Vol. 26, No. 2
INFECTION AND IMMUNITY, Nov. 1979, p. 528-533
0019-9567/79/11-0528/06$02.00/0
Synergistic Protection Against Experimental Cholera by Immunization with Cholera Toxoid and Vaccine JOHNNY W. PETERSON Department of Microbiology, University of Texas Medical Branch, Galveston, Texas 77550 Received for publication 16 August 1979
Rabbits were immunized with two parenteral injections of Wellcome toxoid PX389A, Wyeth toxoid 20101, or Merck bivalent vaccine. Other groups of rabbits were immunized with combinations of the Merck vaccine and each of the two toxoids. Antitoxin responses were monitored in each group of rabbits before livecell challenge of each animal by the ligated intestinal loop assay. Inaba and Ogawa strains of Vibrio cholerae were used for challenge experiments. Basically, the data indicate that the toxoids were equivalent in antigenic potency and antitoxin responses were unaffected by combination of the toxoids with the wholecell vaccine. The 50-,ug doses of each toxoid as well as the 4 x 109 cells of the bivalent vaccine provided the same magnitude of protection against live-cell challenge with either Inaba or Ogawa vibrios. Immunization with either toxoid in combination with the bivalent vaccine resulted in a synergistic protective response against live-cell challenge of intestinal loops with V. cholerae. Synergistic protection was observed when toxoid and vaccine were administered together by the oral and parenteral routes. Maximum protection was obtained when rabbits were immunized with the combined toxoid-whole-cell vaccine administered by both oral and parenteral routes. The objective of this study was to compare the antigenicity of two purified cholera toxoids prepared by different methods and to determine whether parenteral or oral immunization or both with cholera toxoid in combination with wholecell cholera vaccine would provide superior protection against experimental cholera than either agent alone. Germanier et al. (8) reported that use of such a toxoid-vaccine combination did not significantly increase antibody titers to either component above that obtained with the separate components. Similarly, Svennerholm and Holmgren (19) described a synergistic protective effect in rabbits immunized with Vibrio cholerae lipopolysaccharide and cholera toxin. A lesser synergistic effect was observed between lipopolysaccharide and choleragenoid. It was postulated that the synergism resulted from interference by antibody in two separate mechanisms in the pathogenesis of cholera. For V. cholerae to cause loss of fluid and electrolytes from the small intestinal epithelium, the bacteria must migrate through a layer of mucus covering the intestinal villi and become associated with the epithelium in the crypt areas (3). Once attached to the epithelial cells (7, 14), small amounts of cholera toxin secreted from the bacteria can effectively bind to cells of the microvilli via the GM1 ganglioside receptor (10, 16). The subsequent effects of the toxin on ade-
nylate cyclase have been intensely studied (2, 9). Interference by antibody in the sequence of events in the pathogenesis of cholera could occur in two stages. Antibodies to the somatic antigen appear to prevent the migration of V. cholerae cells through the intestinal mucous layer, thereby blocking epithelial surface colonization. The second stage of defense depends upon the capacity of antitoxin to neutralize cholera toxin secreted by those V. cholerae cells surpassing the first stage of defense and colonizing the intestinal epithelium. The current study indicates that synergistic protection against experimental cholera can be achieved by immunization with chemically treated toxoids in combination with whole-cell vaccine. Markedly superior protection was obtained when rabbits were immunized with toxoid-whole-cell vaccine combinations administered by both the oral and parenteral routes. MATERIALS AND METHODS Antigens. Toxoids PX389A and PX389B3 were experimental cholera toxoids provided by Wellcome Research Laboratories, Beckenham, England, and were prepared by immunoaffinity chromatography of
528
Formalin-treated culture filtrates of V. cholerae. Toxoids PX389A and PX389B3 were equivalent, being two preparations made to the same formulation with similar constituents. Toxoid 20101 was prepared by Rappaport et al. (17) at Wyeth Laboratories and consisted
VOL. 26, 1979
,e1
IMMUNITY TO EXPERIMENTAL CHOLERA
of glutaraldehyde treatment of purified cholera toxin. The Wyeth toxoid contained aluminum phosphate and protamine as adjuvants. Wellcome vaccine PX389B8 was a bivalent whole-cell vaccine containing aluminum hydroxide (0.4%, wt/vol) obtained from Carl E. Miller at the National Institutes of Health in Bethesda, Md. Merck bivalent vaccine was prepared by mixing equal parts of Merck monovalent (Ogawa or Inaba) freeze-dried vaccines, which were also acquired from Carl E. Miller. Immunization of animals. Sixteen adult New Zealand rabbits weighing 4 to 5 pounds (ca. 1,814 to 2,2680 g) each were immunized by subcutaneous injection of each antigen in the nape of the neck. A second subcutaneous injection was given to each rabbit after 4 weeks. The dose of each toxoid consisted of 50 fig of protein contained in 0.5 ml of saline, whereas the dose of whole-cell vaccine was a 0.5-ml suspension of 4 x 109 V. cholerae cells. The first and second injection of some rabbits consisted of a 1-ml dose containing both 50 ,g of toxoid and 4 x 109 cells. The portion of this study concerned with route of immunization involved immunization ofrabbits with two 40-,ug doses of toxoid, or 4 x 108 cells or both administered by the parenteral route, whereas oral immunization was accomplished by carefully feeding 400-pg doses of toxoid or 4 x 109 cells or both through a syringe and catheter tube inserted in the rabbit's mouth. Blood samples were taken every 2 weeks, and serum was stored at -20'C before assay. Serological titrations. Cholera antitoxin titrations were assayed by the passive hemagglutination test (6). All titers were expressed as antitoxin units per milliliter based on the Swiss Serum and Vaccine Institute reference serum. Intestinal loop challenge. The basic challenge assay described by De (5) was performed 6 weeks after primary immunization as described below. Lyophilized cultures of V. cholerae (El Tor) Inaba strain V-86 or V. cholerae (Classical) Ogawa strain 395 were suspended in 1.0 ml of heart infusion broth (Difco) and streaked out on a heart infusion agar plate for incubation at 370C. Four or five isolated colonies were streaked for confluent growth on heart infusion plates on the morning of the challenge experiments and incubated at 370C for 4 h. The organisms were collected by washing the plate with 5.0 ml of sterile phosphate-buffered saline with 0.1% gelatin (PBSG), pH 7.4. This was adjusted to read 170 Klett units (green filter, Klett-Summerson photometer) corresponding to a viable cell count of about 1.3 x 109 cells per ml. This stock cell suspension was diluted by 10fold dilutions to a final dilution of 1.3 x 103 cells per ml, and dilutions were kept in an ice bath during the animal surgeries. Animals were given water but were not allowed food for 48 h before surgery. Immediately before surgery, the rabbits were anesthetized by a 3.0ml injection of Ketaset (Bristol-Myers), and their abdomens were shaved. After cleaning the abdomen with a 70% solution of ethanol, an abdominal incision was made approximately 7 cm in length. Beginning about 6 to 10 cm away from the appendix, a series of eight intestinal loops, about 10 cm apart, was made with surgical silk (Ethicon no. 1). Interspaces 2 cm in length were tied between each loop through which the 1-ml
529
injections of live vibros were made. The sequence of injections was reversed in alternate animals, and immunized and control rabbits were randomly challenged. The proximal and distal loops always received 1.0 ml of PBSG as a control. The rabbits were sacrificed 18 h after surgery by injection of 10 ml of air into the marginal ear vein. The loop responses were measured by recording the ratio of fluid accumulation (milliliters) to the length of the loop (centimeters).
