The n e w e ng l a n d j o u r na l of m e dic i n e

original article

An Inactivated Enterovirus 71 Vaccine

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.

A BS T R AC T

BACKGROUND
Enterovirus 71 (EV71) is a major cause of hand, foot, and mouth disease in children From Guangxi Province Center for Disease
and may be fatal. A vaccine against EV71 is needed. Control and Prevention, Nanning (R.L., Z.M.,
Y. Li, T.H., Y.N.), Yunnan Key Laboratory of
Vaccine Research and De­velopment on Se­
METHODS vere Infectious Dis­eases, Institute of Med­
ical Biology, Chinese Academy of Med­i­cal
We conducted a randomized, double-blind, placebo-controlled phase 3 trial involv- Sciences and Peking Union Medical Col­
ing healthy children 6 to 71 months of age in Guangxi Zhuang Autonomous Region, lege, Kunming (L.L., Y.Z., Jingjing Wang,
China. Two doses of an inactivated EV71 vaccine or placebo were administered in- 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.,
tramuscularly, with a 4-week interval between doses, and children were monitored H.S., Q.L.), Key Laboratory Medical Mo­
for up to 11 months. The primary end point was protection against hand, foot, and lec­ular Virology, Ministries of Education
mouth disease caused by EV71. and Health, and the Institutes of Bio­
medical Science, Shanghai Medical Col­
lege, Fudan University, Shanghai (X.W.),
RESULTS Department of Health Statistics, Fourth
A total of 12,000 children were randomly assigned to receive vaccine or placebo. Military Medical University, Xi’an (J.X.,
Z.J.), and National Institutes for Food and
Serum neutralizing antibodies were assessed in 549 children who received the vac- Drug Control, Beijing (Z.L., Q.M., Junzhi
cine. The seroconversion rate was 100% 4 weeks after the two vaccinations, with a Wang) — all in China. Address reprint
geometric mean titer of 170.6. Over the course of two epidemic seasons, the vaccine requests to Dr. Qihan Li at the Institute
of Medical Biology, Chinese Academy of
efficacy was 97.4% (95% confidence interval [CI], 92.9 to 99.0) according to the Medical Sciences and Peking Union Med­
intention-to-treat analysis and 97.3% (95% CI, 92.6 to 99.0) according to the per- ical College, 935 Jiaoling Rd., Kunming,
protocol analysis. Adverse events, such as fever (which occurred in 41.6% of the Yunnan, 650118 China, or at liqihan@
imbcams.com.cn, or to Dr. Junzhi Wang at
participants who received vaccine vs. 35.2% of those who received placebo), were the National Institutes for Food and Drug
significantly more common in the week after vaccination among children who re- Control, No. 2 Tiantanxili, Beijing, 100050
ceived the vaccine than among those who received placebo. China, or at wangjz@nicpbp.org.cn.

Mr. R. Li, Dr. Liu, Mr. Mo, and Drs. X. Wang,

CONCLUSIONs Xia, and Liang contributed equally to this
article.
The inactivated EV71 vaccine elicited EV71-specific immune responses and protec-
N Engl J Med 2014;370:829-37.
tion against EV71-associated hand, foot, and mouth disease. (Funded by the National DOI: 10.1056/NEJMoa1303224
Basic Research Program and others; ClinicalTrials.gov number, NCT01569581.) Copyright © 2014 Massachusetts Medical Society.

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 The n e w e ng l a n d j o u r na l
E
pidemics 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 inci-
dences each year, in May and October.2-5 An im-
portant 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
40.1 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 double-
blind, randomized, placebo-controlled, phase 3
clin­ical 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.
ME THODS
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 alumi-
num hydroxide and suspended in 0.5 ml of buff-
ered 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 com-
pliant 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
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of m e dic i n e
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 neutral-
izing 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 Supple­
mentary Appendix). Any other symptoms or signs
occurring during a 28-day follow-up period after
each injection were recorded as unsolicited symp-
toms 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-
 Inactivated Enterovirus 71 Vaccine in Healthy Children

