Zika Virus with Increased CpG Dinucleotide Frequencies Shows Oncolytic Activity in Glioblastoma Stem Cells

Abstract

We studied whether cytosine phosphate-guanine (CpG) recoding in a viral genome may provide oncolytic candidates with reduced infection kinetics in nonmalignant brain cells, but with high virulence in glioblastoma stem cells (GSCs). As a model, we used well-characterized CpG-recoded Zika virus vaccine candidates that previously showed genetic stability and safety in animal models. In vitro, one of the CpG-recoded Zika virus variants had reduced infection kinetics in nonmalignant brain cells but high infectivity and oncolytic activity in GSCs as represented by reduced cell proliferation. The recoded virus also efficiently replicated in GSC-derived tumors in ovo with a significant reduction of tumor growth. We also showed that some GSCs may be resistant to Zika virus oncolytic activity, emphasizing the need for personalized oncolytic therapy or a strategy to overcome resistance in GSCs. Collectively, we demonstrated the potential of the CpG recoding approach for oncolytic virus development that encourages further research towards a better understanding of host-tumor-CpG-recoded virus interactions.

Keywords: CAM; CpG recoding; Zika virus; egg; glioblastoma; oncolytic virus; stem cells.

Figure 1

The genome of Zika virus (ZIKV) and the CpG (cytosine–phosphate–guanine)-recoding strategy. ZIKV genomic regions encoding envelope (E) and non-structural 1 (NS1) proteins were recoded to increase the number of CpG dinucleotides. A barcode schematically represents the number of CpG dinucleotides. The actual number of CpG dinucleotides are in Supplementary Table S1.

Figure 2

Infection kinetics in nonmalignant human brain cells (HMC3 (a) and NPC (b)) and tumor glioblastoma stem cells (GSC 528 (e) and GSC 157 (f)) after inoculation at multiplicity of infection (MOI) of 0.01. Cell culture supernatants in 96-well plates were collected and viral titers were measured using the endpoint dilution assay. The dotted line represents the limit of detection. Cell proliferation assay after inoculation of cells (HMC3 (c) and NPC (d), GSC 528 (g), and GSC 157 (h)) with MOI of 1. Whiskers represent the standard error of the mean (SE) from three biologically independent replicates with three technical replicates. “dpi”—days post-inoculation. The asterisk (*) indicates p < 0.05 vs. WT (a,b,e,f) and control (c,d,g,h): (c) WT and E+32CpG at 3–7 dpi, permuted control at 5–7 dpi; (e) E/NS1+176CpG at 3 dpi; (f) E+32CpG and E/NS1+176CpG at 4 dpi; (g) WT, permuted control, E+102CpG at 3–7 dpi.

Figure 3

Morphology of glioblastoma stem cells (a: GSC 528; b: GSC 157) in vitro; phase-contrast microscopy. (c) Positive TGM2 and negative SOX2 staining in GSC 528. (d) Negative TGM2 and positive SOX2 staining in GSC 157. Morphology of GSC tumors (e: GSC 528; f: GSC 157) in in ovo cultures by bright-field microscopy. (g) Vascularization of a GSC tumor. (h) Intact chorioallantoic membrane in a control egg. (i) The volume of tumors formed at sampling (day 19 of embryonic development (ED); Mann–Whitney test). Scale bars are 0.1 (a–d) and 1 mm (e–h). Implantation efficiency of GSC cell cultures (GSC 528: n = 27, GSC 157: n = 26) on chicken embryo CAM (GSC 528: 96%, GSC 157: 88%) and egg viability at ED 19 (GSC 528: 92%, GSC 157: 93%) were comparable in both GSC models.

Figure 4

Hematoxylin and eosine staining in glioblastoma stem cells (GSC 528 (a) and GSC 157 (b) tumors at day 19 of embryonic development. CE: chorionic epithelium, AE: allantoic epithelium, M: intermediate vascularized mesenchyme, BV: blood vessel, and T: tumor. TGM2 (c: GSC 528; d: GSC 157) and SOX2 (e: GSC 528; f: GSC 157) protein expression in tumor cells; TGM2-positive staining is in red (c). Scale bars are 1 (a,b) and 0.1 mm (c–f).

Figure 5

Zika virus (ZIKV) quantification in tumors ((a) glioblastoma stem cells (GSC) 528; (b) GSC 157). The dotted line (a,b) represents limit of detection. The volume of tumors inoculated with ZIKV variants (c: GSC 528; d: GSC 157). Relative reduction of tumors ((e) GSC 528; (f) GSC 157); FC: fold change. *: p < 0.05; tumor volumes in ZIKV groups were compared to volumes in the control group. The dashed line (e,f) represents the base tumor volume in the control group. Sampling was performed at day 19 of embryonic development.

Figure 6

Immunohistochemistry of the Zika virus (ZIKV) antigen: (a) Isotype control staining of the glioblastoma stem cells (GSC) 528 tumor; (b) Mock-inoculated GSC 157 tumor; (c) GSC 528 inoculated with ZIKV E+102CpG; (d) GSC 157 inoculated with ZIKV E+102CpG (arrows). H&E staining of mock (e: GSC 528; f: GSC 157) and ZIKV-inoculated tumors (g: GSC 528 inoculated with ZIKV E+102CpG; h: GSC 157 inoculated with ZIKV E+102CpG). Scale bars are 0.1 mm.