INFECTION AND IMMUNITY, Feb. 1974, p. 294-303 Vol. 9, No. 2Copyright © 1974 American Society for Microbiology Printed in U.S.A.

Development of a Purified Cholera ToxoidI. Purification of Toxin

RUTH S. RAPPAPORT, BENJAMIN A. RUBIN, AND HOWARD TINT

Wyeth Laboratories, P.O. Box 8299, Philadelphia, Pennsylvania 19101

Received for publication 15 February 1973

The enterotoxin from Vibrio cholerae is selectively concentrated from cell-freeculture supernatant by co-precipitation with hexametaphosphate and is furtherpurified by adsorption on aluminum hydroxide powder. The bulk of residualsomatic antigen becomes insoluble upon lyophilization of the toxin preparationand is removed by centrifugation of the rehydrated material. Other contaminantsare eliminated by treatment with activated carbon. Preparations of toxin,purified by this method, have been characterized by: (i) a single immuno-precipitin line against polyvalent antisera; (ii) homogeneity on acrylamide gels;(iii) specific activities on the order of 22 limit-of-bluing doses/Ag; (iv) ultravioletspectra characteristic of pure protein; and (v) overall yields on the order of 50%,irrespective of purification scale. Such preparations, however, have been shownto contain trace amount$ of somatic antigen when they are intensively testedeither for their ability to elevate serum vibriocidal antibody titers in immunizedrabbits or for their ability to increase resistance of immunized mice to live vibriochallenge. In the latter test system, the level of residual somatic antigen per 50jig of toxin (toxoid) antigen generally did not exceed 0.025% of the Division ofBiological Standards reference vaccine, V. cholerae Inaba IN-12. Methods forelimination of this small amount of somatic antigen have been investigated andare discussed. The particular combination of purification steps which arepresently described have been easily and reproducibly applied on a productionscale to prepare gram amdunts of toxin with a high degree of purity, even under avariety of initial conditions.

At present, a convincing body of evidencesupports the view that the symptoms of clinicalcholera are caused by an enterotoxin secretedinto the gut by proliferating Vibrio cholerae (2,18). On the basis of this view and becauseconventional bacterial vaccines do not provideadequate long-term protection (1, 24), interesthas been aroused in determining whether an-titoxic immunity alone can provide effectiveprotection against the clinical manifestations ofcholera.The availability of V. cholerae strains, most

notably Inaba 569B, that can produce toxin invitro, together with reliable methods for itsproduction (8, 13, 20), purification (12, 21, 22),and routine assay (3-5), have greatly advancedthe possibility of testing this hypothesis.

Further, the development of experimentalcholera models (2, 18) has made it possible tostudy the efficacy of antitoxic immunity inpreventing or controlling symptoms of the dis-ease. In the canine model, for example, paren-teral immunization with crude cholera toxin,

which stimulates the production of both anti-bacterial and antitoxic antibody, protected dogsagainst subsequent intraintestinal challengewith viable V. cholerae (6). This protectioncorrelated more significantly with serum an-titoxin titers than with serum vibriocidal titers(6). More recently, dogs immunized parenter-ally with pure cholera toxin (plus Freund adju-vant) exhibited elevated serum antitoxin titersand were protected against challenge with via-ble V. cholerae for at least 10 months (19). Inthe same study, parenteral immunization withformalinized toxoid (without adjuvant), al-though resulting in lower antitoxin titers, gaverise to significant protection which endured fora minimum of 5 months (19). This resultsuggested that antitoxic antibody elicited bychemically detoxified toxin (toxoid) may alsocorrelate with protection in experimental ca-nine cholera. It remains to be determined,however, whether or not appropriate parenteralimmunization with a suitable cholera toxoidwill provide effective protection in experimental

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human cholera, and such studies have alreadybeen initiated (R. B. Hornick, personal commu-nication).To determine unambiguously whether an-

titoxic antibodies alone can provide effectiveand prolonged protection in the field, a puretoxin, possessing little or no ability to inducevibriocidal antibodies, is required. To producepurified toxin in sufficient quantity for poten-tial use as a prophylactic against cholera, a

simple, large-scale purification scheme hasbeen developed. The details of this method are

described in this report. The problems sur-rounding the preparation of a stable, antigenictoxoid are the subject of a subsequent commu-nication.

MATERIALS AND METHODSProduction of toxin. V. cholerae serotype Inaba,

strain 569B, was obtained from John C. Feeley of theNational Institutes of Health, Bethesda, Md. Approx-imately 250 liters ofTRY medium (20) was inoculatedwith vibrios from an overnight nutrient agar slant,and the medium was incubated with aeration at 26 Cfor 48 h. In later studies, incubation time was changedfrom 48 to 24 h to minimize components arising fromlysis of the vibrios. After incubation, the organismswere harvested by centrifugation and discarded, andthe supernatant, containing toxin, was filteredthrough a membrane filter (0.22 Mm pore size, Mil-lipore Corp.) and held at 4 C.

