Efficacy, Safety, and Immunogenicity of an Enterovirus 71 Vaccine in China

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1 The new england journal of medicine original article Efficacy, Safety, and Immunogenicity of an Enterovirus 71 Vaccine in China Fengcai Zhu, M.D., Wenbo Xu, M.D., Jielai Xia, Ph.D., Zhenglun Liang, Ph.D., Yan Liu, M.P.H., Xuefeng Zhang, M.D., Xiaojuan Tan, Ph.D., Ling Wang, Ph.D., Qunying Mao, M.Sc., Junyu Wu, Ph.D., Yuemei Hu, M.D., Tianjiao Ji, M.P.H., Lifei Song, M.Sc., Qi Liang, M.P.H., Baomin Zhang, M.P.H., Qiang Gao, M.Sc., Jingxin Li, M.Sc., Shenyu Wang, M.Sc., Yuansheng Hu, M.P.H., Shanru Gu, M.D., Jianhua Zhang, M.D., Genhong Yao, M.D., Jianxiang Gu, M.D., Xushan Wang, M.D., Yuchun Zhou, M.D., Changbiao Chen, M.D., Minglei Zhang, M.D., Minquan Cao, M.D., Junzhi Wang, Ph.D., Hua Wang, M.D., and Nan Wang, M.Sc. ABSTRACT From the Jiangsu Provincial Center for Disease Control and Prevention, Nanjing (F.Z., X.Z., Yuemei Hu, Q.L., J.L., S.W., H.W.); National Institutes for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (W.X., X.T., T.J., B.Z.), National Institutes for Food and Drug Control (Z.L., Q.M., J. Wang), and Sinovac Biotech (Y.L., J. Wu, L.S., Q.G., Yuansheng Hu, N.W.), Beijing; the Fourth Military Medical University, Xi an (J.X., L.W.); Sheyang County Center for Disease Control and Prevention, Yancheng City (S.G., J.G., C.C.); Ganyu County Center for Disease Control and Prevention, No. 98, Lianyungang City (J.Z., X.W., M.Z.); and Taixing County Center for Disease Control and Prevention, No. 224, Taizhou City (G.Y., Y.Z., M.C.) all in China. Address reprint requests to Ms. N. Wang at Sinovac Biotech, 39 Shangdi Western Rd., Haidian District, Beijing, China, or at to Dr. H. Wang at Jiangsu Provincial Center for Disease Control and Prevention, 172 Jiangsu Rd., Nanjing, Jiangsu Province, China, or at or to Dr. J. Wang at the National Institute for Food and Drug Control, No. 2, Tiantanxili, Beijing, China, or at Drs. F. Zhu, W. Xu, and J. Xia contributed equally to this article. N Engl J Med 2014;370: DOI: /NEJMoa Copyright 2014 Massachusetts Medical Society. Background Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease or herpangina worldwide. This phase 3 trial was designed to evaluate the efficacy, safety, and immunogenicity of an EV71 vaccine. Methods We conducted a randomized, double-blind, placebo-controlled, multicenter trial in which 10,007 healthy infants and young children (6 to 35 months of age) were randomly assigned in a 1:1 ratio to receive two intramuscular doses of either EV71 vaccine or placebo, 28 days apart. The surveillance period was 12 months. The primary end point was the occurrence of EV71-associated hand, foot, and mouth disease or herpangina. Results During the 12-month surveillance period, EV71-associated disease was identified in 0.3% of vaccine recipients (13 of 5041 children) and 2.1% of placebo recipients (106 of 5028 children) in the intention-to-treat cohort. The vaccine efficacy against EV71-associated hand, foot, and mouth disease or herpangina was 94.8% (95% confidence interval [CI], 87.2 to 97.9; P<0.001) in this cohort. Vaccine efficacies against EV71-associated hospitalization (0 cases vs. 24 cases) and hand, foot, and mouth disease with neurologic complications (0 cases vs. 8 cases) were both 100% (95% CI, 83.7 to 100 and 42.6 to 100, respectively). Serious adverse events occurred in 111 of 5044 children in the vaccine group (2.2%) and 131 of 5033 children in the placebo group (2.6%). In the immunogenicity subgroup (1291 children), an anti-ev71 immune response was elicited by the two-dose vaccine series in 98.8% of participants at day 56. An anti-ev71 neutralizing antibody titer of 1:16 was associated with protection against EV71-associated hand, foot, and mouth disease or herpangina. Conclusions The EV71 vaccine provided protection against EV71-associated hand, foot, and mouth disease or herpangina in infants and young children. (Funded by Sinovac Biotech; ClinicalTrials.gov number, NCT ) 818

