Evaluating Immunogenicity and Safety Effect on Combined Immune Effect of EV71 Inactivated Vaccine and HepB、MPSV-A、MR、JE-L：A Multi-center Randomized Controlled Trial
Phase: PHASE4
Conditions: Enterovirus 71 Inactivated Vaccine, HFMD
Sponsor: Shandong Province Centers for Disease Control and Prevention

Evaluating Immunogenicity and Safety Effect on 
Combined Immune Effect of EV71 Inactivated 
Vaccine and HepB、MPSV-A、MR、JE-L：A Multi-
center Randomized Controlled Trial 
 
Registered classification: prevention of biological products 
Clinical stage: stage IV 
Program number: EV71-2017-03 
Version date: 05 01 2018 
 
Bidding unit:  
China Biotechnology Limited by Share Ltd  
Wuhan biological products research institute limited liability company  
Research unit:  
Shandong Center for Disease Control and Prevention  
Hunan Center for Disease Control and Prevention  
Shaanxi Center for Disease Control and Prevention  
Shanxi Center for Disease Control and Prevention  
 

Background 
To prevent and control the epidemic of HFMD and related diseases caused by 
EV71 infection, the development of EV71 vaccine has been developed in many 
countries or regions. According to the requirements of drug registration approval, 
we need to evaluate immunogenicity and safety effect on combined immune 
effect of EV71 inactivated vaccine and other vaccines (HepB、MPSV-A、MR、JE-
L).  
Method 
Four experimental groups (HepB:3+EV71, MPSV-A:1+EV71, MR +EV71, JE-
L+EV71) were included in this clinical trail. In addition to the meningococcal 
vaccine research group, the other three groups were followed up for the 4 times. 
The initial blood samples were collected, and the first dose vaccine was inoculated 
at the same time. EV71 second doses of vaccine were inoculated at 30 day, the 
blood was collected after 30 days of immunization with second doses. 6 months 
of safety follow-up was carried out in the whole clinical trial after vaccination. The 
meningococcal vaccine research group increased 1 follow-up after the second 
dose of MPSV-A vaccine. The index of immunogenicity and safety effect in four 
experimental groups need to be evaluated.  
Study design and participants  
The study was conducted in four provinces (Shandong, Shanxi, Shaanxi and Hunan) 
of China, which contains Haiyang city, Rushan city, Shimen County, Chen Cang 
District, Qishan County, Taigu County and Qi County. 6/8 months children were 

recruited in this study, individuals were randomly assigned to experimental group 
and control group. Demographic information (age, sex, height and weight) was 
made and blood samples were collected for each individual in the same way.  
Inclusion Criteria 
Inclusion criteria for group Ⅰ（HepB:3+EV71）were as follows: (1)aged ≥ 6 
months; (2)sign the informed consent form; (3)the legal guardian participate in all 
the planned follow-up and be able to comply with all research procedures; (4)the 
subjects have completed the basic immunization of 2 needle recombinant 
hepatitis B vaccine, there is no inoculation history of EV71 vaccine, and there is no 
history of EV71 infection ; (5)the last vaccination intervals ≥ 14 days ； 
(6)temperature ≤ 37℃ Inclusion criteria for group Ⅱ（MPSV-A:1+EV71）were 
as follows: (1)aged ≥ 6 months; (2)sign the informed consent form; (3)the legal 
guardian participate in all the planned follow-up and be able to comply with all 
research procedures; (4)there is no inoculation history of EV71 vaccine, and there 
is no history of EV71 infection; (5)the last vaccination intervals ≥ 14 days；
(6)temperature ≤ 37℃ Inclusion criteria for group Ⅲ（MR+EV71）were as follows: 
(1)aged ≥ 8 months; (2)sign the informed consent form; (3)the legal guardian 
participate in all the planned follow-up and be able to comply with all research 
procedures; (4)there is no inoculation history of EV71 vaccine, and there is no 
history of EV71 infection; (5)the last vaccination intervals ≥ 14 days and the last 
attenuated live vaccine intervals ≥ 28 days；(6)temperature ≤ 37℃ Inclusion 
criteria for group Ⅳ（JE-L+EV71）were as follows: (1)aged ≥ 8 months; (2)sign 

the informed consent form; (3)the legal guardian participate in all the planned 
follow-up and be able to comply with all research procedures; (4)there is no 
inoculation history of EV71 vaccine, and there is no history of EV71 infection; 
(5)the last vaccination intervals ≥ 14 days and the last attenuated live vaccine 
intervals ≥ 28 days；(6)temperature ≤ 37℃  
Exclusion Criteria 
Exclusion criteria for first needle: (1) the history or family history of anaphylaxis, 
convulsion, epilepsy, encephalopathy and psychosis; (2) the history of severe 
inoculation allergies; (3)patients with immunodeficiency and malignant tumors 
during the treatment period, receiving immunosuppressive therapy (oral steroid) 
or HIV due to low immunity, or family members have congenital immune disease; 
(4)Nonspecific immunoglobulin was injected within one month; (5)temperature 
＞37.1℃ and infectious diseases; (6)the history of thrombocytopenia or other 
thrombocytopenia with a definite diagnosis; (7)respiratory disease, acute infection 
or chronic disease activity period; (8)severe cardiovascular disease (CVD, 
pulmonary edema, hypertension can not be controlled by drugs to the normal 
range), liver and kidney disease, and complications of diabetes; (9)infectious, 
suppurative and allergic dermatosis; (10)other conditions that may affect the 
evaluation of the trail Exclusion criteria for follow-up needle: (1)any serious 
adverse events that have a causal relationship with the inoculation of the upper 
dose of the vaccine; (2)the abnormality of 4 levels (local, systemic adverse 
reactions and vital signs) was judged to be related to vaccination; (3)other new 

