The geometric mean titres are shown above the bar. this vaccine based upon clinical diagnosis. == Results == This sub-cohort showed a >60 % drop-out rate over 2 years. The seroconversion rates among the 161 immunized subjects remained >95 % at the end of study. The geometric mean titres of neutralizing antibodies (anti-genotype C4) 360 days after vaccination in 350 subjects were 81.0 (subjects aged 611 months), 98.4 (1223 months), 95.0 (2435 months), and 81.8 (3671 months). These titres subsequently increased to 423.1, 659.0, 545.0, and 321.9, respectively, at 540 days post-immunization (d.p.i.), and similar levels were maintained at 720 d.p.i. Higher IFN-/IL-4-specific responses to the C4 genotype of EV71 and cross-neutralization reactivity against major EV71 genotype strains were observed in the vaccine group compared to those in the placebo group. Five EV71-infected subjects were observed in the placebo-treated control group and none in the vaccine-immunized group in per-protocol analysis. == Conclusion == These results are consistent with the induction of dynamic immune responses and protective efficacy of the vaccine against most circulating EV71 strains. == Trial registration number == Clinicaltrials.gov,NCT01569581, Trial registration date: March 2012 == Electronic supplementary material == The online version of this article (doi:10.1186/s12916-015-0448-7) contains supplementary material, which is available to authorized users. Keywords:Cross-neutralization, Enterovirus 71, Hand, foot, and mouth disease, Inactivated vaccine, Long-term effect == Background == Hand, foot, and mouth disease (HFMD) ALK inhibitor 2 has recently emerged in the Asian-Pacific region as the most severe epidemic disease affecting children [1,2]. The effective prevention and control of HFMD epidemics is widely recognized as an essential public health issue [3,4]. Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two major pathogens causing HFMD. Of these two viruses, EV71 infection is associated with a higher death rate and is primarily responsible for fatalities [57]. Attempts to develop inactivated EV71 vaccines have made significant progress over a short period [810]. In a published report of a 1-year phase III clinical trial, an inactivated EV71 vaccine derived from a C4 genotype strain (the predominant strain circulating on the Chinese mainland) displayed adequate safety, immunogenicity, and efficacy, showing an efficacy of 97.4 % and geometric mean titres (GMTs) of 224.4 and 118.0 at 56 and 180 days post-immunization (d.p.i.), respectively [8]. However, the long-term effects of a vaccine on immunity (specifically, its cross-protection against circulating strains of various genotypes) are generally considered critical characteristics for its licensing and clinical application. Herein, we report the results of a continued evaluation of the immunogenicity, immune memory effects, and efficacy of this EV71 vaccine in a sub-cohort of subjects. Initially, 1,100 subjects were selected for the random administration of either the vaccine or a placebo, and 350 subjects provided a complete series of blood samples within the 2-year study period. In the present study, we performed a cross-neutralization ALK inhibitor 2 assay with nine individual EV71 strains of genotypes A, B (B3B5), and C (C1C5), using 160 serum samples obtained from the 350 subjects from the immunized and placebo groups, in a 1:1 ratio. The results show the efficacy of the immune response induced by the ALK inhibitor 2 inactivated Rabbit polyclonal to RABAC1 EV71 vaccine and ALK inhibitor 2 provide substantial data on the potential utility of this vaccine. == Methods == == Vaccine and vaccination == The inactivated EV71 vaccine was developed in a good manufacturing practice-compliant facility at the Institute of Medical Biology, Chinese Academy of Medical Sciences, and was tested at the National Institutes for Food and Drug Control before this study. The 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. Each dose of the vaccine contained 100 U of inactivated EV71 viral antigen adsorbed onto 0.5 mg of aluminum hydroxide, suspended in 0.5 mL of buffered saline. Both the vaccine and placebo were administered intramuscularly at 0 and 28 days. == Subjects and study design == The primary outcome of this trial was the determination of the efficacy of an inactivated EV71 vaccine, and the secondary outcome was the evaluation of the long-term immune persistence of this vaccine over a 2-year observation period. The study was conceived and performed with a randomized, double-blind, placebo-controlled cohort and registered at Clinicaltrials.gov asNCT01569581[8]. The study was proposed by the Centres for Disease Control and Prevention of Guangxi Province in association with a professional statistics group from the Fourth Military Medical University, and was approved by the China Food and Drug Administration and an independent ethics committee of Guangxi Zhuang Autonomous Region, China (Additional file1). A subset of 1 1,100 children, whose legal guardian provided written informed consent (Additional.