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.
Highlights
Viral vectors have been used for cancer immunotherapy to intratumorally deliver and express transgenes and to thereby increase therapeutic protein delivery while simultaneously reducing systemic toxicity [1]
The pSFV-IFN-γ-coding gene (Ifng) vector was made by inserting the mIFN-γ gene (Ifng) into the pSFV1/Enh vector downstream and in-frame with the Semliki Forest virus (SFV) capsid translation enhancer sequence (Enh) that was fused to the 2A auto-protease sequence of foot and mouth disease virus (FMDV) (Figure 1A)
Replication-deficient Recombinant Semliki Forest virus (rSFV) particles were produced by baby hamster kidney fibroblasts (BHK-21) cells that were co-transfected with RNA transcribed from the SFV-Helper1 vector and RNA transcribed from a vector encoding a heterologous genes (HGs) (Tnfa, Ifng, or Discosoma sp. red fluorescent protein (DsRed)) (Figure 1D)
Summary
Viral vectors have been used for cancer immunotherapy to intratumorally deliver and express transgenes and to thereby increase therapeutic protein delivery while simultaneously reducing systemic toxicity [1]. Recombinant Semliki Forest virus (rSFV) has been successfully used as a cancer immunotherapy in preclinical tumor models for cytokine gene delivery [2,3,4,5,6,7,8,9,10,11,12,13,14,15] and for immunization with tumor-associated antigens against mastocytoma [2, 16] and human papilloma virus (HPV)-associated cervical cancer [17, 18]. The replication-deficient SFV vector system delivers genes of interest by infecting the cells with viral particles and thereby providing a transiently high level of transgene expression without further virus replication [20]. SFV vector elicits endogenous type-I interferon (IFN) responses, which may be required for the therapeutic effect of a vector-encoded cytokine [3]
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