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. 2023 May 15:10:1190669.
doi: 10.3389/fmolb.2023.1190669. eCollection 2023.

Construction of VSVΔ51M oncolytic virus expressing human interleukin-12

Rwaa H Abdulal  1   2 , Jana S Malki  1 , Ezdehar Ghazal  1 , Ahdab A Alsaieedi  1   3 , Sarah A Almahboub  1 , Muhammad Yasir Khan  1   2 , Reem M Alsulaiman  1 , Mazen M Ghaith  4 , Turki S Abujamel  1   3 , Magdah Ganash  2 , Ahmad Bakur Mahmoud  5   6 , Almohanad A Alkayyal  7 , Anwar M Hashem  1   8
Affiliations

Construction of VSVΔ51M oncolytic virus expressing human interleukin-12

Rwaa H Abdulal et al. Front Mol Biosci. .

Abstract

The use of oncolytic viruses (OVs) in combination with cytokines, such as IL-12, is a promising approach for cancer treatment that addresses the limitations of current standard treatments and traditional cancer immunotherapies. IL-12, a proinflammatory cytokine, triggers intracellular signaling pathways that lead to increased apoptosis of tumor cells and enhanced antitumor activity of immune cells via IFN-γ induction, making this cytokine a promising candidate for cancer therapy. Targeted expression of IL-12 within tumors has been shown to play a crucial role in tumor eradication. The recent development of oncolytic viruses enables targeted delivery and expression of IL-12 at the tumor site, thereby addressing the systemic toxicities associated with traditional cancer therapy. In this study, we constructed an oncolytic virus, VSVΔ51M, based on the commercially available VSV wild-type backbone and further modified it to express human IL-12. Our preclinical data confirmed the safety and limited toxicity of the modified virus, VSV-Δ51M-hIL-12, supporting its potential use for clinical development.

