CA1053152A - Isolation and purification of e. coli enterotoxin - Google Patents

Abstract

Abstract of the Disclosure:
The invention relates to the purification of E. coli enterotoxin and the use of the purified material in vaccines for active immunisation and in the production of sera for passive protection against E. coli infections, and for immunoprophylaxis against cholera.

Description

Case 900-9112 IMPROVF~:NTS IN OR RELAl'ING TO ORGANIC COMPOUNDS
:

~ his invention relates to the purification of E. coli enterotoxin and the use of purified heat-labile Eo coli enterotoxin in vaccines for active immunisation and fvr the production of sera for passive protection a~ainst Eo coli inectionsl and for immunoprophylaxis ~gains~ cholera~

E~ coli enterctoxins have long been impl~cated ~ ~
a~ signi~icant factors in Eo coli infections, in partic- :
ular with regard to th~ severe losses of water and 10 electrolytes that occur in diarrhetic illnesses caused ~y coli b~cteria. Th~ abili~y to synthesise these entero- :
toxins is not strain specific but is controlled by a transmissible plasmid which can be transferred by con~
jugation from a pathogenic E. coli strain to non-pathogenic ;
1~ ~trains; *he appearance of new enteropathogenic serotypes ~``
can thus be explained.

Coli infections have hitherto been treated ;
~ith ooli vaccines or chemotherapeutically, in particular with antibiotics. The use of coli vaccines æe, ~ver, in genera~ ~
~0 directed against particular ser~types of E. coll and not ~ ;
against their exotoxins, in particular enterotoxins 7 ' `'~

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_ _ _ _ . __ . _ _ __ .___ .__ _ _. .

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- 2 - ~0~-~112 This method thus provides an antlbacterial protectiOn but -~ not an ant~-toxic protection. The plasmid-controlled mechanism of synthesis also explains the frequent appearance of antibiotic resistance among entaropatho-genic E~, coli strains, since R-factors are transferred by the same mechanism as enterotoxin plasmids, The plasmid controlled synth~sls o~ twv forms of E. coll en~erotoxin, namely heat-labile ~LT~ enterotoxin and heat-stable ~ST) enterotoxin has been described ln the literature [for example , Smi~h, H.W. and ~les , C .L., Jr. Med. Microbiol. 3, 387 ~ls70~ ] . These orms differ :
primarily in their acid-lability, heat-lability and antigenicity, as indicated, for example, in the follow~n~
table, which al~o shows a number of reported common properties:
...
__ __ . _ _ _ _ .
Property L~ ST
~ _ _ .
. Heat-lability (65C, 15 minutes~ : ~ _ Acid-labi lity (pH 6 ) ~ .
~tigerlicity + _ ~0 Dialysability _ .
High molecular weight Activity in rabbit .
1ntestinal loop model . ;~:~
Act~ity in pig ~+ .
intestinal loop model .

ProduGtion controlled by a plasmi~
Capability to induce aiarrhea + ~
~rl = . _ .. .

, _ ~
- -.
. ' ' ' ~

.. . . . , , , .. .. ~ , - .. -. . .

3~

~ 3 ~ 90~-gll2 Attempts to further ~nve~tigate coli entero-toxins and their role in coli infections have hitherto, howeverl been hindered by an inability to obtain the .
enterotoxins in a suficiently high degree of purity.
A prerequisite for obtalning the necessary purity is a reproduc~ble and accurate assay system for 1QCa1iSing and :
quantifying enterotoxin activity r for exampl~ after chromatographic separation, but the methods used hitherto, for example the lntestinal loop models mPntioned above, . have not provea adequate in these respects, enterotoxins ~dentifled by such methoas ha~ing been found, for example, : ;
to have a wide variety of molecular weights. These known test systems are also time-consuming and can only be used ~ ~
for smallscale investigations. .
1~ ~ more accurate and simpler assay enterotoxin activity is employed in connection with the present i~ven-.. , .. , .. ~
~ion and is based on the fact that the stimulation of adenylate cyclase activity, as reflected by an accumulation of cyclic 3',5'-adenosine monophosphate ~cAMP),in cat myocardial adenylate cyclase preparations is an accurate measure of enterotoxin activity and c~ncentration~ . :
~h$s method for the detection and/or determina- :
tio~ o E. col~ enterotoxin activity comprises incubating a preparation of cat myocardial adenylate cyclase 'or a '~ .
.

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53~ o~gll2 predetermllled time in the presence o~ a material to be - tested for E. coli enterotoxln ac~ivity, and meAsuxing thQ
~ncrease ln adenylate cycla~e activlty in the resulting product in relation to a control.

More particularly,the incubatlon may be effected ~n the presence of a substrate whose conversiGn ~o cyclic 3'~5'-adenosine monophosphate is catalysed by adenylate cyclase, the 1ncrease in adenylate cyclase activity being dete~mined by measuring the increase in concen.tration of cyclic 3t ,S'-adenosine monophosphate in the resulting product in relation to a control.

The cat myocardial adenylate cyclase preparation may be obtained from cat myocardial tissue, in known manner~ for example as des¢ribed by Levey et al. ~G.S.Levey a~d E. Epstein, Biochem. Biophys. Res.- Commu~. 33. 990-995 (1968), and 38, 86-92 ~1970)]. The preparatio~ may ~e in ~:
: particulate form,~or in solubilised form obtained by treat-me~ with a non-ionic deter~ent, in particular Lubrol-PX
lICI ~merica.Inc.] as described by Levey ~suE~a), and taken up in, for examp~e r a sucrose huffer. In the case o solubilised preparations D however~ it is necessary to restore the adenylate cyc~ase acti~ity which is appar en~ly ~estroyed by the detergent. This can readily be achieved by the addition of phospholiplds~ for example phosphatidylserine.

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. 5 ~ 1C1 ~3~52 9~0~9112 .
The adenylate cyclase catalysed ¢onversion of substrates to cyclic 3'~5'-adenosine monophosphate is well ~nown. Suitable substrates include adenosine triphosphate (ATP) or~ preerably, 5'-adenyl-imidodi-phosphate (AMæ-PNP). The incubation may be carrled out in conventional manner t for example as described in the above-mentioned Levey references. The reaction medium suitably comprises magnesium ionsO for example in the orm of magneslum chloride, as well as o~her materials known to assist in the conversion of ATP to c~MP
and in the subsequent assay o~ c~MP,.~or exa~ple bovin~ serum albumin and t~eophyllin plus buffer, for ex~mple ~ris-HCl buffer, as well as the substrate, eAg. AMP-PNP, enzyme and test material or contx~l. The ~ncubation :~
.
ls suitably initiated by addition of the enP.yme, in particulate or solubilised form as described above, : ~~
. to the remai~ing reaction medium,~and may sui~ably be . ~ :
arried out at 37C and or a period of 3 to 45 min~
.
-~ utes preferably 5 to 30 minutesO The conversion can then be stopped ~n known manner, for example by add- :
ition of trichloroacetic acid and maintaining the mixture at 4~C, for example for 10 minutes ox more.
The cAMP in the resulting product may be lsolated in .
conventional manner. ~or example~ ~he precipi~ate '~, .
. ::
,~
-- - , ~

5~z ~00-9112 may be removed by centrifugatlc)n and khe supernatant esctxacted several times with diethyl ether/water, and the aqueous layers evaporated to dryness. The residue may suitably be taken up in buffer, for example acetate buffer pH 4 and aliquots then used for subsequ-ent cA~lP assay.

Prlor to the work-up of cAMæ, a known amount o~ triti~ed ~AMæ is suitably added to the mixture ~or at least one ~ample thereof~) in order to determine the correction for extraction recovery by dètermin.ing the amount of tritiated c~Mæ in the isolated product.

The cAMP can be assayed in accordance with an~of the many known methods ~herefor, ~he preferred methoa being the radio isotope dilutlon te~t system as described by Gilman A.G., Proc.. Nat. Acad.. Sci. 67 3~05-312 (1970) and as ~.odified as ~escribed above, ~his system being co~mercially available in kit form . ;~
~Bo~hringer, Mannheim, Germany).

