INTRODUCTION

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Cholera continues to be a major public health problem in nearly all developing countries, including countries in the Western hemisphere (2, 8, 26, 31). Cholera afflicts both children and adults and exists as an endemic disease in over 100 countries. Cholera does occur in the United States, but cases and deaths are rare. Travelers from the United States to areas where the disease is endemic are at some risk; this risk has been estimated to be about 1 per 30,000 travelers (37), although with active surveillance and use of optimal medium for isolation of Vibrio cholerae, the incidence of cholera in this population has been estimated to be much higher (36). For example, the incidence of cholera among U.S. citizens living in Lima, Peru, was 5.3 cases per 1,000 between 1991 and 1993 (36). The current parenteral vaccine, which provides about 50% protection, commonly elicits systemic and local adverse reactions (21). A more effective, better tolerated vaccine that could be administered orally is therefore desirable, and such new oral vaccines have been developed, including both killed vaccines and live attenuated strains (12, 13, 18, 19, 21). An ideal new vaccine would provide a high level of long-term protection to even those at high risk for severe illness, e.g., individuals with blood group type O. This protection would begin shortly after administration of a single oral dose (18, 19).

CVD 103-HgR is a vaccine strain constructed by deleting 94% of the gene encoding the enzymatically active A subunit of cholera toxin (ctxA) from classical Inaba V. cholerae 569B, leaving intact the gene encoding the immunogenic B (binding) subunit of cholera toxin (ctxB). A gene encoding resistance to Hg²⁺ ions was inserted into the El Tor hemolysin gene to provide a marker to differentiate the vaccine strain from wild-type V. cholerae classical Inaba strains, and an undefined, spontaneous mutation apparently arose during strain construction (14). This vaccine is well tolerated and immunogenic in diverse populations (5, 11, 15-17, 20, 25, 27, 32-34, 38); moreover, fecal excretion of this derivative is low, which should minimize environmental spread of the vaccine (20).

A single dose (5 × 10⁸ CFU) of CVD 103-HgR has provided a high level of protection against cholera in groups of U.S. volunteers challenged with classical biotype V. cholerae O1 of either Inaba or Ogawa serotype (19, 20, 35). In the volunteer challenge model, significant efficacy against challenge with the homologous (classical) biotype persisted for at least 6 months after vaccination (the longest interval tested) and was present as soon as 8 to 10 days after vaccination (the shortest interval tested) (35). The current pandemic of cholera is due to V. cholerae of the El Tor biotype. In three open-label, unblinded efficacy studies of nonrandomized vaccinees (n = 36) and control volunteers (n = 24) challenged with El Tor V. cholerae, a single dose of CVD 103-HgR provided complete protection against moderate and severe diarrhea (19, 20). Based on these volunteer studies in the United States, CVD 103-HgR was licensed in Canada and a number of European countries as a single-dose oral vaccine to prevent cholera.

We undertook a multicenter volunteer challenge study by using a randomized, placebo-controlled, double-blind method to determine the protective efficacy and confidence intervals for protection against moderate and severe diarrhea (3.0-liter purge) due to El Tor Inaba V. cholerae O1 at least 3 months after immunization with CVD 103-HgR.

	MATERIALS AND METHODS

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Study design. A total of 85 volunteers (50 at the University of Maryland and 35 at Children's Hospital Medical Center, University of Cincinnati) were enrolled and randomized to receive CVD 103-HgR or placebo, with the understanding that they would return for challenge with wild-type V. cholerae El Tor Inaba at least 3 months later. All volunteers received prestudy counseling and gave informed, written consent. To ensure comprehension of the study and to document that informed consent had been elicited, the volunteers had to pass a written examination before inoculation with the challenge strain. Volunteers were thoroughly screened to document their mental and physical health before challenge. The inclusion criteria for the challenge study were as follows: healthy man or woman, age 18 to 40 years; normal medical history and physical examination; and no clinically significant abnormalities of urinalysis, complete blood count, serum hepatic transaminases, glucose, creatinine, blood urea nitrogen, electrolytes, or electrocardiogram. The exclusion criteria were as follows: travel to a cholera endemic area in the previous 5 years; abnormal stool pattern or regular use of laxatives; failure to pass a psychological examination; allergy to tetracycline or ciprofloxacin; history of cholera or enterotoxigenic E. coli challenge; history of recent antibiotic use; pregnancy or nursing; positive serology for HIV, hepatitis B antigen, or hepatitis B; stool culture positive for an enteric pathogen; or failure to pass the written examination.

