HSV Amplicon Transduction Activates an Innate Immune Response in CLL B Cells;Implications for Immune Therapy and Vaccine Development.

Abstract

Since gene therapy vectors are designed to shuttle therapeutic genes into various tissues, efficiency transduction is often viewed as the most critical measure of a vector's clinical usefulness. However, other variables such as activation of the host's innate immunity may play an equally important role shaping the clinical outcome. We have studied the application of helper-free HSV (hf-HSV) amplicon vectors to deliver costimulatory ligands into chronic lymphocytic leukemia (CLL) B cells for the purpose of enhancing their antigen presenting capacity. In this work, we examine the innate immune response of CLL B cells to transduction with helper-virus free HSV (hf-HSV) amplicon vectors. We screened culture supernatant of CLL B cells transduced with helper-free HSV amplicon for secretion of IL-6, TNF-α and interferon β by ELISA and observed significant induction of these cytokines upon hf-HSV transduction as compared to non-transduced and CLL cells transduced with helper virus-containing (H+-HSV) HSV amplicons. We also compared the effects of hf-HSV and H+-HSV amplicon vector transduction on surface expression of NKG2D ligand. CLL B cells transduced with hf-HSV but not H+-HSV amplicons upregulated the costimulatory NKG2D ligands MICA and MICB. To investigate mechanisms of HSV activation of innate immunity, we screened a panel of human TLR receptors for recognition of HSV amplicons using 293T cells transfected with individual human TLR receptors and assaying NF-κB driven luciferase activity as a read-out. We found that HSV amplicon activates TLR3 (receptor for double stranded RNA), TLR8 (receptor for single stranded RNA) and TLR9 (receptor for unmethylated CpG motifs). CLL B cells express predominantly TLR9 and to a lesser extent TLR8. TLR3, on the other hand is not expressed on resting B cells and is upregulated upon exposure to interferon β. In 293T cells lacking TLR receptors, viable hf-HSV but not UV-inactivated amplicons up-regulated surface expression of both MICA and MICB and activated interferon-β promoter driven luciferase, indicating the presence of a TLR-independent HSV activated innate response. To establish the presence of TLR-dependent NKG2D ligand induction, CLL B cells (expressing TLR7, 8 and 9) were incubated with their specific ligands (Loxaribine and CpG ODN motifs) for 24 hours and MICA and MICB expression confirmed by flow-cytometry. In summary, our data shows that hf-HSV amplicon vectors activate multiple aspects of innate immunity involving both TLR dependent and independent pathways, which translates into expression of cytokines and costimulatory ligands by transduced cells. The presence of helper virus on the other hand completely abolishes this response. In the setting of a vaccine development, the evoked innate response may prime the adaptive T cell response leading to improved clinical outcome.