RESULTS Antitoxin titrations. Geometric mean passive hemagglutination antitoxin titers for the first set of immunized animals are presented in Fig. 1. It is evident that the addition of Merck whole-cell vaccine to Wellcome toxoid PX389A and Wyeth toxoid 20101 had no effect on the antigenicity of either toxoid, since equivalent antitoxin titers were observed. The potency of the two toxoids is also very similar, giving rise to virtually identical antitoxin response curves. No antitoxin titers were observed in the group ofrabbits immunized with Merck whole-cell vaccine.
-7
2
6
Thme in weeks FIG. 1. Cholera antitoxin dose-response curves as determined by passive hemagglutination. Rabbits received two subcutaneous injections of the respective cholera toxoid with or without combination with cholera whole-cell vaccine. Symbols: 0-----0, Wyeth toxoid 20101 and Merck bivalent vaccine; 0--, Wyeth toxoid 20101; 0-v-., Wellcome toxoid PX389A; *..0.., Wellcome toxoid PX 389A and Merck bi, Merck bivalent vaccine. valent vaccine; *
530
PETERSON
INFECT. IMMUN.
Intestinal loop challenge. Since nonimmunized control rabbits were purchased at the same time as the experimental rabbits, housed under the same conditions, and integrated with the immunized animals at the time of challenge, direct comparisons can be made between each group. Examination of the slope and height of the response curves of the Inaba- and Ogawachallenged control rabbits, shown in Fig. 2 and 4, reveals that the Ogawa strain elicited more fluid accumulation than the Inaba strain, particularly at the higher challenge doses. Generally, it can be stated that Wellcome toxoid PX389A conferred protection against Ogawa and Inaba challenge comparable to that observed with Wyeth toxoid 20101. This conclusion is supported by the relative location of the two curves of toxoid-immunized animals in Fig. 2 and 3 (Ogawa challenge) as well as in Fig. 4 and 5 (Inaba challenge). The relative degree of protection conferred by either toxoid is depicted by the downward shift in the dose-response curves. Merck bivalent vaccine alone provided approximately the same degree of protection against both Inaba and Ogawa challenge strains. Rabbits immunized with a combination of Merck bivalent vaccine and either cholera toxoid resulted in the maximum amount of protection as depicted by the lowest curves in Fig. 2 through 5. The degree of protection observed as a result
I E -a
a 9 Bo QC
LOOP CHALLENGE DOSE FIG. 3. Fluid accumulation responses of rabbits challenged with V. cholerae Ogawa 395. Rabbits were immunized with Wyeth toxoid 20101 or Merck bivalent vaccine or both. Symbols: 0-----0, nonimmunized control; *-4, Wyeth toxoid 20101; 0.....0, Merck bivalent vaccine; 0-0, Wyeth toxoid 20101.
K
E S
/
-
/
/
O/
/
'I
,
,' I
.
,
t-
/
E
= 2
-I
//5
iiz /VF/
LWOP CHALLENGE DOSE FIG. 2. Fluid accumulation responses of rabbits challenged with V. cholerae Ogawa 395. Rabbits were immunized with Wellcome toxoid PX389A or Merck bivalent vaccine or both. Symbols: 0-----0, nonimmunized control; 0---, Wellcome toxoid PX389A; *o..0, Merck bivalent vaccine; 0-.Wellcome toxoid PX389A and Merck bivalent vaccine. ,
LOOP CHALLENGE BOSE FIG. 4. Fluid accumulation responses of rabbits challenged with V. cholerae Inaba V86. Rabbits were immunized with Wellcome toxoid PX 389A or Merck bivalent vaccine or both. Symbols as in Fig. 2.
