Interchangeability of two Enterovirus 71 inactivated vaccines in Chinese children: A phase IV, open-label, and randomized controlled trial

Vaccine xxx (xxxx) xxx

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Interchangeability of two Enterovirus 71 inactivated vaccines in Chinese children: A phase IV, open-label, and randomized controlled trial Qing Xu a,b,1, Qingfan Cao c,1, Wanqi Yang d,1, Xiaodong Liu a, Haidong Liu c, Xiaohui Tian d, Jing Li d, Xueqiang Fang a, Ningning Jia d, Gang Zeng d,⇑, Aiqaing Xu a,b,⇑ a

Shandong Provincial Key Laboratory of Infectious Disease Control and Prevention, Shandong Center for Disease Control and Prevention, Jinan, China Academy of Preventive Medicine, Shandong University, Jinan, China Rushan City Center for Disease Control and Prevention, Rushan, Shandong, China d Sinovac Biotech Co., Ltd., Beijing, China b c

a r t i c l e

i n f o

Article history: Received 28 October 2019 Received in revised form 11 January 2020 Accepted 5 February 2020 Available online xxxx Keywords: Interchangeability EV71 vaccine Immunogenicity Safety

a b s t r a c t Background: In China, three inactivated Enterovirus 71 (EV71) vaccines have been approved. Although the vaccines in an immunization series should be from a single manufacture, children sometimes have to receive EV71 vaccines from more than one manufacturers. The aim of this study was to evaluate the interchangeability and safety of vaccination with EV71 vaccines from two manufacturers among Chinese children. Methods: We conducted an open label and randomized controlled study among children aged 6–35 months from November 2018 to January 2019. The participants were randomly assigned (1:1:1:1) to receive EV71 vaccines in one of the four different schedules (two using a single vaccine for all doses from one manufacture, and two mixed schedules using vaccines from two manufactures). Blood samples were collected pre-vaccination (Day 0) and one month after the second dose (Day 60) for neutralizing antibody assay. Immunogenicity was assessed in the per-protocol cohort and safety was assessed in the total vaccinated cohort. Results: A total of 300 children were enrolled and randomized, of whom 89.0% (267/300) were included in the per-protocol cohort for immunogenicity analysis. The seroconversion rates of the EV71 neutralizing antibody in four groups ranged from 98.4% to 100.0%, and were not significantly different among the groups. Compared with other groups, geometric mean titer was higher in group D, in which the participants received Institute of Medical Biology Chinese Academy of Medical Sciences (CAMS) vaccine in the first dose and the Sinovac vaccine in the second dose. Safety profiles were similar among the four groups and no serious adverse events related to the vaccination were reported. Conclusions: Interchangeability of EV71 vaccines from two manufactures to complete an immunization series showed good immunogenicity and safety. The antibody response levels may vary by vaccination sequences of EV71 vaccines from the two manufacturers. Trial registration: ClinicalTrials.govNCT03873740. Ó 2020 Elsevier Ltd. All rights reserved.

1. Introduction Entervoirus 71 (EV71) is a non-enveloped, positive, singlestranded RNA virus, which belongs to the Picornaviridae family. Hand, Foot and Mouth Disease (HFMD) is a common childhood ill⇑ Corresponding authors at: Sinovac Biotech Co., Ltd., No. 39 Shangdi Western Rd, Haidian District, Beijing, China (Z. Gang). Shandong Provincial Key Laboratory of Infectious Disease Control and Prevention, Shandong Center for Disease Control and Prevention, No. 16992 Jingshi Road, Jinan City, Shandong, China (A. Xu). E-mail addresses: [email protected] (G. Zeng), [email protected] (A. Xu). 1 The first three authors contributed equally to the article.

ness, which is mainly caused by coxsackievirus A16 (CVA16) and EV71. Children younger than five years are especially prone to HFMD [1]. Since 2007, the HFMD has been prevalent in children of mainland China [2]. More than 20 million probable cases of HFMD and 838,000 laboratory-confirmed cases had been reported during 2009–2018. The majority of severe (68%) and fatal (91%) HFMD cases were caused by EV71 [3]. It is estimated that the annual cost of severe and mild HFMD cases caused by EV71 infection is about $29 million and $161–323 million, respectively [4]. The HFMD is raising a growing public concern with a considerable health and economic burden in affected areas.