Method of randomization. For those of blood group O and non-O within each clinical center, subjects were randomized in blocks of four (two to receive vaccine and two to receive placebo) by using SAS PROC PLAN. Volunteers were stratified by blood group (O versus non-O), because persons of blood group O are predisposed to develop more severe forms of cholera diarrhea (3, 9). The code was held by the study sponsor until the database was complete and unalterable.

Vaccination. Volunteers were randomized in a double-blind manner to receive, with buffer, a single oral dose of either CVD 103-HgR (2 × 10⁸ to 8 × 10⁸ CFU) (n = 43) or placebo (killed E. coli K-12) (n = 42). For 3 days after vaccination as outpatients, the volunteers kept a symptom diary to record all stools and to determine the occurrence of adverse reactions such as diarrhea, nausea, vomiting, abdominal cramps, malaise, anorexia, headache, and fever.

Vaccine and placebo formulations. The vaccine strain was prepared and packaged by the Swiss Serum and Vaccine Institute as a lyophilized preparation. The formulation consisted of a pair of attached sachets, one containing a single dose (2 × 10⁸ to 8 × 10⁸ viable organisms) of lyophilized vaccine and the other containing effervescent buffer powder to be mixed with vaccine in 100 ml of water. Each sachet of buffer contained NaHCO₃ (2.5 g), ascorbic acid (1.65 gm), and aspartame (25 mg). The placebo formulation consisted of an identical-appearing pair of sachets, one containing killed E. coli K-12 and the other containing buffer. When suspended in the buffer solution, the placebo was identical in appearance to the vaccine suspension.

Challenge. Approximately 3 months after immunization (mean, 113 days; median, 109 days; range, 85 to 143 days), 51 volunteers (28 vaccinees and 23 placebo recipients) were admitted to Kernan Hospital in Baltimore or to the General Clinical Research Center at Children's Hospital Medical Center in Cincinnati to be orally challenged with 10⁵ CFU of virulent V. cholerae O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum (30). During a period of 1 to 2 days before ingestion of the challenge inoculum, the volunteers were acclimated to the ward while medical screening was completed. Baseline samples of serum were collected for the measurement of antibody.

Definitions of illness. For outpatient volunteers after administration of vaccine or placebo, diarrhea was defined as four loose stools in a 24-h period. For subjects under inpatient surveillance after challenge, diarrhea was defined as the passage of two or more unformed (grades 3 to 5) stools over a 48-h period that equaled or exceeded 200 ml or a single stool of 300 ml or greater.

Serology. Blood was collected before and 10 days after vaccination to provide sera for measurement of vibriocidal antibodies and antibodies to cholera toxin. Blood was collected before and on days 9 and 14 after challenge for serologic studies. Coded sera from each subject were tested for vibriocidal antibodies by using V. cholerae O1 El Tor Inaba strain 89 and for immunoglobulin G (IgG) and IgA antitoxin by enzyme-linked immunosorbent assay (ELISA) (1, 4, 23, 24, 29). A fourfold or greater rise in serum vibriocidal titer over that of the baseline specimen was considered significant. For anti-CT antibody, a 0.20 rise in optical density units between pre- and postvaccination or challenge specimens was considered significant.