standards of "Exclusion criteria for first needle";(4)other conditions that may affect 
the evaluation of the trail 
Vaccine inoculation and follow-up  
TestⅠ(HepB:3+EV71):  
Experimental group - 6 month old HepB third dose and EV71 first dose were 
inoculated at the same time, 7 month old EV71 second dose was inoculated, blood 
samples from the participants were collected at 8 month old;  
Control group 1- 6 month old HepB third dose was inoculated separately, blood 
samples from the participants were collected after two months;  
Control group 2 - 6 month old EV71 first doses was inoculated, 7 month old EV71 
second dose was inoculated, blood samples from the participants were collected 
at 8 month old.  
TestⅡ(MPSV-A:1+EV71):  
Experimental group - 6 month old MPSV-A first dose and EV71 first dose were 
inoculated at the same time, 7 month old EV71 second dose was inoculated, 9 
month old MPSV-A second dose was inoculated, blood samples from the 
participants were collected at 10 month old;  
Control group 1- 6 month old MPSV-A first dose was inoculated separately, 9 
month old MPSV-A second dose was inoculated separately, blood samples from 
the participants were collected at 10 month old;  
Control group 2 - 6 month old EV71 first doses was inoculated, 7 month old EV71 
second dose was inoculated, blood samples from the participants were collected 

at 10 month old.  
TestⅢ (MR+EV71): 
Experimental group - 8 month old MR first dose and EV71 first dose were 
inoculated at the same time, 9 month old EV71 second dose was inoculated, blood 
samples from the participants were collected at 10 month old.  
Control group 1- 8 month old MR first dose was inoculated separately, blood 
samples from the participants were collected at 10 month old;  
Control group 2 - 8 month old EV71 first doses was inoculated, 9 month old EV71 
second dose was inoculated, blood samples from the participants were collected 
at 10 month old.  
TestⅣ(JE-L+EV71):  
Experimental group - 8 month old JE-L first dose and EV71 first dose were 
inoculated at the same time, 9 month old EV71 second dose was inoculated, blood 
samples from the participants were collected at 10 month old.  
Control group 1- 8month old JE-L first dose was inoculated separately, blood 
samples from the participants were collected at 10 month old;  
Control group 2 - 8 month old EV71 first doses was inoculated, 9 month old EV71 
second dose was inoculated, blood samples from the participants were collected 
at 10 month old.  
EV71 vaccine 0.5ml per dose, Wuhan Biological Products Co., Ltd., Wuhan, Hubei 
Province, China; 10μg HepB 0.5ml per dose, Wuhan Biological Products Co., Ltd., 
Wuhan, Hubei Province, China; 30ug MPSV-A 0.5ml per dose, Wuhan Biological 

Products Co., Ltd., Wuhan, Hubei Province, China; MR 0.5ml per dose, Beijiing 
Biological Products Co., Ltd., Beijing, China; JE-L 0.5ml per dose, Chengdu 
Biological Products Co., Ltd., Chengdu,Sichuan Province, China  
Immunogenicity end point  
Detection of serum antibody EV71 vaccine by culture neutralization test, the 
definition of positive for neutralizing antibody titers of <1:8 before inoculation, 
inoculation after neutralizing antibody titers than 1:8; or before inoculation 
neutralizing antibody titer is above 1:8, the titer of neutralizing antibody after 
vaccination appeared more than 4 times the growth. Hepatitis B vaccine 
seroconversion was defined as Anti-HBs<10mIU/ml before inoculation, Anti-HBs 
after inoculation was more than 10mIU/ml. Leprosy vaccine using measles rubella 
immunoglobulin ELISA detection test, measles >200U/ml positive for 
rubella >20U/ml positive or positive before inoculation; after inoculation, antibody 
positive growth is more than 4 times. Japanese encephalitis vaccine serum 
samples by using PRNT test before immunization antibody titer after inoculation 
was less than 1:5, at 1:10, or after vaccination antibody titer than before 
inoculation is no less than 4 times of growth is positive. Meningococcal vaccine 
samples A meningococcal bactericidal antibody level in serum by micro 
bactericidal antibody test, the antibody titer after inoculation than before 
inoculation is no less than 4 times of growth is positive.  
Safety assessment  
Safety assessment Participants were provided with diary cards to record the 

occurrence and severity of solicited local reactions at the injection site (pain, 
induration, erythema, edema, pruritus) during 7 days after vaccination, solicited 
systemic reactions (fever, headache, fatigued, cough, myalgia, asthenia, vertigo, 
diarrhea), and any unsolicited adverse during 29 days after vaccination.