Vaccine

The inactivated EV71 vaccine was developed by the Institute of Medical Biology, Chinese Academy of Medical Sciences.14,15 Briefly, the vaccine was prepared from an EV71 strain of genotype C4 isolated during a pandemic in Fuyan, China, in 2008.16 It was cultured in a human diploid-cell line (KMB17 strain) for proliferation,15 followed by purification and inactivation.

The vaccine contained 100 U of inactivated EV71 viral antigen adsorbed to 0.5 mg of aluminum hydroxide and suspended in 0.5 ml of buffered saline. The same volume of aluminum hydroxide in buffered saline without antigen was used as a placebo. Both the vaccine and placebo were prepared in a facility that was compliant with Good Manufacturing Practices and were tested by the National Institutes for Food and Drug Control before the start of the study.

Study Design and Participants

This randomized, double-blind, placebo-controlled clinical study was designed by the Institute of Medical Biology, the Center for Drug Evaluation in the State Food and Drug Administration, and the Center for Disease Control and Prevention of the Guangxi Zhuang Autonomous Region (Guangxi CDC). The study protocol was approved by an independent ethics committee of Guangxi Zhuang Autonomous Region. Data were collected by the Guangxi CDC with the use of EpiData software, version 3.1 (EpiData Association).

All the authors and trial collaborators vouch for the accuracy and completeness of the data presented and for the fidelity of this report to the study protocol, available with the full text of this article at NEJM.org. Data were transferred to the Fourth Military Medical University and were analyzed by biostatisticians at the Fourth Military Medical University and Fudan University. The study was conducted from March 2012 through February 2013 in seven counties in the Guangxi Zhuang Autonomous Region.

Healthy children, 6 to 71 months of age, whose parent or legal guardian provided written informed consent, were eligible for enrollment in this study. The exclusion criteria are listed in the Supplementary Appendix, available at NEJM.org. All eligible participants were stratified according to age (6 to 23 months vs. 24 to 71 months). Randomization was performed in a 1:1 ratio in blocks of eight. Identical labels with computer-generated random numbers were used on all study-agent vials. Two doses of either vaccine or placebo were administered intramuscularly, with a 4-week interval between doses.

A subgroup of participants in the large-scale study was randomly selected for the evaluation of immunogenicity. Blood samples were obtained from each selected participant at baseline and on days 56 and 180 after the initial injection to evaluate the production of the anti-EV71 neutralizing antibody.

Safety Assessment

The safety observation included close monitoring for immediate adverse events after each injection, and reports of the local and systemic reactions were solicited and recorded daily on diary cards until 7 days after each injection (see the Supplementary Appendix). Any other symptoms or signs occurring during a 28-day follow-up period after each injection were recorded as unsolicited symptoms or signs.

A serious adverse event was defined as any new health-related problem that resulted in death, was life-threatening, necessitated hospitalization or prolongation of existing hospitalization, or resulted in disability or incapacity. Serious adverse events were recorded throughout the entire study period. Parents and legal guardians were asked to contact the investigator immediately in the event of a serious adverse event. An independent data and safety monitoring board whose members were unaware of the study assignments periodically reviewed all reports of serious adverse events to assess the causality of the events and to determine associated secondary diagnoses and other underlying conditions.

Evaluation of Efficacy

A suspected case of hand, foot, and mouth disease was defined as febrile illness (body temperature >37.5°C) accompanied by a papular or vesicular rash in the characteristic distribution on the oral mucosa, hands, feet, or buttocks. A case of EV71-associated hand, foot, and mouth disease was defined as a suspected case of hand, foot, and mouth disease with EV71 detected in a throat swab or stool specimen with the use of a quantitative reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay. A severe case of EV71-associated hand, foot, and mouth disease was defined as a suspected case of hand, foot, and mouth disease caused by EV71 that was associated with neurologic, respiratory, or circulatory complications, on the basis of the diagnostic criteria for hand, foot, and mouth disease published by the Ministry of Health of China (see the Supplementary Appendix).17

A health care center–based surveillance system was set up to facilitate case-finding efforts. Suspected cases of hand, foot, and mouth disease were reported by means of the surveillance system or by parents. In addition, village doctors contacted all the participants once every 2 weeks, either by means of a home visit or telephone call. Participants with a suspected case of hand, foot, and mouth disease were referred to the county or city hospital for confirmation.