Purification of toxin. The procedure adopted forpurification was as follows. Sodium hexametaphos-phate (2.55 g/liter) (Fisher Scientific Co.) was addedat room temperature to cell-free toxin supernatantprecooled to 4 C. When the metaphosphate was

dissolved, the pH of the solution was adjusted slowly,with stirring, to pH 4.6 by the addition of concen-

trated HCl. The solution was stirred under theseconditions until optical density readings at 640 nmindicated no additional precipitation. When equili-bration was complete, filter aid (0.5 g/liter) (Celite-Johns-Manville) was added, and the mixture was

stirred for an additional 10 min. The precipitate was

collected on a filter bed (Whatman no. 1 filter paperplus filter aid) by suction filtration, and the filtratewas discarded. The precipitate was washed on thefilter with 20% of the initial volume (of toxin superna-tant) of cold 0.2 M NaCl, pH 4.5 to 4.6, and the washwas discarded. The washed precipitate plus filter aidwere resuspended in one-half of the desired finalvolume (that volume which concentrated toxin to avalue of approximately 3,000 limit-of-bluing [Lb]doses/ml) of 0.15 M Na2HPO4, pH 8.0; the precipitatewas dissolved by stirring at room temperature for 1 h.The solution was collected by suction filtration, andthe filtrate was set aside. The filter aid was resus-

pended in the remaining 0.5 volume of buffer, stirredfor 1 h, and collected as before. The first and secondfiltrates were combined. This solution was designatedmetaphosphate concentrate and was held at 4 C untilfurther use.The metaphosphate concentrate, at or near room

temperature, was adjusted to pH 5.6 by the dropwiseaddition, with stirring, of concentrated HCl. To each100 ml, 30 g of Al(OH)8-3H2O (Mallinckrodt Chemi-cal Works) was added and dispersed uniformly bystirring at room temperature for 2 h. The adsorbentwas collected by suction filtration with Whatman no.42 filter paper. The filtrate, designated aluminumhydroxide supernatant, was discarded, and the ad-sorbent was washed on the filter with a volume ofdistilled water, pH 5.3 to 5.5, equal to twice the initialvolume of metaphosphate concentrate. The adsorbentwas resuspended in one-half to three-quarters of theinitial volume (metaphosphate concentrate) of 0.2 MNH4HCOS, pH 7.9 to 8.3, and the suspension wasstirred for 1 h. The suspension was suction-filtered, asbefore, and the adsorbent was discarded. Alterna-tively, toxin could be eluted from the adsorbent in twosequential extractions by using one-half of the desiredfinal volume of buffer for each extraction. The filtrate,designated bicarbonate eluant, was held at 4 C untilfurther use.The bicarbonate eluant was lyophilized and then

redissolved in a volume of 0.067 M phosphate-buf-fered saline, pH 7.8 to 8.0, so that the proteinconcentration was approximately 1 mg/ml. Acid-washed activated carbon (Pfanstiehl Laboiatories,Inc.) was added to a final concentration of 1 mg/mland dispersed uniformly throughout the solution bystirring for 1 h at room temperature. The carbon wasremoved by suction filtration, and the concentratedtoxin solution was centrifuged either in a Spinco 30rotor at 78,140 x g for a minimum of 6 h or in a Spinco21 rotor at 44,330 x g for a minimum of 16 h. Thesupernatant was removed from the centrifuge tubesby aspiration, filtered aseptically through a membranefilter (0.22 um pore size, Millipore Corp.) and storedat 4 C. The sediment was discarded.

Preparation of antisera. Antiserum was preparedin goats. Toxin preparations were mixed 1:2 withAmphogel (Wyeth Laboratories, Inc.), and 2.5 ml ofthe mixture was injected intramuscularly into each offour legs. Boosters were given 2 and 4 weeks after theinitial injection by using 1.25 ml of the mixture ineach of four legs. The goats were bled 2 weeks after thelast booster injection. Antisera against two prepara-tions of toxin were obtained. One preparation, con-taining approximately 3,000 Lb doses/ml, was a 10 xAmicon PM-10 concentrate of the antigens in crudecell-free culture fluid. The second preparation, con-taining approximately 1,300 Lb doses/ml, was con-centrated from cell-free supernatant by precipitationwith ammonium sulfate (60% saturation), adsorbedonto and eluted from aluminum hydroxide, andfiltered through an Amicon XM-100 membrane. Thelatter preparation contained some sedimentable so-matic antigen as determined retrospectively by analy-sis of both the immune sera and the antigen prepara-tion. Antiserum to the first preparation was labeledWyeth goat no. 1 antitoxin, and antiserum to thesecond preparation was labeled Wyeth goat no. 2antitoxin.Measurement of toxin potency. Toxin content of

preparations was estimated by the limit of bluingmethod in rabbit skin as described by Craig (3, 4),

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with the exception that 1% Evans Blue dye (Al-lied Chemical Corp.) in saline, given intravenouslyin a dose of 0.5 ml/lb (about 0.45 kg), was substitutedfor Pontamine Sky Blue 6BX for visualization ofzones of increased vascular permeability. For titrationof Lb potency of the toxin, 1 volume of provisionalstandard cholera antitoxin (Swiss Serum and VaccineInstitute-purified cholera antitoxin [horse] with anassigned value of 4,470 antitoxin units [AU] per ml,supplied by John Seal, National Institute of Allergyand Infectious Diseases) containing 1 AU/ml wasincubated with an equal volume of appropriate serial0.15 log toxin dilutions (in 0.067 M phosphate buffer,with 0.1% gelatin, pH 7.4) for 1 h at 37 C before quad-ruplicate intracutaneous inoculations of 0.1 ml intoeach of two rabbit skins. The results were basedon the Lb end point as redefined by Craig (4). One Lbis that amount of toxin which, in the presence of 1AU/ml, gives a 4-mm bluing lesion in rabbit skin (4).Unless otherwise specified, all Lb titrations werecarried out at the Lb/20 level, and potencies areexpressed in Lb doses per milliliter.

