa been prepared according to the Examples of the present invention, were administered in different doses as adsorDate-free vaccins and the antitoxin titer was determined 10 days later. Animals which h~d been given 0.2 to 0.4 ~g/animal of the product according to Example 2, showed a 3~ to 60-fold increase of the antitoxin titer.
The following Examples illus~rate the invention:
EXAI~LE 1:
-Diphtheria toxin (100,000 floculation units e Lf-units , corresponding to about 270 mg of protein) was combined, as a 0.1%-protein solution in a 0.1 M phosphate buffer, at pH 7.8, with iormaldehyde up to a final concentration of 0 015 M of aldehyde and allowed to stand for 16 hours at 4 C. The solution was filled into a dialysis hose and dialyzed against the 10-fold quantity of 0.1 M trishydroxymethylaminome~hane-HCl buf~er (TRIS) havin~
a pH of 8.0, ~ ina~ted with t~in(3.4.21.4) (2 u~) in the presence of dithiothreitol (DTT) (1.5 mg/ml) for 60 minutes at 37 C and evenb~ly oxbined with a t~ypsin (3.4.21.4) inh~bitorfrom bovine lun~s (3 ~g/ml). After concentration of the solution to a final con-centration of 10 mg of protein per ml on an ultrafilter, urea was added to a concentration of 6 ~Vl und the solution was intro-duced into a column (4.5 x 100 c.~) of Sephadex(~) G-150 (Trade-mark of ~less rs. Pharmacia, Uppsala), equilibrated with 6 ~ of urea in 0.1 ~ of Tris buffer, at p~ 8.0, and DTT 0.001 ~. Elu- -tion was effected with the equilibration mixture. ~pon chrom~-_ 9 _ ' ,, , '.. ' '' ' ' ' ', " ' ', ' ', ' ' '' " ~
.. . . . . . . .
.. , . , , , . ~ - -tography, three protein peaks were found by measuring the UV-absorption at 280 nm. The first, smallest peak represented high molecular material which was rejected. The second peak represented the derivative of fragment B. It could be subjected to further purification by a repeated chromatography. Peak 3 represented fragment A.
The solution of the isolated derivative of fragment B was diluted with 0.1 M phosphate buffer, at pH 7.8, to 500 ml and then dialyzed againt the same buffer~until a qualitative urea analysis became negative. The formaldehyde concentration of the urea-free solution of the derivative of fragment B was then adjusted to 0.07 M and incubated for 21 days at 37 C. After a finaly dialysis against 0.15 M of NaCl to remove any free form-aldehyde, the modified derivative of fragment B was isolated.
It was concentrated by ultra-filtration and processed into a . . .
vaccin in known manner. As stabilizer, glycin (0.1 M) or lysin (0.05 M) could be used.
Instead of formaldehyde, there could also be used, re-~ferred to the molar quantity of aldehyde, glutardialdehyde, pro-pionaldehyde or butyraldehyde, either in both steps or in onei step only in order to bbtain a product with the same properties of easy solubility and good antigenicity.
EXAMPLE 2:
Diphtheria toxin (100,000 floculation units = Lf-units, corresponding to about 270 mg of protein) was dialyzed, as 0.1%
solution in 0.1 M trishydroxymethylaminomethane-HCl buffer, having a pH of 8.0, then incubated with trypsin (2 ug/ml) in the pres-ence of dithioerythritol (DTE) 1.5 mg/ml) for 60 minutes at 37C
and eventually combined with a trypsin (3.4.21.4) inhibitor of bovine lungs :
:
-. . . : . :: . . : . , 1063~Z0 (3 ~g/ml). Formaldehyde was theD added up to a concentration of 0.015 M oi aldehyde and the whole was allowed to stand ior -16 ho~rs at 4 C. Urea was added to this solution to a concen-tration of 6 M/l and the derivat~v;e of fragment B was isolated as described in Example 1. If desired, the following reaction with 0.07 N of iormaldehyde could be carried out as described in Example 1.
EXA~LE 3:
Diphtheria toxin (100,000 Iloculation units ~ Lf-units, corresponding to about 270 mg of protein) was treated, as a 0.1~ protein solution, without the pre-treatment with 0.015 M of aldehyde, was treated with ~qpsm (3.4.21.4) in the:pre ~ oe of di-thiothreitol as described in ExaD~le 1 and then further treated with 0.07 M oi aldehyde as described for the reaction of the deri.vative of fragment B;eventual~y, the modified derivative of the iragment B was isolated. .
.
This application is a division of Serial No. 237,446 filed October 10, 1975.
~ ' , , , . . , , , ~ . . . . . .. .. .
- , ~ ;, - , :,:, ~,:
,- .. - , . .
Claims (3)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of a modified derivative of fragment B of diphtheria toxin in which fragment B which is free from denaturing agent is combined with an aliphatic mono- or di-aldehyde having a chain length of 1 to 6 carbon atoms at an aldehyde concentration of 0.035 M to 0.35 M and the mixture is allowed to stand for 5 minutes to 50 hours at 1° to 20°C, the aldehyde is removed and the modified derivative of fragment B of diphtheria toxin is recovered.
2. A process as claimed in claim 1 in which the aldehyde is formaldehyde.
3. A modified derivative of fragment B of diphtheria toxin, whenever obtained according to a process as claimed in claim 1 or claim 2 or by an obvious chemical equivalent thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA313,332A CA1063020A (en) | 1974-10-11 | 1978-10-13 | Derivatives of diphtheria toxin, process for preparing them and agents containing them |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2448530A DE2448530C3 (en) | 1974-10-11 | 1974-10-11 | Process for the preparation of derivatives of diphtheria toxin and compositions containing them |
| CA237,446A CA1059026A (en) | 1974-10-11 | 1975-10-10 | Dialdehyde treatment of diphtheria toxin and derivative obtained |
| CA313,332A CA1063020A (en) | 1974-10-11 | 1978-10-13 | Derivatives of diphtheria toxin, process for preparing them and agents containing them |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1063020A true CA1063020A (en) | 1979-09-25 |
Family
ID=27164153
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA313,332A Expired CA1063020A (en) | 1974-10-11 | 1978-10-13 | Derivatives of diphtheria toxin, process for preparing them and agents containing them |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1063020A (en) |
-
1978
- 1978-10-13 CA CA313,332A patent/CA1063020A/en not_active Expired
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