Science.gov

Sample records for oncolytic vaccinia virus

  1. Theranostic Potential of Oncolytic Vaccinia Virus

    PubMed Central

    Rojas, Juan J; Thorne, Steve H

    2012-01-01

    Biological cancer therapies, such as oncolytic, or replication-selective viruses have advantages over traditional therapeutics as they can employ multiple different mechanisms to target and destroy cancers (including direct cell lysis, immune activation and vascular collapse). This has led to their rapid recent clinical development. However this also makes their pre-clinical and clinical study complex, as many parameters may affect their therapeutic potential and so defining reason for treatment failure or approaches that might enhance their therapeutic activity can be complicated. The ability to non-invasively image viral gene expression in vivo both in pre-clinical models and during clinical testing will considerably enhance the speed of oncolytic virus development as well as increasing the level and type of useful data produced from these studies. Further, subsequent to future clinical approval, imaging of reporter gene expression might be used to evaluate the likelihood of response to oncolytic viral therapy prior to changes in tumor burden. Here different reporter genes used in conjunction with oncolytic viral therapy are described, along with the imaging modalities used to measure their expression, while their applications both in pre-clinical and clinical testing are discussed. Possible future applications for reporter gene expression from oncolytic viruses in the phenotyping of tumors and the personalizing of treatment regimens are also discussed. PMID:22509200

  2. Oncolytic vaccinia virus synergizes with irinotecan in colorectal cancer.

    PubMed

    Ottolino-Perry, Kathryn; Acuna, Sergio A; Angarita, Fernando A; Sellers, Clara; Zerhouni, Siham; Tang, Nan; McCart, J Andrea

    2015-10-01

    Metastatic colorectal cancer (CRC) is complex clinical challenge for which there are limited treatment options. Chemotherapy with or without surgery provides moderate improvements in overall survival and quality of life; nevertheless the 5-year survival remains below 30%. Oncolytic vaccinia virus (VV) shows strong anti-tumour activity in models of CRC, however transient delays in disease progression are insufficient to lead to long-term survival. Here we examined the efficacy of VV with oxaliplatin or SN-38 (active metabolite of irinotecan) in CRC cell lines in vitro and VV with irinotecan in an orthotopic model of metastatic CRC. Synergistic improvements in in vitro cell killing were observed in multiple cell lines. Combination therapy was well tolerated in tumour-bearing mice and the median survival was significantly increased relative to monotherapy despite a drug-dependent decrease in the mean tumour titer. Increased apoptosis following in vitro and in vivo combination therapy was observed. In vitro cell cycle analysis showed increases in S-phase cells following infection occurred in both infected and uninfected cell populations. This corresponded to a 4-fold greater increase in apoptosis in the uninfected compared to infected cells following combination therapy. Combination treatment strategies are among the best options for patients with advanced cancers. VV is currently under clinical investigation in patients with CRC and the data presented here suggest that its combination with irinotecan may provide benefit to a subset of CRC patients. Further, investigation of this combination is necessary to determine the tumour characteristics responsible for mediating synergy. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Antigen profiling analysis of vaccinia virus injected canine tumors: oncolytic virus efficiency predicted by boolean models.

    PubMed

    Cecil, Alexander; Gentschev, Ivaylo; Adelfinger, Marion; Nolte, Ingo; Dandekar, Thomas; Szalay, Aladar A

    2014-01-01

    Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for cancer therapy. In this study we describe for the first time the use of dynamic boolean modeling for tumor growth prediction of vaccinia virus GLV-1h68-injected canine tumors including canine mammary adenoma (ZMTH3), canine mammary carcinoma (MTH52c), canine prostate carcinoma (CT1258), and canine soft tissue sarcoma (STSA-1). Additionally, the STSA-1 xenografted mice were injected with either LIVP 1.1.1 or LIVP 5.1.1 vaccinia virus strains.   Antigen profiling data of the four different vaccinia virus-injected canine tumors were obtained, analyzed and used to calculate differences in the tumor growth signaling network by type and tumor type. Our model combines networks for apoptosis, MAPK, p53, WNT, Hedgehog, TK cell, Interferon, and Interleukin signaling networks. The in silico findings conform with in vivo findings of tumor growth. Boolean modeling describes tumor growth and remission semi-quantitatively with a good fit to the data obtained for all cancer type variants. At the same time it monitors all signaling activities as a basis for treatment planning according to antigen levels. Mitigation and elimination of VACV- susceptible tumor types as well as effects on the non-susceptible type CT1258 are predicted correctly. Thus the combination of Antigen profiling and semi-quantitative modeling optimizes the therapy already before its start.

  4. Polymeric Cups for Cavitation-mediated Delivery of Oncolytic Vaccinia Virus

    PubMed Central

    Myers, Rachel; Coviello, Christian; Erbs, Philippe; Foloppe, Johann; Rowe, Cliff; Kwan, James; Crake, Calum; Finn, Seán; Jackson, Edward; Balloul, Jean-Marc; Story, Colin; Coussios, Constantin; Carlisle, Robert

    2016-01-01

    Oncolytic viruses (OV) could become the most powerful and selective cancer therapies. However, the limited transport of OV into and throughout tumors following intravenous injection means their clinical administration is often restricted to direct intratumoral dosing. Application of physical stimuli, such as focused ultrasound, offers a means of achieving enhanced mass transport. In particular, shockwaves and microstreaming resulting from the instigation of an ultrasound-induced event known as inertial cavitation can propel OV hundreds of microns. We have recently developed a polymeric cup formulation which, when delivered intravenously, provides the nuclei for instigation of sustained inertial cavitation events within tumors. Here we report that exposure of tumors to focused ultrasound after intravenous coinjection of cups and oncolytic vaccinia virus , leads to substantial and significant increases in activity. When cavitation was instigated within SKOV-3 or HepG2 xenografts, reporter gene expression from vaccinia virus was enhanced 1,000-fold (P < 0.0001) or 10,000-fold (P < 0.001), respectively. Similar increases in the number of vaccinia virus genomes recovered from tumors were also observed. In survival studies, the application of cup mediated cavitation to a vaccinia virus expressing a prodrug converting enzyme provided significant (P < 0.05) retardation of tumor growth. This technology could improve the clinical utility of all biological therapeutics including OV. PMID:27375160

  5. Preclinical Evaluation of Oncolytic Vaccinia Virus for Therapy of Canine Soft Tissue Sarcoma

    PubMed Central

    Josupeit, Rafael; Rudolph, Stephan; Ehrig, Klaas; Donat, Ulrike; Weibel, Stephanie; Chen, Nanhai G.; Yu, Yong A.; Zhang, Qian; Heisig, Martin; Thamm, Douglas; Stritzker, Jochen; MacNeill, Amy; Szalay, Aladar A.

    2012-01-01

    Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS. PMID:22615950

  6. Antiangiogenic Arming of an Oncolytic Vaccinia Virus Enhances Antitumor Efficacy in Renal Cell Cancer Models▿ †

    PubMed Central

    Guse, Kilian; Sloniecka, Marta; Diaconu, Iulia; Ottolino-Perry, Kathryn; Tang, Nan; Ng, Calvin; Le Boeuf, Fabrice; Bell, John C.; McCart, J. Andrea; Ristimäki, Ari; Pesonen, Sari; Cerullo, Vincenzo; Hemminki, Akseli

    2010-01-01

    Oncolytic vaccinia viruses have shown compelling results in preclinical cancer models and promising preliminary safety and antitumor activity in early clinical trials. However, to facilitate systemic application it would be useful to improve tumor targeting and antitumor efficacy further. Here we report the generation of vvdd-VEGFR-1-Ig, a targeted and armed oncolytic vaccinia virus. Tumor targeting was achieved by deletion of genes for thymidine kinase and vaccinia virus growth factor, which are necessary for replication in normal but not in cancer cells. Given the high vascularization typical of kidney cancers, we armed the virus with the soluble vascular endothelial growth factor (VEGF) receptor 1 protein for an antiangiogenic effect. Systemic application of high doses of vvdd-VEGFR-1-Ig resulted in cytokine induction in an immunocompromised mouse model. Upon histopathological analysis, splenic extramedullary hematopoiesis was seen in all virus-injected mice and was more pronounced in the vvdd-VEGFR-1-Ig group. Analysis of the innate immune response after intravenous virus injection revealed high transient and dose-dependent cytokine elevations. When medium and low doses were used for intratumoral or intravenous injection, vvdd-VEGFR-1-Ig exhibited a stronger antitumor effect than the unarmed control. Furthermore, expression of VEGFR-1-Ig was confirmed, and a concurrent antiangiogenic effect was seen. In an immunocompetent model, systemic vvdd-VEGFR-1-Ig exhibited superior antitumor efficacy compared to the unarmed control virus. In conclusion, the targeted and armed vvdd-VEGFR-1-Ig has promising anticancer activity in renal cell cancer models. Extramedullary hematopoiesis may be a sensitive indicator of vaccinia virus effects in mice. PMID:19906926

