A phase 1 randomized open-label clinical study to evaluate the safety and tolerability of a novel recombinant hepatitis E vaccine.

BACKGROUND: This study aimed to evaluate the safety and tolerability for variable dosages of a novel hepatitis E vaccine p179. METHODS: The randomized open-label parallel control phase 1 clinical trial enrolled 120 eligible participants aged 16-65years in Jiangsu Province, China. The experimental groups were randomized to receive different dosages of 20µg, 30µg, and 40µg Hepatitis E Virus (HEV) p179 vaccines, with the 30µgHEV vaccine p239 Hecolin as control, and vaccinated at 0, 1 and 6month intervals. Participants were observed for solicited local and systemic adverse reactions (ARs) occurring within 7days after each vaccination, and any serious adverse events (SAEs) occurring within 6months post-vaccination. Blood samples were collected from participants 3days before and after each injection, to determine the blood routine and serum biochemical indexes. RESULTS: The solicited local ARs incidence in experimental groups were significantly lower than that of the control group (P=0.027). The difference between solicited total and systemic ARs incidence of experimental groups and the control group were not significant (P>0.05). Similar patterns were observed when the analyses were performed on the group having ARs of varying grades and symptoms. All changes in blood biochemical indexes and routine blood tests before and after different vaccinations were mild (grade 1) or moderate (grade 2), and the difference in experimental groups and the control group were not statistically significant. No vaccine related SAEs occurred in any of the subjects during the study. CONCLUSION: Three different dosages of HEV p179 vaccine were deemed safe and well tolerated. No vaccine-associated SAEs were identified, and the 30µg dosage formulation was selected for further investigation for efficacy. Clinical trials registration number: 2012L01657.

Establishing the 1st Chinese National Standard for inactivated hepatitis A vaccine.

A reference standard calibrated in the International Units is needed for the quality control of hepatitis A vaccine. Thus, National Institutes for Food and Drug Control launched a project to establish a non-adsorbed inactivated hepatitis A vaccine reference as the working standard calibrated against the 1st International Standard (IS). Two national standard candidates (NSCs) were obtained from two manufacturers, and designated as NSC A (lyophilized form) and NSC B (liquid form). Six laboratories participated in the collaborative study and were asked to use their in-house validated enzyme-linked immunosorbent assay methods to detect hepatitis A vaccine antigen content. Although both exhibited good parallelism and linear relationship with IS, NSC B showed a better agreement among laboratories than NSC A. And based on suitability of the candidates, NSC B was selected. The accelerated degradation study showed that NSC B was stable at the storage temperature (≤-70 °C). Therefore NSC B was approved as the first Chinese national antigen standard for inactivated hepatitis A vaccine, with an assigned antigen content of 70 IU/ml.

The immunogenicity in healthy infants and efficiency to prevent mother to child transmission of Hepatitis B virus of a 10µg recombinant yeast-derived Hepatitis B vaccine (Hep-KSC).

BACKGROUND AND AIMS: To evaluate immunogenicity and efficacy of a 10µg recombinant Saccharomyces cerevisiae-derived hepatitis B vaccine (Kangtai Biological Products Co. Ltd, Shenzhen, China) (Hep-KSC) in newborns. METHODS: Overall 1197 infants born to mothers negative for HBV markers (NM group) and 534 born to HBsAg-positive mothers (PM Group) were enrolled. Infants in NM group were given 10µg Hep-KSC, 10µg Engerix-B or 5µg Hep-KSC and those in PM group received 10µg Hep-KSC or 10µg Engerix-B at 0, 1 and 6 months, with an additional 200IU HBIG at birth for the latter. RESULTS: For NM Group, 10µg Hep-KSC paralleled 10µg Engerix-B but outperformed 5µg Hep-KSC regarding seroprotective rate (95.06% vs 94.83% vs 89.67%, p=0.0077) and anti-HBs geometric mean concentration (GMC) (798.87mIU/ml vs 790.16mIU/ml vs 242.04mIU/ml, p<0.0001) at 7 months. The proportion of infants with anti-HBs greater than 1000mIU/ml was higher in 10µg Hep-KSC than 5µg Hep-KSC group (45.77% vs 11.93%, p<0.0001) at 7 and 12 months. For PM Group, the HBsAg positivity rate in 10µg Hep-KSC and 10µg Engerix-B group was 1.60% and 4.27% at 7 months, respectively. In 10µg Hep-KSC group, 93.61% and 91.29% achieved seroprotection at 7 and 12 months, respectively, and correspondingly 90.24% and 86.96% in 10µg Engerix-B group. The anti-HBs GMC was comparable between 10µg Hep-KSC and 10µg Engerix-B group at 7 and 12 months (575.31mIU/ml vs 559.64mIU/ml; 265.79mIU/ml vs 264.48mIU/ml). CONCLUSIONS: 10µg Hep-KSC might be appropriate for neonatal immunization with good immunogenicity and efficacy, especially for infants born to HBsAg-positive mothers.