2 Enterovirus 71 (EV71), an enterovirus that is not associated with poliomyelitis, was one of the major causative agents of outbreaks of hand, foot, and mouth disease or herpangina in Europe, 1-3 Australia, 4,5 and Japan 6,7 between 1972 and 1988, and it has been implicated in a series of outbreaks across the Asia Pacific region since the 1990s The largest Asia Pacific epidemic occurred in China in 2008, when approximately 490,000 infections and 126 deaths in infants and young children were reported. 12 The vast majority of severe cases and fatal cases occurred in children younger than 3 years of age. EV71 infection can cause a wide spectrum of disease, including hand, foot, and mouth disease, herpangina, aseptic meningitis, and nonspecific illnesses such as febrile illness, viral exanthema, and airway infection There are currently no approved vaccines against EV71, but by analogy with poliomyelitis, vaccination may offer the best option for disease control. The Vero cell based EV71 inactivated vaccine with aluminum hydroxide has consistently been shown to induce immune responses to EV71 in infants and young children, 6 to 35 months of age, in phase 1 and 2 trials, 16,17 and no safety concerns have been identified in these trials. Here, we report the results of a phase 3 trial. Methods STUDY DESIGN AND OVERSIGHT This double-blind, randomized, placebo-controlled trial was designed by the Jiangsu Provincial Center for Disease Control and Prevention (JSCDC), Sinovac Biotech (the study sponsor and manufacturer of the vaccine), the Chinese National Institutes for Food and Drug Control, and the Chinese National Institute for Viral Disease Control and Prevention. Data were collected by investigators at the JSCDC. An independent data and safety monitoring board consisting of five members (for details, see Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org) monitored updated safety data, evaluated the risk to participants during the trial, and made the final determination of the efficacy end points. This trial was performed at three centers (in Ganyu, Taixing, and Sheyang Counties) comprising 35 sites in Jiangsu Province, China. The trial protocol and the informed-consent form were approved by the ethics committee of the JSCDC. Before enrollment, written informed consent was obtained from a parent or guardian of each participant. The trial was conducted in accordance with the principles of the Declaration of Helsinki, the standards of Good Clinical Practice (as defined by the International Conference on Harmonization), and Chinese regulatory requirements. The first three authors and last three authors vouch for the accuracy and completeness of the reported data and the fidelity of the study to the protocol. All authors made the decision to submit the manuscript for publication. The study sponsor had no role in data collection, analysis, or interpretation or in manuscript preparation. PARTICIPANTS Healthy infants and young children, 6 to 35 months of age, were recruited and enrolled in January 2012, before the anticipated seasonal peak of hand, foot, and mouth disease. Children with a history of hand, foot, and mouth disease or vaccination with EV71 vaccine and those with acute febrile disease on the day of enrollment were excluded (for additional exclusion criteria, see Table S2 in the Supplementary Appendix). The participants were randomly assigned in a 1:1 ratio to receive EV71 vaccine or placebo, according to a randomization list (with a block size of 10) that was generated by an independent statistician. For further details of the study conduct, see the protocol, available at NEJM.org. VACCINE The Vero cell based inactivated human EV71 vaccine was developed with the use of EV71 strain H07 (subgenotype C4) as the seed virus. 16 The EV71 vaccines contained 400 U of EV71 antigen with alum adjuvant, whereas the placebo contained aluminum hydroxide diluents with no EV71 antigen; both were packaged in syringes (0.5 ml per vial). Vaccine and placebo were supplied in coded, identical-appearing, single-dose vials and were administered intramuscularly in the deltoid region on days 0 and 28. SURVEILLANCE AND CASE DEFINITION The efficacy of EV71 vaccine was evaluated during a 12-month surveillance period from day 57 to month 14 (Fig. S1 in the Supplementary Appendix). The children s parents or guardians were instructed to seek treatment for illness at desig- 819