of immunization with each of these preparations can best be analyzed by examination of protection factors which are the ratios of the 50% effective dose of the immunized groups to the nonimmunized controls. A 50% effective dose is that dose of bacteria that causes the accumulation of 1 ml of fluid per cm. The data in Table 1 indicate that each immunogen provided pro-
VOL. 26, 1979
IMMUNITY TO EXPERIMENTAL CHOLERA
tection against fluid loss that was greater against
with V. cholerae Inaba than with the Ogawa serotype isolate. Both the Wyeth and Wellcome toxoids provided protection that was approximately of the same magnitude. The most remarkable feature noted was the synergistic increase in protection observed after immunization with the combination of cholera whole-cell vaccine and each of the toxoids. The increase in protection was in excess of 100 times that achieved by immunization with either toxoid or vaccine alone. Effect of immunization route. Table 2 summarizes an experiment to determine whether route of immunization would affect the relative protective response of cholera toxoid, or vaccine, or both. The data indicate that oral inmunization with toxoid or vaccine provided the poorest protective responses (approximately twofold) to challenge
531
live-cell challenge with V. cholerae Inaba V86. Parenteral immunization with toxoid increased protection 10-fold, whereas parenteral administration of the whole-cell vaccine resulted in a 32fold increase in protection. When rabbits were immunized by both routes, the toxoid group exhibited only 10-fold protection, whereas the comparable group of animals receiving oral and parenteral doses of whole-cell vaccine exhibited 56-fold protection. When a combination of toxoid and whole-cell vaccine was administered orally, 16.4-fold protection was observed. This level of protection is synergistic, since it is at least four times the predicted amount of protecTABLE 2. Analysis of the relative degree of protection conferred to rabbits immunized with Wellcome toxoid or vaccine or both by the oral, parenteral, and combination routes Inaba V86 challenge
Group /
Wellcome toxoid PX389B3 Nonimmunized control Oral Parenteral Oral and parenteral
12
e~
~ ~
Wellcome vaccine PX389B8 Nonimmunized control Oral Parenteral Oral and parenteral
1205 107 100 CNALLENGE OM FIG. 5. Fluid accumulation responses of rabbits challenged with V. cholerae Inaba V86. Rabbits were immunized with Wyeth toxoid 20101 or Merck bivalent vaccine or both. Symbols as in Fig. 3.
Combination Wellcome toxoid PX389B3-Wellcome whole-cell vaccine PX389B8 Norummunized control Oral Parenteral Oral and parenteral a ED50, 50% effective dose.
EDs@
Protection factor
1x 1.8 x 1x 1x
104 104 105 105
1 1.8 10 10
1x 2.5 x 3.2 x 5.6 x
105 105 106 106
1 2.5 32 56
2.2 x 3.6 x 3.6 x 4.4 x
104 105 107
1 16.4 1,636 10" 20,000
TABLE 1. Analysis of the relative degree of protection conferred to rabbits imnmunized parenterally with Wellcome and Wyeth toxoids with and without combination with Merck whole-cell vaccine Ogawa challenge Protection factor 1 12.5 25 25
Group GrupEDEN 2 x 104 Control (nonimmu:ized) 2.5 x 105 Wyeth toxoid 20101 Wellcome toxoid PX389A 5 x 105 Merck bivalent vaccine 5 x 105 Combination (Wellcome toxoid PX389-Merck biva1.4 x 108 lent vaccine) Combination (Wyeth toxoid 20101-Merck bivalent 5 x 107 vaccine) a ED50, 50% effective dose.
Inaba challenge Protection EDso factor 7 x 103 1 1.4 x 106 200 6 x 105 85
7,000
1.3 x 106 2.5 x 107
186 3,571
2,500
2 x 108
28,571
532
PETERSON
tion as judged by the product of the protection factors obtained with toxoid or vaccine alone. The amount of synergistic protection observed with the toxoid-vaccine combination administered parenterally was 1,636-fold, although it had been two- to fourfold greater in the previous experiments. Finally, when the toxoid-vaccine combination was administered by both oral and parenteral routes, 20,000-fold protection was achieved. This level of protection was only slightly less than the expected product of the protection obtained by administration of the toxoid-vaccine combination via the oral or parenteral routes.
INFECT. IMMUN.
not propose to know the solution to the problem, it would be interesting to know whether toxoidvaccine combinations administred in adequate dosage via the oral and parenteral routes would provide humans with protection against cholera. The rabbit intestinal loop model employed in this study may or may not predict the degree of protection that could be expected in humans. It is distinctly different from the actual disease situation because by design it is a closed loop system. One could argue that the closed loop, after injection of large numbers of V. cholerae, is a more rigorous challenge situation than would be expected to occur in the intact small intestines of hns. In the loop, the bacteria can multiply and colonize without the disadvantage of the normal clearance action of the open intestinal tract. On the other hand, the model is subject to criticism because surgical manipulations and ligations may cause leakage of small amounts of blood plasma into the loops, providing protection that otherwise would not have occurred. In defense of the model, should leakage of antibody containing plasma into loops be a major problem, we would not have expected to obtain such uniform dose-response curves. The amount of leakage probably would vary from loop to loop and yield erratic results which have not been observed. Furthermore, antisomatic immunity has been proven effective in huns~nR in numerous field trials (1, 12, 13), and protection with whole-cell vaccines is easily achieved in the rabbit loop model as shown here. Regardless of possible limitations, the data presented here provide a basis for further research to improve immunity against cholera.
