INTRODUCTION

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Cholera continues to be a major public health problem in nearly all developing countries (2, 6, 15). Until 1992, all epidemic strains of Vibrio cholerae were of the O1 serotype. However, in that year, a new serogroup of epidemic cholera, V. cholerae O139, synonym Bengal, appeared in Bangladesh and India along the Bay of Bengal (1). Although the pathogenesis and clinical features of O139 cholera are identical to those of O1 cholera, persons having immunity to serotype O1 are not immune to the Bengal serotype. This lack of immunity is primarily due to the unique O139 cell surface, which functions as a major protective antigen in cholera infection. An effective vaccine against V. cholerae O139 would therefore provide an important public health tool with which to control the disease.

An important step in the evaluation of cholera and related vaccines has been volunteer studies in which nonimmune healthy adult volunteers are immunized with the candidate vaccine and are then challenged with a typical cholera strain. A major constraint in these studies has been the difficulty of preparing a consistent inoculum. In the past it has been difficult to obtain reproducible results between different centers. Differences between the inoculum strains and/or between volunteers have been proposed to explain these inconsistencies. In order to eliminate differences between inoculum preparations, we sought to validate a standardized frozen inoculum for use in challenging volunteers with virulent V. cholerae O139 bacteria. The primary objectives of this study were to test the following assumptions: (i) volunteers would be susceptible to illness due to V. cholerae O139 if the bacteria were reconstituted from a frozen preparation; (ii) the resulting illness would be typical of serotype O139 cholera in terms of symptoms, excretion of the organism in feces, and the serologic response; (iii) there would be a dose-response effect to the frozen inoculum; and (iv) the challenge would provide a model with sufficient illness to determine protective efficacy in a vaccine study.

	MATERIALS AND METHODS

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Preparation and validation of the inoculum. The challenge lot (V. cholerae O139 4260B) was prepared from a human strain initially isolated from the International Center for Diarrhoeal Diseases Research, Bangladesh, Dhaka, Bangladesh, in early 1993. This strain helped identify the outbreak as one caused by an atypical non-O1 V. cholerae strain. The strain was initially sent to the Swedish Biological Laboratories by J. Holmgren and from the Swedish Biological Laboratories to D. A. Sack, who provided the seed lot. This isolate had been previously used as a fresh inoculum in volunteers at The Johns Hopkins University (13). From a master seed lot, organisms were prepared at the Salk Institute, West Point, Pa., as a 5-liter fermentor batch, harvested, and washed to remove exogenous toxin. Vials containing 2-ml aliquots of 9.1 × 10⁹ viable organisms in 20% glycerol were frozen (70°C). The biochemical phenotypes and antibiograms were identical between the frozen bacteria and the master seed lot. We used the same validation approach for purity, stability, and virulence as was employed previously for the V. cholerae O1 El Tor Inaba strain N16961 (14). Vials were kept frozen at 70°C at a National Institute of Allergy and Infectious Diseases repository and shipped on dry ice to Cincinnati, Ohio. Vials were reconstituted immediately before use, with no further incubation or bacterial growth. Stability has been validated 2.5 years following manufacture.

Study plan. In order to establish a model for vaccine studies, the aim of the study was to identify a dose of bacteria which (i) would consistently induce diarrhea in the volunteers with an attack rate of 50% (95% lower confidence interval [CI], 50%) and (ii) would induce a purge in the affected volunteers that had a geometric mean volume of at least 3 kg. Based on previous data for O139 strains (10, 13, 16), the initial plan was to begin these experiments with 10⁵ CFU and increase the inoculum strength by a log order as needed to achieve the desired attack rate and severity of illness.

Volunteer experiments. By established exclusion and inclusion criteria, groups of volunteers (n = 5 per group) between 18 and 40 years of age were recruited under an Internal Review Board-approved protocol from the community in Cincinnati, Ohio, as previously described (14). Informed consent was obtained from all subjects, and experimentation followed the guidelines of the U.S. Department of Health and Human Services as well as the Children's Hospital Medical Center Internal Review Board. Briefly, healthy, eligible, and willing volunteers were screened by taking a history and performing a physical examination and were required to pass a written examination to document their knowledge of cholera and the study design. Volunteers underwent a series of clinical laboratory examinations to rule out occult illness or pregnancy. Exclusion criteria included chronic illness; an immunosuppressive condition; an abnormal stool pattern (e.g., history of persistent diarrhea, blood in stool, or regular laxative use); travel to an area where cholera is endemic within the past 5 years; the presence of serum markers indicating hepatitis C or B infection; a determination of pregnancy from taking the subject's history or by a positive urine pregnancy test; a clinically significant abnormality in an electrocardiogram, screening hematology, or chemistry tests; and allergy to tetracycline or use of antibiotics within 7 days prior to challenge. In order to compare this study with the previous O1 validation model (14), and because persons with blood group O are known to have a more severe diarrheal response to certain forms of cholera (5), within each group of five volunteers, two (40%) volunteers were selected to have blood group O.

