Istvan Fodor

Lister strain of vaccinia virus armed with endostatin–angiostatin fusion gene as a novel therapeutic agent for human pancreatic cancer

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.

Low-dose vaccinia virus-mediated cytokine gene therapy of glioma.

Recombinant viruses can produce cytokines in tumors mobilizing an immune response to tumor cells. In this study, the authors investigated gene expression, in vivo antitumor efficacy, and safety of attenuated recombinant vaccinia virus (rVV) carrying murine cytokine genes interleukin (IL)-2 (rVV–mIL-2), IL-12 (rVV–mIL-12), and both IL-2 and IL-12 (rVV-2–12) in an athymic nude mice model. Significant tumor inhibition (p < 0.05) was observed in a preestablished subcutaneously implanted C6 glioma model using rVVs at doses ranging from 102 to 107 plaque forming units (PFU). An antitumor effect did not depend on the dose of the rVV–mIL-2 and rVV–mIL-12 viruses. All constructed rVVs induced a high level of cytokine expression in vitro and in vivo. Most groups injected with high doses of recombinant viruses encoding cytokine genes (105 to 107 PFU) showed signs of cytokine toxicity, whereas in the low-dose treatment groups (102 to 103 PFU) toxicity was greatly reduced. The antitumor activity of rVV–mIL-12 was associated with increases in both the percentage and number of natural killer T cells in the spleen. Local detection of interferon-&ggr; and tumor necrosis factor-&agr; was also correlated with tumor growth arrest induced by the treatment. High-dose VV control vector per se induced tumor inhibition by activating Mac-1+ cells in blood, but the antitumor effect was less pronounced compared with rVV-carrying cytokine genes (p < 0.05). These results suggest that attenuated recombinant strains of VV at low doses may potentially be efficient vectors for cancer immunotherapy.

Evaluation of combined vaccinia virus-mediated antitumor gene therapy with p53, IL-2, and IL-12 in a glioma model.

Our previous studies have shown that vaccinia virus (VV) expressing p53, interleukin-2 (IL-2), and interleukin-12 (IL-12) results in an effective inhibition of subcutaneous glioma growth in mice. We propose that combination therapy of tumors with virus-mediated p53 and cytokine genes offers the prospect of synergistic antitumor response. In this work, the antitumor efficacy of VV-mediated combination of p53, IL-2, and IL-12 genes was evaluated in a nude mouse model. To minimize cytokine-associated toxicity, a virus dose as low as 10 plaque-forming units of VV expressing IL-2 and IL-12 per animal was used alone and together with 2×107 plaque-forming units of VV expressing p53. Intratumoral treatment of established C6 glioma with recombinant viruses rVV-p53, rVV-mIL2, rVV-mIL12, and rVV-2-12 induced the prolonged expression of p53, IL-2, IL-12, and both cytokines simultaneously. The combination of rVV-p53/rVV-mIL12 or rVV-p53/rVV-2-12 resulted in significant tumor inhibition compared to single modality treatment (P<.05). rVV-p53/rVV-2-12 therapy was associated with significant elevation of natural killer, Mac-1+, and NKT cells in blood and interferon-γ and tumor necrosis factor-α expression in tumors. The difference in the inhibition of tumor growth between the rVV-p53/rVV-mIL2 combination and rVV-p53 was statistically insignificant. These data demonstrate that gene therapy based on VV-mediated combination of p53, IL-2, and IL-12 treatment may be a promising adjunctive strategy for glioma treatment. Cancer Gene Therapy (2000) 7, 1437–1447

Vaccinia virus preferentially infects and controls human and murine ovarian tumors in mice

Vaccinia virus has been shown to efficiently infect tumor cells. Therefore, vaccinia virus represents a potentially safe and effective antitumor agent against ovarian cancer. Here, we assessed the ability of vaccinia virus to preferentially infect and control both human and murine ovarian tumors 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 vaccinia was able to effectively infect and kill both human and murine ovarian tumors. Vaccinia virus administered to mice intraperitoneally was specifically targeted to the murine or human ovarian tumors and led to antitumor responses. These findings suggest that 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.

Replication-deficient vaccinia virus gene therapy vector: evaluation of exogenous gene expression mediated by PUV-inactivated virus in glioma cells

Mild psoralen and UV (PUV) treatments inactivate viral DNA replication, but the virus retains its ability to infect cells. Thus, PUV treatment of vaccinia virus (VV) vectors may increase the safety of gene delivery and extend the duration of gene expression. Although the first studies on PUV‐inactivated VV (PUV‐VV) for the delivery of suicide or cytokine genes to cancer cells were promising, the efficiency and kinetics of exogenous gene expression have not been fully evaluated. Furthermore, these studies should be extended to other gene therapy strategies, e.g. tumor suppressor genes.