3 The new england journal of medicine nated health care services (including 640 village clinics, 32 township hospitals, and 7 county hospitals). In addition, study staff visited participants at least once a week to keep abreast of their health status and to determine whether their parents or guardians had sought medical care for them. Participants with any illness were eligible for screening, except conditions requiring surgery (e.g., hernias, fractures, or burns), congenital diseases, tumors, and mental or behavioral disorders with a clear cause unrelated to EV71. Throat and anal swabs were collected from these children as soon as possible and then transferred to the on-site laboratories of the study centers for assessment. A real-time, fluorescence-based, quantitative polymerase-chain-reaction (PCR) assay was used to detect EV71 RNA and discriminate it from other enteroviruses. After the first positive test for EV71 RNA, study staff performed clinical and epidemiologic evaluation at visits and collected a series of throat and anal swabs and stool samples from patients with suspected disease for laboratory assays at an interval of 3 days in the acute stage and 7 days in the recovery stage. Blood samples were collected at the acute stage. All specimens were sent to the central laboratory for case confirmation. Virologically confirmed EV71-associated disease was defined as cases with two consecutive positive results for EV71 RNA on real-time PCR assay or positive results for EV71 on viral isolation and analysis of the VP1 sequence in stool samples or throat and anal swabs. 18,19 EFFICACY END POINTS The primary efficacy end point was the occurrence of EV71-associated hand, foot, and mouth disease or herpangina. Secondary efficacy end points were severe hand, foot, and mouth disease (with neurologic or other serious complications), EV71-associated hospitalization, and all EV71-associated diseases. For the detailed case definitions, see Table S4 in the Supplementary Appendix. Before unblinding, the data and safety monitoring board reviewed all the epidemiologic data and laboratory-assay results for each patient with laboratory-confirmed EV71 infection, then made the final determination of cases of EV71- associated disease and classified the cases as hand, foot, and mouth disease, herpangina, or diseases other than hand, foot, and mouth disease and herpangina. SAFETY ASSESSMENT The parents or guardians of all participants who received the EV71 vaccine or placebo were asked to fill out diary cards that listed injection-site adverse reactions (e.g., pain, redness, and swelling) and systemic adverse reactions (e.g., fever, irritability, and loss of appetite). Safety data were collected on solicited adverse events that occurred within 7 days after an injection and unsolicited adverse events (those reported spontaneously by a parent or guardian) that occurred within 28 days after an injection. Data on serious adverse events were collected throughout the trial. For grading of adverse events, see Table S3 in the Supplementary Appendix. The relationship of the adverse event or serious adverse event with receipt of an injection was decided by investigators before unblinding. IMMUNOGENICITY The immunogenicity subgroup included participants recruited from four sites at three centers. Blood samples were collected before the first injection and at day 56, month 8, and month 14 for immunogenicity evaluation. All serum samples were assessed for EV71 neutralizing antibody by means of a modified cytopathogenic effect assay. 16 A titer of 1:8 or higher indicated seropositivity. STATISTICAL ANALYSIS We calculated that a sample of 5000 participants per group was required for 80% statistical power to show that the lower bound of the 95% confidence interval for vaccine efficacy exceeded that observed with placebo at a significance level of 0.05, assuming a vaccine efficacy rate of 80% and an incidence density of 8 cases of EV71-associated hand, foot, and mouth disease or herpangina per 1000 person-years among unvaccinated children. The vaccine efficacy rate was calculated as follows: [1 (incidence density of the vaccine group incidence density of the placebo group)] 100. We collected 3-ml blood samples on day 56 from all participants who received at least one dose of EV71 vaccine or placebo for detection of neutralizing antibodies. All participants who received at least one dose and entered the surveillance period were included in the intention-totreat cohort for the primary efficacy analysis. Efficacy was also calculated in the per-protocol efficacy cohort, which included participants who 820

