An Inactivated Enterovirus 71 Vaccine in Healthy Children

original article An Inactivated Enterovirus 71 in Healthy Children Rongcheng Li, B.S., Longding Liu, Ph.D., Zhaojun Mo, M.Sc., Xuanyi Wang, M.D., Ph.D., Jielai Xia, Ph.D., Zhenglun Liang, M.D., Ph.D., Ying Zhang, Ph.D., Yanping Li, B.S., Qunying Mao, M.Sc., Jingjing Wang, M.Sc., Li Jiang, B.S., Chenghong Dong, B.S., Yanchun Che, M.Sc., Teng Huang, M.Sc., Zhiwei Jiang, Ph.D., Zhongping Xie, B.S., Lichun Wang, B.S., Yun Liao, B.S., Yan Liang, Ph.D., Yi Nong, B.S., Jiansheng Liu, M.Sc., Hongling Zhao, B.S., Ruixiong Na, B.S., Lei Guo, Ph.D., Jing Pu, B.S., Erxia Yang, B.S., Le Sun, M.Sc., Pingfang Cui, B.S., Haijing Shi, M.Sc., Junzhi Wang, Ph.D., and Qihan Li, M.D., Ph.D. ABSTRACT BACKGROUND Enterovirus 71 (EV71) is a major cause of hand, foot, and mouth disease in children and may be fatal. A vaccine against EV71 is needed. METHODS We conducted a randomized, double-blind, placebo-controlled phase 3 trial involving healthy children 6 to 71 months of age in Guangxi Zhuang Autonomous Region, China. Two doses of an inactivated EV71 vaccine or placebo were administered intramuscularly, with a 4-week interval between doses, and children were monitored for up to 11 months. The primary end point was protection against hand, foot, and mouth disease caused by EV71. RESULTS A total of 12, children were randomly assigned to receive vaccine or placebo. Serum neutralizing antibodies were assessed in 549 children who received the vaccine. The seroconversion rate was 1% 4 weeks after the two vaccinations, with a geometric mean titer of 17.6. Over the course of two epidemic seasons, the vaccine efficacy was 97.4% (95% confidence interval [CI], 92.9 to 99.) according to the intention-to-treat analysis and 97.3% (95% CI, 92.6 to 99.) according to the perprotocol analysis. Adverse events, such as fever (which occurred in 41.6% of the participants who received vaccine vs. 35.2% of those who received placebo), were significantly more common in the week after vaccination among children who received the vaccine than among those who received placebo. CONCLUSIONs The inactivated EV71 vaccine elicited EV71-specific immune responses and protection against EV71-associated hand, foot, and mouth disease. (Funded by the National Basic Research Program and others; ClinicalTrials.gov number, NCT1569581.) From Guangxi Province Center for Disease Control and Prevention, Nanning (R.L., Z.M., Y. Li, T.H., Y.N.), Yunnan Key Laboratory of Research and De velopment on Severe Infectious Dis eases, Institute of Medical Biology, Chinese Academy of Med i cal Sciences and Peking Union Medical College, Kunming (L.L., Y.Z., Jingjing Wang, L.J., C.D., Y.C., Z.X., L.W., Y. Liao, Y. Liang, J.L., H.Z., R.N., L.G., J.P., E.Y., L.S., P.C., H.S., Q.L.), Key Laboratory Medical Molec ular Virology, Ministries of Education and Health, and the Institutes of Biomedical Science, Shanghai Medical College, Fudan University, Shanghai (X.W.), Department of Health Statistics, Fourth Military Medical University, Xi an (J.X., Z.J.), and National Institutes for Food and Drug Control, Beijing (Z.L., Q.M., Junzhi Wang) all in China. Address reprint requests to Dr. Qihan Li at the Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, 935 Jiaoling Rd., Kunming, Yunnan, 65118 China, or at liqihan@ imbcams.com.cn, or to Dr. Junzhi Wang at the National Institutes for Food and Drug Control, No. 2 Tiantanxili, Beijing, 15 China, or at wangjz@nicpbp.org.cn. Mr. R. Li, Dr. Liu, Mr. Mo, and Drs. X. Wang, Xia, and Liang contributed equally to this article. N Engl J Med 214;37:829-37. DOI: 1.156/NEJMoa133224 Copyright 214 Massachusetts Medical Society. n engl j med 37;9 nejm.org february 27, 214 829 Downloaded from nejm.org on July 12, 218. For personal use only. No other uses without permission. Copyright 214 Massachusetts Medical Society. All rights reserved.

