INFECTION AND IMMUNITY, Sept. 1976, p. 687-693Copyright © 1976 American Society for Microbiology

Vol. 14, No. 3Printed in U.S.A.

Development of a Purified Cholera Toxoid. III. Refinementsin Purification of Toxin and Methods for the Determination

of Residual Somatic AntigenR. S. RAPPAPORT,* W. A. PIERZCHALA, G. BONDE, T. McCANN AND B. A. RUBIN

Wyeth Laboratories Inc., Department ofBiological Product Development,P.O. Box 8299, Philadelphia, Pennsylvania 19101

Received for publication 12 April 1976

The addition of an ultrafiltration step to the purification procedure previouslydescribed for cholera toxin (Rappaport et al., 1974) permitted the preparation ofhighly purified antigenic toxoids essentially free of somatic antigen(s). Thepurity of such toxoids is established: (i) by the absence of more than about onepart limulus amebocyte lysate (LAL)-positive endotoxin per 105 parts toxoid and(ii) by the inability of the toxoids to elicit a significant rise in rabbit vibriocidalantibody. The antigenicity of the toxoids is demonstrated by their ability toproduce the same high levels of rabbit serum antitoxin as are produced bycomparable toxoids containing small amounts of somatic antigen. The resultsalso indicate that amounts of somatic antigen on the order of <1 ,ug/1OO jig oftoxoid do not exert an adjuvant effect on the toxoid, at least with respect tocirculating antitoxin. Other data show that, where present, the ability ofsomatic antigen to elicit vibriocidal antibody is influenced by the immunizationschedule employed and that a correlation exists between the LAL-determinedendotoxin content of the toxoids and their ability to stimulate vibriocidal anti-body. Somatic antigen-free toxoids, purified and tested by the refinementsherein described, were prepared for use in National Institutes of Health-spon-sored field trials, and data pertaining to their purity and antigenic propertiesare presented.

The large-scale production and purification ofcholera toxin and its detoxification by glutaral-dehyde to yield a stable, antigenic toxoid havebeen described previously (8, 9). The purifiedtoxoid was shown to contain small amounts ofsomatic antigen which gave rise to low, butmeasurable, levels of vibriocidal antibody inimmunized rabbits (9). To prepare a toxoid withwhich to study the role of pure antitoxic immu-nity in both clinical and experimental cholera,methods for the removal of residual somaticantigen were investigated. The addition of anultrafiltration step to the purification methodpreviously reported (9) resulted in the virtualelimination of somatic antigen from productionscale preparations of cholera toxin and toxoid.

This report describes details of the refine-ment in the purification procedure as well asimproved methods for detection of somatic anti-gen. In addition, evidence of the purity andantigenicity of glutaraldehyde toxoids, pre-pared and tested by the refined procedures, ispresented.

MATERIALS AND METHODSProduction of toxin. Cell-free culture filtrates

were prepared from 24-h broth cultures of Vibrio

cholerae serotype Inaba, strain 569B, as describedearlier (9).

Purification of toxin. Toxin was purified from250-liter batches of cell-free culture filtrate by themethod previously employed (9), with the exceptionthat toxin so purified was passed through a series of35-nm pore size Sartorius cellulose acetate mem-branes (SM 11730) employing a Sartorius membranefilter unit (SM 16525) and peristaltic pump (SM16650) (all of these components were obtained fromScience Essentials Co., Anaheim, Calif.). Two mem-brane filters were used for each liter of fluid. With amaximum of 15 membranes in the unit, flow rateswere about 1.2 liters/h, when the positive pressurewas set between 20 and 25 lb/in2. After passagethrough the membranes, the toxin-containing fil-trate was filtered aseptically through a 0.22-,umpore size membrane filter (Millipore Corp., Bedford,Mass.) and stored at 4°C until it was detoxified withglutaraldehyde.Assay of toxin. The toxin content of various prep-

arations was estimated by the limit-of-bluingmethod in rabbit skin (2), employing conditionsidentical to those described earlier (9).