This test me~hod enables frac~ions of material from which enterotoxin is to be isolated to be separated and to be readily tested for enterotoxin activity. I necessary, in order to achieve greater purity, further separationsmay then be carried out .
- ' ' ' .. - . . ~: . : . ... . . :
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~05~3~5Z ~0-9ll2 on the acti~e ~ractions, suita~ly after poollng thexeof, and the active fractions again identified. The separation and identi~icatlon ~teps can thus be carried out until a homogenous or sufficiently pure product is :~
obtained. ;

The use of the enterotoxin assay descr.ibed has , more particularly, assisted in the development,in accordance with the invention t of methods of obtaining E. coli enterotoxin in a high degree of purity. In particular, lt has been found that E. coli`enterotoxin can be isolated and ~ighly purified by isotachophoresis or affinity chromatography.

The present invention provides a method for :~
;` the isolatio~ and purification of E. coli ente~otoxln ~`
. from a crude or pre-purlfied cell-free culture filtrate of a fermenter culture of~an enteropathogenic E~ coll train :comprising s~ubjecting such culture:~iltrate to - . . isotachophoretic or a~inity chromatographic separation.

: The resulting separated ~ractions, or at ~`
least those containing protein, may then be tested for enterotoxin acti~ity, particularly by. the method -~
described,and subjected to such further separat~on as may be necessary to achieve the xequlred aegree o ~urity.
Naturally, it w~ll be appreciated that ~he activ~ ~
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. - 8 ~53~ 90~-9112 ~ractlons may have been pr~detPrmined, for example in previous separations carried out under identical condi~
t~ons, such that the testing step would not then be neces-sary.

The enterotoxin containing cell~free culture f~ ltrate may be obtai~ed in known manner. For example, the enteropathogenic E. coll strain whose enterotoxin i5 to be isolated, may be cultivated, for example for 3 ~.
genexations and at 37C, in a suitable medium, for example tryticase soy broth tBBL~ Lockeysville, Maryland, USA]
or, preferably, a modified trypticase soy broth obtained ~:
by ultra-filtering the tryticase soy broth, for exa~ple a 30~ ~weight/volume) solution.thereo. The ~irst stage ~ o~ the cultivation is suitably carried out for a-relatively short period of time, for example 4 to;6 hours,.the second.
stage,for example until the culture reaches the mid of the logarithmic phase, and the inaL:~tage,for example for 8 - to 10 hours.

The cells may then be harvested in conventional manner~ for example by cen~rifugation,and the superna~ant filtered and checked for sterility. The crude sterile broth filtrate is then suitably concentrated by ultra-~lltration, for example through Diaflo PM-30 membranes, . ::

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~ S ~ 5~ 9112 desalted by treating with a mixed~bed ion exchange resin, - ~or example AG 501-X8, lyophilized and stored for further us~, for example at -~0 to -70C. Durlng this initial concentration procedure, the bacterial products are desirably protected against proteolytic degradation by addltion o a pxotease inhib~tor, for exàmple pentamidine isothionate~

~he initially conc~ntra~ed lyophilised sterile cell filtxate may suitably be pre-purifled by one or more gel filtration steps prior to ~he main separa~ion step, particularly when this is to be by isotachophoresis~
.
The gel-filtration step(s) may be carried out in conventional mannex, the appropriate gels and eluants .
being determined, for ~xample, in pre-runs aimed at establishing the approximate molecular weights with ~thich enterotoxin activity is associated. In this connection, it has been found con~enient to carry ou~ a first gel . f~ltration separatio~ employing Bio-Gel A-5m as support ~eparatlon range 5,000,000 80,000) and an ammonium .
bicarbonate bu~fer, pH 7.9-8~0, as e~uant. Ater deter- .
minatlon of the enterotox~n active fractions, for exampler by the test method described, the desired fraction~ may then :;
be pooled and a subsequent gel filtra ion separation efecked,.suitably on-a Sephadex G-75 column (separation ' " ' ' ~. ' .
..
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! ~

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range 70yO00-3,000~ using the same buffer or a Tris-HCl buffer ~pH 8.0~. The active ~ractions may then be determined again and pooled.
The pooled fractions are then desirably con~
centrated urther by dialysisl for example agalnst 0.1 M
Tris ~-amino~caproate buffer (pH 8.9~ to facllitate prep~
aration ~orthe preferred subsequent separation by isotacho- ~
phore~is~ ~-The isotachophoretic sepaxation may be carrled out by the method o Svendson and Rose, Science ~ools 17, 13- (1970), uslng.polyacrylamide gel as supporting medium .
- and Ampholine carrler ampholytes as buffer and spacer ubstances. The separation is pre~erabl~ carried out in single buf~ered gel columns, the Ampholine caxrier .
ampholytes being mixed with the sample and the term-inating electxol~te, :which is suitably Tris -amino caproa~e buffer ~pH 8.93. The terminating electrolyte i~ layered on top of the gel and Tris-sulphate .
b~~er (pH 7.1) is suitably employed as the elution :~
~lectrolyte. The sample for separation,in particular th~ co~centrated Sephadex G-75 gel filtration product, is suitably mixed wi~h sucrose to increase its viscosity, `;
- d~luted with ~he terminating electrolyte and mixed with ~
.
~he carrier ampholytes, and introduced via a capillary ~ ~ ~

.~

~ .
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~ 5 3~ ~ g~0~9ll2 through the upper layer of the terminating electrolyte to form a layer on top of the gel. The ~el is suitahly pxepared from stock solutions according to Davis~
Ann. N.Y. Acad. Sci. l2l, 404 (lg64), using, preferably Tris-phosphate pH 8.l as qel buffer, polymerised by photopolymerisation only. ;-The eluted fractions may then tested for entero-toxin activity, for e ~ ple by the method descr ~ d,and the desired ~ ::
fractions suitably pooledand concentrated again by dialysis against Tris ~ aminocaproate buffer pH 8.9.
If desired, a further isotachophoretic separ- :
ation can be carried out to achieve even greater purity, this time using a different ratio of the mol~rities :
of leading ion and counter1on, or, preferably, a dif ferent leading electrolyte, for example Tris-2-(N~
morpholino)ethane sulphonic acid pH 6.2 Again,active fractions may be pooled and concen-trated, suitably ~y dialysis-a~ described above~ The :
final product is then suitably suhjected to gel filtration .
on Sephadex G-50 to separate the low molecular weight Ampholine carrier ampholytes from the isotachophoretic~
ally fractionated protein.

An alternative method of isolating the entero~
toxin from initially concentrated sterile cell filtrate `.
, ~

~;':

S~31,.5i2 900~911~

or ~rom the cell filtrate prepurified bygel filtration is ~y af finity chromatography ~ ~finity chromatography is a known technique for the separation of, for example, proteins, which exploits the biological property of these materials to bind ligands specifically and revexsibly.
A solutio~ containing the material to be puxified is .
passed through a column containing an insoluble polymer or yel to which a specific inhibitor or other ligand has been covalently attached. Proteins having no appreciable af~in~ty for the ligand will pass unretarded through the column, whereas those which recognise the inhibitor will be retarded in proportion to their affinity~ The speci-.
fically adsorbed protein can then be eluted by altering the composit~on o~ the solvent so that dissociation occurs.
The separation in accordance with the invention may be carried out in manner conventional for affini~y ~chromatographic separation, for example as described by . Cuatrecasas et al~, Methods of Enzymology XXII, 345-,-~19713. For the purposes of the present invention, suitable bindin~ ligands include amphipathic substances, such as phospholipids or glycolipids, having ~ree carbox :~
ylic groups, in particular gangliosides. These ll~ands ~ay be bound to an insoluble matrix, which is sui~ably an agarGse d~rivative, such as a deriva~ive produced by .

