Double-deleted Vaccinia Virus Research Articles

Radiovirotherapy at twenty

Radiovirotherapy at twenty

Oncolytic vaccinia virus acts synergistically with anti-PD-L1 antibody to enhance the killing of colon cancer cells by CD8+ T cells

Both oncolytic vaccinia virus (OVV) and anti-PD-L1 antibody hold promise in cancer immunotherapy. Herein, we aimed to explore the possible synergistic effects of OVV and anti-PD-L1 on the growth and metastasis of colon cancer (CC) in mouse models. Microarray profiling of CC-related genes was first conducted. Expression of PD-L1 in CC tissues was predicted by TCGA and verified by flow cytometry and RT-qPCR. Then, mouse CC cell lines stably carrying luciferase MC38-luc and CT26-luc were infected with recombinant double-deleted vaccinia virus (vvDD) to evaluate the effect of vvDD on cell viability. The data indicated that PD-L1 was highly expressed in CC tissues and cells following vvDD infection. MC38-luc cells were inoculated into mice to construct CC-bearing mouse models, which were treated with vvDD or combined with anti-PD-L1, with tumor growth, metastasis, survival, and the immune environment analyzed. It was found that OVV combined with anti-PD-L1 antibody led to lower tumor burden and growth and higher survival rates than individual treatment in CC-bearing mice. In addition, this combination exerted a remote effect on the untreated subcutaneous tumors in the lateral abdomen, thus suppressing the tumor metastasis. Furthermore, combined therapy of OVV with anti-PD-L1 antibody activated CD8+ T cells, reduced exhaustion of CD8+ T cells, and enhanced their immune response, strengthening the killing of CC cells and inhibiting tumor growth and metastasis. In conclusion, our findings provide mechanistic insights into the action and efficacy of OVV as an immunomodulatory agent combined with the anti-PD-L1 antibody for the treatment of CC.

Deletion of immunomodulatory genes as a novel approach to oncolytic vaccinia virus development

Vaccinia virus (VV) has emerged as a promising platform for oncolytic virotherapy. Many clinical VV candidates, such as the double-deleted VV, vvDD, are engineered with deletions that enhance viral tumor selectivity based on cellular proliferation rates. An alternative approach is to exploit the dampened interferon-based innate immune responses of tumor cells by deleting one of the many VV immunomodulatory genes expressed to dismantle the antiviral response. We hypothesized that such a VV mutant would be attenuated in non-tumor cells but retain the ability to effectively propagate in and kill tumor cells, yielding a tumor-selective oncolytic VV with significant anti-tumor potency. In this study, we demonstrated that VVs with a deletion in one of several VV immunomodulatory genes (N1L, K1L, K3L, A46R, or A52R) have similar or improved in vitro replication, spread, and cytotoxicity in colon and ovarian cancer cells compared to vvDD. These deletion mutants are tumor selective, and the best performing candidates (ΔK1L, ΔA46R, and ΔA52R VV) are associated with significant improvement in survival, as well as immunomodulation, within the tumor environment. Overall, we show that exploiting the diminished antiviral responses in tumors serves as an effective strategy for generating tumor-selective and potent oncolytic VVs, with important implications in future oncolytic virus (OV) design.

Fluorescent Tagged Vaccinia Virus Genome Allows Rapid and Efficient Measurement of Oncolytic Potential and Discovery of Oncolytic Modulators.

As a live biologic agent, oncolytic vaccinia virus has the ability to target and selectively amplify at tumor sites. We have previously reported that deletion of thymidine kinase and ribonucleotide reductase genes in vaccinia virus can increase the safety and efficacy of the virus. Here, to allow direct visualization of the viral genome in living cells, we incorporated the ANCH target sequence and the OR3-Santaka gene in the double-deleted vaccinia virus. Infection of human tumor cells with ANCHOR3-tagged vaccinia virus enables visualization and quantification of viral genome dynamics in living cells. The results show that the ANCHOR technology permits the measurement of the oncolytic potential of the double deleted vaccinia virus. Quantitative analysis of infection kinetics and of viral DNA replication allow rapid and efficient identification of inhibitors and activators of oncolytic activity. Our results highlight the potential application of the ANCHOR technology to track vaccinia virus and virtually any kind of poxvirus in living cells.