Short-Fragment DNA Residue from Vaccine Purification Processes Promotes Immune Response to the New Inactivated EV71 Vaccine by Upregulating TLR9 mRNA.

To reduce potential oncogenic long genomic DNA in vaccines, nuclease treatment has been applied in the purification processes. However, this action increased the residue of short-fragment DNA and its effect on vaccine potency was still elusive. In this study, we found residual sf-DNA in an inactivated EV71 vaccine could enhance humoral immune response in mice. Ag stimulation in vitro and vaccine injection in vivo revealed that TLR9 transcription level was elevated, indicating that sf-DNA could activate TLR9. These new findings will help us to understand the molecular mechanism induced by vero-cell culture-derived vaccines.

An epidemic of coxsackievirus B3 infection in infants and children in Jiangsu Province, China: a prospective cohort study.

To investigate the epidemiological data on coxsackievirus B3 (CVB3) infection and its incidence in infants and children, a prospective cohort study was carried out from 2012 to 2014 in Jiangsu Province, China. According to the results of seropositive rates and NTAb titers of CVB3, an epidemic of CVB3 infection was found, and a dynamic change in CVB3 neutralizing antibody was also observed. One case was recorded with CVB3-associated hand, foot and mouth disease (HFMD), and the isolates belonged to the CVB3 D2 subtype. Our data help us to better understand the epidemic characteristics of CVB3 infection in infants and children.

Enterovirus spectrum from the active surveillance of hand foot and mouth disease patients under the clinical trial of inactivated Enterovirus A71 vaccine in Jiangsu, China, 2012-2013.

Epidemiological data from active surveillance on human enterovirus, which could cause hand, foot, and mouth disease, were limited. An active surveillance system was used to investigate the enterovirus spectrum and the incidence of different enteroviruses in infants aged 6-35 months in Jiangsu Province from 2012 to 2013. Fifty-nine infants were randomly selected from 522 non-EV-A71/CV-A16 HFMD patients. We collected 173 throat swabs and 174 rectal swabs from these infants. RT-PCR was used to amplify 5'-UTR and VP1 regions of enteroviruses and the serotypes were determined by the sequence comparison using BLAST. Twenty-one non-EV-A71/CA16 enterovirus serotypes were detected in those infants. E16, E18 were firstly reported in HFMD patients. The four top common non-EV-A71/CV-A enteroviruses among infants were CV-B3, CV-A10, CV-A6, and E9 with the HFMD incidence rates at 1.4%, 0.84%, 0.56%, and 0.47%, respectively. Over 20.8% patients were co-infected with multiple enteroviruses. Neither the course of sickness nor clinical symptoms of the co-infected patients was more severe than those infected with single enterovirus. Two patients were infected different enterovirus successively within 2 months. Several new enterovirus serotypes and multiple models of infection associated with HFMD were discovered through the active surveillance system. These data provide a better understanding of the viral etiology of HFMD.

Echovirus 7 associated with hand, foot, and mouth disease in mainland China has undergone a recombination event.

To investigate the evolution of echovirus 7 (Echo7) strains and the relationship between Echo7 strains and the prototype strain Wallace, phylogenetic analysis of Echo7 strains prevailing in mainland China was performed. The Echo7 strain, DH22G/JS/2012 was isolated from a 32-month-old boy who was clinically diagnosed with HFMD. The complete genome sequence of this isolate was determined after the virus was propagated in cell culture. Phylogenetic analysis showed that the subgroups B1 and C1 prevailed in mainland China from 1998 to 2012 and that the subgroup B2 began to circulate in mainland China in 2009. The result of Simplot analysis showed that the Echo7 strain DH22G/JS/2012 is a recombinant coxsackievirus B4 (CVB4) that circulated in mainland China in 2010.