DISCUSSION The data presented in this report confirm the initial observation of Svennerholm and Holmgren (19) that it is possible to exploit antisomatic and antitoxic immunity by combined immunization to obtain superior protection against experimental cholera. This synergistic protective response is not due to elevated levels of either antibody population, but rather to the increased efficiency of dual mechanisms of antibody action. Antisomatic antibody is effective in reducing the number of bacteria colonizing the mucosal epithelium in the crypt regions (18), whereas antitoxin neutralizes toxin elaborated by those bacterial cells overwhelming the antisomatic antibody blockade. However, both stages of immunity may be overcome as the challenge dose increases. These observations with combinations of experimental toxoids and vaccines, some of which may be used in future field trials, provided a basis for optimism regarding their potential efACKNOWLEDGMENI fectiveness in conferring protection against cholera. The superior protection observed when the I express my appreciation to Howard R. Williams, Jr., Briney for technical assistance. toxoid-vaccine combinations were administered Ronald Roberts, and Carolyn from the U.S. Cholera Panel in the design of these by the parenteral and oral routes simultaneously Advice is gratefully acknowledged. suggests strongly that to maximize immunity, experiments This study was supported by Public Health Service conboth oral and parenteral routes of immunization tract NOl AI02101 from the National Institute of Allergy and Infectious Diseases. should be exploited. At this time, it is somewhat less than popular LITERATURE CITED to propose the extensive use of antitoxic immuJ. nity against cholera. The reason for this air of 1. Azurin, C., A. Cruz, T. P. Pesigan, M. Alvero, T. Camera, R. Suplide, L Ledesma, and C. Z. Gomez. pesiumism is due to the rather poor performance 1967. A controlled field trial of the effectiveness of tested of a glutaraldehyde toxoid (Wyeth 20101) cholera and cholera El Tor vaccines in the Philippines. in Bangladesh in 1975 (3, 4). In that trial, two Bull. W.H.O. 37:703-71. of toxoid were administered with 2. Bennett, V., and P. Cuatrecasa. 1975. Mechanism of 100-ILg doses Another action of Vibrio cholerae enterotoxin. J. Membr. Biol. trial of a Formalin toxoid jet injectors. 22:1-28. in the Philippines was also discouraging (15). 3. Curlin, G. T., R. Levine, K. K A. IAz, A. S. M. Interestingly, the same dose of toxoid was emRahmain and W. F. Verwey. 1975. Field trial of cholera toxoid, p. 314-335. In Proceedings of the Elevployed. Challenge studies with glutaraldehyde toxoid in American volunteers administered higher doses of toxoid via the oral or parenteral routes have not revealed any better protection from antitoxic immunity (11). Although we do
enth Joint Conference of the U.S.-Japan Cooperative Medical Science Program. Department of Health, Education, and Welfare, Washington, D.C. 4. Curlin, G. T., R. L. Levine, K. M. A. AziA, A. S. M. Mizanur Rahman, and W. F. Verwey. 1976. Serolog-
VOL. 26, 1979
5.
6.
7. 8.
9. 10.
11.
12.
IMMUNITY TO EXPERIMENTAL CHOLERA
ical aspects of a cholera toxoid field trial in Bangadesh p. 276-285. In H. Fukumi and Y. Zinnaka (ed.), Symposium on cholera. National Institute of Health, Tokyo. De, S. N., and D. N. Chatterje. 1963. An experimental study of the mechanism of action of Vibrio cholerae on thle intestinal mucous membrane. J. Pathol. Bacteriol. 46:569-62. Finkelstein, R. A, and J. W. Peterson 1970. In vitro detection of antibody to cholera enterotoxin in cholera patients and laboratory animals. Infect. Immun. 1:2129. Freter, R., H. L Smith, and F. J. Sweeney, Jr. 1961. An evaluation of intestinal fluids in the pathogenesis of cholera. J. Infect. Dis. 109:35-42. Germanier, R., E. Furer, S. Varallyay, and T. M. Inderbitzin. 1977. Antigenicity of cholera toxoid in humans. J. Infect. Dis. 135:512-516. Johnson, G. 8S, and V. R. Mukku. 1979. Evidence in intact cells for an involvement of GTP in the activation of adenylate cyclase. J. Biol. Chem. 264:96-100. King, C. A., and W. E. van Heynlngen. 1973. Inactivation of cholera toxin by sialidase resistant monosialoganglioside. J. Infect. Dis. 127:639-647. Levine, M. M., J. P. Craig, N. F. Pierce, D. H. Waterman, E. S. Caplan, and J. P. Libonati. 1976. The immune response to parenteral and oral cholera toxoid in volunteers, p. 338-340. In H. Fukumi and Y. Zinnaka (ed.), Symposium on cholera. National Institute of Health, Tokyo. Mosley, W. H., W. ML McCormack, ML Fahimuddin, K. M A. Aziz A. S. aman, A. K. L Chrowdhury, A. R. Martin, J. C. Feeley, and R. A. Phillips. 1969. Report of the 1966-67 cholera field trials in rural
13.
14.
15.
16.
17.
18.
19.
533
East Pakistan.L. Study design and results of the first year of observation. Bull. W.H.O. 40:117-206. Mosley, W. H, W. E. Woodward, K. M. A. Azr, A. S. M. Rahman, A. K. AL Chrowdhury, A. Ahmed, and J. C. Feeley. 1970. The 1968-69 cholera vaccine field trial in rural East Pakistan. Effectiveness ofmonovalent Ogawa and Inaba vaccine and a purified Inaba antigen, with comparative results of serological and animal protection tests. J. Infect. Dis. 3(Suppl):S1-S19. Nelson, E. T., J. D. Clements, and R. A. Finkelstein. 1976. Vibrio chokrae adherence and colonization in experimental cholera: electron microscopic studies. Infect. Immun. 14:527-647. Noriki, H. 1976. Evaluation of toxoid field trial in the Philippines, p. 302-310. In H. Fukumi and Y. Zinnaka (ed.), Symposium on cholera. National Institute of Health, Tokyo. Peterson, J. W., J. J. LoSpalluto, and R. A. Finkelstein. 1972. Icalization of cholera toxin in vivo. J. Infect. Dis. 126:617-621. Rappaport, R. S., W. A. Pierzchala, G. Bonde, T. McCann, and B. A. Rubin. 1976. Development of a purified cholera toxoid. III. Refinement in purification of toxin and methods for the determination of residual somatic antigen. Infect. Immune. 14:687-693. Sbrank, G. D, and W. F. Verwey. 1976. Distribution of cholera organisms in experimental Vibrio cholerae infections: proposed mechanisms of pathogenesis and antibacterial immunity. Infect. Immune. 13:195-203. Svennerholm, A.-M., and J. Holmgren. 1976. Synergistic protective effect in rabbits of immunization with Vibrio cholerae lipopolysaccharide and toxin/toxoid. Infect. Immun. 13:735-740.
