No abstract

Oncolytic vaccinia virus is an attractive platform for immunotherapy. Oncolysis releases tumor antigens and provides co-stimulatory danger signals. However, arming the virus can improve efficacy further. CD40 ligand (CD40L, CD154) can induce apoptosis of tumor cells and it also triggers several immune mechanisms. One of these is a T-helper type 1 (Th1) response that leads to activation of cytotoxic T-cells and reduction of immune suppression. Therefore, we constructed an oncolytic vaccinia virus expressing hCD40L (vvdd-hCD40L-tdTomato), which in addition features a cDNA expressing the tdTomato fluorochrome for detection of virus, potentially important for biosafety evaluation. We show effective expression of functional CD40L both in vitro and in vivo. In a xenograft model of bladder carcinoma sensitive to CD40L treatment, we show that growth of tumors was significantly inhibited by the oncolysis and apoptosis following both intravenous and intratumoral administration. In a CD40-negative model, CD40L expression did not add potency to vaccinia oncolysis. Tumors treated with vvdd-mCD40L-tdtomato showed enhanced efficacy in a syngenic mouse model and induced recruitment of antigen-presenting cells and lymphocytes at the tumor site. In summary, oncolytic vaccinia virus coding for CD40L mediates multiple antitumor effects including oncolysis, apoptosis and induction of Th1 type T-cell responses.

Oncolytic Western Reserve strain vaccinia virus selective for epidermal growth factor receptor pathway mutations and tumorassociated hypermetabolism was armed with human granulocyte-macrophage colony-stimulating factor (GMCSF) and a tdTomato fluorophore. As the assessment of immunological responses to human transgenes is challenging in the most commonly used animal models, we used immunocompetent Syrian golden hamsters, known to be sensitive to human GMCSF and semipermissive to vaccinia virus. Efficacy was initially tested in vitro on various human and hamster cell lines and oncolytic potency of transgene-carrying viruses was similar to unarmed virus. The hGMCSF-encoding virus was able to completely eradicate subcutaneous pancreatic tumors in hamsters, and to fully protect the animals from subsequent rechallenge with the same tumor. Induction of specific antitumor immunity was also shown by ex vivo co-culture experiments with hamster splenocytes. In addition, histological examination revealed increased infiltration of neutrophils and macrophages in GMCSF-virus-treated tumors. These findings help clarify the mechanism of action of GMCSF-armed vaccinia viruses undergoing clinical trials.Oncolytic vaccinia virus is appealing for cancer gene therapy owing to several characteristics. Wild-type vaccinia viruses have been used in hundreds of millions of humans as a vaccine for the eradication of smallpox. In addition to its solid safety profile in humans, vaccinia virus has a strong oncolytic effect owing to its fast replication cycle 1 and high tropism for cancer tissue, 2 which has led to the design of novel cancer therapeutics based on vaccinia backbones. 3,4 In our studies, we use double-deleted Western Reserve vaccinia virus (vvdd) with deletions in the virally encoded thymidine kinase (TK) and vaccinia growth factor (VGF) genes. [5][6][7] These genes are necessary for replication in normal cells but not in cancer cells, and both genes have been shown to reduce the pathogenicity of the virus. 8,9 Good safety and preliminary evidence of efficacy have been seen with oncolytic vaccinia virus in preclinical models and clinical trials. [10][11][12] The replication of oncolytic virus in the tumor is an immunogenic phenomenon, and their antitumor efficacy

In oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. Vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. However, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI) to boost the innate immune system and to activate adaptive immune cells in the tumor. We showed that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways. Treatment with DAI-armed VV resulted in significant reduction in the size of syngeneic melanoma tumors in mice. When the mice were rechallenged with the same tumor, DAI-VV-treated mice completely rejected growth of the new tumor, which indicates immunity established against the tumor. We also showed enhanced control of growth of human melanoma tumors and elevated levels of human T-cells in DAI-VV-treated mice humanized with human peripheral blood mononuclear cells. We conclude that expression of DAI by an oncolytic VV is a promising way to amplify the vaccine potency of an oncolytic vaccinia virus to trigger the innate—and eventually the long-lasting adaptive immunity against cancer.

