Study of the integrated immune response induced by an inactivated EV71 vaccine

Longding Liu, Ying Zhang, Jingjing Wang, Hongling Zhao, Li Jiang, Yanchun Che, Haijin Shi, Rongcheng Li, Zhaojun Mo, Teng Huang, Zhenglun Liang, Qunying Mao, Lichun Wang, Chenghong Dong, Yun Liao, Lei Guo, Erxia Yang, Jing Pu, Lei Yue, Zhenxin Zhou, Qihan Li, Longding Liu, Ying Zhang, Jingjing Wang, Hongling Zhao, Li Jiang, Yanchun Che, Haijin Shi, Rongcheng Li, Zhaojun Mo, Teng Huang, Zhenglun Liang, Qunying Mao, Lichun Wang, Chenghong Dong, Yun Liao, Lei Guo, Erxia Yang, Jing Pu, Lei Yue, Zhenxin Zhou, Qihan Li

Abstract

Enterovirus 71 (EV71), a major causative agent of hand-foot-and-mouth disease (HFMD), causes outbreaks among children in the Asia-Pacific region. A vaccine is urgently needed. Based on successful pre-clinical work, phase I and II clinical trials of an inactivated EV71 vaccine, which included the participants of 288 and 660 respectively, have been conducted. In the present study, the immune response and the correlated modulation of gene expression in the peripheral blood mononuclear cells (PBMCs) of 30 infants (6 to 11 months) immunized with this vaccine or placebo and consented to join this study in the phase II clinical trial were analyzed. The results showed significantly greater neutralizing antibody and specific T cell responses in vaccine group after two inoculations on days 0 and 28. Additionally, more than 600 functional genes that were up- or down-regulated in PBMCs were identified by the microarray assay, and these genes included 68 genes associated with the immune response in vaccine group. These results emphasize the gene expression profile of the immune system in response to an inactivated EV71 vaccine in humans and confirmed that such an immune response was generated as the result of the positive mobilization of the immune system. Furthermore, the immune response was not accompanied by the development of a remarkable inflammatory response.

Clinical trial registration: NCT01391494 and NCT01512706.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Vaccination with the inactivated EV71…
Figure 1. Vaccination with the inactivated EV71 vaccine modulated gene expression.
A global view of gene modulation in the PBMCs of vaccinated infants (Vac, n = 20), 28 days after the vaccine booster, compared to the placebo cohort (Pla, n = 10) was determined by microarray analysis. The genes were classified according to their function. A heat map representation (a) and principal component analysis (PCA) (b) generated using the significantly modulated genes from at least one comparison versus day 0 are shown. (a) The color scale shows the significantly modulated genes from up- (dark red) to down-regulated (dark blue) to the right of each image. The values are shown on a log2 scale. (b) The Y value is the log2-fold-changes in gene expression of the vaccinated group vs. the placebo group. The up-regulated changes are above the baseline (Y = 0, the same with placebo group), and the down-regulated changes are below the baseline. The bars represent the maximum and minimum. The mean of the fold-changes in gene expression with a 95% CI is shown as the rectangle. The line in the rectangle represents the mean of these fold changes in gene expression. The number of genes that changed after immunization is shown beside the rectangles on the image.
Figure 2. Immune responses were induced by…
Figure 2. Immune responses were induced by the inactivated EV71 vaccine.
The humoral (a) and cellular immune responses (b) against EV71 antigen were induced by the inactivated EV71 vaccine in volunteers (n = 20 for Vaccinated and n = 10 for Placebo). (a) The neutralizing antibodies targeting the EV71 antigen in the serum of experimental subjects. The geometric mean titers (GMTs) of neutralizing antibodies to EV71 were measured by a neutralization test as described in the Methods. **, P values of vaccinated group vs baseline value (at 0 day before primary immunization) and vs placebo group were 2 scale.
Figure 3. Inflammatory responses were induced by…
Figure 3. Inflammatory responses were induced by the inactivated EV71 vaccine.
Measure of serum proinflammatory cytokines (a) and the modulation of the expression of genes associated with the inflammatory response (b). (a) Levels of serum proinflammatory cytokines in vaccinated and placebo subjects. All cytokine (IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL12p70, TNF-α and IFN-γ) levels were measured with an R&D Systems assay kits according to the manufacturer protocol. (b) The genes modulated and associated with the inflammatory response in vaccinated group (Vac) and placebo group (Pla) were classified and fallen under different functional categories: (1) ILs group: IL1A, IL1B, IL2RA, NFKBIZ (activator of IL-6 expression), IL8, IL17C, IL26 and IL32 in the IL group; (2) TNFs group: TNFAIP2 and TNFAIP6; (3) IFNs group: IFNA10 and OAS2 (activator of IFN-γ); (4) chemokines group: CCL3, CCL4, CCL15, CCL23, CCL26, CXCL1, CXCL3 and CXCR7. The levels of gene expression were measured by microarray analysis as described in the Methods. Data are shown in log2 scale.
Figure 4. Network of differentially expressed genes…
Figure 4. Network of differentially expressed genes after EV71 inactivated vaccine inoculation.
The network represents the genes and their inferred association with cell proliferation (a) and immune responses (b) that are significantly modulated. Node colors indicate fold change of gene expression in vaccinated subjects (n = 20). The value of normal control of pre-vaccinated was 1. The color scale shows the levels of gene expression from low (green, blue and purple) to high (red, pink and yellow).
Figure 5. Confirmation of the genes modulated…
Figure 5. Confirmation of the genes modulated in vaccinated subjects by real-time RT-PCR.
Real-time RT-PCR analyses of 30 selected genes of PBMCs from the vaccinated infants (Vac, n = 20) at day 28 after vaccine booster. The y-axis indicates the relative quantity of the specific mRNA in the samples compared with the pre-vaccinated samples. The results are normalised to the level of endogenous GAPDH. The error bars indicate the SD of the relative quantities.

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