Detoxification. Detoxification was accomplishedby incubating toxin (500 ,ug/ml) in 0.067 M phos-phate-buffered saline, pH 7.8, with glutaraldehyde,using a 200:1 molar ratio of reagent to toxin (8).Unless noted otherwise, incubation was carried outat 30°C for 6 days, at which time excess reagent was

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removed by dialysis against phosphate-buffered sa-line, pH 7.8. After dialysis, ,-lactose (Eastman Or-ganic Chemicals Div., Eastman Kodak Co., Roches-ter, N.Y.) at a final concentration of 1% and thime-rosal (Elanco Products Div., Eli Lilly & Co., Indian-apolis, Ind.) at a final concentration of 0.005% wereadded to the toxoid solution, and the solution wasfiltered aseptically through a 0.22-,um pore sizemembrane filter (Millipore). The solutions werethen distributed into vials and lyophilized.

Schedules of rabbit immunization. Two immuni-zation schedules were used. (i) In the standardschedule, groups of at least eight New Zealand al-bino rabbits, each weighing between 2.5 and 4 kg,received two intramuscular inoculations of 100 ,ug oftoxoid, with or without protamine-aluminum adju-vant (8), with a 6-week interval between inocula-tions. Serum samples were prepared from blood ob-tained by heart puncture at 0, 6, and 8 weeks, re-spectively. (ii) In the accelerated schedule, groups ofeight rabbits of the same type and size describedabove received three or four consecutive weekly in-tramuscular inoculations of 100 to 400 ug of toxoidwithout adjuvant. Serum samples were obtained asdescribed above 11 or 14 days after the last inocula-tion.Measurement of vibriocidal antibody. Vibriocidal

antibody titers based on the ability ofvarious sera toinhibit the growth of Inaba VC-13 in the presence ofguinea pig complement were determined by a micro-titer technique described earlier (9). The results ofthe assays are given as the reciprocal of the lasttwofold serum dilution which completely inhibitedbacterial growth. National Institutes of Health ref-erence convalescent antiserum was used to stand-ardize the results of different tests.Measurement of antitoxin. Antitoxin titers of

various sera were determined by the intracutaneousmethod in rabbits (1, 2) as described earlier (8). Thecholera toxin test dose was 1 limit-of-bluing dose/ml,and in each assay this dose was tested against theprovisional standard cholera antitoxin (Swiss Se-rum and Vaccine Institute antitoxin containing4,470 antitoxin units/ml) at 2, 1, and 0.5 antitoxinunits/ml. Results are presented in antitoxin unitsper milliliter determined from the reciprocal of theserum dilution which, in the presence of an equalvolume of 1 limit-of-bluing dose of cholera toxin perml, yielded a 4-mm bluing lesion in rabbit skin 16 to18 h after intradermal inoculation of 0.1 ml.Measurement of endotoxin by the LAL test. The

limulus amebocyte lysate (LAL) test used in thisstudy was a commercial preparation obtained fromMallinckrodt Chemical Works, St. Louis, Mo. Dilu-tions of toxin or toxoid were made with pyrogen-freesaline (McGaw Laboratories Inc., Glendale, Calif.)and sterile, glass disposable pipettes (Corning GlassWorks, Corning, N.Y.). Pyrogen-free test tubes (13by 100 mm) were prepared by treating them accord-ing to the procedure of Yin et al. (12). The LAL testwas performed by mixing 0.1 ml of the lysate with0.1 ml of appropriate serial twofold dilutions of chol-era toxin or toxoid. The mixtures were incubated at37°C for 1 h, at which time they were examined. Afirm gel which adhered to the bottom of the tube

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when inverted was interpreted as a positive test.Negative controls consisted of mixing pyrogen-freesaline with lysate. Positive controls consisted ofmixing the lysate with 0.5 ng (in 0.1 ml) of Esche-richia coli endotoxin provided by the manufacturer.Neither toxin nor toxoid (which themselves did notcontain enough endotoxin to cause gelation) inter-fered with the ability of the endotoxin standard tocause gelation of the lysate. For routine comparisonof various toxin samples, before and after passagethrough Sartorius cellulose acetate membranes, theamount of endotoxin was estimated by reference tothe endotoxin standard, tested at a concentration of5 ng/ml. For precise estimation of endotoxin con-tent, the standard was titrated to an end point, andthe amount of endotoxin contained in various sam-ples was based on the sensitivity of the lysate to thestandard. The lysates employed were generally sen-sitive to between 0.1 to 1.0 ng oftheE. coli endotoxinstandard per ml.

