Development of a Vaccine Against Experimental Cholera and

Transcription

1 INFECTION AND IMMUNITY, May 1981, p /81/ $02.00/0 Vol. 32, No. 2 Development of a Vaccine Against Experimental Cholera and Escherichia coli Diarrheal Disease R. S. RAPPAPORT* AND G. BONDE2 Departments of Biochemistry' and Biological Product Development,2 Wyeth Laboratories, Philadelphia, Pennsylvania The results of the present investigation indicate a simple approach to the development of a single-vaccine formula which may ultimately be used to confer protection against both cholera and certain types of Escherichia coli diarrheal disease in humans and domestic animals. The design of the vaccine is based on the well-documented ability of cholera antitoxin to neutralize both cholera and heat-labile E. coli enterotoxins (CT and LT, respectively) and on the ability of killed E. coli to enhance the immune response to cholera toxoid and, possibly, to conventional cholera vaccine as well. Evidence presented shows that a parenterally administered E. coli vaccine, prepared from an LT-only enterotoxigenic strain, reproducibly elevated rabbit antitoxin responses to cholera toxoid and that such responses correlated with dramatic protection against live cholera vibrios and the homologous E. coli strain in the rabbit ligated loop model of diarrheal disease. The results show also that cholera vaccine acted to suppress the rabbits' immune response to the cholera toxoid and E. coli vaccine formula, even though all three antigens combined still provided significant protection against live organism challenge. On the basis of data presently available, the vaccine formula would be composed simply of cholera toxoid and E. coli vaccine, but may also include cholera vaccine. Since it has already been established that cholera toxoid and cholera vaccine are each safe for human use, additional vaccine development would require investigation of the safety of E. coli vaccine, alone and in combination with the other components. Despite advances in our understanding of the pathogenic mechanisms involved in cholera and enterotoxigenic Escherichia coli (ETEC) diarrheal disease, knowledge of how best to stimulate long-lasting protective immunity against these related, enterotoxin-mediated diseases remains incomplete (2). In the case of cholera, the only immunoprophylactic agents presently available are killed whole-cell vaccines which confer only limited protection for a limited period of time (5); in the case of E. coli diarrheal disease, no immunoprophylactic agents presently exist. Since the recognition that cholera enterotoxin (CT) is the primary agent responsible for cholera pathogenesis, the question of whether pure antitoxic immunity can protect against cholera (just as it does in diphtheria and tetanus) has been raised repeatedly. In an attempt to answer this question, a highly purified, stable cholera toxoid was developed (15, 16) and subsequently tested in a field trial in Dacca, Bangladesh, during the 1974 to 1975 cholera season. The results of the field trial were disappointing: the toxoid, although evoking good levels of circulating antitoxin, afforded only slight and transient protection (3). These findings raised many doubts about the sufficiency of antitoxic immunity and led to the notion, in some quarters, that the glutaraldehyde toxoid used in the field study was an inferior immunogen (7). Subsequently, new approaches to cholera vaccine development have been advanced. One of these involves combined-antigen formulas consisting of toxoid or the B subunit of CT together with conventional cholera vaccine or Vibrio cholerae lipopolysaccharide (6, 7, 12, 18); another involves the isolation of an avirulent mutant of V. cholerae which is a candidate live oral vaccine (8). In the studies reported here, we investigated the ability of glutaraldehyde-inactivated cholera toxin (cholera toxoid) alone and in combination with conventional cholera vaccine or an experimental E. coli vaccine, or both, to simultaneously protect rabbits against challenge with viable cholera vibrios and heat-labile enterotoxin (LT)-only E. coli. E. coli vaccine was included in the study as a result of previous observations which showed that ETEC, by virtue of membrane-bound LT, were capable of eliciting antitoxin specific for LT (14) and, at the same time, were also capable of acting as adjuvants for cholera toxoid (R. S. Rappaport, unpublished 534