The throat swabs and stool specimens from participants with suspected cases were screened with the use of quantitative RT-PCR in the central laboratory at the Guangxi CDC, and the results were confirmed by the National Institutes for Food and Drug Control. Serum specimens were obtained once during the acute phase (≤3 days after onset) and once during convalescence (approximately 10 to 15 days after onset). The data and safety monitoring board reviewed the clinical and laboratory results and confirmed the diagnosis of EV71-associated hand, foot, and mouth disease before the data were unblinded.

Laboratory Analysis

Viral RNA was extracted from stool samples with the use of an RNeasy Mini Kit (Qiagen) for EV71 and coxsackievirus A16 and was identified by means of real-time RT-PCR with standard primers for EV71, coxsackievirus A16, and enterovirus.18 The EV71 genome was sequenced to identify the genotype.1 The level of EV71-specific neutralizing antibodies was measured in serum samples by means of a microneutralization assay on Vero cells grown in 96-well plates with the use of a standard viral strain provided by the National Institutes for Food and Drug Control.18

End Points

The primary end point was efficacy against EV71-associated hand, foot, and mouth disease according to the case definition described above. Efficacy was assessed from 2 weeks after completion of the two-dose schedule until 1 year after receipt of the first dose. The secondary end points were efficacy against severe hand, foot, and mouth disease within the same study period and the proportion of participants in whom EV71-neutralizing antibodies developed at 56 days and 180 days after the initial vaccination.

Data Management

Data were double-entered into custom-made data-entry programs (EpiData). The analysis of the primary end point was performed according to the intention-to-treat principle and included participants who received at least one dose of vaccine or placebo. A per-protocol analysis was also performed that included enrolled participants who received two doses of the vaccine and completed the follow-up observation 12 months after the initial injection.

Adverse events were summarized for all participants who received at least one dose of study agent (vaccine or placebo). For the assessment of immunogenicity, because preexisting EV71-neutralizing antibodies were detected in some participants, the data from all participants were analyzed in the intention-to-treat analysis and the data from those with a titer of EV71-neutralizing antibody that was less than 1:8 were analyzed in the per-protocol analysis.

Statistical Analysis

On the basis of the preceding 3-year surveillance of the entire Guangxi Zhuang Autonomous Region, the incidence of EV71-associated hand, foot, and mouth disease among children younger than 5 years of age was estimated to be 5 cases per 1000 children per year (see the Supplementary Appendix). Assuming a vaccine efficacy of 75%, and allowing for a withdrawal rate of 20%, we calculated that a sample of 5600 participants per group would be needed for the study to have 90% power to detect a difference in the primary end point between the vaccine group and the placebo group, at a two-tailed alpha level of 0.05. Because the withdrawal rate was low and similar in the two groups, the Mantel–Haenszel chi-square method was used for the comparison of vaccine efficacy, which was assessed as the difference in the proportions of children in the two groups with EV71-associated hand, foot, and mouth disease.

Student's t-test or the Mann–Whitney U test (for nonnormally distributed data) was used for the analysis of dimensional outcomes, and the chi-square test or Fisher's exact test (when data were sparse) was used for the analysis of dichotomous outcomes; all tests were two-tailed. For analyses of serum anti-EV71 antibodies, titers of less than 1:8, which was the threshold of detection, were assigned an arbitrary value of 1:4. In the statistical analyses of antibody titers, the titers were logarithmically converted to allow the assessment of geometric mean titers. SAS software, version 9.1 (SAS Institute), was used for statistical analysis.