Trial Oversight

In this phase 3, double-blind, randomized, placebo-controlled trial, we enrolled healthy children and adolescents at 26 sites in which dengue is endemic in Brazil (4 sites), Colombia (4), the Dominican Republic (2), Nicaragua (1), Panama (4), the Philippines (4), Sri Lanka (4), and Thailand (3); participants received their first injections between September 2016 and March 2017. The trial is being conducted in accordance with the Declaration of Helsinki and the International Council for Harmonisation Tripartite Guidelines for Good Clinical Practice, as well as in accordance with applicable local regulations. Informed assent or consent forms and the trial protocol and its amendments (available with the full text of this article at NEJM.org) were reviewed and approved by institutional review boards, independent ethics committees, and health authorities. Written informed assent or consent was obtained from all participants or their parents or legal guardians before enrollment. During the trial, consent was obtained again from participants when they legally became adults. At the time of this analysis, the process of obtaining repeat consent for some participants was ongoing. Should any of these participants decline to provide repeat consent, data that were collected after they had reached legal adult age will be removed from future analyses.

The trial sponsor, Takeda Vaccines, is responsible for the overall trial design (taking into consideration the investigators’ input), trial site selection, and data analysis. The trial investigators are responsible for data collection and day-to-day trial site management. To maintain blinding in this ongoing trial, certain authors employed by the sponsor, including a statistician, and the medical writers had access to group- and individual-level trial data and vouch for the accuracy and completeness of the data. Other authors had access only to the data presented in this article. All the authors vouch for the fidelity of the trial to the protocol. Medical writers at OLC Bioscience who were paid by the sponsor prepared the first draft of the manuscript on the basis of an outline previously agreed on by all the authors. All the authors provided critical input during manuscript preparation and approved the submitted version. An independent data and safety monitoring committee has access to unblinded safety data on request.

Participants, Randomization, and Blinding

Children and adolescents 4 to 16 years of age who met the trial entry criteria were randomly assigned in a 2:1 ratio to receive two doses of vaccine or placebo, 3 months apart. Randomization was stratified according to region (Asia-Pacific region or Latin America) and age (4 to 5 years, 6 to 11 years, or 12 to 16 years). A subpopulation of 4000 of the 20,099 participants who underwent randomization was randomly selected for additional safety and immunogenicity assessments. During the trial, investigators, participants and their parents or guardians, and representatives of the sponsor who advise on trial conduct remain unaware of the trial-group assignments. One or more designated pharmacists or vaccine administrators at each site are aware of the trial-group assignments but have no role in the collection or assessment of participant safety data.

Trial Vaccine and Placebo

The lyophilized vaccine formulation was reconstituted before administration. One 0.5-ml dose of TAK-003 contained approximately 3.6, 4.0, 4.6, and 5.1 log 10 plaque-forming units of TDV-1, TDV-2, TDV-3, and TDV-4, respectively. The placebo was a 0.5-ml injection of saline. Vaccine and placebo were administered subcutaneously into the upper arm. The lyophilized vaccine kits were kept at 2 to 8°C during shipping and storage.

Trial Procedures

This ongoing trial consists of three parts for each participant, with active surveillance during parts 1 and 2 and modified active surveillance during part 3 (Fig. S1 in the Supplementary Appendix, available at NEJM.org). Participants or their parents or guardians are contacted at least weekly for the entire trial duration to remind them to present for evaluation of febrile illness (defined as body temperature ≥38°C in any 2 of 3 consecutive days) to ensure identification of dengue cases. Part 1 was complete after 120 cases of virologically confirmed dengue had been confirmed for the analysis of the primary end point and participants had had 12 months of follow-up after the second vaccination. Data from part 1 are reported here. Part 2 lasts for another 6 months for the assessment of secondary efficacy end points, to be followed by an additional 3 years in part 3 for the evaluation of long-term efficacy and safety.

During active surveillance, participants presenting with febrile illness or clinically suspected dengue have blood samples taken in the acute phase (i.e., as soon as possible and preferably within 5 days after fever onset) and convalescent phase (i.e., 7 to 14 days after the acute-phase specimen is obtained). Testing includes quantitative serotype-specific reverse-transcriptase polymerase chain reaction (RT-PCR); enzyme-linked immunosorbent assay (ELISA) for dengue NS1, IgM, and IgG; and assessment of hematocrit, liver enzymes (aspartate aminotransferase and alanine aminotransferase), and platelet counts. RT-PCR and NS1 ELISA are performed only on the acute-phase specimen. Febrile illnesses are evaluated clinically, and additional tests can be performed in accordance with the local standard of care.

For the efficacy analyses, virologically confirmed dengue is defined as febrile illness or illness clinically suspected to be dengue by the investigator in association with a positive serotype-specific RT-PCR result. The severity of virologically confirmed dengue is assessed with the use of two approaches: blinded review by the dengue case adjudication committee, using predefined criteria, and with a program for analyzing data in accordance with World Health Organization (WHO) 1997 criteria for dengue hemorrhagic fever.7 Blood specimens were obtained from all participants on day 1 (before vaccination) and day 120 to measure levels of dengue-neutralizing antibodies by microneutralization testing. Additional blood specimens for microneutralization testing are obtained on days 30, 90, 270, and 450 and then annually from the participants in the safety subpopulation. Safety assessments in the safety subpopulation included assessment of local reactions and systemic adverse events for 7 or 14 days, respectively, and of unsolicited adverse events for 28 days after each vaccination. Data on serious adverse events were collected for all trial participants. Additional details of our methods and procedures are provided in the Supplementary Appendix and the protocol.

Outcomes

The primary end point was the vaccine efficacy of two doses of TAK-003 for the prevention of virologically confirmed dengue induced by any dengue virus serotype from 30 days after the second injection until the end of part 1 of the trial. Secondary vaccine efficacy end points included efficacy against individual dengue virus serotypes, efficacy according to baseline serostatus, and efficacy for the prevention of dengue leading to hospitalization and the prevention of severe dengue until the end of part 2 of the trial. Although the full assessment of these secondary end points is planned to occur after part 2, we report here on a planned exploratory analysis of vaccine efficacy in subgroups of interest based on cases of virologically confirmed dengue that occurred during part 1.

Statistical Analysis

Analysis of the primary end point was performed with the per-protocol population, which included all participants who did not have any major protocol violations. Vaccine efficacy is defined as 1minus the hazard ratio (vaccine vs. placebo). Hazard ratios and corresponding 95% confidence intervals were estimated with a Cox proportional-hazards model that included trial group as a factor, with adjustment for age and stratification according to region. The primary vaccine efficacy objective was considered to be met if the lower bound of the 95% confidence interval for vaccine efficacy was above 25%. Additional analyses were performed with the per-protocol population, safety population, full analysis population, or safety and immunogenicity subpopulations (definitions are provided in the Supplementary Appendix). The sample size calculation was based on the assumption of a true vaccine efficacy of 60% and a background annual dengue incidence of 1%. We calculated that a sample of 20,100 participants undergoing randomization in a 2:1 ratio (TAK-003:placebo) would enable identification of 120 cases of virologically confirmed dengue from 30 days after the second vaccination to the end of part 1, providing at least 90% power to rule out a vaccine efficacy of 25% or less (with a two-sided significance level of 0.05).