Pseudocowpox virus, a novel vector to enhance the therapeutic efficacy of antitumor vaccination.

Objective: Antitumor viral vaccines, and more particularly poxviral vaccines, represent an active field for clinical development and translational research. To improve the efficacy and treatment outcome, new viral vectors are sought, with emphasis on their abilities to stimulate innate immunity, to display tumor antigens and to induce a specific T-cell response. Methods: We screened for a new poxviral backbone with improved innate and adaptive immune stimulation using IFN-α secretion levels in infected PBMC cultures as selection criteria. Assessment of virus effectiveness was made in vitro and in vivo. Results: The bovine pseudocowpox virus (PCPV) stood out among several poxviruses for its ability to induce significant secretion of IFN-α. PCPV produced efficient activation of human monocytes and dendritic cells, degranulation of NK cells and reversed MDSC-induced T-cell suppression, without being offensive to activated T cells. A PCPV-based vaccine, encoding the HPV16 E7 protein (PCPV-E7), stimulated strong antigen-specific T-cell responses in TC1 tumor-bearing mice. Complete regression of tumors was obtained in a CD8+ T-cell-dependent manner after intratumoral injection of PCPV-E7, followed by intravenous injection of the cancer vaccine MVA-E7. PCPV also proved active when injected repeatedly intratumorally in MC38 tumor-bearing mice, generating tumor-specific T-cell responses without encoding a specific MC38 antigen. From a translational perspective, we demonstrated that PCPV-E7 effectively stimulated IFN-γ production by T cells from tumor-draining lymph nodes of HPV+-infected cancer patients. Conclusion: We propose PCPV as a viral vector suitable for vaccination in the field of personalised cancer vaccines, in particular for heterologous prime-boost regimens.

Sequential administration of MVA-based vaccines and PD-1/PD-L1-blocking antibodies confers measurable benefits on tumor growth and survival: Preclinical studies with MVA-ßGal and MVA-MUC1 (TG4010) in a murine tumor model.

TG4010, a Modified Vaccinia virus Ankara (MVA) expressing human mucin1 (MUC1) has demonstrated clinical benefit for patients suffering from advanced non-small cell lung cancer (NSCLC) in combination with chemotherapy. To support its development, preclinical experiments were performed with either TG4010 or ß-galactosidase-encoding MVA vector (MVA-ßgal) in mice presenting tumors in the lung. Tumor growth was obtained after intravenous injection of CT26 murine colon cancer cells, engineered to express either MUC1 or ßgal. Mice showed increased survival rates after repeated intravenous injections of TG4010 or MVA-ßgal, compared to an empty MVA control vector. Treatment with MVA vectors led to the accumulation of CD3dimCD8dim T cells, with two subpopulations characterized as KLRG1+CD127- short-lived effector cells (SLECs), and KLRG1-CD127- early effector cells (EECs) comprising cells releasing IFNγ, Granzyme B and CD107a upon antigen-specific peptide stimulation. EECs were characterized by an up-regulation of PD-1. Tumor growth in the diseased lung correlated with the appearance of PD1+ Treg cells that partially disappeared after TG4010 treatment. At late stage of tumor development in the lung, PD-L1 was detected on CD45- tumor cells, on CD4+ cells, including Treg cells, on CD3+CD8+ and CD3dimCD8dim T lymphocytes, on NK cells, on MDSCs and on alveolar macrophages. We demonstrated that targeting the PD-1/PD-L1 pathway with blocking monoclonal antibodies several days after TG4010 treatment, at late stage of tumor development, enhanced the therapeutic protection induced by the vaccine, supporting the ongoing clinical evaluation of TG4010 immunotherapy in combination with Nivolumab.

Sequential administration of a MVA-based MUC1 cancer vaccine and the TLR9 ligand Litenimod (Li28) improves local immune defense against tumors.