High Oncolytic Activity of a Double-Deleted Vaccinia Virus Copenhagen Strain against Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a cancer of the pleura that lacks efficient treatment. Oncolytic immunotherapy using oncolytic vaccinia virus (VV) may represent an alternative therapeutic approach for the treatment of this malignancy. Here, we studied the oncolytic activity of VV thymidine kinase (TK)-ribonucleotide reductase (RR)-/green fluorescent protein (GFP) against MPM. This virus is a VV from the Copenhagen strain that is deleted of two genes encoding the TK (J2R) and the RR (I4L) and that express the GFP. First, we show in vitro that VVTK-RR-/GFP efficiently infects and kills the twenty-two human MPM cell lines used in this study. We also show that the virus replicates in all eight tested MPM cell lines, however, with approximately a 10-fold difference in the amplification level from one cell line to another. Then, we studied the therapeutic efficiency of VVTK-RR-/GFP in non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice that bear peritoneal human MPM tumors. One intraperitoneal infection of VVTK-RR-/GFP reduces the tumor burden and significantly increases mice survival compared to untreated animals. Thus, VVTK-RR- may be a promising oncolytic virus (OV) for the oncolytic immunotherapy of MPM.

Human SNF5 arming of double-deleted vaccinia virus shows oncolytic and cytostatic activity against central nervous system atypical teratoid/rhabdoid tumor cells.

Central nervous system (CNS) atypical teratoid/rhabdoid tumor (AT/RT) is a rare, aggressive tumor that most often affects very young children. The common decisive molecular defect in AT/RT has been shown to be a single genetic alteration, i.e., the loss of hSNF5 gene that encodes for a subunit of the SWI/SNF complex that modulates chromatin remodeling activities. As a result, AT/RT cells display unregulated cell proliferation due to the dysfunction of an important epigenetic control. We have previously demonstrated the preclinical efficacy of the oncolytic double-deleted vaccinia virus (VVDD) against AT/RT. Here we report the establishment of a modified VVDD engineered to express wild type hSNF5 gene. We show that this reconstructed vaccinia virus retains comparable infectivity and in vitro cytotoxicity of the parent strain. However, in addition, hSNF5-arming of VVDD results in a decreased cell cycle S phase population and down-regulation of cyclin D1. These findings suggest that hSNF5-arming of VVDD may increase the efficacy in the treatment of AT/RT and validates, as a proof-of-concept, an experimental approach to enhance the effective use of novel modified oncolytic viruses in the treatment of tumors with loss of a tumor suppressor gene function.

Temozolomide resistant human brain tumor stem cells are susceptible to recombinant vesicular stomatitis virus and double-deleted Vaccinia virus in vitro

BackgroundMalignant glioma still has a poor prognosis and remains incurable. Although temozolomide (TMZ) has demonstrated antitumor activity, its use recently has been halted because of some patients’ resistance to this drug. New treatments are desperately needed. An oncolytic virus (virotherapy) is being developed as a novel cancer therapy. We have previously reported that recombinant Vesicular Stomatitis Virus (VSV-ΔM51) and double deleted Vaccinia Virus (vvDD) infected and killed glioma cell lines in vitro and prolonged survival in animal glioma models. As a proposed ex vivo test, the oncolytic potential of VSV-ΔM51 and vvDD in the established human brain tumor stem cells (BTSCs) and the differentiated cells from fresh brain tumor tissues in vitro were further investigated. MethodsBTSCs from fresh surgical glioblastoma multiforme (GBM) specimens were isolated and cultured, and the characterization of BTSCs were tested. The sensitivity of BTSCs to TMZ and the susceptibility of TMZ resistant BTSCs and their differentiated cells to both oncolytic viruses were examined. ResultsThe BTSC spheres cultured had all the characteristics of stem cells. The GFP-labeled VSV-ΔM51 and vvDD could infect TMZ resistant BTSCs and cause cytopathic effects. The VSV-ΔM51and vvDD inhibited the self-renewal activity of TMZ resistant BTSCs. And the VSV-ΔM51and vvDD also infected and caused cytopathic effects in differentiated BTSCs. ConclusionVSV-ΔM51and vvDD could infect and kill both the TMZ resistant BTSCs and the differentiated compartments of GBMs in vitro, suggesting that they may be an effective treatment supplement for GBM therapy, particularly for TMZ resistant GBM patients.