4 received two doses and completed the 12-month surveillance period. The safety analysis was performed on data from the total cohort of participants who received at least one dose of EV71 vaccine or placebo, and the immunogenicity analysis was performed on data from the perprotocol immunogenicity cohort. To evaluate the correlation between EV71 neutralizing antibody levels and disease protection, an exploratory analysis was performed in a perprotocol subcohort consisting of the participants with EV71-associated hand, foot, and mouth disease or herpangina and matched case-free participants, in a ratio of 1:5. In the analysis, sensitivity was defined as the proportion of participants with antibody titers below the cutoff value on day 56, for those with EV71-associated hand, foot, and mouth disease or herpangina, and specificity was defined as the proportion of participants with titers greater than or equal to the cutoff value on day 56, for the matched case-free participants. The possible serologic marker for 12,446 Children were assessed for eligibility 2369 Were excluded 2198 Did not meet eligibility criteria 171 Had other reasons 10,077 Underwent randomization 5044 Received first dose of vaccine and were included in safety analysis 4719 Received second dose 325 Did not receive second dose 17 Had adverse event 3 Had severe adverse event 47 Were unavailable 81 Were excluded by investigators 174 Had parental consent withdrawn 2 Were lost to follow-up 1 Moved away 5033 Received first dose of placebo and were included in safety analysis 4711 Received second dose 322 Did not receive second dose 23 Had adverse event 1 Had severe adverse event 38 Were unavailable 62 Were excluded by investigators 194 Had parental consent withdrawn 3 Were lost to follow-up 1 Moved away 3 Discontinued 2 Were lost to follow-up 1 Moved away 5 Discontinued 3 Were lost to follow-up 1 Moved away 1 Died 5041 Entered surveillance beginning at day 57 and were included in primary efficacy analysis (intention-to-treat) 5028 Entered surveillance beginning at day 57 and were included in primary efficacy analysis (intention-to-treat) 132 Discontinued the study 13 Moved away 111 Were lost to follow-up 6 Were excluded by investigators 2 Died 132 Discontinued the study 16 Moved away 105 Were lost to follow-up 8 Were excluded by investigators 3 Died 4587 Were included in efficacy analysis (per-protocol) 4578 Were included in efficacy analysis (per-protocol) Figure 1. Screening, Randomization, and Inclusion in Safety and Efficacy Analyses. 821

5 The new england journal of medicine Table 1. Baseline Characteristics of the Study Participants.* Characteristic EV71 Vaccine Placebo Safety cohort No. of participants Age mo ± ± ± ± ± ±3.5 Male sex no. (%) 2789 (55.3) 2761 (54.9) Height cm 82.7± ±8.1 Weight kg 13.0± ±2.3 Immunogenicity cohort No. of participants Age mo ± ± ± ± ± ±3.7 Male sex no. (%) 321 (55.4) 300 (52.5) Height cm 84.0± ±8.2 Weight kg 13.2± ±2.4 Efficacy cohort No. of participants Age mo ± ± ± ± ± ±3.5 Male sex no. (%) 2787 (55.3) 2759 (54.9) Height cm 82.7± ±8.1 Weight kg 13.0± ±2.3 * Plus minus values are means ±SD. There were no significant differences between the groups in any of the characteristics listed here (P>0.05). Although 1291 children recruited from four sites at three centers were included in the immunogenicity subgroup, only 1150 of them were eligible for the per-protocol analysis of immunogenicity. protection was the lowest titer with the maximum sum of the specificity and sensitivity corresponding to each cutoff value. A chi-square test or Fisher s exact test was used to compare categorical data, and Student s t-test was used to compare log-transformed neutralizing antibody values. Vaccine efficacy and case-free survival were estimated with the use of a Cox proportional-hazards model and the Kaplan Meier method, respectively. Hypothesis testing was two-sided with an alpha value of