Protein determinations. The protein content ofvarious toxin or toxoid samples was determinedeither by the spectrophotometric method ofWarburgand Christian (10) or by the Lowry modification ofthe Folin-Ciocalteu method (5), using crystallinebovine serum albumin as a standard.

RESULTSEndotoxin content of cholera toxin prepa-

rations before and after ultrafiltration. Pas-sage of from 1 to 10 liters of cholera toxin,purified as described earlier (9), through a se-ries of Sartorius cellulose acetate membranes(with a molecular weight cutoff of 160,000) re-sulted in a substantial reduction in endotoxincontent (Table 1). Based on the LAL test, thereduction in endotoxin content was on the orderof 1,000-fold. Since the E. coli endotoxin stand-ard was assayed only at a concentration of 5 ng/ml, and since the limulus lysates employedwere generally sensitive to between 0.1 to 1.0ng of endotoxin per ml, the values presented inTable 1 may be overestimated by a factor of atleast five. Taking this into account, the endo-toxin content of the toxin fluids was thereforeestimated to vary between 1 part per 25 to 400parts toxin before ultrafiltration and 1 part per2 x 104 to 2 x 105 parts toxin after ultrafiltra-tion (Table 1). In addition, a comparison of thepotency of the toxin fluids before and after fil-tration showed that the specific activity of thetoxin was unaffected by the filtration processand that toxin recoveries varied from 76 to 91%(Table 1). In most cases, unrecovered toxin wasaccounted for in the retentate volume and inthe hoses of the filter apparatus. When theretentate was diluted to a suitable volume orcombined with other retentates, additionaltoxin of similar quality to the initial filtratewas recovered after passage through the mem-branes.

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Relationship of endotoxin content and abil-ity to elicit vibriocidal antibody. Since it wasnot known whether endotoxin measured by theLAL test was identical to the somatic anti-gen(s) which elicited vibriocidal antibody inimmunized rabbits, portions of toxin were de-toxified with glutaraldehyde, before and afterultrafiltration. Groups of rabbits were immu-nized with 100 jig of each toxoid antigen usingtwo immunization schedules: (i) a standardschedule which supported the development ofhigh levels of serum antitoxin (R. Rappaport,unpublished observations) and (ii) an acceler-ated schedule which preliminary studiesshowed was about 10-fold more sensitive to thedevelopment of vibriocidal antibody than was

the standard schedule. The data in Table 2demonstrate that toxoid prepared from toxinwhich had been passed through the Sartoriusmembranes did not elicit vibriocidal antibodyby either immunization schedule, whereas tox-oid prepared from unfiltered toxin elicited de-tectable vibriocidal antibody in both immuniza-tion schedules. Further, the data (Table 2)showed that the accelerated schedule, which inthis instance included four weekly spaced inoc-ulations, was approximately seven times moresensitive to the development of vibriocidal anti-body than was the standard two-dose schedule(with a 6-week interval between inoculations).In contrast, the standard schedule supportedthe development of greater antitoxin titers

TABLE 1. Properties of cholera toxin before and after ultrafiltrationaBefore ultrafiltration After ultrafiltration

Preprn Toxn toxin/TEndoxin Sp act Endotoxin Endotoxinl(AgImI)b (Lb/AgY (,±g/ml)d toxin (jgIml)b (Lb/Lg)c (/Ag/Ml)d toxIn (tg

a 1,400 24.5 NT - 1,070 25.7 NT -

b 990 19.2 41 1/24 750 21.5 0.04 1/18,750c 830 24.1 10 1/83 790 22.9 <0.04 <1/19,750d 1,080 21.2 41 1/26 850 20.9 <0.02 <1/42,500e 1,450 20.2 50 1/29 1,150 20.6 <0.05 1/23,000

a Toxin preparations were tested before and after passage through a series of 35-nm pore size Sartoriuscellulose acetate filters.

b Determined spectrophotometrically by the method of Warburg and Christian (10).c Lb, Limit of bluing.d Determined in relation to an E. coli endotoxin standard, tested at 5 ng/ml. The determinations may be

overestimated by a factor of at least five (see text).