Evidence for differential viral oncolytic efficacy in an in vitro model of epithelial ovarian cancer metastasis

Epithelial ovarian cancer is unique among most carcinomas in that metastasis occurs by direct dissemination of malignant cells traversing throughout the intraperitoneal fluid. Accordingly, we test new therapeutic strategies using an in vitro three-dimensional spheroid suspension culture model that mimics key steps of this metastatic process. In the present study, we sought to uncover the differential oncolytic efficacy among three different viruses—Myxoma virus, double-deleted vaccinia virus, and Maraba virus—using three ovarian cancer cell lines in our metastasis model system. Herein, we demonstrate that Maraba virus effectively infects, replicates, and kills epithelial ovarian cancer (EOC) cells in proliferating adherent cells and with slightly slower kinetics in tumor spheroids. Myxoma virus and vaccinia viruses infect and kill adherent cells to a much lesser extent than Maraba virus, and their oncolytic potential is almost completely attenuated in spheroids. Myxoma virus and vaccinia are able to infect and spread throughout spheroids, but are blocked in the final stages of the lytic cycle, and oncolytic-mediated cell killing is reactivated upon spheroid reattachment. Alternatively, Maraba virus has a remarkably reduced ability to initially enter spheroid cells, yet rapidly infects and spreads throughout spheroids generating significant cell killing effects. We show that low-density lipoprotein receptor expression in ovarian cancer spheroids is reduced and this controls efficient Maraba virus binding and entry into infected cells. Taken together, these results are the first to implicate the potential impact of differential viral oncolytic properties at key steps of ovarian cancer metastasis.

121 A modified double-deleted vaccinia virus combining viral oncolysis and potential gene therapy as a novel therapeutic for atypical teratoid/rhabdoid tumors

121 A modified double-deleted vaccinia virus combining viral oncolysis and potential gene therapy as a novel therapeutic for atypical teratoid/rhabdoid tumors

Regional Delivery of Oncolytic Vaccinia Virus: It’s Time for Clinical Trials

Regional Delivery of Oncolytic Vaccinia Virus: It’s Time for Clinical Trials

Double-deleted vaccinia virus in virotherapy for refractory and metastatic pediatric solid tumors

BackgroundPrevious studies have shown successful antitumor effects of systemically delivered double-deleted vaccinia virus (vvDD) against a number of adult tumor models, including glioma, colon and ovarian cancers. The purpose of this study was to investigate the oncolytic potential of vvDD against a panel of cell lines representative of pediatric solid tumors that are currently difficult to cure. MethodsCell lines derived from central nervous system atypical teratoid rhabdoid tumor (AT/RT) (BT12, BT16 and KCCF1), sarcoma (143B, HOS, RD and RH30), and neuroblastoma (SKNAS, SKNBE2, IMR-5 and IMR-32) were examined for vvDD mediated cytotoxicity defined by virus expansion followed by loss of tumor cell viability. The normal human fibroblast cell line HS68 was used as a control. Next, relevant orthotopic, subcutaneous and lung metastasis xenograft models were treated with intravenous doses of live vvDD or killed virus controls (DV). Tumor growth inhibition and viral replication were quantified and survival outcomes of these animals were assessed. ResultsvvDD was able to infect and kill nine of eleven of the pediatric tumor cells (81.8%) in vitro. In xenograft models, intravenous administration of a single dose of vvDD significantly inhibited the growth of tumors and prolonged the survival of intracranial and metastatic tumors. ConclusionsOncolytic vvDD administered i.v. shows activity in preclinical models of pediatric malignancies that are resistant to many currently available treatments. Our data support further evaluation of vvDD virotherapy for refractory pediatric solid tumors.