INFECTION AND IMMUNITY, Nov. 1979, p. 528-533
0019-9567/79/11-0528/06$02.00/0
Synergistic Protection Against Experimental Cholera by Immunization with Cholera Toxoid and Vaccine JOHNNY W. PETERSON Department of Microbiology, University of Texas Medical Branch, Galveston, Texas 77550 Received for publication 16 August 1979
Rabbits were immunized with two parenteral injections of Wellcome toxoid PX389A, Wyeth toxoid 20101, or Merck bivalent vaccine. Other groups of rabbits were immunized with combinations of the Merck vaccine and each of the two toxoids. Antitoxin responses were monitored in each group of rabbits before livecell challenge of each animal by the ligated intestinal loop assay. Inaba and Ogawa strains of Vibrio cholerae were used for challenge experiments. Basically, the data indicate that the toxoids were equivalent in antigenic potency and antitoxin responses were unaffected by combination of the toxoids with the wholecell vaccine. The 50-,ug doses of each toxoid as well as the 4 x 109 cells of the bivalent vaccine provided the same magnitude of protection against live-cell challenge with either Inaba or Ogawa vibrios. Immunization with either toxoid in combination with the bivalent vaccine resulted in a synergistic protective response against live-cell challenge of intestinal loops with V. cholerae. Synergistic protection was observed when toxoid and vaccine were administered together by the oral and parenteral routes. Maximum protection was obtained when rabbits were immunized with the combined toxoid-whole-cell vaccine administered by both oral and parenteral routes. The objective of this study was to compare the antigenicity of two purified cholera toxoids prepared by different methods and to determine whether parenteral or oral immunization or both with cholera toxoid in combination with wholecell cholera vaccine would provide superior protection against experimental cholera than either agent alone. Germanier et al. (8) reported that use of such a toxoid-vaccine combination did not significantly increase antibody titers to either component above that obtained with the separate components. Similarly, Svennerholm and Holmgren (19) described a synergistic protective effect in rabbits immunized with Vibrio cholerae lipopolysaccharide and cholera toxin. A lesser synergistic effect was observed between lipopolysaccharide and choleragenoid. It was postulated that the synergism resulted from interference by antibody in two separate mechanisms in the pathogenesis of cholera. For V. cholerae to cause loss of fluid and electrolytes from the small intestinal epithelium, the bacteria must migrate through a layer of mucus covering the intestinal villi and become associated with the epithelium in the crypt areas (3). Once attached to the epithelial cells (7, 14), small amounts of cholera toxin secreted from the bacteria can effectively bind to cells of the microvilli via the GM1 ganglioside receptor (10, 16). The subsequent effects of the toxin on ade-
nylate cyclase have been intensely studied (2, 9). Interference by antibody in the sequence of events in the pathogenesis of cholera could occur in two stages. Antibodies to the somatic antigen appear to prevent the migration of V. cholerae cells through the intestinal mucous layer, thereby blocking epithelial surface colonization. The second stage of defense depends upon the capacity of antitoxin to neutralize cholera toxin secreted by those V. cholerae cells surpassing the first stage of defense and colonizing the intestinal epithelium. The current study indicates that synergistic protection against experimental cholera can be achieved by immunization with chemically treated toxoids in combination with whole-cell vaccine. Markedly superior protection was obtained when rabbits were immunized with toxoid-whole-cell vaccine combinations administered by both the oral and parenteral routes. MATERIALS AND METHODS Antigens. Toxoids PX389A and PX389B3 were experimental cholera toxoids provided by Wellcome Research Laboratories, Beckenham, England, and were prepared by immunoaffinity chromatography of
528
Formalin-treated culture filtrates of V. cholerae. Toxoids PX389A and PX389B3 were equivalent, being two preparations made to the same formulation with similar constituents. Toxoid 20101 was prepared by Rappaport et al. (17) at Wyeth Laboratories and consisted
VOL. 26, 1979
,e1
IMMUNITY TO EXPERIMENTAL CHOLERA
of glutaraldehyde treatment of purified cholera toxin. The Wyeth toxoid contained aluminum phosphate and protamine as adjuvants. Wellcome vaccine PX389B8 was a bivalent whole-cell vaccine containing aluminum hydroxide (0.4%, wt/vol) obtained from Carl E. Miller at the National Institutes of Health in Bethesda, Md. Merck bivalent vaccine was prepared by mixing equal parts of Merck monovalent (Ogawa or Inaba) freeze-dried vaccines, which were also acquired from Carl E. Miller. Immunization of animals. Sixteen adult New Zealand rabbits weighing 4 to 5 pounds (ca. 1,814 to 2,2680 g) each were immunized by subcutaneous injection of each antigen in the nape of the neck. A second subcutaneous injection was given to each rabbit after 4 weeks. The dose of each toxoid consisted of 50 fig of protein contained in 0.5 ml of saline, whereas the dose of whole-cell vaccine was a 0.5-ml suspension of 4 x 109 V. cholerae cells. The first and second injection of some rabbits consisted of a 1-ml dose containing both 50 ,g of toxoid and 4 x 109 cells. The portion of this study concerned with route of immunization involved immunization ofrabbits with two 40-,ug doses of toxoid, or 4 x 108 cells or both administered by the parenteral route, whereas oral immunization was accomplished by carefully feeding 400-pg doses of toxoid or 4 x 109 cells or both through a syringe and catheter tube inserted in the rabbit's mouth. Blood samples were taken every 2 weeks, and serum was stored at -20'C before assay. Serological titrations. Cholera antitoxin titrations were assayed by the passive hemagglutination test (6). All titers were expressed as antitoxin units per milliliter based on the Swiss Serum and Vaccine Institute reference serum. Intestinal loop challenge. The basic challenge assay described by De (5) was performed 6 weeks after primary immunization as described below. Lyophilized cultures of V. cholerae (El Tor) Inaba strain V-86 or V. cholerae (Classical) Ogawa strain 395 were suspended in 1.0 ml of heart infusion broth (Difco) and streaked out on a heart infusion agar plate for incubation at 370C. Four or five isolated colonies were streaked for confluent growth on heart infusion plates on the morning of the challenge experiments and incubated at 370C for 4 h. The organisms were collected by washing the plate with 5.0 ml of sterile phosphate-buffered saline with 0.1% gelatin (PBSG), pH 7.4. This was adjusted to read 170 Klett units (green filter, Klett-Summerson photometer) corresponding to a viable cell count of about 1.3 x 109 cells per ml. This stock cell suspension was diluted by 10fold dilutions to a final dilution of 1.3 x 103 cells per ml, and dilutions were kept in an ice bath during the animal surgeries. Animals were given water but were not allowed food for 48 h before surgery. Immediately before surgery, the rabbits were anesthetized by a 3.0ml injection of Ketaset (Bristol-Myers), and their abdomens were shaved. After cleaning the abdomen with a 70% solution of ethanol, an abdominal incision was made approximately 7 cm in length. Beginning about 6 to 10 cm away from the appendix, a series of eight intestinal loops, about 10 cm apart, was made with surgical silk (Ethicon no. 1). Interspaces 2 cm in length were tied between each loop through which the 1-ml
529
injections of live vibros were made. The sequence of injections was reversed in alternate animals, and immunized and control rabbits were randomly challenged. The proximal and distal loops always received 1.0 ml of PBSG as a control. The rabbits were sacrificed 18 h after surgery by injection of 10 ml of air into the marginal ear vein. The loop responses were measured by recording the ratio of fluid accumulation (milliliters) to the length of the loop (centimeters).