  7. Oncolytic and immunologic cancer therapy with GM-CSF-armed vaccinia virus of Tian Tan strain Guang9.

    PubMed

    Deng, Lili; Fan, Jun; Guo, Mingming; Huang, Biao

    2016-03-28

    Targeted oncolytic vaccinia viruses are being developed as a novel strategy in cancer therapy. Arming vaccinia viruses with immunostimulatory cytokines can enhance antitumor efficacy. Such engineered oncolytic viruses, like JX-594, a Wyeth strain vaccinia virus modified with human granulocyte-macrophage colony-stimulating factor (GM-CSF), have shown promising results and have proceeded rapidly in clinical trials. However, the oncolytic potential of the Chinese vaccine strain Tian Tan (VTT) has not been explored. In this study, we constructed a targeted oncolytic vaccinia virus of Tian Tan strain Guang9 (VG9) expressing murine GM-CSF (VG9-GMCSF) and evaluated the antitumor effect of this recombinant vaccinia virus in a murine melanoma model. In vitro, viral replication and cytotoxicity of VG9-GMCSF was as potent as VG9; in vivo, VG9-GMCSF significantly inhibited the growth of subcutaneously implanted melanoma tumors, prolonged the survival of tumor-bearing mice, and produced an antitumor cytotoxic response. Such antitumor effect may be due to the lytic nature of virus as well as the stimulation of immune activity by GM-CSF production. Our results indicate that VG9-GMCSF induces strong tumoricidal activity, providing a potential therapeutic strategy for combating cancer.

  8. Silk-elastin-like protein polymer matrix for intraoperative delivery of an oncolytic vaccinia virus

    PubMed Central

    Price, Daniel L.; Li, Pingdong; Chen, Chun-Hao; Wong, Danni; Yu, Zhenkun; Chen, Nanhai G.; Yu, Yong A.; Szalay, Aladar A.; Cappello, Joseph; Fong, Yuman; Wong, Richard J.

    2016-01-01

    Background Oncolytic viral efficacy may be limited by the penetration of the virus into tumors. This may be enhanced by intraoperative application of virus immediately after surgical resection. Methods Oncolytic vaccinia virus GLV-1h68 was delivered in silk-elastin-like protein polymer (SELP) in vitro and in vivo in anaplastic thyroid carcinoma cell line 8505c in nude mice. Results GLV-1h68 in SELP infected and lysed anaplastic thyroid cancer cells in vitro equally as effectively as in phosphate-buffered saline (PBS), and at 1 week retains a thousand fold greater infectious plaque-forming units. In surgical resection models of residual tumor, GLV-1h68 in SELP improves tumor control and shows increased viral β-galactosidase expression as compared to PBS. Conclusion The use of SELP matrix for intraoperative oncolytic viral delivery protects infectious viral particles from degradation, facilitates sustained viral delivery and transgene expression, and improves tumor control. Such optimization of methods of oncolytic viral delivery may enhance therapeutic outcomes. PMID:25244076

  9. Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer.

    PubMed

    Mansfield, D C; Kyula, J N; Rosenfelder, N; Chao-Chu, J; Kramer-Marek, G; Khan, A A; Roulstone, V; McLaughlin, M; Melcher, A A; Vile, R G; Pandha, H S; Khoo, V; Harrington, K J

    2016-04-01

    Oncolytic strains of vaccinia virus are currently in clinical development with clear evidence of safety and promising signs of efficacy. Addition of therapeutic genes to the viral genome may increase the therapeutic efficacy of vaccinia. We evaluated the therapeutic potential of vaccinia virus expressing the sodium iodide symporter (NIS) in prostate cancer models, combining oncolysis, external beam radiotherapy and NIS-mediated radioiodide therapy. The NIS-expressing vaccinia virus (VV-NIS), GLV-1h153, was tested in in vitro analyzes of viral cell killing, combination with radiotherapy, NIS expression, cellular radioiodide uptake and apoptotic cell death in PC3, DU145, LNCaP and WPMY-1 human prostate cell lines. In vivo experiments were carried out in PC3 xenografts in CD1 nude mice to assess NIS expression and tumor radioiodide uptake. In addition, the therapeutic benefit of radioiodide treatment in combination with viral oncolysis and external beam radiotherapy was measured. In vitro viral cell killing of prostate cancers was dose- and time-dependent and was through apoptotic mechanisms. Importantly, combined virus therapy and iodizing radiation did not adversely affect oncolysis. NIS gene expression in infected cells was functional and mediated uptake of radioiodide both in vitro and in vivo. Therapy experiments with both xenograft and immunocompetent Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse models showed that the addition of radioiodide to VV-NIS-infected tumors was more effective than each single-agent therapy, restricting tumor growth and increasing survival. In conclusion, VV-NIS is effective in prostate cancer models. This treatment modality would be an attractive complement to existing clinical radiotherapy practice.

  10. Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer

    PubMed Central

    Mansfield, D C; Kyula, J N; Rosenfelder, N; Chao-Chu, J; Kramer-Marek, G; Khan, A A; Roulstone, V; McLaughlin, M; Melcher, A A; Vile, R G; Pandha, H S; Khoo, V; Harrington, K J

    2016-01-01

    Oncolytic strains of vaccinia virus are currently in clinical development with clear evidence of safety and promising signs of efficacy. Addition of therapeutic genes to the viral genome may increase the therapeutic efficacy of vaccinia. We evaluated the therapeutic potential of vaccinia virus expressing the sodium iodide symporter (NIS) in prostate cancer models, combining oncolysis, external beam radiotherapy and NIS-mediated radioiodide therapy. The NIS-expressing vaccinia virus (VV-NIS), GLV-1h153, was tested in in vitro analyzes of viral cell killing, combination with radiotherapy, NIS expression, cellular radioiodide uptake and apoptotic cell death in PC3, DU145, LNCaP and WPMY-1 human prostate cell lines. In vivo experiments were carried out in PC3 xenografts in CD1 nude mice to assess NIS expression and tumor radioiodide uptake. In addition, the therapeutic benefit of radioiodide treatment in combination with viral oncolysis and external beam radiotherapy was measured. In vitro viral cell killing of prostate cancers was dose- and time-dependent and was through apoptotic mechanisms. Importantly, combined virus therapy and iodizing radiation did not adversely affect oncolysis. NIS gene expression in infected cells was functional and mediated uptake of radioiodide both in vitro and in vivo. Therapy experiments with both xenograft and immunocompetent Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse models showed that the addition of radioiodide to VV-NIS-infected tumors was more effective than each single-agent therapy, restricting tumor growth and increasing survival. In conclusion, VV-NIS is effective in prostate cancer models. This treatment modality would be an attractive complement to existing clinical radiotherapy practice. PMID:26814609

  11. Engineering of double recombinant vaccinia virus with enhanced oncolytic potential for solid tumor virotherapy

    PubMed Central

    Kochneva, Galina; Sivolobova, Galina; Tkacheva, Anastasiya; Grazhdantseva, Antonina; Troitskaya, Olga; Nushtaeva, Anna; Tkachenko, Anastasiya; Kuligina, Elena; Richter, Vladimir; Koval, Olga

    2016-01-01

    Vaccinia virus (VACV) oncolytic therapy has been successful in a number of tumor models. In this study our goal was to generate a double recombinant vaccinia virus (VV-GMCSF-Lact) with enhanced antitumor activity that expresses exogenous proteins: the antitumor protein lactaptin and human granulocyte-macrophage colony-stimulating factor (GM-CSF). Lactaptin has previously been demonstrated to act as a tumor suppressor in mouse hepatoma as well as MDA-MB-231 human adenocarcinoma cells grafted into SCID mice. VV-GMCSF-Lact was engineered from Lister strain (L-IVP) vaccinia virus and has deletions of the viral thymidine kinase and vaccinia growth factor genes. Cell culture experiments revealed that engineered VV-GMCSF-Lact induced the death of cultured cancer cells more efficiently than recombinant VACV coding only GM-CSF (VV-GMCSF-dGF). Normal human MCF-10A cells were resistant to both recombinants up to 10 PFU/cell. The selectivity index for breast cancer cells measured in pair cultures MCF-7/MCF-10A was 200 for recombinant VV-GMCSF-Lact coding lactaptin and 100 for VV-GMCSF-dGF. Using flow cytometry we demonstrated that both recombinants induced apoptosis in treated cells but that the rate in the cells with active caspase −3 and −7 was higher after treatment with VV-GMCSF-Lact than with VV-GMCSF-dGF. Tumor growth inhibition and survival outcomes after VV-GMCSF-Lact treatment were estimated using immunodeficient and immunocompetent mice models. We observed that VV-GMCSF-Lact efficiently delays the growth of sensitive and chemoresistant tumors. These results demonstrate that recombinant VACVs coding an apoptosis-inducing protein have good therapeutic potential against chemoresistant tumors. Our data will also stimulate further investigation of coding lactaptin double recombinant VACV in clinical settings. PMID:27708236

  12. Oncolytic virotherapy for ovarian carcinomatosis using a replication-selective vaccinia virus armed with a yeast cytosine deaminase gene.