https://doi.org/10.1016/j.vaccine.2020.02.013 0264-410X/Ó 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: Q. Xu, Q. Cao, W. Yang et al., Interchangeability of two Enterovirus 71 inactivated vaccines in Chinese children: A phase IV, openlabel, and randomized controlled trial, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.013

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Q. Xu et al. / Vaccine xxx (xxxx) xxx

Currently, vaccination is considered as the most effective and promising strategy to prevent HFMD caused by EV71 [4–6]. There are three inactivated EV71 vaccines having been approved by the China Food and Drug Administration. They are produced by three manufacturers respectively: Vigoo, Sinovac Biotech Co., Ltd. (Sinovac), and the Institute of Medical Biology Chinese Academy of Medical Sciences (CAMS). All the three vaccines were developed by using the C4 subgenotype as the seed virus; and they all have a schedule of two doses being administered one month apart [4]. The three vaccines showed good safety profiles and provided protective effects against EV71-associated HFMD in children in phase III trails, with a vaccine efficacy of 90.9% for Vigoo [7], 94.6% for Sinovac [8], and 97.4% for CAMS [9]. As each EV71 vaccine is considered to be distinct with regard to the source of strains, cell lines, antigen content, and so on, Chinese Center for Disease Control and Prevention (China CDC) recommends that a single manufacturer’s vaccine should be given, whenever feasible, for all the two doses of an immunization series. The main reason for this recommendation is that no data are available about the safety, immunogenicity and efficacy of interchanging EV71 vaccines from different manufactures in one immunization series [10]. However, vaccination with EV71 vaccines from different manufactures is usually unavoidable if the vaccine used in the first dose is not known or the supply of EV71 vaccine from the same manufacturer is disrupted. Therefore, it is critical to assess the interchangeability of EV71 vaccines from different manufacturers to provide support for practical application. The current study was to evaluate the immunogenicity and safety of interchanging two licensed EV71 vaccines (Sinovac and CAMS) in a vaccination schedule, with a comparison to using the vaccines alone among Chinese children. 2. Methods 2.1. Study design The open-label and randomized controlled trial was performed in Rushan City, Shandong Province from November 2018 to January 2019. The trial was designed to evaluate the interchangeability of two different EV71 vaccines (Sinovac and CAMS) in a vaccination schedule and its safety among Chinese children aged 6–35 months. The study was approved by the independent ethics committee of Shandong Provincial CDC. Before enrollment, a written informed consent was obtained from a parent or guardian of each participant. This study is reported as per the Consolidated Standards of Reporting Trials (CONSORT) guideline (Checklist S1) and was registered with ClinicalTrials.gov, number NCT03873740. 2.2. Vaccines In the study, two licensed vaccines were used. The Sinovac EV71 vaccine was prepared from the EV71 H07 strain of sub-genotype C4, cultured in a Vero cell for proliferation, and then purified and inactivated [8]. The CAMS EV71 vaccine was prepared from the EV71 FY-23K-B strain of sub-genotype C4, cultured in a human diploid cell line (KMB17) for proliferation, and then purified and inactivated [9]. Both vaccines were administered intramuscularly one month apart. 2.3. Participants A total of ten townships in Rushan city were selected to recruit the participants, including Chengzhong, Chengnan, Wuji, Xiachu, Baishatan, Dagushan, Yazi, Rushanzhai, Yuli, and Xiacun. Eligible participants were in stable healthy conditions and between 6 and

