Skip to main content
NCBI home page
Human Vaccines & Immunotherapeutics logoLink to Human Vaccines & Immunotherapeutics
. 2013 Jun 6;9(8):1701–1705. doi: 10.4161/hv.24949

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

Zheng-Lun Liang 1, Qun-Ying Mao 1, Yi-Ping Wang 1, Feng-Cai Zhu 2, Jing-Xin Li 2, Xin Yao 1, Fan Gao 1, Xing Wu 1, Miao Xu 1, Jun-Zhi Wang 1,*
PMCID: PMC3906269  PMID: 23744508

Abstract

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.

Keywords: enterovirus 71, hand-foot-mouth diseases (HFMD), inactivated whole-virus vaccine, immunogenicity, protective effect, standard

Introduction

The first EV71 case was reported in the United States in 1969.1 In recent years, there has been a continually increasing trend of EV71 infection-related epidemics in the Western Pacific region.2 To effectively control the prevalence of diseases caused by EV71 infection, different types of EV71 vaccine candidates are under active R&D, including inactivated whole-virus vaccines,3-8 live attenuated vaccines,9,10 recombinant vaccines9,11-14 and peptide vaccines15,16; among these approaches, research on inactivated whole-virus vaccines has shown rapid progress due to the advantages of immunogenicity and mature manufacturing technologies.3,4,6,17,18 Good immunogenicity and preventive effects were demonstrated for several inactivated vaccines in animals.5-8 In order to promote the R&D process of inactivated EV71 vaccines, a series of studies on quality control standards and standard references were performed, leading to the establishment of a quality control and evaluation system for inactivated EV71 vaccines that laid a solid foundation for clinical research and approval of new vaccines.19-23 At present, five organizations, located in Mainland China, Taiwan and Singapore, have reported rapid progress on the development of inactivated EV71 vaccines. Phase I and II clinical trial results have shown that the inactivated vaccines have good safety and immunogenicity.4,18,24 Recently published Phase III reports showed that the EV71 vaccines developed by three enterprises in Mainland China all have good safety and mediate protective effects against EV71 infection-related diseases.25,26 In this article, the R&D of inactivated EV71 vaccines, relevant quality control standards and the perspectives of EV71 vaccine research are summarized.

R&D Progress of Inactivated EV71 Vaccines

R&D progress of inactivated EV71 vaccines in Mainland China

In the R&D process of inactivated EV71 vaccines, Vigoo, Sinovac and CAMS established their respective C4 genotype vaccine strains, the seed lot system and cell banks of Vero or diploid cells according to WHO guidelines and the current edition of the Chinese Pharmacopoeia and Technical Guideline for Pre-Clinical Research of Preventive Vaccines issued by the State Food and Drug Administration, P.R. China (SFDA). They also completed cross-protection studies of the vaccine strains and conducted research on process optimization, animal immunogenicity and protective effects in animal models.5-8,27-31 All three vaccine candidates entered clinical trials between December 2010 and February 2011. In Phase I and II trials, the doses of the EV71 vaccine developed by Vigoo were 160, 320 and 640 U/dose (0.25, 0.5, 1.0 μg protein/dose), while the Phase III dose was 320 U/dose. Phase I and II doses of the EV71 vaccine developed by Sinovac were 100, 200 and 400 KU/dose, while the Phase III dose was 400 U/dose. The Phase I and II doses of the vaccine developed by CAMS were 160, 320 and 640 EU/dose, while the Phase III dose was 100 U/dose. Sinovac was the first to publish its Phase I clinical trial results, which indicated that the vaccine was safe and elicited protective antibody responses in adults, children and infants.18 Subsequently, Vigoo reported its Phase I trial results, confirming that the vaccine has good safety and immunogenicity in the target populations of adults, children and infants.24,32 The Phase II clinical trial results reported by Vigoo suggest that 320 U is the optimal dose for EV71 vaccination; while their vaccine were not found interference with immune response due to poliovirus vaccination induced crossimmunity, and the neutralizing antibody titer against EV71 significantly declined at month 8 compared with day 56, suggesting a need for a booster shot.33 The EV71 vaccine developed by CAMS using diploid cells has a clinical safety and immunogenicity profile consistent with the other two vaccines developed using Vero cells as a substrate.4 Taken together, the results of Phase I and II clinical trials demonstrate that the three inactivated EV71 whole-virus vaccines have good safety and immunogenicity in humans, especially children and infants. Moreover, the three companies recently announced the results of Phase III clinical trials on vaccine safety and the protection rate against EV71 infection-related diseases. In each trial, about 10,000 healthy infants and young children were immunized with an EV71 vaccine following the d0/d28 two-vaccination procedure, and the protective effect was observed in the following year (two epidemiological seasons). The report by Sinovac showed a protection rate of 95%,26 while the vaccine developed by CAMS showed a protection rate of 95% or more for the diseases caused by EV71 infection.34 A study by Vigoo also demonstrated that the vaccine has similarly good safety and efficacy profiles.35 The three vaccine clinical trials all showed good protective effects against EV71 infection-related diseases, which will advance the vaccine evaluation and licensing process.