Epidemics of hand, foot, and mouth disease in children have emerged recently in Asia and have been caused primarily by enterovirus 71 (EV71) and coxsackievirus A16, 1 which typically show two peak epidemic incidences each year, in May and October. 2-5 An important clinical concern regarding hand, foot, and mouth disease is central nervous system injury, which occurs during the disease course in some severe cases and may result in a poor outcome. 6-11 Infection with the EV71 C4 genotype accounts for 4.1 to 55.4% of cases of hand, foot, and mouth disease, with considerable associated mortality, including thousands of deaths in China. 3,9,12,13 To reduce morbidity, an EV71 vaccine is needed. 2-5 A human diploid-cell based inactivated EV71 C4 genotype vaccine is being developed and has shown evidence of safety and immunogenicity in phase 1 and 2 studies. 14 We conducted a doubleblind, randomized, placebo-controlled, phase 3 clin ical trial to evaluate the protection induced by this vaccine against EV71-associated hand, foot, and mouth disease in children 6 to 71 months of age. METHODS 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 28. 16 It was cultured in a human diploid-cell line (KMB17 strain) for proliferation, 15 followed by purification and inactivation. The vaccine contained 1 U of inactivated EV71 viral antigen adsorbed to.5 mg of aluminum hydroxide and suspended in.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 212 through February 213 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 computergenerated 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 18 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 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 ad- 83 n engl j med 37;9 nejm.org february 27, 214 Downloaded from nejm.org on July 12, 218. For personal use only. No other uses without permission. Copyright 214 Massachusetts Medical Society. All rights reserved.

Inactivated Enterovirus 71 in Healthy Children verse 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 reversetranscriptase 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 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 1 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 18 days after the initial vaccination. DATA MANAGEMENT Data were double-entered into custom-made dataentry 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 n engl j med 37;9 nejm.org february 27, 214 831 Downloaded from nejm.org on July 12, 218. For personal use only. No other uses without permission. Copyright 214 Massachusetts Medical Society. All rights reserved.

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 1 children per year (see the Supplementary Assuming a vaccine efficacy of 75%, and allowing for a withdrawal rate of 2%, we calculated that a sample of 56 participants per group would be needed for the study to have 9% 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.5. 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 14,445 Patients were assessed for eligibility 2445 Were excluded 713 Had parent or guardian who did not give consent 1732 Did not fulfill eligibility criteria 12, Underwent randomization 11 Underwent randomization in immunogenicity subgroup 6 Were assigned to vaccine group (first dose) 6 Were assigned to placebo group (first dose) 1 Was excluded owing to no serum sample provided at baseline 514 Discontinued study 2 Had protocol violation 469 Had parent or guardian who declined second dose 43 Missed second dose 5486 Received second dose 498 Discontinued study 2 Had protocol violation 47 Had parent or guardian who declined second dose 26 Missed second dose 552 Received second dose 549 Were assigned to vaccine group 148 Discontinued study 57 Were infected before enrollment 91 Had only one sample after baseline 55 Were assigned to placebo group 148 Discontinued study 65 Were infected before enrollment 83 Had only one sample after baseline 5 Were excluded owing to ineligible age at enrollment 3 Were excluded owing to ineligible age at enrollment 41 Were included in the immunogenicity analysis 42 Were included in the immunogenicity analysis 5481 Were included in per-protocol analysis (second dose) 5499 Were included in per-protocol analysis (second dose) Figure 1. Screening, Randomization, and Follow-up. A total of 14,445 children, 6 to 71 months of age, were assessed for eligibility, and 12, eligible participants were randomly assigned to receive vaccine or placebo. After an 11-month follow-up, 5481 participants in the vaccine group and 5499 in the placebo group were included in the final per-protocol analysis. In the immunogenicity subgroup, the procedure was the same as that for the entire study population, except that blood samples were obtained at baseline and at days 56 and 18 to evaluate the production of the anti-ev71 neutralizing antibody. 832 n engl j med 37;9 nejm.org february 27, 214 Downloaded from nejm.org on July 12, 218. For personal use only. No other uses without permission. Copyright 214 Massachusetts Medical Society. All rights reserved.