RESULTS Antitoxin titrations. Geometric mean passive hemagglutination antitoxin titers for the first set of immunized animals are presented in Fig. 1. It is evident that the addition of Merck whole-cell vaccine to Wellcome toxoid PX389A and Wyeth toxoid 20101 had no effect on the antigenicity of either toxoid, since equivalent antitoxin titers were observed. The potency of the two toxoids is also very similar, giving rise to virtually identical antitoxin response curves. No antitoxin titers were observed in the group ofrabbits immunized with Merck whole-cell vaccine.
-7
2
6
Thme in weeks FIG. 1. Cholera antitoxin dose-response curves as determined by passive hemagglutination. Rabbits received two subcutaneous injections of the respective cholera toxoid with or without combination with cholera whole-cell vaccine. Symbols: 0-----0, Wyeth toxoid 20101 and Merck bivalent vaccine; 0--, Wyeth toxoid 20101; 0-v-., Wellcome toxoid PX389A; *..0.., Wellcome toxoid PX 389A and Merck bi, Merck bivalent vaccine. valent vaccine; *
530
PETERSON
INFECT. IMMUN.
Intestinal loop challenge. Since nonimmunized control rabbits were purchased at the same time as the experimental rabbits, housed under the same conditions, and integrated with the immunized animals at the time of challenge, direct comparisons can be made between each group. Examination of the slope and height of the response curves of the Inaba- and Ogawachallenged control rabbits, shown in Fig. 2 and 4, reveals that the Ogawa strain elicited more fluid accumulation than the Inaba strain, particularly at the higher challenge doses. Generally, it can be stated that Wellcome toxoid PX389A conferred protection against Ogawa and Inaba challenge comparable to that observed with Wyeth toxoid 20101. This conclusion is supported by the relative location of the two curves of toxoid-immunized animals in Fig. 2 and 3 (Ogawa challenge) as well as in Fig. 4 and 5 (Inaba challenge). The relative degree of protection conferred by either toxoid is depicted by the downward shift in the dose-response curves. Merck bivalent vaccine alone provided approximately the same degree of protection against both Inaba and Ogawa challenge strains. Rabbits immunized with a combination of Merck bivalent vaccine and either cholera toxoid resulted in the maximum amount of protection as depicted by the lowest curves in Fig. 2 through 5. The degree of protection observed as a result
I E -a
a 9 Bo QC
LOOP CHALLENGE DOSE FIG. 3. Fluid accumulation responses of rabbits challenged with V. cholerae Ogawa 395. Rabbits were immunized with Wyeth toxoid 20101 or Merck bivalent vaccine or both. Symbols: 0-----0, nonimmunized control; *-4, Wyeth toxoid 20101; 0.....0, Merck bivalent vaccine; 0-0, Wyeth toxoid 20101.
K
E S
/
-
/
/
O/
/
'I
,
,' I
.
,
t-
/
E
= 2
-I
//5
iiz /VF/
LWOP CHALLENGE DOSE FIG. 2. Fluid accumulation responses of rabbits challenged with V. cholerae Ogawa 395. Rabbits were immunized with Wellcome toxoid PX389A or Merck bivalent vaccine or both. Symbols: 0-----0, nonimmunized control; 0---, Wellcome toxoid PX389A; *o..0, Merck bivalent vaccine; 0-.Wellcome toxoid PX389A and Merck bivalent vaccine. ,
LOOP CHALLENGE BOSE FIG. 4. Fluid accumulation responses of rabbits challenged with V. cholerae Inaba V86. Rabbits were immunized with Wellcome toxoid PX 389A or Merck bivalent vaccine or both. Symbols as in Fig. 2.