R&D progress of inactivated EV71 vaccines by the NHRI of Taiwan

NHRI employed a B4 genotype vaccine strain36 and established the Vero cell bank in accordance with the relevant requirements of the FDA.17,36 Based on a comparative study of the vaccine strains,37,38 research with regards to process optimization,39,40 serum-free culture techniques for inactivated vaccines,41 quality control methods20,21 and immunogenicity assays38 has been conducted. The vaccine entered clinical trials in 2010, and the doses employed in the clinical trials were 5 μg and 10μg protein/dose. At present, Phase I clinical trials have been completed, and the result showed that the EV71 vaccine can induce a 4-fold or greater increase in neutralization antibody titers (NT) in healthy adults after only the first vaccination.42

R&D progress of inactivated EV71 vaccines by Inviragen of Singapore

The vaccine developed by Inviragen using a B3 genotype vaccine strain and Vero cells as the substrate entered clinical trials in 2011. High-dose and low-dose groups were included in the clinical trials, and Phase I trials have been completed.43

Research on Vaccine Quality Control Standards and Reference Standard Preparation

The establishment of vaccine quality control standards and reference standards are major steps in vaccine development and evaluation. Three EV71 vaccine candidates began registered clinical trials around the same time. In order to facilitate comparisons of the quality and efficacy of EV71 vaccines, the Chinese Drug Administration unified the quality control requirements for EV71 vaccines. Specifically, the establishment of vaccine strains, seed lot system and cell banks must meet the requirements of the Chinese Pharmacopoeia and WHO procedures, which mandate that the VP1 nucleotide sequence in the major protective area must remain unchanged from the primary seed lots to the working seed lots to ensure genetic stability. Among the EV71 vaccines that have entered into clinical trials, four of them have used Vero cells as the substrate for production, so the purity of the vaccine was key point for quality control. The content of EV71 antigen should not be less than 95.0% using the HPLC method, residual cell-substrate DNA should be less than 100 pg/dose and host contaminating proteins should be no more than 10% of the total protein content.

