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

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.

Magnetic nanoparticles for efficient cell transduction with Semliki Forest virus.

Semliki Forest virus (SFV) is a potential cancer gene therapy vector capable of providing high and transient expression of heterologous proteins in mammalian cells. However, SFV has shown suboptimal transduction levels in several cancer cell types as well as wide biodistribution of SFV has been observed after in vivo applications. Magnetic nanoparticles (MNPs) have been shown to increase cell transduction with several viral vectors in vitro under an external magnetic field and enhance magnetically guided viral vector delivery. Here, we examined a panel of MNPs for enhanced cancer cell transduction with SFV vector. Magneto-transduction using positively charged MNPs increased Semliki Forest virus transduction in TS/A mouse mammary carcinoma cells in vitro in the presence of fetal bovine serum. Positively charged MNPs efficiently captured SFV particles independently of capturing medium, and MNPs-SFV complexes were successfully separated from suspension by magnetic precipitation. These results reveal the potential application of MNPs for enhanced gene delivery by SFV vector as well as proposes magnetic precipitation for efficient concentration of SFV particles from different media.

Comparative protein profiling of B16 mouse melanoma cells susceptible and non-susceptible to alphavirus infection: Effect of the tumor microenvironment.

Alphavirus vectors are promising tools for cancer treatment. However, relevant entry mechanisms and interactions with host cells are still not clearly understood. The first step toward a more effective therapy is the identification of novel intracellular alterations that could be associated with cancer aggressiveness and could affect the therapeutic potential of these vectors. In this study, we observed that alphaviruses efficiently infected B16 mouse melanoma tumors/tumor cells in vivo, whereas their transduction efficiency in B16 cells under in vitro conditions was blocked. Therefore, we further aimed to understand the mechanisms pertaining to the differential transduction efficacy of alphaviruses in B16 tumor cells under varying growth conditions. We hypothesized that the tumor microenvironment might alter gene expression in B16 cells, leading to an up-regulation of the expression of virus-binding receptors or factors associated with virus entry and replication. To test our hypothesis, we performed a proteomics analysis of B16 cells cultured in vitro and of B16 cells isolated from tumors, and we identified 277 differentially regulated proteins. A further in-depth analysis to identify the biological and molecular functions of the detected proteins revealed a set of candidate genes that could affect virus infectivity. Importantly, we observed a decrease in the expression of interferon α (IFN-α) in tumor-isolated cells that resulted in the suppression of several IFN-regulated genes, thereby abrogating host cell antiviral defense. Additionally, differences in the expression of genes that regulate cytoskeletal organization caused significant alterations in cell membrane elasticity. Taken together, our findings demonstrated favorable intracellular conditions for alphavirus transduction/replication that occurred during tumor transformation. These results pave the way for optimizing the development of strategies for the application of alphaviral vectors as a potent cancer therapy.