35 months of age. Children were excluded if they (a) were previously vaccinated or had HFMD; (b) were febrile (axillary temperature＞37.0 ) before vaccination or acutely ill in the past 7 days; (c) had a history of vaccine allergy or severe adverse effects; (d) had a history of epilepsy, convulsions, family psychosis autoimmune diseases or immunodeficiency; (e) had received any blood products, immunoglobulin or hormones within 30 days and received live attenuated vaccine within 14 days or subunit and inactivated vaccine within 7 days; (f) showed obvious abnormalities in heart, lung, skin, and pharynx at the physical examination; and (g) had any factors that tendered them unsuitable for the trial. 2.4. Randomization Eligible participants were randomly assigned (1:1:1:1) to groups A, B, C, and D, according to a randomization list (with a block size of 4) generated by the sponsor. Group A received two doses of Sinovac EV71 vaccine on Day 0 and 30; group B received two doses of CAMS EV71 vaccine on Day 0 and 30; group C received the first dose of Sinovac EV71 vaccine on Day 0 and the second dose of CAMS EV71 vaccine on Day 30; and group D received the first dose of CAMS EV71 vaccine on Day 0 and the second dose of Sinovac EV71 vaccine on Day 30. Randomization-code numbers were assigned to the subjects in a chronological order by the investigators. 2.5. Safety assessment All participants were observed for at least 30 min after each vaccination to monitor the immediate adverse reactions. The parents or guardians of the participants were asked to fill out the diary cards that listed injection-site adverse reactions (e.g., pain, redness, and swelling) and systemic adverse reactions (e.g., fever, irritability, and anorexia). Safety data were collected on solicited adverse events that occurred within 7 days and unsolicited adverse events (those reported spontaneously by a parent or guardian) that occurred within 30 days after each dose. Data on serious adverse events were collected throughout the trial. The relationship between the adverse event or serious adverse event and the receipt of an injection was decided by the investigators of the study. 2.6. Immunogenicity Blood samples (3.0 ml) were collected before vaccination (Day 0) and one month after the second dose of the vaccination (Day 60). The neutralizing antibody titers against EV71 were measured by the fixed virus-diluted serum method in Sinovac Biotech Co., Ltd. Laboratory procedures are described in Protocol S1. The immunogenicity outcomes were the seroconversion rate, seropositivity rate, geometric mean titer (GMT), and geometric mean increase (GMI) of the study groups at Day 60. A titer of 1:8 or higher indicated seropositivity. Seroconversion in a subject was defined by either a pre-vaccination antibody titer < 1:8 and a Day 60 titer
1:8 or by a pre-vaccination titer
1:8 and a minimum 4-fold increase at Day 60. 2.7. Statistical analysis We calculated the sample size based on the results of the previous clinical trials conducted by the two manufacturers [8,9,11,12] via the PASS software (version 11.0). Assuming that the seroconversion rate of the interchangeable vaccine group was 95%, the typeⅠerror was 5%, and the permissible error was 6% between the sample rate and population rate, we obtained a sample size of 51 subjects per group. Assuming a 20% dropout of participants,

Please cite this article as: Q. Xu, Q. Cao, W. Yang et al., Interchangeability of two Enterovirus 71 inactivated vaccines in Chinese children: A phase IV, openlabel, and randomized controlled trial, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.013

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approximately 75 participants were to be enrolled for each vaccine group. The comparison of demographic characteristics among groups was performed based on the full analysis set (FAS) and the per-protocol set (PPS). The FAS comprised participants who were randomized, received at least one dose of vaccination, and had one pre- or post-vaccination valid serum value. The PPS included participants who met the eligibility criteria, complied with the protocol, and had immunogenicity results before and after the vaccination. Immunogenicity was analyzed in PPS. The neutralizing antibody titer distributions were described with reverse cumulative distribution maps. Seroconversion rate and seropositivity rate were calculated with 95% confidence intervals (CI); and the Pearson v2 test or the Fisher exact test was performed to compare the differences among different groups. The antibody titers and its increase were described by geometric means and 95% CIs, and their between-group differences were tested by the Student’s t-test or Wilcoxon rank sum test after log-transforming antibody titers. Safety analyses were performed based on the total vaccinated cohort. Solicited and unsolicited AEs were described with 95% CIs; and the Pearson v2 test or the Fisher exact test was conducted to compare the differences among different groups. Statistical significance was defined as a P-value < 0.05 (two-sided). The SAS 9.4 software (SAS Institute, Cary, NC, USA) was used to conduct the statistical analyses.