Bacteriology. All stools after challenge were plated directly onto thiosulfate citrate bile salts sucrose (TCBS) agar, as well as inoculated into alkaline peptone water enrichment broth, for overnight incubation before plating onto TCBS agar (28). Up to two stools each day were cultured quantitatively to determine the number of vaccine organisms per gram of stool. A rectal swab was obtained if no stool was passed. Suspicious colonies were agglutinated with specific V. cholerae O1 Inaba antiserum.

Statistical analysis. The frequency of adverse reactions among vaccinees and placebo recipients was compared by use of the Fisher's exact test (FET). The maximum recorded severity for each of these reactions was compared between groups by use of the Wilcoxon test. The total number of days of diarrhea and peak temperature among vaccinees and placebo recipients were compared by using Wilcoxon tests.

	RESULTS

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Volunteers. The 43 vaccinees and 42 placebo recipients had similar demographic profiles. The participants included 48 men and 37 women with an average age of 30.1 years (range, 18 to 40 years). A total of 40% of the vaccinees and 41% of the placebo recipients were of blood group O. Three months after vaccination, 56 of the 85 volunteers were available, willing, and medically and psychologically eligible to participate in the cholera challenge study. Of these, 51 were randomized to participate in the challenge phase; 33 were challenged at the University of Maryland, and 18 were challenged at the University of Cincinnati. Among the 51 subjects, 54% of the 28 vaccinees and 35% of the 23 placebo recipients were of blood group O (P was not significant). There were no statistically significant demographic differences between subjects who participated in the challenge study and those who did not.

Reactions to vaccine. The vaccine was well tolerated. There were no significant differences in the rates or severity of anorexia, malaise, abdominal cramps, headache, nausea, vomiting, diarrhea, or fever reported by subjects who had ingested CVD 103-HgR and those who had ingested killed E. coli K-12 placebo (Table 1).

Immune response to vaccine. Overall, 91% of the vaccine recipients developed significant increases in serum Inaba vibriocidal antibody titers. The geometric mean reciprocal titer (GMT) was 3,056 (range, 20 to 20,480) for all vaccinees after vaccination, a 113-fold rise over the baseline GMT (Table 2). Among vaccinees who participated in the challenge phase, 93% had significant rises in Inaba vibriocidal titers with a GMT of 1,470 (range, 20 to 20,480) after vaccination. One volunteer experienced a rise in vibriocidal titer after receiving placebo; he remained clinically well during this period. This response may represent cross-reacting antibodies to some subclinical infection (6) or, less likely, a specimen collection, laboratory, or protocol error. After immunization, serum IgG anti-cholera toxin rose in 61% of vaccinees who subsequently underwent challenge.

Clinical and bacteriologic responses to challenge. A total of 9 (39%) of the 23 placebo recipients and 1 (4%) of the 28 vaccinees developed moderate or severe cholera (3.0-liters of total diarrheal stool volume) after challenge (P = 0.003; protective efficacy, 91%; lower 95% confidence limit, 51%) (Table 3). The one vaccinee who became ill had severe cholera with a total diarrheal stool volume of 6.8 liters. She was of blood group O and had had a vigorous vibriocidal antibody response to vaccine (titer of 1:20,480 on day 10 after vaccination) and, immediately before challenge 3 months later, she had a vibriocidal titer of 1:1,280.

Relationship between immune responses to vaccination and protection against cholera challenge. Among the 28 vaccinees, the vibriocidal antibody titers measured 10 days after immunization and those measured immediately before challenge were examined in relation to whether or not the vaccinee had diarrhea and to the severity of diarrhea after challenge. Neither the 10-day postvaccination vibriocidal titer nor the vibriocidal titer drawn immediately before challenge clearly correlated with incidence or severity of illness after challenge among the vaccinees (Table 4).