Analyses were conducted by statisticians at the Fourth Military Medical University with the use of SAS software, version 9.2 (SAS Institute). Results STUDY POPULATION In January 2012, a total of 10,077 children were enrolled and received at least one dose of EV71 vaccine or placebo, and 9430 of these children (4719 in the vaccine group and 4711 in the placebo group) received the second dose (93.6% of all participants). On day 57, a total of 5041 participants in the vaccine group and 5028 in the placebo group entered the surveillance period. A total of 9165 participants (90.9%) completed the 12-month surveillance period in accordance with the protocol. Information on study enrollment, randomization, and surveillance is shown in Figure 1. The vaccine and placebo groups were well balanced in terms of sex and mean age, height, and weight (Table 1, and Table S7 in the Supplementary Appendix). VACCINE EFFICACY A total of 119 participants (13 [0.3%] in the vaccine group and 106 [2.1%] in the placebo group) had EV71-associated disease during the surveillance period. Of these participants, 99 had confirmed hand, foot, and mouth disease or her pangina (83.2%), 15 presented with res piratory symptoms (12.6%), 3 presented with gastro intestinal symptoms (2.5%), and 2 appeared to have only a febrile syndrome (1.7%). The efficacy of the vaccine against EV71-associated hand, foot, and mouth disease or herpangina was 94.8% (95% confidence interval [CI], 87.2 to 97.9) during the 12-month surveillance period in the intention-to-treat cohort (Table 2). The vaccine was effective in preventing EV71- associated hospitalization (0 cases in the vaccine group vs. 24 cases in the placebo group, P<0.001) and severe hand, foot, and mouth disease (0 cases in the vaccine group vs. 8 cases [all with neurologic complications] in the placebo group, P = 0.004), resulting in vaccine efficacies of 100% (95% CI, 83.7 to 100 and 42.6 to 100, respectively). The overall efficacy of the EV71 vaccine against EV71-associated disease was 88.0% (95% CI, 78.6 to 93.2) during the 12-month period. The efficacy was slightly higher during the first 822

6 Table 2. Efficacy of the EV71 Vaccine against EV71-Associated HFMD or Herpangina during the 12-Month Surveillance Period in the Intention-to-Treat Population.* End Point EV71 Vaccine Placebo Protective Efficacy No. of Cases Incidence Density cases/1000 person-yr No. of Cases Incidence Density cases/1000 person-yr % (95% CI) At 6 Mo EV71-associated HFMD or herpangina (90.0 to 99.4) HFMD (89.5 to 99.4) Herpangina ( 49.0 to 100) EV71-associated hospitalization (83.7 to 100) EV71-associated HFMD with neurologic complications (42.4 to 100) All EV71-associated diseases (79.5 to 94.4) At 1 Yr EV71-associated HFMD or herpangina (87.2 to 97.9) HFMD (86.6 to 97.8) Herpangina ( 48.4 to 100) EV71-associated hospitalization (83.7 to 100) EV71-associated HFMD with (42.6 to 100) neurologic complications All EV71-associated diseases (78.6 to 93.2) * CI denotes confidence interval, EV71 enterovirus 71, and HFMD hand, foot, and mouth disease. In the vaccine group, there were person-years of follow-up at 6 months and person-years at 1 year. In the placebo group, there were person-years of follow-up at 6 months and person-years at 1 year. P< All cases of EV71-associated hospitalization were in patients with HFMD. P< months, a difference that was not significant. Similar efficacies were also observed in the perprotocol analysis (Table S8 and Fig. S3 in the Supplementary Appendix). Among patients with EV71 infection, the average EV71 shedding period did not differ significantly between the vaccine and placebo groups (5.0 days and 7.9 days, respectively) (Fig. S5 in the Supplementary Appendix). We also estimated vaccine efficacy against EV71- associated hand, foot, and mouth disease or herpangina in an analysis with stratification according to age and study center (Tables S10 and S11, respectively, in the Supplementary Appendix). According to the Cox proportionalhazards model, children 12 to 23 months of age and those at the study centers in Sheyang and Taixing Counties had an increased risk of EV71- associated hand, foot, and mouth disease or herpangina (Table S13 in the Supplementary Appendix). In the 12-month