2 VOL. 32, 1981 data). These findings, together with the fact that antitoxin elicited by cholera toxoid neutralizes both LT and CT (11, 13, 14), led us to compare the antigenicity and immunogenicity of cholera toxoid, cholera vaccine, and E. coli vaccine alone and in all possible combinations. (This paper was presented in part at the International Symposium on Bacterial Vaccines, Bethesda, Md., September 1980.) MATERIALS AND METHODS Antigens. Wyeth cholera toxoid (lot no ) was prepared as previously described (15, 16). Whole-cell antigens consisted of Wyeth cholera vaccine (lot no ) containing equal numbers of phenol (0.5%)- inactivated Inaba and Ogawa organisms (grown on Trypticase soy agar; BBL Microbiology Systems, Cockeysville, Md.) and three experimental E. coli vaccines. The E. coli vaccines were prepared from the following strains: E. coli KL320, a nontoxigenic auxotrophic mutant of E. coli K-12, obtained from Werner K. Maas, New York, N.Y.; E. coli KL320-1, identical to 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. coli H (08:H9), an LT-only strain isolated from an infant 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 used by Evans et al. (4), were inoculated with approximately 105 organisms per ml of each strain grown to mid-log phase in 2% peptone broth. The flasks were incubated at 37 C on a rotary shaker (150 rpm) for 16 h. The cultures, ranging in density from 3 x 109 to 1.2 x 10' colony-forming units per ml, were then centrifuged and 'washed once with M phosphatebuffered saline (PBS), ph 7.8. Washed organisms were then suspended at their original density in PBS containing 0.01% glutaraldehyde (Aldrich Chemical Co., Milwaukee, Wis.). In most instances, treatment with glutaraldehyde proceeded for 4 h at room temperature, followed by an 18- to 24-h incubation at 4 C. After treatment, the organisms were centrifuged from the inactivation medium, resuspended at the same concentration in sterile PBS, and then checked for viability. Once it was established that the cultures were completely nonviable, thimerosal (0.01%) was added as a preservative and the vaccine suspensions were stored at 4 C until used. In some instances, when the concentration 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 was repeated. Gram stains of the vaccines showed that the treatment did not alter their morphology, nor did it result in cellular aggregation. In addition, intramuscular inoculation of rabbits with 2 x 1010 organisms did not result in overt local or systemic distress. Immunization of animals. For antigenicity studies, groups of five to seven male New Zealand albino rabbits weighing between 2.3 and 5.0 kg were twice immunized intramuscularly in the right posterior thigh, with an interval of 6 weeks between inoculations. Cholera toxoid was administered at a dose of VACCINE AGAINST DIARRHEAL DISEASE ,g in 1 ml of PBS, ph 7.8, containing 0.01% thimerosal (PBS-T), and the whole-organism vaccines were administered at doses of 4 x 109 organisms (cholera vaccine); 5 x 109 organisms (E. coli H74-114); and 1 x 1010 organisms (E. coli strains KL320 and KL320-1), each contained in 1 ml of PBS-T. When antigens were combined, the concentration of individual components was adjusted so that the doses specified above were all contained in a final volume of 1 ml. Blood samples were obtained before and at 6 and 8 weeks after primary immunization, and the sera were stored at -20 C until assayed. For protection studies, groups of five to eight recently weaned rabbits weighing between 0.68 and 1.36 kg were used so that they would be a suitable size at the time of challenge. Groups of these animals were immunized with the antigens described above, each administered alone and in various combinations, using the same doses, route, and immunization schedule. Control animals were similarly immunized with 1 ml of PBS-T. Blood samples were obtained 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 intracutaneous method in rabbits as described earlier (14-16). One limit-of-bluing dose of CT or LT (of approximately 68,000 daltons; isolated by Sephadex G-150 chromatography from E. coli H culture filtrates [14]) per ml was the test dose in each assay, and each toxin dose was determined by titration against standard cholera antitoxin (Swiss Serum and Vaccine Institute serum containing 4,470 antitoxin units [AU]/ml). All titers are expressed in AU per milliliter. Measurement of vibriocidal antibody. Complement-dependent vibriocidal antibody titers, based on the ability of various sera to inhibit the growth of Inaba VC-13, were determined by a microtiter technique as described previously (16). National Institutes of Health reference convalescent antiserum was used as a standard. Titers are expressed as the last twofold dilution which completely inhibited bacterial growth. Protection