TABLE 2. Serum vibriocidal and antitoxin responses to glutaraldehyde toxoids prepared from cholera toxinbefore and after ultrafiltration, respectively: comparison of two rabbit immunization schedulesa

Dose Immuni- Vibriocidal response (VAU/ml)a Antitoxin response (AU/ml)aper in zationAntigen ocula- shdltion scheaule 0 wkb 5 wk 8 wk 0 wk 5 wk 8 wk(j.g) (wk)

Toxoid-1 (be- 100 0, 1, 2, 3 <2 (8)d 52 (8) <2 (8) 178 (8)fore ultra- (<2-512)e (100-409)filtration)c 100 0, 6 <2 (7) 7 (7) <2 (7) 805 (7)

(<2-96) (345-1,817)

Toxoid-2 100 0, 1, 2, 3 <2 (7) <2 (7) <2 (7) 420 (7)(after ul- (194-749)trafiltra- 100 0, 6 <2 (7) <2 (7) <2 (7) 935 (7)tion)C (764-1,914)a Abbreviations: VAU, VibriocidalIantibody units, determined as described in Materials and Methods.

AU, Antitoxin units, determined as described in Materials and Methods.b Titers were determined on sera obtained at time indicated. Zero weeks represents preimmunization

titers of aera obtained within 10 days before immunization.c Cholera toxin (preparation b, Table 1) was passed through a series of 35-nm pore size Sartorius cellulose

acetate filters. A portion of the toxin was detoxified with glutaraldehyde before (toxoid-1) and after (toxoid-2) passage through the filters.

d Numbers represent mean values. Numbers in parentheses represent the number of rabbits.eRange of values.

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than did the accelerated schedule. Since bothtoxoids (filtered and unfiltered) produced simi-lar levels of circulating antitoxin in each of theimmunization schedules, it was tentativelyconcluded that endotoxin concentrations on theorder of 1 to 4 ug/100 ,g of toxoid did not exertan adjuvant effect on the toxoid.On the basis of these and other studies, the

ultrafiltration procedure was adopted as a finalstep in the purification process previously de-scribed (9).

Properties of field trial toxoids. Several pro-duction lots of cholera toxin were purified bythe refined procedure and detoxified with glu-taraldehyde. The endotoxin content of each tox-oid, as measured by the LAL test, was less thanor equal to 1 part per 105 parts toxoid, whereasearlier toxoid lots, the parent toxins of whichhad not been ultrafiltered, contained no more

than 1 part endotoxin per 800 parts toxoid (Ta-ble 3). Most of the toxoids described in Table 3were compared for their ability or inability toelicit rabbit vibriocidal antibody, using the ac-

celerated immunization schedule, and for theirability to elicit antitoxin, using the standardimmunization schedule. In the case ofthe accel-erated schedule (Table 4), toxoids prepared bythe refined procedure did not elicit significantrises in vibriocidal antibody when they were

administered at doses ranging from 100 to 400p,g. (Only rises in vibriocidal antibody whichwere greater than twofold and which increasedwith antigen dose were considered significant.)In contrast, toxoids containing about 0.1 ,ug ofendotoxin per 100 ,ug of toxoid produced rises invibriocidal antibody of at least 26-fold (Tables 3

and 4). A comparison of the antitoxin titerselicited by the various toxoids (in the acceler-ated schedule) indicated that the refined tox-oids were just as antigenic as toxoids whichcontained small amounts of endotoxin.

