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. 2019 May 28;17:126–137. doi: 10.1016/j.omtn.2019.05.010

Figure 3.

Figure 3

LV Challenge of Pre-immunized Mice Strengthens the Neutralizing Activity

(A) The strength of VSVind.G-LV neutralization activity elicited by Gth immunization, LV challenge, and the combination of both. The calculated IC50 values were as follows: immunization (IO sera pooled), 6.96 × 10−3; vector challenge (CO sera pooled), 1.20 × 10−3; and immunization + challenge (IC sera pooled), 1.51 × 10−4. (B) Cross-neutralizing activity of sera isolated from IC terminal bleeds. Solid lines signify the neutralization effect observed, while the dotted lines indicate the lack of neutralization. Data shown represent the mean ± SD of three repeats performed in duplicates. To calculate IC50 values, curves were fitted using the software GraphPad Prism 5 modeled as an [inhibitor] versus response curve with variable hill slopes. (C) Isotypes of antibodies produced following Gth immunization versus VSVind.G-LV challenge. Wells were coated overnight with 50 ng/well recombinant VSVind.G, and they were incubated with pooled serum samples isolated from pre- and terminal bleeds. The absorbances measured from pre-bleed serum samples were regarded as background and subtracted from terminal bleed sera. One-way ANOVA with Tukey’s multiple comparison post-test was performed to compare absorbance levels within the experimental groups (****p < 0.0001, **p < 0.01; n.s., not significant). Data shown represent mean ± SD of one experiment performed in triplicates. (D) Anti-VesG antibodies induced by Gth immunization-boost regimen. Sera obtained from pre-bleed and pre-challenge bleeds (i.e., post-immunization) were pooled, and the amounts of antibodies directed against the four VesGs were determined via LV-based ELISA. The amount of antibodies recognizing PIRYV.G was significantly less compared to the other three VesGs. One-way ANOVA followed by Holm-Sidak’s multiple comparison post-test was performed to analyze the significance of the amount of anti-VesG antibodies produced via Gth immunization (***p < 0.001). Data shown represent mean ± SD from two experiments performed in duplicates.