Research has shown that while Vero cells can induce tumors after more than 170 passages, such tumorigenicity was not found in passages 134–150.44,45 Currently, Vero cells have been approved for IPV and rotavirus vaccine production. Several live viral vaccines produced in Vero cells have been licensed in the United States over the last decade: the smallpox vaccine (2007) and two rotavirus vaccines (2006 and 2008).46 The WHO recommends that vaccine production organizations use 150 passages as qualified passage as far as possible. In addition, WHO recommends that the final DNA content per dose of the vaccine product is less than 10 ng.47 The US FDA mandates that the residual DNA content of each dose of biological products be no more than 100 pg. The Chinese Pharmacopoeia (third edition, 2010) requires the residual exogenous DNA in Vero cell culture-derived vaccines be no more than 100 pg per dose. The IPV vaccine manufactured by Pasteur Co., Ltd, using Vero cells requires residual DNA content to be below 10 pg.48 For the two EV71 vaccine candidates developed by Mainland Chinese companies using Vero cells as the substrate, cell passages were kept below 150, which is in line with the WHO requirement, and residual DNA was kept less than 100 pg/dose. Taking into account that the EV71 vaccine target populations are mainly healthy infants and young children, the residual DNA standard should be further improved to 10 pg/dose. It is very important to accurately measure the residual cellular DNA content in EV71 vaccine products. The National Institutes for Food and Drug Control (NIFDC) has established the first batch of Vero cell DNA reference standards (Approval No: 2011-NRS0044). The successful establishment of a Vero cell DNA quantitative reference will facilitate the standardization of dot blot hybridization for measuring residual host cell DNA contamination.49

The detection of vaccine antigens and neutralizing antibodies is important for vaccine development. To enable accurate detection of antigen content and the level of neutralizing antibodies, a series of studies were performed in China. CAMS and Sinovac developed reagents for quantitative detection of EV71 vaccine antigen.22,23 A collaborative study on determination method of EV71 neutralizing antibody was performed by NIFDC.50 NIFDC also developed a national EV71 vaccine antigen reference standard and neutralizing antibody standards (Approval No: 20100023, 0024).19 The vaccine antigen standard has a set value of 1600 U/ml, while the neutralizing antibody standards include 3 references: strongly positive (N12: 1000 U/ml), weakly positive (N3) and negative (J10), and these are used for vaccine quality control, vaccine dosage determination, immunogenicity and efficacy evaluation. Establishment of national EV71 antigen and neutralizing antibody quantitative standards facilitates vaccine quality control and comparative analysis of the immunogenicity of vaccines developed by Mainland Chinese enterprises, while also promoting the EV71 vaccine development process. However, the epidemic EV71 strain varies among different countries and regions while the dominant strain in Mainland China since 1998 has maintained a C4 genotype, so determining whether these standards are representative and appropriate as the global antigen and neutralizing antibody standards deserves further investigation.

In order to provide sufficient data on immunogenicity and antibody-response dynamic characteristics for clinical trials, Mao unified the units of dose (U/ml) of the three vaccines from Mainland China with the antigen reference standard and compared their immunogenicity in mice.51 The results showed that despite the differences in vaccine strains used, the cell substrates used, the production process employed and the differences in immunogenicity existing between the vaccine strains and original extracts, the final aluminum-adjuvant vaccines have similar immunogenicity.51 This result was consistent with the results of human clinical trials,18,24 suggesting that the immunogenicity of vaccines prepared from different strains, cell substrates and production processes can be compared on the basis of a unified dose. In order to solve the shortcomings of traditional neutralizing antibody assays, such as being labor-intensive, subjective and time-consuming, NIFDC has developed a pseudovirus-luciferase assay for rapid and quantitative detection of EV71 neutralizing antibody (NTAB) levels after vaccination.52

NHRI investigated the methodology for quantifying EV71 antigens (VP2-based Q-ELISA) to develop surrogate markers for vaccine efficacy studies. The Q-ELISA method to detect the EV71 VP2-28 antigen (residues 136–150 of VP2) has been developed, and the test results demonstrated a dose-response relationship with vaccine immunogenicity.20 In addition, they also completed the development and qualification of an in-house standardized virus neutralization assay (RD cell micro-neutralization assay). The structure and immunogenicity of empty particles (E-particles) and full particles (F-particles) prepared by different processes were compared in order to guarantee the vaccine efficacy by establishing corresponding quality control standards for the manufacturing process.21

Future Perspective of EV71 Vaccine Research

After evaluation of vaccine safety, immunogenicity and protective effects, future efforts should focus on international vaccine applicability, strengthening the monitoring of prevalent strains and variation, development of EV71/CA16 combination vaccines and promoting international cooperation for establishment of global vaccine standards.