In the case of the standard immunizationschedule (Table 5), no rise in vibriocidal anti-body was produced by the refined toxoids irre-spective of whether they were administeredwith or without protamine-aluminum adjuvant(8). In contrast, vibriocidal antibody rises onthe order of four- (without adjuvant) to tenfold(with adjuvant) were observed for the toxoid(lot no. 11201) which contained 0.12 ,ug of endo-toxin per 100 ,ug of toxoid (Tables 3 and 5).Again, each toxoid elicited comparable anti-toxin levels irrespective of their ability to elicitvibriocidal antibody, and in each instance anti-toxin titers rose four- to fivefold when the tox-oids were administered with adjuvant. A com-parison of the data presented in Tables 4 and 5(for toxoids administered without adjuvant)showed that the accelerated immunizationschedule was at least ten times more sensitiveto the development of vibriocidal antibody thanwas the standard schedule, whereas the latterschedule was at least four times more sensitiveto the development of antitoxin. The data fur-ther demonstrate that although repeated dosesof endotoxin on the order of 0.1 ,ug are capableof eliciting measurable vibriocidal antibody,they do not exert an adjuvant effect on thetoxoid. Finally, the magnitude and uniformityof serum antitoxin titers observed after immu-nization with glutaraldehyde toxoids (Table 5)indicate that, at least in the rabbit, such tox-

TABLE 3. Endotoxin content of various glutaraldehyde toxoids

Toxoid lot nlo.a Toxin purification method Protein Endotoxin (,Ig/100 Endotoxin/toxoid (jAg/(,ug/ml)b ,ug of toxoid)c /.g)

11201d Previous (reference 9) 100 0.12 (5)e 1/83311491d Previous (reference 9) 400 0.07 (2) 1/142920101 Refined 200 0.0008 (4) 1/1.3 x 10520201 Refined 200 0.00007 (4) 1/1.4 x 10620301 Refined 200 0.00125 (4) 1/0.8 x 10520401f Refined 500 0.0011 (3) 1/0.9 x 105

a Five-digit numbers indicate toxoid was dispensed into vials and lyophilized.b Determinations were made on samples rehydrated with pyrogen-free water to give the indicated toxoid

concentrations in 0.067 M phosphate-buffered saline, pH 7.8. Protein was determined by the Lowry methodbefore lyophilization.

c Determined by the LAL test according to the instructions of the manufacturer. Determinations werebased on the sensitivity of the lysate to the E. coli endotoxin standard, which varied in different testsbetween 0.1 and 1 ng/ml.

d Toxoid lots no. 11201 and no. 11491 were prepared by reacting toxin with glutaraldehyde at 30°C for 5and 7 days, respectively. All other toxoids were prepared as described in Materials and Methods.

e Numbers represent the average of two or more determinations. Numbers in parentheses indicate thenumber of determinations.

f Lot no. 20401 was identical to lot no. 2030L except that it was lyophilized, without preservative, at 2.5times the concentration of lot no. 20301.

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TABLE 4. Rabbit serum vibriocidal and antitoxin responses to various glutaraldehyde toxoids: acceleratedimmunization schedule

Toxoid lot Dose per Immunization Vibriocidal response (VAU/ml)a Antitoxin response (AU/ml)'inocula- scheduleno. tion (gg) (wk) 0 daysb 25 days 0 days 25 days

11201 100 0, 1, 2 2.4 (18)d 62.2 (18) <2 (18) 95 (18)(<2-24)e (4-512) (16-306)

11491 100 0, 1, 2 1.4 (8) 59.0 (8) <2 (8) 153 (8)(<2-4) (16-512) (32-716)

201C 100 0, 1, 2 2.0 (6) 5.0 (6) <2 (6) 96 (6)(<2-16) (<2-192) (79-121)

200 0, 1, 2 <2 (8) 1.4 (8) <2 (8) 160 (8)(<2-8) (100-274)

202 200 0, 1, 2 2.5 (6) 4.8 (6) <2 (6) 366 (6)(<2-24) (<2-192) (238-461)

203 100 0, 1, 2 2.1 (7) 2.0 (7) <2 (7) 185 (7)(<2-8) (<2-16) (100-303)

200 0, 1, 2 1.1 (8) 1.3 (8) <2 (8) 241 (8)(<2-3) (<2-4) (58-817)

204 400 0, 1, 2 2.0 (8) 1.4 (8) <2 (8) 346 (8)(<2-16) (<2-16) (119-1,606)

a. b See Table 2.C Three-digit numbers indicate toxoid was tested before lyophilization.d,e See Table 2.

oids are both reproducible and effective anti-gens.