    PubMed

    Chalikonda, S; Kivlen, M H; O'Malley, M E; Eric Dong, X D; McCart, J A; Gorry, M C; Yin, X-Y; Brown, C K; Zeh, H J; Guo, Z S; Bartlett, D L

    2008-02-01

    In this study, we assessed the ability of a highly tumor-selective oncolytic vaccinia virus armed with a yeast cytosine deaminase gene to infect and lyse human and murine ovarian tumors both in vitro and in vivo. The virus vvDD-CD could infect, replicate in and effectively lyse both human and mouse ovarian cancer cells in vitro. In two different treatment schedules involving either murine MOSEC or human A2780 ovarian carcinomatosis models, regional delivery of vvDD-CD selectively targeted tumor cells and ovarian tissue, effectively delaying the development of either tumor or ascites and leading to significant survival advantages. Oncolytic virotherapy using vvDD-CD in combination with the prodrug 5-fluorocytosine conferred an additional long-term survival advantage upon tumor-bearing immunocompetent mice. These findings demonstrate that a tumor-selective oncolytic vaccinia combined with gene-directed enzyme prodrug therapy is a highly effective strategy for treating advanced ovarian cancers in both syngeneic mouse and human xenograft models. Given the biological safety, tumor selectivity and oncolytic potency of this armed oncolytic virus, this dual therapy merits further investigation as a promising new treatment for metastatic ovarian cancer.

  13. Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy

    PubMed Central

    Weibel, Stephanie; Langbein-Laugwitz, Johanna; Härtl, Barbara; Escobar, Hugo Murua; Nolte, Ingo; Chen, Nanhai G.; Aguilar, Richard J.; Yu, Yong A.; Zhang, Qian; Frentzen, Alexa; Szalay, Aladar A.

    2014-01-01

    Virotherapy on the basis of oncolytic vaccinia virus (VACV) infection is a promising approach for cancer therapy. In this study we describe the establishment of a new preclinical model of feline mammary carcinoma (FMC) using a recently established cancer cell line, DT09/06. In addition, we evaluated a recombinant vaccinia virus strain, GLV-5b451, expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as an oncolytic agent against FMC. Cell culture data demonstrate that GLV-5b451 virus efficiently infected, replicated in and destroyed DT09/06 cancer cells. In the selected xenografts of FMC, a single systemic administration of GLV-5b451 led to significant inhibition of tumor growth in comparison to untreated tumor-bearing mice. Furthermore, tumor-specific virus infection led to overproduction of functional scAb GLAF-2, which caused drastic reduction of intratumoral VEGF levels and inhibition of angiogenesis. In summary, here we have shown, for the first time, that the vaccinia virus strains and especially GLV-5b451 have great potential for effective treatment of FMC in animal model. PMID:25093734

  14. Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory Beagles

    PubMed Central

    Autio, Karoliina; Knuuttila, Anna; Kipar, Anja; Pesonen, Sari; Guse, Kilian; Parviainen, Suvi; Rajamäki, Minna; Laitinen-Vapaavuori, Outi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

    2014-01-01

    We evaluated adverse events, biodistribution and shedding of oncolytic vaccinia virus encoding CD40 ligand in two Beagles, in preparation for a phase 1 trial in canine cancer patients. Dog 1 received one dose of vaccinia virus and was euthanized 24 hours afterwards, while dog 2 received virus four times once weekly and was euthanized 7 days after that. Dogs were monitored for adverse events and underwent a detailed postmortem examination. Blood, saliva, urine, feces, and organs were collected for virus detection. Dog 1 had mild fever and lethargy while dog 2 experienced a possible seizure 5.5 hours after first virus administration. Viral DNA declined quickly in the blood after virus administration in both dogs but was still detectable 1 week later by quantitative polymerase chain reaction. Only samples taken directly after virus infusion contained infectious virus. Small amounts of viral DNA, but no infectious virus, were detected in a few saliva and urine samples. Necropsies did not reveal any relevant pathological changes and virus DNA was detected mainly in the spleen. The dogs in the study did not have cancer, and thus adverse events could be more common and viral load higher in dogs with tumors which allow viral amplification. PMID:27119092

  15. Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

    PubMed Central

    Liu, Zuqiang; Ravindranathan, Roshni; Kalinski, Pawel; Guo, Z. Sheng; Bartlett, David L.

    2017-01-01

    Both anti-PD1/PD-L1 therapy and oncolytic virotherapy have demonstrated promise, yet have exhibited efficacy in only a small fraction of cancer patients. Here we hypothesized that an oncolytic poxvirus would attract T cells into the tumour, and induce PD-L1 expression in cancer and immune cells, leading to more susceptible targets for anti-PD-L1 immunotherapy. Our results demonstrate in colon and ovarian cancer models that an oncolytic vaccinia virus attracts effector T cells and induces PD-L1 expression on both cancer and immune cells in the tumour. The dual therapy reduces PD-L1+ cells and facilitates non-redundant tumour infiltration of effector CD8+, CD4+ T cells, with increased IFN-γ, ICOS, granzyme B and perforin expression. Furthermore, the treatment reduces the virus-induced PD-L1+ DC, MDSC, TAM and Treg, as well as co-inhibitory molecules-double-positive, severely exhausted PD-1+CD8+ T cells, leading to reduced tumour burden and improved survival. This combinatorial therapy may be applicable to a much wider population of cancer patients. PMID:28345650

  16. Replication efficiency of oncolytic vaccinia virus in cell cultures prognosticates the virulence and antitumor efficacy in mice

    PubMed Central

    2011-01-01

    Background We have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding Renilla luciferase-Aequorea GFP fusion protein, β-galactosidase, and β-glucuronidase into the F14.5L, J2R, and A56R loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV-1h68. This strain shows tumor-specific replication and is capable of eradicating tumors with little or no virulence in mice. This study aimed to distinguish the contribution of added VACV promoter-driven transcriptional units as inserts from the effects of insertional inactivation of three viral genes, and to determine the correlation between replication efficiency of oncolytic vaccinia virus in cell cultures and the virulence and antitumor efficacy in mice Methods A series of recombinant VACV strains was generated by replacing one, two, or all three of the expression cassettes in GLV-1h68 with short non-coding DNA sequences. The replication efficiency and tumor cell killing capacity of these newly generated VACV strains were compared with those of the parent virus GLV-1h68 in cell cultures. The virus replication efficiency in tumors and antitumor efficacy as well as the virulence were evaluated in nu/nu (nude) mice bearing human breast tumor xenografts. Results we found that virus replication efficiency increased with removal of each of the expression cassettes. The increase in virus replication efficiency was proportionate to the strength of removed VACV promoters linked to foreign genes. The replication efficiency of the new VACV strains paralleled their cytotoxicity in cell cultures. The increased replication efficiency in tumor xenografts resulted in enhanced antitumor efficacy in nude mice. Similarly, the enhanced virus replication efficiency was indicative of increased virulence in nude mice. Conclusions These data demonstrated that insertion of VACV promoter-driven transcriptional units into the viral genome for the

  17. Phase 1 Study of Intratumoral Pexa-Vec (JX-594), an Oncolytic and Immunotherapeutic Vaccinia Virus, in Pediatric Cancer Patients

    PubMed Central

    Cripe, Timothy P; Ngo, Minhtran C; Geller, James I; Louis, Chrystal U; Currier, Mark A; Racadio, John M; Towbin, Alexander J; Rooney, Cliona M; Pelusio, Adina; Moon, Anne; Hwang, Tae-Ho; Burke, James M; Bell, John C; Kirn, David H; Breitbach, Caroline J

    2015-01-01

    Pexa-Vec (pexastimogene devacirepvec, JX-594) is an oncolytic and immunotherapeutic vaccinia virus designed to destroy cancer cells through viral lysis and induction of granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven tumor-specific immunity. Pexa-Vec has undergone phase 1 and 2 testing alone and in combination with other therapies in adult patients, via both intratumoral and intravenous administration routes. We sought to determine the safety of intratumoral administration in pediatric patients. In a dose-escalation study using either 106 or 107 plaque-forming units per kilogram, we performed one-time injections in up to three tumor sites in five pediatric patients and two injections in one patient. Ages at study entry ranged from 4 to 21 years, and their cancer diagnoses included neuroblastoma, hepatocellular carcinoma, and Ewing sarcoma. All toxicities were ≤ grade 3. The most common side effects were sinus fever and sinus tachycardia. All three patients at the higher dose developed asymptomatic grade 1 treatment-related skin pustules that resolved within 3–4 weeks. One patient showed imaging evidence suggestive of antitumor biological activity. The two patients tested for cellular immunoreactivity to vaccinia antigens showed strong responses. Overall, our study suggests Pexa-Vec is safe to administer to pediatric patients by intratumoral administration and could be studied further in this patient population. PMID:25531693

  18. Combination treatment with oncolytic Vaccinia virus and cyclophosphamide results in synergistic antitumor effects in human lung adenocarcinoma bearing mice