Toxoid units. To quantify preparations of inacti-vated toxin (toxoid), toxoid units (TU), a measure ofantitoxin combining power, were determined accord-ing to a method devised by Craig (4; personal commu-nication). Based on the ability of toxoid to combinewith antitoxin in vitro, the assay measures the appar-ent decrease in neutralizing capacity of a knownamount of antitoxin after it has been incubated withtoxoid. A series of tubes containing equal volumes ofantitoxin (2 AU/ml) and an appropriate dilution oftoxoid were incubated at 37 C for 30 min. Anotherseries of tubes containing equal volumes of antitoxin(2 AU/ml) and buffer were incubated under the sameconditions. After incubation, an equal volume ofserial 0.15 log dilutions of toxin were added to eachseries of tubes, and the assay was carried out in amanner identical to the Lb assay described above.The displacement of the bluing end point was used tocalculate the amount of free and bound antitoxin inthe antitoxin-toxoid mixture. One TU was thatamount of toxoid which could effectively bind 1 unitof antitoxin.Mouse protection test for determination of so-

matic antigen. Protective activities of residual so-matic antigen in various toxin preparations againstInaba NIH 35A-3 challenge were determined relativeto the DBS reference vaccine, V. cholerae InabaIN-12, by the mouse protection test described byFeeley and Pittman (9). For immunization, toxin wasconverted into a Formalin toxoid by a standardprocedure (see Results). Results were determined asthe ratio of the mean effective doses (in milliliters) ofthe reference to the toxoid preparation per 1,000 TU(approximately 50 gg of toxoid antigen).

Vibriocidal assay. Vibriocidal activity of variousimmune rabbit sera was determined by using Feeley'smodification (personal communication) of the bacte-ricidal assay described by Muschel and Treffers (16).Inaba VC-13, obtained from Dr. Feeley, was the vibriostrain used. Sera were obtained from rabbits 4 or 6weeks after primary intramuscular inoculation with100 gg of Formalin or glutaraldehyde toxoid (seeResults) and 2 weeks after identical booster inocula-

tions given at 4 or 6 weeks. In some instances, serawere also obtained at 6 and 12 weeks after boosterinoculation. The results of the assay were recorded asthe reciprocal of the last twofold serum dilution whichcompletely inhibited bacterial growth.

Immunodiffusion and immunoelectrophoresis.Ouchterlony-type immunodiffusion tests were per-formed at room temperature in IDF-I cells obtainedfrom the Cordis Corp. (Miami, Fla.). For quantitativeanalysis of sedimentable somatic antigen, a radialimmunodiffusion technique similar to that describedby Mancini et al. (15) was used. Uniform agar layers(1% Noble agar in 0.1 M barbiturate buffer, pH 8.6),1-mm thick and containing a final dilution of 1:64Wyeth goat no. 2 antitoxin, were prepared on glassslides (83 by 102 mm). A single immunodiffusionplate accommodated 36 wells, each of which had aninternal diameter of 2.8 mm and held 5 gliters. Serialtwofold dilutions of antigen were assayed in sextupli-cate, and zones of precipitation were allowed todevelop at room temperature for 24 h. Each immuno-precipitin plate was photographed with a Cordiscamera, and the diameters of the precipitin rings,magnified 1.9 times by photography, were measureddirectly from the photograph.

Immunoelectrophoresis was performed with aShandon electrophoresis apparatus according to theprocedure described by Finkelstein and LoSpalluto(12) with the exceptions that: (i) electrophoresis wasperformed for 2 h at 4 C; and (ii) polyvalent antiserum(Wyeth goat no. 1 antitoxin) was used for develop-ment of the immunoprecipitin pattern. Samples wereconcentrated to approximately 10 mg/ml by dialysisagainst Ficoll (Pharmacia, Piscataway, N. J.) beforeelectrophoresis.

Acrylamide gel electrophoresis. Acrylamide gelelectrophoresis was performed at pH 9.3 by using thestandard two-gel system described by Ornstein (17)and Davis (7), and by using Canalco equipment(Canal Industrial Corp., Rockville, Md.). Before elec-trophoresis, samples were dialyzed against 0.025 Mtris(hydroxymethyl)aminomethane, 0.19 M glycine,pH 8.3. Between 100 and 200 gg of protein in 0.2 to 0.3ml was layered on top of the spacer gel with a drop ofglycerol. Current was 1 mA per gel until the trackingdye entered the separating gel; then the current wasincreased to 2 mA per gel for the remainder of the run.In some instances, electrophoresis was allowed toproceed 30 min beyond the clearance of the trackingdye from the gel.

Electron microscopy. Microdroplets of purifiedtoxin (90 gg/ml) were deposited on carbon-coatedcopper specimen grids and negatively stained with2.0% uranyl acetate, pH 4.1. Specimens were observedat 50 kV with an RCA EMU-3H electron microscopeequipped with double condenser illumination.

Protein determinations. Protein was determinedby the Lowry modification of the Folin-Ciocalteaumethod (14) with crystalline bovine serum albumin asstandard.