studies. Protection was assessed on the basis of secretory responses elicited in immunized animals when they were challenged by a modification of the ligated ileal loop technique (1). During a 12-day period beginning on the 12th day after booster immunization, laparotomy was performed on each rabbit (ca. four per day), and approximately 21 5-cm loops were ligated and inoculated with five graded doses of live cholera vibrios (Ogawa 395), ranging from 103 to 108 organisms (in 1 ml), and four graded doses of live E. coli (strain H74-114), ranging from 107 to 1010 organisms (in 1 ml). On each day, challenge organisms were collected from brain heart infusion (Difco Laboratories, Detroit, Mich.) agar slants after two passages from lyophilized stocks. The cultures were suspended in Evans medium (4) and adjusted to 2.0 x 1010 (Ogawa 395) and 4.0 x 1010 (E. coli H'74-114) organisms/ml on the basis of optical density readings at 650 nm, using a Bausch & Lomb spectrophotometer. For precise quantitation of challenge inocula, colony-forming units were also determined by direct plating on brain heart infusion agar. Each dilution of vibrios or E. coli (in Evans medium) was tested in duplicate, each of which was randomly positioned in the upper and lower halves

3 536 RAPPAPORT AND BONDE of the small intestines. Negative control loops positioned proximally, in the middle, and at the distal ends of the test loops were inoculated with medium alone. Group 50% effective dose (ED50) values (the number of organisms required to elicit a mean secretory response of 1 ml/cm) were determined for Ogawa 395 and E. coli H Protection factors (estimates of the degree of protection) were calculated from the ratio of group ED50 values for antigen-immunized versus vehicle (PBS-T)-immunized animals. The secretory data were evaluated by analysis of variance, and then Duncan's multiple-range test (17) was applied. Data from animals succumbing as a result of challenge were excluded. RESULTS Comparison of the antitoxin response to cholera toxoid alone and in combination with a toxigenic or a nontoxigenic E. coli vaccine strain. Previous studies comparing the antigenicity of cholera toxoid in combination with either high-molecular-weight LT (thought to represent fragments of outer membrane) or E. coli endotoxin (055:B5) revealed that both LT and endotoxin acted as adjuvants for the toxoid (Rappaport, unpublished data). These findings led us to consider that suitably inactivated ETEC should, by virtue of membranebound LT and outer membrane lipopolysaccharide, be capable of both eliciting specific LT antitoxin and acting as an adjuvant for the toxoid. To test these possibilities, groups of rabbits were twice immunized intramuscularly with TABLE 1. INFECT. IMMUN. cholera toxoid, alone and in combination with a nontoxigenic E. coli strain (KL320) or a toxigenic strain of identical serotype (KL320-1), using a 6-week interval between inoculations. Two other groups of rabbits were similarly immunized with each whole-cell vaccine alone. The results of antitoxin determinations performed on pretreatment sera and sera obtained at 6 and 8 weeks showed that the toxigenic strain (KL320-1) was indeed capable of stimulating antitoxin. Like the antitoxin produced by high-molecularweight LT (14), it was specific for LT, showing only a slight ability to neutralize CT (Table 1). In contrast, the nontoxigenic strain (KL320) did not produce antitoxin to either LT or CT. Both strains, however, acted as adjuvants for toxoid, since mean antitoxin titers elicited by the toxoid were increased from three- to fivefold at 6 weeks and from four- to sixfold at 8 weeks when it was combined with either strain (Table 1). Mean LT antitoxin titers produced by the toxoid in combination with the toxigenic strain were greater than in combination with the nontoxigenic strain, possibly reflecting the contribution of both LT and CT antitoxin. Comparative antigenicity of cholera toxoid, cholera vaccine, and E. coli vaccine alone and in combination. Since current efforts to develop a vaccine against cholera point to a need to elicit both antibacterial and antitoxic immunity (6-8, 12, 18), it was of interest to investigate the ability of E. coli vaccine to act as Comparative antigenicity of cholera toxoid and toxigenic and nontoxigenic E. coli vaccines alone and 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 antitoxin titers, and the dose of each toxin was determined by titration against standard cholera antitoxin. Titers represent the geometric mean for each group of sera obtained before treatment and at 6 and 8 weeks. c Number of sera. d Range of values.

