Vol. 32, No. 2INFECTION AND IMMUNITY, May 1981, p. 534-5410019-9567/81/050534-08$02.00/0

Development of a Vaccine Against Experimental Cholera andEscherichia coli Diarrheal Disease

R. S. RAPPAPORT* AND G. BONDE2Departments of Biochemistry' and Biological Product Development,2 Wyeth Laboratories, Philadelphia,

Pennsylvania 19101

The results of the present investigation indicate a simple approach to thedevelopment of a single-vaccine formula which may ultimately be used to conferprotection against both cholera and certain types of Escherichia coli diarrhealdisease in humans and domestic animals. The design of the vaccine is based onthe well-documented ability of cholera antitoxin to neutralize both cholera andheat-labile E. coli enterotoxins (CT and LT, respectively) and on the ability ofkilled E. coli to enhance the immune response to cholera toxoid and, possibly, toconventional cholera vaccine as well. Evidence presented shows that a parenter-ally administered E. coli vaccine, prepared from an LT-only enterotoxigenicstrain, reproducibly elevated rabbit antitoxin responses to cholera toxoid and thatsuch responses correlated with dramatic protection against live cholera vibriosand the homologous E. coli strain in the rabbit ligated loop model of diarrhealdisease. The results show also that cholera vaccine acted to suppress the rabbits'immune response to the cholera toxoid and E. coli vaccine formula, even thoughall three antigens combined still provided significant protection against liveorganism challenge. On the basis of data presently available, the vaccine formulawould be composed simply of cholera toxoid and E. coli vaccine, but may alsoinclude cholera vaccine. Since it has already been established that cholera toxoidand cholera vaccine are each safe for human use, additional vaccine developmentwould require investigation of the safety of E. coli vaccine, alone and in combi-nation with the other components.

Despite advances in our understanding of thepathogenic mechanisms involved in cholera andenterotoxigenic Escherichia coli (ETEC) diar-rheal disease, knowledge of how best to stimu-late long-lasting protective immunity againstthese related, enterotoxin-mediated diseases re-mains incomplete (2). In the case of cholera, theonly immunoprophylactic agents presentlyavailable are killed whole-cell vaccines whichconfer only limited protection for a limited pe-riod of time (5); in the case of E. coli diarrhealdisease, no immunoprophylactic agents pres-ently exist.

Since the recognition that cholera enterotoxin(CT) is the primary agent responsible for cholerapathogenesis, the question of whether pure an-titoxic immunity can protect against cholera(just as it does in diphtheria and tetanus) hasbeen raised repeatedly. In an attempt to answerthis question, a highly purified, stable choleratoxoid was developed (15, 16) and subsequentlytested in a field trial in Dacca, Bangladesh,during the 1974 to 1975 cholera season. Theresults of the field trial were disappointing: thetoxoid, although evoking good levels of circulat-ing antitoxin, afforded only slight and transientprotection (3). These findings raised many

doubts about the sufficiency of antitoxic immu-nity and led to the notion, in some quarters, thatthe glutaraldehyde toxoid used in the field studywas an inferior immunogen (7).

Subsequently, new approaches to cholera vac-cine development have been advanced. One ofthese involves combined-antigen formulas con-sisting of toxoid or the B subunit ofCT togetherwith conventional cholera vaccine or Vibriocholerae lipopolysaccharide (6, 7, 12, 18); an-other involves the isolation of an avirulent mu-tant of V. cholerae which is a candidate live oralvaccine (8).

In the studies reported here, we investigatedthe ability of glutaraldehyde-inactivated choleratoxin (cholera toxoid) alone and in combinationwith conventional cholera vaccine or an experi-mental E. coli vaccine, or both, to simultane-ously protect rabbits against challenge with vi-able cholera vibrios and heat-labile enterotoxin(LT)-only E. coli. E. coli vaccine was includedin the study as a result of previous observationswhich showed that ETEC, by virtue of mem-brane-bound LT, were capable of eliciting anti-toxin specific for LT (14) and, at the same time,were also capable of acting as adjuvants forcholera toxoid (R. S. Rappaport, unpublished

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data). These findings, together with the fact thatantitoxin elicited by cholera toxoid neutralizesboth LT and CT (11, 13, 14), led us to comparethe antigenicity and immunogenicity of choleratoxoid, cholera vaccine, and E. coli vaccine aloneand in all possible combinations.

(This paper was presented in part at the In-ternational Symposium on Bacterial Vaccines,Bethesda, Md., 15-18 September 1980.)