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attaching an amino copolymer, e.g. poly L-lysyl-DL-alanine to cyanobromide activated agarose in kno~n mannex [for example Cuatrecasas. P., J. Biol. Chem. 245, 3059 (1970~ and Sica V. et alO, Nature ~London~, New Biology 244, 36 (1973)~ or alhumin agarose. The ligand may be bound to the matrix in conventional manner. In the case of gangliosides, for example, the carboxylic group of khe terminal sialic acid is coupled to ~he amino groups of the agarose dexivatives mentioned above, in the pres~
ence o a water~soluble carbodiimide reagen~, for ex~mple l-ethyl-3-dimethyl-3-(3-dimethylaminopropyl)carbodiimide, or dicyclohexylcarbodiimide. For the coupling, the ' gangliosides are suitably employed in the form Oî their ~ N-hydroxysuccinimide esters or of their activat~d mixed anhydrides. The reaction is suitably effected in a solvent or solvent mixture, for exam~le a cyclic et~er, eOg. dioxane or a water/dioxane mixture.
- Other suitable ligand-matrix systems for -use as affinity resins in the separation inciude fatty acid-aminoalkylamino-agarose complexes. These may be prepared in conventional manner, for example as des- -cribed in the first Cuatrecasas reerence mentioned above. Suitable alkylenediamines for forming the amino-:' ,:
alkylaminoagarose derivatives include hexanediamine, ,' ~' ' '.
.
,., - - .

' . - - : . .. - ,. .. .. .. ,,. ~

- 14 ~ 3~ 5~ 900-911~

decanedlamine and ethylenediamune. Suitable fatt~ acids lnclude palmitic acid.
The affinity resins described above are then suitably placed in an affinity column and the initially concentrated lyophilised sterile cell filtrate is prPf-erably taken up in a buffer, for example Krebs-Ringer blcarbonate bufer pH 7.4, transferred to the column and developed, suitably under cooling at 4Co The elution of the enterotoxins may, for example, be effected with sodium dodecyl sulphate. The subsequent removal of the sodium dodecyl sulphate and the necessary - rena~uration o~ the eluted coli entero~oxin may be effected in known manner (for example, K. Weber et al., J. Biol. Chem. 246, 4504 [1971~

For the production of enterotoxins in accordance ~ v with the invention any enteropathogenic E. coli strains, or non-pathogenic strains rendered pathogenic by plasmid transfex, may be employed. Preferred strains include porcine pathogenic E. colL strains P 263, serotype 08:K87, K88ab:Hl9; P 155, serotype 0149:K91:88a,c; and P 307, ;~
serotype 08:K87,K88ab; human pathogenic strains H 10407, ~erotype 078K? and H 19, serotype 026:K60:H11; as well as all Ent E. coli K12 strain, to which the Ent plasmid from human ~train H 19 has been ~ransferred.

;.

By the isola~ion process o the invention, i~
1~ possible to ~btain E. coli enterotoxin in a purity of at least 99%. The process also enables thP partic-ul~x foxms ofenterotoxint i.e. the heat~lablle (LT) form and the heat~stable (ST) form to be isolated since, while these forms both show enteroxigenie activity in the test described, theY differ in physical properties, for example molecular weight, and thus appear ... ~n different fractions after the separation step, for ex~mple the gel filtration or affinity chromatographic separations.

The invention also proYides a heatrlabile (LT) E. coli enterotoxin having a molecular weight of 102,000 + 3, 000 which has been lsolated by the process of the .. . .
invention, in particular from cultures of E. coli ;.
stra~n P263, and which has not previously been reported. . ::

E. coli strains are known to promote diarrhetlc .
lllnesses in man and animals, particularly in young .
animals such as gilts, lambs a~d calves. Coli infections are in particular held responsible for infant enteritis, ~:~
~ost forms of travell~r's diæase,and acute tropical dlarrhea.
In domestic animals, particularly in young animals, as indicated, coli infections are among the most sign~ficant Illnesses. In pigs, col1 in~ect1ons occur mainly in ' . ' - , ' - .
~ ' ' . ' . .............................. ~ . :, , . .

, ~ , - :

~ 16 ~ 3~ 9~0-9112 connec~ion with disturbances o~ the walls of the gastro-~ lntestinal tract, particularly in new-born piglets and ln weaned piglets. There then arises a massive multipli-cation of certain serotypes of haemolysing coli bacteria~

The purlfied heat-labile (LT) E.coli ~nteroboxlns ~:
of the i~vention, in particular that having a molecular welght of 102,000 ~ 3~000, are useful in protecting against E, coli infections as indicated by their activ-lty in the rabbit and pig lntestinal loop test (1~. Burrows, G.H. Musteikis~ J. Infect. Dis. 116~ 183 [1966] and -;
.W. Smith, C.h. Gyles, J. Med. Microbiol 3, 403 [1570]), ln the rabbit skin te~t (J.O. Craig, Proc. Cholera Res.
Symp. Honolulu 24, U~S. Government Printing Office [1965]) and in the fat cell lipase test (P. Cuatrecasas, Bioch~m.
. 12, 3567 [1973]) and in the adenylate cyclase stimulating t~st of the invention.

. . ~he purifled heat-labile (L~) enterotoxins of : . . ~the invention ~are also use~ul in protecting against cholera as indlcated by the fact t~at a close sexological relat1onship or cross-immun1ty exists ~et~Jeen the E. coli ~LT) enterotoxin and cholera enterotoxin, which has pre~
~iously been produced ln pure orm and comprehensively characterised by physico-chemical chaxacterist~cs.
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' ' 17 ~ 5~ ~0-9112 ~he invention accordingly also relates to the use of the purif~ed heat-la~lle (LT~ E. coli enterotoxins o~ the lnvention in vaocines for acti~e protection and in the production of sera for passive protection against E. coli in~ectlons and in the immunopxophylaxis against cholera. In this connection~ it has been found that thP enterotoxins from various E. coli ~trains are immu~ologically identical or so closely related that a :~
cross-immunity exists, which is a requislte for some immunological uses, for example for the passive obtention of antisera, obtained in a different animal :
species. .
For some purposes~ in particular fox oral application and for the production of a serum, it ls necessary that the hea~-labile (~T) E. coli ente.rotoxin be in the form o~ a stable toxoid, that is a preparati on which retains the antigenicity of the enterotoxin but - whose toxicity is diminished or eliminated. Such tox-oids may be produced in conventlonal manner (for example R.S. Rappaport, et a~., Inf, Im~. 9, 304 ~1974]).
~or example, conventional cross-linking agents, such as : formaldehyde or, preferably, glutaraldehyde may b~ added~
care being taken that the immunological properties are retained during the treatmen~O For example, the puri- :

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fied heat-labile ~LT) enterotoxin of the inventlon may be lncubated with formalaehyde at an elevated temper-ature, for example 35 to 40C,over a lengthy period, for example 2 to 6 ~eeks, or with glutaraldehyde at room temperature to 40C~ employing ~ glutaralde~.yde concentration of 0.001 to 0.05 Mo The invention accordingly provides a vaccine comprising an immunologically effertive amount of a . purified heat-labile (LT~ E. coli enterotoxln of the invention or a stable toxoid thereof, as well as a method :~
of immunisation against E. coli infections or cholera . .
cQmprising administering an immunologically effective . amount o~ a purlfied heat-labile (LT) E. coli entero-toxin of the invention or a stable toxoid thereo.

The invention also provides a serum compris-.
ing antibodies induced by the administration of a purified heat-labile (LT) E. coli enterotoxin of the `~
~nvention or a stable toxoid thereof~ as well as a method of immunisation against E. coli lnfections or cholera comprlsing administering an immunologically ef~ective ~ ;
amount o~ such serum.