	DISCUSSION

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Multiple previous studies of the efficacy of CVD 103-HgR against challenge with El Tor and classical V. cholerae O1 have been conducted among U.S. volunteers (19, 20, 35). These studies were open-label, unblinded, nonrandomized studies in which vaccinees were enrolled and vaccinated 1 to 6 months before challenge and, for logistical reasons, unvaccinated control volunteers were enrolled at a later time just before challenge. In these studies, the prospectively designated outcome measure was the development of moderate or severe cholera, which was defined by specific volumes of diarrheal stool; moderate cholera was defined as 3 liters of diarrheal stool, and severe cholera was defined as 5 liters of diarrheal stool. The entire blood volume of an average adult male is ca. 5 liters, so these volumes represent potentially life-threatening fluid losses if such diarrhea were untreated. Because of the objective outcome measure used in this study design, it was considered unlikely that the outcome was significantly affected by investigator or volunteer bias.

However, the rigorous randomized double-blind design of the current study allays concerns about any possible biases that might have been inadvertently introduced in the previous studies. Moreover, the results of this trial increase the experience in protecting against El Tor cholera, the biotype which is currently prevalent throughout the world. In this rigorous examination of vaccine efficacy, CVD 103-HgR performed well; the vaccine induced 91% protective efficacy against moderate and severe El Tor cholera, the clinical syndrome that is responsible for mortality associated with the disease, and provided 80% efficacy against any diarrhea after El Tor V. cholerae O1 challenge in these U.S. volunteers, a level higher than that observed in previous studies (19). Previous studies have shown no difference in protection after rechallenge with V. cholerae of different serotypes (Ogawa or Inaba) (22). In addition, this trial demonstrates that the duration of protection against El Tor cholera provided by CVD 103-HgR extends to at least 3 months. The vaccine, as expected, reduced the extent of excretion of vibrios in the stools of challenged volunteers by 100,000-fold.

For the first time in an efficacy study of CVD 103-HgR, a vaccinated volunteer developed severe cholera after challenge. This was not due to a failure of the subject to respond serologically to the vaccine. This volunteer, a healthy 32-year-old woman of blood group O, had vigorous anti-cholera toxin and Inaba vibriocidal antibody responses 10 days after immunization, in which her vibriocidal titer rose from 1:10 to 1:20,480. The vibriocidal titer immediately before challenge 3 months later was 1:1,280. Serum vibriocidal titer has been considered a surrogate marker of protective immunity, and a titer of this level would have been expected to protect her from at least severe diarrhea (10). The absence of protection shows that the vibriocidal response is an imperfect surrogate measure of protection at the mucosa in any individual patient. Retrospectively, total serum and stool IgA levels were measured in this volunteer and were within the normal range, so that IgA deficiency did not contribute to the severe clinical syndrome she experienced after challenge.

In reviewing the entire experience over several years, only 1 (1%) of 103 CVD 103-HgR vaccinees developed moderate or severe cholera after challenge with classical or El Tor biotype V. cholerae compared to 21 (24%) of 86 control volunteers. When challenges with El Tor strains only are reviewed in combination, only 1 (2%) of 64 vaccinees but 21 (45%) of 47 control volunteers experienced moderate or severe cholera. Although strict statistical analysis of these data is problematic since the studies were designed differently and conducted over a period of many years, the overwhelming impression is supportive of the strong protective effect of vaccination.

The collective experience of experimental cholera in U.S. volunteers vaccinated with CVD 103-HgR is in direct contrast to the only field trial of the efficacy of CVD 103-HgR carried out in an area where the disease is endemic. In Jakarta, Indonesia, protection could not be detected for 6 to 53 months after vaccination, and too few cases occurred in the first 6 months to determine whether or not protection occurred during this period (29). Experimental cholera challenge is arguably a better model for estimating efficacy among immunologically naive travelers from developed countries to countries where the disease is endemic than a direct measurement of field efficacy among residents of such an area. Therefore, we think the weight of evidence gathered through challenge studies suggests that CVD 103-HgR may be a suitable vaccine for protecting travelers against cholera.