The study drug, consists of bivalent WC bulks of formaldehyde-inactivated V. cholerae O139 bacteria and heat- or formaldehyde-inactivated V. cholerae O1 bacteria, was manufactured by processes of fermentation, concentration, washing and inactivation. The formulation of this vaccine has been developed by the IVI, Seoul, Korea considering the fact to meeting the requirements of WHO. The final bulk was prepared in EuBiologics Co., Ltd., Chuncheon, Korea by mixing of five individual bulks obtained through the fermentation of different strains of V. cholerae with phosphate buffer, preservative and purified water. The study drug is a liquid suspension of inactivated V. cholerae and does not contain the B subunit of cholera toxin that is available in Dukoral. Finally, 1.6 mL of final bulk (i.e., a yellow to yellowish color) was filled into each glass vial of 2 mL, which was closed with a rubber stopper covered with an aluminum flip-off seal. The study drug was stored in a refrigerator at 2℃ to 8℃. The detailed composition of this vaccine is mentioned in Table 1. In order to comply with the requirements of WHO, the antigens content (expressed as Lipopolysaccharide ELISA Units, L.E.U.) in OCV were standardized using ELISA specific for V. cholerae O1 and V. cholerae O139 LPS antigens (9). The study drug was also tested for the content of toxin where no detectable toxin (limit of detection 1 ng/mL) was found. The LPS, toxin assays and all other lot release assays were performed at EuBiologics Co., Ltd. The Lot number of study drug is "Euvichol-12001".

A total of 25 healthy adult male volunteers were screened in this study; and their written consent was taken prior to enrolment. Subjects who satisfied all the inclusion and exclusion criteria of the protocol were finally selected for this clinical trial. Briefly, subjects with history of hypersensitivity to other vaccination, immunization with other vaccine within 2 months, diarrhea or abdominal pain within 1 week, using anti-diarrheal and antibiotics within 2 weeks prior to the start of the study were excluded from the study. However, five of them were failed to meet the eligibility criteria assessed by clinical laboratory tests. Therefore, only 20 subjects were considered at the end.

Twenty screened subjects were admitted to the CTC, CNUH by 4 pm on the day (D-1) before the first dosing day (D0, Visit 2). They were remained fasting from 10 pm on D-1. Drinking water was also prohibited between 1 hr of pre-dose and 2 hr of post-dose. At around 8 am on D0, the study drug (1.5 mL) was orally administered into the subjects. The OCV was personally administered by a sub-investigator (physician) or clinical trial pharmacist, and the dosing was confirmed by an oral examination. Afterward, the vital signs and electrocardiogram (ECG) in subjects were measured as scheduled. At 8 am on D1 (after 24 hr of first dosing), subjects who completed the hospitalization schedule were discharged as they were found to have no safety problem. They were again admitted to the CTC, CNUH by 4 pm on the day (D13, Visit 3) before the second dosing day (D14) and remained fasting from 10 pm on D13. At around 8 am on D14, the OCV was transferred into a syringe and administered orally into the subjects. The vital signs and ECG were measured thereafter. At 8 am on D15 (after 24 hr of second dosing), subjects were discharged after completing the hospitalization schedule as no safety problem was observed. Subsequently, they made outpatient visits to the CTC, CNUH for Visit 4 (D28±2 days) and Visit 5 (D42±5 days) and were performed the above-mentioned tests. The visiting schedule for each subject is given in Fig. 1.

The safety was assessed in subjects who received at least 1 dose of investigational product. The symptoms and signs of adverse events, duration (onset and completion dates), severity (mild, moderate, severe), causal relationship with the study drug, the actions taken for the adverse events, and outcome were recorded by the sub-investigators and principal investigator. In the case of any adverse events those are not demonstrated by the medical diagnosis terms, the symptoms and signs occurred in subjects were recorded by preferred terms (PTs) listed in the Medical Dictionary for Regulatory Activities (MedDRA, version 15.1). They are presented by system organ class (SOC) in the clinical study report. However, the symptoms and signs observed in the subjects prior to the participation in this study were not recorded as an adverse event. The principal investigator or sub-investigators followed the subjects with an adverse event until the event was resolved or abnormal clinical laboratory findings were returned to baseline or the observed change was satisfactorily explained.