Complete Genome Sequence Analysis of Echovirus 24 Associated with Hand-Foot-and-Mouth Disease in China in 2012.

Echovirus 24 belongs to human enterovirus B species in the family Picornaviridae. Here, we report the whole-genome sequences of a novel complete genome sequence of a recombinant (echovirus 24) Echo 24 strain, PZ18/JS/2012, which was isolated from a patient with hand-foot-and-mouth disease in China.

Immune response to hepatitis B vaccine with high antigen content in non-responders after standard primary vaccination in Chinese adults.

CONTEXT: Alternative schedules are needed to provide greater immunogenicity in adults who fail to respond to the standard hepatitis B (HB) vaccine regimen. OBJECTIVE: To evaluate the immunogenicity and safety of high antigen content HB vaccine formulations administered to non-responders after routine primary vaccination. DESIGN SETTING, AND PARTICIPANTS: This was a phase III, double-blind, controlled clinical trial in China. We enrolled healthy participants (16-60 years old) seronegative for HB surface (HBs) antigen after primary vaccination, who had HBs antibody (anti-HBs) titres <10 mIU/ml at 28 days following routine vaccination with licensed HB vaccine containing 10 µg of antigen. Participants were randomised (2:2:1) to receive three booster doses of HB vaccine formulations containing 60 µg, 30 µg or 10 µg of antigen per dose 28 days apart. Blood samples were obtained pre-vaccination and 28 days after each dose to assess immunogenicity. Reactogenicity and safety were evaluated up to 28 days after each vaccine dose. RESULTS: Seroconversion rates were ≥ 92.1% and ≥ 87.1% as from the second dose of the 60 µg and 30 µg HB vaccine formulations, respectively, with geometric mean concentrations (GMCs) of ≥ 286.0 mIU/ml and ≥ 164.0 mIU/ml. In the 10 µg HB vaccine group the seroconversion rates were ≥ 83.0% and the GMCs ≥ 110.1 mIU/ml as from the second vaccine dose. All HB vaccine formulations were well tolerated: 352/1091 (32.3%) participants reported at least one injection-site or systemic adverse reaction (145/434 [33.4%] from the 60µg group; 138/435 [31.7%] from the 30 µg group and 69/222 [31.1%] from the 10 µg group). Most reactions were mild or moderate and resolved within 24h. No serious adverse events were reported. CONCLUSION: Booster vaccination with a three-dose schedule of a high antigen content HB vaccine formulation was immunogenic and well tolerated in healthy adults. Clinicaltrialsgov Identifier: NCT01203319.

Development of enterovirus 71 vaccines: from the lab bench to Phase III clinical trials.

The widespread epidemics of enterovirus 71 (EV71) seriously affected the Western Pacific Region. Young children, especially those younger than 3 years are the most susceptible population to the EV71-associated diseases. Several Asian countries have begun to focus on the research and development of EV71 vaccines. Five inactivated whole-virus EV71 candidate vaccines (three were manufactured in mainland China based on a C4 genotype strain, one in Taiwan based on a B4 genotype strain and one in Singapore based on a B2 genotype strain) have been assessed in clinical trials. Three candidate vaccines developed in mainland China have already completed Phase III clinical trials recently. The tested EV71 vaccine could provide good efficacy, satisfactory safety, and high immunogenicity. Thus, inactivated EV71 vaccines are expected to become the first available vaccines against EV71 in the near future.

Clinical evaluation for batch consistency of an inactivated enterovirus 71 vaccine in a large-scale phase 3 clinical trial.

