Study On The Immunologic Mechanism Of EV71 Inactivated Vaccine Using Systems Biology Approachs

Enterovirus 71(EV71) belongs to the Enterovirus genus of the Picornaviridae family and is one of the major pathogens associated with human hand, foot, and mouth disease(HFMD). EV71 is neurotropic and may lead to complications in the central nervous system, such as encephalitis, aseptic meningitis, even death in serious cases. EV71 is now considered as the most dangerous neurotropic enterovirus of the post-polio era. EV71 was epidemical worldwide, especially in the west Pacific region. There are no efficient drugs available for EV71 treatment, so vaccine is regarded as an essential way to control the EV71 outbreak.Research and development of EV71 vaccine was carried out in several Asian countries. Currently three inactivated EV71 whole-virus vaccines in mainland China have completed Phase III clinical trials in more than 30,000 infants and children. Results showed that these vaccines were safe and there were over 90% efficacy in preventing EV71-associated HFMD, 80% efficacy in preventing EV71-associated diseases. Two manufacturers has been approved to market in 2015. However, EV71 vaccine is novel, so a comprehensive understanding of immune responses to this new vaccine is still lacking. Exploring the effective protective immune mechanism will contribute greatly to the development of novel vaccines and the improvement of existing vaccines.Traditional vaccine development mainly depended on experience, which showed unconcern to mechanism of vaccine. This leaded to bottleneck in development of new vaccine, such as HCV and HIV. In 2009, foreign scholars applied systems biology in vaccinology firstly and introduced a new concept, “systems vaccinology”. The advancement of high-throughput technologies, together with the extensive identification of genomics, proteomics, etc. have been successfully applied to the field of vaccine research for the predicition of immunogenicity, efficacy and immune persistence of vaccines, such as HPV, Yellow Fever and Influenza vaccine.This study was conducted base on the success of former scholars. PBMC from recipients vaccination with EV71 vaccine were collected and their gene expression signatures were tested by microarray, and profiles of transcriptome were obtained. Then we tried to find regular results using the computer model, molecular biology methods and to verify in gene knockout mice, which helped to explore EV71 vaccine immune mechanism preliminarily.Part 1: Identify the common immune mechanism on the early stage after vaccination by meta-analysis of microarray dataHigh throughput technology such as microarray has been applied to explore the immune mechanisms of different kinds of vaccines, such as HPV, Yellow Fever and Influenza vaccine.While individual microarray study can be informative in identifying genes or significant biological pathways of a single vaccine, it is still difficult to obtain a comprehensive understanding of vaccine immune mechanisms since kinds of vaccines, microarray platforms and analysis methods differed appreciably. Meta analysis can increase the reliability and generalizability of results and obtain a more precise estimate of gene expression through combining information from multiple existing studies to get common immune mechanism.In this study, a meta-analysis of microarray datasets was performed using publicly available microarray Gene Expression Omnibus(GEO) and Array Express datasets of vaccination records. Five studies(out of 35) were selected for this metaanalysis. The results indicated that the type I IFN related genes and corresponding pathways were common in early immune responses for different kinds of vaccines. This finding was further validated through the examination of EV71 vaccinees using RT-PCR. Further application of this discovery may be of interest to the design of new vaccines and the screening of predictors of vaccine immunogenicity.Part 2: Comparing the primary and recall immune response induced by a new EV71 vaccine using systems biology approachesSystems biology approach has been pioneered in the studies of yellow fever vaccine, influenza vaccine and HPV virus-like particles vaccine. In these studies, genes involved in innate immune response pathways were enriched after vaccination, and specific biomarkers were found to predict the immune effect of vaccines with high accuracy. Since most of these researches were focused on the primary responses after initial immunization, little is known about the recall response. The recall response is important due to its close relationship with neutralizing antibodies titer and immune persistence.PBMC from 19 recipients before and after vaccination with EV71 vaccine are collected and their gene expression signatures after stimulation with EV71 antigen were compared. Primary response were investigated by comparing gene expression levels in pre-immunization PBMCs stimulated by EV71 Ag, and recall immune responses were investigated by comparing post-vaccination gene expression patterns of PBMCs stimulated by EV71 Ag.The results showed that primary and recall response to EV71 antigen have both activated an IRF7 regulating type I interferon and antiviral immune response network. However, up-regulated genes involved in T cell activation regulated by IRF1, inflammatory response, B-cell activation and humoral immune response were only observed in recall response. The specific secretion of IL-10 in primary response and IL-2, IP-10, CCL14 a, CCL21 in recall response was consistent with the activation of immune response process found in genes.Part 3: Short-fragment DNA residue from vaccine purification processes promotes immune response to the new inactivated EV71 vaccine by activating TLR9Results from meta analysis and microarray assay both revealed EV71 vaccine injection induced the activation of IFN-I signal pathway and which was regulated by TLR signal pathway. Besides, recent studies have revealed TLRs modulated the immune response after vaccination and single nucleotide polymorphisms of TLRs have been reported to influence the immune effect of several vaccines. Therefore, we suspected TLR might be involved in the immune response process of EV71 vaccine.In this study, we explored the function of TLR4 and TLR9 in response to the new EV71 vaccine, and our results showed TLR4 and TLR9 were both activated after incubation in vitro and vaccination in vivo. And there was a clear dose-response relationship between TLR4,9 and EV71 antigens. Cytokines profiles and the humoral immune level in TLR4 and TLR9 gene knock-out mice supported our conclusion in genes. Further research revealed the residual sf-DNA in the EV71 vaccine induced the activation of TLR9 and then promoted the generation of EV71 NTAbs.This is the first report about function study of TLR in response to the EV71 inactivated vaccine, and the first report about immune function of residul sf-DNA.