Vital signs (blood pressure, pulse rate and body temperature) were measured throughout the study in order to screen the eligibility of subjects as well as on the days of administrating the study drug i.e., after 0.5 hr, 1 hr, 2 hr, 3 hr, 4 hr, and 6 hr of post-dose. 12-lead ECG was conducted in all subjects during screening, just before the pre-dose, and after 6 hr of post-dose. The parameters i.e., ventricular rate (VR) (beats/min), PR interval (msec), QRS duration (msec), and QT/QTc interval (msec) were measured by an autoanalyzer. The results were based on the comprehensive reading findings calculated through the internal algorithm of the autoanalyzer and the clinical findings observed by a physician. Safety of subjects was also assessed by the clinical laboratory tests and physical examination. Blood was collected as scheduled from the subjects using an aseptic technique, and samples were tested by hematology/blood chemistry, blood coagulation test, urine test, serology (Hepatitis B/C & HIV). However, serology was conducted only during screening. All the test results were recorded in the Case Report Form (CRF) with the consent of the principal investigator. The investigator's opinion was also noted in the case of any findings that were outside the reference range. All the analysis was performed at the relevant department of the study site that has been received the quality control certification from the Korean Association of Quality Assurance for Clinical Laboratory. The analytical personnel were well experienced about similar kind of study and performed all the tests according to the standard operating procedures (SOPs) of the study site.

The immunogenicity of investigational drug was evaluated by serum vibriocidal assay. In this study, the anti-V. cholerae O1 and O139 antibodies was measured in the sera of subjects administered with the study drug at day 0, day 14, and day 28. Approximately 7 mL venous blood of each subject participated in this study was collected into a plain (non-heparinized) tube. After separating the serum from blood, 0.5 mL aliquots were transferred into sterile micro-centrifuge tubes. All the serum samples were shipped frozen to the IVI and stored -70℃ until the vibriocidal assay was performed. Serum vibriocidal antibodies to V. cholerae O1 (T19479 [El Tor Inaba] and X25049 [El Tor Ogawa]) and O139 (CIRS 134-SR) strains were performed in the laboratory of Clinical Immunology, IVI using a microtiter technique (10, 11). Briefly, the mentioned strains of V. cholerae O1 and O139 were grown in Brain Heart Infusion (BHI) and Luria-Bertani (LB) plus streptomycin (Sm) media, respectively, at 37℃ until reaching mid-log phase. V. cholerae O1 and O139 were harvested by centrifugation, and cells were resuspended in sterile saline (0.85% NaCl) solution and PBS, respectively. Two different mixtures containing the resuspended bacteria and the guinea pig complement (C300-500; Rockland, Gilbertsville, PA, USA) were prepared. The bacterial mixtures were thereafter added to a 96-well microtiter plate (Nunc, Roskilde, Denmark) with the test sera diluted properly by sterile saline and PBS, respectively, and incubated the plates for 60-70 min at 37℃. The initial dilution of all test sera were at 1 in 2.5 for V. cholerae O1 and at 1 in 10 for V. cholerae O139, respectively, and 2-fold serially further diluted.

Human serum (HSQC) and rabbit serum (anti-4260B) were used as the control sera (with known range of vibriocidal titer) for the determination of serum vibriocidal antibodies to V. cholerae O1 and O139 strains, respectively. All the test sera and controls were placed at 56℃ for 30 min to inactivate complement before adding them into the microtiter plates. BHI and 4*LB (+ Sm) media were added to each well of microtiter plates and incubated at 37℃ for 3-5 hr in order to determine the vibriocidal antibodies against V. cholerae O1 and O139, respectively. Finally, the optical densities of resulting suspensions were measured at 600 nm with a microplate reader (Spectramax 190, Molecular Device, Sunnyvale, CA, USA). The titers of vibriocidal O1 and O139 antibodies were defined as the reciprocal of the highest dilution of serum that gave complete inhibition of visible growth of V. cholerae O1 and the dilution of serum that caused 70% killing V. cholerae O139, respectively. The serum vibriocidal antibody titers were determined with mean of the two tests and the assay was repeated whenever more than two-fold difference was noted between the results of duplicates. The statistical analysis (e.g., geometric mean, standard deviation [SD], min, max, median, 95% confidence interval [CI] for antibody titers) was conducted to compare the antibody titers and fold increase in regards to the baseline titers. Vibriocidal titers <2.5 for V. cholerae O1 and <20 for V. cholerae O139 were considered as 1.25 and 10, respectively, for the statistical analysis. Antibody titers in sera collected on day 14 and 28 were divided by the baseline titers (D0) in order to obtain the fold-rise. Seroconversion was defined as a four-fold or greater increase in titer between pre- and post-vaccination sera.

The data management for this study was implemented according to the guidelines of International Conference on Harmonization - Good Clinical Practice (ICH-GCP) and Korea Good Clinical Practice (KGCP). The adverse events and various test findings were reviewed, incorporated and comprehensively judged by a sub-investigator. For safety assessment, the probability of observing at least 1 adverse event with the incidence of 15% in 20 subjects is 95%. In the case of antibody titers, geometric mean, SD, min, max, median and 95% CI are calculated. The means of titers are calculated as geometric mean titer (GMT), and after log-transformation when necessary, analyzed and exponentiated. CRFs and all the relevant data were monitored by the study coordinator as designated by the EuBiologics Co., Ltd.