verse events were recorded throughout the entire LABORATORY ANALYSIS

study period. Parents and legal guardians were Viral RNA was extracted from stool samples
asked to contact the investigator immediately in with the use of an RNeasy Mini Kit (Qiagen) for
the event of a serious adverse event. An indepen- EV71 and coxsackievirus A16 and was identified
dent data and safety monitoring board whose by means of real-time RT-PCR with standard
members were unaware of the study assignments primers for EV71, coxsackievirus A16, and entero-
periodically reviewed all reports of serious ad- virus.18 The EV71 genome was sequenced to
verse events to assess the causality of the events identify the genotype.1 The level of EV71-specific
and to determine associated secondary diagno- neutralizing antibodies was measured in serum
ses and other underlying conditions. samples by means of a microneutralization as-
say on Vero cells grown in 96-well plates with
EVALUATION OF EFFICACY the use of a standard viral strain provided by
A suspected case of hand, foot, and mouth disease the National Institutes for Food and Drug
was defined as febrile illness (body temperature Control.18
>37.5°C) accompanied by a papular or vesicular rash
in the characteristic distribution on the oral mu- END POINTS
cosa, hands, feet, or buttocks. A case of EV71- The primary end point was efficacy against EV71-
associated hand, foot, and mouth disease was de- associated hand, foot, and mouth disease ac-
fined as a suspected case of hand, foot, and mouth cording to the case definition described above.
disease with EV71 detected in a throat swab or stool Efficacy was assessed from 2 weeks after com-
specimen with the use of a quantitative reverse- pletion of the two-dose schedule until 1 year after
transcriptase–polymerase-chain-reaction (RT-PCR) receipt of the first dose. The secondary end points
assay. A severe case of EV71-associated hand, foot, were efficacy against severe hand, foot, and mouth
and mouth disease was defined as a suspected case disease within the same study period and the
of hand, foot, and mouth disease caused by EV71 proportion of participants in whom EV71-
that was associated with neurologic, respiratory, neutralizing antibodies developed at 56 days and
or circulatory complications, on the basis of the 180 days after the initial vaccination.
diagnostic criteria for hand, foot, and mouth dis-
ease published by the Ministry of Health of China DATA MANAGEMENT
(see the Supplementary Appendix).17 Data were double-entered into custom-made data-
A health care center–based surveillance sys- entry programs (EpiData). The analysis of the
tem was set up to facilitate case-finding efforts. primary end point was performed according to
Suspected cases of hand, foot, and mouth disease the intention-to-treat principle and included par-
were reported by means of the surveillance sys- ticipants who received at least one dose of vac-
tem or by parents. In addition, village doctors cine or placebo. A per-protocol analysis was also
contacted all the participants once every 2 weeks, performed that included enrolled participants
either by means of a home visit or telephone call. who received two doses of the vaccine and com-
Participants with a suspected case of hand, foot, pleted the follow-up observation 12 months after
and mouth disease were referred to the county the initial injection.
or city hospital for confirmation. Adverse events were summarized for all
The throat swabs and stool specimens from ­participants who received at least one dose of
participants with suspected cases were screened study agent (vaccine or placebo). For the assess-
with the use of quantitative RT-PCR in the central ment of immunogenicity, because preexisting
laboratory at the Guangxi CDC, and the results EV71-neutralizing antibodies were detected in
were confirmed by the National Institutes for some participants, the data from all partici-
Food and Drug Control. Serum specimens were pants were analyzed in the intention-to-treat
obtained once during the acute phase (≤3 days analysis and the data from those with a titer of
after onset) and once during convalescence (ap- EV71-neutralizing antibody that was less than 1:8
proximately 10 to 15 days after onset). The data were analyzed in the per-protocol analysis.
and safety monitoring board reviewed the clini-
cal and laboratory results and confirmed the STATISTICAL ANALYSIS
diagnosis of EV71-associated hand, foot, and On the basis of the preceding 3-year surveillance
mouth disease before the data were unblinded. of the entire Guangxi Zhuang Autonomous

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 The n e w e ng l a n d j o u r na l of m e dic i n e

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 dichoto-
mous 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