MATERIALS AND METHODSAntigens. Wyeth cholera toxoid (lot no. 20301) was

prepared as previously described (15, 16). Whole-cellantigens consisted of Wyeth cholera vaccine (lot no.104701) containing equal numbers of phenol (0.5%)-inactivated Inaba and Ogawa organisms (grown onTrypticase soy agar; BBL Microbiology Systems,Cockeysville, Md.) and three experimental E. colivaccines. The E. coli vaccines were prepared from thefollowing strains: E. coli KL320, a nontoxigenic auxo-trophic mutant of E. coli K-12, obtained from WernerK. Maas, New York, N.Y.; E. coli KL320-1, identicalto strain KL320 except that it carried a plasmid(pCG86) for LT and heat-stable toxin (ST) production(also obtained from Werner K. Maas); and E. coliH74-114 (08:H9), an LT-only strain isolated from aninfant in Brooklyn, N.Y., and obtained from John P.Craig, Brooklyn.Production ofE. coli vaccines. Erlenmeyer flasks

(500 ml), each containing 80 ml of the medium usedby Evans et al. (4), were inoculated with approxi-mately 105 organisms per ml of each strain grown tomid-log phase in 2% peptone broth. The flasks wereincubated at 37°C on a rotary shaker (150 rpm) for 16h. The cultures, ranging in density from 3 x 109 to 1.2x 10' colony-forming units per ml, were then centri-fuged and 'washed once with 0.067 M phosphate-buffered saline (PBS), pH 7.8. Washed organisms werethen suspended at their original density in PBS con-taining 0.01% glutaraldehyde (Aldrich Chemical Co.,Milwaukee, Wis.). In most instances, treatment withglutaraldehyde proceeded for 4 h at room temperature,followed by an 18- to 24-h incubation at 4°C. Aftertreatment, the organisms were centrifuged from theinactivation medium, resuspended at the same concen-tration in sterile PBS, and then checked for viability.Once it was established that the cultures were com-pletely nonviable, thimerosal (0.01%) was added as apreservative and the vaccine suspensions were storedat 4°C until used. In some instances, when the concen-tration of viable cells was greater than 1010 organisms/ml, a small number of viable organisms (ca. 500/ml)were detected; in such instances, the procedure wasrepeated. Gram stains of the vaccines showed that thetreatment did not alter their morphology, nor did itresult in cellular aggregation. In addition, intramus-cular inoculation of rabbits with 2 x 1010 organismsdid not result in overt local or systemic distress.Immunization of animals. For antigenicity stud-

ies, groups of five to seven male New Zealand albinorabbits weighing between 2.3 and 5.0 kg were twiceimmunized intramuscularly in the right posteriorthigh, with an interval of 6 weeks between inocula-tions. Cholera toxoid was administered at a dose of

100 ,g in 1 ml of PBS, pH 7.8, containing 0.01%thimerosal (PBS-T), and the whole-organism vaccineswere administered at doses of 4 x 109 organisms (chol-era vaccine); 5 x 109 organisms (E. coli H74-114); and1 x 1010 organisms (E. coli strains KL320 and KL320-1), each contained in 1 ml of PBS-T. When antigenswere combined, the concentration of individual com-ponents was adjusted so that the doses specified abovewere all contained in a final volume of 1 ml. Bloodsamples were obtained before and at 6 and 8 weeksafter primary immunization, and the sera were storedat -20°C until assayed. For protection studies, groupsof five to eight recently weaned rabbits weighing be-tween 0.68 and 1.36 kg were used so that they wouldbe a suitable size at the time of challenge. Groups ofthese animals were immunized with the antigens de-scribed above, each administered alone and in variouscombinations, using the same doses, route, and im-munization schedule. Control animals were similarlyimmunized with 1 ml of PBS-T. Blood samples wereobtained at the time of challenge (see text and below),and sera were stored at -20°C until assayed.Measurement of antitoxin. Antitoxin titers of

various sera were determined by the intracutaneousmethod in rabbits as described earlier (14-16). Onelimit-of-bluing dose of CT or LT (of approximately68,000 daltons; isolated by Sephadex G-150 chroma-tography from E. coli H74-114 culture filtrates [14])per ml was the test dose in each assay, and each toxindose was determined by titration against standardcholera antitoxin (Swiss Serum and Vaccine Instituteserum containing 4,470 antitoxin units [AU]/ml). Alltiters are expressed in AU per milliliter.Measurement of vibriocidal antibody. Comple-

ment-dependent vibriocidal antibody titers, based onthe ability of various sera to inhibit the growth ofInaba VC-13, were determined by a microtiter tech-nique as described previously (16). National Institutesof Health reference convalescent antiserum was usedas a standard. Titers are expressed as the last twofolddilution which completely inhibited bacterial growth.