    PubMed Central

    2014-01-01

    Background The capacity of the recombinant Vaccinia virus GLV-1h68 as a single agent to efficiently treat different human or canine cancers has been shown in several preclinical studies. Currently, its human safety and efficacy are investigated in phase I/II clinical trials. In this study we set out to evaluate the oncolytic activity of GLV-1h68 in the human lung adenocarcinoma cell line PC14PE6-RFP in cell cultures and analyzed the antitumor potency of a combined treatment strategy consisting of GLV-1h68 and cyclophosphamide (CPA) in a mouse model of PC14PE6-RFP lung adenocarcinoma. Methods PC14PE6-RFP cells were treated in cell culture with GLV-1h68. Viral replication and cell survival were determined by plaque assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Subcutaneously implanted PC14PE6-RFP xenografts were treated by systemic injection of GLV-1h68, CPA or a combination of both. Tumor growth and viral biodistribution were monitored and immune-related antigen profiling of tumor lysates was performed. Results GLV-1h68 efficiently infected, replicated in and lysed human PC14PE6-RFP cells in cell cultures. PC14PE6-RFP tumors were efficiently colonized by GLV-1h68 leading to much delayed tumor growth in PC14PE6-RFP tumor-bearing nude mice. Combination treatment with GLV-1h68 and CPA significantly improved the antitumor efficacy of GLV-1h68 and led to an increased viral distribution within the tumors. Pro-inflammatory cytokines and chemokines were distinctly elevated in tumors of GLV-1h68-treated mice. Factors expressed by endothelial cells or present in the blood were decreased after combination treatment. A complete loss in the hemorrhagic phenotype of the PC14PE6-RFP tumors and a decrease in the number of blood vessels after combination treatment could be observed. Conclusions CPA and GLV-1h68 have synergistic antitumor effects on PC14PE6-RFP xenografts. We strongly suppose that in the PC14PE6-RFP model the

  19. Isolated limb perfusion with melphalan, tumour necrosis factor-alpha and oncolytic vaccinia virus improves tumour targeting and prolongs survival in a rat model of advanced extremity sarcoma.

    PubMed

    Pencavel, Tim D; Wilkinson, Michelle J; Mansfield, David C; Khan, Aadil A; Seth, Rohit; Karapanagiotou, Eleni M; Roulstone, Victoria; Aguilar, Richard J; Chen, Nanhai G; Szalay, Aladar A; Hayes, Andrew J; Harrington, Kevin J

    2015-02-15

    Isolated limb perfusion (ILP) is a treatment for advanced extremity sarcoma and in-transit melanoma. Advancing this procedure by investigating the addition of novel agents, such as cancer-selective oncolytic viruses, may improve both the therapeutic efficacy of ILP and the tumour-targeted delivery of oncolytic virotherapy. Standard in vitro assays were used to characterise single agent and combinatorial activities of melphalan, tumour necrosis factor-alpha (TNF-α) and Lister strain vaccinia virus (GLV-1h68) against BN175 rat sarcoma cells. An orthotopic model of advanced extremity sarcoma was used to evaluate survival of animals after ILP with combinations of TNF-α, melphalan and GLV-1h68. We investigated the efficiency of viral tumour delivery by ILP compared to intravenous therapy, the locoregional and systemic biodistribution of virus after ILP, and the effect of mode of administration on antibody response. The combination of melphalan and GLV-1h68 was synergistic in vitro. The addition of virus to standard ILP regimens was well tolerated and demonstrated superior tumour targeting compared to intravenous administration. Triple therapy (melphalan/TNF-α/GLV-1h68) resulted in increased tumour growth delay and enhanced survival compared to other treatment regimens. Live virus was recovered in large amounts from perfused regions, but in smaller amounts from systemic organs. The addition of oncolytic vaccinia virus to existing TNF-α/melphalan-based ILP strategies results in survival advantage in an immunocompetent rat model of advanced extremity sarcoma. Virus administered by ILP has superior tumour targeting compared to intravenous delivery. Further evaluation and clinical translation of this approach is warranted.

  20. Oncolytic virus therapy for cancer

    PubMed Central

    Goldufsky, Joe; Sivendran, Shanthi; Harcharik, Sara; Pan, Michael; Bernardo, Sebastian; Stern, Richard H; Friedlander, Philip; Ruby, Carl E; Saenger, Yvonne; Kaufman, Howard L

    2013-01-01

    The use of oncolytic viruses to treat cancer is based on the selection of tropic tumor viruses or the generation of replication selective vectors that can either directly kill infected tumor cells or increase their susceptibility to cell death and apoptosis through additional exposure to radiation or chemotherapy. In addition, viral vectors can be modified to promote more potent tumor cell death, improve the toxicity profile, and/or generate host antitumor immunity. A variety of viruses have been developed as oncolytic therapeutics, including adenovirus, vaccinia virus, herpesvirus, coxsackie A virus, Newcastle disease virus, and reovirus. The clinical development of oncolytic viral therapy has accelerated in the last few years, with several vectors entering clinical trials for a variety of cancers. In this review, current strategies to optimize the therapeutic effectiveness and safety of the major oncolytic viruses are discussed, and a summary of current clinical trials is provided. Further investigation is needed to characterize better the clinical impact of oncolytic viruses, but there are increasing data demonstrating the potential promise of this approach for the treatment of human and animal cancers. PMID:27512656

  1. Oncolytic virotherapy with an armed vaccinia virus in an orthotopic model of renal carcinoma is associated with modification of the tumor microenvironment.

    PubMed

    Fend, Laetitia; Remy-Ziller, Christelle; Foloppe, Johann; Kempf, Juliette; Cochin, Sandrine; Barraud, Luc; Accart, Nathalie; Erbs, Philippe; Fournel, Sylvie; Préville, Xavier

    2016-02-01

    Oncolytic virotherapy is an emergent promising therapeutic approach for the treatment of cancer. We have constructed a vaccinia virus (WR strain) deleted for thymidine kinase (TK) and ribonucleotide reductase (RR) genes that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. We evaluated this construct (VV-FCU1) in the orthotopic model of renal carcinoma (RenCa). Systemic administration of VV-FCU1 resulted in orthotopic tumor growth inhibition, despite temporary expression of viral proteins. VV-FCU1 treatment was associated with an infiltration of tumors by CD8(+) T lymphocytes and a decrease in the proportion of infiltrating Tregs, thus modifying the ratio of CD8(+)/CD4(+) Treg in favor of CD8(+)cytotoxic T cells. We demonstrated that VV-FCU1 treatment prolonged survival of animals implanted with RenCa cells in kidney. Depletion of CD8(+) T cells abolished the therapeutic effect of VV-FCU1 while depletion of CD4(+) T cells enhanced its protective activity. Administration of the prodrug 5-fluorocytosine (5-FC) resulted in a sustained control of tumor growth but did not extend survival. This study shows the importance of CD4(+) and CD8(+) T cells in vaccinia virus-mediated oncolytic virotherapy and suggests that this approach may be evaluated for the treatment of human renal cell carcinoma.

  2. Oncolytic vaccinia virus combined with radiotherapy induces apoptotic cell death in sarcoma cells by down-regulating the inhibitors of apoptosis

    PubMed Central

    Wilkinson, Michelle J.; Smith, Henry G.; McEntee, Gráinne; Kyula-Currie, Joan; Mansfield, David C.; Khan, Aadil A.; Roulstone, Victoria

    2016-01-01

    Advanced extremity melanoma and sarcoma present a significant therapeutic challenge, requiring multimodality therapy to treat or even palliate disease. These aggressive tumours are relatively chemo-resistant, therefore new treatment approaches are urgently required. We have previously reported on the efficacy of oncolytic virotherapy (OV) delivered by isolated limb perfusion. In this report, we have improved therapeutic outcomes by combining OV with radiotherapy. In vitro, the combination of oncolytic vaccinia virus (GLV-1h68) and radiotherapy demonstrated synergistic cytotoxicity. This effect was not due to increased viral replication, but mediated through induction of intrinsic apoptosis. GLV-1h68 therapy downregulated the anti-apoptotic BCL-2 proteins (MCL-1 and BCL-XL) and the downstream inhibitors of apoptosis, resulting in cleavage of effector caspases 3 and 7. In an in vivo ILP model, the combination of OV and radiotherapy significantly delayed tumour growth and prolonged survival compared to single agent therapy. These data suggest that the virally-mediated down-regulation of anti-apoptotic proteins may increase the sensitivity of tumour cells to the cytotoxic effects of ionizing radiation. Oncolytic virotherapy represents an exciting candidate for clinical development when delivered by ILP. Its ability to overcome anti-apoptotic signals within tumour cells points the way to further development in combination with conventional anti-cancer therapies. PMID:27783991

  3. Viral warfare! Front-line defence and arming the immune system against cancer using oncolytic vaccinia and other viruses.