DISCUSSION

By a simple refinement, namely the additionof an ultrafiltration step to our previous purifi-cation process (9), it has been possible to pre-pare, on a production scale, highly purified chol-era toxoid which contains no more than 1 partendotoxin per 105 parts toxoid (Tables 1 and 3)and which shows no ability to elicit significantlevels ofvibriocidal antibody in immunized rab-bits (Tables 2, 4, and 5). These results havebeen confirmed independently by other investi-gators both with regard to the absence of LAL-endotoxin (C. A. Miller, personal communica-tion) and with regard to the inability of therefined toxoids to elicit significant vibriocidalantibody in immunized rabbits (6) and man (4,6).The ultrafiltration step described in this re-

port has proven to be a reliable and rapidmethod for achieving a 1,000-fold reduction inthe endotoxin content of toxin fluids. In ourexperience, the Sartorius ultrafiltration unitwith cellulose acetate filters (which retainedmolecules greater than 160,000 daltons) wassuperior to other ultrafiltration systems inachieving the reduction of endotoxin fromabout 1 part per 102 parts toxin to about 1 partper 105 parts toxin (toxoid). It is possible thatthe design of the unit, which continually recy-cles fluid across sandwich-layered filters under

positive pressure, together with the composi-tion of the filters, may both contribute to theefficiency of the procedure. This method, whichappears to work equally well on a small or largescale, may be useful for removal of endotoxinfrom other soluble biological or pharmacologi-cal preparations.The sensitivity of the LAL test, which meas-

ures picogram amounts of endotoxin (12), pro-vided a rapid method for monitoring the resultsof ultrafiltration. Otherwise, the most reliablemethod for detecting small amounts of endo-toxin was rabbit immunization, a procedurewhich, depending on the immunization sched-ule employed, took from 4 to 9 weeks. In com-paring the results of both tests (LAL and rabbitimmunization), the LAL test was found to be areliable indicator of the ability or inability of aparticular toxoid to elicit vibriocidal antibodyin immunized rabbits. Toxoids which containedlittle or no endotoxin by the LAL test did notelicit significant vibriocidal antibody, whereastoxoids with measurable LAL-endotoxin did.Although insufficient data are available to es-tablish that a linear relationship exists be-tween LAL-endotoxin content and the ability(of a toxoid) to elicit vibriocidal antibody, it isnoteworthy that toxoids with similar levels ofLAL-endotoxin produced similar levels of vi-briocidal antibody (for example, see toxoid lots11201 and 11491, Tables 3 and 4). Thus, in thisparticular system, LAL-endotoxin appears torepresent an estimate of the somatic antigencontent of various toxoids.

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TABLE 5. Rabbit serum vibriocidal and antitoxin responses to various glutaraldehyde toxoids: standardimmunization schedule

Dose Immu- Vibriocidal response (VAU/ml)a Antitoxin response (AU/ml)aper in- nixationToxoid lot no. ocula- scheduletion swkl 0 wk" 6 wk 8 wk O wk 6 wk 8 wk(g) (wk)

11201, with- 100 0, 6 <2 (8)d 1.5 (8) 3.7 (8) <2 (8) 16 (8) 455 (8)out adju- (<2-6)e (<2-12) (6-28) (388-891)vantc

11201, with 100 0, 6 <2 (8) 1.4 (8) 10.1 (8) <2 (37) 75 (37) 4280 (37)adjuvantc (<2-8) (<2-64) (14-111) (1,136-16,384)

20101, with- 100 0, 6 <2 (8) 1.1 (8) <2.0 (8) <2 (8) 20 (8) 795 (8)out adju- (<2-3) (<2) (8-128) (478-1,663)vant

20101, with 100 0, 6 <2 (6) 1.2 (6) 1.6 (6) <2 (6) 79 (6) 4,150 (6)adjuvant (<2-3) (<2-6) (56-188) (1,845-8,790)