For use in vaccines, the dosage of the puri-fled heat-labile (LT) E. coli enterotoxin of the invention or stable toxoid thereof will of couxse ~ary ;;

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~j3~ ~, 900-9ll2 ~ ~.
dependlng on the mode o~ adminlstration, the host and --the tre~tment desired. However, in gener~l satls~
factory results are obtained with a dosage of from about 00015 ~g to 10 ~g per kilogram of animal body weight.
For the largQr mammals, the total daily dosage is in the xange of from about 1 ~g to 1 mg~ and dosage forms suitabl~ for oral application comprise from about 100 ~g to 1 mg of the stable toxoid, The toxin or toxoid ls ;;~
æuitably freeze-dried.

The amounts of serum ~o be administered for - passive immunisation will of course be those equivalentto the effective doses of toxin and toxoid indicated above and will of course depend on the antibody content of the serum. These can be determlned by measurement of the quantity of serum necessary to neutralise the appropriate quantities of ~oxintresidual activit~ being determined in the intestinal loop tests mentioned above ~ the adenylate cyclase stimulating ~ te~t of the invention, the passive haemagglutination microtest (S.V. Boydent J. Exp. Med. 93, 107 [1951~) and A.B. Stavitski, J. Immunol. 72, 368 [19$4]) and the Bentonite flocculation microtest (E~.C. Goodman, J. Bozicevich, Immunochemical Methods,1964~ 43).

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2~ 3~ 9~0~gll2 The vaccines of the ~nvention may be ormulat~d in conventio~al m~nner, for example with or without adjuvants. Where adjuvants are used~ howev~r, suitable adjuvants lnclude aluminium compounds ~ for example aluminium hydroxide or phosphate or "Alhydrogel'~, and water-in-oil emulsions, such as Adju~ant 65 (a ~ater-in- -oil ~mulsion of antigen in ground nut oil, which is emulsi fied with mannitol monooleate and stabilised wi~h aluminium stearate~ and, for animal usage, Freund's adjuvant.
~. ~
The vaccines and s~ra of the invention may be usea for protection of E. Coli infections and cholera~
ln conventional manner. For example protection of infants and adults against coli-induced diarrhea can ~
be achieved for infan~s by maternal vaccin. ~ ;
ation coupled with a vaccine programme for the infants before the end of the passive~protection, or generally by active immunisation with toxoid~

- The protection of animals against coli ini~ec-~0 tions is particularly important in young animals.
Because the new-born of agriculturally important animals ., .
¢an take antlbodies with the colostrum of the mother only in the first few hours or days after birth, it .: :
ls important for their protec~ion that this colostrum ::

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21 ~ 3~ 00-9112 has a high content of 5peciic antibodie~. Thi~ can be achieved by repeated immunisation of the mothex with the suitable enterotoxin. I~mediately in the first few weeks o~ life, this protection by maternal antibodies ~ :
S ls necessary because, at this time, infectlons in young anlmals by enterotoxin producing E~ coli strains very requently occur and can be fatal~ A further possi.bil-ity for protecting young animals, is the administrat~on of specific heterologous antibodies, for example in the form of a serum, obtained from coli enterotoxin i~unised ~:
animals ~
The following Examples illustrate tbe invention.

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, - 22 - ~S~5~ 900-9112 EX~MPLE 1~

a) Medium _ _ _ _ _ _ "Trypticase soy broth" is used as medium. The pep- :
tone water medlumcontaLns, per litre, 17 g of ~pt}case peptone, 3 g of phyton peptone, 5 g of NaC1, 2.5 g of dextrose and 2.5 g of K2HPO4.3H2O and has a pH of ~.3.
AlternativPly, a "modified trypticase soy broth", ;~
having the same compositlon as described above, but the peptone having prevlously been ultrafiltered .
through Diaflo PM-10 membranes (Amicon), can be used as medium, A 30% ~weight/volume) solution of the "trypticase soy broth" is ultrafiltered, the residue discarded and the filtrate used further. It is advantageous to employ these relati~ely small peptone ~.olecules in place of the untreated product, ~ ;~
' to facilita~e subsequent separation o unaltered : .
medium from the bacterial products in the crude supernatant of the E. coli cultures.

b~ Cu~tivation :~
__ ___ _ _ __ __ .
~ The freeze-dried E. coli strain P. 263, Sero~ype ~
.

08~K87~ K88ab:Hl9 is ~aken up in water, s~reaked on blood agar plates and cultivated at 37C for ~2 hours. Cells from the surface growth are trans-.

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.

- 23 ~0-9112 3~

~erred by means of a platinun loop to 1 lltre Erlenme~er flasks containing 200 ml of the ~.edium (unmodified or modifled trypticase soy broth) on a rotating shaker.
The bacteria are cultivated at 37 for 4 hours and are then used as inoculum for a 2 l~tre shaker cul~ure~
When this culture reaches the mid of the logarithmic phase t it is used as inoculum for a 20 litre fermenter in which the medium is stirred a~ S00 rev/min and aerated ~1~ l/min~ To prevent foaming, 1 ml of a 25% ~:~
(weight/volume) solution of an anti-foaming agent (Glanapon 2000, ~ross-Busettit Vienna), i5 added.
The fermenter is incubated at 37C for 9 hours and the ~:
cells are harvested by ~entrifugation ln a cooled ultra oentri-fuge at 4C. The supernatant fluid is filtered through 1~ 0.45 ~m membrane filter and checked for sterility(by streaking on blood agar plates~ The sterile filtrate is desalted by treatment wi~h a mixed bed ian exchange re~in ~for example AG-501-`X8~,lyophilised and stored for - further use at -20 to -70C

. Ih manner analogous to th~t described ~ ve, the cell-free culture filtrates of other E. ooli strains, for example ~ cine patho geNic stx ~ s P.155, sero~ 0149:K91:88a,c and P307, ser~ 08: -K87 K38a, bt as well as the human pathogenic strains H104Q7, ~erotype 078K? and Hl9~ serotype 026 K60 :Hll; and also an Ent and an Ent E. coll X12 strain, which has received :~
the Ent plasmid from human pathogenic straln Hl9.

.. . ........ . .. , ,. . - . . . .

~4 - ` 50~-~3~q~

EXP~LE ~: ~urification of the col~ -enterotoxin b el . ., _ ~
fi ltratlon The lyophilisate obtained in Example 1 (from any of the strains mentioned) is taken up in 0.1 M NH4HC0 buiffer (pH 7. 9~ (eluant) and the chromatographic separ at$on is carxied out on a ~io Gel ~-5mi colwnni (separation ~;
ranige 5,000,000 - 80,0003 . By use OI this coluTnn (2.5 x 100 om) with a total volume of 500 ml, and approximate empty volume of 190 ml~ a substance peak elute~ at approxi~iately 220 ml, which coxresponds to a theoretical molecular wei~ht of approximately 106 daltons ;~n ~he basis of Ev/Eo = 1.15. A fur~her substance peak with a distinct shoulder elutes at 420 to 480 ml, which ~;
corresponas~ on the samie basis r to a molecular weight of about 9 x 104 - 1~2 x 105 daltons. The heat-labile activity can clearly be ~ound, for exa~ple by the adenylate ~yclase test, in the shoulder of thisi peak.

The lyophiilised active fractions may be pooled and taken up in 0.1 M NH4HCO3 buf er (pH 7~9) anda f~ier gel ; , filtration carried out using a Sephadex G~-75 column ~separation range 70~000 - 3,000). The eluted material in the empty volumeiis clearly separated fro~ other proteins and shows biological activity. The active fractions ~as identified by~ or example, the adenylate cyclase test) are pooled ana lyophilised.

:

, . . . . . ... . . _ . . ., : _ , . . ., .. . ., .. . .. .. . . .. , . . _ .. , _, . _ _ .. . . .... _ _ . ,, . . . .. .. . _ .