The role of the preepithelial mucus-HCO-3 layer in protection against intracellular acidosis was investigated in isolated Necturus gastric antral mucosa exposed to luminal acid by simultaneous measurement of intracellular pH (pH(i)) and extracellular surface pH (pHs) in surface epithelium with microelectrode technique. Acidification of the luminal perfusate to pH 2.5 acidified pH(i) in surface epithelial cells from 7.33 +/- 0.02 to 7.20 +/- 0.04, whereas pHs fell from 6.75 +/- 0.21 to 5.20 +/- 0.25 (P < 0.01; n = 9), followed by a steady state for at least 2 h. Inhibition of epithelial HCO-3 secretion and transport by removal of serosal HCO-3 and CO2 (HEPES and O2 substitution) during acid exposure provoked a progressive acidification of pHs from 5.60 +/- 0.41 to 2.74 +/- 0.14 in 30 min (P < 0.01; n = 9), which was accompanied, after a 5- to 10-min delay, by acidification of pH(i) from 7.21 +/- 0.03 to 5.68 +/- 0.26 (P < 0.01). Digestion of the surface mucus gel by pepsin (5% wt/vol) at pH 2.5 caused a slow acidification of pHs from 5.22 +/- 0.59 to 3.60 +/- 0.46 within 2 h. This was followed by a more rapid acidification to 2.53 +/- 0.38 (P < 0.01; n = 7), with concomitant acidification of pH(i) from 7.19 +/- 0.05 to 6.03 +/- 0.33 (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer is one of the most common reasons for death in dogs. One promising approach is oncolytic virotherapy. We assessed the oncolytic effect of genetically modified vaccinia viruses in canine cancer cells, in freshly excised tumour biopsies, and in mice harbouring canine tumour xenografts. Tumour transduction efficacy was assessed using virus expressing luciferase or fluorescent marker genes and oncolysis was quantified by a colorimetric cell viability assay. Oncolytic efficacy in vivo was evaluated in a nude mouse xenograft model. Vaccinia virus was shown to infect most tested canine cancer cell lines and primary surgical tumour tissues. Virus infection significantly reduced tumour growth in the xenograft model. Oncolytic vaccinia virus has antitumour effects against canine cancer cells and experimental tumours and is able to replicate in freshly excised patient tumour tissue. Our results suggest that oncolytic vaccinia virus may offer an effective treatment option for otherwise incurable canine tumours.

The effect of vagotomy and pyloroplasty on the density of nerve fibers containing bombesin/gastrin-releasing peptide (GRP), calcitonin gene-related peptide (CGRP), and galanin as well as histamine-, 5-hydroxytryptamine (5-HT)-, and somatostatin-containing cells in the oxyntic mucosa of the rat stomach was studied. Ten days after vagotomy and pyloroplasty the density of histamine-containing cells in the oxyntic mucosa was increased by 70% (P < 0.05), and these cells were larger and showed more extensive cell processes than in control animals. The density of 5-HT-immunoreactive (IR) cells and somatostatin-IR cells were not affected. A marked decrease in the density of CGRP-IR nerve fibers and a slighter decrease in the density of GRP-IR nerve fibers was observed in the mucosal layer, while only a minor reduction of CGRP-IR fibers, and no reduction of GRP-IR fibers was seen in the muscular layer. The density of galanin-IR nerve fibers was not affected. The height of the oxyntic mucosa was reduced by about 25% (P < 0.05). Thus, a striking effect on the histamine-IR cells was seen, supporting the view that these cells are regulated by the vagus nerve. The study also indicates that a major portion of the CGRP-IR nerve fibers, and part of the GRP-IR nerve fibers, in the mucosal layer of the fundic region are of vagal origin or regulated by normal vagus nerve activity.

In this study, the ontogenetic appearance of three neuronal markers, tyrosine hydroxylase (TH), neurofilament (NF) proteins and acetylcholinesterase (AChE), have been compared in the neural tube and derivatives of the neural crest with special consideration on developing rat sympathetic tissues. The tree markers appeared for the first time on embryonic day E 12.5. At this age, NF immunoreactivity was located in the cells on the ventro- and dorsolateral edges of the neural tube, i.e., in the regions where the cells had reached the postmitotic stage. In addition, on day E 12.5, NF-immunoreactive fibers were located in the dorsal and ventral roots and the spinal and sympathetic ganglia. This suggests rapid extension of neurites. In contrast to NF, AChE first appeared on day E 12.5 in cell somata of spinal and sympathetic ganglia and only after that in axons. Thus, it can be considered as a marker of differentiating neuronal cell bodies. In the developing sympathoadrenal cells, TH is expressed before NF and AChE. However, the migrating TH immunoreactive sympathetic cells are constantly followed by NF immunoreactive fibers, suggesting that sympathetic tissues may receive innervation from preganglionic axons at the very beginning of their ontogeny. During the later development, all sympathetic tissues contain two major cell groups: 1) one with a moderate TH immunoreactivity, NF immunoreactivity and AChE activity and 2) the other with an intense TH immunoreactivity but lacking NF immunoreactivity or AChE activity. The former includes principal neurons, neuron-like cells of the paraganglia and noradrenaline cells of the adrenal medullae, and the latter includes ganglionic small intensely fluorescent (SIF) cells, paraganglionic cells and medullary adrenaline cells.