The protocol for this clinical study (UBC101) was also approved by the Korea Food and Drug Administration (KFDA) and institutional review board (No. CNUH-2012-08-012) of the Clinical Trials Center, Chungnam National University Hospital on 31 July, 2012 and 29 October 2012, respectively. This study was registered with the Clinical Trials Registry (http://clinicaltrials.gov/, Identifier: NCT01707537).

The safety of study drug was assessed based on the adverse events measured by the clinical laboratory tests, vital signs, ECGs, and physical examinations in all the subjects who took part in this study. Since this drug is orally administered unlike other vaccines, adverse events were not classified as local and systemic events. However, a total of 7 adverse events occurred in 6 subjects i.e., 2 events of headache, 1 event each of toothache, blood creatine phosphokinase increase, liver function test abnormality, musculoskeletal pain, and tooth extraction. Based on severity, toothache and blood creatine phosphokinase increase were considered as Grade 3, tooth extraction was Grade 2, and the rest of the events were Grade 1. The causal relationship for 2 events of headache and 1 event of other adverse events was designated as 'Probably related' and 'Possibly related', respectively. For other events not mentioned above, the causal relationship was 'Probably not related'. However, no serious adverse event occurred.

Vital signs (blood pressure, pulse rate and body temperature) were measured in each subject from the start of screening and during the study according to the planned schedule. However, no clinically relevant change was found in systolic/diastolic blood pressure and pulse rate while reviewing individually. The parameters of ECG (VR, PR interval, QRS duration, QT interval, and QTc interval) measured at each point were compared with the baseline data. However, there was no difference found before/after vaccination, and no clinically significant change (for example, arrhythmia) was observed from the comprehensive judgment based on the automatic ECG tracing and investigator's findings. In addition, all subjects had clinical laboratory tests during screening, Visit 4 and Visit 5. No clinically relevant change was observed in hematology, blood chemistry, blood coagulation test, and urology parameters before/after vaccination. Physical examinations were also performed during the study. However, no clinically meaningful change was noted, suggesting that there is no major safety problems related to this investigational drug.

The vibriocidal antibody titers to 3 different strains of V. cholerae measured prior to the first dose (baseline, D0), before the second dose (D14), and 14 days after the second dose (D28) of OCV were ranged from <2.5-5,120, <2.5-10,240, and <2.5-480 for O1 Inaba, O1 Ogawa and O139, respectively. The geometric mean serum vibriocidal antibody titers (GMT), 95% CI and number of subjects were also measured at the above time points, which are presented in Fig. 2. In the case of all antibodies, a noticeable increase of GMT was observed in sera collected at D14 and D28 as comapred to D0. The descriptive statistics of antibody titers measured at the above-mentioned days are presented in Table 2. The antibody titers to V. cholerae O1 Inaba, O1 Ogawa, and O139 measured on D14 and D28 were divided by the baseline titers (D0) in order to obtain the geometric mean fold rise (GMF-rise). The antibody titers from baseline value measured in this study ranges from 1-4,096 for O1 Inaba, 1-8,192 for O1 Ogawa, and 1-384 for O139. When the baseline titer was <2.5, it was substituted with 1.25. Fig. 3 shows the GMF-rise from baseline titer to V. cholerae O1 Inaba, O1 Ogawa, and O139 measured on D14 and D28. The descriptive statistics of GMF-rise to mentioned 3 strains of V. cholerae is mentioned in Table 3.

We calculated the percentages of subjects seroconverted (≥ 4-fold rise in serum vibriocidal titers) on day 14 and 28 after the study drug administration. In the case of vibriocidal antibodies against both O1 Inaba and O1 Ogawa strains of V. cholerae, 19 subjects (95%) showed a ≥4 fold increase on D28. The number of subjects with a ≥4 fold increase against V. cholerae O139 was 10 (50%) and 9 (45%) on D14 and D28, respectively (Table 4). The statistical analysis was also conducted to compare the antibody titers, fold rise from baselines and the titers between the dosing points. In the case of all three antibodies, the geometric mean titers (GMT) against V. cholerae measured on D14 showed a statistically significant (P value, <0.001) increase from the baseline, whereas no statistical difference was obtained in antibody titers measured on D28 from that of D14 (Table 2). The GMF-rise on D28 also exhibited no statistically significant difference from the GMF-rise on D14 (Table 3), indicating that the antibody titers significantly increased on D14 and maintained at a constant level till D28.