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3. Results 3.1. Study participants A total of 300 participants were enrolled and randomly assigned to four groups in a 1:1:1:1 ratio. There were 298 participants who received the first dose and 289 who received the second dose. Among all participants, 270 (90.0%, 270/300) received two doses, attended all visits, and provided planned blood samples. A total of 297(99%, 297/300) participants were included in the safety set for safety assessment. The per-protocol set for immunogenicity analysis included 267 (89%, 267/300) participants (Fig. 1). The baseline characteristics of the participants among the four groups were similar in terms of sex, mean age, height, weight, and ethnicity (Table 1). 3.2. Immunogenicity At baseline, the EV71 antibody titers were comparable among the four groups, with the seropositive rates ranging 0–5.9% and GMTs ranging 4.0–4.7 (Table 2). The seroconversion rates of the EV71 neutralizing antibody at Day 60 ranged from 98.4% to 100.0% and were not significantly different among the four groups. A significant increase in antibody titers at Day 60 was achieved in all groups. The GMT of group D

Fig. 1. Flow of participants through screening, random assignment, and analysis. ITT, Intention-to-treat; PPS, per-protocol set. Group A received two doses of Sinovac EV71 vaccine on day 0 and 30, group B received two doses of CAMS EV71 vaccine on day 0 and 30, group C received the first dose of Sinovac EV71 vaccine on day 0 and the second dose of CAMS EV71 vaccine on day 30, and group D received the first dose of CAMS EV71 vaccine on day 0 and the second dose of Sinovac EV71 vaccine on day 30.

Please cite this article as: Q. Xu, Q. Cao, W. Yang et al., Interchangeability of two Enterovirus 71 inactivated vaccines in Chinese children: A phase IV, openlabel, and randomized controlled trial, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.013

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Table 1 Demographic characteristic of study participants.

Total cohort No. of participants Age(Months) Male sex-no. Height(cm) Weight(kg) Ethnicity Han Per-protocol cohort No. of participants Age(years) Male sex-no.(%) Height(cm) Weight(kg) Ethnicity Han

Group A

Group B

Group C

Group D

75 19.6 ± 9.1 41(54.7) 83.0 ± 9.7 12.2 ± 2.6

75 19.6 ± 8.4 42(56.0) 82.9 ± 8.9 12.3 ± 2.3

74 19.7 ± 9.4 39(52.7) 82.1 ± 9.9 12.0 ± 2.5

74 20.3 ± 8.7 35(47.3) 83.8 ± 9.0 12.3 ± 2.6

75(100.0)

75(100.0)

73(98.6)

74(100.0)

68 19.8 ± 8.9 38(55.9) 83.4 ± 9.9 12.3 ± 2.7

67 19.6 ± 8.4 39(58.2) 83.1 ± 8.6 12.2 ± 2.3

68 19.9 ± 9.4 35(51.5) 82.3 ± 10.2 12.1 ± 2.5

64 20.8 ± 8.4 32(50.0) 84.3 ± 9.0 12.4 ± 2.7

68(100.0)

67(100.0)

67(98.5)

64(100.0)

Data are presented as n (%) mean ± standard deviation unless otherwise indicated.

Table 2 Antibody responses to EV71 pre- and post- vaccination in the per-protocol set. Group A (N = 68)

Group B (N = 67)

Group C (N = 68)

Group D (N = 64)

P value

Pre-vaccination (Day 0) SPR*, No (%) (95% CI) GMT (95% CI)

4 (5.9) (1.6, 14.4) 4.7 (3.8, 5.8)

1 (1.5) (0.0, 8.0) 4.2 (3.9, 4.5)

2 (2.9) (0.4, 10.2) 4.7 (3.8, 5.9)

0 (0.0) (0.0, 5.6) 4.00 (-)–

0.2216

Post-vaccination(Day 60) SCR**, No (%) (95% CI) SPR, No (%) (95% CI) GMT (95% CI) GMI (95% CI)

68 (100.0) (94.7, 100.0) 68 (100.0) (94.7, 100.0) 255.7 (187.7, 348.4) 54.5 (41.8, 71.3)

67 (100.0) (94.6, 100.0) 67 (100.0) (94.6, 100.0) 220.6 (167.3, 290.9) 53.2 (40.4, 69.9)