The applicability of EV71 vaccines

EV71 has one serotype and three genotypes, which can be further divided into 11 genetic subtypes, including A, B1-B5 and C1-C5. Gene mutation and recombination frequently occur during EV71 epidemics, with the exception of those occurring Mainland China, where the dominant epidemic strain has remained C4 since 1998.53 The common epidemic strains observed after 2000 are C2, C4, C5, B4 and B5. Recent studies have discovered new genotypes, and this poses challenges in the selection and application of vaccine strains (i.e., the applicability of the vaccines).54 Thus, due to the presence of different genotypes, subtypes, and potentially emerging new genotypes, current R&D should focus on the cross-protection elicited by current vaccines against EV71 strains of subgenotype C4, B3 or B4 after the safety, immunogenicity and protective effects of these vaccines are obtained. Our recent research using serum from vaccine-immunized populations suggests that the single C4-strain derived vaccine confers a high level of cross-protection [unpublished data].

The need for the development of EV71/CA16 combination vaccines

CA16 is one of the main pathogens causing HFMD. Although the number of severe cases and deaths caused by CA16 is lower than caused by EV71, the clinical symptoms of EV71 and CA16 infections are difficult to distinguish. There is an urgent need to develop CA16-specific or combined CA16/EV71 vaccines.17,38,55 Several enterprises in Mainland China have completed pre-clinical studies on EV71/CA16 combination vaccines. Animal experiments showed that the bivalent vaccine could elicit a neutralizing antibody response to a similar extent as monovalent vaccines [unpublished data].

The international demand for EV71 vaccine development

The recently announced protective effects of EV71 vaccines in clinical trials highlight the promising potential of these vaccines as a new vaccine at the stage closest to approval and application. In the past, R&D as well as the promotion of new vaccines (e.g., HPV, rotavirus and pneumonia vaccines) has been dominated by large international companies such as Merck Sharp and Dohme (MSD) or GlaxoSmithKline (GSK). These companies are well experienced in the development of quality control standards for the R&D of new vaccines, and have strong capabilities in carrying out international clinical trials and application of new vaccines. However, the EV71 vaccine is a new vaccine led by developing countries, which have limited experience in establishing international reference standards. Therefore, successful R&D and promotion of vaccines to control EV71 epidemics requires cooperation between governments and vaccine manufacturers to be strengthened.56 In the meantime, international standardization of vaccine quality control should be performed under the guidance of the WHO.55 Recently, the WHO ECBS approved a joint research project between the NIFDC and National Institute for Biological Standards and Control (NIBSC) to develop EV71 vaccine quantitative standards for detection of neutralizing antibodies and antigens, which are expected to help promote the internationalization of the EV71 vaccine development.

Conclusion

The development of EV71 vaccines will play an important role in controlling EV71-related epidemics. In addition to the evaluation of the safety, immunogenicity and protective effects of the inactivated vaccines, future research should also focus on the cross-protective effects of vaccines developed from strains of different genotypes, strengthening the monitoring of prevalent strains and strain variation, development of EV71/CA16 combination vaccines and promoting international cooperation for establishment of global vaccine standards. In particular, the WHO-guided formulation of vaccine quality control standards and international cooperation for establishment of reference standards will greatly promote the R&D and application of EV71 vaccines worldwide.

