Figure 2.

Efficacy of the oHSV detargeting and retargeting

(A) Detargeting was evaluated by infection of J1.1–2, J/A (J1.1 HVEM⁺), and J/C (J1.1 nectin-1⁺) cells infected for 72 h at different MOIs with the recombinant oHSVs expressing either wild-type (WT) gD (oHSV/gD) or gD modified by the insertion of an anti-hCXCR4 nanobody (oHSV/Nb-gD). Both viruses express EGFP under the control of pICP6, allowing the visualization of infected cells by epifluorescence microscopy. Scale bars represent 5 mm. (B and C) Retargeting was evaluated on U87MG (B) and U87MG CXCR4⁺ (C) cells. Cells were plated in 96-well plates, infected with oHSV/gD or oHSV/Nb-gD (MOI 0.1) and incubated in Incucyte S3 for real-time analyses during 72 h. EGFP expression and cell confluency were quantified every 6 h. Circles represent the ratio between the green and the phase area expressed as the mean ± SEM of four wells. Statistical significance was determined by ordinary two-way ANOVA with Bonferroni multiple comparisons of means with a single pooled variance (ns, non-significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001). Images were taken every 6 h, and representative images taken at 72 hpi are shown. Scale bars represent 2 mm. Additional representative whole-well images taken at 24, 48, and 72 h are shown in Figure S2. See also growth curve of oHSV/gD and oHSV/Nb-gD in U87MG CXCR4⁺ cells in Figure S3