A Vaccinia Virus Armed with Interleukin-10 Is a Promising Therapeutic Agent for Treatment of Murine Pancreatic Cancer

Purpose: Vaccinia virus has strong potential as a novel therapeutic agent for treatment of pancreatic cancer. We investigated whether arming vaccinia virus with interleukin-10 (IL10) could enhance the antitumor efficacy with the view that IL10 might dampen the host immunity to the virus, increasing viral persistence, thus maximizing the oncolytic effect and antitumor immunity associated with vaccinia virus. Experimental Design: The antitumor efficacy of IL10-armed vaccinia virus (VVLΔTK-IL10) and control VVΔTK was assessed in pancreatic cancer cell lines, mice bearing subcutaneous pancreatic cancer tumors and a pancreatic cancer transgenic mouse model. Viral persistence within the tumors was examined and immune depletion experiments as well as immunophenotyping of splenocytes were carried out to dissect the functional mechanisms associated with the viral efficacy. Results: Compared with unarmed VVLΔTK, VVLΔTK-IL10 had a similar level of cytotoxicity and replication in vitro in murine pancreatic cancer cell lines, but rendered a superior antitumor efficacy in the subcutaneous pancreatic cancer model and a K-ras-p53 mutant-transgenic pancreatic cancer model after systemic delivery, with induction of long-term antitumor immunity. The antitumor efficacy of VVLΔTK-IL10 was dependent on CD4+ and CD8+, but not NK cells. Clearance of VVLΔTK-IL10 was reduced at early time points compared with the control virus. Treatment with VVLΔTK-IL10 resulted in a reduction in virus-specific, but not tumor-specific CD8+ cells compared with VVLΔTK. Conclusions: These results suggest that VVLΔTK-IL10 has strong potential as an antitumor therapeutic for pancreatic cancer. Clin Cancer Res; 21(2); 405–16. ©2014 AACR.

Lister Vaccine Strain of Vaccinia Virus Armed with the Endostatin–Angiostatin Fusion Gene: An Oncolytic Virus Superior to dl1520 (ONYX-015) for Human Head and Neck Cancer

Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC.

Distribution of B-cell epitopes on the pseudorabies virus glycoprotein B.

In order to map antigenically important regions of glycoprotein B (gB) of pseudorabies virus (PrV), a panel of recombinant fragments of gB expressed in E. coli and truncated fragments of gB generated by cleavage of purified native gB with trypsin and cyanogen bromide was analysed by using 26 monoclonal antibodies directed against gB. Three continuous epitopes were localized in the vicinity of the N terminus of gB, between amino acids (aa) 59 and 126. One continuous epitope mapped between residues 214 and 279. The residues involved in the assembly of eight discontinuous epitopes were located between aa 540 and 734. The constituents of two discontinuous epitopes were harboured in a segment encompassing aa 540-646. The clustering of continuous epitopes at the extreme N terminus of PrV gB and the locations of residues involved in the assembly of discontinuous epitopes of PrV gB are in good agreement with data on epitope locations in gB homologues from other herpesviruses.

A putative latency promoter/enhancer (P(LAT2)) region of pseudorabies virus contains a virulence determinant

Contradictory data have recently been reported on the role of the unique long-internal repeat junction area of pseudorabies (Aujeszkys disease) virus (PrV) genome in the virulence of the virus. To investigate the basis of the difference, four recombinant PrVs mutated at the outer region of inverted repeats that involved a putative latency promoter (P(LAT2)) were constructed in this study. Propagation characteristics of mutant viruses in cultured cells were similar to those of the wild-type virus. However, a 757 bp deletion at this location caused significant reduction in the virulence of PrV after intraperitoneal inoculation of mice and a moderate decrease in the virulence after intracranial inoculation. These results indicate that the P(LAT2) region is an important virulence determinant that may be implicated in the neuroinvasive capability of the virus.

Glycoprotein B of Aujeszky's disease virus : topographical epitope mapping and epitope-specific antibody response

A panel of 26 monoclonal antibodies (mAbs) against glycoprotein B (gB) of Aujeszkys disease (pseudorabies) virus (ADV), a glycoprotein complex consisting of three glycoproteins, gBa, gBb, and gBc, was produced by two research groups and was used for the topographical epitope mapping of gB. An epitope map was constructed in which the identified epitopes of gB were situated in 14 topologically distinct antigenic domains; ten antigenic domains represented by 22 mAbs were localized on gBc, while four antigenic domains represented by four mAbs resided on gBb of the gB complex. All the epitopes located on gBc appeared to be conformation-dependent, whereas all the epitopes on gBb were conformation-independent. The identified epitopes of gB were conserved among laboratory, vaccine and field ADV strains. Conformation-dependent epitopes were shown to contribute largely to the overall antibody response to gB in naturally infected swine and immunized mice. Moreover, it was found that most of the infected animals responded relatively weakly to the identified conformation-independent epitopes of gB, while a group of immunodominant epitopes that induced a strong antibody response was represented exclusively by conformation-dependent epitopes from different antigenic domains. The results clearly demonstrated that conformation-dependent epitopes of gBc play a crucial role in inducing the humoral immune response to gB of ADV during the natural infection of swine and immunization of mice. The application of mAbs of our panel as research and diagnostic tools is discussed.