1100 Underwent randomization

12,000 Underwent randomization
in immunogenicity subgroup

1 Was excluded owing to no

serum sample provided
at baseline
6000 Were assigned to 6000 Were assigned to
vaccine group (first dose) placebo group (first dose)

514 Discontinued 498 Discontinued 549 Were assigned to 550 Were assigned to
study study vaccine group placebo group
2 Had protocol violation 2 Had protocol violation
469 Had parent or guardian 470 Had parent or guardian
who declined second who declined second 148 Discontinued 148 Discontinued
dose dose study study
43 Missed 26 Missed 57 Were infected 65 Were infected
second dose second dose before enroll- before enroll-
ment ment
91 Had only one 83 Had only one
5486 Received second 5502 Received second sample after sample after
dose dose baseline baseline

401 Were included in the 402 Were included in the

5 Were excluded 3 Were excluded immunogenicity analysis immunogenicity analysis
owing to ineligible age owing to ineligible age
at enrollment at enrollment

5481 Were included in 5499 Were included in

per-protocol analysis per-protocol analysis
(second dose) (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 12,000 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 180 to evaluate the production of the anti-EV71
neutralizing antibody.

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 Inactivated Enterovirus 71 Vaccine in Healthy Children

less than 1:8, which was the threshold of detec-
tion, were assigned an arbitrary value of 1:4. In
the statistical analyses of antibody titers, the titers
were logarithmically converted to allow the as-
sessment of geometric mean titers. SAS soft-
ware, version 9.1 (SAS Institute), was used for
statistical analysis.
R E SULT S
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 de-
clined to have them participate. We randomly as-
signed 12,000 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 2012 through February 2013, 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
Table 1. Demographic Characteristics of the Participants Included in the Intention-to-Treat Analysis.*
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 vac-
cine group and 5499 in the placebo group were
included in the per-protocol analysis (Fig. 1).
The demographic characteristics of the partici-
pants were similar in the two groups (Table 1,
and Table S1 in the Supplementary Appendix).
A total of 594 suspected cases of hand, foot,
and mouth disease were reported by the surveil-
lance system or by parents or guardians over the
course of the study period in the catchment area;
70.1% 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 202 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), 102 caused by coxsackievirus A16 (48 and

Characteristic Efficacy Cohort Immunogenicity Subgroup

Vaccine Placebo Vaccine Placebo
Group Group Group Group
(N = 6000) (N = 6000) (N = 549) (N = 550)
Sex — no. (%)
Male 3151 (52.5) 3099 (51.6) 279 (50.8) 296 (53.8)
Female 2849 (47.5) 2901 (48.4) 270 (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 6.0–71.9 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 4.5–30.0 5.0–22.0 5.0–23.0
Ethnic group — no (%)†
Han 5278 (88.0) 5201 (86.7) 494 (90.0) 477 (86.7)
Zhuang 434 (7.2) 485 (8.1) 28 (5.1) 38 (6.9)
Yao 207 (3.4) 227 (3.8) 22 (4.0) 27 (4.9)
Miao 27 (0.4) 31 (0.5) 1 (0.2) 5 (0.9)
Other 54 (0.9) 56 (0.9) 4 (0.7) 3 (0.5)

* Plus–minus values are means ±SD. There were no significant between-group differences at baseline. The intention-to-
treat 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.

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 The n e w e ng l a n d j o u r na l of m e dic i n e

54, respectively), and 234 caused by other entero-
viruses (106 and 128, respectively) (Table 2, and
Table S2 in the Supplementary Appendix). The
vaccine efficacy against EV71-associated hand,
foot, and mouth disease was 97.4% (95% confi-
dence interval [CI], 92.9 to 99.0) in the intention-
to-treat analysis (Table 2) and 97.3% (95% CI,
92.6 to 99.0) in the per-protocol analysis (Table
S2 in the Supplementary Appendix). 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 Appendix). Among the 155 cases
of EV71-associated hand, foot, and mouth dis-
ease, 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
Appendix).
The cumulative incidence over the study
­period is shown in Figure 2. The genotypes of
87 viral genomes from 155 samples were iden-
tified 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
phylo­genetic analysis (Fig. S3 in the Supple­men­
tary Appendix).
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 pla-
cebo recipients, as compared with none among
514 vaccine recipients. Three patients in the pla-
cebo 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-proto-
col analyses (Fig. 2, and Fig. S1 in the Supple­
mentary Appendix).
ANTIBODY ASSESSMENTS
Antibody responses were measured in a prespec-
ified subgroup of participants. After the neutral-
izing-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 180 were significantly
higher among participants who received the vac-
cine than among those who received placebo
(intention-to-treat analyses: 87.6% vs. 4.1% at day
56, and 88.0% vs. 26.4% at day 180; per-protocol
analyses: 100% vs. 3.5% at day 56, and 99.3% vs.
30.4% at day 180; P<0.001 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 107.1

