Attenuated vaccinia viruses (VV) selectively replicate in malignant cells and confer oncolytic effect in vivo. Here we demonstrate that oncolytic VV may also be used as a diagnostic agent for tumor-bearing mice. A series of recombinant vaccinia viruses has been constructed expressing optical reporters to mediate emission of bioluminescent and fluorescent light which can be visualized. Data show that following systemic virus delivery the developing tumors can be non-invasively visualized in mice in vivo. Renilla luciferase and Aquoria GFP have been effective in imaging xenografted PC-3 prostate and orthotopic MB-49 bladder tumors. Brighter reporters, Gaussia luciferase and Renilla GFP have been used for imaging TRAMP prostate cancer and C6 subcutaneous model of glioma. The C6 imaging data have been corroborated by traditional MRI. We are also developing a VV-mediated system for tumor detection in far red or near infra red fluorescent light. Results suggest that VV-mediated imaging is a promising alternative for early diagnosis of various human cancers.

The vaccinia virus has been shown to efficiently infect tumor cells. Therefore, the vaccinia virus represents a potentially safe and effective anti-tumor agent against ovarian cancer. Here, we assessed the ability of the vaccinia virus to preferentially infect and control both human and murine ovarian tumors in vitro and in vivo. We used the non-invasive luminescence imaging system to monitor the infection and suppression of ovarian tumors by vaccinia in live mice. Our data indicated that the vaccinia virus was able to preferentially infect and kill both human and murine ovarian tumors. In addition, vaccinia virus administered to mice intraperitoneally was specifically targeted to the established murine or human ovarian tumors and led to antitumor responses. Taken together, these findings suggest that the vaccinia virus is capable of selectively targeting and controlling ovarian tumors. Thus, intraperitoneal injection with vaccinia virus may provide a potentially effective strategy for treating advanced stage ovarian cancers.

In autoimmune (type 1) diabetes, autoreactive lymphocytes destroy pancreatic cells responsible for insulin synthesis. To assess the feasibility of gene therapy for type 1 diabetes, recombinant vaccinia virus (rVV) vectors were constructed expressing pancreatic islet autoantigens proinsulin (INS) and a 55-kDa immunogenic peptide from glutamic acid decarboxylase (GAD), and the immunomodulatory cytokine interleukin (IL)-10. To augment the beneficial effects of recombinant virus therapy, the INS and GAD genes were fused to the C terminus of the cholera toxin B subunit (CTB). Five-week-old non-obese diabetic (NOD) mice were injected once with rVV. Humoral antibody immune responses and hyperglycemia in the infected mice were analyzed. Only 20% of the mice inoculated with rVV expressing the CTB::INS fusion protein developed hyperglycemia, in comparison to 70% of the mice in the uninoculated animal group. Islets from pancreatic tissues isolated from euglycemic mice from this animal group showed no sign of inflammatory lymphocyte invasion. Inoculation with rVV producing CTB::GAD or IL-10 was somewhat less effective in reducing diabetes. Humoral antibody isotypes of hyperglycemic and euglycemic mice from all treated groups possessed similar IgG1/IgG2c antibody titer ratios from 19 to 32 wk after virus inoculation. In comparison with uninoculated mice, 11-wk-old NOD mice injected with virus expressing CTB::INS were delayed in diabetes onset by more than 4 wk. The experimental results demonstrate the feasibility of using rVV expressing CTB::INS fusion protein to generate significant protection and therapy against type 1 diabetes onset and progression.

Background. Interferons (IFN) play an important role in host antiviral responses, but viruses, including vaccinia viruses (VV), employ mechanisms to disrupt IFN activities, and these viral mechanisms are often associated with their virulence. Here, we explore an attenuation strategy with a vaccine strain of VV lacking a virus-encoded IFN-γ receptor homolog (viroceptor). Methods. To facilitate the monitoring of virus properties, first we constructed a Lister vaccine strain derivative VV-RG expressing optical reporters. Further, we constructed a VV-RG derivative, VV-RG8, which lacks the IFN-gammaR viroceptor (B8R gene product). Replication, immunological and pathogenic properties of the constructed strains were compared. Results. Viruses did not show significant differences in humoral and cellular immune responses of immune-competent mice. Replication of constructed viruses was efficient both in vitro and in vivo, but showed marked difference in kinetics of propagation. In cultured CV-1 epithelial cells, the VV-RG8 retained the propagation potential of the parental virus, while in the C6 glial cells, significant delay was observed in the kinetics of the VV-RG8 replication cycle compared to VV-RG. The pathogenesis of the viruses was tested by survival assay and biodistribution in nude mice. High dose inoculation of nude mice with VV-RG8 caused less pronounced virus dissemination, improved weight gain, and increased survival rate, as compared with the VV-RG strain. Conclusions. The replication-competent virus VV-RG8 carrying mutation at B8R gene is less pathogenic for mice than the parental vaccine virus. We anticipate that step-wise inactivation of VV vaccine genes involved in evasion of host immune response may provide an alternative approach for generation hyper-attenuated replication-competent vaccines.

The study of neurons in culture would be facilitated by the development of a gene transduction system capable of delivering foreign genes at high efficiency, as transduction of primary neurons with existing systems is inefficient. The efficacy of lytic vaccinia virus (VV) infection of dissociated retinal cultures and PC12 cells was examined in order to determine the efficiency of using VV in neuronal primary culture. VV was able to infect retinal cells and PC12 cells in a virus dose-dependent manner. 50-100% of retinal cells were positive for virus infection at multiplicities of infection (MOI) between 10 and 100 while over 50% of PC12 cells were infected at a MOI of 10. The production of foreign mRNA and protein by VV following infection was verified, as was the simultaneous production of two foreign transgenes from a dual promoter viral construct. Because VV is a lytic virus, cytopathic effects were examined. Retinal cultures maintained for 0.5 days in vitro showed greater than 90% survival at 24 h post-infection, while 14 day cultures were equally viable for 48 h. VV was specifically effective to infect retinal ganglion cells with high efficiency. These data suggest that VV may be a useful vector for delivering foreign genes to neuronal cells with a high degree of efficiency of transient gene expression.