RESULTSPurification. Table 1 summarizes the results

of purification in a step-by-step analysis of therecovery of protein and toxin activity in a

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TABLE 1. Summary of purification: specific activity and recovery

FoldStage of Vol Lb/mI Total Protein Total Sp act increase RecoveryOD22purification (liters) Lb x 10' (g/ml) ug x 10 (Lb/psg) in sp act| ( 20/260

Cell-free, culture filtrate 255 282 71.9 829 211.4 0.34 1 100 0.8Metaphosphate concentrate 30 2,692 80.8 175 5.25 15.4 45 112 1.2Bicarbonate eluant, fromaluminum hydroxide 21 2,291 48.1 121 2.54 18.9 56 67 1.4

After lyophilization.2.4 18,620 44.7 1000 2.40 18.6 55 62 1.4After activated carbon 2.4 18,410 44.2 840 2.02 21.9 64 61 1.8After centrifugation.2.4 17,990 43.2 820 1.97 21.9 65 60 1.8After 0.22-am filtration 2.4 19,720 47.3 800 1.92 24.6 72 66 1.8

a Optical density at 280 nm/optical density at 260 nm.

representative large-scale preparation. In thefirst step of the procedure, toxin was concenrtrated from cell-free culture filtrate by co-precipitation with sodium hexametaphosphateat pH 4.6. Figure 1 shows the distribution ofprecipitating species as a function of pH in thepresence and absence of metaphosphate. Theaddition of metaphosphate to the culture fil-trate enhanced precipitation throughout the pHgradient and partitioned the precipitating spe-cies into three distinguishable zones. Activityassays and immunodiffusion tests showed thattoxin precipitated in the first zone between pH5.4 and 4.6. When toxin was concentrated bythe metaphosphate procedure, increases in spe-cific activity on the order of 15-fold or greaterand recoveries of 90% or more were reproduciblyobtained in four consecutive large-scale prepa-rations.

In the second step of the procedure, toxin wasfurther purified by adsorption onto, and elutionfrom, aluminum hydroxide powder. Adsorptionwas pH dependent, with maximal binding oftoxin occurring at or near pH 5.6. Elutionappeared to be independent of the buffer systememployed as long as the pH was alkaline. In thisprocedure, toxin was eluted from the adsorbentwith 0.2 M ammonium bicarbonate, pH 8.0.Under conditions of maximal binding, toxin

recoveries were governed by the relative concen-tration of toxin and adsorbent. Figure 2 showsthe dependence of recovery on percent concen-tration of adsorbent suspension for the case inwhich the amount of toxin exceeded the adsorp-tion capacity of the powder. When conditionswere such that the ratio of total Lb units pergram of adsorbent was on the order of 10', toxinrecoveries ranging from 60 to 72% were reprodu-cibly obtained in four consecutive large-scalepreparations.When examined by agar gel double-diffusion

assays, toxin preparations at this stage of purifi-cation exhibited a single immunoprecipitin lineupon diffusion against antiserum containing

0.5r

0.4

- 0.3

J

0.2

0.1

-H

I*V i

6.6 6. 5.8 5.4 5.0I 4. 4.2 3. 3. 3.06.6 6.2 5.8 5.4 5.0 4.6 4.2 3.8 3.4 3.0

pH

FIG. 1. Distribution of precipitating species incrude culture filtrates as a function of pH in thepresence (a) and absence (0) of metaphosphate (2.55glliter).

50

z40

8 20a

5 10 I5 20 25 30

SCOCENTRATION OF ALUMINUM HYDROXIDE

FIG. 2. Dependence of toxin recovery on concentra-tion of aluminum hydroxide suspension at pH 5.6when the total Lb doses per gram of adsorbent weregreater than or equal to 3.2 x 10', i.e., for the case inwhich the toxin concentration exceeded the capacityof the adsorbent.

antibodies against toxin and several somaticantigens (Wyeth goat no. 1 antitoxin). Suchpreparations, however, were able to protectimmunized mice against live vibrio challenge,thus indicating the presence of residual somaticantigen. Further studies showed that the levelof somatic antigen could be significantly re-duced if toxin preparations were centrifuged athigh speed.Table 2 gives representative data showing the

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TABLE 2. Comparison of somatic antigen content of arepresentative toxin preparation before and afterhigh-speed centrifugation as determined by the

mouse protection test

Relative potencyED, to standard

Material TU/mla ED;m0 tstdrdml5 As Pertested 1,000 TU

Aluminum 3,497 0.0152 0.06310 0.018hydroxide-purifiedtoxin: beforecentrifugation

Aluminum 3,472 0.6070 0.00158 0.00046hydroxide-purifiedtoxin: aftercentritugation

aToxoid units contained in starting material; mice wereimmunized with undiluted material and four serial fivefolddilutions.

b ED50, mean effective dose; determined by the method ofWilson and Worcester (Proc. Nat. Acad. Sci. 29:207, 1943).

results of mouse protection tests performed onan aluminum hydroxide-purified toxin prepara-tion before and after centrifugation. The har-vested sediment, after concentration by centrif-ugation, was identified by immunodiffusionwith one of the somatic antigens present incrude culture fluids and was found to be highlyeffective in protecting mice against challengewith live vibrios. The same material also gaverise to high vibriocidal antibody titers in immu-nized rabbits.

Conditions for sedimenting optimal amountsof somatic antigen were determined by cen-trifuging samples of crude culture filtrate (fromwhich all detectable toxin had been removed bymetaphosphate precipitation) and determiningby radial immunodiffusion the amount of so-matic antigen sedimented as a function of time.For this purpose, antiserum specific for boththis particular somatic antigen and toxin(Wyeth goat no. 2 antitoxin) was employed.Since toxin had been removed from the culturefiltrate, only precipitin rings due to the interac-tion of somatic antigen with its antibody wereobserved. The amount of somatic antigen whichcould be sedimented reached a maximum aftercentrifugation at 78,410 x g for 6 h in a Spinco30 rotor (Fig. 3). In scaling up the procedure toaccommodate larger volumes, equivalent re-sults were obtained when the Spinco 21 rotorwas substituted for the 30 rotor and sedimenta-tion was performed at 44,330 x g for a minimumof 16 h.To achieve additional purity, aluminum hy-

droxide-purified toxin preparations were lyoph-ilized, dissolved in a reduced volume of buffer,

treated with activated carbon, and centrifugedunder the conditions described above. Lyophili-zation was employed to reduce volumes forcentrifugation but, more importantly, becauseresidual somatic antigen tended to becomeinsoluble after lyophilization. Activated carbonremoved variable (from lot to lot) amounts ofyellow-brown contaminant(s), which becameapparent only after concentration. After carbontreatment, toxin preparations were generallywater-clear and they exhibited ultraviolet spec-tra characteristic of pure protein (Fig. 4 andTable 1).