~, - - ~: - - , '- ~ ' ' ~ 25 - gO0-9112 EXAMPLE 3: Purification of the coli enterotoxin by ~ isotachophoresis __ -The cell filtrate (from Example 1) or the coli~(LT) enterotoxin prepaxation obtained in Example 2 may then be subjected to preparative isotachophoresis. For the isotachophoretic separation, a vertlcal electrophoresis ~pparatus with 3.3% polyacrylamide gel (for example in accoxdance with P~J. Svendson `and Carsten Rose~ Science Tools 17 ~1~ t 13 [1970]) ~s s~rting m~dlum may be ~sed. A
- 10 sin~le buffer gel column is suitabl~ employed, the A~pholine ~ rier ~holy~s being mixed wi~h the protein sample as well as the terminating electrolyte [Tris--aminocaproate buffer pH 8.9~. The terminating elec~rolyte, which connects the system with the upper electrode (oathode) is layered above the gel. In the lower electrode compartment of the column (anode), Tris-sulphate buffer (pH 7.1) is used as elution electrolyte. The buffer solution is ~.
- . included in an external reservoir and is maintained in . circula~ion with an electric differential pump..The sample, ;~
2~ mixed with the Tris-~-aminocaproate buf~er is introduced lnto the colu~n above the gel with the help of a capillary protruding ~hrough the upper layer of Tris--aminocaproate buffer. In order to ~acilitate $ntroduction o~f the sample, the viscos~ty thereof is increased by addition of 3%
~5 ~ucrose.

... . ' ' '~ ' ... ' ..... .......

--- -. : :.. . .. - . . . : . . . . - : ,.
. .. . ;: : . . . . . .. . . . . . ~

- 2~ ~ ~00-9112 Th~ gel is prepared fxom a stoc~ solution in accordance with the method of B.J. Davies f~nn. N.Y. Acad. Sci. 121, 404 [1964]) using Tris-phosphate (pH ~.1) as gel buffer, and is polymerised only by photopolymerlsation. The active material is eluted in the Ampholyte carrier ampholyte system, shortly before or with termi~atiny electrolyt~ `

The eluate is txansferred vla a W absorptionmeter to a fraction collecter.to provide an initial estim~
ation of the separation (proteln containing fractions).
Th~ active fractions are determined finally by the adeno~
lyte cyclase test.

EXAMPLE 4: Purification of coli enterotoxin by affinity ~1_A~ ' `
The column (10 ml of gel in a glass column ~1 x 20 cm]) is w~sh~d at room temperature, firstly for ~ days with 50~ ~/V) ~ethanol, then for 6 hours wlth 200 ml of G M guanidine hydr~oride and then for 3 houxs ~lith 100 ml of Krebs-Ringer bicarbonate buf~er pH 7.4. 50 g of l~ophilis~aculture filtrate is taken up m 150 ml of Krebs-Ringer bicar-bonate buffer and i5 dialysed exhaustively at 4C agaLns-t the buffer. ~ , Ihe sa~ple is applied to the colu~n which is develc~ed with a ~low speed of 5 ml/hour at 4C. It is washed with 500 ml of 0.1 M
N~4NCO3 buffer pH 8Ø The elution of the ~iological ac:tlvlty is effected at room temperature with : :-.. ... ,. .. , . . , . . . , ., . _, , .. . .. .. ., ~ . . . . ........ . . ... . . .... .

. ~ . . ; . , OO-gll2 i3~

0.1 ~ NH4HCO3 bufer p~ 8. a, o . 1% sodium dodecyl ~ulphate (SDS). The SDS eluted material 15 separately subjected to the following treatment,for separation of the SDS and renaturatio~ with an urea solution (K. Weber, D.J. Kuter, J. Biol. Chem~ 246 4504 [1971])~

The protein solution is brought to 6 M urea by add-ition of solid urea, incubated at 30 minutesl and dialysed against buf~er solution A [0.05 M Tris-acetate pH 7.8, 6 M urea] . The SDS is removed on a column of Dowex 1 x2, equilibrated ~Jith buffer A~ The development .
of the column is then efected with the same buffer.
The recovery of the protein from th~ urea solution is effected at room temperature by dropwise dilution 1D a 0.05 M Tris-acetate buffer pH 7.8, so that the end dil- :
ution is at least 10-fold. The solution is then concentrated over ~iaflo UM 10 filter at 4C in an Amicon~
C~ll, and dialysed at 4~C against 0~05 MTris~acetate buffer pH 7.8, to ~emove the remainder of the urea.
~he column gel can be obtained in the following manner~-. 20 A) Cou~lin~_of qanqlioside ... ..... .. . . .. . . .

~Albumin agarose (25 ml) is washed wi~h wa~er ~;
and suspended in a 50% tV/V] aqueous dioxane solution.

- : ' - ., . ~:

:
. . .... . .... .... . , . , ., .... . .,.. ........ .. . . . , . .. . .. , .~ . . .... . .. _ . . . . .. ...

. ' , . . . . .

~ ~8 - 900-9112 ~53~S~

~o mg o the des~red ganglioside, for example monoslalosyl-ganglioside GMl rGGNSLC] or GGtet3 [SGGNSS~C] is added to this suspension and the mixture is shaken for 15 minutes at room temperature. After the addition of 100 mg of 1-ethyl-3-dimethyl-3-(3-dimethylamino-propyl)carbodiimide, ~h~ suspension is shaken fox 6 hours at room ~emperature, ~
whereafter a further 100 mg of the carbodiimide is added. ~ `
After shaking ~or a further 12 hours, the gel is washed with 500 ml of water, 500 ml of 75% ~V/V) a~ueous methanol~ 250 ml of 6M guanidine hydrochloride and a further 500 ml of water. The ganglioside content, determined from the difference hetween the added ganglio-side and the recovered, unreacted ganglioside, is xoughly 0.5 mg/ml of gel.

15The coupling can also be effected by reacting 20 mg of the ganglloside and 5 mg of dicyclohexylcarbo~iimide in 10 ml of dioxane for 30 minutes at 15C. The mlxture ;;~
is then added to 20 ml of albuminagarose suspended in d~oxane to a total volume of 40 ml. ~fter 15 hours at xoom temperature, the gel is washed with 500 ml of diox-ane, 500 ~1 of 90~ (V/V) methanol and; finally~ with 250 ml of 6M guanidlne hydrochloride.

... .. ..... .. . ... . . .... . . . .. .. .. . . ..
--''' ' ' ~ :

'~ ' ' . , ' B) Coupling of N-Hydrox~succinimide ester of qanglioside __________________________ ________________.___~_____ 20 mg of the ganglioside/ for example as under A), `.~.
abo~e, is reacted wlth 2.5 mg of N-hydroxysuccinimide and 2.5 mg of dicyclohexyl carbodiimide, ln 10 ml of S dioxane, for 30 minutes at room temperature. ~he solution is then added to 20 ml vf albuminagarose suspended in d~oxane to an end volume of 40 ml. After 15 hours shaking at 4C~ the gel is washed with 500 ml of dioxane, 500 ml of 90~ (V/V) methanol and with 250 ml of 6M guanidine ;
hydrochloride.

C) Usin~ mixed anhydride of gan~lioside 100 ~1 of 0.1 M N-methylmorpholine in tetrahydrofurin i~ added to a solution of 20 mgof the ganglioside, for example as under A), above, in anhydrous tetrahydrofuran.
The mlxture is stirred for 10 minutes at 0~, ana 100 of 0.1 M isobutylchloroformate in tetrahydrofurin is added. The mixture ~s maintained at 0C for 20 minutes and is then a~ded to 20 ml of albuminagarose suspended in dioxane to an end volume of 40 ml. The mixture is then maintained at room temperature for 15 hours and ~he re-~ulting gel is washed as described under A), abcve.
In manner analogous to E~amples A, B and C, ~x~e! gels may be prepared using a copolymer of polyl-L-l.ysyl-DL-alanine ~`~
~2.'~

....... .. . . . . , _ .. . . . _ .. _ _ . _ . . ..... ` . . . . . . .