Outcome measures. (i) Clinical monitoring of volunteers. The volunteers were monitored for the occurrence of nausea, vomiting, diarrhea, and fever (38°C). They were examined at least twice daily, and fluid intake and output were continuously monitored. All stools were passed into a fecal collection hat for weighing, inspection, sampling, and disinfection with sodium hypochlorite. The stools were graded on a scale of 1 to 5 (1, firm variation of a normal stool; 2, soft variation of a normal stool; 3, runny stool that takes the shape of the container; 4, opaque-liquid stool; 5, rice water). A validated grading system model was used for identification of the primary-outcome variable (grade 3 stools) as previously described (14). The hydration of volunteers was maintained by replacing stool losses with a rice-based oral rehydration solution (CeraLyte, Jessup, Md.). Replacement oral rehydration solution was offered at one and one-half times the measured stool loss, e.g., 1.5 ml per gram of stool. When clinically appropriate, intravenous fluids such as Ringer's Lactate, with or without added potassium, were administered.

(ii) Laboratory outcome measures. Stool cultures were obtained daily after challenge to determine the duration of excretion of virulent V. cholerae and to quantitate the number of V. cholerae organisms being excreted as previously described (14). Briefly, stool or a rectal swab was incubated in alkaline peptone water enrichment medium prior to inoculation onto thiosulfate-citrate-bile salts sucrose medium. For quantitative cultures, a measured aliquot of stool was serially diluted onto thiosulfate-citrate-bile salts agar. Suspected colonies were confirmed to be V. cholerae with oxidase reagent and by agglutination with O139 antiserum (Centers for Disease Control and Prevention, Atlanta, Ga.).

Case definition. A cholera case was defined as a positive stool culture for V. cholerae and at least two diarrheal stools (grade 3, 4, or 5) totaling at least 200 g within 48 h or one diarrheal stool totaling 300 g. A moderate case was passage of at least 3 kg of diarrheal stool during the study, and a severe case was passage of at least 5 kg during the study. These are the same definitions that we used for the O1 challenge model (14).

	RESULTS

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Clinical and bacteriological data. A total of 25 volunteers were studied: 10 at a dose of 10⁵ CFU and 15 at dose of 10⁶ CFU. The clinical illnesses observed at both doses were typical of cholera, as shown in Table 1. As expected, the most prominent symptom of the illness was watery diarrhea. The clinical attack rate at 10⁵ CFU was 80%; however, this dose produced a mild to moderate illness (geometric mean purge volume of 2,175 g). Because the geometric mean purge volume was less than 3,000 g and therefore inadequate by the study design, volunteers were challenged at log-unit-higher dose (10⁶ CFU). This dose was sufficient to reproducibly generate moderate to severe diarrheal disease in three groups of volunteers with an average attack rate of 93%, with a CI (95%) of 68 to 100%. The geometric mean purge volume at this dose was 5,621 g. Vomiting was present in 20% of the volunteers at 10⁵ CFU and in 40% of the volunteers challenged with 10⁶ CFU. Fever (>38°C) was not seen in any volunteer.

Comparison between volunteers with the O blood group and volunteers with non-O blood groups. Similar to the increased risk or severity of illness seen with El Tor, but not classical strains, of V. cholerae O1 (3), there was a marked increase in the severity of purging in volunteers with blood group O who received 10⁶ CFU (Table 2). At this dose, of the six volunteers with blood group O, all had moderate (>3 kg) or severe (>5 kg) diarrhea but only three of nine volunteers with other blood groups had moderate or severe diarrhea (P < 0.05). Volunteers with blood group O had significantly more purging than those with other blood groups (10,353 versus 3,555 g, respective geometric mean stool weights; P < 0.01). There was no difference between blood groups in times of onset to purging after inoculation or the fecal shedding of cholera organisms.