Finally, purified toxin was filtered asepticallythrough a membrane filter (0.22 um, Milli-pore Corp.) and stored at 4 C without loss inactivity for periods of at least 6 months. Overalltoxin yields on the order of 50% or more, andspecific activities on the order of 22 Lb doses/

14

13

E- 12

11 _ j 000 0_11 11

5 10 15 20 25TIME (HOURS}

FIG. 3. Quantitative radial immunodiffusion assaymonitoring the removal of sedimentable somatic anti-gen from crude toxin-free filtrates by high-speedcentrifugation. Sediment was assayed undiluted (0)and at dilutions of 1:2 (0) and 1:4 (-).

0.34

0.32

0.30

0.28

0.26

0.24

0.22

,0.200.18

0.16CL

0.14

0.12

0.10

0.08

0.06

/

0

--- 230 250 270 290 310 330WAVELENGTH X

FIG. 4. Ultraviolet spectrum of purified toxin.

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,ug, were reproducibly obtained in four consecu-tive large-scale preparations.Immunodiffusion and immunoelectropho-

resis. Ouchterlony-type double diffusion wasused as a qualitative method for monitoringpurification. Figure 5 shows immunoprecipitinlines which were observed when various frac-tions obtained during the first two steps of thepurification procedure were diffused againstWyeth goat no. 1 antitoxin. With this anti-serum, it was possible to distinguish reproduc-ibly at least four antigens in cell-free -culturefiltrates, three of which were identified assomatic in origin and one of which was identi-fied as toxin. The three somatic antigenswere invariably observed in the metaphos-phate supernatant fraction, and a fourth,very weak, unidentified antigen, which was notapparent in crude culture filtrates, was gener-ally observed along with toxin antigen in themetaphosphate concentrate fraction. (Theweak, unidentified antigen may not be clearlyvisible in Fig. 5; its location in wells no. 2, 4, and5 is between the toxin-antitoxin immuno-precipitin line and the antiserum well.) Occa-sionally, some proportion of one of the threesomatic antigens described above (second pre-cipitin line from center well) also appeared inthe metaphosphate concentrate fraction. Theweak unidentified antigen and the somaticantigen, when present, did not bind to alumi-num hydroxide under the conditions that wereemployed and therefore appeared in the alumi-num hydroxide supernatant fraction. The bicar-bonate eluant from aluminum hydroxide exhib-ited a single immunoprecipitin line, which was

FIG. 5. Agar gel double immunodiffusion of vari-ous fractions obtained during purification. Clockwisefrom the top: well 1, cell-free culture fluid; well 2,metaphosphate concentrate; well 3, bicarbonate elu-ant; well 4, aluminum hydroxide supernatant; well 5,same as well 2; and well 6, metaphosphate superna-tant. Center well contained polyvalent antiserum(Wyeth goat no. 1 antitoxin).

identified as toxin and which could be moni-tored quantitatively by radial immunodiffusionthroughout subsequent steps of the purificationprocedure. Purified toxin preparations, contain-ing on the order of 1 mg of toxin per ml,continued to exhibit a single immunoprecipitinline when they were diffused against the afore-mentioned polyvalent antiserum. The somaticantigen nearest to the antigen wells in thecell-free culture filtrate and metaphosphatesupernatant fractions (see Fig. 5), respectively,was identical to the antigen which was subse-quently removed from toxin preparations bysedimentation.The discovery and isolation by Finkelstein

and LoSpalluto (12, 13) of a degradation prod-uct of toxin (11), which they named cholerage-noid and which they showed was electrophoreti-cally distinguishable but immunologically in-distinguishable from toxin (12), prompted aninvestigation of its possible presence in prepara-tions of toxin purified by the present procedure.Immunoelectrophoretic analysis comparing tworepresentative toxin preparations with a prepa-ration of choleragenoid provided by R. A. Fin-kelstein revealed that toxin purified by thepresent method does not contain significantamounts, if any, of choleragenoid (Fig. 6).Polyvalent antiserum (Wyeth goat no. 1 an-titoxin) was used in the development of theimmunoprecipitin pattern because Ouchter-lony-type immunodiffusion tests had shown thatcholeragenoid and the toxin preparations exhib-ited a single, identical immunoprecipitin linewhen reacted against this antiserum.Acrylamide gel electrophoresis. After elec-

trophoresis and staining, toxin appeared as abroad, somewhat diffuse band which could beresolved into one major and one (or two) minorbands (Fig. 7). The possibility that the minorband(s) were contaminants was dismissed whenthe major band, after excision from unstainedgels, exhibited the same electrophoretic pattern(as before) upon re-electrophoresis. In addition,when toxin was preincubated at 37 C in thepresence of 0.035% ammonium persulfate (thecatalyst for polymerization of the separatinggel), a change in the distribution of proteinsfrom the major to the minor species was ob-served. Whether or not the minor bands oc-curred exclusively as a result of electrophoresis(because of oxidation due to excess persulfate)or occurred during production and/or purifica-tion as well, has not been determined.