Protection studies. Protection was assessed onthe basis of secretory responses elicited in immunizedanimals when they were challenged by a modificationof the ligated ileal loop technique (1). During a 12-dayperiod beginning on the 12th day after booster im-munization, laparotomy was performed on each rabbit(ca. four per day), and approximately 21 5-cm loopswere ligated and inoculated with five graded doses oflive cholera vibrios (Ogawa 395), ranging from 103 to108 organisms (in 1 ml), and four graded doses of liveE. coli (strain H74-114), ranging from 107 to 1010organisms (in 1 ml). On each day, challenge organismswere collected from brain heart infusion (Difco Labo-ratories, Detroit, Mich.) agar slants after two passagesfrom lyophilized stocks. The cultures were suspendedin Evans medium (4) and adjusted to 2.0 x 1010 (Ogawa395) and 4.0 x 1010 (E. coli H'74-114) organisms/ml onthe basis of optical density readings at 650 nm, usinga Bausch & Lomb spectrophotometer. For precisequantitation ofchallenge inocula, colony-forming unitswere also determined by direct plating on brain heartinfusion agar. Each dilution of vibrios or E. coli (inEvans medium) was tested in duplicate, each of whichwas randomly positioned in the upper and lower halves

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536 RAPPAPORT AND BONDE

of the small intestines. Negative control loops posi-tioned proximally, in the middle, and at the distal endsof the test loops were inoculated with medium alone.Group 50% effective dose (ED50) values (the numberof organisms required to elicit a mean secretory re-sponse of 1 ml/cm) were determined for Ogawa 395and E. coli H74-114. Protection factors (estimates ofthe degree of protection) were calculated from theratio of group ED50 values for antigen-immunized ver-sus vehicle (PBS-T)-immunized animals. The secre-

tory data were evaluated by analysis of variance, andthen Duncan's multiple-range test (17) was applied.Data from animals succumbing as a result of challengewere excluded.

RESULTSComparison of the antitoxin response to

cholera toxoid alone and in combinationwith a toxigenic or a nontoxigenic E. colivaccine strain. Previous studies comparing theantigenicity of cholera toxoid in combinationwith either high-molecular-weight LT (thoughtto represent fragments of outer membrane) orE. coli endotoxin (055:B5) revealed that bothLT and endotoxin acted as adjuvants for thetoxoid (Rappaport, unpublished data). Thesefindings led us to consider that suitably inacti-vated ETEC should, by virtue of membrane-bound LT and outer membrane lipopolysaccha-ride, be capable of both eliciting specific LTantitoxin and acting as an adjuvant for the tox-oid. To test these possibilities, groups of rabbitswere twice immunized intramuscularly with

cholera toxoid, alone and in combination with anontoxigenic E. coli strain (KL320) or a toxi-genic strain of identical serotype (KL320-1), us-ing a 6-week interval between inoculations. Twoother groups of rabbits were similarly immu-nized with each whole-cell vaccine alone. Theresults of antitoxin determinations performed onpretreatment sera and sera obtained at 6 and 8weeks showed that the toxigenic strain (KL320-1) was indeed capable of stimulating antitoxin.Like the antitoxin produced by high-molecular-weight LT (14), it was specific for LT, showingonly a slight ability to neutralize CT (Table 1).In contrast, the nontoxigenic strain (KL320) didnot produce antitoxin to either LT or CT. Bothstrains, however, acted as adjuvants for toxoid,since mean antitoxin titers elicited by the toxoidwere increased from three- to fivefold at 6 weeksand from four- to sixfold at 8 weeks when it wascombined with either strain (Table 1). Mean LTantitoxin titers produced by the toxoid in com-

bination with the toxigenic strain were greaterthan in combination with the nontoxigenicstrain, possibly reflecting the contribution ofboth LT and CT antitoxin.Comparative antigenicity of cholera tox-

oid, cholera vaccine, and E. coli vaccinealone and in combination. Since current ef-forts to develop a vaccine against cholera pointto a need to elicit both antibacterial and anti-toxic immunity (6-8, 12, 18), it was of interest toinvestigate the ability of E. coli vaccine to act as

TABLE 1. Comparative antigenicity of cholera toxoid and toxigenic and nontoxigenic E. coli vaccines aloneand in combination: serum antitoxin responses in rabbits

Cholera antitoxin (AU/ml)b E. coli LT antitoxin (AU/mrl)

Antigen' Pretreat- Pre-Pentra 6 wk 8 wk treat- 6 wk 8 wk

ment

Cholera toxoid <2 (6)c 39 (6) 955 (6) <2 (6) 60 (6) 2,038 (6)(17-79)d (375-1,910) (23-222) (549-4,543)

E. coli KL320 <2 (7) <2 (7) <2 (6) <2 (7) <2 (7) <2 (6)

E. coli KL320-1 <2 (6) <5 (6) 31 (6) <2 (6) 20 (6) 312 (6)(<2-227) (6-316) (<2-359) (71-1,024)

Cholera toxoid + E. coli <2 (6) 166 (6) 6,828 (4) <2 (6) 201 (6) 8,244 (4)KL320 (52-445) (3,821-11,189) (52-990) (5,790-16,945)

Cholera toxoid + E. coli <2 (6) 185 (6) 6,208 (4) <2 (6) 282 (6) 11,830 (4)KL320-1 (66-477) (2,895-36,364) (87-830) (5,313-16,385)

'Two identical intramuscular inoculations spaced 6 weeks apart. The dose of cholera toxoid was 100 ,ug/inoculation, and the dose of each E. coli vaccine was 1010 organisms/inoculation.

b One limit-of-bluing dose of cholera toxin or E. coli enterotoxin was used in the measurement of antitoxintiters, and the dose of each toxin was determined by titration against standard cholera antitoxin. Titers representthe geometric mean for each group of sera obtained before treatment and at 6 and 8 weeks.

c Number of sera.d Range of values.