4 VOL. 32, 1981 an adjuvant for toxoid in the presence or absence of cholera vaccine. To examine the interaction of these antigens, groups of rabbits were twice immunized with each of the antigens alone and in 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 was used for both. In this study, however, the E. coli vaccine was prepared from strain H The results of antitoxin determinations performed on pretreatment sera and sera obtained at 6 and 8 weeks showed that E. coli vaccine acted as an adjuvant for toxoid both in the presence and absence of cholera vaccine (Table 2). As expected from the results of previous investigations (6, 7, 12), cholera vaccine did not act as an adjuvant for toxoid, since antitoxin titers (against both CT and LT) did not vary significantly with the addition or omission of the vaccine (Table 2). When toxoid was combined with E. coli vaccine, however, mean antitoxin titers were elevated from 7.5- to 12-fold in the case of LT antitoxin and from 13- to 20-fold in the case of CT antitoxin 8 weeks after primary immunization (Table 2). In addition, the study showed that strain H was at least as effective an adjuvant as were strains KL320 and KL320-1, TABLE 2. VACCINE AGAINST DIARRHEAL DISEASE 537 suggesting that many, if not all, E. coli strains of different serotype may exhibit this property to one extent or another. The results also confirmed that both LT/ST and LT-only ETEC evoke antitoxin specific for LT, since only LT antitoxin was elicited by strains KL320-1 and H (Tables 1 and 2). In contrast, cholera vaccine produced no antitoxin whatsoever. Comparative immunogenicity of cholera toxoid, cholera vaccine, and E. coli vaccine alone and in combination. Since E. coli vaccine acted as an adjuvant for toxoid in the presence or absence of cholera vaccine, it seemed likely that a combination of all three antigens might provide superior protection against challenge with both viable vibrios and LT-producing E. coli. To evaluate the relative immunogenicity of such a combination of antigens, groups of infant rabbits were twice immunized with each of these antigens alone and in various combinations, using conditions identical to those of the previous studies. Rabbits from each group were randomly challenged with vibrios and LT-only E. coli during a 12-day period beginning on the 12th day after booster immunization. ED5o values and protection factors, therefore, reflect the average level of protection observed over a 12- day period (approximately 7.5 to 9.5 weeks after Comparative antigenicity of cholera toxoid, cholera vaccine, and E. coli vaccine alone and in combination: serum antitoxin and vibriocidal responses in rabbits Cholera antitoxin (AU/ml)b E. coli LT antitoxin (AU/ml)b Vibriocidal antibody' Antigen' Pre- Pre- Pretreat- 6 wk 8 wk treat- 6 wk 8 wk treat- 8 wk ment 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) ( ) 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) ( ) (1, ,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) ( ,000) E. coli vaccine a 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. f ND, Not done.