Efforts to distinguish between the major andminor species either by immunological tech-niques or by electron microscopy proved unsuc-cessful. When unstained gels were sliced into

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FIG. 6. Comparison of immunoelectrophoretic pat-tern of two representative purified toxin preparations(top and bottom wells) with a preparation of Finkel-stein's choleragenoid (center well). Precipitin lineswere developed with polyvalent antiserum (Wyethgoat no. 1 antitoxin).

2-mm disks and the disks were placed on agarcontaining antitoxin, immunoprecipitin ringswere observed surrounding gel slices from re-gions corresponding to both the major andminor species. Similarly, when protein waseluted from such slices and assayed for activity,a symmetrical profile (of activity) with a shoul-der in the region occupied by the minor bandswas observed. Further, electron microscopy ofexcised fractions levealed similar particle typesin the eluants associated with both the majorand minor species.Electron microscopy. Examinati6n of a rep-

resentative toxin preparation by electron mi-croscopy revealed a quasi-crystalline array ofuniform, closely packed particles (Fig. 8). Indi-vidual particles appeared to be hollow withmostly rectangular, but sometimes circular,outlines. The diameter of the particles rangedfrom 5 to 6 nm.Somatic antigen analyses. Because the im-

munological and biophysical results presentedthus far did not reveal the presence of traceamounts of extraneous somatic antigen, it wasnecessary to use sensitive in vivo assays todemonstrate its presence. In general, the level ofsomatic antigen was determined in two ways: (i)by measuring resistance of immunized mice tolive vibrio challenge in a mouse protection test;and (ii) by measuring vibriocidal antibody ti-ters in the sera of immunized rabbits. Becausethe concentration of toxin which would havebeen required to reliably determine residuallevels of somatic antigen was either lethal orcaused reactions at the site of inoculation, toxinwas converted into a Formalin toxoid for mouse

immunization and into either a Formalin orglutaraldehyde toxoid for rabbit immunization.The conditions surrounding the preparation ofthese toxoids are presented in the subsequentreport.With regard to the mouse protection test, it

should be emphasized that, unless mice wereimmunized with dilutions of toxoid containingat least 10,000 TU (approximately 500 gg oftoxoid antigen) in the lowest dilution, mostpreparations did not exhibit statistically signifi-cant protection. Even at these doses, survivalcurves were often erratic or nonlinear; it isnoteworthy that mice immunized with placebo(phosphate-buffered saline) also exhibitedsurvival curves similar to those observed withvarious toxoid preparations. Nevertheless, anestimate of the amount of residual somaticantigen in representative toxoid preparationswas obtained, and it generally did not exceed0.025% of the DBS reference vaccine, V.cholerae Inaba IN-12, per 1,000 TU.As determined by the microtiter vibriocidal

assay, geometric mean serum titers from groupsof rabbits immunized with representative For-malin toxoids ranged from 10 to 40 vibriocidalantibody units per ml 4 weeks after primary in-oculation with approximately 2,000 TU (100 ,gof toxoid antigen). Like antitoxin titers, vibrio-

FIG. 7. Acrylamide gel electrophoretic patterns ofpurified toxin. Electrophoresis was performed untilthe tracking dye reached the bottom of the gel (left)and 30 min beyond the clearance of the dye from thegel (right) using approximately 100 ,ug of toxin per gel.

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DEVELOPMENT OF PURIFIED CHOLERA TOXOID. I.

FIG. 8. Electron micrograph of purified toxin.Magnification of column-sized picture = x398,000.

cidal titers rose 2 weeks after booster inocula-tion, achieving levels approximately one-thirdof the NIH reference (convalescent) antiserum;but they declined towards the titers cited abovewithin several weeks (Table 3). However, rab-bits twice immunized with 100 pg of glutaralde-hyde toxoid (with a 6-week interval betweeninoculations) exhibited vibriocidal titers whichwere significantly reduced relative to titerselicited by Formalin toxoid (Table 4). Evenwhen this toxoid was administered with adju-vant (Table 4), post-booster titers were approxi-mately 100-fold less than the NIH referenceantiserum.

DISCUSSIONAn effective and comparatively simple

method for the large-scale production of puri-fied cholera toxin has been developed. Startingwith cell-free culture filtrates, the method reliesentirely upon batch procedures, each of which

results in a reduction in volume and a concomi-tant increase in specific activity. The methodhas proven reliable regardless of whether theinitial volume of culture filtrate was 1 or 250liters.The metaphosphate precipitation technique

employed in the first step of the procedure wasdeveloped some years ago by one of us (H. Tint,U.S. patent 2,772,201, 1956) for the concentra-tion and purification of tetanus and diphtheriatoxoids. Involving the formation of complexesbetween the phosphate polymers and proteinsand the subsequent precipitation of these com-plexus at their respective isoelectric pHs, themethod was designed to purify as well asconcentrate. In its original application, all ofthe protein complexes having an isoelectric pHequal to or higher than that of the desiredprotein were precipitated together and dis-

TABLE 3. Serum antitoxin and vibriocidal titers afterimmunization of rabbits with 100X g of Formalin

toxoid at 0 and 4 weeks, respectively

Antitoxin titer Vibriocidal titerWeeks post- (AU/ml)a (VU/ml)"immunization

Prepn 1 Prepn 2 Prepn 1 Prepn 2

0 <2 (6)b <2 (8) <2 (6) <2 (8)4 367 (6) 285 (8) 38 (6) 11 (8)6 1,392 (6) 1,011 (7) 121 (6) 74 (7)10 469 (6) 341 (7) 51 (6) 31 (7)16 268 (6) 93 (6) 34 (6) 13 (6)

NIH reference re- 362 256agent (convales-cent antiserum)

a AU, Antitoxin units; VU, vibriocidal antibody units.'Numbers represent geometric means. Numbers in paren-

theses indicate number of rabbits.