Table 2. Efficacy of the Enterovirus 71 (EV71) Vaccine 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.

Vaccine Group Placebo Group

Cases of Hand, Foot, and Mouth Disease (N = 6000) (N = 6000) Vaccine Efficacy* P Value
Participants Incidence Participants Incidence
no. of cases/ no. of cases/
1000 1000
participants/yr participants/yr % (95% CI)
Clinically diagnosed and pathogenically
confirmed cases
Caused by EV71 — no. 4 0.7 151 25.2 97.4 (92.9 to 99.0) <0.001
Age 6–23 mo — no./total no. 2/3500 0.6 94/3500 26.9 97.9 (91.4 to 99.5) <0.001
Age 24–72 mo — no./total no. 2/2500 0.8 57/2500 22.8 96.5 (85.6 to 99.1) <0.001
Caused by coxsackievirus A16 — no. 48 8.0 54 9.0 11.1 (−30.8 to 39.6) 0.55
Caused by other enterovirus — no. 106 17.7 128 21.3 17.2 (−6.0 to 35.8) 0.15
Clinically diagnosed cases — no. 202 33.7 392 65.3 48.5 (39.2 to 56.3) <0.001

* The calculation of overall vaccine efficacy was adjusted for study center.

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 Inactivated Enterovirus 71 Vaccine in Healthy Children

(intention-to-treat analysis) and 170.6 (per-pro- cebo group (P<0.001). All the patients recovered
tocol analysis). At day 180, levels had declined to within 3 days after hospitalization. One partici-
65.8 (intention-to-treat analysis) and 88.3 (per- pant in the placebo group died owing to severe
protocol analysis). There were no significant EV71-associated hand, foot, and mouth disease;
differences in anti-EV71 antibody titers between this child died within 24 hours after arrival at the
vaccine recipients who were 6 to 23 months of hospital and was not considered to be hospital-
age and those who were 24 to 71 months of age ized. Two injection-associated serious adverse
(P = 0.92) either at day 56 or at day 180 (Table S4 events were identified in each group. One patient
in the Supplementary Appendix). in the vaccine group died owing to a traffic ac-
cident (Table 3).
ADVERSE EVENTS Within 7 days after each injection, systemic
Most of the adverse events were mild. A total of adverse events (according to solicited reports)
68 serious adverse events were reported in the occurred in 48.6% of the participants in the vac-
vaccine group, as compared with 125 in the pla- cine group, as compared with 42.9% of those in
cebo group (P<0.001); 41 participants in the vac- the placebo group (P<0.001). The most common
cine group were hospitalized for hand, foot, and reactions were fever, diarrhea, nausea, and vom-
mouth disease, as compared with 88 in the pla- iting. The proportion of local adverse events

1.00

0.90
Proportion of Participants without EV71-Associated

1.00
0.80 Vaccine
0.99
Hand, Foot, and Mouth Disease

0.70 0.98

0.97 Placebo
0.60
0.96
0.50 0.95

0.94
0.40
0.93
0.30
0.92

0.20 0.91 Vaccine efficacy, 97.4% (95% CI, 92.9–99.0)

P<0.001
0.90
0.10 0.00
0 1 2 3 4 5 6 7 8 9 10 11 12

0.00
0 1 2 3 4 5 6 7 8 9 10 11 12
Months since First Dose
No. at Risk
Vaccine 6000 6000 6000 5999 5997 5997 5997 5997 5997 5996 5996 5996
Placebo 6000 6000 5980 5914 5880 5862 5854 5854 5853 5850 5849 5849

No. of Events
Vaccine 0 0 1 2 0 0 0 0 1 0 0
Placebo 0 20 66 34 18 8 0 1 3 1 0

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 transforma­
tion 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.