, ~S3~ g~-9112 and cyanobromide activated agarose ln place of the albumina~arose~

D). Preparation of a fatty acid - a~arose derivative com~lex Amlnoethylamino -, aminohexylamino - and aminodecyl-aminoagarose d~r~vatives may be produced in accordance ::
with the method of Cuatrecasas and Anrinsen Methods in Enzymology 22, 345 E1971], by activation of Sepharose 4B
with 200 mg of CNBr/ml of resin at pH 11-11.5, and stirring at pH 10 and 3C for 15 hours with the corresponding diamine. The molar proportion of hexane-or decane-diamine to CNBr is 1, while that of ethylenediamine to CNBr is 5. The aminoalkylamino-agarose derivative is - aged for S days and reacted with lM 2i~minoethanol at room temperature, in order to saturate any remaining activated groups of the Sepharose. The resulting derivative contains about 20 moles of free amino groups /ml of agarose. The desired fatty acLd, (for exmaple palmitic arid) is coupled by stirring, for three days, a mixtuxe of the .
agarose deri~ative in 1.5 times its volume of a 0.1 M ~ :
solution of the sodium soap of the atty acid at pH 10 and at 37~, in the presence of 50 mg of ethyl -3-(3-dimethylaminopropyl)carbodiimideper ml of Sepharose. 25%
ethanol is added to raise the solubility of the soap. The product is washed at 37C wit~ 50% (V/V) ethanol, ethanol~
0.07~ M sodium phosphate (1:1), pH 2.4, and with ethanol-0.05 N NaOH. Remainlng anreacted groups are satura~ed .:
... ~ . . .~ ..................... .. .... ............ ....... .

.
-- , ~

- 3~ S3~ goo~9ll2 by acetylatin with acetic anhydride.

Example 5 Molecular wel~ht determination by ~el filtrQtion For determinatlon of th~ molecular weiyht of E. coli ~.
P263 (LT) ente~otoxin, the method of gel filtratiQn may be :~
used. A Sephadex G 150 column is calibrated by the method of P. Andrews ~Biochem~ J. 96, 595 [1965~) using horse heart cytochrcme C (MW 1.3 x 104), bovine pancreas chymo~
slnogen A, ~2.4 x 10 ), ~ cken aIb ~ n (4.5 x 10 ), bcvine serum ~ . :
alb~n (6.8 x 104), rabbit muscle addolase (1.47 x 105) and bovine liver catal ~ (2.2 x 105) as comparative protems tmarkersj.
The proteins are monitored by their transmission at 280 mH.

Further for the preparation of the standard curve, tritiated water ~1.11 104 dpm) is monitored by liquid scintillation and a saturated solution of blue dextrane is used and its optical density at 620 ~ is ut1li~ed in its detection.
The Sephadex columIl is equilibrated with 50 mM of Tris-chloride, pH 7.5, ~OOmM KCl and 0.01 M 2-mercaptoetha- .
nol. All of these materials, in total ~olume of 0.~ ml are transferred to the column and eluted in 0.9 ml fractions. The experiment is caxried out a~ 4C. The molecular weight of E. coll P263 (LT~ e~terotoxin is determined as 102.000 + 3.000 (the limits of experimental error).

.- . ' ' - ' ' -' ', ' , , ,, , ', ,, ~ ' ' .......
..

.

~ 32 - ~5~LS~ goo~gll2 E ~ 6 ~lo ~=~ YL~5 m~

~ Polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulphate is carried out at a pH of 7.2 in 7.5~ acryl ~ de by the method of A~L. Shapiro, E. Vmuela, J~Maizei.(Biochem. ~oph~s.
R~S. ~nLn. 28, 215 ~1967]), and J.~eber, M. Osborn (J.B.C. ~44, 4006 [1~69]~. The foll~wing proteins æe used as st~ndards:-bovine pancreas trypsinogen (mo~ecular weight of poly peptide chain - 2 . 4 . 10 ), bo~ine heart lactate dehydrogenase ~.
lQ (3.6.104), bovine serum albumin (6.8.10 ) t rabbit muscle phosphorylase (9 .4~104) and E. coli ~-g~actosidase (1.3.105).
A 1% solution of the sample (E. coli P263 (LT) entero- ~`
toxin) in sodium dodecyl sulphate and 2-mercaptoethanol is boiled or 3 minutes to eliminate the possibility o proteolytic degxadation. The sample is then dialysed against a solution consisting of 0.1% in sodium dodecyl sulpha e and 2-mercaptoethanol, 0.OlM sodium phosphate, pH 7.1. The comparative proteins are treatedin the same way.

io The molecular ~7eight of E. coli (LT) enterotoxin is determined by this method as 102,000 ~ 3,000 tLimits of experimental error). `-., ' ' ' ' ~'~'' .. , -~ i. ~.`'.

:

_ ~0-9112 D ~

To determine the isoelectric point of the purified enterotoxin, isoelectric ocusing is carried out in a glass column ~Type 8100, ll0 ml) according to the method of O. Vesterberg and H. Svensson (Chem. Scand. 20, ~20 tl966~. The anode electrolyte (phosphoric acid) is located at the bottom oE the apparatus. The Ampholine carried ampholytes cover a pH range of 3 to l0. Optical density i.s measured at 280mm by emptying the column thxough the measuring cell of an Uvicord II - W- absorp- ;:
tiometer. The isoelectric point of the protein is determined at 4C by measuring the pH o~ the eluted fractions with a combined microelectode.

The isoelectric point of E. coli P263 (LT~ enterotoxin is determined by this method as 6.95.

Enterotoxin is isofocused on a 5~ polyacrylamide gel plate according Z.1. Awdeh et.al. (Nature [London] 219, 66 tl966])as modified by J. Bours and W.J. van Doorenmaalen ~Science Tools 17, 36 tl970]. A polyacrylamide gel of 26 x 12.5 om and 1 mm thickness is produced wi~h an end ', :~ ' ' - ,. . . .. ,....... ... ... ~

~s~ s~

.. concentration of 2~ "~mpholine sarrlex ampholytes" with a pH range of 3 5, 5-7, and 7-10 in the proporti.on 1 The gel is polymerised for 2 hours with rlboflavin as photo- `~
c~talyser. 10 to 25 ml of a 0.1% solution of the sample in 2% Ampholine, pH3-10, is placed on 10 x 5 mm ~hatman 3 MM paper strips which are layed on ~he gel. The iso-electric focusing is carried out at lO~C in an electropho-- resis apparatus. Before the gel plates are fi~ed to the electrodes, the ele¢trodes are covered with 0.5 x 25 cm filter-paper strips, which are moistened with 0.1% (V/~
ethylenediamine solution (cathode). The initial power is 50mA (and is decreased to 28mA) for 2~5 hours at 210 volts ~raised t~ 1080 volts). The pH gradient is established :~
with a combined microsurface electrode. Af~er isofocusing, 1~ the proteins are precipitate~ith 14% trichloroacetic acid at 60C. The gel is then washed at room temperature with 10%, 5~ and 3~ trichloroacetic acid over 24 hours, in ~rder to remove the carrier ampholytes. The gel is then ;:
stained with a 0.05% solution of Coomassie Brllliant Blue ~; -R-~50 ~dissoived in methanol/acetic acid/water-45/9/46) over 2 hours, followea by washing in the same solvent. : .

For re-swelling, the gel is placed in acetic acid/water ~, ~9:91), and is then photographed.

The isoelectric point of E. coli P263 (LT) enterotoxin by this method is 6.95. .

` ' ~ .

,, ;~

. ' ' '. ' ' ' ' . ' . ' .. ' ' ' ' ~' "' . ~'' ., '' " .',. ' . .' . . ,' ' ' '. '' . ' ' ' ' . ' . , ~ 3~ ~ 900-9ll2 E~ I ~tnrr 7f lU
6 mg amounts of coli enterotoxin (in partlcular E. coli P263 ~LT) enterotoxin) in 24 ml of tyrode solutlon are placed in 6 test tubes. ~he contents in the tubes are brought to pH 1,3,5,6,7 and 9 respectively, with concentrated HC1 or 6N NaOH. The test tubes are incuba-ted at 4C and each tube then brought to p~l 7. The contents of each tube are then tested in the rabbit and pig intestual loop testsat 2 ml (0.5 mg) per intesti~al loop.