    PubMed

    Dave, R V; Jebar, A H S; Jennings, V A; Adair, R A; West, E J; Errington-Mais, F; Toogood, G J; Melcher, A A

    2014-08-01

    Despite mankind's many achievements, we are yet to find a cure for cancer. We are now approaching a new era which recognises the promise of harnessing the immune system for anti-cancer therapy. Pathogens have been implicated for decades as potential anti-cancer agents, but implementation into clinical therapy has been plagued with significant drawbacks. Newer 'designer' agents have addressed some of these concerns, in particular, a new breed of oncolytic virus: JX-594, a genetically engineered pox virus, is showing promise. To review the current literature on the use of oncolytic viruses in the treatment of cancer; both by direct oncolysis and stimulation of the immune system. The review will provide a background and historical progression for the surgeon on tumour immunology, and the interplay between oncolytic viruses, immune cells, inflammation on tumourigenesis. A literature review was performed using the Medline database. Viral therapeutics hold promise as a novel treatment modality for the treatment of disseminated malignancy. It provides a multi-pronged attack against tumour burden; direct tumour cell lysis, exposure of tumour-associated antigens (TAA), induction of immune danger signals, and recognition by immune effector cells. Copyright © 2014 Royal College of Surgeons of Edinburgh (Scottish charity number SC005317) and Royal College of Surgeons in Ireland. Published by Elsevier Ltd. All rights reserved.

  4. Vaccinia virus, a promising new therapeutic agent for pancreatic cancer.

    PubMed

    Al Yaghchi, Chadwan; Zhang, Zhongxian; Alusi, Ghassan; Lemoine, Nicholas R; Wang, Yaohe

    2015-01-01

    The poor prognosis of pancreatic cancer patients signifies a need for radically new therapeutic strategies. Tumor-targeted oncolytic viruses have emerged as attractive therapeutic candidates for cancer treatment due to their inherent ability to specifically target and lyse tumor cells as well as induce antitumor effects by multiple action mechanisms. Vaccinia virus has several inherent features that make it particularly suitable for use as an oncolytic agent. In this review, we will discuss the potential of vaccinia virus in the management of pancreatic cancer in light of our increased understanding of cellular and immunological mechanisms involved in the disease process as well as our extending knowledge in the biology of vaccinia virus.

  5. Preclinical Testing Oncolytic Vaccinia Virus Strain GLV-5b451 Expressing an Anti-VEGF Single-Chain Antibody for Canine Cancer Therapy.

    PubMed

    Adelfinger, Marion; Bessler, Simon; Frentzen, Alexa; Cecil, Alexander; Langbein-Laugwitz, Johanna; Gentschev, Ivaylo; Szalay, Aladar A

    2015-07-20

    Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for canine cancer therapy. Here we describe, for the first time, the characterization and the use of VACV strain GLV-5b451 expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as therapeutic agent against different canine cancers. Cell culture data demonstrated that GLV-5b451 efficiently infected and destroyed all four tested canine cancer cell lines including: mammary carcinoma (MTH52c), mammary adenoma (ZMTH3), prostate carcinoma (CT1258), and soft tissue sarcoma (STSA-1). The GLV-5b451 virus-mediated production of GLAF-2 antibody was observed in all four cancer cell lines. In addition, this antibody specifically recognized canine VEGF. Finally, in canine soft tissue sarcoma (CSTS) xenografted mice, a single systemic administration of GLV-5b451 was found to be safe and led to anti-tumor effects resulting in the significant reduction and substantial long-term inhibition of tumor growth. A CD31-based immuno-staining showed significantly decreased neo-angiogenesis in GLV-5b451-treated tumors compared to the controls. In summary, these findings indicate that GLV-5b451 has potential for use as a therapeutic agent in the treatment of CSTS.

  6. Preclinical Testing Oncolytic Vaccinia Virus Strain GLV-5b451 Expressing an Anti-VEGF Single-Chain Antibody for Canine Cancer Therapy

    PubMed Central

    Adelfinger, Marion; Bessler, Simon; Frentzen, Alexa; Cecil, Alexander; Langbein-Laugwitz, Johanna; Gentschev, Ivaylo; Szalay, Aladar A.

    2015-01-01

    Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for canine cancer therapy. Here we describe, for the first time, the characterization and the use of VACV strain GLV-5b451 expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as therapeutic agent against different canine cancers. Cell culture data demonstrated that GLV-5b451 efficiently infected and destroyed all four tested canine cancer cell lines including: mammary carcinoma (MTH52c), mammary adenoma (ZMTH3), prostate carcinoma (CT1258), and soft tissue sarcoma (STSA-1). The GLV-5b451 virus-mediated production of GLAF-2 antibody was observed in all four cancer cell lines. In addition, this antibody specifically recognized canine VEGF. Finally, in canine soft tissue sarcoma (CSTS) xenografted mice, a single systemic administration of GLV-5b451 was found to be safe and led to anti-tumor effects resulting in the significant reduction and substantial long-term inhibition of tumor growth. A CD31-based immuno-staining showed significantly decreased neo-angiogenesis in GLV-5b451-treated tumors compared to the controls. In summary, these findings indicate that GLV-5b451 has potential for use as a therapeutic agent in the treatment of CSTS. PMID:26205404

  7. Phase I Trial of Intravenous Oncolytic Vaccinia Virus (GL-ONC1) with Cisplatin and Radiotherapy in Patients with Locoregionally Advanced Head and Neck Carcinoma.

    PubMed

    Mell, Loren K; Brumund, Kevin T; Daniels, Gregory A; Advani, Sunil J; Zakeri, Kaveh; Wright, Mary E; Onyeama, Sara-Jane; Weisman, Robert A; Sanghvi, Parag R; Martin, Peter J; Szalay, Aladar A

    2017-10-01

    Purpose: Preclinical models have shown that the effectiveness of GL-ONC1, a modified oncolytic vaccinia virus, is enhanced by radiation and chemotherapy. The purpose of this study was to determine the safety of GL-ONC1 when delivered intravenously with chemoradiotherapy to patients with primary, nonmetastatic head and neck cancer.Experimental Design: Patients with locoregionally advanced unresected, nonmetastatic carcinoma of the head/neck, excluding stage III-IVA p16-positive oropharyngeal cancers, were treated with escalating doses and cycles of intravenous GL-ONC1, along with radiotherapy and chemotherapy. The primary aims were to define the MTD and dose-limiting toxicities, and to recommend a dose for phase II trials.Results: Between May 2012 and December 2014, 19 patients were enrolled. The most frequent adverse reactions included grade 1-2 rigors, fever, fatigue, and rash. Grade 3 adverse reactions included hypotension, mucositis, nausea, and vomiting. In 2 patients, the rash was confirmed as viral in origin by fluorescence imaging and viral plaque assay. In 4 patients, viral presence in tumor was confirmed on midtreatment biopsy by quantitative PCR. In 1 patient, live virus was confirmed in a tongue tumor 7 days after receiving the first dose of virus. The MTD was not reached. With median follow-up of 30 months, 1-year (2-year) progression-free survival and overall survival were 74.4% (64.1%) and 84.6% (69.2%), respectively.Conclusions: Delivery of GL-ONC1 is safe and feasible in patients with locoregionally advanced head/neck cancer undergoing standard chemoradiotherapy. A phase II study is warranted to further investigate this novel treatment strategy. Clin Cancer Res; 23(19); 5696-702. ©2017 AACR. ©2017 American Association for Cancer Research.

  8. The mechanisms of genetically modified vaccinia viruses for the treatment of cancer.

    PubMed

    Jefferson, Artrish; Cadet, Valerie E; Hielscher, Abigail

    2015-09-01

    The use of oncolytic viruses for the treatment of cancer is an emerging field of cancer research and therapy. Oncolytic viruses are designed to induce tumor specific immunity while replicating selectively within cancer cells to cause lysis of the tumor cells. While there are several forms of oncolytic viruses, the use of vaccinia viruses for oncolysis may be more beneficial than other forms of oncolytic viruses. For example, vaccinia viruses have been shown to exert their anti-tumor effects through genetic engineering strategies which enhance their therapeutic efficacy. This paper will address some of the most common forms of genetically modified vaccinia viruses and will explore the mechanisms whereby they selectively target, enter and destroy cancer cells. Furthermore, this review will highlight how vaccinia viruses activate host immune responses against cancer cells and will address clinical trials evaluating the tumor-directed and killing efficacy of these viruses against solid tumors.

