We have generated a thymidine kinase gene-deleted vaccinia virus (VV) (Copenhagen strain) that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. Intratumoral inoculation of this thymidine kinase genedeleted VV encoding FCU1 (VV-FCU1) in the presence of systemically administered prodrug 5-fluorocytosine (5-FC) produced statistically significant reductions in the growth of subcutaneous human colon cancer in nude mice compared with thymidine kinase gene-deleted VV treatments or with control 5-fluorouracil alone. A limitation of prodrug therapies has often been the requirement for the direct injection of the virus into relatively large, accessible tumors. Here we demonstrate vector targeting of tumors growing subcutaneously following systemic administration of VV-FCU1. More importantly we also demonstrate that the systemic injection of VV-FCU1 in nude mice bearing orthotopic liver metastasis of a human colon cancer, with concomitant administration of 5-FC, leads to substantial tumor growth retardation. In conclusion, the insertion of the fusion FCU1 suicide gene potentiates the oncolytic efficiency of the thymidine kinase gene-deleted VV and represents a potentially efficient means for gene therapy of distant metastasis from colon and other cancers.

BackgroundAdvanced non-small cell lung cancer patients receiving TG4010, a therapeutic viral vaccine encoding human Mucin 1 and interleukin-2 in addition to standard chemotherapy, displayed longer overall survival in comparison to that of patients treated with standard chemotherapy alone. Our study intended to establish the association between overall survival and vaccine-induced T cell responses against tumor associated antigens (TAA) targeted by the vaccine.MethodThe TIME trial was a placebo-controlled, randomized phase II study aimed at assessing efficacy of TG4010 with chemotherapy in NSCLC. 78 patients from the TIME study carrying the HLA-A02*01 haplotype were analyzed using combinatorial encoding of MHC multimers to detect low frequencies of cellular immune responses to TG4010 and other unrelated TAA.ResultsWe report that improvement of survival under TG4010 treatment correlated with development of T cell responses against MUC1. Interestingly, responses against MUC1 were associated with broadening of CD8 responses against non-targeted TAA, thus demonstrating induction of epitope spreading.ConclusionOur results support the causality of specific T-cell response in improved survival in NSCLC. Additionally, vaccine induced epitope spreading to other TAA participates to the enrichment of the diversity of the anti-tumor response. Hence, TG4010 appears as a useful therapeutic option to maximize response rate and clinical benefit in association with other targeted immuno-modulators.Trial registrationRegistered on ClinicalTrials.gov under identifier NCT01383148 on June 23rd, 2011.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-017-0274-x) contains supplementary material, which is available to authorized users.

This combination of immunotherapies strongly increased at the injection sites the percentage of infiltrating natural killer (NK) cells and plasmacytoid dendritic cells (pDCs), cell types which can modulate innate and adaptive immune responses. IMPORTANCEVirus-based cancer vaccines offer a good alternative to the treatment of cancer but could be improved. Starting from a screening approach, we have identified and characterized an unexplored biological molecule with immunomodulatory characteristics which augments the efficacy of an MVA-based immunotherapeutic agent. The immune modulator consists of the purified dsRNA genome isolated from a commercially used yeast strain, NAB2, mixed with a cationic lipid, Lipofectin. NAB2-Lipofectin stimulates the immune system via TLR3 and MDA-5. When it was injected at the MVA vaccination site, the immune modulator increased survival in a preclinical tumor model. We could demonstrate that NAB2-Lipofectin augments the MVA-induced infiltration of natural killer and plasmacytoid dendritic cells. We suggest indirect mechanisms of activation of these cell types by the influence of NAB2-Lipofectin on innate and adaptive immunity. Detailed analysis of cell migration at the vaccine injection site and the appropriate choice of an immune modulator should be considered to achieve the rational improvement of virus vectorbased vaccination by immune modulators.

Oncolytic virus therapy has recently been recognized as a promising new therapeutic approach for cancer treatment. In this study, we are proposing for the first time to evaluate the in vitro and in vivo oncolytic capacities of the Cowpox virus (CPXV). To improve the tumor selectivity and oncolytic activity, we developed a thymidine kinase (TK)-deleted CPXV expressing the suicide gene FCU1, which converts the non-toxic prodrug 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) and 5-fluorouridine-5′-monophosphate (5-FUMP). This TK-deleted virus replicated efficiently in human tumor cell lines; however, it was notably attenuated in normal primary cells, thus displaying a good therapeutic index. Furthermore, this new recombinant poxvirus rendered cells sensitive to 5-FC. In vivo, after systemic injection in mice, the TK-deleted variant caused significantly less mortality than the wild-type strain. A biodistribution study demonstrated high tumor selectivity and low accumulation in normal tissues. In human xenograft models of solid tumors, the recombinant CPXV also displayed high replication, inducing relevant tumor growth inhibition. This anti-tumor effect was improved by 5-FC co-administration. These results demonstrated that CPXV is a promising oncolytic vector capable of expressing functional therapeutic transgenes.

The delivery of stimulatory signals to dendritic cells (DCs) in the tumor microenvironment could be an effective means to break tumorinduced tolerance. The work presented here evaluates the immunostimulatory properties of pathogen-associated molecular patterns (PAMPs), microbial molecules which bind Toll-like receptors and deliver activating signals to immune cells, when expressed in tumor cells using adenoviral (Ad) vectors. In vitro, transduction of A549 tumor cells with Ad vectors expressing either flagellin from Listeria monocytogenes or P40 protein from Klebsiella pneumoniae induced the maturation of human monocyte-derived DCs in co-cultures. In mixed lymphocyte reactions (MLRs), Ad-flagellin and Ad-P40 transduction of tumor cells stimulated lymphocyte proliferation and the secretion of IFN-g. In vivo, these vectors were used either as stand-alone immunoadjuvants injected intratumorally or as vaccine adjuvants combined with a tumor antigen-expressing vector. When Ad-PAMPs were administered intratumorally to mice bearing subcutaneous syngeneic B16F0-CAR (cocksackie-adenovirus receptor) melanomas, tumor progression was transiently inhibited by Ad-P40. In a therapeutic vaccine setting, the combination of Ad-MUC1 and Ad-PAMP vectors injected subcutaneously delayed the growth of implanted RenCa-MUC1 tumors and improved tumor rejection when compared with vaccination with Ad-MUC1 alone. These results suggest that Ad-PAMPs could be effective immunoadjuvants for cancer immunotherapy.