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VACCINE AGAINST DIARRHEAL DISEASE 537

an adjuvant for toxoid in the presence or absenceof cholera vaccine. To examine the interactionof these antigens, groups of rabbits were twiceimmunized with each of the antigens alone andin all possible combinations, using the same

conditions as in the previous study (Table 1).Since the two studies were carried out in parallel,the same group of toxoid-immunized rabbits wasused for both. In this study, however, the E. colivaccine was prepared from strain H74-114. Theresults of antitoxin determinations performed onpretreatment sera and sera obtained at 6 and 8weeks showed that E. coli vaccine acted as an

adjuvant for toxoid both in the presence andabsence of cholera vaccine (Table 2). As ex-

pected from the results of previous investiga-tions (6, 7, 12), cholera vaccine did not act as anadjuvant for toxoid, since antitoxin titers(against both CT and LT) did not vary signifi-cantly with the addition or omission of the vac-cine (Table 2). When toxoid was combined withE. coli vaccine, however, mean antitoxin titerswere elevated from 7.5- to 12-fold in the case ofLT antitoxin and from 13- to 20-fold in the caseof CT antitoxin 8 weeks after primary immuni-

zation (Table 2). In addition, the study showedthat strain H74-114 was at least as effective an

adjuvant as were strains KL320 and KL320-1,

suggesting that many, if not all, E. coli strains ofdifferent serotype may exhibit this property toone extent or another. The results also con-

firmed that both LT/ST and LT-only ETECevoke antitoxin specific for LT, since only LTantitoxin was elicited by strains KL320-1 andH74-114 (Tables 1 and 2). In contrast, choleravaccine produced no antitoxin whatsoever.Comparative immunogenicity of cholera

toxoid, cholera vaccine, and E. coli vaccinealone and in combination. Since E. coli vac-cine acted as an adjuvant for toxoid in the pres-

ence or absence of cholera vaccine, it seemedlikely that a combination of all three antigensmight provide superior protection against chal-lenge with both viable vibrios and LT-producingE. coli. To evaluate the relative immunogenicityof such a combination of antigens, groups ofinfant rabbits were twice immunized with eachof these antigens alone and in various combina-tions, using conditions identical to those of theprevious studies. Rabbits from each group wererandomly challenged with vibrios and LT-onlyE. coli during a 12-day period beginning on the12th day after booster immunization. ED5o val-ues and protection factors, therefore, reflect theaverage level of protection observed over a 12-day period (approximately 7.5 to 9.5 weeks after

TABLE 2. Comparative antigenicity of cholera toxoid, cholera vaccine, and E. coli vaccine alone and incombination: serum antitoxin and vibriocidal responses in rabbits

Cholera antitoxin (AU/ml)b E. coli LT antitoxin (AU/ml)b Vibriocidal antibody'

Antigen' Pre- Pre- Pre-treat- 6 wk 8 wk treat- 6 wk 8 wk treat- 8 wkment ment ment

Cholera toxoid <2 (6)d 39 (6) 955 (6) <2 (6) 60 (6) 2,038 (6) <2 (6) <2 (6)(17-79)' (375-1,910) (23-222) (549-4,543)

Cholera vaccine <2 (6) <2 (6) <2 (6) <2 (6) NDf <2 (6) <2 (6) 1,821 (6)(512-32,768)

E. coli vaccine <2 (5) <2 (5) <4 (5) <2 (5) ND 248 (5) <2 (5) <2 (5)(<4-29) (115-920)

Cholera toxoid + <2 (6) 30 (6) 1,573 (6) <2 (6) ND 1,771 (6) <2 (6) 813 (6)cholera vaccine (19-60) (496-3,113) (955-3,442) (256-2,048)

Cholera toxoid + E. <2 (5) 77 (5) 13,131 (5) <2 (5) ND 15,390 (5) <2 (5) <2 (5)coli vaccine (40-158) (8,188-47,941) (5,400-80,600)

Cholera vaccine + E. <2 (6) <2 (6) <7 (5) <2 (6) ND 245 (5) <2 (5) 4,077 (5)coli vaccine (<2-20) (111-430) (1,024-256,000)

Cholera toxoid + <2 (6) 91 (6) 20,169 (5) <2 (5) ND 24,500 (5) <2 (5) 5,379 (5)cholera vaccine + (44-367) (14,252-33,899) (15,270-30,120) (512-128,000)E. coli vaccinea Two identical intramuscular inoculations spaced 6 weeks apart. The dose of cholera toxoid was 100 ,g/inoculation, and the

doses per inoculation of the whole-cell vaccines were 4 x 10' organisms (cholera vaccine) and 5 x 10' organisms (E. coli vaccine,strain H74-114).

b, d. 'See Table 1.'The reciprocal of the last twofold dilution of serum which completely inhibited growth of Inaba VC-13. Titers represent the

geometric mean for each group of sera.fND, Not done.

