Virus replication is not required for oncolytic bovine herpesvirus-1 immunotherapy

Abstract

Oncolytic viruses are a promising approach for cancer treatment where viruses selectively target and kill cancer cells while also stimulating an immune response. Among viruses with this ability, bovine herpesvirus-1 (BoHV-1) has several advantages, including observations suggesting it may not require viral replication for its anti-cancer effects. We previously demonstrated that binding and penetration of enveloped virus particles are sufficient to trigger intrinsic and innate immune signaling in normal cells, while other groups have published the efficacy of non-replicating viruses as viable immunotherapies in different cancer models. In this work, we definitively show that live and UV-inactivated (UV) (non-replicating) BoHV-1-based regimens extend survival of tumor-bearing mice to similar degrees and induce infiltration of similar immune cell populations, with the exception of neutrophils. Transcriptomic analysis of tumors treated with either live or UV BoHV-1-based regimens revealed similar pathway enrichment and a subset of overlapping differentially regulated genes, suggesting live and UV BoHV-1 have similar mechanisms of activity. Last, we present a gene signature across our in vitro and in vivo models that could potentially be used to validate new BoHV-1 therapeutics. This work contributes to the growing body of literature showing that replication may not be necessary for therapeutic efficacy of viral immunotherapies.

Keywords: MT: Regular Issue; bovine herpesvirus-1; gene signature; inactivated virus; oncolytic virus; transcriptome profiling; viral immunotherapy.

Graphical abstract

Figure 1

Differential gene expression in C10 cells infected with UV and live BoHV-1 (A) Comparison of genes with differential expression (≥3-fold) induced by live BoHV-1 and UV BoHV-1 at 6 and 12 hpi. (B) Overlap between genes differentially regulated by UV and live BoHV-1 at 6 hpi, with nine shared genes, six of which remain differentially expressed at 12 hpi. (C) Kinetics of relative gene expression between live and UV virus treatments (from (B)), showing similar expression at 6 hpi, but higher expression with live BoHV-1 at 12 hpi. Points on the graphs represent the mean fold change relative to mock and the bars represent the standard deviation. (D) Fold expression values of genes induced by both treatments.

Figure 2

UV-BoHV-1 is as effective as live BoHV-1 at extending the survival of mice bearing C10 melanoma tumors (A) Experimental schematic outlining the treatment protocol for C57/Bl6 mice with C10 melanoma tumors, treated either with PBS (n = 5), live (n = 10) or UV BoHV-1 (n = 10) as part of a triple-combination therapeutic regimen including mitomycin and checkpoint inhibitors. (B) Survival curve indicating no significant difference in survival between live and UV-BoHV-1-based regimes. (C) Average tumor volumes between groups treated with PBS, live or UV BoHV-1. (D) Individual mice tumor growth curves for PBS, live and UV BoHV-1-treated mice. ∗∗∗p < 0.001.

Figure 3

Immune cell infiltration profile of tumors treated with either live or UV BoHV-1-based regimes Tumors from PBS (n = 5), live (n = 5), or UV BoHV-1-treated (n = 5) regimes were harvested on day 10 (Figure 2A) and analyzed by flow cytometry for the presence of immune cell populations shown as percent (frequency) of immune cell populations per CD45⁺ cells in tumors. Unpaired t test was used for pairwise data comparison. Ns, not significant. ∗p < 0.05, ∗∗ p < 0.01. Gating strategy is seen in Figure S1.

Figure 4

Pathway enrichment profiles and differentially regulated genes of tumors treated with mitomycin C and either live or UV BoHV-1 (A) Histograms of the top 10 pathway enrichment profiles in tumors treated with live BoHV-1 and mitomycin C (Mito) and (B) UV BoHV-1 and Mito. Histograms shaded in blue represent pathways that are in common in both groups. Gray-shaded pathways were identified in both groups, but with ordinal ranks outside the top 10. (C) Ranking of the top 30 differentially regulated genes (>3-fold change) common between tumors treated with Mito and either live or UV BoHV-1. Numbers represent the rank of the relative fold change of each gene. The fold change value for each group is the mean value of five tumors per group (n = 5). Green boxes represent upregulation of gene expression and red boxes represent downregulation.

Figure 5

Schematic diagram of differential gene expression patterns in C10 cells and tumors infected in vitro (12 hpi) or in vivo (5 d post infection) with live or UV BoHV-1 in the presence of low dose mitomycin C Values within a given set of the Venn diagram represent the number of genes differentially regulated between the indicated group and its respective mock-infected control. Signature genes are the intersection of genes differentially regulated across live BoHV-1 and UV BoHV-1 treatments, and across in vitro vs. in vivo experiments all treated with mitomycin C. The table lists the 16 signature genes and indicates their fold-change ranking within their group. ∗ISG. #p53-related.