of immunization with each of these preparations can best be analyzed by examination of protection factors which are the ratios of the 50% effective dose of the immunized groups to the nonimmunized controls. A 50% effective dose is that dose of bacteria that causes the accumulation of 1 ml of fluid per cm. The data in Table 1 indicate that each immunogen provided pro-
VOL. 26, 1979
IMMUNITY TO EXPERIMENTAL CHOLERA
tection against fluid loss that was greater against
with V. cholerae Inaba than with the Ogawa serotype isolate. Both the Wyeth and Wellcome toxoids provided protection that was approximately of the same magnitude. The most remarkable feature noted was the synergistic increase in protection observed after immunization with the combination of cholera whole-cell vaccine and each of the toxoids. The increase in protection was in excess of 100 times that achieved by immunization with either toxoid or vaccine alone. Effect of immunization route. Table 2 summarizes an experiment to determine whether route of immunization would affect the relative protective response of cholera toxoid, or vaccine, or both. The data indicate that oral inmunization with toxoid or vaccine provided the poorest protective responses (approximately twofold) to challenge
531
live-cell challenge with V. cholerae Inaba V86. Parenteral immunization with toxoid increased protection 10-fold, whereas parenteral administration of the whole-cell vaccine resulted in a 32fold increase in protection. When rabbits were immunized by both routes, the toxoid group exhibited only 10-fold protection, whereas the comparable group of animals receiving oral and parenteral doses of whole-cell vaccine exhibited 56-fold protection. When a combination of toxoid and whole-cell vaccine was administered orally, 16.4-fold protection was observed. This level of protection is synergistic, since it is at least four times the predicted amount of protecTABLE 2. Analysis of the relative degree of protection conferred to rabbits immunized with Wellcome toxoid or vaccine or both by the oral, parenteral, and combination routes Inaba V86 challenge
Group /
Wellcome toxoid PX389B3 Nonimmunized control Oral Parenteral Oral and parenteral
12
e~
~ ~
Wellcome vaccine PX389B8 Nonimmunized control Oral Parenteral Oral and parenteral
1205 107 100 CNALLENGE OM FIG. 5. Fluid accumulation responses of rabbits challenged with V. cholerae Inaba V86. Rabbits were immunized with Wyeth toxoid 20101 or Merck bivalent vaccine or both. Symbols as in Fig. 3.
Combination Wellcome toxoid PX389B3-Wellcome whole-cell vaccine PX389B8 Norummunized control Oral Parenteral Oral and parenteral a ED50, 50% effective dose.
EDs@
Protection factor
1x 1.8 x 1x 1x
104 104 105 105
1 1.8 10 10
1x 2.5 x 3.2 x 5.6 x
105 105 106 106
1 2.5 32 56
2.2 x 3.6 x 3.6 x 4.4 x
104 105 107
1 16.4 1,636 10" 20,000
TABLE 1. Analysis of the relative degree of protection conferred to rabbits imnmunized parenterally with Wellcome and Wyeth toxoids with and without combination with Merck whole-cell vaccine Ogawa challenge Protection factor 1 12.5 25 25
Group GrupEDEN 2 x 104 Control (nonimmu:ized) 2.5 x 105 Wyeth toxoid 20101 Wellcome toxoid PX389A 5 x 105 Merck bivalent vaccine 5 x 105 Combination (Wellcome toxoid PX389-Merck biva1.4 x 108 lent vaccine) Combination (Wyeth toxoid 20101-Merck bivalent 5 x 107 vaccine) a ED50, 50% effective dose.
Inaba challenge Protection EDso factor 7 x 103 1 1.4 x 106 200 6 x 105 85
7,000
1.3 x 106 2.5 x 107
186 3,571
2,500
2 x 108
28,571
532
PETERSON
tion as judged by the product of the protection factors obtained with toxoid or vaccine alone. The amount of synergistic protection observed with the toxoid-vaccine combination administered parenterally was 1,636-fold, although it had been two- to fourfold greater in the previous experiments. Finally, when the toxoid-vaccine combination was administered by both oral and parenteral routes, 20,000-fold protection was achieved. This level of protection was only slightly less than the expected product of the protection obtained by administration of the toxoid-vaccine combination via the oral or parenteral routes.
INFECT. IMMUN.
not propose to know the solution to the problem, it would be interesting to know whether toxoidvaccine combinations administred in adequate dosage via the oral and parenteral routes would provide humans with protection against cholera. The rabbit intestinal loop model employed in this study may or may not predict the degree of protection that could be expected in humans. It is distinctly different from the actual disease situation because by design it is a closed loop system. One could argue that the closed loop, after injection of large numbers of V. cholerae, is a more rigorous challenge situation than would be expected to occur in the intact small intestines of hns. In the loop, the bacteria can multiply and colonize without the disadvantage of the normal clearance action of the open intestinal tract. On the other hand, the model is subject to criticism because surgical manipulations and ligations may cause leakage of small amounts of blood plasma into the loops, providing protection that otherwise would not have occurred. In defense of the model, should leakage of antibody containing plasma into loops be a major problem, we would not have expected to obtain such uniform dose-response curves. The amount of leakage probably would vary from loop to loop and yield erratic results which have not been observed. Furthermore, antisomatic immunity has been proven effective in huns~nR in numerous field trials (1, 12, 13), and protection with whole-cell vaccines is easily achieved in the rabbit loop model as shown here. Regardless of possible limitations, the data presented here provide a basis for further research to improve immunity against cholera.