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538 RAPPAPORT AND BONDE

primary immunization).In the case of vibrio challenge, each group of

animals exhibited some degree of protection rel-ative to controls (Table 3). Of these, the leastprotective antigen was E. coli vaccine, and thenext least protective antigen was cholera vac-cine. The former result was not surprising sinceantibodies directed against E. coli would not beexpected to inactivate cholera vibrios, and thelatter result indicated that vibriocidal antibodyalone was not sufficient to provide superior pro-tection against vibrio challenge under the con-ditions used. When the two whole-cell vaccineswere combined, however, the protection factorrose to a level greater than or equal to theproduct of the individual vaccines, indicating a

synergistic response. Even though mean vibrio-cidal titers did not show a significant statisticaldifference between groups, there was some in-dication that the enhanced protection mighthave been due to an adjuvant effect of E. colivaccine on the immune response to cholera vac-

cine (Tables 2 and 3). Despite differences inprotection exhibited by these three groups, sta-tistical analysis of the secretory responses byDuncan's multiple-range test indicated consid-erable overlap between groups (Table 3).

In the case of the remaining four groups ofanimals, all were statistically significantly differ-ent from controls and from the three groupsimmunized with whole-cell vaccines, alone or in

combination. Of these, the group immunizedwith a combination of cholera toxoid and E. colivaccine exhibited the highest level of protection,a level not commonly evoked by the parenteralroute. Furthermore, this group exhibited thehighest antitoxin response as well (Table 3).Surprisingly, this combination of antigens was

less protective when it was combined with chol-era vaccine, and the decrease in protection was

associated with a reduction in circulating anti-toxin (Table 3). These results suggested thatunder certain conditions cholera vaccine mayact as an immunosuppressant. The triple com-

bination of antigens, nevertheless, still providedthe second-highest level of protection observedamong the various groups. In addition, there wasvery little difference in the level of protectionexhibited by the group of animals immunizedwith toxoid alone versus the group immunizedwith toxoid and cholera vaccine, each of whichshowed comparable antitoxin responses (Table3). In the latter comparison, the absence of a

synergistic response due to a combination ofantitoxic and antibacterial immunity was unex-

pected. Nevertheless, all groups immunized withtoxoid alone or in combination with other anti-gens were significantly protected against livevibrio challenge, and there appeared to be acorrelation between circulating antitoxin andprotection. Statistically, the level of protectionexhibited by the group immunized with the com-

TABLE 3. Comparative immunogenicity of cholera toxoid, cholera vaccine, and E. coli vaccine alone and incombination: antibody responses and protection of immunized rabbits against intestinal challenge with V.

cholerae (Ogawa 395)

Immunogen gropStatistical rED5tiefac- Cholera anti- VibriocidalImmunogen'groupingb ED5o titorn toxin' (AU/ml) antibody'PBS-T (control) A 7.5 x 103 (8)f 1 <2 (8) <2 (8)Cholera vaccine A 2.7 x 105 (3) 36 <4 (3) 256 (3)

(32-1,024) RE. coli vaccine A 3.0 x 104 (6) 4 <6 (6) <2 (6)

(<2-80)Cholera vaccine + E. coli vaccine A 1.3 x 106 (5) 173 <4 (5) 588 (5)

(256-2,048)Cholera toxoid B 1.3 x 10' (6) 1,733 828 (6) <2 (6)

(249-2,207)Cholera toxoid + cholera vaccine B, C 1.2 x 107 (5) 1,600 480 (5) 338 (5)

(124-1,007) (128-1,024)Cholera toxoid + E. coli vaccine C 1.2 x 108 (5) 16,000 3,310 (4) <2 (5)

(1,547-5,694)Cholera toxoid + cholera vaccine + E. B 2.5 x 107 (7) 3,333 1,216 (7) 624 (7)

coli vaccine (256-2,355) (128-2,048)a See Table 2.b Groups with the same letter were not significantly different from each other at P < 0.05 when secretory

data were evaluated by analysis of variance and Duncan's multiple-range test.c Number of organisms required to elicit a mean secretory response of 1 ml/cm.d Ratio of EDs, values for antigen-immunized versus control (PBS-T-immunized) groups.e g, h See footnotes b through d, Table 1.f See Table 2, footnote c.

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VACCINE AGAINST DIARRHEAL DISEASE 539

bination of toxoid and E. coli vaccine was sig-nificantly different from levels of all othergroups, although there was some overlap withthe group immunized with toxoid and choleravaccine (Table 3).