TABLE 4. Serum vibriocidal titers after immunizationof rabbits with 100 pg of glutaraldehyde toxoid at

0 and 6 weeks, respectively

Vibriocidal titer (VU/ml)aWeeks post-immunization Prepn 1 +Prepn 1 Prepn 2c

+Adjuvant"

0 1.0 (8)' 1.0 (8) 1.2 (8)6 1.5 (8) 1.4 (7) 2.2 (7)8 3.7 (7) 10.1 (8) 6.2 (8)

NIH reference re- 1,024.0 1,024.0 1,024.0agent (convales-cent antiserum)

'VU, Vibriocidal antibody units.'Adjuvant was protamine-aluminum phosphate. Details

of its preparation and use are given in the subsequent report.c Prepared from the same toxin as preparation 2 in Table 3."Numbers represent geometric means. All serum titers

<2 were arbitrarily assigned the value of 1 VU/ml. Numbersin parentheses indicate number of rabbits.

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RAPPAPORT, RUBIN, AND TINT

solved in an appropriate buffer, and the desiredprotein was then fractionated according to itssolubility characteristics. In this particular sys-tem, the toxin-metaphosphate complexes wereamong the first species to precipitate as the pHwas lowered to 4.6, so that concentration as wellas a substantial increase in specific activity wasaccomplished in a single step. It may be ofinterest that the somatic antigens remaining inthe metaphosphate supernatant fraction (seeFig. 5) could also be concentrated and purifiedby selective precipitation below pH 4.6.

In the purification of toxin by aluminumhydroxide adsorption, the powder form of theadsorbent was used exclusively. This was be-cause the powder form proved to have greaterselectivity for toxin than did aluminum hydrox-ide gels such as those used by Spyrides andFeeley (22) in their original observations. Theone disadvantage of the powder adsorbent wasthat approximately 2 g of adsorbent was used topurify as little as 1 mg of toxin. A possibleexplanation for this observation may be thatother ions in the medium are competing withtoxin for binding sites on the adsorbent.Although the specific activity of toxin prepa-

rations was relatively high after aluminumhydroxide purification, subsequent lyophiliza-tion and centrifugation consistently resulted ina substantial reduction in the amount of resid-ual somatic antigen. In a few instances whereresidual somatic antigen levels were higher thanusual (as, for example, when the starting mate-rial was 48-h culture filtrate), recycling thepurified toxin preparation through the lyophili-zation and centrifugation steps resulted in re-moval of approximately 70% of the residualsomatic antigen. Also, in cases where theamount of pigment contamination was greaterthan usual, a repetition of the carbon treatmentresulted in the complete removal of these con-taminants.On the basis of mass, the amount of residual

somatic antigen recovered by sedimentationwas small. Although no detailed chemical anal-yses were performed, this antigen exhibitedcharacteristics similar to the protein-lipopolysaccharide complex isolated from Inabaserotypes by Watanabe et al. (23), since it couldbe described as a polydisperse, fine suspensionwhich tended to become insoluble upon freezingand lyophilization. The observation that toxinpreparations continued to exhibit very low lev-els of somatic antigen after lyophilization andcentrifugation suggested that, even when pres-ent in trace amounts, this antigen was eitherhighly immunogenic or its immunogenicity wasgreatly enhanced in the presence of toxin ortoxoid.

In terms of specific activity (of toxin), thepreparations described here compare favorablywith other purified preparations described bythe most often cited investigators, Finkelsteinand LoSpalluto (12) and Richardson et al. (21),in that the amount of toxin required to producea 7-mm bluing lesion in rabbit skin is on theorder of 0.1 to 0.5 ng. With regard to content ofsomatic antigen, it is more difficult to compareour material with that of other investigatorsbecause either insufficient data are available ordifferent methods of analysis have been em-ployed.Richardson et al. (21) reported the presence of

"low but consistent vibriocidal antibody titerrise" in rabbits immunized with some of theirpreparations, and Finkelstein and LoSpallutoemployed an in vitro neutralization test (10) todemonstrate the absence of somatic antigenfrom their toxin preparations (13). Recently, agroup of rabbits immunized in this laboratorywith 100 ,g of a Formalin toxoid prepared froma toxin preparation purified by R. A. Finkel-stein (lot 1071; prepared under contract for theNational Institute of Allergy and InfectiousDiseases essentially according to proceduresdescribed in reference 12) showed no significantrise in serum vibriocidal antibody after primaryand secondary immunizations.

Although the ultimate objective of this investi-gation was to develop a reliable production-scalemethod for the preparation of purified toxin(toxoid) with no ability to elicit vibriocidal anti-body, the data clearly show that representa-tive toxoid preparations stimulate low levels ofvibriocidal antibody in mice and rabbits whenthey are suitably immunized with a higher thananticipated human dose (100 to 200 ug). Thereason that glutaraldehyde toxoids elicited sig-nificantly less vibriocidal antibody than didFormalin toxoids is thought to be related moreto the different stability characteristics of thetoxoids (the subject of the subsequent manu-script) than to a direct effect of glutaraldehydeon somatic antigen.

