Some attenuated variants of vesicular stomatitis virus show enhanced oncolytic activity against human glioblastoma cells relative to normal brain cells

Abstract

Vesicular stomatitis virus (VSV) has been shown in laboratory studies to be effective against a variety of tumors, including malignant brain tumors. However, attenuation of VSV may be necessary to balance the potential toxicity toward normal cells, particularly when targeting brain tumors. Here we compared 10 recombinant VSV variants resulting from different attenuation strategies. Attenuations included gene shifting (VSV-p1-GFP/RFP), M protein mutation (VSV-M51), G protein cytoplasmic tail truncations (VSV-CT1/CT9), G protein deletions (VSV-dG-GFP/RFP), and combinations thereof (VSV-CT9-M51). Using in vitro viability and replication assays, the VSV variants were grouped into three categories, based on their antitumor activity and non-tumor-cell attenuation. In the first group, wild-type-based VSV-G/GFP, tumor-adapted VSV-rp30, and VSV-CT9 showed a strong antitumor profile but also retained some toxicity toward noncancer control cells. The second group, VSV-CT1, VSV-dG-GFP, and VSV-dG-RFP, had significantly diminished toxicity toward normal cells but showed little oncolytic action. The third group displayed a desired combination of diminished general toxicity and effective antitumor action; this group included VSV-M51, VSV-CT9-M51, VSV-p1-GFP, and VSV-p1-RFP. A member of the last group, VSV-p1-GFP, was then compared in vivo against wild-type-based VSV-G/GFP. Intranasal inoculation of young, postnatal day 16 mice with VSV-p1-GFP showed no adverse neurological effects, whereas VSV-G/GFP was associated with high lethality (80%). Using an intracranial tumor xenograft model, we further demonstrated that attenuated VSV-p1-GFP targets and kills human U87 glioblastoma cells after systemic application. We concluded that some, but not all, attenuated VSV mutants display a favorable oncolytic profile and merit further investigation.

FIG. 1.

Schematic display of wild-type VSV and 10 variants. The VSV genome consists of a single RNA strand encoding five genes. The top displays a schematic of the VSV wild-type genome and a color-matched illustration of the virus structure. The next genome shows a recombinant variant, VSV-G/GFP, in which an additional copy of the G gene is inserted, tagged to a GFP reporter gene. VSV-dG-GFP has the whole gene order shifted through insertion of the GFP reporter gene at the first position and has the complete sequence for the G protein deleted. VSV-CT9 has a truncated G protein, shortening the 27-amino-acid chain of the cytoplasmic G protein tail down to 9 amino acids; the GFP reporter gene is inserted between the G and L genes. VSV-CT1 has the G gene truncated to a single amino acid in its cytoplasmic tail. VSV-M51 has a methionine deletion at position 51 of the M protein, leading to more susceptibility of the virus to the interferon-mediated antiviral defense; the GFP reporter gene is inserted between the G and L genes. VSV-CT9-M51 combines the mutation of M51 with the CT9 mutation. VSV-p1-GFP has a wild-type-related genome that is completely shifted by the insertion of the GFP reporter gene at position 1 of the gene order, leading to decreased viral transcription of downstream genes. VSV-rp30 is based on recombinant VSV-G/GFP, with one mutation each in the P and L proteins, leading to enhanced infection and oncolysis.

FIG. 2.

VSV variants infecting U87 human glioblastoma cells. A panel of representative photomicrographs shows phase-contrast and GFP fluorescence microscopy images of cultured U87 cells after infection with VSV variants at an MOI of 0.1, at 36 h postinfection. The left two columns show control conditions, and the right two columns show experiments performed after interferon preincubation. Interferon does not protect U87 cells from VSV infection. Cytopathic effects were observed in phase-contrast mode. Replication-deficient VSV-dG-GFP and VSV-dG-RFP show significantly less infection. VSV-CT1 contains no GFP reporter, and thus no fluorescence image is shown for it.

FIG. 3.