Inactivated Enterovirus 71 in Healthy Children 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. RESULTS STUDY PARTICIPANTS A total of 14,445 children, 6 to 71 months of age, were evaluated for inclusion in the study. Of these, 2445 children were excluded: 1732 children were ineligible, and 713 had parents who declined to have them participate. We randomly assigned 12, participants in a ratio of 1:1 to receive the vaccine or placebo; these participants composed the data set for the intention-to-treat analysis (Fig. 1). From March 212 through February 213, all the participants were monitored for hand, foot, and mouth disease after receipt of the study agent. The withdrawal rate was 8.4%; a total of 514 participants in the vaccine group and 498 in the placebo group missed the second dose, had a protocol violation, or were lost to follow-up. After the exclusion of 5 children in the vaccine group and 3 in the placebo group who did not meet the age criteria, 5481 children in the vaccine group and 5499 in the placebo group were included in the per-protocol analysis (Fig. 1). The demographic characteristics of the participants were similar in the two groups (Table 1, and Table S1 in the Supplementary A total of 594 suspected cases of hand, foot, and mouth disease were reported by the surveillance system or by parents or guardians over the course of the study period in the catchment area; 7.1% of the cases occurred between May and July and were confirmed by the data and safety monitoring board before unblinding of the data. A total of 22 cases were identified in the vaccine group and 392 in the placebo group. END POINTS Using sequencing, we identified 155 cases of hand, foot, and mouth disease caused by EV71 (4 cases in the vaccine group and 151 in the placebo group), 12 caused by coxsackievirus A16 (48 and Table 1. Demographic Characteristics of the Participants Included in the Intention-to-Treat Analysis.* Characteristic Efficacy Cohort Immunogenicity Subgroup (N = 6) (N = 6) (N = 549) (N = 55) Sex no. (%) Male 3151 (52.5) 399 (51.6) 279 (5.8) 296 (53.8) Female 2849 (47.5) 291 (48.4) 27 (49.2) 254 (46.2) Age at entry mo Mean 23.7±15.2 23.7±15.2 21.2±12.9 21.3±12.8 Range 6. 71.9 6.1 71.9 6.1 71.3 6.1 69.1 Weight at entry kg Mean 11.9±3.1 11.9±3.1 11.3±2.7 11.4±2.8 Range 4.2 33.6 4.5 3. 5. 22. 5. 23. Ethnic group no (%) Han 5278 (88.) 521 (86.7) 494 (9.) 477 (86.7) Zhuang 434 (7.2) 485 (8.1) 28 (5.1) 38 (6.9) Yao 27 (3.4) 227 (3.8) 22 (4.) 27 (4.9) Miao 27 (.4) 31 (.5) 1 (.2) 5 (.9) Other 54 (.9) 56 (.9) 4 (.7) 3 (.5) * Plus minus values are means ±SD. There were no significant between-group differences at baseline. The intention-totreat analysis included data from all participants who received at least one dose of vaccine or placebo. Ethnic group was reported by a parent or guardian and verified by a study investigator. n engl j med 37;9 nejm.org february 27, 214 833 Downloaded from nejm.org on July 12, 218. For personal use only. No other uses without permission. Copyright 214 Massachusetts Medical Society. All rights reserved.

