Newly identified oncolytic VSV-GP-specific CD8+ T cell epitopes for monitoring of anti-viral immune responses in the BALB/c mouse model

The vesicular stomatitis virus variant VSV-GP is an oncolytic virus (OV) platform extensively studied in preclinical settings, which recently entered clinical trial testing. For oncolytic virotherapy, innate and adaptive immune system activation are considered major contributors. However, upon OV treatment, in addition to potential anti-tumor, anti-viral T cells are also raised, and comprehensively monitoring these anti-viral T cells presents a major challenge. Therefore, we aimed to identify anti-viral CD8⁺ T cells upon VSV-GP treatment in the widely utilized BALB/c mouse model using a multi-level adapted bioinformatics viral epitope prediction approach. Predicted viral epitopes presented on BALB/c major histocompatibility complex class I (MHC-I) alleles H2-Kd, H2-Dd, and H2-Ld were validated using ELISpot assay and intracellular cytokine staining. Subsequently, custom peptide-MHC-I multimers generated with the newly identified epitopes were used to directly detect virus-specific CD8⁺ T cells. Additionally, anti-viral CD8⁺ T cell dynamics of different treatment routes and multivirus exposure status in CT26.CL25 tumors and the spleen were analyzed. Taken together, the 11 newly identified epitopes facilitate the monitoring of anti-viral CD8⁺ T cells, which will aid the preclinical development of novel VSV-GP variants. This epitope-specific monitoring also serves as proof of concept for the potential future application of anti-viral immunomonitoring in clinical trial settings.