The enterotoxigenic activity of E. coli P263 ~LT) enterotoxin is highly acid labile. The activity is fully ~:~
destroyed at pH4 or less and is already influenced at pH6.

Exam~le 10 Influence of tem~erature 5 mg quantities of the purified enter~toxin (in particular P263 (L~) enterotoxin~ are each taken up ln 20 ml of tyrode solution and heated at 40, 50, 60, 65.
70 and 100C, respectively for 30 minutes. The heated solutions are tested in the rabbit and pig intestinal `
loop tests with an lmtreated solution as control. 2.0 ml (0,5 mg) are administered per intestinal loop.

~he biological activity of the enterotoxin is fully destroyed by heating for~ 30 minutes at 65C. The heat-lability ls already evident at 50C, and the complete destruction of the act~lty occurs mainly already at 60F.

.
- ; ' ;

~ : . . ~ ,. ; . . -~ 36 ~ 3~2 g~o-gll~

E ~ ~vidence of :lmmunol~
8~
~he fre~ze-dried E. coli strain P. 155, serotype 0149:K01, K88a, c or human pathogenic E. coll strain H 10407, or the Ent E. coli strain K 12 and Ent E. coli strain K 1~ are used to produce a cell-free culture filtrate as described above for strain P 263. rrhe cell ~:
~iltrate serves as sample with unknown co~position of Antigen Ag-X~

~he identification and quantification is earried out employing a reference antigen, Ref-Ag (the pure coli enterotoxin ~rom strain P 263 or H 19) and a reference antiserum Ref-Ab (monovalent antiserum against colientero-toxin3. .

The two questions, whether Ag X contains an antigen ;~
which is "identical'l to Ref-Ag, and, i~ so, how does the . ~ .
concentration of this antigen compare with the known ' concentratLon of antigen Ref-Ag, can ~e resolved by "crossed immunoelectrophoresis [H.G.M. Clarke and i:
T. Freeman, Clin. Sci. 35, 403 (1968)] and"tandem crossed electrophoresis"~Kroll J., A manuel of Quantitative Immunoelectrophoresis, edit. N.H. Axelsen et.al.
Universitetsforlagetr Oslo, page 57 (1973), Suppl. 1/1973, Scandinavian J. Immun~
;:

'''' ::

.

~S31~ 900-9112 With ref~rence to the accompanying drawings, the drawing Abb. 1 shows a "crossed lmmu~oelectxophoresis"
picture of Ref-Ag, developed against Ref-~b. Only one precipitate occurs.

In drawing Abb. 2, Ag X is used in place of Re-Ag.
On khe basis of the position of the precipi~ate in Abh. 2, lt can be taken that Ag-X contains an anti~en which is identical with Ref-Ag. This conclusion can be reinforced :~
~y reaction of the 'lidentity" using "fusing precipitates'i.

This experiment can be caxried out with the help o "tan-dem crossed immunoelectrophersis (Abb. 3).

By comparing Abb. 3 with Abb. 1, it can he seen that ~.
Ag-X con~ains Ref-Ag and that the concentratlon of the latter in Ag-X is less than in Ref-Ag. The quantifying of the fused peaks can be e~fected by standard methods (J. Kxoll, J. Clin. Lab. Invest 22, 112 [1968]).

If the culture f~ltrate:Ent E~ coli K 12 strain is -used in this test in place of Ag-X, no precipitate occurs in Abb. 2. ~his confirms that the precipitate occurring 29 in Abb. 2 is associated with the enterotoxins of pathogeric E. coli strains.
, ~ The same experimentation can be carried out to .
esta~lish th~ immunologic identity or cross-~mmunity of human pathogenic and porcine pathogenLc E~ coli enterotoxins.

`
... ~. ........... ..

. ~ . - -.

~ ~ S3~ ~ g~0-9112 In th~s test, pure E. coli P 263 ~LT) enterotoxin serves as Ref-Ag, and the monovalent antiserum against the P 263 enterotoxin as Ref-Ab. The human pathogenic enteroto~in H 19 serves as the sample wlth unknown composition of Ag-X. As descrlbed for Abb. 2, a single precipitate also occurs in the plcture of "crossed ;~
lmmunoelectrophoresis" of Ag-X developed agai~st ~ef-Ab in the same position as in Abb. 1. On the basis of the position of the precipitate, it can be taken that Ag-X : ~
contains an antigen whlch is identical to Ref-AgO ~ -~he reaction of the "identity" using fusing precipitates ~ :
in "tandem crossed immunoelectrophoresis" shows (as in Abb. 3) that Ag-X and Ref-Ag are serologicall~ identical. ~.
In view of these findings, human pathogenic or porcine pathogenic coli esterotoxins may be used as antigens for immunisation or for preparing toxoids.

Employing the same expeximentation, the question of whether there is,between E. coli enterotoxin and V.
cholerae enterotoxin,a partial or cc~plete1mmunological ` .
identity can be investigated. The pure E. coll P 263 ~LT) enterotoxin serves as Ref-Ag, while the E. coli P 263 antitoxin is used as Re.Ab and the pure V. cholerae toxin sexves as antigen sample Ag-X.
Th- results are show- in drawings Abb. 4, 5 and 6.

~, ' ', '. ' . ... ;:

,' `~ ~ ' - ' ~
:. -, ,, . :
- .

9~0~9112 '~S~

In fi~ure Abb. ~, as before, a single precipltate occurs. In Abb. 5 ~gain a single precipitate occurs, but in a different posltion and haviny a different form ~o that in Abb. 4.

From Abb. 6 ~"tandem-crossed immunoelectrophoresis") it is clear that there is a distinct and at least partial ~dentity bet~Jeen Ref-Ag and Ag-X (E.coli enterotoxin and V. Cholera- enteroto~in~O

Exam~le 12 Active immun~sation l~ Breeding sows are artificially inseminated and immunised for the first time one month before the estima-ted litter time: 0.95 mg of purified E. coli enterotoxin (in particular P 263 (LT) enterotoxin) together with 15 ml of complete Freund's adjuvant (7.5 ml of adjuvant ~ 7.5 ml NcCl-buffer 0~05 MJ 10 2M ~mercaptoethanol) is given i.m. After 2 weeks, a further 0.35 mg of enterotoxin in S ~.l of 0.9~ NaC1 solution is given i.vo ,~

Because of the serological identity between porcine and human pathogenic coli enterotoxins, the purified ~LT) en~erotoxin of strain ~ 19 may, for example, alternatively be used.

6~ 12 and 24 hours ater the littering of the hreeding sows, colostrum is drawn off and pooled. ~6 hours after the litter time, blood is taken for the ob~ention of ~erum.

- -- - , - , .

.. , - ,...... , , . :, . :

~ ~o ~" gOO-9112 ~s~

The determination o ~he antibody tltre may be carried out by the B~nto~it-flocculation-microtest (SBF-test). The following results are obtained. :~

. .
Time Titre .
Colostrum 6 h l : 512 12 h l : 512 24 h l : 64 ~:
,~: ' Serum 1 : 64 _ _ . __ . ~

, .'.:
~ A 1:2 diluted plg serum bay be used ln neutralisation studies in the rabbit intestinal loop test. Upon use of a ;:
toxin dose of 0.25 mg/loop (i.e. 3 toxin units) more than 60% inhibition is obtained. Correspondlng tests with colostrum (6 hours post partem) shows,in the rabbit model, ~.
a complete neutralisation of the toxin activity.
~l toxin unit is defined as the ED53 in the intestinal ~:
~oop test or in th~ adenylate cyclase system] ~
:
~33~ P~D~L~O~ ' .
In order to achieve op~imum conditions for the lnact~vation, on the basis of a molecular weight of 102,000 for the toxin molecule, concentrations of glutaraldehyde are initially varied f~om 50 to lO00 mol/
mol of toxin. The reaction is carried out using a toxin .