Keywords: B16F10; MCF-7; VSVΔ51M; cancer immunotherapy; interleukin-12; oncolytic virus.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Construction and generation of VSV viruses. (A) Schematic diagram of the recombinant VSVΔ51M-hIL-12 virus. The hIL-12 gene is indicated in red and was inserted between the M and P genes of the parental vector VSVΔ51M using the AscI and AvrII restriction sites. (B) Schematic representation of the generation process for the VSV, VSVΔ51M and VSVΔ51M-hIL-12 viruses. BHK-21 cells were infected with vaccinia T7; transfected with the targeted plasmid VSV, VSVΔ51M or VSVΔ51M-hIL-12 and VSV-system accessory plasmids; and then primary supernatant (Passage 1) was used for sequential passaging in Vero-E6 cells to propagate recovered viruses. (C) Negative staining of electron micrographs of purified VSVΔ51M virions. Fifty microliters of the purified virus were collected, fixed in 4% glutaraldehyde, and imaged using transmission electron microscopy. Arrows indicate the magnified area of the virus with a bullet-shaped morphology. (D) Western blot analysis of recovered VSV virus-infected cells. MCF-7 cells were infected at an MOI of 10 with VSV, VSVΔ51M or VSVΔ51M-hIL-12 or transfected with each virus control plasmid. Cell lysates were prepared, separated by 8% SDS‒PAGE, and electrophoretically transferred to a PVDF membrane. The VSV-G protein was detected with anti-VSV G antibody. (E) Production of hIL-12 by MCF-7 or Vero-E6 cells infected with VSVΔ51M or VSVΔ51M-hIL-12 at different time points, as measured by ELISA. The expression level of hIL-12 was detected at 18, 24, and 48 h post-infection with VSVΔ51M-hIL-12 and is reported as pg/ml of hIL-12. Mock-infected control cells and cells infected with parental VSVΔ51M failed to produce a detectable level of hIL-12. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test, n = 3; the graph represents the mean ± SD (****p < 0.0001). (F) Recombinant VSV protein expression and localization. Immunofluorescence staining of Vero-E6 cells infected with VSV, VSVΔ51M or VSVΔ51M-hIL-12 or transfected with control plasmids. Infected and transfected cells were stained with anti-VSV N, M, or G mouse monoclonal antibodies (green), and nuclei were counterstained with DAPI (blue). Scale bars are included in each image.
FIGURE 2
FIGURE 2
Efficacy of VSV and VSVΔ51M in C57Bl/6 mice bearing B16F10 tumors. (A) Timeline of animal experiment. Six-to eight-week-old female C57Bl/6 mice were subcutaneously injected with 100 μL of 5 × 105 B16F10 cells in the right flank on Day 0. Tumors were established by day 7, and mice were randomly divided into 3 groups (n = 7 mice per group). One group served as a control (no treatment), and three intratumoral doses of VSV or VSVΔ51M (5 × 108 PFU/50 μL), starting on day 7 (days 7, 10, and 13), were administered to the other two groups. (B) Tumor size was monitored in each group by caliper measurements until a mouse in the group was euthanized due to its tumor burden. Tumor size was calculated using the following formula: tumor volume (mm2) = length × width2. (C) Mice were weighed every 3 days, and the percentage of weight change of each mouse was determined. (D) The survival times of treated and untreated groups were plotted as a Kaplan‒Meier survival curve. Statistical analysis of tumor volume was performed by two-way ANOVA, followed by survival analysis by the log-rank (Mantel‒Cox) test (**p = 0.0028). *p < 0.03; **p < 0.002; ***p < 0.0002; ****p < 0.0001.
FIGURE 3
FIGURE 3
Growth and spreading kinetics and cytotoxicity of the rescued VSVs. To assess (A) single-step and (B) multistep viral growth kinetics, Vero-E6 cell monolayers were infected in triplicate with VSV, VSVΔ51M, or VSVΔ51M-hIL-12 at a multiplicity of infection of 10 or 0.01. Supernatants were collected at 6, 12, 18, 24, and 48 h after infection. The titer of each virus was determined in Vero-E6 cells (in triplicate) using standard plaque assay. The mean ± SD of log-transformed titers is shown. Statistical significance was determined using two-way ANOVA with Tukey’s multiple comparison test. (C) Effects of VSV viruses on the morphological characteristics of normal and cancerous cells. Light microscopy images of MCF-7, A549, GM-38, and B16F10 cells that were mock-infected or infected with VSV, VSVΔ51M, or VSVΔ51M-hIL-12 (MOI = 10) for 24 h. The cytotoxic effects of the viruses were assessed by examining representative photomicrographs of cell monolayers at a magnification of ×20 following infection. (D) Cytotoxicity of the VSV, VSVΔ51M, and VSVΔ51M-hIL-12 viruses to MCF-7, A549, GM-38, and B16F10 cells. Cells were infected with the VSV viruses at multiplicities of 10, 1, 0.1, 0.01, and 0.001 PFU per cell. At the indicated time points post-infection, cell viability was measured by an AlamarBlue assay. Data are expressed as the percentage of the cell viability of mock-infected cells and represent the means ± SD of 6 technical replicates. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The statistical significance calculations for the multiple comparisons have been color-coded by lines as illustrated in the figure legend underneath panel (D).
FIGURE 4
FIGURE 4
VSVΔ51M-hIL-12 enhances the production of IFN-γ by stimulated PBMCs ex vivo. (A) Schematic illustration of the experimental setup. Isolated PBMCs were cocultured with mock-infected MCF-7 cells or MCF-7 cells infected with VSVΔ51M or VSVΔ51M-hIL-12. (B) The percentage of intracellular IFN-γ+ expression by NK (CD56+CD3−) cells for each condition was quantified by flow cytometry. (C) The concentration of secreted IFN-γ in supernatant for each condition was quantified by ELISA. Graph represents the mean ± SD of five replicate values from two independent experiments. Differences between treatment arms were analyzed using Tukey’s multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
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Grants and funding

This research work was funded by the Institutional Fund Project under grant no (IFPRC-161-290-2020). Therefore, authors gratefully acknowledge technical and financial support from the Ministry of Education and King Abdulaziz University, Jeddah, Saudi Arabia.

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