Serologic test results. A total of 10 (67%) of the 15 volunteers who received 10⁶ CFU and 6 (60%) of the 10 who received 10⁵ CFU had a significant (fourfold) rise in their vibriocidal titers when prechallenge sera were compared with sera collected on days 9 and 14 after challenge. Reciprocal prechallenge titers were <20; postchallenge reciprocal titers ranged from 20 to 160 (geometric mean peak reciprocal titer, 70). All of the volunteers who received either 10⁶ or 10⁵ CFU had a significant rise in their antitoxin titers compared to prechallenge values.

	DISCUSSION

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This study demonstrated that ingestion of an inoculum prepared directly from a frozen vial of V. cholerae O139, with no further incubation, resulted in a consistent, acute watery diarrheal illness typical of O139 cholera. The incubation period, clinical attack rate, colonization rate, and severity of illness at 10⁶ CFU in this challenge were similar to those parameters of previously published challenge studies using freshly harvested V. cholerae O139 AI1837 (10, 16) (Table 4). However, using freshly harvested AI1837 inoculum, other investigators were unable to reproduce this clinical response in Thai volunteers even at doses up to 10⁸ CFU (11). Validation of a large batch of frozen strain 4260B for human challenge studies will permit further testing to determine whether host susceptibility differences between North American and Thai volunteers or loss of pathogen virulence was responsible for this discrepancy.

We demonstrated a significant rise in the serum vibriocidal antibody responses of the majority of volunteers by using a modified unencapsulated 2L mutant target strain as previously described (9). When an encapsulated organism is used as the target strain, no vibriocidal response is seen after challenge with O139 organisms (Table 4). The vibriocidal responses we observed are similar to those seen in natural infection with O139 organisms but much more modest than those seen in natural infection or volunteer challenge studies with O1 organisms (4). While the vibriocidal response is the best correlate to protection against subsequent O1 infection, it is likely that protective immunity is conferred by a mucosal response. As previously observed (12), the magnitudes and isotypes of the ASC responses were similar between O139- and O1-infected volunteers (14). All volunteers manifested an antitoxin response, which was similar to what occurred with volunteers challenged with frozen O1 organisms. In this study, 27 of 30 volunteers had a significant rise in antitoxin antibodies (14).

We recently validated a human challenge model using a frozen O1 strain (14). The designs of the experiments were identical, as were the desired minimums of illness severity (attack rate with a 95% Cl of >50% and a geometric mean purge volume of >3,000 g). In order to achieve this threshold of illness, a 1-log-unit-higher dose of V. cholerae O139 was required than was needed with frozen O1 organisms. However, the disease produced by V. cholerae O139 was more severe than that seen with the frozen O1 challenge. Although we tested only two doses for each strain, it is possible that the clinical response to an increased O139 inoculum was greater than the clinical response to an increased O1 inoculum. For the O1 challenge, a 1-log-unit increase in inoculum from 10⁴ to 10⁵ CFU produced a 2.1-fold increase in geometric mean purge, from 1,608 to 3,416 g. For the O139 organisms, a 1-log-unit increase in inoculum from 10⁵ to 10⁶ CFU produced a 2.6-fold increase in geometric mean purge, from 2,175 to 5,621 g. However, it appears that much of the increase in purge volume with the frozen O139 strain resulted from more severe disease in the volunteers with blood group O. This difference is qualitatively similar to, but quantitatively greater than, differences observed in previous studies of El Tor O1 organisms (14). These data demonstrate that moderate or severe illness due to V. cholerae O139 is more common in persons with blood group O. As is the case for certain but not all O1 strains (5), the mechanism for this increased risk is unknown. It has been suggested that the increased severity is due to enhanced adherence of vibrios to the human intestinal epithelium, which is rich in blood group antigens (7).

We have recently used identically prepared frozen O1 inocula to test a live oral V. cholerae vaccine (17). Based on the results of this study, we conclude that the ability to use easily prepared, identical vials of frozen organisms and the degree of cholera illness, the predicted clinical attack rate, and the immunological responses we observed validate this model for use in human-volunteer challenge studies to test the efficacies of O139 or combined O1-O139 cholera vaccines against moderate and severe illness.