surveillance period, a total of 1950 cases of hand, foot, and mouth disease or herpangina were observed. Of those, 627 cases (32.2%) were caused by coxsackievirus A16. The vaccine showed no protection against hand, foot, and mouth disease or herpangina caused by coxsackievirus A16 (protective efficacy, 6.6% [95% CI, 9.2 to 20.1]) and limited protection against all hand, foot, and mouth disease or herpangina (protective efficacy, 19.8% [95% CI, 11.0 to 27.7]) (Table S9 in the Supplementary Appendix). ADVERSE REACTIONS During the entire study period, 242 serious adverse events were reported (111 in the vaccine group and 131 in the placebo group), with a similar rate of occurrence in the two groups (Table 3, 823

7 The new england journal of medicine and Table S15 in the Supplementary Appendix). Eleven serious adverse events that occurred within 28 days after an injection were considered to be related to receipt of an injection: 5 in the vaccine group (4 cases of infection or infestation and 1 case of a gastrointestinal disorder) and 6 in the placebo group (4 cases of infection or infestations, 1 case of a gastrointestinal disorder, and 1 case of a general disorder with injection-site reaction) (Table S16 in the Supplementary Appendix). During the study period, 5 deaths due to drowning (1 in the vaccine group and 4 in the placebo group) and 1 death due to a traffic accident (in the vaccine group) were recorded (Table S17 in the Supplementary Appendix). The frequencies of both solicited and unsolicited adverse events were similar in the two study groups (Table 3, and Tables S18, S19, and S20 in the Supplementary Appendix). The most common systemic adverse reactions were fever, diarrhea, and loss of appetite, and the most common injection-site adverse reactions were redness, induration, and pain. A total of 150 participants in the vaccine group (3.0%) and 158 participants in the placebo group (3.1%) had grade 3 adverse events within 7 days after an injection. IMMUNOGENICITY The baseline EV71 neutralizing antibody titers in the 1291 participants in the immunogenicity sub- Table 3. Serious Adverse Events and Injection-Site or Systemic Adverse Reactions in the Safety Population.* Event EV71 Vaccine (N = 5044) participants % (95% CI) Placebo (N = 5033) participants % (95% CI) Serious adverse events Events within 28 days after an injection ( ) ( ) Events from day 0 to month ( ) ( ) Deaths ( ) ( ) Events related to receipt of an injection ( ) ( ) Solicited adverse reactions within 7 days after an injection Any ( ) ( ) Grade ( ) ( ) Injection-site reactions Redness Any ( ) ( ) Grade ( ) ( ) Induration Any ( ) ( ) Grade ( ) ( ) Pain Any ( ) ( ) Grade ( ) ( ) Swelling Any ( ) ( ) Grade ( ) ( ) Pruritus Any ( ) ( ) Grade ( ) ( ) 824

8 Table 3. (Continued.) Event Systemic reactions Fever participants EV71 Vaccine (N = 5044) % (95% CI) participants Placebo (N = 5033) % (95% CI) Any ( ) ( ) Grade ( ) ( ) Diarrhea Any ( ) ( ) Grade ( ) ( ) Loss of appetite Any ( ) ( ) Grade ( ) ( ) Nausea and vomiting Any ( ) ( ) Grade ( ) ( ) Irritability Any ( ) ( ) Grade ( ) ( ) Fatigue Allergy Any ( ) ( ) Grade ( ) ( ) Any ( ) ( ) Grade ( ) ( ) Adverse reactions within 28 days after an injection ( ) ( ) * Participants could have more than one adverse event. Data on serious adverse events were collected throughout the trial. For serious adverse events classified according to the Medical Dictionary for Regulatory Activities, see Table S15 in the Supplementary Appendix. Five participants died from drowning, and one participant died from a traffic accident. Details of the serious adverse events considered to be related to receipt of an injection are available in Table S16 in the Supplementary Appendix. Solicited adverse reactions were those listed on diary cards that were filled out by the parents or guardians of the participants. For all solicited reactions, those that occurred in more than 1% of participants in either group and that were considered to be related to receipt of an injection are listed here. For the criteria used to grade solicited adverse reactions, see Table S3 in the Supplementary Appendix. These data include unsolicited adverse reactions (those spontaneously reported by a parent or guardian). group were low and similar in the two study groups (Table 4). A significant neutralizing antibody response was elicited by two-dose vaccination: 98.8% of participants in the vaccine group were seropositive on day 56, with a geometric mean titer of (95% CI, to 188.5). During the 1-year observation period, 11 participants in the immunogenicity subgroup had confirmed EV71-associated hand, foot, and mouth disease or herpangina. All 11 participants were in the placebo group and did not have EV71 infection at baseline. With respect to the duration of EV71 neutralizing antibody titers, a moderate decrease be- 825