  9. Evolution of oncolytic viruses.

    PubMed

    Sanjuán, Rafael; Grdzelishvili, Valery Z

    2015-08-01

    Owing to their replicative capacity, oncolytic viruses (OVs) can evolve under the action of natural selection. Reversion to virulence and recombination with wild-type strains may compromise OV safety, therefore requiring evolutionary risk assessment studies. On the other hand, evolution can be directed in the laboratory to create more potent and safer OVs. Previous work in the experimental evolution field provides a background for OV directed evolution, and has identified interesting exploitable features. While genetic engineering has greatly advanced the field of oncolytic virotherapy, this approach is sometimes curtailed by the complexity and diversity of virus-host interactions. Directed evolution provides an alternative approach that may help to obtain new OVs without prejudice toward the underlying molecular mechanisms involved. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition

    PubMed Central

    Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

    2016-01-01

    ABSTRACT We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8+ and CD4+). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs. PMID:27853644

  11. Group 2 Vaccinia Virus, Brazil

    PubMed Central

    Assis, Felipe Lopes; Borges, Iara Apolinario; Ferreira, Paulo César Peregrino; Bonjardim, Cláudio Antônio; Trindade, Giliane de Souza; Lobato, Zélia Inês Portela; Guedes, Maria Isabel Maldonado; Mesquita, Vaz; Kroon, Erna Geessien

    2012-01-01

    In 2011, vaccinia virus caused an outbreak of bovine vaccinia, affecting dairy cattle and dairy workers in Brazil. Genetic and phenotypic analyses identified this isolate as distinct from others recently identified, thereby reinforcing the hypothesis that different vaccinia virus strains co-circulate in Brazil. PMID:23171598

  12. Vaccinia virus, a promising new therapeutic agent for pancreatic cancer

    PubMed Central

    Yaghchi, Chadwan Al; Zhang, Zhongxian; Alusi, Ghassan; Lemoine, Nicholas R; Wang, Yaohe

    2015-01-01

    The poor prognosis of pancreatic cancer patients signifies a need for radically new therapeutic strategies. Tumor-targeted oncolytic viruses have emerged as attractive therapeutic candidates for cancer treatment due to their inherent ability to specifically target and lyse tumor cells as well as induce antitumor effects by multiple action mechanisms. Vaccinia virus has several inherent features that make it particularly suitable for use as an oncolytic agent. In this review, we will discuss the potential of vaccinia virus in the management of pancreatic cancer in light of our increased understanding of cellular and immunological mechanisms involved in the disease process as well as our extending knowledge in the biology of vaccinia virus. PMID:26595180

  13. Phase 1b Trial of Biweekly Intravenous Pexa-Vec (JX-594), an Oncolytic and Immunotherapeutic Vaccinia Virus in Colorectal Cancer

    PubMed Central

    Park, Se Hoon; Breitbach, Caroline J; Lee, Jeeyun; Park, Joon Oh; Lim, Ho Yeong; Kang, Won Ki; Moon, Anne; Mun, Jae-Hee; Sommermann, Erica M; Maruri Avidal, Liliana; Patt, Rick; Pelusio, Adina; Burke, James; Hwang, Tae-Ho; Kirn, David; Park, Young Suk

    2015-01-01

    Fifteen patients with treatment-refractory colorectal cancer were enrolled on a phase 1b study of Pexa-Vec (pexastimogene devacirepvec; JX-594), an oncolytic and immunotherapeutic vaccinia designed to selectively replicate in cancer cells. Pexa-Vec was administered intravenously every 14 days, at dose levels of 1 × 106, 1 × 107, or 3 × 107 plaque-forming units (pfu)/kg. The primary endpoint was to determine the maximum tolerated dose. Secondary endpoints were pharmacokinetics and pharmacodynamics as well as antitumor activity. Patients were heavily pretreated (mean 4.5 lines of therapy). All patients received at least two Pexa-Vec doses (median = 4; range = 2–4). No dose-limiting toxicities were reported, and the maximum tolerated dose was not reached. The most common adverse events were grade 1/2 flu-like symptoms, generally lasting <24 hours. During the first and last cycles, genome pharmacokinetics were unchanged. Infectious pfu could be detected in plasma up to 2 hours after cycle 1 and up to 30 minutes after cycle 4 (when antivaccinia antibody titers are known to have peaked). Ten patients (67%) had radiographically stable disease. Given the acceptable safety profile of multiple intravenous Pexa-Vec infusions in patients with treatment-refractory colorectal cancer, further trials evaluating efficacy of intravenous Pexa-Vec, as monotherapy or in combination with chemotherapeutic agents, is warranted in this patient population. PMID:26073886

  14. Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

    NASA Astrophysics Data System (ADS)

    Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

    1985-09-01

    The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

  15. Designing herpes viruses as oncolytics

    PubMed Central

    Peters, Cole; Rabkin, Samuel D

    2015-01-01

    Oncolytic herpes simplex virus (oHSV) was one of the first genetically-engineered oncolytic viruses. Because HSV is a natural human pathogen that can cause serious disease, it is incumbent that it can be genetically-engineered or significantly attenuated for safety. Here, we present a detailed explanation of the functions of HSV-1 genes frequently mutated to endow oncolytic activity. These genes are nonessential for growth in tissue culture cells but are important for growth in postmitotic cells, interfering with intrinsic antiviral and innate immune responses or causing pathology, functions dispensable for replication in cancer cells. Understanding the function of these genes leads to informed creation of new oHSVs with better therapeutic efficacy. Virus infection and replication can also be directed to cancer cells through tumor-selective receptor binding and transcriptional- or post-transcriptional miRNA-targeting, respectively. In addition to the direct effects of oHSV on infected cancer cells and tumors, oHSV can be “armed” with transgenes that are: reporters, to track virus replication and spread; cytotoxic, to kill uninfected tumor cells; immune modulatory, to stimulate antitumor immunity; or tumor microenvironment altering, to enhance virus spread or to inhibit tumor growth. In addition to HSV-1, other alphaherpesviruses are also discussed for their oncolytic activity. PMID:26462293

  16. Current status of clinical trials assessing oncolytic virus therapy for urological cancers.

    PubMed

    Taguchi, Satoru; Fukuhara, Hiroshi; Homma, Yukio; Todo, Tomoki

    2017-03-21

    Oncolytic virus therapy has recently been recognized as a promising new option for cancer treatment. Oncolytic viruses replicate selectively in cancer cells, thus killing them without harming normal cells. Notably, T-VEC (talimogene laherparepvec, formerly called OncoVEX(GM)(-)(CSF) ), an oncolytic herpes simplex virus type 1, was approved by the US Food and Drug Administration for the treatment of inoperable melanoma in October 2015, and was subsequently approved in Europe and Australia in 2016. The efficacies of many types of oncolytic viruses against urological cancers have been investigated in preclinical studies during the past decade, and some have already been tested in clinical trials. For example, a phase I trial of the third-generation oncolytic Herpes simplex virus type 1, G47Δ, in patients with prostate cancer was completed in 2016. We summarize the current status of clinical trials of oncolytic virus therapy in patients with the three major urological cancers: prostate, bladder and renal cell cancers. In addition to Herpes simplex virus type 1, adenoviruses, reoviruses, vaccinia virus, Sendai virus and Newcastle disease virus have also been used as parental viruses in these trials. We believe that oncolytic virus therapy is likely to become an important and major treatment option for urological cancers in the near future.

  17. Virus, Oncolytic virus and Human Prostate Cancer.

    PubMed

    Liu, Guang Bin; Zhao, Liang; Zhang, Lifang; Zhao, Kong-Nan

    2016-12-15

    Prostate cancer (PCa), a disease, is characterized by abnormal cell growth in the prostate - a gland in the male reproductive system. PCa is one of the leading causes of cancer death among men of all races. Although older age and a family history of the disease have been recognized as the risk factors of PCa, the cause of this cancer remains unclear. Mounting evidence suggests that infections with various viruses are causally linked to PCa pathogenesis. Published studies have provided strong evidence that at least two viruses (RXMV and HPV) contribute to prostate tumourigenicity and impact on the survival of patients with malignant PCa. Traditional therapies including chemotherapy and radiotherapy are unable to distinguish cancer cells from normal cells, which are a significant drawback and leads to toxicities for PCa patients undergoing treatment. So far, few other options are available for treating patients with advanced PCa. Virotherapy is being developed to be a novel therapy for cancers, which uses oncotropic and oncolytic viruses with their abilities to find and destroy malignant cells in the body. For PCa treatment, oncolytic virotherapy appears to be much more attractive, which uses live viruses to selectively kill cancer cells. Oncolytic viruses can be genetically engineered to induce cancer cell lysis through virus replication and expression of cytotoxic proteins. As oncolytic viruses are a relatively new class of anti-cancer immunotherapy agents, several important barriers still exist on the road to the use of oncolytic viruses for PCa therapy. In this review, we first discuss the controversy of the contribution of virus infection to PCa, and subsequently summarize the development of oncolytic virotherapy for PCa in the past several years.