20201, with- 100 0, 6 <2 (8; 9 0 (8) 2.1 (8) <2 (8) 51 (8) 1,008 (8)out adju- (<2-b, (<2-12) (7-108) (362-3,383)vant

20201, with 100 0, 6 1.8 (9) 2.3 (9) 2.2 (9) <2 (9) 117 (9) 4,283 (9)adjuvant (<2-12) (<2-4) (<2-8) (27-211) (3,324-6,794)

20301, with- 100 0, 6 7.0 (8) 7.0 (8) 10.8 (7) <2 (8) 28 (8) 1,525 (7)out adju- (2-24) (2-32) (3-128) (8-52) (1,025-2,355)vant

20301, with 100 0, 6 2.1 (6) 2.8 (6) 1.9 (6) <2 (6) 172 (6) 6,155 (6)adjuvant (2-4) (<2-16) (<2-12) (97-543) (1,979-18,842)a b See Table 2.c Dried toxoid (plus sodium phosphate and chloride salts) was rehydrated to 100 ,ug/ml either with water

(without adjuvant) or with protamine sulfate (0.5 mg/ml)-aluminum chloride (3.75 mg/ml) diluent (withadjuvant).

d, e See Table 2.

A comparison of the endotoxin content of sev-eral toxoids with that of their respective parenttoxins did not resolve the question of whethertreatment with glutaraldehyde altered the abil-ity of endotoxin to cause gelation of LAL. Insome instances, LAL titers were lowered ap-proximately fivefold after detoxification, but inother instances no significant reduction wasobserved. It would be of some interest to deter-mine the effect of glutaraldehyde treatment onthe biological and antigenic properties of de-fined endotoxin preparations. In the presentcase, data pertaining to the chemical composi-tion of the contaminating endotoxin, whosefunctional properties are herein described,were not obtained. However, it appeared toexhibit physical properties similar to the pro-tein-lipopolysaccharide complex isolated fromInaba serotypes by Watanabe et al. (11).The observation that toxoids elicited compa-

rable levels of circulating antitoxin, irrespec-

tive of the presence or absence of residual so-matic antigen, indicates that small amounts ofsomatic antigen do not exert an adjuvant effecton toxoid, at least with regard to serum anti-toxin. This does not rule out the possibility,however, that residual somatic antigen mayexert some effect on intestinal antitoxin re-sponses to parenteral immunization with tox-oid. The fact that amounts of endotoxin on theorder of 0.1 ,ug/100 ug of toxoid are capable ofeliciting serum vibriocidal antibody suggests,also, the possibility of an intestinal vibriocidalresponse. In this event, toxoids which containsmall amounts of endotoxin may provide betterprotection than pure toxoid in animal protec-tion studies involving live vibrio challenge.

In determining the ability of various toxoidsto elicit vibriocidal antibody in immunized rab-bits, two immunization schedules were em-ployed. The accelerated schedule (which, in itsfinal form, involved three weekly doses of tox-

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oid) proved to be about 10-fold more sensitive tosmall amounts of somatic antigen than thestandard schedule and had the additional ad-vantage of permitting data to be obtained inabout 4 weeks instead of 9 weeks, the timerequired in the case of the standard schedule.The latter schedule, however, proved to bemore sensitive to the production of antitoxin byfluid toxoid, resulting in up to 10-fold moreantitoxin than was observed in the acceleratedimmunization schedule (Tables 4 and 5). Theseresults suggest that the efficacy of combinedsomatic antigen and toxoid vaccines may beinfluenced by or depend on the choice of anappropriate immunization schedule.In the rabbit studies reported here, the

amount of toxoid antigen employed was 10 to 20times greater on a milligram per kilogrambasis than has, up to this time, been evaluatedin man. It is therefore important to considerthis fact when comparing serological data fromrabbits and man and when evaluating the re-