54, respectively), and 234 caused by other enteroviruses (16 and 128, respectively) (Table 2, and Table S2 in the Supplementary The vaccine efficacy against EV71-associated hand, foot, and mouth disease was 97.4% (95% confidence interval [CI], 92.9 to 99.) in the intentionto-treat analysis (Table 2) and 97.3% (95% CI, 92.6 to 99.) in the per-protocol analysis (Table S2 in the Supplementary The vaccine showed no efficacy against hand, foot, and mouth disease caused by coxsackievirus A16 or other enteroviruses (Table 2, and Table S2 in the Supplementary Among the 155 cases of EV71-associated hand, foot, and mouth disease, 2 cases were severe, both of which occurred in the placebo group; 1 of the participants with a severe case died (Table S3 in the Supplementary The cumulative incidence over the study period is shown in Figure 2. The genotypes of 87 viral genomes from 155 samples were identified as C4 by means of sequencing, and the rest remained unidentified owing to failure of routine RT-PCR to detect the entire sequence of VP1, the gene encoding a capsid protein, for phylo genetic analysis (Fig. S3 in the Supple mentary In a subgroup analysis, the vaccine efficacy against EV71-associated hand, foot, and mouth disease was similar in children 6 to 23 months of age and in those 24 to 71 months of age (Table 2). Among participants who received only one dose of study agent (vaccine or placebo), 2 patients with EV71-associated hand, foot, and mouth disease were identified among 498 placebo recipients, as compared with none among 514 vaccine recipients. Three patients in the placebo group who received both doses (and who did not meet the age criteria) had infectious EV71-associated hand, foot, and mouth disease within 56 days after receipt of the first injection; these patients were excluded from the per-protocol analyses (Fig. 2, and Fig. S1 in the Supplementary ANTIBODY ASSESSMENTS Antibody responses were measured in a prespecified subgroup of participants. After the neutralizing-antibody assessment at baseline, 57 children in the vaccine group and 65 in the placebo group who were positive for anti-ev71 antibodies were excluded from the immunogenicity analysis. The seropositive rates for serum EV71-neutralizing antibodies at days 56 and 18 were significantly higher among participants who received the vaccine than among those who received placebo (intention-to-treat analyses: 87.6% vs. 4.1% at day 56, and 88.% vs. 26.4% at day 18; per-protocol analyses: 1% vs. 3.5% at day 56, and 99.3% vs. 3.4% at day 18; P<.1 for all comparisons). Similarly, children in the vaccine group had higher titers of neutralizing antibody than did children in the placebo group. The antibody levels in vaccine recipients peaked 4 weeks after the administration of the second dose, with geometric mean titers of 17.1 Table 2. Efficacy of the Enterovirus 71 (EV71) against Overall Hand, Foot, and Mouth Disease and EV71-Associated Hand, Foot, and Mouth Disease over an 11-Month Period, According to the Intention-to-Treat Analysis. Cases of Hand, Foot, and Mouth Disease Clinically diagnosed and pathogenically confirmed cases (N = 6) Participants Incidence Participants Incidence no. of cases/ 1 participants/yr (N = 6) Efficacy* P Value no. of cases/ 1 participants/yr % (95% CI) Caused by EV71 no. 4.7 151 25.2 97.4 (92.9 to 99.) <.1 Age 6 23 mo no./total no. 2/35.6 94/35 26.9 97.9 (91.4 to 99.5) <.1 Age 24 72 mo no./total no. 2/25.8 57/25 22.8 96.5 (85.6 to 99.1) <.1 Caused by coxsackievirus A16 no. 48 8. 54 9. 11.1 ( 3.8 to 39.6).55 Caused by other enterovirus no. 16 17.7 128 21.3 17.2 ( 6. to 35.8).15 Clinically diagnosed cases no. 22 33.7 392 65.3 48.5 (39.2 to 56.3) <.1 * The calculation of overall vaccine efficacy was adjusted for study center. 834 n engl j med 37;9 nejm.org february 27, 214 Downloaded from nejm.org on July 12, 218. For personal use only. No other uses without permission. Copyright 214 Massachusetts Medical Society. All rights reserved.

