Comparing the Primary and Recall Immune Response Induced by a New EV71 Vaccine Using Systems Biology Approaches

<div><p>Three inactivated EV71 whole-virus vaccines have completed Phase III clinical trials in mainland China, with high efficacy, satisfactory safety, and sustained immunogenicity. However, the molecular mechanisms how this new vaccine elicit potent immune response remain poorly understood. To characterize the primary and recall responses to EV71 vaccines, 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. 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,CCL14a, CCL21 in recall response was consistent with the activation of immune response process found in genes. Furthermore, the expression of MX1 and secretion of IP-10 in recall response were strongly correlated with NTAb level at 180d after vaccination (r = 0.81 and 0.99). In summary, inflammatory response, adaptive immune response and a stronger antiviral response were indentified in recall response.</p></div

Cytokines profiling of <i>in vitro</i> EV71 antigen stimulated PBMCs which were from recipients after vaccination.

<p>PBMC were incubated with either EV71 antigen or medium as indicated in Materials and Methods. Up-regulated cytokines are shown in red and down-regulated cytokines in green. Each row represents a probeset and each column represents an individual.</p

Heat map of DEGs in primary and recall response.

<p>Colors ranging from blue to red corresponded represent the DEGs’ average fold change among the subjects (n = 19). (a) Common genes identified in primary and recall response. However, the fold change of these genes in recall response was higher than that in primary response. (b) Pathways that were only observed in recall response,including inflammatory response, antigen processing and presentation, B cell activation, T cell activation and humoral immune response.</p

Molecular signatures of immune reponses to EV71 vaccine.

<p>(a) Ingenuity Pathways Analysis of DEGs in primary response. IRF7 was found as a hub of transcriptional regulation. (b) Gene network analysis of recall response. IRF7 was also found as a major node of transcriptional regulation. There was another key node, IRF1, which upregulated the genes related to activation of MHC-I as shown in the solid box.</p

Cytokines profiling of <i>in vitro</i> EV71 antigen stimulated PBMCs which were from recipients before vaccination.

<p>PBMC were incubated with either EV71 antigen or medium as indicated in Materials and Methods. Five were up-regulated cytokines (GDF-15, MMP-13, IL-10, TIMP-2 and Angiopoietin-2) and 2 were down-regulated cytokines (PDGF-R alpha and GRO). Seven cytokines were significantly induced by EV71 antigen compared with control (P <0.05).</p

Titers of neutralizing antibodies responses to EV71 vaccine on days 0, 56 and 180 post vaccination.

<p>Each symbol represents an individual donor; median value and standard deviation were indicated. There were 19 subjects in the EV71 vaccine group and 13 in the placebo group. After vaccination (the first vaccination on day 0 and boosted on day 28), seroconversion was 100% in EV71 vaccine group.</p

Verification of selective genes with RT-PCR.

<p>Correlation analysis of gene expressions changes with microarray and RT-PCR (r = 0.7253). 17 genes were selected to confirm within 60 samples. Each point represents a single gene at a given sample.</p