Development of a Surrogate Neutralization Assay for Norovirus Vaccine Evaluation at the Cellular Level

Figures

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  Figure 1. Expression of α1,2-fucosyltransferase 2 (FUT2) in 293T-FUT2 cells. Total RNAs from cells of wild-type 293T, mixed-cell population, and 293T-FUT2 cell lines were extracted and analyzed for FUT2 expression. The expression in wild-type 293T cells was set to 1 and used as the calibrator. Data are the means ± standard deviation (SD) of triplicate experiments. Mixed, mixed-cell population. Statistical significance was determined by the Student’s t-test using GraphPad Prism version 5, and values are indicated as follows: ** p < 0.01; *** p < 0.001.

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  Figure 2. Expression of histo-blood group antigens (HBGAs) in 293T-FUT2 cells. Wild-type 293T cells and transduced 293T-FUT2 cells were examined for their HBGA expression through immunofluorescence analysis and cellular enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies specific to each of six types of HBGAs (H1, Leb, H2, Ley, A, and B) as described in Materials and Methods. (A) Immunofluorescence analysis of HBGA expression in 293T-FUT2 cells. Scale bar, 10 μm. (B) Cellular ELISA analysis of HBGA expression in 293T-FUT2 cells. Data are the means ± SD of the optical density at 450 nm (OD450nm) values subtracted from the blank value (cells without incubation with primary antibody) for quadruple wells. The dash line indicates the cutoff, which is defined as 0.1 OD unit above the blank. All of the experiments were replicated three times.

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  Figure 3. Binding of norovirus virus-like particles (NoVs VLPs) to 293T-FUT2 cells. NoV VLPs were incubated with 293T and 293T-FUT2 cells for 1 h at 4 °C and subjected to immunofluorescence and Western blot analyses with mouse antisera against GI.1, GII.4, or GII.17 VLPs. β-Actin was chosen as an internal control. (A) Immunofluorescence analysis of NoV VLPs binding to 293T-FUT2 cells. Scale bar, 10 μm. (B–D) Western blot analysis of NoV VLPs binding to 293T-FUT2 cells. All of the experiments were replicated three times.

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  Figure 4. Norovirus virus-like particles (NoVs VLPs) attachment to 293T-FUT2 cells in a dose-dependent manner. Various doses of NoV VLPs were incubated with 293T-FUT2 cells for 1 h at 4 °C and detected by cellular ELISA with rabbit antisera against GI.1, GII.4, or GII.17 VLPs. (A) GI.1VLP attachment to 293T-FUT2 cells; (B) GII.4 VLP attachment to 293T-FUT2 cells; (C) GII.17 VLP attachment to 293T-FUT2 cells. Data are the means ± SD of OD450nm values for triple wells. Statistical significance was determined by the Student’s t-test using GraphPad Prism version 5, and values are indicated as *** p < 0.001. All of the experiments were replicated three times.

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  Figure 5. Inhibition of the norovirus virus-like particle (NoV VLP) attachment to 293T-FUT2 cells. NoV VLPs were preincubated with serially diluted mouse antisera for 1 h at 37 °C, and the resulting VLP/antisera mixtures were added to 293T-FUT2 cells. The attached VLPs were detected with VLP-specific rabbit antisera. (A) GI.1 VLP-specific antisera inhibited GI.1 VLP attachment to 293T-FUT2 cells; (B) GII.4 VLP-specific antisera inhibited GII.4 VLP attachment to 293T-FUT2 cells; (C) GII.17 VLP-specific antisera inhibited GII.17 VLP attachment to 293T-FUT2 cells. The enterovirus 71 (EV71) VLP-specific antisera were used as negative control sera. Data are the means ± SD of OD450nm values for triple wells. All of the experiments were replicated three times.

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  Figure 6. Evaluation of norovirus vaccines by a cellular ELISA-based attachment inhibition assay. Four groups of mice were immunized intraperitoneally with GI.1 virus-like particles (VLPs), GII.4 VLPs, GII.17 VLPs, or PBS, respectively, at weeks 0 and 2. Sera were collected from all immunized mice at 2 weeks after the last immunization and subjected to attachment inhibition assay and histo-blood group antigen blocking assay. (A) The 50% neutralization titers (NT50s) and (D) 50% blocking titers (BT50s) of sera from the GI.1 VLP group against GI.1; (B) NT50s; and, (E) BT50s of sera from the GII.4 VLP group against GII.4; (C) NT50s; and, (F) BT50s of sera from the GII.17 VLP group against GII.17. Samples that showed less than 50% blockade or inhibition at a 1:100 dilution (the lowest dilution tested) were assigned a titer of 50 for geometric mean titer computation. Each symbol represents one mouse, and the line indicates the geometric mean value of the group. Statistical significance was determined by the Student’s t-test using GraphPad Prism version 5, and values are indicated as follows: ** p < 0.01; *** p < 0.001; (G) Spearman’s correlation between NT50s and BT50s. Each square represents…