5 538 RAPPAPORT AND BONDE primary immunization). In the case of vibrio challenge, each group of animals exhibited some degree of protection relative to controls (Table 3). Of these, the least protective antigen was E. coli vaccine, and the next least protective antigen was cholera vaccine. The former result was not surprising since antibodies directed against E. coli would not be expected to inactivate cholera vibrios, and the latter result indicated that vibriocidal antibody alone was not sufficient to provide superior protection against vibrio challenge under the conditions used. When the two whole-cell vaccines were combined, however, the protection factor rose to a level greater than or equal to the product of the individual vaccines, indicating a synergistic response. Even though mean vibriocidal titers did not show a significant statistical difference between groups, there was some indication that the enhanced protection might have been due to an adjuvant effect of E. coli vaccine on the immune response to cholera vaccine (Tables 2 and 3). Despite differences in protection exhibited by these three groups, statistical analysis of the secretory responses by Duncan's multiple-range test indicated considerable overlap between groups (Table 3). In the case of the remaining four groups of animals, all were statistically significantly different from controls and from the three groups immunized with whole-cell vaccines, alone or in INFECT. IMMUN. combination. Of these, the group immunized with a combination of cholera toxoid and E. coli vaccine exhibited the highest level of protection, a level not commonly evoked by the parenteral route. Furthermore, this group exhibited the highest antitoxin response as well (Table 3). Surprisingly, this combination of antigens was less protective when it was combined with cholera vaccine, and the decrease in protection was associated with a reduction in circulating antitoxin (Table 3). These results suggested that under certain conditions cholera vaccine may act as an immunosuppressant. The triple combination of antigens, nevertheless, still provided the second-highest level of protection observed among the various groups. In addition, there was very little difference in the level of protection exhibited by the group of animals immunized with toxoid alone versus the group immunized with toxoid and cholera vaccine, each of which showed comparable antitoxin responses (Table 3). In the latter comparison, the absence of a synergistic response due to a combination of antitoxic and antibacterial immunity was unexpected. Nevertheless, all groups immunized with toxoid alone or in combination with other antigens were significantly protected against live vibrio challenge, and there appeared to be a correlation between circulating antitoxin and protection. Statistically, the level of protection exhibited by the group immunized with the com- Downloaded from TABLE 3. Comparative immunogenicity of cholera toxoid, cholera vaccine, and E. coli vaccine alone and in combination: antibody responses and protection of immunized rabbits against intestinal challenge with V. cholerae (Ogawa 395) Immunogen' gropstatistical groupingb ED5o red5tiefac- titorn Cholera antitoxin' (AU/ml) Vibriocidal 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) E. coli vaccine A 3.0 x 104 (6) 4 <6 (6) (32-1,024) R <2 (6) Cholera vaccine + E. coli vaccine A 1.3 x 106 (5) 173 (<2-80) <4 (5) 588 (5) (256-2,048) Cholera toxoid B 1.3 x 10' (6) 1, (6) <2 (6) (249-2,207) Cholera toxoid + cholera vaccine B, C 1.2 x 107 (5) 1, (5) 338 (5) Cholera toxoid + E. coli vaccine C 1.2 x 108 (5) 16,000 (124-1,007) 3,310 (4) (128-1,024) <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. on May 11, 2018 by guest