67 (98.5) (92.1, 100.0) 68 (100.0) (94.7, 100.0) 211.4 (155.1, 288.1) 45.1 (33.5, 60.8)

63 (98.4) (91.6, 100.0) 63 (98.4) (91.6, 100.0) 460.5 (356.0, 595.7) 115.1 (89.0, 148.9)

0.6132

0.1814

0.2397 ＜0.0001 ＜0.0001

Abbreviations: SPR, Seropositive rate; SCR, Seroconversion rate; GMT, geometric mean titer; GMI, geometric mean increase. * Seropositive was defined as a EV71 neutralizing antibody titer of 1:8 or higher. ** Seroconversion was defined by either a pre-vaccination antibody titer <1:8 and a Day 60 titer
1:8 or by a pre-vaccination titer
1:8 and a minimum 4-fold increase at Day 60. – GMT were 4.00 in pre-immunization in subjects of group D.

(the first dose of CAMS EV71 vaccine and the second dose of Sinovac EV71 vaccine) was the highest (460.5; 95%Confidence interval (CI):356.0, 595.7) among all the groups, with the GMI of 115.1 (95% CI: 89.0,148.9). The GMTs of group A, group B, and group C were 255.7 (95% CI:187.7, 348.4), 220.6 (95% CI:167.3, 290.9), and 211.4 (95% CI:155.1, 288.1), respectively; and the GMIs were 54.5 (95% CI: 41.8, 71.3), 53.2 (95% CI: 40.4, 69.9), and 45.1 (95% CI: 33.5, 60.8), respectively. The differences in GMT and GMI among the four groups were statistically significant (P < 0.001) (Table 2). Furthermore, based on the pairwise comparison, there was a significant difference (P < 0.001) in GMT between group D and the other three groups, respectively (Table S1). The proportion of the participants with an antibody titer of
1:128 one month after the second dose was 92.2% in group D, while that in other three groups were 69.1–72.1% (Fig. 2). Stratified analysis by gender and age group showed a similar response pattern that the GMT in group D was about 1.5 times that of other three groups in each stratum (Table S2 and Table S3). 3.3. Safety Participants who took at least one dose of EV71 vaccine were included in safety analysis set. The vaccine-related adverse events including solicited local and systemic reactions and unsolicited

adverse events after any vaccination are shown in Table 3. The safety profiles were similar between the study groups and all vaccination schedules were well tolerated. Most adverse events were mild in severity. The incidence of grade 3 adverse events were 2.7% (2/75) in group A, 2.7% (2/74) in group B, 1.4% (1/74) in group C, and 0.0% in group D, respectively. The incidence of solicited adverse events was 25.3% (19/75) in group A, 32.4% (24/74) in group B, 24.3% (18/74) in group C, and 24.3% (18/74) in group D, presenting no significant difference among the four groups. Pain (1.0%, 3/297) was the most frequently reported local symptom, and all local symptoms were in grade 1. The most common systemic symptoms were fever (21.6%, 64/297), diarrhea (4.7%, 14/297) and anorexia (4.4%, 13/297). The grade 3 systemic adverse events were mainly fever, which means that the axillary temperature exceeds 39 °C. There were no significant differences between the four groups in all local and systemic reactions. Only one unsolicited adverse event considered related to vaccination was reported, which was in group B; and the symptom was abdominal pain. In addition, the incidence of adverse events after the first dose was higher than that after the second dose (Supplementary Table 4). Ten serious adverse events were reported during 60 days observation period by a total of 7 participants: 1 of 75 (1.3%) in group A, 3 of 74 (4.1%) in group B, 2 of 74 (2.7%) in group C, and 1 of

Please cite this article as: Q. Xu, Q. Cao, W. Yang et al., Interchangeability of two Enterovirus 71 inactivated vaccines in Chinese children: A phase IV, openlabel, and randomized controlled trial, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.013

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Fig. 2. Reverse cumulative curves for enterovirus 71 (EV71) neutralizing antibody titers in the per-protocol population. (a). Pre-vaccination (Day 0). (b) Post-vaccination (Day 60).