Inactivated Enterovirus 71 in Healthy Children (intention-to-treat analysis) and 17.6 (per-protocol analysis). At day 18, levels had declined to 65.8 (intention-to-treat analysis) and 88.3 (perprotocol analysis). There were no significant differences in anti-ev71 antibody titers between vaccine recipients who were 6 to 23 months of age and those who were 24 to 71 months of age (P =.92) either at day 56 or at day 18 (Table S4 in the Supplementary ADVERSE EVENTS Most of the adverse events were mild. A total of 68 serious adverse events were reported in the vaccine group, as compared with 125 in the placebo group (P<.1); 41 participants in the vaccine group were hospitalized for hand, foot, and mouth disease, as compared with 88 in the placebo group (P<.1). All the patients recovered within 3 days after hospitalization. One participant in the placebo group died owing to severe EV71-associated hand, foot, and mouth disease; this child died within 24 hours after arrival at the hospital and was not considered to be hospitalized. Two injection-associated serious adverse events were identified in each group. One patient in the vaccine group died owing to a traffic accident (Table 3). Within 7 days after each injection, systemic adverse events (according to solicited reports) occurred in 48.6% of the participants in the vaccine group, as compared with 42.9% of those in the placebo group (P<.1). The most common reactions were fever, diarrhea, nausea, and vomiting. The proportion of local adverse events 1..9 Proportion of Participants without EV71-Associated Hand, Foot, and Mouth Disease.8.7.6.5.4.3.2.1 1..99.98.97.96.95.94.93.92.91.9 efficacy, 97.4% (95% CI, 92.9 99.) P<.1. 1 2 3 4 5 6 7 8 9 1 11 12 No. at Risk. 1 2 3 4 5 6 7 8 9 1 11 12 Months since First Dose 6 6 6 6 6 598 5999 5914 588 5862 5854 5854 5853 5996 585 5996 5849 5996 5849 No. of Events 2 1 66 2 34 18 8 1 1 3 1 Figure 2. Cumulative Hazard of Hand, Foot, and Mouth Disease Caused by Enterovirus 71 (EV71), According to an Intention-to-Treat Analysis. The cumulative hazard of EV71-associated hand, foot, and mouth disease was estimated as a minus-log transformation of the Kaplan Meier survival curve during the period from the receipt of the first dose until 11 months after the initial dose among all participants who received at least one dose of either vaccine or placebo. The inset shows the same data on an enlarged y axis. n engl j med 37;9 nejm.org february 27, 214 835 Downloaded from nejm.org on July 12, 218. For personal use only. No other uses without permission. Copyright 214 Massachusetts Medical Society. All rights reserved.

(solicited reports) was higher in the vaccine group than in the placebo group (5.9% vs. 2.3%, P<.1). Local adverse events included pain, redness, swelling, and itching at the injection site (Table 3, and Table S5 in the Supplementary DISCUSSION In this randomized, double-blind, placebo-controlled trial, which included 12, children 6 to 71 months of age, the inactivated EV71 vaccine was protective against EV71-associated hand, foot, and mouth disease after two doses of the vaccine, with 97.4% efficacy over an observation period of 11 months, which covered two peak epidemic seasons, in May and October. These epidemic peaks were similar to the epidemic trends of hand, foot, and mouth disease reported during the previous 3 years in the Guangxi region (Fig. S2 in the Supplementary No significant difference in efficacy was identified in children 6 to 23 months of age versus those 24 to 71 months of age. Children who received only a single dose of vaccine may have benefited (no cases of EV71-associated hand, foot, and mouth disease in the vaccine group vs. Table 3. Adverse Events and Serious Adverse Events. Event All Adverse Events Adverse Events of Grade 3 or Higher* (N = 6) (N = 6) P Value (N = 6) (N = 6) P Value no. of participants with event (%) no. of participants with event (%) Adverse event 7 days after injection Systemic event 2916 (48.6) 2574 (42.9) <.1 Fever 2498 (41.6) 2111 (35.2) <.1 147 (2.4) 149 (2.5).95 Diarrhea 498 (8.3) 535 (8.9).24 8 (.1) 12 (.2).5 Nausea, vomiting, or anorexia 53 (8.8) 475 (7.9).8 6 (.1) 7 (.1) 1. Irritability, drowsiness, or weakness 36 (6.) 33 (5.).3 3 (<.1) 7 (.1).34 Allergy 166 (2.8) 156 (2.6).61 2 (<.1).5 Local event 356 (5.9) 138 (2.3) <.1 Pain 211 (3.5) 8 (1.3) <.1 1 (<.1) 1. Redness 13 (2.2) 34 (.6) <.1 1 (<.1) 1. Itching 59 (1.) 