sults of field studies. In the first cholera toxoidfield trial, for example, volunteers in Dacca,Bangladesh, received two 100-,g inoculationsof toxoid lot no. 20101 (Tables 3-5) spaced 6weeks apart (3). Curlin et al. (4), in theiranalysis of the field trial, reported that a briefbut real protection against Inaba cholera waselicited by the toxoid. This represents the firstevidence from the field that pure antitoxic im-munity may play a role in protection againstcholera. However, the study pointed to the needfor markedly enhancing the duration and levelof protection (4). Therefore, before it is possibleto fully evaluate what role antitoxic immunitymay play in prevention of cholera, it will benecessary to establish those immunization pa-rameters which best promote an optimal im-mune response. It remains to be determined,for example, which routes, parenteral or oral(or a combination of both, as indicated by re-cent studies of Pierce et al. [7]), and whichdoses and schedules elicit prolonged, protectivecirculating and/or local antitoxin. In the ab-sence of optimal conditions of immunization(for either the case ofpure toxoid or toxoid plusV. cholerae somatic antigens), it may not bepossible to effectively combine antibacterialand antitoxic immunity in the control of thedisease. The availability of a pure, stable, anti-genic toxoid which does not elicit vibriocidalantibody in rabbits or man and which does nototherwise exhibit toxic side effects should con-

tribute to solving the problem of establishinglong-term cholera immunity.

ACKNOWLEDGMENTSWe express our appreciation to David Hoover for super-

vising and participating in the production phase of thiswork and to Terry Schaffer for typing the manuscript.

This investigation was supported by Public Health Serv-ice contract NIH 70-2102 from the National Institute ofAllergy and Infectious Diseases.

LITERATURE CITED1. Craig, J. P. 1965. A permeability factor (toxin) found in

cholera stools and culture filtrates and its neutraliza-tion by convalescent cholera sera. Nature (London)207:614-616.

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

3. Curlin, G. 1975. Current progress in the cholera toxoidfield trial in Bangladesh, p. 98-102. In H. Fukumiand M. Ohashi (ed.), Proc. 10th Joint Conference ofthe U.S.-Japan Cooperative Medical Science Pro-gram Symposium on Cholera. Kyoto, Japan, 1974.

4. Curlin, G., R. Levine, K. M. A. Aziz, A. S. M. Rehman,and W. F. Verwey. 1976. Field trial of cholera toxoid,p. 314-329. In Proc. 11th Joint Conference of theU.S.-Japan Cooperative Medical Science ProgramSymposium on Cholera. New Orleans, 1975.

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

6. Peterson, J. W., W. F. Verwey, J. P. Craig, J. C.Guckian, H. R. Williams, and N. F. Pierce. 1975. Theresponse to glutaraldehyde toxoid in human volun-teers. A progress report, p. 89-97. In H. Fukumiand M. Ohashi (ed.), Proc. 10th Joint Conference ofthe U.S.-Japan Cooperative Medical Science Pro-gram Symposium on Cholera. Kyoto, Japan, 1974.

7. Pierce, N. F., R. B. Sack, and B. K. Sircar. 1976.Immunity to experimental cholera. m. Enhanced du-ration of protection after sequential parenteral-oraltoxoid administration to dogs. J. Infect. Dis. (inpress).

8. Rappaport, R. S., G. Bonde, T. McCann, B. A. Rubin,and H. Tint. 1974. Development of a purified choleratoxoid. H. Preparation ofa stable, antigenic toxoid byreaction oftoxin with glutaraldehyde. Infect. Immun.9:304-317.

9. Rappaport, R. S., B. A. Rubin, and H. Tint. 1974.Development of a purified cholera toxoid. I. Purifica-tion of toxin. Infect. Immun. 9:294-303.

10. Warburg, O., and W. Christian. 1942. Isolation andcrystallization ofenolase. Biochem. Z. 310:384-421.

11. 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 ofmouse protection anti-gens derived from Vibrio cholerae. Tex. Rep. Biol.Med. 27:275-298.

12. Yin, E. T., C. Galanos, S. Kinsky, R. A. Bradshaw, S.Wessler, 0. Lideritz, and M. E. Sariento. 1972.Picogram-sensitive assay for endotoxin: gelationof Limulus polyphemus blood cell lysate induced bypurified lipopolysaccharides and lipid A from gramnegative bacteria. Biochim. Biophys. Acta 26:284-289.

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