6 VOL. 32, 1981 bination of toxoid and E. coli vaccine was significantly different from levels of all other groups, although there was some overlap with the group immunized with toxoid and cholera vaccine (Table 3). In the case of E. coli challenge, the dose range studied was comparatively limited due to the fact that nearly 109 organisms were required to produce a secretory response (in control animals) equivalent to that produced by about 104 cholera vibrios (Tables 3 and 4). Despite the difference in pathogenicity of the two challenge organisms, it was still possible to ascertain the relative abilities of the various antigens and antigen combinations to protect against E. coli challenge. With the exception of cholera vaccine, all of the antigens or antigen combinations conferred some degree of statistically significant protection against challenge with strain H (Table 4). Of these, the least protective antigen was E. coli vaccine. When the cholera and E. coli whole-cell vaccines were combined, however, the protection evoked by the combination was greater than or equal to the product of the individual vaccines, just as it was in the case of vibrio challenge. Of the four remaining groups, the group immunized with the combination of toxoid and E. coli vaccine exhibited the highest level of protection. Statistically, the secretory responses produced in rabbits immunized with this combination of antigens were significantly different from the responses of any other group (Table 4). The toxoid in combination with E. coli vaccine also elicited the highest antitoxin VACCINE AGAINST DIARRHEAL DISEASE 539 response, evoking titers eightfold greater than those produced by toxoid alone (Table 4). Again, as in the case of vibrio challenge, the protection afforded by these antigens was diminished when they were administered together with cholera vaccine, adding further support to the notion of a cholera vaccine-mediated immunosuppression. Similarly, the protection conferred by toxoid alone, the second most protective antigen, was also diminished when the toxoid was combined with cholera vaccine, but this difference was not statistically significant (Table 4). DISCUSSION Because of the obvious advantages of developing a single-vaccine formula which might provide protection against both cholera and LTmediated E. coli diarrheal disease, we investigated the comparative antigenicity and immunogenicity of cholera toxoid, conventional cholera vaccine, and E. coli vaccine, alone and in various combinations. The results show that glutaraldehyde toxoid alone is both antigenic and protective in the rabbit ligated loop model, especially for cholera (Ogawa 395), but also for diarrheal disease evoked by an LT-only E. coli strain (H74-114). Although further studies are required to establish the efficacy and safety of the toxoid in combination with other antigens, the present results also show that a vaccine formula which includes cholera toxoid and E. coli vaccine is capable of conferring remarkable protection against both cholera vibrio and homologous E. coli challenge in the rabbit model. TABLE 4. Comparative immunogenicity of cholera toxoid, cholera vaccine, and E. coli vaccine alone and in combination: antitoxin responses and protection of immunized rabbits against intestinal challenge with E. coli H Statistical 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) (6) (8-256) B Cholera vaccine + E. coli vaccine C 5.2 x 109 (5) (5) (<2-64) Cholera toxoid B, C 7.2 x 109 (6) (6) (205-2,321) Cholera toxoid + cholera vaccine B, C 3.3 x 109 (5) (5) ( ) Cholera toxoid + E. coli vaccine D 1.0 x 10" (5)h ,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 all challenge doses. The ED50 value is therefore an extrapolation of the dose-response curve.

7 540 RAPPAPORT AND BONDE Of all the antigen formulas tested, those including toxoid and E. coli vaccine generally provided greater protection than those which did not, and it is noteworthy that the combination of cholera vaccine and E. coli vaccine also provided greater protection than did either whole-cell vaccine alone. This observation suggests that E. coli may be an effective adjuvant not only for toxoid, but for cholera vaccine as well. Furthermore, those antigen formulas which provided the greatest protection also produced the highest antitoxin responses, and those formulas which provided the least protection produced little or no antitoxin. In the case of the combined toxoid- E. coli vaccine formula, mean CT and LT antitoxin titers were at least 4-fold and as high as 20-fold greater than CT and LT antitoxin titers elicited by toxoid alone approximately 8 weeks after primary immunization (Tables 1 through 4). The elevation in antitoxin titer appears to be due to an adjuvant effect of E. coli on the immune response to toxoid, and such an effect can be evoked by both toxigenic and nontoxigenic strains (Table 1). Since E. coli endotoxin (055:B5) also acted as an adjuvant for toxoid (Rappaport, unpublished data), the adjuvant action of the E. coli