The combination of immunosuppression and carrier cells significantly enhances the efficacy of oncolytic poxvirus in the pre-immunized host

Pre-existing anti-poxvirus immunity in cancer patients presents a severe barrier to poxvirus-mediated oncolytic virotherapy. We have explored strategies of immunosuppression (IS) and/or immune evasion for efficient delivery of an oncolytic vaccinia virus (vvDD) to tumors in the pre-immunized mice. Transient IS using immunosuppressive drugs, including tacrolimus, mycophenolate mofetil and methylprednisolone sodium succinate, have been used successfully in organ transplantation. This drug cocktail alone did not enhance viral recovery from subcutaneous tumor after systemic viral delivery. Using B cell knockout mice, we confirmed that the neutralizing antibodies played a significant role in preventing poxvirus infection. Using a MC38 peritoneal carcinomatosis (PC) model, we found that the combination of IS and tumor cells as carriers led to the most effective viral delivery, viral replication and viral spread inside the tumor mass. We found that our immunosuppressive drug cocktail facilitated recruitment of tumor-associated macrophages and conversion into an immunosuppressive M2 phenotype (IL-10hi/IL-12low) in the tumor microenvironment. A combination of IS and carrier cells led to significantly prolonged survival in the tumor model. These results demonstrated the feasibility of treating pre-vaccinated patients with peritoneal carcinomatosis using an oncolytic poxvirus and a combined immune intervention strategy.

Antiproliferative Effects of 111In- or 177Lu-DOTATOC on Cells Exposed to Low Multiplicity-of-Infection Double-Deleted Vaccinia Virus Encoding Somatostatin Subtype-2 Receptor

Oncolytic viruses may be limited in their ability to infect and lyse tumor cells because of penetration barriers and viral elimination by the immune system. Combining virotherapy with targeted radiotherapy that uses (111)In- or (177)Lu-DOTATOC may address such issues by spatially enhancing antitumor effects through bystander and/or cross-fire phenomena. In this study, a double-deleted vaccinia virus (vvDD) encoding the gene for somatostatin subtype-2 receptor (sstr-2) infected MC-38 murine colon adenocarcinoma cells and increased their sstr-2 expression by 2-fold. A low multiplicity-of-infection (MOI = 0.1) of vvDD and short exposure time (48 hours) preserved MC-38 viability (>80%-90%) for up to 3 days, permitting targeting of sstr-2 by (111)In- or (177)Lu-DOTATOC. (111)In-DOTATOC, alone or in combination with vvDD, was less effective than (177)Lu-DOTATOC at decreasing the growth of sstr-2-gene-transfected human embryonic kidney (HEK)-293 cells or MC-38 cells in monolayer. However, (111)In- or (177)Lu-DOTATOC combined with vvDD provided equivalent growth inhibition of HEK-293 or MC-38 cells as spheroids, suggesting a bystander effect from (111)In-DOTATOC. Growth of the cells was reduced 4-fold (from 20% to <5%) at 8 days in this case. Further evaluation of low-MOI vvDD in combination with (111)In- or (177)Lu-DOTATOC for the treatment of MC-38 tumors in mice is planned.

Efficacy of Systemically Administered Oncolytic Vaccinia Virotherapy for Malignant Gliomas Is Enhanced by Combination Therapy with Rapamycin or Cyclophosphamide