  18. Targeting cancer stem cells with oncolytic virus

    PubMed Central

    Tong, Yin

    2014-01-01

    Cancer stem cells (CSCs) represent a distinct subpopulation of cancer cells which are shown to be relatively resistant to conventional anticancer therapies and have been correlated to disease recurrence. Oncolytic viruses utilize methods of cell killing that differ from traditional therapies and thus are able to elude the typical mechanisms that CSCs use to resist current chemotherapies and radiotherapies. Moreover, genetically engineered oncolytic viruses may further augment the oncolytic effects. Here we review the recent data regarding the ability of several oncolytic viruses to eradicate CSCs. PMID:27358866

  19. Oncolytic Poxviruses

    PubMed Central

    Chan, Winnie M.; McFadden, Grant

    2015-01-01

    Current standard treatments of cancer can prolong survival of many cancer patients but usually do not effectively cure the disease. Oncolytic virotherapy is an emerging therapeutic for the treatment of cancer that exploits replication-competent viruses to selectively infect and destroy cancerous cells while sparing normal cells and tissues. Clinical and/or preclinical studies on oncolytic viruses have revealed that the candidate viruses being tested in trials are remarkably safe and offer potential for treating many classes of currently incurable cancers. Among these candidates are vaccinia and myxoma viruses, which belong to the family Poxviridae and possess promising oncolytic features. This article describes poxviruses that are being developed for oncolytic virotherapy and summarizes the outcomes of both clinical and preclinical studies. Additionally, studies demonstrating superior efficacy when poxvirus oncolytic virotherapy is combined with conventional therapies are described. PMID:25839047

  20. Antigen profiling analysis of vaccinia virus injected canine tumors

    PubMed Central

    Cecil, Alexander; Gentschev, Ivaylo; Adelfinger, Marion; Nolte, Ingo; Dandekar, Thomas; Szalay, Aladar A

    2014-01-01

    Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for cancer therapy. In this study we describe for the first time the use of dynamic boolean modeling for tumor growth prediction of vaccinia virus GLV-1h68-injected canine tumors including canine mammary adenoma (ZMTH3), canine mammary carcinoma (MTH52c), canine prostate carcinoma (CT1258), and canine soft tissue sarcoma (STSA-1). Additionally, the STSA-1 xenografted mice were injected with either LIVP 1.1.1 or LIVP 5.1.1 vaccinia virus strains.   Antigen profiling data of the four different vaccinia virus-injected canine tumors were obtained, analyzed and used to calculate differences in the tumor growth signaling network by type and tumor type. Our model combines networks for apoptosis, MAPK, p53, WNT, Hedgehog, TK cell, Interferon, and Interleukin signaling networks. The in silico findings conform with in vivo findings of tumor growth. Boolean modeling describes tumor growth and remission semi-quantitatively with a good fit to the data obtained for all cancer type variants. At the same time it monitors all signaling activities as a basis for treatment planning according to antigen levels. Mitigation and elimination of VACV- susceptible tumor types as well as effects on the non-susceptible type CT1258 are predicted correctly. Thus the combination of Antigen profiling and semi-quantitative modeling optimizes the therapy already before its start. PMID:25482233

  1. Neutrophil uptake of vaccinia virus in vitro

    SciTech Connect

    West, B.C.; Eschete, M.L.; Cox, M.E.; King, J.W.

    1987-10-01

    We studied human neutrophils for uptake of vaccinia virus. Uptake was determined radiometrically and by electron microscopy. Vaccinia virus was labeled with /sup 14/C or /sup 3/H, incubated with neutrophils, and quantified in neutrophil pellets in a new radiometric phagocytosis assay. Better results were obtained from assays of (/sup 3/H)thymidine-labeled virus; uptake increased through 1 hr and then plateaued. Phagocytosis of 3H-labeled Staphylococcus aureus was normal. Uptake of virus was serum dependent. Hexose monophosphate shunt activity was measured by two methods. No /sup 14/CO/sub 2/ from (/sup 14/C)1-glucose accompanied uptake of vaccinia virus, in contrast to the respiratory burst accompanying bacterial phagocytosis. Electron microscopy showed intact to slightly digested intraphagolysosomal vaccinia virus. Pock reduction assay showed a decrease in viral content due to neutrophils until 6 hr of incubation, when a modest but significant increase was observed. Thus, neutrophil uptake of vaccinia virus is distinguished from bacterial phagocytosis.

  2. Oncolytic virus therapy: A new era of cancer treatment at dawn.

    PubMed

    Fukuhara, Hiroshi; Ino, Yasushi; Todo, Tomoki

    2016-10-01

    Oncolytic virus therapy is perhaps the next major breakthrough in cancer treatment following the success in immunotherapy using immune checkpoint inhibitors. Oncolytic viruses are defined as genetically engineered or naturally occurring viruses that selectively replicate in and kill cancer cells without harming the normal tissues. T-Vec (talimogene laherparepvec), a second-generation oncolytic herpes simplex virus type 1 (HSV-1) armed with GM-CSF, was recently approved as the first oncolytic virus drug in the USA and Europe. The phase III trial proved that local intralesional injections with T-Vec in advanced malignant melanoma patients can not only suppress the growth of injected tumors but also act systemically and prolong overall survival. Other oncolytic viruses that are closing in on drug approval in North America and Europe include vaccinia virus JX-594 (pexastimogene devacirepvec) for hepatocellular carcinoma, GM-CSF-expressing adenovirus CG0070 for bladder cancer, and Reolysin (pelareorep), a wild-type variant of reovirus, for head and neck cancer. In Japan, a phase II clinical trial of G47∆, a third-generation oncolytic HSV-1, is ongoing in glioblastoma patients. G47∆ was recently designated as a "Sakigake" breakthrough therapy drug in Japan. This new system by the Japanese government should provide G47∆ with priority reviews and a fast-track drug approval by the regulatory authorities. Whereas numerous oncolytic viruses have been subjected to clinical trials, the common feature that is expected to play a major role in prolonging the survival of cancer patients is an induction of specific antitumor immunity in the course of tumor-specific viral replication. It appears that it will not be long before oncolytic virus therapy becomes a standard therapeutic option for all cancer patients. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  3. Oncolytic Myxoma Virus: The path to clinic

    PubMed Central

    Chan, Winnie M.; Rahman, Masmudur M.; McFadden, Grant

    2013-01-01

    Many common neoplasms are still noncurative with current standards of cancer therapy. More therapeutic modalities need to be developed to significantly prolong the lives of patients and eventually cure a wider spectrum of cancers. Oncolytic virotherapy is one of the promising new additions to clinical cancer therapeutics. Successful oncolytic virotherapy in the clinic will be those strategies that best combine tumor cell oncolysis with enhanced immune responses against tumor antigens. The current candidate oncolytic viruses all share the common property that they are relatively nonpathogenic to humans, yet they have the ability to replicate selectively in human cancer cells and induce cancer regression by direct oncolysis and/or induction of improved anti-tumor immune responses. Many candidate oncolytic viruses are in various stages of clinical and preclinical development. One such preclinical candidate is myxoma virus (MYXV), a member of the Poxviridae family that, in its natural setting, exhibits a very restricted host range and is only pathogenic to European rabbits. Despite its narrow host range in nature, MYXV has been shown to productively infect various classes of human cancer cells. Several preclinical in vivo modeling studies have demonstrated that MYXV is an attractive and safe candidate oncolytic virus, and hence, MYXV is currently being developed as a potential therapeutic for several cancers, such as pancreatic cancer, glioblastoma, ovarian cancer, melanoma, and hematologic malignancies. This review highlights the preclinical cancer models that have shown the most promise for translation of MYXV into human clinical trials. PMID:23726825

  4. Oncolytic myxoma virus: the path to clinic.

    PubMed

    Chan, Winnie M; Rahman, Masmudur M; McFadden, Grant

    2013-09-06

    Many common neoplasms are still noncurative with current standards of cancer therapy. More therapeutic modalities need to be developed to significantly prolong the lives of patients and eventually cure a wider spectrum of cancers. Oncolytic virotherapy is one of the promising new additions to clinical cancer therapeutics. Successful oncolytic virotherapy in the clinic will be those strategies that best combine tumor cell oncolysis with enhanced immune responses against tumor antigens. The current candidate oncolytic viruses all share the common property that they are relatively nonpathogenic to humans, yet they have the ability to replicate selectively in human cancer cells and induce cancer regression by direct oncolysis and/or induction of improved anti-tumor immune responses. Many candidate oncolytic viruses are in various stages of clinical and preclinical development. One such preclinical candidate is myxoma virus (MYXV), a member of the Poxviridae family that, in its natural setting, exhibits a very restricted host range and is only pathogenic to European rabbits. Despite its narrow host range in nature, MYXV has been shown to productively infect various classes of human cancer cells. Several preclinical in vivo modeling studies have demonstrated that MYXV is an attractive and safe candidate oncolytic virus, and hence, MYXV is currently being developed as a potential therapeutic for several cancers, such as pancreatic cancer, glioblastoma, ovarian cancer, melanoma, and hematologic malignancies. This review highlights the preclinical cancer models that have shown the most promise for translation of MYXV into human clinical trials.

  5. Vaccinia virus infection in monkeys, Brazilian Amazon.

    PubMed

    Abrahão, Jônatas S; Silva-Fernandes, André T; Lima, Larissa S; Campos, Rafael K; Guedes, Maria I M C; Cota, Marcela M G; Assis, Felipe L; Borges, Iara A; Souza-Júnior, Milton F; Lobato, Zélia I P; Bonjardim, Cláudio A; Ferreira, Paulo C P; Trindade, Giliane S; Kroon, Erna G

    2010-06-01

    To detect orthopoxvirus in the Brazilian Amazon, we conducted a serosurvey of 344 wild animals. Neutralizing antibodies against orthopoxvirus were detected by plaque-reduction neutralizing tests in 84 serum samples. Amplicons from 6 monkey samples were sequenced. These amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in Brazil.