9 The new england journal of medicine Table 4. Antibody Response in the Per-Protocol Cohort.* Variable EV71 Vaccine Placebo Day 0 No. of participants Geometric mean titer value (95% CI) 7.5 ( ) 8.2 ( ) Participants with titer 1:8 % (95% CI) 15.0 ( ) 18.0 ( ) Participants with titer 1:16 % (95% CI) 14.3 ( ) 17.3 ( ) Participants with titer 1:32 % (95% CI) 13.8 ( ) 16.1 ( ) Day 56 No. of participants Geometric mean titer value (95% CI) ( ) 8.9 ( ) Factor increase from day 0 no. (95% CI) 22.1 ( ) 1.1 ( ) Seroconversion rate % (95% CI) 88.1 ( ) 2.8 ( ) Participants with titer 1:8 % (95% CI) 98.8 ( ) 19.6 ( ) Participants with titer 1:16 % (95% CI) 97.1 ( ) 18.4 ( ) Participants with titer 1:32 % (95% CI) 91.4 ( ) 16.8 ( ) Month 8 No. of participants Geometric mean titer value (95% CI) 88.8 ( ) 12.5 ( ) Factor increase from day 0 no. (95% CI) 12.0 ( ) 1.5 ( ) Seroconversion rate % (95% CI) 68.2 ( ) 12.0 ( ) Participants with titer 1:8 % (95% CI) 99.3 ( ) 28.1 ( ) Participants with titer 1:16 % (95% CI) 92.7 ( ) 25.7 ( ) Participants with titer 1:32 % (95% CI) 75.3 ( ) 24.8 ( ) Month 14 No. of participants Geometric mean titer value (95% CI) 92.1 ( ) 13.2 ( ) Factor increase from day 0 no. (95% CI) 12.1 ( ) 1.6 ( ) Seroconversion rate % (95% CI) 71.8 ( ) 12.3 ( ) Participants with titer 1:8 % (95% CI) 99.2 ( ) 34.7 ( ) Participants with titer 1:16 % (95% CI) 92.4 ( ) 27.4 ( ) Participants with titer 1:32 % (95% CI) 80.7 ( ) 25.3 ( ) * Seroconversion was defined by a titer of less than 1:8 before any injections of EV71 vaccine or placebo with a titer of 1:8 or more after any injections or by an increase in the antibody titer by a factor of four or more. P<0.001 for all comparisons between the study groups at day 56 and months 8 and 14. tween day 56 and month 8 was observed, followed by a leveling off between month 8 and month 14 (Fig. S4 and Table S21 in the Supplementary Appendix), even after exclusion of the confirmed cases of EV71-associated disease and the subclinical cases of EV71 infection during the surveillance period (Tables S22 and S23 in the Supplementary Appendix). In the evaluation of the correlation between EV71 neutralizing antibody levels and disease protection, little variation was observed in the sum of specificity and sensitivity values for neutralizing antibody titers of 1:8 to 1:32. A titer of 1:16 had the maximum summed sensitivity and specificity values (Table S24 in the Supplementary Appendix), and this titer may be considered as a 826

10 possible serologic marker for protection against EV71-associated hand, foot, and mouth disease or herpangina in vaccinated children. Discussion The EV71 vaccine showed efficacy against EV71- associated hand, foot, and mouth disease or herpangina and EV71-associated hospitalization or severe cases of hand, foot, and mouth disease (i.e., those with neurologic complications). The vaccine did not have efficacy against EV71-associated diseases other than hand, foot, and mouth disease and herpangina, an observation that may reflect the small number of cases and insufficient power. One limitation of this trial is that no assessment of etiologic factors other than EV71 was performed in participants with EV71-associated disease, and we could therefore not exclude the possibility of coinfection with other pathogens. In addition, 15.0 to 18.0% of participants in the immunogenicity subgroup were seropositive at baseline, a finding that suggests that some participants may have already had immunity against EV71 before receiving EV71 vaccine or placebo. Because blood samples were collected before the first injection only in the immunogenicity subgroup, we could not identify all participants in the study cohort who were seropositive at baseline. Assuming, however, that the numbers of participants with seropositivity at baseline were small and were evenly distributed between the study groups through randomization, the assessment of vaccine efficacy was not likely to have been substantially biased by baseline seropositivity. As expected, data in this trial indicated that the EV71 vaccine offered no protection against hand, foot, and mouth disease or herpangina caused by coxsackievirus A16 or other enterovirus serotypes, a finding that is consistent with the results of a preclinical study. 