The oncolytic effects of a systemically delivered, replicating, double-deleted vaccinia virus has been previously shown for the treatment of many cancers, including colon, ovarian, and others. The purpose of this study was to investigate the oncolytic potential of double-deleted vaccinia virus alone or in combination with rapamycin or cyclophosphamide to treat malignant gliomas in vitro and in vivo. Rat (RG2, F98, C6) and human (A172, U87MG, U118) glioma cell lines were cultured in vitro and treated with live or UV-inactivated vaccinia virus. Viral gene [enhanced green fluorescent protein (EGFP)] expression by fluorescence-activated cell sorting, relative cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and assays for cytopathic effects were examined. S.c. murine tumor xenografts (U87MG, U118, C6) and i.c. (RG2, F98) tumor models in immunocompetent rats were treated with systemic administration of EGFP-expressing vaccinia virus (vvDD-EGFP), alone or in combination with rapamycin or cyclophosphamide, or controls. Tumor size, viral biodistribution, and animal survival were assessed. Lastly, the oncolytic effects of vvDD-EGFP on human malignant glioma explants were evaluated. vvDD-EGFP was able to infect and kill glioma cells in vitro. A single systemic dose of vvDD-EGFP significantly inhibited the growth of xenografts in athymic mice. Systemic delivery of vvDD-EGFP alone was able to target solitary and multifocal i.c. tumors and prolong survival of immunocompetent rats, whereas combination therapy with rapamycin or cyclophosphamide enhanced viral replication and further prolonged survival. Finally, vvDD-EGFP was able to infect and kill ex vivo primary human malignant gliomas. These results suggest that vvDD-EGFP is a promising novel agent for human malignant glioma therapy, and in combination with immunosuppressive agents, may lead to prolonged survival from this disease.

Oncolytic virotherapy for multiple myeloma using a tumour-specific double-deleted vaccinia virus

Oncolytic virotherapy is a tumour-specific strategy, in which viruses selectively kill cancer cells, either through targeted alterations in the cancer cell,1, 2 viral deletions,3, 4 tissue-specific transcriptional control5 or tumour-specific receptors.6, 7 Normal cells are generally left intact. Haematologic malignancies such as multiple myeloma (MM) are particularly attractive for this approach as patients generally have less bulky disease and may have immune system dysfunction favouring viral replication. To date, MM has demonstrated pre-clinical responses to oncolytic measles virus,6, 7 vesicular stomatitis virus8 and coxsackievirus A21.9 Our lab has previously developed an attenuated, recombinant double-deleted vaccinia virus (VV) that infects, replicates and expresses genes preferentially in rapidly dividing tumour cells.4 We asked whether VV would be an effective oncolytic virus against MM.

Oncolytic Virotherapy for Multiple Myeloma.

Oncolytic virotherapy for cancer employs viruses which specifically lyse tumour cells, leaving normal cells intact. Double-deleted vaccinia virus (vvDD) is tumour-specific due to mutations that restrict viral replication to dividing cells. This is the first use of vvDD to treat a hematologic malignancy. vvDD expressing enhanced green fluorescent protein (EGFP) was used to infect multiple myeloma (MM) cell lines. We used flow cytometry (FACS) analysis to quantify EGFP positive cells in parallel to colorimetric (MTT) and microscopic cell viability assays. FACS results indicated vvDD efficiently (27–73%) infected the MM cell lines RPMI8226, MY5, H929, U266 and LP1 by 72 hours post infection. MTT assays revealed a 7–8 fold decrease in cell viability of infected cells compared to controls. MM cell lines were grown as subcutaneous xenografts (My5) or systemically (RPMI8226) in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, and tumour size/paralysis was monitored after systemic delivery of vvDD. My5 tumours responded to ip vvDD, resulting in a significant delay of tumour growth compared to controls, while 80% of mice treated with vvDD 3 days after iv RPMI8226 tumour injection were cured. Analysis of mouse tissues for viral biodistribution and pathogenicity showed that the ovary was the second highest viral containing tissue though it was 4 logs lower than tumour. Ovaries showed some evidence of follicular necrosis in treated tissues. All other tissues were unaffected. Primary human MM (CD138+) cells obtained from bone marrow aspiration were readily (50–60%) infected with vvDD while normal mononuclear cells from the same samples were not infected. At the same time primary human CD138+ cells were infected with vvDD at an MOI of 1 or mock infected and then plated in matrigel. vvDD treated cells showed significantly reduced colony formation compared to mock-infected controls. These results suggest that vvDD is a safe and effective treatment for multiple myeloma and should be considered for phase I testing in these patients.