Development of a novel EV-A71 monoclonal antibody for monitoring vaccine potency

Figures

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  Fig 1. The neutralizing efficacy of NHRI2016-1 (A) Schematic of EV-A71 hybridoma supernatants generation. After the final boost, splenocytes isolated from immunized mice were fused with a myeloma cell line to create hybridoma supernatants, followed by neutralization assays to select suitable candidates. This schematic is original and created by the authors using Procreate. (B) The neutralizing activity of five hybridoma supernatants (NHRI2016-1 to NHRI2016-5) against B5-141 was assessed by a neutralization assay. Neutralizing titers are presented as the mean ± standard deviation (SD) of triplicates. Statistical significance was determined using a two-way ANOVA, comparing the neutralizing titers between preserum and the hybridoma supernatants (****P < 0.0001, ns no significant difference (P ≥ 0.05)). (C) Neutralizing efficacy of NHRI2016-1 and MAB979 was evaluated against various EV-A71 strains (A/BrCr, B4-E59, B5-2008, C4-2008) by a neutralization assay. Data are presented as the mean ± SD of triplicates. Statistical significance was assessed by two-way ANOVA, comparing the neutralizing titers of MAB979 and NHRI2016-1 against each virus strain (****P < 0.0001). LD limit of detection.

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  Fig 2. Characterization of NHRI2016-1 and generation of NHRI2016-1-escape mutant. (A) Denatured purified EV-A71 B5 E + F particles were detected by western blotting using either MAB979 or (B) NHRI2016-1; MAB979 recognized linear epitopes, while NHRI2016-1 did not. M: marker. (C) Outline of serial passage experiments of B5-141 in the presence of NHRI2016-1. This image is original and created by the authors using Procreate. (D) Binding comparison of MAB979 and NHRI2016-1 to B5-141 and B5-Mut3-1 using IFA. Infected Vero cells were stained with primary antibody (either MAB979 or NHRI2016-1), then incubated with Alexa Fluor 488 goat anti-mouse IgG secondary antibody. NHRI2016-1 did not recognize B5-Mut3-1. Scale bar: 100 μm.

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  Fig 3. Epitope mapping of B5-Mut3-1. The front view of the EV-A71 pentamer is shown using PyMOL. Black, gray, white, and orange represent the EV-A71 viral capsid proteins VP1, VP2, VP3, and VP4, respectively. (A) The surface view showing the location of external mutations. (B) The cartoon diagram of EV-A71 pentamer illustrating both external and internal mutations.

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  Fig 4. Mutant virus bearing mutation at VP3-S64R effectively abolishes NHRI2016-1 recognition. (A) Dot blot data for rgHG-B5 and other mutant viruses. A series of two-fold dilutions of each inactivated virus was loaded onto the nitrocellulose (NC) membrane. After blocking, the NC membrane was probed with either MAB979 or NHRI2016-1, followed by an HRP-conjugated anti-mouse IgG secondary antibody to detect the bound target. (B) Vero cells were infected with either rgHG-B5 or mutant viruses; then, IFA was carried out to detect the green fluorescent signal. Scale bar: 100 μm.

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  Fig 5. Sequence comparison of the VP3 region (residues 38 to 77) among multiple EV-A71 genotypes and genogroups. The VP3-64 residue is highlighted in red, and conserved residues are depicted in black characters on a gray background. The VP3 sequence of the EV-A71 genogroup G is not yet available on NCBI.

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  Fig 6. Dot blot data and rat immunization study using NHRI2016-1 to quantify different EV-A71 immunogenic forms. Ineffective antigens refer to those that have lost their integrity (e.g., 90°C and 60°C heated antigens). Effective antigens maintain their potency and integrity (e.g., 30°C heated, 4°C unheated, UV-C exposed). (A) Dot blot results of antigens recognized by MAB979 compared to NHRI2016-1. Red boxes: 90°C heated antigen; brown boxes: 60°C heated antigen; black boxes: 30°C heated, 4°C unheated and UV-C-exposed antigens. (B) TEM images of effective and ineffective antigens. (C) NHRI2016-1 was employed in ELISA to quantify antigen concentrations. Specific pathogen-free female Wistar rats (n = 4 for each group) were immunized with a prime dose on day 0 and a booster injection on day 14, with 400 IU of the respective antigens. This schematic is original and created by the authors using Procreate. (D) Immunogenicity of effective and ineffective antigens in rats at day 28 post-primary immunization against homologous virus. LD: limit of detection. The box and whisker diagram represents neutralizing antibody titers for each group. The bar in the box indicates median antibody titers, and…