Acknowledgments

This work was supported by the National 12th Five Major Special Projects Funding Program (No. 2012ZX10004701) and National High Technology Research and Development Program (“863” program, No. 2012AA02A402) from the Ministry of Science and Technology of the People’s Republic of China.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Footnotes

References

  • 1.Schmidt NJ, Lennette EH, Ho HH. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis. 1974;129:304–9. doi: 10.1093/infdis/129.3.304. [DOI] [PubMed] [Google Scholar]
  • 2.Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis. 2010;10:778–90. doi: 10.1016/S1473-3099(10)70194-8. [DOI] [PubMed] [Google Scholar]
  • 3.Dong C, Wang J, Liu L, Zhao H, Shi H, Zhang Y, et al. Optimized development of a candidate strain of inactivated EV71 vaccine and analysis of its immunogenicity in rhesus monkeys. Hum Vaccin. 2010;6:1028–37. doi: 10.4161/hv.6.12.12982. [DOI] [PubMed] [Google Scholar]
  • 4.Liu L, Zhang Y, Wang J, Zhao H, Jiang L, Che Y, et al. Study of the integrated immune response induced by an inactivated EV71 vaccine. PLoS One. 2013;8:e54451. doi: 10.1371/journal.pone.0054451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Dong C, Liu L, Zhao H, Wang J, Liao Y, Zhang X, et al. Immunoprotection elicited by an enterovirus type 71 experimental inactivated vaccine in mice and rhesus monkeys. Vaccine. 2011;29:6269–75. doi: 10.1016/j.vaccine.2011.06.044. [DOI] [PubMed] [Google Scholar]
  • 6.Li XL, Zhang ZY, Wang XX, Hao CS, Yang YJ, Li Y, et al. Immunogenicity and protective efficacy of the inactivated EV71 vaccine. Chinese Journal of Viral Diseases. 2012;6:415–8. [Google Scholar]
  • 7.Bek EJ, Hussain KM, Phuektes P, Kok CC, Gao Q, Cai F, et al. Formalin-inactivated vaccine provokes cross-protective immunity in a mouse model of human enterovirus 71 infection. Vaccine. 2011;29:4829–38. doi: 10.1016/j.vaccine.2011.04.070. [DOI] [PubMed] [Google Scholar]
  • 8.Zhao HL, Wang JJ, Zhang Y, et al. Immunoprotection of Inactivated EV71 Vaccine Against Enterovirus in Neonatal Rhesus Monkey. SCIENTIA SINICA Vitae. 2011;41:439–48. [Google Scholar]
  • 9.Chiu CH, Chu C, He CC, Lin TY. Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71. Microbes Infect. 2006;8:1671–8. doi: 10.1016/j.micinf.2006.01.021. [DOI] [PubMed] [Google Scholar]
  • 10.Arita M, Nagata N, Iwata N, Ami Y, Suzaki Y, Mizuta K, et al. An attenuated strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys. J Virol. 2007;81:9386–95. doi: 10.1128/JVI.02856-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Chung YC, Ho MS, Wu JC, Chen WJ, Huang JH, Chou ST, et al. Immunization with virus-like particles of enterovirus 71 elicits potent immune responses and protects mice against lethal challenge. Vaccine. 2008;26:1855–62. doi: 10.1016/j.vaccine.2008.01.058. [DOI] [PubMed] [Google Scholar]
  • 12.Wu CN, Lin YC, Fann C, Liao NS, Shih SR, Ho MS. Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine. 2001;20:895–904. doi: 10.1016/S0264-410X(01)00385-1. [DOI] [PubMed] [Google Scholar]
  • 13.Chung CY, Chen CY, Lin SY, Chung YC, Chiu HY, Chi WK, et al. Enterovirus 71 virus-like particle vaccine: improved production conditions for enhanced yield. Vaccine. 2010;28:6951–7. doi: 10.1016/j.vaccine.2010.08.052. [DOI] [PubMed] [Google Scholar]