Table 3 Vaccine-related adverse events and serious adverse events after any vaccination in total Vaccinated cohort. Event

Total vaccine-related adverse events Any Grade 3** Solicited adverse events§ Local reactions Pain Redness Swelling Pruritus Induration Systemic reactions Feverk Diarrhea Nausea and vomiting Anorexia Allergy Fatigue I rritability Unsolicited adverse events– Serious adverse eventy

Group A (N = 75) n(%)

Group B (N = 74*) n(%)

Group C (N = 74) n(%)

Group D (N = 74) n(%)

P value

19(25.3) 2(2.7) 19(25.3) 1 (1.3) 1(1.3) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 19(25.3) 14(18.7) 3(4.0) 1(1.3) 3(4.0) 2(2.7) 2(2.7) 1(1.3) 0(0.0) 1(1.3)

25(32.8) 2(2.7) 24(32.4) 2(2.7) 1(1.4) 0(0.0) 0(0.0) 1(1.4) 0(0.0) 23(31.1) 20(27.0) 5(6.8) 3(4.1) 6(8.1) 2(2.7) 0(0.0) 5(6.8) 1(1.4) 3(4.1)

18(24.3) 1(1.4) 18(24.3) 3(4.1) 1(1.4) 1(1.4) 2(2.7) 1(1.4) 1(1.4) 17(23.0) 15(20.3) 3(4.1) 1(1.4) 2(2.7) 0(0.0) 0(0.0) 1(1.4) 0(0.0) 2(2.7)

18(24.3) 0(0.0) 18(24.3) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 18(24.3) 15(20.3) 3(4.1) 1(1.4) 2(2.7) 2(2.7) 0(0.0) 1(1.4) 0(0.0) 1(1.4)

0.5243 0.7608 0.6415 0.3454 1.0000 0.7475 0.1843 0.6212 0.7475 0.7094 0.6289 0.8863 0.6998 0.3809 0.6117 0.2475 0.2028 0.7475 0.6838

*

A total of 75 participants were vaccinated in group B, but one moved from the study area and no safety data were collected. Adverse events of grade 3 were defined as those severe enough to prevent activity. For future classifications see Protocol S1. k Fever: Axillary temperature
37.1 °C; Grade 3 fever, temperature >39 °C. § Solicited adverse reactions within 7 days following any vaccination. – Unsolicited adverse reaction within 30 days following any vaccination. y Serious adverse event during entire study period. Serious adverse events were defined as events that resulted in death, were life threatening, required hospitalization or prolongation of existing hospitalization, resulted in disability or incapacity, or were a congenital anomaly/birth defect in offspring of a participant. All were considered to be unrelated to the vaccine by the study investigators. **

74 (1.4%) in group D (P = 0.6834) (Table 3). All serious adverse events were considered to be unrelated to the vaccine by the study investigators.

4. Discussion To the best of our knowledge, this is the first study to evaluate the immunogenicity and safety of a mixed use of EV71 vaccines from two manufacturers in an immunization series in the world. The results showed that interchanging Sinovac EV71 vaccine and CAMS EV71 vaccine in a series did not affect the number of chil-

dren achieving protective concentrations of antibody after the vaccination or increase the rate of adverse reactions. Data are available regarding the interchangeability of some other vaccines, including hepatitis A vaccines [13–15], hepatitis B vaccines [16], H. influenza type b conjugate vaccines [17,18], and rotavirus vaccines [19]. These studies demonstrated that mixed schedules had good immunogenicity and safety compared with using vaccines from a single manufacturer alone. In our study, 98.4–100% of the participants achieved seroconversion and 98.4– 100% o achieved antibody titer
1:8 one month after vaccination in all study groups, which are similar to previous trials evaluating these vaccines in children [8,9,20]. In addition, the high antibody

Please cite this article as: Q. Xu, Q. Cao, W. Yang et al., Interchangeability of two Enterovirus 71 inactivated vaccines in Chinese children: A phase IV, openlabel, and randomized controlled trial, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.013

