HSV Encoded ICP-0 Inhibits TLR Signaling in CLL Cells by Targeting TRAF-6.
Authors: Daubeuf Sandrine , Rosenblatt Joseph D. , Bowers William J. , Federoff Howard J. , Tolba Khaled A. 
Published: April 2020

Abstract
Innate immunity to viral vectors has largely been overlooked in development of gene therapy strategies and may play a key role in shaping immune responses and potentially clinical outcomes. In the setting of cancer immune therapy, the innate response may influence subsequent evolution of an anti-tumor adaptive immune response. We have studied two forms of HSV based amplicon vectors packaged in the presence or absence of HSV helper virus for use in immune therapy of chronic lymphocytic leukemia (CLL). Our previous work showed that the presence of helper-virus in HSV amplicon stocks impedes the development of tumor-specific adaptive immunity, and inhibits CTL development. We asked whether treatment outcomes could be explained by differences in the innate immune response elicited by the two vectors. We characterized innate immune response in CLL B cells transduced by helper-virus free (HF-HSV) vs. helper-virus containing (H+-HSV) amplicon vectors. Transduction of CLL B cells with HF-HSV induced robust innate cytokine/chemokine response that was not seen when transduction at similar efficiency was performed with H+-HSV vector. We profiled mRNA expression of TLR 1–10 in CLL B cells by quantitative PCR and confirmed the expression of TLR 2 and 9, which play a key role in mediating HSV innate response. Using 293 stably expressing TLR2/6 and TLR9, we show that the discrepancy in innate response of CLL B cells to transduction with HF-HSV and H+-HSV corresponded to differences seen in these viruses’ capacity to activate TLR-2 and TLR-9. Pre-incubation with cyclohexamide restored H+-HSV capacity to activate TLR2 and 9 suggesting that H+-HSV encodes an inhibitory protein that interferes with TLR signaling. A screen of the four HSV immediate early (IE) proteins (ICP-0, ICP-22, ICP-27 and ICP-47) encoded by HSV Helper-virus identified ICP-0 as a likely candidate for inhibition of TLR signaling. Ectopic expression of ICP-0 inhibited innate response to HF-HSV in TLR2/6. In addition, ICP-0 inhibited other TLRs (TLR3, 4, 7, and 8) and IL-1 receptor mediated NF-κB activation, suggesting that ICP-0 targets a common signaling protein downstream of these receptors. Over-expression of various TLR signaling components identified TRAF-6 as a target for ICP-0 mediated inhibition of the NF-κB response. Finally, we correlated HSV vector-mediated innate response to capacity to prime an adaptive anti-CLL T cell response. A comparison of immune function of CD40L and B7.1 encoded by either hf-HSV or H+-HSV to prime autologous T cell response in vitro showed a clear advantage for HF-HSV amplicons encoding CD40L and B7.1 compared to H+-HSV amplicons. Our results highlight a novel mechanism through which HSV suppresses host innate immunity through a hitherto un-described ICP-0 inhibition of TLR/IL-1R signaling. The work also establishes a potential link between vector mediated innate immunity and ability to prime an adaptive anti-tumor immune response against CLL cells, which has important implications in relation to development of gene therapy vectors for clinical testing.