  • 14.Tung WS, Bakar SA, Sekawi Z, Rosli R. DNA vaccine constructs against enterovirus 71 elicit immune response in mice. Genet Vaccines Ther. 2007;5:6. doi: 10.1186/1479-0556-5-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Foo DG, Alonso S, Chow VT, Poh CL. Passive protection against lethal enterovirus 71 infection in newborn mice by neutralizing antibodies elicited by a synthetic peptide. Microbes Infect. 2007;9:1299–306. doi: 10.1016/j.micinf.2007.06.002. [DOI] [PubMed] [Google Scholar]
  • 16.Liu JN, Wang W, Duo JY, Hao Y, Ma CM, Li WB, et al. Combined peptides of human enterovirus 71 protect against virus infection in mice. Vaccine. 2010;28:7444–51. doi: 10.1016/j.vaccine.2010.08.080. [DOI] [PubMed] [Google Scholar]
  • 17.Chong P, Hsieh SY, Liu CC, Chou AH, Chang JY, Wu SC, et al. Production of EV71 vaccine candidates. Hum Vaccin Immunother. 2012;8 doi: 10.4161/hv.21739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.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;30:3295–303. doi: 10.1016/j.vaccine.2012.03.010. [DOI] [PubMed] [Google Scholar]
  • 19.Liang Z, Mao Q, Gao Q, Li X, Dong C, Yu X, et al. Establishing China’s national standards of antigen content and neutralizing antibody responses for evaluation of enterovirus 71 (EV71) vaccines. Vaccine. 2011;29:9668–74. doi: 10.1016/j.vaccine.2011.10.018. [DOI] [PubMed] [Google Scholar]
  • 20.Liu CC, Chang HW, Yang G, Chiang JR, Chow YH, Sai IH, et al. Development of a quantitative enzyme linked immunosorbent assay for monitoring the Enterovirus 71 vaccine manufacturing process. J Virol Methods. 2011;176:60–8. doi: 10.1016/j.jviromet.2011.06.001. [DOI] [PubMed] [Google Scholar]
  • 21.Liu CC, Guo MS, Lin FH, Hsiao KN, Chang KH, Chou AH, et al. Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system. PLoS One. 2011;6:e20005. doi: 10.1371/journal.pone.0020005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Long RX, Xie ZP, Yang R, Li H, Bai HZ, Dong CH, et al. Development and Application of BA-ELISA Method for EV71 Antigen. Chinese Journal of Biological. 2009;12:1230–2. [Google Scholar]
  • 23.Jia H, Cai F, Gao Q, Zhang JS, Jing SR. Development of A Quantitative ELISA Method for EV71 Antigen. Chinese Journal of Biologicals. 2010;23:87–90. [Google Scholar]
  • 24.Meng FY, Li JX, Li XL, Chu K, Zhang YT, Ji H, et al. Tolerability and immunogenicity of an inactivated enterovirus 71 vaccine in Chinese healthy adults and children: an open label, phase 1 clinical trial. Hum Vaccin Immunother. 2012;8:668–74. doi: 10.4161/hv.19521. [DOI] [PubMed] [Google Scholar]
  • 25.A Protected Study of Inactivated EV71 Vaccine (Human Diploid Cell, KMB-17) in Chinese Infants and Children. Mar 10, 2013 2013. http://www.deepdyve.com/lp/clinical-trials/a-protected-study-of-inactivated-ev71-vaccine-human-diploid-cell-kmb-679kOYq0nc/1.
  • 26.Positive topline results from Phase III trial of Enterovirus 71 vaccine (Sinovac Biotech) against Hand, Foot and Mouth Disease. 2013. http://www.epgonline.org/news/2013/Mar/Positive-topline-results-fromPhase.cfm.
  • 27.Liang Y, Zhao HL, Liu LD, Liao Y, Dong CH, Na RX, et al. Large scale preparation of enterovirus type 71 inactivated vaccine in human diploid cells and its immunogenic study. Chinese Journal of Viral Diseases. 2012;2:409–14. [Google Scholar]
  • 28.Liu L, Zhao H, Zhang Y, Wang J, Che Y, Dong C, et al. Neonatal rhesus monkey is a potential animal model for studying pathogenesis of EV71 infection. Virology. 2011;412:91–100. doi: 10.1016/j.virol.2010.12.058. [DOI] [PubMed] [Google Scholar]
  • 29.Chen H, Zhang Y, Yang E, Liu L, Che Y, Wang J, et al. The effect of enterovirus 71 immunization on neuropathogenesis and protein expression profiles in the thalamus of infected rhesus neonates. Virology. 2012;432:417–26. doi: 10.1016/j.virol.2012.06.026. [DOI] [PubMed] [Google Scholar]
  • 30.Liang Y, Zhou X, Yang E, Pu J, Che Y, Wang J, et al. Analysis of the Th1/Th2 reaction in the immune response induced by EV71 inactivated vaccine in neonatal rhesus monkeys. J Clin Immunol. 2012;32:1048–58. doi: 10.1007/s10875-012-9690-3. [DOI] [PubMed] [Google Scholar]
  • 31.Mao Q, Li N, Yu X, Yao X, Li F, Lu F, et al. Antigenicity, animal protective effect and genetic characteristics of candidate vaccine strains of enterovirus 71. Arch Virol. 2012;157:37–41. doi: 10.1007/s00705-011-1136-3. [DOI] [PubMed] [Google Scholar]
  • 32.Zhu FC, Wang JZ, Li XL, Liang ZL, Ge HM, Meng FY, et al. Reactogenicity and immunogenicity of an enterovirus 71 vaccine in Chinese healthy children and infants. Pediatr Infect Dis J. 2012;31:1158–65. doi: 10.1097/INF.0b013e31826eba74. [DOI] [PubMed] [Google Scholar]
  • 33.Zhu FC, Liang ZL, Li XL, Ge HM, Meng FY, Mao QY, et al. Immunogenicity and safety of an enterovirus 71 vaccine in healthy Chinese children and infants: a randomised, double-blind, placebo-controlled phase 2 clinical trial. Lancet. 2013 doi: 10.1016/S0140-6736(12)61764-4. [DOI] [PubMed] [Google Scholar]
  • 34.http://wb.yunnan.cn/html/2013/ducheng_0312/64349.html
  • 35.EV71 Inactivated Vaccine Which Independently Researched and Developed by China has Achieved Important Progress – Phase III Clinical Research Demonstrated Good Efficacy and Safety. 2013. http://en.cnbg.com.cn/html/news/2013/0326/768.html
  • 36.Chang JY, Chang CP, Tsai HH, Lee CD, Lian WC, Ih-Jen-Su, et al. Selection and characterization of vaccine strain for Enterovirus 71 vaccine development. Vaccine. 2012;30:703–11. doi: 10.1016/j.vaccine.2011.11.087. [DOI] [PubMed] [Google Scholar]
  • 37.Lin YC, Wu CN, Shih SR, Ho MS. Characterization of a Vero cell-adapted virulent strain of enterovirus 71 suitable for use as a vaccine candidate. Vaccine. 2002;20:2485–93. doi: 10.1016/S0264-410X(02)00182-2. [DOI] [PubMed] [Google Scholar]
  • 38.Chou AH, Liu CC, Chang JY, Lien SP, Guo MS, Tasi HP, et al. Immunological evaluation and comparison of different EV71 vaccine candidates. Clin Dev Immunol. 2012;2012:831282. doi: 10.1155/2012/831282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Chou AH, Liu CC, Chang CP, Guo MS, Hsieh SY, Yang WH, et al. Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates. PLoS One. 2012;7:e34834. doi: 10.1371/journal.pone.0034834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Wu SC, Liu CC, Lian WC. Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development. Vaccine. 2004;22:3858–64. doi: 10.1016/j.vaccine.2004.05.037. [DOI] [PubMed] [Google Scholar]
  • 41.Liu CC, Lian WC, Butler M, Wu SC. High immunogenic enterovirus 71 strain and its production using serum-free microcarrier Vero cell culture. Vaccine. 2007;25:19–24. doi: 10.1016/j.vaccine.2006.06.083. [DOI] [PubMed] [Google Scholar]