In the case of E. coli challenge, the dose range

studied was comparatively limited due to thefact that nearly 109 organisms were required toproduce a secretory response (in control ani-mals) equivalent to that produced by about 104cholera vibrios (Tables 3 and 4). Despite thedifference in pathogenicity of the two challengeorganisms, it was still possible to ascertain therelative abilities of the various antigens and an-

tigen combinations to protect against E. colichallenge. With the exception of cholera vaccine,all of the antigens or antigen combinations con-

ferred some degree of statistically significantprotection against challenge with strain H74-114(Table 4). Of these, the least protective antigenwas E. coli vaccine. When the cholera and E.coli whole-cell vaccines were combined, how-ever, the protection evoked by the combinationwas greater than or equal to the product of theindividual vaccines, just as it was in the case ofvibrio challenge. Of the four remaining groups,the group immunized with the combination oftoxoid and E. coli vaccine exhibited the highestlevel of protection. Statistically, the secretoryresponses produced in rabbits immunized withthis combination of antigens were significantlydifferent from the responses of any other group(Table 4). The toxoid in combination with E.coli vaccine also elicited the highest antitoxin

response, evoking titers eightfold greater thanthose produced by toxoid alone (Table 4). Again,as in the case of vibrio challenge, the protectionafforded by these antigens was diminished whenthey were administered together with choleravaccine, adding further support to the notion ofa cholera vaccine-mediated immunosuppression.Similarly, the protection conferred by toxoidalone, the second most protective antigen, was

also diminished when the toxoid was combinedwith cholera vaccine, but this difference was notstatistically significant (Table 4).

DISCUSSIONBecause of the obvious advantages of devel-

oping a single-vaccine formula which might pro-vide protection against both cholera and LT-mediated E. coli diarrheal disease, we investi-gated the comparative antigenicity and immu-nogenicity of cholera toxoid, conventional chol-era vaccine, and E. coli vaccine, alone and invarious combinations. The results show that glu-taraldehyde toxoid alone is both antigenic andprotective in the rabbit ligated loop model, es-

pecially for cholera (Ogawa 395), but also fordiarrheal disease evoked by an LT-only E. colistrain (H74-114). Although further studies are

required to establish the efficacy and safety ofthe toxoid in combination with other antigens,the present results also show that a vaccineformula which includes cholera toxoid and E.coli vaccine is capable of conferring remarkableprotection against both cholera vibrio and ho-mologous E. coli challenge in the rabbit model.

TABLE 4. Comparative immunogenicity of cholera toxoid, cholera vaccine, and E. coli vaccine alone and incombination: antitoxin responses and protection of immunized rabbits against intestinal challenge with E.

coli H74-114Statistical ED c Protection E. coli LT antitoxin'Immunogena groUpingb 50 factord (AU/ml)

PBS-T (control) A 7.5 x 108 (8)f 1.0 <2 (8)Cholera vaccine A 1.2 x 109 (3) 1.6 <2 (3)E. coli vaccine A, B, C 2.7 x 109 (6) 3.6 84 (6)

(8-256) BCholera vaccine + E. coli vaccine C 5.2 x 109 (5) 6.9 19 (5)

(<2-64)Cholera toxoid B, C 7.2 x 109 (6) 9.6 882 (6)

(205-2,321)Cholera toxoid + cholera vaccine B, C 3.3 x 109 (5) 4.4 538 (5)

(431-892)Cholera toxoid + E. coli vaccine D 1.0 x 10" (5)h 133.0 7,134 (4)

(3,823-17,446)Cholera toxoid + cholera vaccine A, B 2.9 x 109 (7) 3.9 1,205 (7)+ E. coli vaccine (462-7,645)

See Table 2.bd See Table 3.e- See footnotes b through d, Table 1.h In this case, mean secretory responses were: 0 ml/cm (107 organisms); 0.04 ml/cm (10' organisms); 0.25 ml/

cm (109 organisms); and 0.45 ml/cm (1010 organisms), with three of five animals exhibiting negative loops at allchallenge doses. The ED50 value is therefore an extrapolation of the dose-response curve.

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540 RAPPAPORT AND BONDE

Of all the antigen formulas tested, those includ-ing toxoid and E. coli vaccine generally providedgreater protection than those which did not, andit is noteworthy that the combination of choleravaccine and E. coli vaccine also provided greaterprotection than did either whole-cell vaccinealone. This observation suggests that E. colimay be an effective adjuvant not only for toxoid,but for cholera vaccine as well. Furthermore,those antigen formulas which provided thegreatest protection also produced the highestantitoxin responses, and those formulas whichprovided the least protection produced little orno antitoxin. In the case of the combined toxoid-E. coli vaccine formula, mean CT and LT anti-toxin titers were at least 4-fold and as high as20-fold greater than CT and LT antitoxin titerselicited by toxoid alone approximately 8 weeksafter primary immunization (Tables 1 through4). The elevation in antitoxin titer appears to bedue to an adjuvant effect of E. coli on theimmune response to toxoid, and such an effectcan be evoked by both toxigenic and nontoxi-genic strains (Table 1). Since E. coli endotoxin(055:B5) also acted as an adjuvant for toxoid(Rappaport, unpublished data), the adjuvant ac-tion of the E. coli vaccines is presumed to bedue primarily to outer membrane lipopolysac-charide, but other surface antigens may be im-portant as well. In the case of diarrheal diseasemediated wholly or in part by LT, the contri-bution of specific LT antitoxin by an enterotox-igenic vaccine strain may be an advantage. LTantitoxin titers, for example, were generallygreater than CT antitoxin titers, and this maybe a result of the development of both "type"-specific (anti-LT) and "group"-specific (anti-CT) antitoxin.Another interesting observation concerns the

