Fig 1.
CDV OL can infect cells lacking SLAMF1 and nectin-4 receptors.
(A) Assessment of infection of Vero cells by CDV isolates in comparison with the OL strain. Cells were infected at an MOI of 0.1 (determined on Vero-dogSLAMF1 cells) and stained with Hema-Quick 48 hours later for visualization. (B) A panel of CHO cells expressing different relevant receptors was infected with an eGFP reporter MeV comprising the CDV H/F OL glycoproteins. Infectivity was reported using a fluorescence microscope. Magnification 40X.
Fig 2.
Heterologous combination of wild-type CDV H/F with a shortened signal peptide results in enhanced receptor-dependent fusion due to a weaker H-F interaction.
(A) Syncytia formation in cells cotransfected with CDV-F, CDV-H and eGFP. The signal peptides of CDV-F were replaced with the homolog from MeV-F, as indicated by the black boxes in the schematic. Twenty-four hours after cotransfection, fusion score was assessed under the GFP channel. (B) Quantitative fusion assay. Effector BHK cells were transfected with the indicated combination of attachment (CDV-H or Nipah-G) and fusion (F) proteins plus one of the dual-split reporter plasmids. Target CHO cells and CHO cells expressing CD38 (CHO-CD38) were transfected with the other dual-split reporter plasmids. 16 hours post-transfection cells were overlaid and Renilla luciferase activity was determined (RLU) 8 hours later. Values represent the mean ± standard deviation (SD) of one representative experiment performed in triplicate. Statistical significance was determined using one-way ANOVA with Holm-Sidak’s multiple comparison test (ns, not significant *, p<0.05; **, p<0.002; ***, p<0.0001). (C) CDV-H/F coimmunoprecipitation. HEK293T cells transiently expressing either wt or mutant HIS-tagged CDV-H proteins together with FLAG-tagged CDV-F proteins were lysed and immunoprecipitated (IP) with an anti-FLAG antibody. The signal intensity was determined using an anti-HIS antibody. (D) Quantitative fusion assay of fully retargeted CDV-H and MeV-H proteins onto CHO cells and CHO cells derivates. Either HIS-tagged or HIS-tagged and CD38-retargeted MeV-H/F and CDV-H/F complexes were transfected into effector cells and luminescence signal was determined over time. MeV-Haals = MeV-H blinded for CD46, nectin-4 and SLAMF1 via amino acid substitutions Y481A, R533A, S548L and F549S.
Fig 3.
CD46 binding affinity of the displayed scFv determines CD46-dependent cell-to-cell fusion of the retargeted CDV H/F complex.
(A) Representative sensogram (in resonance units, RU) for the binding of CD46 to biosensor surfaces containing (continuous line) or lacking (discontinuous line) single-chain antibody fragments (scFv). Experimental data represents the injection for 300s of scFv K2 followed by buffer injection. 1 μM of CD46 was subsequently flown over both biosensor surfaces and the signal was recorded during (association) and after injection (dissociation). The surfaces were finally regenerated at the end of the cycle as described in Materials and Methods. (B) Binding of CD46 to scFvs as assessed by surface plasmon resonance. Sensograms showing the response units (black lines) of various concentrations of CD46 to the scFvs. Best fit 1:1 binding model is shown as discontinuous red lines. Binding affinity (Kd) was determined from the association and dissociation rates (Table 1). (C) Quantitative fusion assay for the MeV-H and CDV-H variants on CHO cells. The experiment was carried out in duplicate and repeated twice with similar results (see S5 Fig). The data are shown as the mean ± SD.
Table 1.
Affinity and kinetic rate constants for single-chain variable fragment binding to CD46.
Fig 4.
CD46-retargeted CDV envelope glycoproteins determine virus tropism.