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levels were achieved in single-vaccine groups or interchangeable vaccine groups. The antibody level of the interchangeable group was found to be between the levels of the single-vaccine groups in several studies [13–19]. In our study, there were significant differences in antibody levels between the two interchangeable vaccine groups; and the group D (children received CAMS EV71 vaccine followed by Sinovac EV71 vaccine) had significantly higher antibody responses compared to the other three groups. The reason for the difference has been unknown. At present, the immune response mechanism of EV71 vaccine is still unclear. Differences in vaccine epitopes, antigen content, and activated immune response pathways of the two vaccines may result in different immune response strengths, given the different sequence of interchangeable vaccination schedules. We will carry out further studies to explore the influence of interchanging EV71 vaccines on immune responses in mice. Overall, the safety profiles of the interchangeable vaccine groups were similar to that of the single-vaccine groups in the study. There were no serious adverse events associated with the vaccination and most reactions were mild and transient. The incidence of solicited local or systemic adverse reactions in each group was comparable with the previous clinical trials of the two vaccines [8,9,20]. The study has some limitations. Long-term immunogenicity of interchanging the two EV71 vaccines has not been explored. In our next step, we will design a 1-year follow-up study of children to evaluate the immunity persistence of interchanging the two EV71 vaccines. In addition, there are three approved EV71 vaccines in China, but only two were selected in the study. Additional studies are recommended to assess the interchangeability of the three vaccines. In conclusion, vaccination with EV71 vaccines from CAMS and Sinovac to complete an immunization series showed good immunogenicity and safety when compared with using EV71 vaccines from a single manufacturer alone. The antibody response levels may vary by different vaccination sequences of EV71 vaccines from different manufacturers to complete an immunization series. The results of the study may provide practical implications for vaccination doctors faced with situations in which vaccination with EV71 vaccines from different manufactures (CAMS and Sinovac) cannot be avoided such as the vaccine previously used is not known or not available anymore. In addition, the different immune responses caused by the sequence of interchangeable vaccination schedules also provide a new direction for future immune mechanism research. CRediT authorship contribution statement Qing Xu: Conceptualization, Data curation, Formal analysis, Investigation, Supervision, Writing - original draft, Writing review & editing. Qian Cao: Conceptualization, Investigation, Supervision, Writing - review & editing. Wanqi Yang: Data curation, Formal analysis, Investigation, Writing - review & editing. Xiaodong Liu: Data curation, Investigation, Supervision, Writing - review & editing. Haidong Liu: Data curation, Investigation, Supervision, Writing - review & editing. Xiaohui Tian: Investigation, Writing - review & editing. Jing Li: Investigation, Writing review & editing. Xueqiang Fang: Investigation, Writing - review & editing. Ningning Jia: Investigation, Writing - review & editing. Gang Zeng: Conceptualization, Formal analysis, Investigation, Supervision, Writing - review & editing. : . Aiqaing Xu: Conceptualization, Investigation, Supervision, Writing - review & editing. Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing

interests: WQ Yang, XH Tian, J LI, NN Jia and G Zeng are employed by the Sinovac Biotech Co., LTD. All other authors report no potential conflicts. Acknowledgments We thank all the investigators from Rushan City Center for Disease Control and Prevention, who contributed to the site work for the trial. We thank all the investigators from ten healthy clinics in towns selected for this study in Rushan, including Chengzhong, Chengnan, Wuji, Xiachu, Baishatan, Dagushan, Yazi, Rushanzhai, Yuli, Xiacun. Financial support This study was supported by Sinovac Biotech Co., LTD and the Taishan Scholar Project of Shandong Province (No. ts20151105). Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2020.02.013. References [1] Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Experim Vaccine Res 2017;6(1):4–14. [2] Huang J, Liao Q, Ooi MH, Cowling BJ, Chang Z, Wu P, et al. Epidemiology of recurrent hand, foot and mouth disease, China, 2008–2015. Emerg Infect Dis. 2018;24(3). [3] Chinese Center for Disease Control and Prevention. Analysis of epidemic characteristics of hand, foot and mouth disease in China from 2009 to 2018. Infectious Diseases Report. 2019;7(1):17. [4] Li L, Yin H, An Z, Feng Z. Considerations for developing an immunization strategy with enterovirus 71 vaccine. Vaccine 2015;33(9):1107–12. [5] Ng Q, He F, Kwang J. Recent Progress towards Novel EV71 Anti-Therapeutics and Vaccines. Viruses 2015;7(12):6441–57. [6] Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 2010;10(11):778–90. [7] Zhu FC, Meng FY, Li JX, Li XL, Mao QY, Tao H, et al. Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebocontrolled, phase 3 trial. Lancet 2013;381(9882):2024–32. [8] Zhu F, Xu W, Xia J, Liang Z, Liu Y, Zhang X, et al. Efficacy, safety, and immunogenicity of an enterovirus 71 vaccine in China. New Engl J Med 2014;370(9):818–28. [9] Li R, Liu L, Mo Z, Wang X, Xia J, Liang Z, et al. An inactivated enterovirus 71 vaccine in healthy children. New Engl J Med 2014;370(9):829–37. [10] Chinese Center for Disease Control and Prevention. Technical guidelines for the use of inactivated enterovirus 71 vaccines (2016). http://wwwchinacdccn/ zxdt/201606/t20160608_131032html. 2016. [11] Li YP, Liang ZL, Gao Q, Huang LR, Mao QY, Wen SQ, et al. Safety and immunogenicity of a novel human Enterovirus 71 (EV71) vaccine: a randomized, placebo-controlled, double-blind, Phase I clinical trial. Vaccine 2012;30(22):3295–303. [12] Li YP, Liang ZL, Xia JL, Wu JY, Wang L, Song LF, et al. Immunogenicity, safety, and immune persistence of a novel inactivated human enterovirus 71 vaccine: a phase II, Randomized, double-blind, placebo-controlled Trial. J Infect Dis 2014;209(1):46–55. [13] Abarca K, Ibanez I, Perret C, Vial P, Zinsou JA. Immunogenicity, safety, and interchangeability of two inactivated hepatitis A vaccines in Chilean children. Int J Infect Dis 2008;12(3):270–7. [14] Zhang ZL, Zhu XJ, Wang X, Liang M, Sun J, Liu Y, et al. Interchangeability and tolerability of two inactivated hepatitis A vaccines in Chinese children. Vaccine 2012;30(27):4028–33. [15] Bryan JP, Henry CH, Hoffman AG, South-Paul JE, Smith JA, Cruess D, et al. Randomized, cross-over, controlled comparison of two inactivated hepatitis A vaccines. Vaccine 2000;19(7–8):743–50. [16] Tregnaghi M, Ussher J, Baudagna AM, Calvari M, Grana G. Comparison of two recombinant hepatitis B vaccines and their interchangeability in Argentine infants. Revista panamericana de salud publica = Pan American Journal of Public Health 2004;15(1):35–40. [17] Anderson EL, Decker MD, Englund JA, Edwards KM, Anderson P, McInnes P, et al. Interchangeability of conjugated Haemophilus influenzae type b vaccines in infants. JAMA 1995;273(11):849–53.

Please cite this article as: Q. Xu, Q. Cao, W. Yang et al., Interchangeability of two Enterovirus 71 inactivated vaccines in Chinese children: A phase IV, openlabel, and randomized controlled trial, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.013

Q. Xu et al. / Vaccine xxx (xxxx) xxx [18] Bewley KM, Schwab JG, Ballanco GA, Daum RS. Interchangeability of Haemophilus influenzae type b vaccines in the primary series: evaluation of a two-dose mixed regimen. Pediatrics 1996;98(5):898–904. [19] Libster R, McNeal M, Walter EB, Shane AL, Winokur P, Cress G, et al. Safety and Immunogenicity of Sequential Rotavirus Vaccine Schedules. Pediatrics 2016;137(2):e20152603.

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[20] Zhang Z, Liang Z, Zeng J, Zhang J, He P, Su J, et al. Immunogenicity and safety of an inactivated enterovirus 71 vaccine administered simultaneously with recombinant hepatitis B vaccine and group A meningococcal polysaccharide vaccine: a phase IV, open-label, single-center, randomized, non-inferiority trial. J Infect Dis 2019.

Please cite this article as: Q. Xu, Q. Cao, W. Yang et al., Interchangeability of two Enterovirus 71 inactivated vaccines in Chinese children: A phase IV, openlabel, and randomized controlled trial, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.013