comparison of the toxoid and E. coli vaccineformula with the same antigens combined withcholera vaccine. In the case of both choleravibrio and E. coli challenge, inclusion of choleravaccine resulted in a reduction in both protec-tion and circulating antitoxin (Tables 3 and 4).A similar reduction in antitoxin titer did notoccur after immunization of adult rabbits withthe same antigen formula (Table 2), suggestingthat the age of the animals may be a relevantfactor. Under the conditions used in the protec-tion studies, however, cholera vaccine appearsto have acted as an immunosuppressant. It isnoteworthy that subcutaneously administeredcholera vaccine also suppressed the immune re-sponse to perorally administered live polio vac-cine (A. M. Svennerholm, L. A, Hanson, J.Holmgren, B. S. Lindblad, S. R. Khan, A. Nils-son, and B. Svennerholm, J. Infect. Dis., inpress).

The substantial antitoxin response producedby cholera toxoid in combination with E. colivaccine appears to be a major factor in elicitingprotection against both cholera and E. coli.Other factors, such as antibodies directedagainst pili, adhesion, or colonization factors,may be important as well. But, contrary to cur-rent views of the importance of both antitoxicand antibacterial immunity in cholera (6-8, 12,18) or of the importance of antibacterial immu-nity alone (9), the present results do not supporta major role for vibriocidal antibody, at least inthe rabbit model of diarrheal disease. The ob-servation that toxoid plus E. coli vaccine (whichelicited no vibriocidal antibody) protected rab-bits against vibrio challenge to at least the sameextent as the same antigens plus cholera vaccine(or toxoid plus cholera vaccine) suggests anti-toxic immunity as the predominant factor inlong-term cholera immunity, just as it is in diph-theria and tetanus.Our failure to demonstrate synergistic protec-

tion due to both antitoxic and antibacterial (vi-briocidal) immunity conflicts with the findingsof Holmgren et al. (7) and Peterson (12). Theabsence of such synergism may be due to differ-ences in the potencies of the cholera vaccines ormay stem from the choice of immunization pa-rameters used in the different studies. Holmgrenet al. used a 2-week interval between immuni-zations and challenged 5 and 21 days later,whereas Peterson used a 4-week interval andchallenged 2 weeks later. In the study of Holm-gren et al., there was a rapid decline in antibac-terial immunity between days 5 and 21 afterbooster immunization (7), indicating that thevibriocidal component of immunity was short-lived. In the present study, a 6-week interval wasused, and any synergism between antitoxic andantibacterial immunity in the days after boosterimmunization was not apparent at challenge, 12to 23 days later. A previous study has, in fact,shown that the ability of toxoid to elicit maximallevels of antitoxin and the ability of V. choleraesomatic antigen to elicit vibriocidal antibody areboth influenced by the choice of immunizationschedule, the 6-week interval favoring the for-mer and disfavoring the latter (16). The com-paratively poor performance of glutaraldehydetoxoid versus B (or L) subunit in the study ofHolmgren et al., therefore, may not necessarilybe due to an inherent immunogenic inferiorityof the toxoid antigen, but rather to the choice ofimmunization schedule and to the possibility ofthe two antigens not being equally inert.(Batches of B [or L] subunit were reported tohave less than 0.1% the toxicity of intact toxin[7], whereas glutaraldehyde toxoids reproduc-ibly exhibited less than 0.003% the toxicity of

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VACCINE AGAINST DIARRHEAL DISEASE 541

parent toxin [15]).The present studies support the sufficiency of

antitoxic immunity in protection, but do notestablish the duration of either; nor do theyaddress the question of the type and distributionof antibodies elicited by the toxoid-E. coli vac-cine formula. Whether the high levels of anti-toxin evoked by the combined antigens will per-sist in sufficient quantity to penetrate the lumenof the gut and whether local antitoxin can beevoked by this combination of antigens remainto be determined. Since the diseases in questionare confined to the intestinal tract, it wouldclearly be desirable to determine the efficacy ofthe present formulation when it is administeredby the oral route or a combination of parenteraland oral routes. The antigenicity of orally ad-ministered toxoid alone has already been estab-lished in humans (10), but thus far protectionhas not been demonstrated under the conditionsused (9). If the combined vaccine were to confersignificant protection when administered peror-ally, then it would obviate many of the problemsassociated with the development of parenteralfornulations. The present usage of gram-nega-tive bacteria in parenteral vaccines, however,provides a precedent for considering the poten-tial inclusion of a suitably detoxified E. colivaccine (or component thereof) in a parenteraltoxoid fornulation. Moreover, the present re-sults do not preclude the incorporation of avariety of enteropathogenic (E. coli) serotypes(including invasive, nontoxigenic E. coli) and anappropriate heat-stable enterotoxin antigen inthe vaccine.