In addition, it has been demonstrated thatresidual somatic antigen can be eliminated bymolecular sieve chromatography (using Sepha-dex G-150, Sephadex G-200, or Bio-Gel A-5Mgels) or, alternatively, by affinity chromatogra-phy (using purified vibriocidal antibody boundto Sepharose 4B); these techniques are pres-ently being evaluated on a production scale.Whether or not material of the quality pres-

ently achieved can be utilized to determine,unambiguously, the efficacy of antitoxic immu-nity in endemic areas is the subject of continu-ing investigation. Irrespective of the outcome ofsuch investigations, this material should be

302 INFECT. IMMUNITY

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DEVELOPMENT OF PURIFIED CHOLERA TOXOID. I.

useful in studying the protein chemistry andmechanism of action of cholera toxin in variousbiological systems. Ultimately, material of thequality presently described should, without fur-ther purification, be a suitable candidate forlarge-scale immunization, provided that an-titoxic efficacy is established.

ACKNOWLEDGMENTSWe express our appreciation to the following people: Alan

Bernstein, for his cooperation in providing facilities andpersonnel for the production phase of this work and forsupervising the performance of mouse protection tests; D.Hoover, G. Bonde, T. McCann, and L. Repsher for theirinvaluable and outstanding technical assistance in variousphases of this investigation; S. Vernon for electron micros-copy, T. Schaffer for secretarial assistance, and J. P. Craig forvaluable criticism of the manuscript.

This investigation was supported by the National Instituteof Allergy and Infectious Diseases under Public HealthService contract NIH 70-2102.

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0. Oseasohn. 1968. Cholera vaccine field trials in EastPakistan. 2. Effectiveness in the field. Bull. W.H.O.38:359-372.

2. Burrows, W. 1968. Cholera toxins. Annu. Rev. Microbiol.22:245-268.

3. Craig, J. P. 1966. Preparation of the vascular permeabil-ity factor of Vibrio cholerae. J. Bacteriol. 92:793-795.

4. Craig, J. P. 1971. Cholera toxins, p. 189-254. In S. Kadis,C. Montie, and S. Ajl (ed.), Microbial toxins, vol. 2A.Academic Press Inc., New York.

5. Craig, J. P., E. R. Eichner, and R. B. Hornick. 1972.Cutaneous responses to cholera skin toxin in man. I.Responses in unimmunized American males. J. Infect.Dis. 125:203-215.

6. Curlin, G. Y., J. P. Craig, A. Subong, and C. C. J.Carpenter. 1970. Antitoxin immunity in experimentalcanine cholera. J. Infect. Dis. 121:463-470.

7. Davis, B. J. 1964. Disc electrophoresis. I. Background andtheory. Ann. N.Y. Acad. Sci. 121:321-349.

8. Evans, D. J., Jr., and S. H. Richardson. 1968. In vitroproduction of choleragen and vascular permeabilityfactor by Vibrio cholerae. J. Bacteriol. 96:126-130.

9. Feeley, J. D., and M. Pittman. 1962. A mouse protectiontest for assay of cholera vaccine, p. 92-94. In SEATOconference on cholera. SEATO, Bangkok, Thailand.

10. Finkelstein, R. A. 1962. Vibriocidal antibody inhibition(VAI) analysis: a technique for the identification of thepredominant vibriocidal antibodies in serum and foithe detection and identification of Vibrio choleraeantigens. J. Immunol. 89:264-271.

11. Finkelstein, R. A., K. Fujita, and J. J. LoSpalluto. 1971.Procholeragenoid: an aggregated intermediate in theformation of choleragenoid. J. Immunol. 107:1043-1051.

12. Finkelstein, R. A., and J. J. LoSpalluto. 1969. Pathogen-esis of experimental cholera: preparation and isolationof choleragen and choleragenoid. J. Exp. Med.130:185-202.

13. Finkelstein, R. A., and J. J. LoSpalluto. 1970. Productionof highly purified choleragen and choleragenoid. J.Infect. Dis. 121(Suppl.):563-572.

14. Lowry, 0. H., N. J. Rosebrough, A. C. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

15. Mancini, G., A. 0. Carbonara, and J. F. Heremans. 1965.Immunochemical quantitation of antigens by singleradial immunodiffusion. Immunochemistry 2:235-254.

16. Muschel, L. H., and H. P. Treffers. 1956. Quantitativestudies on the bactericidal actions of serum and com-plement. I. A rapid photometric growth assay forbactericidal activity. J. Immunol. 76:1-27.

17. Ornstein, L. 1964. Disc electrophoresis. I. Backgroundand theory. Ann. N.Y. Acad. Sci. 121:404-427.

18. Pierce, N. F., W. B. Greenough, and C. C. J. Carpenter,Jr. 1971. Vibrio cholerae enterotoxin and its mode ofaction. Bacteriol. Rev. 35:1-13.

19. Pierce, N. F., E. A. Kaniecki, and R. S. Northrup. 1972.Protection against experimental cholera by antitoxin.J. Infect. Dis. 126:606-616.

20. Richardson, S. H. 1969. Factors influencing in vitro skinpermeability factor production by Vibrio cholerae. J.Bacteriol. 100:27-34.

21. Richardson, S. H., D. G. Evans. and J. C. Feeley. 1970.Biochemistry of Vibrio cholerae virulence. I. Purifica-tion and biochemical properties of PF/cholera entero-toxin. Infect. Immunity 1:546-554.

22. Spyrides, G. J., and J. C. Feelev. 1970. Concentration andpurification of cholera exotoxin by adsorption on alu-minum compound gels. J. Infect. Dis. 121S:96-99.

23. Watanabe, Y., W. F. Verwey, J. C. Guckian, H. R.Williams, Jr., P. E. Phillips. and S. S. Rocha, Jr. 1965.Some of the properties of mouse protection antigensderived from Vibrio cholerae. Texas Rep. Biol. Med.,27:275-298.

24. W.H.O. Scientific Group Report on Cholera Immunology.1969. W.H.O. Technical Report Series no. 414, p. 1-21.World Health Organization, Geneva.

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