- ' - - ~ -- ~ .

90~-9112 ~ ~ 5 ~ 5 ~
concentra~ion range of 100 to 1000 ~g/ml, at 30~C in a 0.065M Pss-buff~r pH 7.8 and or various incubation times.
The reactionsare stopped by dialysls twice against 100-fold volumes of PBS-buffer pH 7.8. At a toxin concentra~
tion of over 600 ~g/ml3, the reaction product begins, with increaslng concentration of glutaraldehyde, to be indoluble. If the toxin concentration is maintained between 100 and 600 ~g/ml and the glutaraldehyde concen-tration varied between 50 and 400 mol/mol toxin, the reaction product remains in soluble form. ~.
The adenylate cyclase test shows that the optimal detoxification is achieved in the pH range 7.8 to 8~2 If a toxin concentration of 400 to 600 mg/ml is selected, the residual toxicity (measured after incubation or 72 hours at 30C) decreases ten-fold on two-fold increase of the glutaralde concentration between 50 and 200 mol/mol toxin. If the glutaraldehyde concentration is raised fxom 200 to 400 mol/mol of toxin~ the residual activity is not detectable even wi~h the most sensitive methods tadenylate cyclase last).
In order to detect preparations of inactivated ~oli enterotoxin (toxoid) ~uantitatively, toxin units are - determined by the method of Crai~ (J.P. Craig, 1971, : ~holera toxins, pp 189-254 in S. Kadis et.al.ledi~), Microbial Toxins 2, Academic Press Inc. New York~. :

:` :
. ~ :

,. .

. ' ' ~

. . .

4~ ~ S 3~ 00-~112 ~his determination is based on the abillt~ of toxins to reactin vitro wlth the antitoxin, The test determines , the decrease in neutralisation capacity of a known quantity of antitoxln, after lncub~tion with toxoid. A
series of test tubes, each containlng the same quantity :~.
of antitoxin (2AU/ml) and a corresponding dilution of toxoid are each incuba~ed at 37C for 30 minu~es. A ~:
urthex series of test tubes serves as control, each tube containing ~he same quantltiy o~ anti~oxin (2AU /ml) to which buffer has been added, and being incubated undex the same conditions. After th~ incubation, identical volumes of a 0.151Og series dilution of the toxin are added to the test tubes and ~he de~exmination is effected with the adenylate cyclase test.

The displacement.of the 50% v~lues in the dose-actiYity curve is then used to dete~nine the quantity of free and bound antitoxins in the antitoxin - toxoid mlxture. 1 TU is the amount of toxoid whLch binds 1 AU
of the antitoxin. (AU --antitoxin unit).

2~ E ~ Adenylate~yclase test_ The production of the adenylate cyclase preparation in.partlculate form is effected by the method of Levey tG.S. Levey and E. Epstein, Biochem. Biophys. Res.
Commun. 33, 990-995 ~1968) and 38, 86-9~ (1970)] using ~ ~3 ~ 900-9112 ~ S~ 5~2 cat myocordial ~issuP. The activation of the adenylate cyclase b~ the coli enterotoxin is determinPd by the radioisotope dilutlon test ~A.G. ~ilman, Proc. Nat. Acad.
Slc. 67, 305-312 ~1~70] uslng 5-adenylimidodiphosphate ~AMP-PNP) as the substrate in place of ATP~ The ~MP-PNP
is purified before use by passage through a column of Dowex 50 x 2 in the H form.

~he reaction medium comprises 50 n~l Tris-HCl buffer ~pH7.4), 5mM of M~Cl~, lOmM of theophylline, 0~1~ bovine 0 serum albumin and 2mM of AMP-PNP. The incubation volume amounts to 0.1 ml in total and the enzyme concen-tration is 0.15 mg of protein per test. The incubation is started by addition of the enzyme and is carried out for 30 minutes at 37C. To terminate the reaction, 500 ml of 7.5~ trichloroacetic acid is added and the mixture maintained at 4~C for 10 minutes. ~he preci-pitate is separated ~y cen~ifugation and the supenatant ~s added to 5ml of diethyl etler saturated with water.
The layers are separated by centrifuging and the ether phase ~ removed. After 3 ether extractions in all, ~he aqueous solution is reduced to dryness at 60-70C on a water bath with an air-stream. The residue is dissol~ed ln 1 ml of 0.~ M acetate buffer (pH 4.0). The quantity o cAMP formed, a direct measure of the degree of activation of adenylate cyc1ase by ~oll enterOtOXin,iL ~ ;

~ ':
.... ........

~ ~00-9112 SZ

determined by means of the above mentioned test (available as a test kit from Boehringer Mannheim, W. Germa~y), Exam~ Production of a vaccine An enterotoxoid solution ~in particular E. coli P 263 ~ :
(LT) enterotoxoid) (lmg/ml) is dialysed exhaustively against a solution of Na HPO4 (0~07M) and filtered on a : .
Seitz-sterilisation filter. The sa~e volume of a sterile 0.07M CaC12 solution is then added to produce a vaccine absorbed on calcium phosphate with a pH o 6.8 to 7. For inoculation,the vaccine is diluted and a~NI~stered in a dosage of 1 to 100 ~g, for example.

E~m~ o~ e~ 8e_l9=~

Freeze-dried ~uantities of enterotoxo.idtin particular P263(LT) entexotoxoid) (for example 100 ~g-lmg) are pxessed lnto a dragee core ~ith a suitable filter (which can :
also serve as an adjuvant) and coated wlth an anionic ~:
: polymer ~rom methacrylic acid and methacrylic acid esters.
gfor example Eudragit L and S~, with ~hich a softening agent has been mixed~ Coatings of this type protect against the a tion of gastric juices because they are insoluble in the acid pH range and water-impermeable; thus they are resistant to gastric juices. After passaye through th~ -; ~tomach, the coatings dissolv~ Fox example Eudragit C -' ~ .

.

... , . ... . .. . .. . ~ ........... . .. ......

-' , ' . : ' _ ,~5 _ 900-9112 1~53~

~ lms dlssolve in the neutxal pH range ~rom pH6 (duo~
~enal juice5), while Eudragit S - ~ilms are ~irst s~ ble in the law-er small intestin~l regions ~ from pH7 ) .

,' ' - ' . ~'' ' .
;.~ ' , - . - - :

Claims (9)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for the isolation and purification of E. coli enterotoxin from a crude or pre-purified cell-free culture filtrate of a fermenter culture of an entero-pathogenic E. coli strain, comprising subjecting such culture filtrate to isotachophoretic or affinity chroma-tographic separation.

2. The method according to claim 1 wherein the culture filtrate is subjected to isotachophoretic separation.

3. The method according to claim 1 wherein the culture filtrate is subjected to affinity chromotographic separation.

4. The method according to claim 1, 2 or 3, wherein the E. coli strain is selected from porcine pathogenic E. coli strains P 263, serotype 08:K87, K88ab:H19; P155, serotype 0149:K91:88a,c; and P 307, serotype 08:K87,K88ab;
human pathogenic strains H10407, serotype 078K? and H19, serotype 026:K60:H11 and an Ent+ E. coli K12 strain to which the Ent plasmid from human strain H19 has been transferred.

5. The method according to claim 1 wherein the sep-aration includes the step of isolating a heat-labile (LT) E. coli enterotoxin.

6. The method according to claim 1 wherein the sep-aration includes the step of isolating a heat-labile (LT) E. coli enterotoxin of molecular weight 102,000 ? 3,000.

7. The method according to claim 5 which includes the step of reacting the heat-labile (LT) E. coli enterotoxin with formaldehyde or glutaraldehyde.

8. The method according to claim 1, 2 or 3 wherein the free-cell culture filtrate is pre-purified by gel filtration.

9. A heat-labile (LT) E. coli enterotoxin having a molecular weight of 102,000 ? 3,000 which has been isolated and purified by the process of claim 6.