ACKNOWLEDGMENTSThis study was supported in part by contract DAMD 17-

74-C-4007 from the U.S. Army Medical Research and Devel-opment Command.

LITERATURE CITED

1. Burrows, W., and G. M. Musteikis. 1966. Cholera in-fection and toxin in the rabbit ileal loop. J. Infect. Dis.116:183-190.

2. Craig, J. P. 1980. A survey of the enterotoxic enteropa-thies, p. 15-25. In 0. Ouchterlony and J. Holmgren(ed.), Cholera and related diarrheas, 43rd Nobel Sym-posium. S. Karger, Basel

3. Curlin, G., R. Levine, K. M. A. Aziz, A. S. M. Rahman,and W. F. Verwey. 1976. Field trial of cholera toxoid,p. 314-335. In Proceedings of the 11th Joint Conferenceof the U.S.-Japan Cooperative Medical Science Pro-

gram Symposium on Cholera. Department of Health,Education, and Welfare, Washington, D.C.

4. Evans, D. J., D. G. Evans, and S. L Gorbach. 1973.Production of vascular permeability factor by entero-toxigenic Escherichia coli isolated from man. Infect.Immun. 8:725-730.

5. Feeley, J. C., and E. J. Gangarosa. 1980. Field trials ofcholera vaccine, p. 204-210. In 0. Ouchterlony and J.Holmgren (ed.), Cholera and related diarrheas, 43rdNobel Symposium. S. Karger, Basel.

6. Germanier, R., E. Furer, S. Varallyay, and T. M.Inderbitzin. 1977. Antigenicity of cholera toxoid inhumans. J. Infect. Dis. 135:512-516.

7. Holmgren, J., A. M. Svennerholm, L. Lonnroth, M.Fall-Persson, B. Markman, and H. Lundbeck. 1977.Development of improved cholera vaccine based onsubunit toxoid. Nature (London) 269:602-604.

8. Honda, T., and R. A. Finkelstein. 1979. Selection andcharacteristics of a Vibrio chokerae mutant lacking theA (ADP-ribosylating) portion of the cholera entero-toxin. Proc. Natl. Acad. Sci. U.S.A. 76:2052-2056.

9. Levine, M. M. 1980. Immunity to cholera as evaluated involunteers, p. 195-203. In 0. Ouchterlony and J. Holm-gren (ed.), Cholera and related diarrheas, 43rd NobelSymposium. S. Karger, Basel.

10. Levine, M. M., T. P. Hughes, C. R. Young, S.O'Donnell, J. P. Craig, H. P. Holley, and E. J.Bergquist. 1978. Antigenicity of purified glutaralde-hyde-treated toxoid administered orally. Infect. Immun.21:158-162.

11. Nalin, D. R., A. Al-Mahmud, G. Curlin, A. Ahmen,and J. Peterson. 1974. Cholera toxoid boosts serumEscherichia coli antitoxin in humans. Infect. Immun.10:747-749.

12. Peterson, J. W. 1979. Synergistic protection against ex-perimental cholera by immunization with cholera toxoidand vaccine. Infect. Immun. 26:528-533.

13. Pierce, N. F. 1977. Protection against challenge withEscherichia coli heat-labile enterotoxin by immuniza-tion of rats with cholera toxin/toxoid. Infect. Immun.18:338-341.

14. Rappaport, R. S. 1978. Observations on the mechanismof release of heat-labile E. coli enterotoxin, p. 443-474.In Proceedings of the 13th Joint Conference of the U.S.-Japan Cooperative Medical Science Program Sympo-sium on Cholera. Department of Health, Education,and Welfare, Washington, D.C.

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

16. Rappaport, R. S., W. A. Pierzchala, G. Bonde, T.McCann, and B. A. Rubin. 1976. Development of apurified cholera toxoid. III. Refinements in purificationof toxin and methods for the determination of residualsomatic antigen. Infect. Immun. 14:687-693.

17. Steele, R. G. D., and J. H. Tome. 1980. Principles andprocedures of statistics. McGraw-Hill Book Co., NewYork.

18. Svennerholm, A. M., and J. Holmgren. 1976. Synergis-tic protective effect in rabbits immunized with Vibriocholerae lipopolysaccharide and toxin/toxoid. Infect.Immun. 13:735-740.

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