(A) Schematic representation of Stealth: a vaccine-derived measles virus pseudotyped with CD46-retargeted CDV H/F envelope proteins. Created with BioRender.com. (B) Role of the CD46 binding affinity into virus entry. Cells were infected at the indicated MOI with Stealth viruses displaying scFv with different affinity to CD46. eGFP expression was monitored 48 h post infection. (C) CHO cells and derivates expressing the HIS-pseudoreceptor or CD46 were infected with Fluc-expressing Stealth viruses (K1 and A09) at MOI 0.5. Luciferase expression was measured 48 h postinfection. n = 2 except for CHO-CD46 (n = 3), *, p-value<0.05 (two-tailed t-test). (D) Protein composition of Vero cells lysates. Western blot analysis was determined with similar amounts of virus particles and probed with the relevant antibodies. The molecular weight of the standard (colored lanes) is indicated. (E) Multistep growth kinetics of Stealth-A09 in Vero or Vero-αHIS cells. At the indicated time-point, both the supernatant and the cell pellet were collected and virus titers were determined on Vero-αHIS cells. Values and error bars (SD) were determined for a representative experiment performed in triplicate. (F) Virus tropism. CHO cell derivatives were infected with the eGFP-expressing viruses as indicated. eGFP autofluorescence was determined 48 h later. Scale bar, 200 μm. (G) Genetic stability of Stealth. Vero-hSLAMF1 cells were infected with Stealth and passaged multiple times. After 8 passages, the recovered virus was used to infect Vero cells expressing human or dog SLAMF1. Representative microphotographs are shown after infection for three (Vero dog SLAMF1) or six days (Vero and Vero human SLAMF1).
Fig 5.
High CD46 binding affinity governs oncolytic activity of CD46-targeted Stealth virus in a mouse model of ovarian cancer.
(A) Study schematic of the experimental design. SKVOv3ip.1 tumor cells encoding the firefly luciferase gene (SKOV3ip.Fluc) were implanted intraperitoneally into athymic mice. At day 10, 1x106 TCID50 particles of Stealth were administered following the same route. Tumor burden was then monitored at 7 days interval through bioluminescence imaging (BLI). (B) Kaplan-survival curves of SKOV3ip.Fluc-bearing mice treated with Stealth-N1E and Stealth-A09 viruses (n = 5). Statistical significance defined by long-rank test. (C) Representative BLI showing the dorsal view of treated animals. Radiance (photons per second per cm per steradian, p/s/cm2/sr) was translated to colors to indicate tumor burden in the mice, according to the legend shown on the right. (D) Quantification of the total body luminescence in photons per second per cm per steradian (p/s/cm2/sr). n = 5. Ns, not significant; *, p-value <0.05; **, p<0.005.
Fig 6.
Stealth-A09 virus achieves oncolysis indistinguisible from the parental MeV in a mouse model of multiple myeloma.
(A) SCID mice bearing subcutaneous U266.B1 cell tumors were treated intravenously with a suboptimal dose of virus. Tumor growth was measured with a caliper (n = 5) and animals were euthanized when the tumors ulcerated or when the tumor size reached 20% of the body weight. (B) Kaplan-Meier survival curves (n = 5). Significant differences among the groups were determined by the long-rank test (*, p<0.05). (C) Virus trafficking to subcutaneous tumor cells after systemic administration. eGFP expression was evaluated by immunohistochemistry of two representative samples from each group collected at euthanasia. Scale, 200 nm.
Fig 7.
Stealth virus remains oncolytic in the presence of MeV-immune serum.
(A) SKOV3ip.Fluc cells were injected into athymic nude mice and allowed to establish for 10 days. Next, mice in the relevant groups received 600 mIU of anti-MeV IgG antibody intraperitoneally three hours before virus treatment via the same route. (B) Kaplan-Meier survival curves (n = 5 mice per group). Significant differences among the groups were determined by the long-rank test (ns, not significant; *, p<0.05; **, p<0.002). (C) Representative BLI showing the dorsal view of treated animals. Radiance (photons per second per cm per steradian, p/s/cm2/sr) was translated to colors to indicate tumor burden in the mice, according to the legend shown on the right. (D) Quantification of the total body luminescence in photons per second per cm per steradian (p/s/cm2/sr). n = 5. Statistical significance was determined by one-way ANOVA with Dunnetts’ multiple comparison test. Ns, no significant; nd, not done.