vaccines is presumed to be due primarily to outer membrane lipopolysaccharide, but other surface antigens may be important as well. In the case of diarrheal disease mediated wholly or in part by LT, the contribution of specific LT antitoxin by an enterotoxigenic vaccine strain may be an advantage. LT antitoxin titers, for example, were generally greater than CT antitoxin titers, and this may be 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 vaccine formula with the same antigens combined with cholera vaccine. In the case of both cholera vibrio and E. coli challenge, inclusion of cholera vaccine resulted in a reduction in both protection and circulating antitoxin (Tables 3 and 4). A similar reduction in antitoxin titer did not occur after immunization of adult rabbits with the same antigen formula (Table 2), suggesting that the age of the animals may be a relevant factor. Under the conditions used in the protection studies, however, cholera vaccine appears to have acted as an immunosuppressant. It is noteworthy that subcutaneously administered cholera vaccine also suppressed the immune response to perorally administered live polio vaccine (A. M. Svennerholm, L. A, Hanson, J. Holmgren, B. S. Lindblad, S. R. Khan, A. Nilsson, and B. Svennerholm, J. Infect. Dis., in press). INFECT. IMMUN. The substantial antitoxin response produced by cholera toxoid in combination with E. coli vaccine appears to be a major factor in eliciting protection against both cholera and E. coli. Other factors, such as antibodies directed against pili, adhesion, or colonization factors, may be important as well. But, contrary to current views of the importance of both antitoxic and antibacterial immunity in cholera (6-8, 12, 18) or of the importance of antibacterial immunity alone (9), the present results do not support a major role for vibriocidal antibody, at least in the rabbit model of diarrheal disease. The observation that toxoid plus E. coli vaccine (which elicited no vibriocidal antibody) protected rabbits against vibrio challenge to at least the same extent as the same antigens plus cholera vaccine (or toxoid plus cholera vaccine) suggests antitoxic immunity as the predominant factor in long-term cholera immunity, just as it is in diphtheria and tetanus. Our failure to demonstrate synergistic protection due to both antitoxic and antibacterial (vibriocidal) immunity conflicts with the findings of Holmgren et al. (7) and Peterson (12). The absence of such synergism may be due to differences in the potencies of the cholera vaccines or may stem from the choice of immunization parameters used in the different studies. Holmgren et al. used a 2-week interval between immunizations and challenged 5 and 21 days later, whereas Peterson used a 4-week interval and challenged 2 weeks later. In the study of Holmgren et al., there was a rapid decline in antibacterial immunity between days 5 and 21 after booster immunization (7), indicating that the vibriocidal component of immunity was shortlived. In the present study, a 6-week interval was used, and any synergism between antitoxic and antibacterial immunity in the days after booster immunization was not apparent at challenge, 12 to 23 days later. A previous study has, in fact, shown that the ability of toxoid to elicit maximal levels of antitoxin and the ability of V. cholerae somatic antigen to elicit vibriocidal antibody are both influenced by the choice of immunization schedule, the 6-week interval favoring the former and disfavoring the latter (16). The comparatively poor performance of glutaraldehyde toxoid versus B (or L) subunit in the study of Holmgren et al., therefore, may not necessarily be due to an inherent immunogenic inferiority of the toxoid antigen, but rather to the choice of immunization schedule and to the possibility of the two antigens not being equally inert. (Batches of B [or L] subunit were reported to have less than 0.1% the toxicity of intact toxin [7], whereas glutaraldehyde toxoids reproducibly exhibited less than 0.003% the toxicity of