Purpose: To determine if vaccinia virus (VV) mediated delivery of human tumor suppressor p53 is safe and effective for bladder tumor therapy in an orthotopic murine model. Materials and Methods: We have used recombinant VV vectors (rVV) to express transgenes in murine bladder cancer MB-49 cells in culture, and growing orthotopically in syngeneic mice. Cultured MB-49 cells were infected with rVV expressing reporter genes (rVV-L15) or p53 (rVV-TK-53) to measure virus infection and apoptosis induction. Orthotopic MB-49 tumors in C57/Bl6 mice were treated by intravesical instillation of rVV, and tumor incidence, survival and transgene expression were determined. Results: Productive virus infection in vitro was observed in MB-49 cells, though at somewhat lower efficiency than in African Green Monkey kidney CV-1 cells. Expression of transgenes in vitro correlated with the virus dose. Cells infected with rVV underwent apoptosis, with rVV-TK-53 inducing far greater cell death than rVV-L15.The rVV-L15 virus had no effect on tumor incidence, but increased the mean survival compared to control. Instillation of the rVV-TK-53 decreased tumor incidence, and 33% of mice survived the treatment. At necropsy, all succumbed mice had bladder tumor, whereas two survivors of the rVV-TK-53-treated group were tumor-free. Immunohistochemistry of tumors detected expression of the human p53 gene product in tumor cells. Conclusions: For the first time we show that recombinant vaccinia virus expressing human p53 can induce death of MB-49 tumor cells in vivo, not only through lytic effect of the virus, but also through expression of the death-inducing p53 transgene. Further studies are needed to shed light on mechanisms of rVV-TK-53 -mediated tumor apoptosis and anti-tumor immune response.

Oral administration of autoantigens and allergens can delay or suppress clinical disease in experimental autoimmune and allergic disorders. However, repeated feeding of large amounts of the tolerogens is required over long periods and is only partially effective in animals systemically sensitized to the ingested antigen. Enhanced suppression of Type 1 autoimmune diabetes insulitis and hyperglycemia was demonstrated in both naive and immune animals following oral inoculation with plant-based antigens coupled to the cholera toxin B subunit (CTB). Thus, plant-synthesized antigens linked to the CTB adjuvant, can enhance suppression of inflammatory Th1 lymphocyte mediated autoreactivity in both naive and immune animals. To stimulate adjuvant-autoantigen fusion protein biosynthesis in the gut mucosae, we evaluated oral inoculation of juvenile NOD mice with recombinant vaccinia virus (rVV) expressing fusion genes encoding CTB linked to the pancreatic islet autoantigens proinsulin (INS) and a 55 kDa C-terminal peptide from glutamate decarboxylase (GAD55). Oral inoculation with rVV carrying the CTB::GAD fusion gene generated a significant reduction in insulitis. Hyperglycemia in the rVV-CTB::GAD inoculated mice was reduced by 40% in comparison with rVV-GAD inoculated mice. An increase in IgG1 antibody isotype titers in comparison with IgG2c isotype titers in rVV infected mice suggested possible activation of autoantigen specific Th2 lymphocytes. The experimental results demonstrate the feasibility of using vaccinia virus oral delivery of adjuvanted autoantigens to the mucosae of prediabetic mice for suppression and therapy of Type 1 autoimmune diabetes.

Targeting cells that support tumor growth by administering potent angiogenesis inhibitors is currently an area of intense interest. In the present study, a unique plasmid vector for the mouse endostatin gene, pXLG-mEndo, was constructed and evaluated with and without radiation using the Lewis lung carcinoma (LLC) cell line. The physical properties of the expressed endostatin protein were validated by PCR, gel electrophoresis, and Western blot. Enzyme-linked immunosorbent and immunocytochemical analyses for the therapeutic gene demonstrated that transfected LLC cells secreted the protein into the medium. Exposure of the cells to 2 gray (Gy) -rays reduced the time to reach the maximum expression level of the endostatin gene and also increased the amount of secreted endostatin protein (P

Abstract. Survival after pancreatic cancer remains poor despite incremental advances in surgical and adjuvant therapy, and new strategies for treatment are needed. Oncolytic virotherapy is an attractive approach for cancer treatment. In this study, we have evaluated the effectiveness of the Lister vaccine strain of vaccinia virus armed with the endostatin–angiostatin fusion gene (VVhEA) as a novel therapeutic approach for pancreatic cancer. The Lister vaccine strain of vaccinia virus was effective against all human pancreatic carcinoma cells tested in vitro, especially those insensitive to oncolytic adenovirus. The virus displayed inherently high selectivity for cancer cells, sparing normal cells both in vitro and in vivo, with effective infection of tumors after both intravenous (i.v.) and intratumoral (i.t.) administrations. The expression of the endostatin–angiostatin fusion protein was confirmed in a pancreatic cancer model both in vitro and in vivo, with evidence of inhibition of angiogenesis. This novel vaccinia virus showed significant antitumor potency in vivo against the Suit-2 model by i.t. administration. This study suggests that the novel Lister strain of vaccinia virus armed with the endostatin–angiostatin fusion gene is a potential therapeutic agent for pancreatic cancer.