Phase III Clinical Trials Comparing the Immunogenicity and Safety of the Vero Cell-Derived Japanese Encephalitis Vaccine Encevac with Those of Mouse Brain-Derived Vaccine by Using the Beijing-1 Strain

Vaccines.The CC-JEV was manufactured according to the method described by Sugawara et al. (6). Briefly, Vero cells were passaged in a culture medium containing 2% fetal bovine serum and microcarriers. When cell numbers reached approximately 2 × 10⁶ cells/ml, cells cultured by microcarriers were isolated and inoculated with the Beijing-1 strain. A serum-free medium was added after virus adsorption, and thereafter, the cells were cultured for 4 days at 37°C. The culture supernatant was then harvested and concentrated by ultrafiltration. After inactivation with formalin, the vaccine antigens were purified by sucrose density gradient centrifugation, in addition to the manufacturing method used for the production of the MB-JEV. For the production of the CC-JEV, additional affinity column chromatography using Cellufine sulfate (JNC Corporation, Tokyo, Japan) was added to achieve further purification by removing residual host cell-derived proteins and DNAs from the final bulk vaccine. Two series of phase III clinical trials, trials 1 and 2, were conducted using the CC-JEV as a test vaccine. In trial 1, formulation H, which was a liquid-type vaccine containing 17 μg/dose of CC-JEV in 0.5 ml, was used. In trial 2, two freeze-dried types of the CC-JEV vaccine instead of the liquid-type vaccine were used to increase the stability; these were formulations L and M, containing 4 μg/dose and 8 μg/dose of CC-JEV in 0.5 ml, respectively. The MB-JEV, used as a comparator vaccine in both trials, was manufactured by the Chemo-Sero-Therapeutic Research Institute (Kaketsuken), Kumamoto, Japan, as follows: the Beijing-1 strain was inoculated into the brains of 4-week-old ddy strain mice. The brains of the mice showing symptoms of encephalitis were collected to prepare a brain emulsion. After centrifugation, the supernatant of the emulsion was treated with protamine sulfate to remove the mouse brain-derived materials. The virus fluid was recovered from the supernatant, and the supernatant was subjected to the sucrose density gradient centrifugation. After centrifugation, the virus particle faction was collected and then treated with formalin for inactivation in order to prepare an inactivated purified virus preparation. Finally, the total amount of protein of the MB-JEV was adjusted to 34 μg/ml. In both trials, the liquid MB-JVE containing 17 μg/dose of vaccine antigen in 0.5 ml was used as a comparator vaccine. The vaccine was administered subcutaneously.

Vaccination design.Two series of phase III clinical trials, trials 1 and 2, were conducted as double-blinded randomized parallel-group studies in multiple facilities. The subjects were children 6 to 89 months of age without a history of JE vaccine or of JEV infection. The main exclusion criteria were: (i) subjects with acute serious diseases, (ii) subjects who clearly presented anaphylaxis to vaccine components or excipients, (iii) subjects who had received a blood transfusion or administration of gamma-globulin preparation within 3 months before the start of the trial or who had received a massive dose therapy of gamma-globulin preparation (≥200 mg/kg of body weight) within 6 months before the start of the trial, and (iv) subjects for whom enrollment in the trial was judged by the principal investigator or subinvestigators as not appropriate. Written informed consent was obtained from a parent or guardian of each subject before trial enrollment. Prior to the conduction of the trials, the trial-related forms, such as the trial protocols, were investigated by the institutional review boards/independent ethics committees of the facilities conducting the trials or of the base hospital of the relevant network. The trials were conducted in compliance with the ethical doctrine based on the Helsinki Declaration, the good clinical practice regulations, and other relevant regulations.

Trial 1.Trial 1 was conducted from February 2003 to August 2004. A flowchart depicting the dispositions of the subjects in trial 1 is shown in Fig. 1. After obtaining informed consent, 468 qualified subjects were recruited for trial 1; among them, 235 and 233 subjects were assigned to two groups and were injected subcutaneously with a series of 2 doses of 0.5 ml of formulation H or the MB-JEV, respectively, at an interval of 1 to 4 weeks, and an additional injection was given 6 to 15 months after the 2nd injection. For the children <3 years of age, doses of 0.25 ml were given using the same vaccination schedule.

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FIG 1

Disposition of the subjects in trial 1. ^a, 8 subjects withdrew: 2 because of cancellations of the trial by parent or guardian of the subjects, 2 for the occurrence of adverse events, 1 because of change of residence, and 3 because of judgments by the primary investigator. ^b, 4 subjects withdrew: 3 because of cancellations of the trial by parent or guardian of the subjects and 1 because of a positive antibody titer before injection.

Trial 2.Trial 2 was conducted from June 2008 to May 2009. A flowchart depicting the dispositions of the subjects in trial 2 is shown in Fig. 2. After obtaining informed consent, 480 qualified subjects were recruited for trial 2; among them, 163, 158, and 159 subjects were assigned to three groups, and were injected subcutaneously with a dose of 0.5 ml of formulation M, formulation L, or the MB-JEV, respectively, at an interval of 2 to 4 weeks, and an additional injection was given 1 to 15 months after the 2nd injection. For the children <3 years of age, doses of 0.25 ml were given using the same vaccination schedule.

