Myxoma Virus-Loaded Mesenchymal Stem Cells in Experimental Oncolytic Therapy of Murine Pulmonary Melanoma.

Oncolytic viruses can target neoplasms, triggering oncolytic and immune effects. Their delivery to melanoma lesions remains challenging. Bone-marrow-derived mesenchymal stem cells (MSCs) were shown to be permissive for oncolytic myxoma virus (MYXV), allowing its transfer to melanoma cells, leading to their killing. Involvement of progeny virus was demonstrated in the transfer from MSCs to co-cultured melanoma cells. The inhibitory effect of virus on melanoma foci formation in murine lungs was revealed using melanoma cells previously co-cultured with MYXV-infected MSCs. Virus accumulation and persistence in lungs of lesion-bearing mice were shown following intravenous administration of MSC-shielded MYXV construct encoding luciferase. Therapy of experimentally induced lung melanoma in mice with interleukin (IL)-15-carrying MYXV construct delivered by MSCs led to marked regression of lesions and could increase survival. Elevated natural killer (NK) cell percentages in blood indicated robust innate responses against unshielded virus only. Lung infiltration by NK cells was followed by inflow of CD8+ T lymphocytes into melanoma lesions. Elevated expression of genes involved in adaptive immune response following oncolytic treatment was confirmed using RT-qPCR. No adverse pathological effects related to MSC-mediated oncolytic therapy with MYXV were observed. MSCs allow for safe and efficient ferrying of therapeutic MYXV to pulmonary melanoma foci triggering immune effects.

Oncolytic Viruses and the Immune System: The Dynamic Duo.

Oncolytic viruses (OVs) constitute a new and promising immunotherapeutic approach toward cancer treatment. This therapy takes advantage of the natural propensity of most tumor cells to be infected by specific OVs. Besides the direct killing potential (oncolysis), what makes OV administration attractive for the present cancer immunotherapeutic scenario is the capacity to induce two new overlapping, but distinct, immunities: anti-tumoral and anti-viral. OV infection and oncolysis naturally elicit both innate and adaptive immune responses (required for long-term anti-tumoral immunity); at the same time, the viral infection prompts an anti-viral response. In this review, we discuss the dynamic interaction between OVs and the triggered responses of the immune system. The anti-OV immunological events that lead to viral clearance and the strategies to deal with such potential loss of the therapeutic virus are discussed. Additionally, we review the immune stimulatory actions induced by OVs through different inherent strategies, such as modulation of the tumor microenvironment, the role of immunogenic cell death, and the consequences of genetically modifying OVs by arming them with therapeutic transgenes. An understanding of the balance between the OV-induced anti-tumoral versus anti-viral immunities will provide insight when choosing the appropriate virotherapy for any specific cancer.

Ex Vivo Virotherapy with Myxoma Virus to Treat Cancer.

Myxoma virus (MYXV) is a member of the Poxviridae family and the genus Leporipoxvirus. In nature MYXV tropism is restricted to lagomorphs, and is specifically pathogenic only for European rabbits (Oryctolagus cuniculus), in which this virus causes the lethal systemic disease called myxomatosis. Importantly, although MYXV cannot cause any disease pathology in humans, mice, or any other domestic animals other than rabbit, this virus can productively infect and kill a variety of human and murine cancer cells, from either primary sources or cultured cancer cell lines. Therefore, in the last decade, MYXV has emerged as a novel oncolytic virus against hematologic malignancies and various solid cancers. One novel aspect of MYXV virotherapy is a new systemic virus delivery strategy to cancer sites in the recipient, by which adsorption of the virus to isolated leukocytes is conducted prior to reinfusion of the virus-infected cells back into the recipient, via a procedure called ex vivo virotherapy (EVV, or simply EV2). The EV2 delivery strategy thus exploits the inherent migratory properties of leukocytes to ferry MYXV to tissue sites bearing cancer cells that are accessible to leukocyte chemotaxis. Here, we describe EV2 procedures with MYXV to systemically deliver the virus to sites of disseminated and/or metastatic cancer in situ via infected leukocytes derived from either bone marrow or peripheral blood.

Sequence-specific detection of different strains of LCMV in a single sample using tentacle probes.

BACKGROUND: Virus infections often result in quasispecies of viral strains that can have dramatic impacts on disease outcomes. However, sequencing of viruses to determine strain composition is time consuming and often cost-prohibitive. Rapid, cost-effective methods are needed for accurate measurement of virus diversity to understand virus evolution and can be useful for experimental systems. METHODS: We have developed a novel molecular method for sequence-specific detection of RNA virus genetic variants called Tentacle Probes. The probes are modified molecular beacons that have dramatically improved false positive rates and specificity in routine qPCR. To validate this approach, we have designed Tentacle Probes for two different strains of Lymphocytic Choriomeningitis Virus (LCMV) that differ by only 3 nucleotide substitutions, the parental Armstrong and the more virulent Clone-13 strain. One of these mutations is a missense mutation in the receptor protein GP1 that leads to the Armstrong strain to cause an acute infection and Clone-13 to cause a chronic infection instead. The probes were designed using thermodynamic calculations for hybridization between target or non-target sequences and the probe. RESULTS: Using this approach, we were able to distinguish these two strains of LCMV individually by a single nucleotide mutation. The assay showed high reproducibility among different concentrations of viral cDNA, as well as high specificity and sensitivity, especially for the Clone-13 Tentacle Probe. Furthermore, in virus mixing experiments we were able to detect less than 10% of Clone-13 cDNA diluted in Armstrong cDNA. CONCLUSIONS: Thus, we have developed a fast, cost-effective approach for identifying Clone-13 strain in a mix of other LCMV strains.

Feasibility Study of an Optical Caustic Plasmonic Light Scattering Sensor for Human Serum Anti-Dengue Protein E Antibody Detection.

Antibody detection and accurate diagnosis of tropical diseases is essential to help prevent the spread of disease. However, most detection methods lack cost-effectiveness and field portability, which are essential features for achieving diagnosis in a timely manner. To address this, 3D-printed oblate spheroid sample chambers were fabricated to measure green light scattering of gold nanoparticles using an optical caustic focus to detect antibodies. Scattering signals of 20-200 nm gold nanoparticles using a green laser were compared to green light emitting diode (LED) light source signals and to Mie theory. The change in signal from 60 to 120 nm decreased in the order of Mie Theory > optical caustic scattering > 90° scattering. These results suggested that conjugating 60 nm gold nanoparticles and using an optical caustic system to detect plasmonic light scattering, would result in a sensitive test for detecting human antibodies in serum. Therefore, we studied the light scattering response of conjugated gold nanoparticles exposed to different concentrations of anti-protein E antibody, and a feasibility study of 10 human serum samples using dot blot and a handheld optical caustic-based sensor device. The overall agreement between detection methods suggests that the new sensor concept shows promise to detect gold nanoparticle aggregation in a homogeneous assay. Further testing and protocol optimization is needed to draw conclusions on the positive and negative predictive values for this new testing system.