Substitution of Coxsackievirus A16 VP1 BC and EF Loop Altered the Protective Immune Responses in Chimera Enterovirus A71

Figures

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  Figure 1. Schematic representation of EV-A71 genome and architecture of capsid loops. Structural protein consists of VP1–VP4. Blue-colored regions represent the location of each capsid loop constructed in this study, and the numbers indicate the amino acid position within each viral protein.

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  Figure 2. (A). Schematic representation of location of capsid loops involved in this study. The figure illustrates pentamer and protomer model of EV-A71 (PDB ID: 3VBS) with highlighted capsid loops involved in this study, including VP1 BC, DE, EF, GH and HI loops (red), VP2 EF loop (green) and VP3 GH loop (purple). (B). Loop sequences of ChiE71 substituted by CV-A16. Columns from left to right represent the multiple sequence alignments and stereo view superpositions of corresponding loop regions of VP1 of WT EV-A71 (blue) and ChiE71 (red). The ChiE71 designed in this study include ChiE71-1-BC, ChiE71-1-DE, ChiE71-1-EF, ChiE71-1-GH, ChiE71-1-HI, ChiE71-2-EF and ChiE71-3-GH. The alignments were performed using Clustal Omega. ((∗)—identical conserved residue; (:)— strongly similar residues; (.)—residues have weakly similar properties; none—residues that are not conserved). The superpositions of ChiE71-1-BC, ChiE71-1-DE, ChiE71-1-EF, ChiE71-1-GH, ChiE71-1-HI, ChiE71-2-EF and ChiE71-3-GH (red) with WT EV-A71 (blue) showing structural variations upon replacement with the corresponding CV-A16 VP1 BC, DE, EF, GH, HI, VP2 EF and VP3 GH loops, respectively.

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  Figure 3. Characteristics of ChiE71. (A) One-step growth curves of the ChiE71. The RD cells were infected with 0.1 MOI of WT EV-A71 and ChiE71. The cell supernatants were harvested at 0, 12, 24, 48 and 96 h post-infection and viral titers were determined. The data are presented as mean of three replicates with standard deviation as the error bar. (B) Plaque morphology and plaque diameters of the WT EV-A71 and ChiE71 at passage 1 in RD cells. Data are presented as a mean of at least five replicates with standard deviation. (C) Heat map of neutralization capacity of each human anti-EV-A71 seropositive or seronegative serum against ChiE71, EV-A71 and CV-A16. The nine serum samples are shown on the left vertical axis, samples 1–6 are anti-EV-A71 positive and 7–9 are seronegative. The color gradient represents anti-log of base 2 neutralization titer. The lowest serum dilution was 1:23.

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  Figure 4. Type of interactions and sites of docked chimeras with anti-EV-A71 or anti-CV-A16 mono-clonal antibodies. WT EV-A71, WT CV-A16 and ChiE71-1-BC and ChiE71-1-EF models were docked with anti-EV-A71 D5 (PDB: 3JAU), in (A–D); or anti-CV-A16 D110 (PDB: 6LHQ), in (E–H). Each ChiE71 is presented as a protomer with four capsid proteins VP1 (blue), VP2 (cyan), VP3 (green) and VP4 (orange). Docked antibodies are presented as red strands.

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  Figure 5. Characterization of ChiE71-1-BC and ChiE71-1-EF. (A) Western blot of partially purified WT CV-A16 (lane 1), WT EV-A71 (lane 2), ChiE71-1-BC (lane 3) and ChiE71-1-EF (lane 4). (B) Particle stability thermal release assay (PaSTRY). Partially purified viruses were mixed with SYBR green II and heated from 25 to 95 °C in a StepOne Plus Real-Time PCR System. Derivative melt curves for ChiE71-1-BC, ChiE71-1-EF, EV-A71 and CV-A16 were plotted.

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  Figure 6. Immunogenicity of inactivated viruses in adult mice. (A) Schematic of the in vivo experiment to evaluate immune responses following ChiE71-1-BC and ChiE71-1-EF immunization. Each group of female adult BALB/C mice (n = 5) received prime injection and one booster of 10 μg of inactivated virus at indicated times. (B) Virus-specific IgG analysis of immunized mice sera on days 0, 14 and 28 using ELISA. (C) Heat map of EV-A71 and CV-A16 neutralization titers of immunized mice sera (after booster immunization). The lowest dilution of serum was 1:23. (D) EV-A71 VP1-specific IgG analysis of immunized mouse serum. The day 28 immunized mice sera were subjected to EV-A71 VP1-coated ELISA. (E) Inactivated ChiE71, WT EV-A71 and WT CV-A16 cellular immune responses in mice. Immunized mouse splenocytes were stimulated by 1μg of inactivated WT EV-A71 or WT CV-A16. The cytokine (IFN-γ, IL-2, IL-4 and IL-10) concentrations in the cell culture supernatant were detected using ELISA. Each dot represents serum from an individual mouse. Data are presented as mean (±SD) of mice in each group. *** p<0.001, ** p < 0.01; * p < 0.05.

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  Figure 7. In vivo protection following ChiE71 immunization of neonatal mice. (A) Schematic of in vivo experiment involving passive transfer of one-day-old neonatal mice with ChiE71-1-BC-, ChiE71-1-EF-, WT EV-A71- or WT CV-A16-immunized mouse sera followed by lethal MP4 EV-A71 or CV-A16 challenge. (B) Survival curves of the mice after passive transfer (C) Schematic of in vivo experiment involving active immunization of one-day-old neonatal mice with live ChiE71-1-BC, ChiE71-1-EF, WT EV-A71 or inactivated CV-A16 followed by lethal MP4 EV-A71 or CV-A16 challenge on day 7. (D) Survival curves of the mice after active immunization. Survival curves were compared, and the significance was determined based on p-values of Gehan-Breslow-Wilcoxon tests with corrected Bonferroni α values. Asterisk (*) indicates a significant difference.