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FIG 2

Disposition of the subjects in trial 2. ^a, of the 484 randomized subjects, 4 subjects withdrew: 2 because of cancellations of the trial by parent or guardian of the subjects and 2 because of judgments by the investigator. ^b, of the 158 formulation M-injected subjects, 1 subject withdrew because a drug with a nonallocated number was injected after the 2nd injection. ^c, of the 157 formulation M-injected subjects, 2 withdrew: 1 because of cancellation of the trial by parent or guardian of the subject and 1 because of a positive antibody titer before injection.

Randomization.In both trials, randomizations were performed according to a computer-generated algorithm. Trial 1 was performed with a permuted-block design. Trial 2 was performed with a stratified randomization as a stratified factor of the vaccine volumes. The eligible subjects were randomly assigned in a 1:1 ratio for trial 1 and a 1:1:1 ratio for trial 2. In both trials, the statistician generating the randomization algorithm was not involved in determining the eligibility, vaccination course, or determining outcomes of the subjects.

Blinding.In trial 1, the test vaccine and the comparator vaccine were liquid-type vaccines with an identical appearance. However, in trial 2, the test vaccine was a freeze-dried type, while the comparator vaccine was a liquid type. Therefore, to ensure blinding, an investigational drug coordinator who prepared the investigational drug for a doctor and cleared it off after the inoculation instead of the doctor was specially assigned at each hospital.

Safety analysis.In trial 1, a parent or guardian of each subject recorded the presence or absence of adverse events in a health diary every day for 7 days after each inoculation. In trial 2, a parent or guardian of each subject recorded the presence or absence of adverse events in a health diary every day for 13 days after each inoculation. Adverse events were recorded until 27 days after inoculation when they were recognized. Regarding local reactions, including erythema or swelling at the injection site, a reaction of ≥2 cm in diameter was recorded as an adverse event in trial 1, and any reaction regardless of its size was recorded as an adverse event in trial 2. A fever of ≥37.5°C was recorded as an adverse event in both trials. In trial 1, the severity of adverse events was judged by the principal investigator or a subinvestigator and was classified into three levels, mild, moderate, and severe, considered for their interference with normal daily activities. Similarly, in trial 2, the criteria were defined in terms of grades 1 to 4, according to severity. Adverse events for which a possible relationship with one of the test vaccines could not be denied were judged as vaccine-related adverse events.

Immunological analysis.In trial 1, blood serum samples for the measurement of antibody titers were obtained from each subject before the 1st injection and 2 to 6 weeks after the 2nd and 3rd injections. In trial 2, serum samples were obtained from each subject before the 1st injection and 4 to 6 weeks after the 2nd and 3rd injections. The sera were stored at −20°C until the time of measurement. Neutralizing antibodies against a JE virus strain, Beijing-1, were measured by a 50% plaque reduction method using Vero cells and calculated using the 3 points least-squares regression method (3LSRM) (12). Briefly, Vero cells grown in six-well plates (Costar six-well cell culture cluster, flat bottom, with lid, catalog no. 3506; Corning Incorporated, Corning, NY, USA) were used. Serial dilutions of the serum samples were carried out routinely to 1:10, 1:40, 1:160, 1:640, 1:2,560, and 1:10,240, and the challenge virus (Beijing-1 strain) was also diluted to give 100 plaques per well. One more dilution was added and the rest repeated if there was not a 50% plaque reduction. The same volume of diluted serum samples and virus were mixed and added to the cell-seeded wells in triplicate. The control virus was added to 12 wells. The plates were then incubated for 90 min at 37°C, and overlay medium was added. Following 5 days at 37°C incubation, 10% formalin was added, followed by methylene blue tetrahydrate to stain the virus plaques. If the average value of the number of plaques in ≥10 control wells was between 50 and 150, the assay was accepted. The neutralizing antibody titer was expressed as the reciprocal of the dilution of serum that caused a 50% reduction of plaque formation compared to the plaque number of the diluted challenge virus in the absence of antiserum. Antibody positive was defined as the neutralizing antibody titer being ≥1:10 (13).

Statistical analysis of antibody titers.In both trials, the primary endpoint was the seroconversion rate after the 3rd injection based on the neutralizing antibody titer. The secondary endpoints were the seroconversion rate after the 2nd injection and the geometric mean antibody titers (GMTs) after the 2nd and 3rd injections. The per-protocol set (PPS) was used to represent the immunogenicity population. Statistical analyses were performed using SAS version 8.2 for trial 1 and SAS version 9.1 for trial 2.

Trial 1.In trial 1, the noninferiority in the seroconversion rate after the 3rd injection for the formulation H-injected group (group H) against the MB-JEV group (control group) was statistically analyzed by the method of Dunnett and Gent (14). A sample size of 100 subjects per group was calculated to verify the noninferiority in the seroconversion rate (assumed to have an expected seroconversion rate of 95%) in each group (α = 0.025 [1-tailed test], β = 0.10, 90% power, and noninferiority limit δ = 10%). Assuming that the seropositive rate was 45% before the trial and the withdrawal rate was 10%, 204 subjects in each group, for a total of 408 subjects, were required.

Trial 2.In trial 2, the noninferiority in the seroconversion rates after the 3rd injection for the formulation L-injected group (group L) and the formulation M-injected group (group M) against the control group were statistically analyzed by the Farrington-Manning method (15). A sample size of 81 subjects for each group was calculated to verify noninferiority (assumed to have an expected seroconversion rate of 98% against JE virus after the 3rd injection in both groups (α = 0.025 [1-tailed test], β = 0.10, 90% power, and noninferiority limit δ = 10%). Assuming that the seropositive rate was 10% before the trial and that the withdrawal rate was 10%, 100 subjects in each group, for a total 300 subjects, were required.