DISCUSSION The data presented in this report confirm the initial observation of Svennerholm and Holmgren (19) that it is possible to exploit antisomatic and antitoxic immunity by combined immunization to obtain superior protection against experimental cholera. This synergistic protective response is not due to elevated levels of either antibody population, but rather to the increased efficiency of dual mechanisms of antibody action. Antisomatic antibody is effective in reducing the number of bacteria colonizing the mucosal epithelium in the crypt regions (18), whereas antitoxin neutralizes toxin elaborated by those bacterial cells overwhelming the antisomatic antibody blockade. However, both stages of immunity may be overcome as the challenge dose increases. These observations with combinations of experimental toxoids and vaccines, some of which may be used in future field trials, provided a basis for optimism regarding their potential efACKNOWLEDGMENI fectiveness in conferring protection against cholera. The superior protection observed when the I express my appreciation to Howard R. Williams, Jr., Briney for technical assistance. toxoid-vaccine combinations were administered Ronald Roberts, and Carolyn from the U.S. Cholera Panel in the design of these by the parenteral and oral routes simultaneously Advice is gratefully acknowledged. suggests strongly that to maximize immunity, experiments This study was supported by Public Health Service conboth oral and parenteral routes of immunization tract NOl AI02101 from the National Institute of Allergy and Infectious Diseases. should be exploited. At this time, it is somewhat less than popular LITERATURE CITED to propose the extensive use of antitoxic immuJ. nity against cholera. The reason for this air of 1. Azurin, C., A. Cruz, T. P. Pesigan, M. Alvero, T. Camera, R. Suplide, L Ledesma, and C. Z. Gomez. pesiumism is due to the rather poor performance 1967. A controlled field trial of the effectiveness of tested of a glutaraldehyde toxoid (Wyeth 20101) cholera and cholera El Tor vaccines in the Philippines. in Bangladesh in 1975 (3, 4). In that trial, two Bull. W.H.O. 37:703-71. of toxoid were administered with 2. Bennett, V., and P. Cuatrecasa. 1975. Mechanism of 100-ILg doses Another action of Vibrio cholerae enterotoxin. J. Membr. Biol. trial of a Formalin toxoid jet injectors. 22:1-28. in the Philippines was also discouraging (15). 3. Curlin, G. T., R. Levine, K. K A. IAz, A. S. M. Interestingly, the same dose of toxoid was emRahmain and W. F. Verwey. 1975. Field trial of cholera toxoid, p. 314-335. In Proceedings of the Elevployed. Challenge studies with glutaraldehyde toxoid in American volunteers administered higher doses of toxoid via the oral or parenteral routes have not revealed any better protection from antitoxic immunity (11). Although we do
enth Joint Conference of the U.S.-Japan Cooperative Medical Science Program. Department of Health, Education, and Welfare, Washington, D.C. 4. Curlin, G. T., R. L. Levine, K. M. A. AziA, A. S. M. Mizanur Rahman, and W. F. Verwey. 1976. Serolog-
VOL. 26, 1979
5.
6.
7. 8.
9. 10.
11.
12.
IMMUNITY TO EXPERIMENTAL CHOLERA
ical aspects of a cholera toxoid field trial in Bangadesh p. 276-285. In H. Fukumi and Y. Zinnaka (ed.), Symposium on cholera. National Institute of Health, Tokyo. De, S. N., and D. N. Chatterje. 1963. An experimental study of the mechanism of action of Vibrio cholerae on thle intestinal mucous membrane. J. Pathol. Bacteriol. 46:569-62. Finkelstein, R. A, and J. W. Peterson 1970. In vitro detection of antibody to cholera enterotoxin in cholera patients and laboratory animals. Infect. Immun. 1:2129. Freter, R., H. L Smith, and F. J. Sweeney, Jr. 1961. An evaluation of intestinal fluids in the pathogenesis of cholera. J. Infect. Dis. 109:35-42. Germanier, R., E. Furer, S. Varallyay, and T. M. Inderbitzin. 1977. Antigenicity of cholera toxoid in humans. J. Infect. Dis. 135:512-516. Johnson, G. 8S, and V. R. Mukku. 1979. Evidence in intact cells for an involvement of GTP in the activation of adenylate cyclase. J. Biol. Chem. 264:96-100. King, C. A., and W. E. van Heynlngen. 1973. Inactivation of cholera toxin by sialidase resistant monosialoganglioside. J. Infect. Dis. 127:639-647. Levine, M. M., J. P. Craig, N. F. Pierce, D. H. Waterman, E. S. Caplan, and J. P. Libonati. 1976. The immune response to parenteral and oral cholera toxoid in volunteers, p. 338-340. In H. Fukumi and Y. Zinnaka (ed.), Symposium on cholera. National Institute of Health, Tokyo. Mosley, W. H., W. ML McCormack, ML Fahimuddin, K. M A. Aziz A. S. aman, A. K. L Chrowdhury, A. R. Martin, J. C. Feeley, and R. A. Phillips. 1969. Report of the 1966-67 cholera field trials in rural
13.
14.
15.
16.
17.
18.
19.
533
East Pakistan.L. Study design and results of the first year of observation. Bull. W.H.O. 40:117-206. Mosley, W. H, W. E. Woodward, K. M. A. Azr, A. S. M. Rahman, A. K. AL Chrowdhury, A. Ahmed, and J. C. Feeley. 1970. The 1968-69 cholera vaccine field trial in rural East Pakistan. Effectiveness ofmonovalent Ogawa and Inaba vaccine and a purified Inaba antigen, with comparative results of serological and animal protection tests. J. Infect. Dis. 3(Suppl):S1-S19. Nelson, E. T., J. D. Clements, and R. A. Finkelstein. 1976. Vibrio chokrae adherence and colonization in experimental cholera: electron microscopic studies. Infect. Immun. 14:527-647. Noriki, H. 1976. Evaluation of toxoid field trial in the Philippines, p. 302-310. In H. Fukumi and Y. Zinnaka (ed.), Symposium on cholera. National Institute of Health, Tokyo. Peterson, J. W., J. J. LoSpalluto, and R. A. Finkelstein. 1972. Icalization of cholera toxin in vivo. J. Infect. Dis. 126:617-621. Rappaport, R. S., W. A. Pierzchala, G. Bonde, T. McCann, and B. A. Rubin. 1976. Development of a purified cholera toxoid. III. Refinement in purification of toxin and methods for the determination of residual somatic antigen. Infect. Immune. 14:687-693. Sbrank, G. D, and W. F. Verwey. 1976. Distribution of cholera organisms in experimental Vibrio cholerae infections: proposed mechanisms of pathogenesis and antibacterial immunity. Infect. Immune. 13:195-203. Svennerholm, A.-M., and J. Holmgren. 1976. Synergistic protective effect in rabbits of immunization with Vibrio cholerae lipopolysaccharide and toxin/toxoid. Infect. Immun. 13:735-740.