  • 42.Cheng A, Fung CP, Liu CC, Lin YT, Tsai HY, Chang SC, et al. A Phase I, randomized, open-label study to evaluate the safety and immunogenicity of an enterovirus 71 vaccine. Vaccine. 2013;31:2471–6. doi: 10.1016/j.vaccine.2013.03.015. [DOI] [PubMed] [Google Scholar]
  • 43.Safety and Immunogenicity Study of an Inactivated Vaccine Against Hand, Foot and Mouth Disease Caused by Enterovirus. http://prsinfo.clinicaltrial.gov/ct2/show/nct01376479?term=inviragen+%28singapore%29+pte+ltd.&rank=1 2013.
  • 44.Requirements for the use of animal cells as in vitro substrates for the production of biologicals. WHO Expert Comminittee on Biological Standardization Forty-seventh Report. Geneva,World Health Organization; 1998. [Google Scholar]
  • 45.Requirements for continuous cell lines used for biological production. WHO Expert Commmitte on Biological Standardiztion Thirty-sixth Report Geneva, World Health Organization: (WHO Technical Report Series, No. 745,Annex 3). 1987. [PubMed] [Google Scholar]
  • 46.Cell Lines Derived from Human Tumors for Vaccine Manufacture. Vaccines and Related Biological Products Advisory Committee Meeting 2012; 2012. [Google Scholar]
  • 47.Safety of biological products prepared from mammalian cell culture. DNA. 1st ed. Basel: S Karger; 1998. [PubMed] [Google Scholar]
  • 48.Theresa M. Finn WE. Vaccine additives and manufacturing residuals in the United States: licensed vaccines. 6th ed. Edinburgh: Elsevier/Saunders; 2013. [Google Scholar]
  • 49.Cao S, Dong G, Tang J, Li J, Liu J, Shi L, et al. Development of a Vero cell DNA reference standard for residual DNA measurement in China. Hum Vaccin Immunother. 2013;9 doi: 10.4161/hv.22699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Mao QY, He P, Yu X, Li N, Hao CS, Gao Q, et al. Laboratory Evaluation of Method for Determination of Neutralizing Antibodyagainst Human Enterovirus 71. Chin J Biologicals. 2010;8:99–102. [Google Scholar]
  • 51.Mao Q, Dong C, Li X, Gao Q, Guo Z, Yao X, et al. Comparative analysis of the immunogenicity and protective effects of inactivated EV71 vaccines in mice. PLoS One. 2012;7:e46043. doi: 10.1371/journal.pone.0046043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Wu X, Mao QY, Yao X, Chen P, Chen XM, Shao J, et al.Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71. PLoS ONE 2013; 8: e64116. [DOI] [PMC free article] [PubMed]
  • 53.Tan X, Huang X, Zhu S, Chen H, Yu Q, Wang H, et al. The persistent circulation of enterovirus 71 in People’s Republic of China: causing emerging nationwide epidemics since 2008. PLoS One. 2011;6:e25662. doi: 10.1371/journal.pone.0025662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Zhang D, Lu J, Lu J. Enterovirus 71 vaccine: close but still far. Int J Infect Dis. 2010;14:e739–43. doi: 10.1016/j.ijid.2009.12.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Lee MS, Tseng FC, Wang JR, Chi CY, Chong P, Su IJ. Challenges to licensure of enterovirus 71 vaccines. PLoS Negl Trop Dis. 2012;6:e1737. doi: 10.1371/journal.pntd.0001737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Xu J, Qian Y, Wang S, Serrano JM, Li W, Huang Z, et al. EV71: an emerging infectious disease vaccine target in the Far East? Vaccine. 2010;28:3516–21. doi: 10.1016/j.vaccine.2010.03.003. [DOI] [PubMed] [Google Scholar]

Articles from Human Vaccines & Immunotherapeutics are provided here courtesy of Taylor & Francis

RESOURCES