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 The n e w e ng l a n d j o u r na l of m e dic i n e

(solicited reports) was higher in the vaccine
group than in the placebo group (5.9% vs. 2.3%,
P<0.001). Local adverse events included pain,
redness, swelling, and itching at the injection
site (Table 3, and Table S5 in the Supplementary
Appendix).
DISCUSSION
In this randomized, double-blind, placebo-con-
trolled trial, which included 12,000 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 observa-
tion period of 11 months, which covered two
peak epidemic seasons, in May and October.
These epidemic peaks were similar to the epi-
demic trends of hand, foot, and mouth disease
reported during the previous 3 years in the
Guangxi region (Fig. S2 in the Supplementary
Appendix).
No significant difference in efficacy was iden-
tified in children 6 to 23 months of age versus
those 24 to 71 months of age. Children who re-
ceived 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*

Vaccine Placebo Vaccine Placebo
Group Group Group Group
(N = 6000) (N = 6000) P Value (N = 6000) (N = 6000) 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) <0.001
Fever 2498 (41.6) 2111 (35.2) <0.001 147 (2.4) 149 (2.5) 0.95
Diarrhea 498 (8.3) 535 (8.9) 0.24 8 (0.1) 12 (0.2) 0.50
Nausea, vomiting, or anorexia 530 (8.8) 475 (7.9) 0.08 6 (0.1) 7 (0.1) 1.00
Irritability, drowsiness, or weakness 360 (6.0) 303 (5.0) 0.03 3 (<0.1) 7 (0.1) 0.34
Allergy 166 (2.8) 156 (2.6) 0.61 2 (<0.1) 0 0.50
Local event 356 (5.9) 138 (2.3) <0.001
Pain 211 (3.5) 80 (1.3) <0.001 1 (<0.1) 0 1.00
Redness 130 (2.2) 34 (0.6) <0.001 1 (<0.1) 0 1.00
Itching 59 (1.0) 31 (0.5) 0.004 0 0 —
Swelling 106 (1.8) 22 (0.4) <0.001 0 0 —
Adverse event ≤28 days after injection 2841 (47.4) 2985 (49.8) 0.009 136 (2.3) 136 (2.3) 1.00
Serious adverse event — — — 68 (1.1) 125 (2.1) <0.001
Death† — — — 1 (<0.1) 1 (<0.1) 1.00
Hospitalization‡ — — — 67 (1.1) 124 (2.1) <0.001
Hand, foot, and mouth disease — — — 41 (0.7) 88 (1.5) <0.001
Injection-related cause§ — — — 2 (<0.1) 2 (<0.1) 1.0
Other cause¶ — — — 24 (0.4) 34 (0.6) 0.34

* 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 Appendix). 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.

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 Inactivated Enterovirus 71 Vaccine 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 dis-
ease were observed among the vaccine recipi-
ents, 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, neutraliz-
ing antibodies against EV71 developed in all vac-
cine recipients. Titers waned slightly at 6 months
after the initial vaccination (geometric mean titer,
170.6 at day 56 and 88.3 at day 180 after vaccina-
tion), 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 swell-
ing at the injection site in vaccine recipients.
Our trial has several limitations. First, the im-
munogenicity 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 ad-
dressed. However, 200 vaccine recipients en-
rolled in the completed phase 1 trial, as well as
the participants included in the immunogenicity
subgroup of this study, are currently being mon­
itored for antibody persistence while cases of
hand, foot, and mouth disease among children
younger than 10 years of age are being moni-
tored. Second, because of limitations associated
with the double-blind design, a detailed charac-
terization 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
(2011CB504903), the National High-Tech Research and Devel­
opment Program (2012AA02A404), and the State Project for
Essential Drug Research and Development (2012ZX09101319).
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.

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