Figure 1.

Cloning and characterization of recombinant Measles virus (MV) vectors. (a) Schematic representation of recombinant MV genomes. X: empty or EGFP, Y: aCTLA-4, aPD-L1, IgG Fc, or EGFP. H: Measles attachment protein hemagglutinin with native tropism; HαCD20: H retargeted to CD20. (b) Transgene expression. Thirty-six hours after infection at MOI 3, cells culture supernatants were collected. After immunoprecipitation with Protein A Sepharose immunoblot analysis was performed with anti-HA antibody. (c) Binding of MV-encoded aCTLA-4 and aPD-L1 to their cognate antigens was assessed by enzyme-linked immunosorbent assay (ELISA). (d) Syncytia formation and lysis of human melanoma cell lines. Sk-Mel-28 and Mel888 cells were infected with recombinant MV encoding EGFP and aCTLA-4 or aPD-L1 at MOI 1 and images were taken 36 hours p.i. (e) Targeted infection. Parental Vero and B16 as well as Vero-CD20 and B16-CD20 which stably express the CD20 surface antigen were infected with MV-EGFP with unmodified tropism and retargeted MV-EGFP HαCD20 at an MOI of 1. Images of cells 24 hours after infection (Vero, Vero-CD20) and 48 hours after infection (B16, B16-CD20) are shown. Scale bars: 100 µm. Replication and cytotoxicity in (f) human and (g) murine cells. Top panels: Viral growth kinetics. One-step growth curves of parental MV ld-EGFP, MV H-EGFP, MV-IgG Fc, MV-aCTLA-4, and MV-aPD-L1. Cells were harvested at designated time points and progeny viral particles were quantified by titration assays. Bottom panels: Cytotoxic effects. Cell viability was assessed by XTT assay. Mean values from triplicate infections are shown. Error bars indicate standard deviations. Viability of mock-treated cells was set to 100%. XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.