8 VOL. 32, 1981 parent toxin [15]). The present studies support the sufficiency of antitoxic immunity in protection, but do not establish the duration of either; nor do they address the question of the type and distribution of antibodies elicited by the toxoid-e. coli vaccine formula. Whether the high levels of antitoxin evoked by the combined antigens will persist in sufficient quantity to penetrate the lumen of the gut and whether local antitoxin can be evoked by this combination of antigens remain to be determined. Since the diseases in question are confined to the intestinal tract, it would clearly be desirable to determine the efficacy of the present formulation when it is administered by the oral route or a combination of parenteral and oral routes. The antigenicity of orally administered toxoid alone has already been established in humans (10), but thus far protection has not been demonstrated under the conditions used (9). If the combined vaccine were to confer significant protection when administered perorally, then it would obviate many of the problems associated with the development of parenteral fornulations. The present usage of gram-negative bacteria in parenteral vaccines, however, provides a precedent for considering the potential inclusion of a suitably detoxified E. coli vaccine (or component thereof) in a parenteral toxoid fornulation. Moreover, the present results do not preclude the incorporation of a variety of enteropathogenic (E. coli) serotypes (including invasive, nontoxigenic E. coli) and an appropriate heat-stable enterotoxin antigen in the vaccine. ACKNOWLEDGMENTS This study was supported in part by contract DAMD C-4007 from the U.S. Army Medical Research and Development Command. LITERATURE CITED VACCINE AGAINST DIARRHEAL DISEASE Burrows, W., and G. M. Musteikis Cholera infection and toxin in the rabbit ileal loop. J. Infect. Dis. 116: Craig, J. P A survey of the enterotoxic enteropathies, p In 0. Ouchterlony and J. Holmgren (ed.), Cholera and related diarrheas, 43rd Nobel Symposium. S. Karger, Basel 3. Curlin, G., R. Levine, K. M. A. Aziz, A. S. M. Rahman, and W. F. Verwey Field trial of cholera toxoid, p In Proceedings of the 11th Joint Conference of the U.S.-Japan Cooperative Medical Science Program Symposium on Cholera. Department of Health, Education, and Welfare, Washington, D.C. 4. Evans, D. J., D. G. Evans, and S. L Gorbach Production of vascular permeability factor by enterotoxigenic Escherichia coli isolated from man. Infect. Immun. 8: Feeley, J. C., and E. J. Gangarosa Field trials of cholera vaccine, p In 0. Ouchterlony and J. Holmgren (ed.), Cholera and related diarrheas, 43rd Nobel Symposium. S. Karger, Basel. 6. Germanier, R., E. Furer, S. Varallyay, and T. M. Inderbitzin Antigenicity of cholera toxoid in humans. J. Infect. Dis. 135: Holmgren, J., A. M. Svennerholm, L. Lonnroth, M. Fall-Persson, B. Markman, and H. Lundbeck Development of improved cholera vaccine based on subunit toxoid. Nature (London) 269: Honda, T., and R. A. Finkelstein Selection and characteristics of a Vibrio chokerae mutant lacking the A (ADP-ribosylating) portion of the cholera enterotoxin. Proc. Natl. Acad. Sci. U.S.A. 76: Levine, M. M Immunity to cholera as evaluated in volunteers, p In 0. Ouchterlony and J. Holmgren (ed.), Cholera and related diarrheas, 43rd Nobel Symposium. 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 Antigenicity of purified glutaraldehyde-treated toxoid administered orally. Infect. Immun. 21: Nalin, D. R., A. Al-Mahmud, G. Curlin, A. Ahmen, and J. Peterson Cholera toxoid boosts serum Escherichia coli antitoxin in humans. Infect. Immun. 10: Peterson, J. W Synergistic protection against experimental cholera by immunization with cholera toxoid and vaccine. Infect. Immun. 26: Pierce, N. F Protection against challenge with Escherichia coli heat-labile enterotoxin by immunization of rats with cholera toxin/toxoid. Infect. Immun. 18: Rappaport, R. S Observations on the mechanism of release of heat-labile E. coli enterotoxin, p In Proceedings of the 13th Joint Conference of the U.S.- Japan Cooperative Medical Science Program Symposium 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 Development of a purified cholera toxoid. II. Preparation of a stable, antigenic toxoid by reaction of purified toxin with glutaraldehyde. Infect. Immun. 9: Rappaport, R. S., W. A. Pierzchala, G. Bonde, T. McCann, and B. A. Rubin Development of a purified cholera toxoid. III. Refinements in purification of toxin and methods for the determination of residual somatic antigen. Infect. Immun. 14: Steele, R. G. D., and J. H. Tome Principles and procedures of statistics. McGraw-Hill Book Co., New York. 18. 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