20 EV71 accounted for only a small proportion of the cases of hand, foot, and mouth disease or herpangina identified in this trial (99 of 1950 cases), a finding that suggests that EV71 was not the predominant virus associated with hand, foot, and mouth disease and herpangina in the study area in 2012, when coxsackievirus A16 was also circulating. Therefore, immunization with EV71 vaccine did not significantly decrease the overall incidence of hand, foot, and mouth disease or herpangina. The EV71 neutralizing antibody titer was monitored over a period of 12 months after day 56; it declined by half during the first 6 months and then remained stable during the next 6 months. A similar trend was reported in a previous study. 17 In this trial, evaluation of the efficacy of the vaccine and the duration of immunogenicity involved a paradoxical situation: vaccine efficacy could be better observed in a population with high EV71 prevalence, but the duration of immunogenicity elicited by the vaccine could be affected by natural infections during EV71 epidemics. Therefore, data from this trial may lead to an overestimation of the duration of neutralizing antibodies. In this trial, an EV71 neutralizing antibody titer of 1:16 was found to correlate with the estimated vaccine efficacy among children. The fact that more than 90% of vaccine recipients had a geometric mean titer of 1:16 or higher up to month 14 after vaccination is consistent with probable protection against EV71-associated hand, foot, and mouth disease or herpangina for 1 year. Longer-term surveillance of the study participants is being performed to evaluate the longevity of serologic responses and vaccine protection and the need for booster injections. The incidence of solicited injection-site or systemic adverse reactions that may have been related to vaccination was consistent with that reported in phase 1 and 2 trials. 16,17 The safety profile of the vaccine is also similar to that of other inactivated EV71 vaccine candidates. 21,22 The current EV71 vaccine candidates in clinical research were developed on the basis of a single subgenotype strain (B3, B4, or C4) of EV71. 23,24 Although some immunogenicity studies in animals indicated that EV71 inactivated vaccine could elicit antibody responses that cross-neutralized with different EV71 subgenotypes, 20,25,26 this has not been tested yet in clinical trials. In conclusion, this study showed that the EV71 vaccine consistently elicited immunogenicity and provided protection against mild-to-severe disease caused by EV71 for at least 1 year in infants and young children. A neutralizing antibody titer of 1:16 was associated with protection against EV71. Supported by Sinovac Biotech. Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. 827

11 We thank all the investigators from the Jiangsu Provincial Center for Disease Control and Prevention, Sheyang County Center for Disease Control and Prevention, Ganyu County Center for Disease Control and Prevention, and Taixing County Center for Disease Control and Prevention who contributed to the site work of the trial. References 1. Blomberg J, Lycke E, Ahlfors K, Johnsson T, Wolontis S, von Zeipel G. New enterovirus type associated with epidemic of aseptic meningitis and-or hand, foot, and mouth disease. Lancet 1974;2: Shindarov LM, Chumakov MP, Voroshilova MK, et al. Epidemiological, clinical, and pathomorphological characteristics of epidemic poliomyelitis-like disease caused by enterovirus 71. J Hyg Epidemiol Microbiol Immunol 1979;23: Nagy G, Takátsy S, Kukán E, Mihály I, Dömök I. 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