TG4010 is an immunotherapeutic vaccine based on Modified Vaccinia virus Ankara (MVA) encoding the human tumor-associated antigen MUC1 and human IL-2. In combination with first-line standard of care chemotherapy in advanced metastatic non-small-cell lung cancer (NSCLC), repeated subcutaneous injection of TG4010 improved progression-free survival in phase 2b clinical trials. In preclinical tumor models, MVATG9931, the research version of TG4010, conferred antigen-specific responses against the weak antigen human MUC1. The combination of a suboptimal dose of MVATG9931 and the type B TLR9 ligand Litenimod (Li28) markedly increased survival in a subcutaneous RMA-MUC1 tumor model compared to the treatment with MVATG9931 or Li28 alone. The requirements for this protection were (i) de novo synthesis of MUC1, (ii) Li28 delivered several hours after MVATG9931 at the same site, (iii) at least two vaccination cycles, and (iv) implantation of MUC1-positive tumor cells in the vicinity to the vaccination site. Subcutaneously injected MVATG9931 allowed transient local gene expression and induced the local accumulation of MCP-1, RANTES, M-CSF, IL-15/IL-15R and IP-10. After repeated injection, CD4+ and CD8+ T lymphocytes, B lymphocytes, NK cells, pDCs, neutrophils, and macrophages accumulated around the injection site, local RANTES levels remained high. Delayed injection of Li28 into this environment, led to further accumulation of macrophages, the secretion of IL-18 and IL-1 beta, and an increase of the percentage of activated CD69+ NK cell. Combination treatment augmented the number of activated CD86+ DCs in the draining lymph nodes and increased the percentage of KLRG1+ CD127-CD8+ T cells at the injection site. In vivo depletion of macrophages around the injection site by Clodronate liposomes reduced local IL-18 levels and diminished survival rates significantly. Thus, sequential administration of MVATG9931 and Li28 improves local innate and adaptive immune defense against tumors, arguing for intratumoral delivery of this peculiar sequential combination therapy.

Yeast virus-derived stimulator of the innate immune system augments the efficacy of virus vector-based immunotherapy.

UNLABELLED: To identify novel stimulators of the innate immune system, we constructed a panel of eight HEK293 cell lines double positive for human Toll-like receptors (TLRs) and an NF-κB-inducible reporter gene. Screening of a large variety of compounds and cellular extracts detected a TLR3-activating compound in a microsomal yeast extract. Fractionation of this extract identified an RNA molecule of 4.6 kb, named nucleic acid band 2 (NAB2), that was sufficient to confer the activation of TLR3. Digests with single- and double-strand-specific RNases showed the double-strand nature of this RNA, and its sequence was found to be identical to that of the genome of the double-stranded RNA (dsRNA) L-BC virus of Saccharomyces cerevisiae. A large-scale process of production and purification of this RNA was established on the basis of chemical cell lysis and dsRNA-specific chromatography. NAB2 complexed with the cationic lipid Lipofectin but neither NAB2 nor Lipofectin alone induced the secretion of interleukin-12(p70) [IL-12(p70)], alpha interferon, gamma interferon-induced protein 10, macrophage inflammatory protein 1ß, or IL-6 in human monocyte-derived dendritic cells. While NAB2 activated TLR3, Lipofectin-stabilized NAB2 also signaled via the cytoplasmic sensor for RNA recognition MDA-5. A significant increase of RMA-MUC1 tumor rejection and survival was observed in C57BL/6 mice after prophylactic vaccination with MUC1-encoding modified vaccinia virus Ankara (MVA) and NAB2-Lipofectin. 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. IMPORTANCE: Virus-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 vector-based vaccination by immune modulators.

Improvement of calcium handling and changes in calcium-release properties after mini- or full-length dystrophin forced expression in cultured skeletal myotubes.

Dystrophin is a cytoskeletal protein normally expressed underneath the sarcolemma of muscle fibers. The lack of dystrophin in Duchenne muscular Dystrophy (DMD) muscles results in fiber necrosis, which was proposed to be mediated by chronic calcium mishandling. The extensive comparison of dystrophic cells from human or mdx mice with normal muscles have suggested that the lack of dystrophin may alter the resting calcium permeability and steady-state levels of calcium, but this latter observation remains controversial. It is also not clear, whether calcium mishandling is resulting from the dystrophic process or if dystrophin can directly regulate calcium handling in muscle cells. This prompted us to determine if transfection of full-length dystrophin or Becker Muscular Dystrophy (BMD) minidystrophin, a candidate for viral-mediated gene therapy, could change calcium handling properties. We took advantage of specific properties of Sol8 cell line showing the absence of dystrophin expression together with a drastic calcium mishandling. Here, we show that full-length dystrophin allowed the recovery of a low resting intracellular-free calcium concentration together with lower calcium transients. We also show for the first time that stable expression of minidystrophin was able to restore normal calcium handling in Sol8 myotubes through a better control of steady-state levels, calcium transients, and subcellular calcium events. It suggests that dystrophin could play a regulatory role on calcium homeostasis apparatus and that functional links exist between calcium signaling and cytoskeleton.