As antiviral drug resistance develops and new viruses emerge there is a pressing need to develop strategies to rapidly develop antiviral therapeutics. Here we use phospho-specific flow cytometry to assess perturbations of many different cellular signaling pathways during treatment with drug combinations that are highly effective in blocking Herpes simplex virus type 1 (HSV-1) infection. We discovered two antiviral drug combinations act on distinct signaling pathways, either STAT1 or S6 phosphorylation, to block HSV-1 infection. We focused on upregulation of S6 phosphorylation by HSV-1 infection, and our subsequent finding that ribavirin antagonizes this upregulation of S6 phosphorylation. We go on to show that the S6 kinase inhibitor SL0101 blocks HSV-1 replication in vitro and in an in vivo animal model of HSV-1 infection. Overall, we have used an unbiased analysis of cellular signaling pathways during treatment by antiviral drug combinations to discover a novel antiviral drug target against HSV-1 infection. The outcomes of the approach we present highlight the importance of analyzing how antiviral drugs modulate cellular and pathogen-induced signaling as a method to discover new drug therapy targets.

As antiviral drug resistance develops and new viruses emerge there is a pressing need to develop strategies to rapidly develop antiviral therapeutics. Here we use phospho-specific flow cytometry to assess perturbations of many different cellular signaling pathways during treatment with drug combinations that are highly effective in blocking Herpes simplex virus type 1 (HSV-1) infection. We discovered two antiviral drug combinations act on distinct signaling pathways, either STAT1 or S6 phosphorylation, to block HSV-1 infection. We focused on upregulation of S6 phosphorylation by HSV-1 infection, and our subsequent finding that ribavirin antagonizes this upregulation of S6 phosphorylation. We go on to show that the S6 kinase inhibitor SL0101 blocks HSV-1 replication in vitro and in an in vivo animal model of HSV-1 infection. Overall, we have used an unbiased analysis of cellular signaling pathways during treatment by antiviral drug combinations to discover a novel antiviral drug target against HSV-1 infection. The outcomes of the approach we present highlight the importance of analyzing how antiviral drugs modulate cellular and pathogen-induced signaling as a method to discover new drug therapy targets.

The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates ≥98% of peripheral immune cells with ≥4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery.