Generation and Functional In Vitro Analysis of Semliki Forest Virus Vectors Encoding TNF-α and IFN-γ

Abstract

Cytokine gene delivery by viral vectors is a promising novel strategy for cancer immunotherapy. Semliki Forest virus (SFV) has many advantages as a delivery vector, including the ability to (i) induce p53-independent killing of tumor cells via apoptosis, (ii) elicit a type-I interferon (IFN) response, and (iii) express high levels of the transgene. SFV vectors encoding cytokines such as interleukin (IL)-12 have shown promising therapeutic responses in experimental tumor models. Here, we developed two new recombinant SFV vectors encoding either murine tumor necrosis factor-α (TNF-α) or murine interferon-γ (IFN-γ), two cytokines with documented immunostimulatory and antitumor activity. The SFV vector showed high infection rate and cytotoxicity in mouse and human lung carcinoma cells in vitro. By contrast, mouse and human macrophages were resistant to infection with SFV. The recombinant SFV vectors directly inhibited mouse lung carcinoma cell growth in vitro, while exploiting the cancer cells for production of SFV vector-encoded cytokines. The functionality of SFV vector-derived TNF-α was confirmed through successful induction of cell death in TNF-α-sensitive fibroblasts in a concentration-dependent manner. SFV vector-derived IFN-γ activated macrophages toward a tumoricidal phenotype leading to suppressed Lewis lung carcinoma cell growth in vitro in a concentration-dependent manner. The ability of SFV to provide functional cytokines and infect tumor cells but not macrophages suggests that SFV may be very useful for cancer immunotherapy employing tumor-infiltrating macrophages.

Both Type I and Type II Interferons Can Activate Antitumor M1 Macrophages When Combined With TLR Stimulation

“…We stimulated BMDMs with Pam3CSK4 in combination with different concentrations of either IFN-γ, IFN-β, or the other major type I IFN, IFN-α (type A), for 24 h before analyzing NO production and growth inhibition of LLC cancer cells. In accordance with a previous report ( 39 ), recombinant IFN-γ synergized with Pam3CSK4 in inducing NO production and BMDM-mediated growth inhibition of cancer cells in a dose-dependent manner starting from the lowest IFN-γ concentration, 0.04 ng/mL (Figures 4A,B ). Likewise, recombinant IFN-β as well as IFN-α were able to synergize in a dose-dependent manner with Pam3CSK4 in inducing NO production (Figures 4C,E ).…”

“…Despite these important functions in anti-tumor immune responses, clinical use of IFN-γ for cancer treatment in general and for antitumor activation of TAMs in particular has so far been hampered by significant dose-limiting toxicities and the complex pleiotropic effects of IFN-γ on a large number of different cell types ( 52 ). These challenges could potentially be met by more targeted therapies, such as IFN-γ gene delivery to tumors by oncolytic viruses ( 39 ). The finding that type I IFNs, in a similar way as IFN-γ, can synergize with TLR agonists to induce antitumor macrophages opens up for further alternative treatment strategies targeting TAMs.…”

SI‐CLP inhibits the growth of mouse mammary adenocarcinoma by preventing recruitment of tumor‐associated macrophages

“…The new pSFVEnh/mSI‐CLP construct was generated by replacing Luc gene in pSFVEnh/Luc vector with full‐length mouse SI‐CLP gene containing FLAG sequence at C‐terminus. The resulting plasmids pSFVEnh/Luc and pSFVEnh/mSI‐CLP were used to produce recombinant virus particles in BHK‐21 cells as previously described . The cell growth medium containing the infectious recSFV particles was then harvested, filtered through 22 μm filters and rapidly frozen in liquid nitrogen, and subsequently used as a source of recSFV particles for cell infection and SI‐CLP containing supernatant production.…”

“…The resulting plasmids pSFVEnh/Luc and pSFVEnh/mSI-CLP were used to produce recombinant virus particles in BHK-21 cells as previously described. 17 The cell growth medium containing the infectious recSFV particles was then harvested, filtered through 22 μm filters and rapidly frozen in liquid nitrogen, and subsequently used as a source of recSFV particles for cell infection and SI-CLP containing supernatant production. Infection of BHK-21 cells was done as previously described.…”

Alphavirus-driven Interferon Gamma (IFNg) Expression Inhibits Tumour Growth in Orthotopic 4T1 Breast Cancer Model

“…Recent studies indicate the synergistic antitumour activity of alphaviral vectors expressing cytokines when combined with checkpoint inhibitors (antibodies) and chemical drugs [32][33][34]. In contrast to other viruses, Semliki Forest virus (SFV) does not infect human and mouse macrophages [35], making the system suitable for functional SFV/IFNg-based programming of macrophages and initiation of downstream M1-related proinflammatory reactions in the TME.…”