The demonstration of batch-to-batch consistency to confirm the reliability of the manufacturing process has become a mandatory step in vaccine development. This is a post-hoc analysis aimed to provide more solid evidence on the immunogenicity and consistency of 3 consecutive batches of a novel inactivated enterovirus 71 (EV71) vaccine. In total 10 245 healthy Chinese children aged 6-35 months had been recruited and randomized to receive one of 3 batches of EV71 vaccine or placebo according to a two-dose immunization schedule in a phase 3 clinical trial. Blood samples were taken just before and 28 days after vaccinations for serological tests of EV71 neutralizing antibody (NTAb) titer from the subjects. Among them, 7263 (70.9%) subjects with seronegative EV71 NTAb at baseline and the data of serological tests post-vaccination available were included for the analysis. The results showed that EV71 vaccine elicited high geometric mean titers (GMTs) of 407.0 U/mL (95% CI, 373.5-443.6) for batch 1, 468.1 U/mL (95% CI, 432.2-507.0) for batch 2, and 520.6 U/mL (95% CI, 481.2-563.3) for batch 3. The two-sided 95% confidence intervals (CIs) for the GMT ratios between each pair of vaccine batches were all within an interval of [0.67, 1.5]. Subjects who received EV71 vaccines demonstrated significant higher GMTs than those received placebos did (P<0.001). In terms of incidence of both local and general adverse reactions, no differences were found among 3 vaccine batches and placebos. EV71 vaccine was highly immunogenic in children, and the 3 consecutive batches were well consistent.

How to understand the efficacy measurements for enterovirus type 71 vaccine?

The choice of endpoint was most important for an efficacy vaccine trial. The objective of this paper is to gear toward answering questions about the rationality and scientificity of the primary endpoints choosing, case capturing and diagnosis strategy in our recently reported EV71 vaccine efficacy phase 3 trial. In order to obtain both high sensitivity and specificity in the case detecting, EV71-associated disease had been chosen as primary endpoint, a broad spectrum of clinical symptoms was surveyed, both the real-time RT-PCR and virus isolation were combined for the laboratory diagnosis, and serial specimens since disease onset were collected for assays. Though, the EV71 vaccine efficacy was well measured in the phase 3 trial, several potential factors could also have influences on the cases confirming. More evidence of EV71 vaccine efficacy will be demanded in post-marketing studies in the future.

Immunogenicity, safety, and immune persistence of a novel inactivated human enterovirus 71 vaccine: a phase II, Randomized, double-blind, placebo-controlled Trial.

BACKGROUND: Vaccination is considered a top priority for the control of human enterovirus 71 (EV71) infection outbreaks. METHODS: On the basis of phase I trial results, we conducted a double-blind, randomized, controlled trial to evaluate the optimal dose, immunogenicity, safety and immune persistence of the vaccine. A total of 480 healthy infants were randomly assigned to receive 2 injections of 100 U of vaccine, 200 U of vaccine, 400 U of vaccine, or placebo. Solicited adverse events (AEs) within 7 days and unsolicited AEs within 28 days after each vaccination were collected for safety evaluation. Blood samples were collected for neutralizing antibody assay. RESULTS: EV71 vaccine was well tolerated, and no dose-related safety concerns were observed. Two doses of the vaccine yielded seropositivity frequencies of 92.3%, 95.9%, and 99.0% (with titers ≥1:8) in the 100 U, 200 U, and 400 U groups, respectively. Geometric mean titers measured by neutralizing antibody assay increased to 60.2 (95% confidence interval [CI], 41.9-86.4), 72.8 (95% CI, 50.8-104.3), and 252.1 (95% CI, 180.8-351.6) for the 100 U, 200 U, and 400 U groups, respectively. The dose-response relationship, with the 400 U dose showing higher immunogenicity than the 100 U and 200 U doses, remained until 13 months after the second vaccination, despite waning antibody levels. CONCLUSIONS: The 400 U dose was recommended as the optimal dose for the phase III trial because of its good safety profile and higher immunogenicity.

Immunogenicity, safety, and lot consistency of a novel inactivated enterovirus 71 vaccine in Chinese children aged 6 to 59 months.

The determination of lot-to-lot consistency in the manufacturing process is a mandatory step in the clinical development of the novel enterovirus 71 (EV71) vaccine. A phase III, randomized, placebo-controlled, double-blind trial assessed the lot consistency, immunogenicity, and safety of the EV71 vaccine in children aged 6 to 59 months. Healthy children (n = 1,400) received one of three lots of the EV71 vaccine containing 400 U of EV71 antigen or a placebo at days 0 and 28. Blood samples were collected before dose 1 and at 28 days after dose 2 (day 56) for an anti-EV71 neutralizing antibody (NTAb) assay. The geometric mean titer (GMT) and the seropositivity rates (with titers of ≥1:8) were compared at day 56. After each dose, the solicited injection site and general adverse events (AEs) were recorded for 7 days, and unsolicited AEs were recorded for 28 days. At day 56, the seropositivity rates ranged from 99.7% to 100% for the vaccine groups. The NTAb GMTs for the vaccine were 140.3 (95% confidence interval [CI], 117.8 to 167.1), 141.5 (95% CI, 118.0 to 169.6), and 146.6 (95% CI, 122.5 to 175.3). The two-sided 95% CI of the log difference in GMTs between the pairs of lots were between -0.176 and 0.176, therefore meeting the predefined equivalence criteria. The percentages of subjects reporting any injection site AEs, general AEs, or serious AEs were similar across the four vaccination groups. In conclusion, the demonstration of consistency between the manufacturing lots confirms for the purposes of clinical development the reliability of the EV71 vaccine production process. (This study has been registered at ClinicalTrials.gov under registration no. NCT01636245.).

Progress on the research and development of inactivated EV71 whole-virus vaccines.

The prevalence of diseases caused by EV71 infection has become a serious public health problem in the Western Pacific region. Due to a lack of effective treatment options, controlling EV71 epidemics has mainly focused on the research and development (R&D) of EV71 vaccines. Thus far, five organizations have completed pre-clinical studies focused on the development of inactivated EV71 whole-virus vaccines, including vaccine strain screening, process optimization, safety and immunogenicity evaluation, and are in different stages of clinical trials. Among these organizations, three companies in Mainland China [Beijing Vigoo Biological Co., Ltd. (Vigoo), Sinovac Biotech Ltd. (Sinovac) and Institute of Medical Biology, Chinese Academy of Medical Science (CAMS)] have recently completed Phase III trials for the vaccines they developed. In addition, the other two vaccines, developed by National Health Research Institutes (NHRI) of Taiwan and Inviragen Pte., Ltd (Inviragen), of Singapore, have also completed Phase I clinical trials. Published clinical trial results indicate that the inactivated EV71 vaccines have good safety and immunogenicity in the target population (infants) and confer a relatively high rate of protection against EV71 infection-related diseases. The results of clinical trials suggest a promising future for the clinical use of EV71 vaccines. Here, we review and highlight the recent progress on the R&D of inactivated EV71 whole-virus vaccines.

Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: A vaccine for enterovirus 71 (EV71) is needed to address the high burden of disease associated with infection. We assessed the efficacy, safety, immunogenicity, antibody persistence, and immunological correlates of an inactivated alum-adjuvant EV71 vaccine. METHODS: We did a randomised, double-blind, placebo-controlled, phase 3 trial. Healthy children aged 6-35 months from four centres in China were randomly assigned (1:1) to receive vaccine or alum-adjuvant placebo at day 0 and 28, according to a randomisation list (block size 30) generated by an independent statistician. Investigators and participants and their guardians were masked to the assignment. Primary endpoints were EV71-associated hand, foot, and mouth disease (HFMD) and EV71-associated disease during the surveillance period from day 56 to month 14, analysed in the per-protocol population. This study is registered with ClinicalTrials.gov, number NCT01508247. FINDINGS: 10,245 participants were enrolled and assigned: 5120 to vaccine versus 5125 to placebo. 4907 (with three cases of EV71-associated HFMD and eight cases of EV71-associated disease) versus 4939 (with 30 cases of EV71-associated HFMD and 41 cases of EV71-associated disease) were included in the primary efficacy analysis. Vaccine efficacy was 90·0% (95% CI 67·1-96·9) against EV71-associated HFMD (p=0·0001) and 80·4% (95% CI 58·2-90·8) against EV71-associated disease (p<0·0001). Serious adverse events were reported by 62 of 5117 (1·2%) participants in the vaccine group versus 75 of 5123 (1·5%) in the placebo group (p=0·27). Adverse events occurred in 3644 (71·2%) versus 3603 (70·3%; p=0·33). INTERPRETATION: EV71 vaccine provides high efficacy, satisfactory safety, and sustained immunogenicity. FUNDING: China's 12-5 National Major Infectious Disease Program, Beijing Vigoo Biological.