31 (.5).4 Swelling 16 (1.8) 22 (.4) <.1 Adverse event 28 days after injection 2841 (47.4) 2985 (49.8).9 136 (2.3) 136 (2.3) 1. Serious adverse event 68 (1.1) 125 (2.1) <.1 Death 1 (<.1) 1 (<.1) 1. Hospitalization 67 (1.1) 124 (2.1) <.1 Hand, foot, and mouth disease 41 (.7) 88 (1.5) <.1 Injection-related cause 2 (<.1) 2 (<.1) 1. Other cause 24 (.4) 34 (.6).34 * Adverse events of grade 3 or higher were defined as those severe enough to prevent activity, according to the common terminology criteria of adverse events from the Ministry of Health in China (see the Supplementary Participants could have had multiple events. Two participants died (one patient in the vaccine group, from a traffic accident; and one in the placebo group, from severe EV71-associated hand, foot, and mouth disease). All serious adverse events except for death were events that required hospitalization. Events that were considered to be associated or most likely associated with injection included fever (in two participants in the vaccine group), vomiting (in one in the placebo group), and allergy (in one in the placebo group). Events that were considered not to be associated with injection (i.e., those that occurred >28 days after injection or for which there was strong evidence against the association) included cold or respiratory infection; inguinal hernia; convulsion; fever; diarrhea; nausea, vomiting, or anorexia; and irritability, drowsiness, or weakness. 836 n engl j med 37;9 nejm.org february 27, 214 Downloaded from nejm.org on July 12, 218. For personal use only. No other uses without permission. Copyright 214 Massachusetts Medical Society. All rights reserved.

Inactivated Enterovirus 71 in Healthy Children two in the placebo group), although statistical significance was not reached, owing to the small number of single-dose recipients. No severe cases of EV71-associated hand, foot, and mouth disease were observed among the vaccine recipients, whereas two severe cases, including one death, were observed in the placebo group. No efficacy against coxsackievirus A16 or other enteroviruses was shown in this trial. In the immunogenicity subgroup, neutralizing antibodies against EV71 developed in all vaccine recipients. Titers waned slightly at 6 months after the initial vaccination (geometric mean titer, 17.6 at day 56 and 88.3 at day 18 after vaccination), although evidence for protection remained during both epidemic seasons. Fewer serious adverse events were observed among vaccine recipients than among placebo recipients. Overall, the side effects observed in this trial were mild. The incidences of systemic adverse events (according to solicited reports) were generally similar in the vaccine and placebo groups, except for the frequency of fever, which was slightly higher in the vaccine group. Local reactions were limited to pain, redness, and swelling at the injection site in vaccine recipients. Our trial has several limitations. First, the immunogenicity follow-up was stopped at 6 months, which was only the middle of the study period. The opportunity to understand the dynamics of neutralizing antibodies was therefore not addressed. However, 2 vaccine recipients enrolled in the completed phase 1 trial, as well as the participants included in the immunogenicity subgroup of this study, are currently being monitored for antibody persistence while cases of hand, foot, and mouth disease among children younger than 1 years of age are being monitored. Second, because of limitations associated with the double-blind design, a detailed characterization of the immune response induced on virus challenge was not conducted among the vaccinated participants; this factor was, however, investigated in a small-scale phase 2 trial. 14 In conclusion, EV71, a major pathogen of hand, foot, and mouth disease, may be controlled with the use of a vaccine. Supported by grants from the National Basic Research Program (211CB5493), the National High-Tech Research and Development Program (212AA2A44), and the State Project for Essential Drug Research and Development (212ZX911319). Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. References 1. Modlin F. Coxsackieviruses, echoviruses, and newer enteroviruses. In: Mandell LG, Bennett EJ, Dolin R, eds. Mandell, Douglas, and Bennett s principles and practice of infectious diseases. 6th ed. Philadelphia: Churchill Livingstone, 25:2148-61. 2. 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Establishing China s national standards of antigen content and neutralizing antibody responses for evaluation of enterovirus 71 (EV71) vaccines. 211;29:9668-74. Copyright 214 Massachusetts Medical Society. n engl j med 37;9 nejm.org february 27, 214 837 Downloaded from nejm.org on July 12, 218. For personal use only. No other uses without permission. Copyright 214 Massachusetts Medical Society. All rights reserved.