Cell viability of human glioblastoma and normal human glia cells after infection with VSV variants. Using an MTT cell viability assay, the cytotoxic effects of 10 VSV variants were tested on human glia control cells and U87 glioblastoma cells. (A) At 36 hpi at an MOI of 0.5, little effect on cell viability was seen for all VSV variants on control cells. (B) VSV-G/GFP, VSV-rp30, and VSV-CT9 showed signs of toxicity after 72 h. The presence of IFN (shaded boxes) completely protected normal human glia cells from infection with all VSV variants. (C) Human U87 glioblastoma cells showed a pronounced loss of viability at 36 hpi with 6 of 10 VSV variants. (D) At 72 hpi, all VSV variants, except the two replication-restricted VSV-dG mutants, completely killed U87 cells. Interferon provided little protection for U87 cells at 72 hpi (shaded box).

FIG. 4.

Replication of VSV variants on U87 cells and normal human glia cells. Virus replication was compared for each variant between brain tumor cells and normal human glia cells, using a plaque assay. Monolayers of each culture were infected at an MOI of 1 with the respective VSV variant, and supernatants were collected at the indicated time points. Virus replication was attenuated on human glia cells compared to that on U87 cells, by about 2 log. Also, VSV-p1-GFP and VSV-p1-RFP showed decreased viral replication. Differences between the other variants were marginal. (A) Note the complete block of viral replication after IFN preincubation. (B) In contrast, viral replication in U87 brain tumor cells was higher for each variant and productive even in the presence of IFN. Graphs for replication-restricted VSV-dG variants display the baseline for the original inoculum.

FIG. 5.

Infection and growth suppression of alternate human glioma cultures and MxA gene induction of VSV variants on control cells. Tumor cell infectivity and growth suppression were tested for human glioblastoma cell types U118, U373, and A-172. VSV variants VSV-rp30, VSV-M51, VSV-CT9-M51, and VSV-p1-GFP were applied at an MOI of 2 and analyzed at 24 hpi. (A) As on U87 cells, VSV-rp30 had the strongest growth-suppressing effect, and VSV-CT9-M51 and VSV-p1-GFP suppressed tumor growth to a lesser extent. (B) GFP fluorescence revealed a similar picture, with VSV-rp30 having the highest rates of infection compared to the attenuated VSV variants. Bars show mean values for five microscopic fields. Error bars indicate standard errors of the means. (C) Using quantitative real-time PCR, the expression of the interferon-induced antiviral gene MxA was compared after infection of cells with VSV-G/GFP, VSV-rp30, VSV-p1-GFP, VSV-M51, and VSV-CT9-M51. M51 mutation-containing mutants induced about 5 to 6 times more MxA than did VSV variants with wild-type M protein. Results are means for triplicate cultures. Error bars indicate standard errors of the means.

FIG. 6.

Intranasal application of VSV-G/GFP and VSV-p1-GFP. VSV has been shown to cause neurotoxicity in young mice when administered through an olfactory entry route. A total of 500,000 PFU of either wild-type-related VSV-G/GFP or attenuated VSV-p1-GFP was inoculated intranasally into young mice. The mice lost body weight after VSV-G/GFP inoculation (B), and 8 of 10 mice ultimately succumbed (A). In contrast, littermates that received VSV-p1-GFP gained weight steadily, and no mortality was seen. n = 10, the number of mice initially infected with the virus.

FIG. 7.

Targeting of human brain tumor transplants after intravenous injection of VSV-p1-GFP. Human glioblastoma cells expressing red fluorescent protein were stereotactically injected into the brains of SCID mice and given 10 days to form sizeable tumors. After a single injection of 10⁷ PFU of VSV-p1-GFP into the tail vein, the virus targeted the tumors, as indicated by GFP fluorescence. (A) Complete spread through the tumor mass at 3 dpi. (B) Initiating focus of viral infection at an early time point (2 dpi). (C) Remote tumor cell clusters were also successfully targeted by VSV-p1-GFP without infecting the surrounding, nontumor brain parenchyma.