  6. Vaccinia Virus Infection in Monkeys, Brazilian Amazon

    PubMed Central

    Abrahão, Jônatas S.; Silva-Fernandes, André T.; Lima, Larissa S.; Campos, Rafael K.; Guedes, Maria I.M.C.; Cota, Marcela M.G.; Assis, Felipe L.; Borges, Iara A.; Souza-Júnior, Milton F.; Lobato, Zélia I.P.; Bonjardim, Cláudio A.; Ferreira, Paulo C.P.; Trindade, Giliane S.

    2010-01-01

    To detect orthopoxvirus in the Brazilian Amazon, we conducted a serosurvey of 344 wild animals. Neutralizing antibodies against orthopoxvirus were detected by plaque-reduction neutralizing tests in 84 serum samples. Amplicons from 6 monkey samples were sequenced. These amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in Brazil. PMID:20507750

  7. Oncolytic Virus: Regulatory Aspects from Quality Control to Clinical Studies.

    PubMed

    Yamaguchi, Teruhide; Uchida, Eriko

    2017-02-22

    Oncolytic viruses, which include both naturally occurring wild-type viruses/attenuated viruses and genetically modified viruses, have recently been developed for use in innovative cancer therapies. Genetically modified oncolytic viruses possess the unique ability to replicate conditionally as a unique gene therapy product. Since oncolytic viruses exhibit prolonged persistence in patients, viral shedding and transmission to third parties should be major concerns for clinical trials, along with the clinical safety and efficacy. Accordingly, studies are now underway to establish the safety and efficacy of oncolytic viruses.

  8. MORPHOLOGICAL STRUCTURE OF THE VIRUS OF VACCINIA

    PubMed Central

    Green, R. H.; Anderson, T. F.; Smadel, J. E.

    1942-01-01

    The pictorial data obtained by means of the electron microscope indicate a remarkable regularity in the morphology of the elementary body of vaccinia. The virus particles apparently have internal structure and some sort of limiting membrane. PMID:19871212

  9. Oncolytic Maraba Virus MG1 as a Treatment for Sarcoma.

    PubMed

    Le Boeuf, Fabrice; Selman, Mohammed; Son, Hwan Hee; Bergeron, Anabel; Chen, Andrew; Tsang, Jovian; Butterwick, Derek; Arulanandam, Rozanne; Forbes, Nicole E; Tzelepis, Fanny; Bell, John C; Werier, Joel; Abdelbary, Hesham; Diallo, Jean-Simon

    2017-09-15

    The poor prognosis of patients with advanced bone and soft-tissue sarcoma has not changed in the past several decades, highlighting the necessity for new therapeutic approaches. Immunotherapies, including oncolytic viral (OV) therapy, have shown great promise in a number of clinical trials for a variety of tumor types. However, the effective application of OV in treating sarcoma still remains to be demonstrated. Although few pre-clinical studies using distinct OVs have been performed and demonstrated therapeutic benefit in sarcoma models, a side-by-side comparison of clinically relevant OV platforms has not been performed. Four clinically relevant OV platforms (Reovirus, Vaccinia virus, Herpes-simplex virus and Rhabdovirus) were screened for their ability to infect and kill human and canine sarcoma cell lines in vitro, and human sarcoma specimens ex vivo. In vivo treatment efficacy was tested in a murine model. The rhabdovirus MG1 demonstrated the highest potency in vitro. Ex vivo, MG1 productively infected more than 80% of human sarcoma tissues tested, and treatment in vivo led to a significant increase in long-lasting cures in sarcoma-bearing mice. Importantly, MG1 treatment induced the generation of memory immune response that provided protection against a subsequent tumor challenge. This study opens the door for the use of MG1-based oncolytic immunotherapy strategies as treatment for sarcoma or as a component of a combined therapy. © 2017 UICC.

  10. Advances in Oncolytic Virus Therapy for Glioma

    PubMed Central

    Haseley, Amy; Alvarez-Breckenridge, Christopher; Chaudhury, Abhik Ray; Kaur, Balveen

    2009-01-01

    The World Health Organization grossly classifies the various types of astrocytomas using a grade system with grade IV gliomas having the worst prognosis. Oncolytic virus therapy is a novel treatment option for GBM patients. Several patents describe various oncolytic viruses used in preclinical and clinical trials to evaluate safety and efficacy. These viruses are natural or genetically engineered from different viruses such as HSV-1, Adenovirus, Reovirus, and New Castle Disease Virus. While several anecdotal studies have indicated therapeutic advantage, recent clinical trials have revealed the safety of their usage, but demonstration of significant efficacy remains to be established. Oncolytic viruses are being redesigned with an interest in combating the tumor microenvironment in addition to defeating the cancerous cells. Several patents describe the inclusion of tumor microenvironment modulating genes within the viral backbone and in particular those which attack the tumor angiotome. The very innovative approaches being used to improve therapeutic efficacy include: design of viruses which can express cytokines to activate a systemic antitumor immune response, inclusion of angiostatic genes to combat tumor vasculature, and also enzymes capable of digesting tumor extra cellular matrix (ECM) to enhance viral spread through solid tumors. As increasingly more novel viruses are being tested and patented, the future battle against glioma looks promising. PMID:19149710

  11. Intelligent design: combination therapy with oncolytic viruses.

    PubMed

    Ottolino-Perry, Kathryn; Diallo, Jean-Simon; Lichty, Brian D; Bell, John C; McCart, J Andrea

    2010-02-01

    Metastatic cancer remains an incurable disease in the majority of cases and thus novel treatment strategies such as oncolytic virotherapy are rapidly advancing toward clinical use. In order to be successful, it is likely that some type of combination therapy will be necessary to have a meaningful impact on this disease. Although it may be tempting to simply combine an oncolytic virus with the existing standard radiation or chemotherapeutics, the long-term goal of such treatments must be to have a rational, potentially synergistic combination strategy that can be safely and easily used in the clinical setting. The combination of oncolytic virotherapy with existing radiotherapy and chemotherapy modalities is reviewed along with novel biologic therapies including immunotherapies, in order to help investigators make intelligent decisions during the clinical development of these products.

  12. High-throughput screening to enhance oncolytic virus immunotherapy

    PubMed Central

    Allan, KJ; Stojdl, David F; Swift, SL

    2016-01-01

    High-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. Although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. Oncolytic viruses (OVs) are biotherapeutic agents with an inherent specificity for treating malignant disease. Certain OV platforms – including those based on herpes simplex virus, reovirus, and vaccinia virus – have shown success against solid tumors in advanced clinical trials. Yet, many of these OVs have only undergone minimal engineering to solidify tumor specificity, with few extra modifications to manipulate additional factors. Several aspects of the interaction between an OV and a tumor-bearing host have clear value as targets to improve therapeutic outcomes. At the virus level, these include delivery to the tumor, infectivity, productivity, oncolysis, bystander killing, spread, and persistence. At the host level, these include engaging the immune system and manipulating the tumor microenvironment. Here, we review the chemical- and genome-based high-throughput screens that have been performed to manipulate such parameters during OV infection and analyze their impact on therapeutic efficacy. We further explore emerging themes that represent key areas of focus for future research. PMID:27579293

  13. Vaccinia virus infections in martial arts gym, Maryland, USA, 2008.

    PubMed

    Hughes, Christine M; Blythe, David; Li, Yu; Reddy, Ramani; Jordan, Carol; Edwards, Cindy; Adams, Celia; Conners, Holly; Rasa, Catherine; Wilby, Sue; Russell, Jamaal; Russo, Kelly S; Somsel, Patricia; Wiedbrauk, Danny L; Dougherty, Cindy; Allen, Christopher; Frace, Mike; Emerson, Ginny; Olson, Victoria A; Smith, Scott K; Braden, Zachary; Abel, Jason; Davidson, Whitni; Reynolds, Mary; Damon, Inger K

    2011-04-01

    Vaccinia virus is an orthopoxvirus used in the live vaccine against smallpox. Vaccinia virus infections can be transmissible and can cause severe complications in those with weakened immune systems. We report on a cluster of 4 cases of vaccinia virus infection in Maryland, USA, likely acquired at a martial arts gym.

  14. Vaccinia Virus Infections in Martial Arts Gym, Maryland, USA, 2008

    PubMed Central

    Blythe, David; Li, Yu; Reddy, Ramani; Jordan, Carol; Edwards, Cindy; Adams, Celia; Conners, Holly; Rasa, Catherine; Wilby, Sue; Russell, Jamaal; Russo, Kelly S.; Somsel, Patricia; Wiedbrauk, Danny L.; Dougherty, Cindy; Allen, Christopher; Frace, Mike; Emerson, Ginny; Olson, Victoria A.; Smith, Scott K.; Braden, Zachary; Abel, Jason; Davidson, Whitni; Reynolds, Mary; Damon, Inger K.

    2011-01-01

    Vaccinia virus is an orthopoxvirus used in the live vaccine against smallpox. Vaccinia virus infections can be transmissible and can cause severe complications in those with weakened immune systems. We report on a cluster of 4 cases of vaccinia virus infection in Maryland, USA, likely acquired at a martial arts gym. PMID:21470473

  15. Environmental persistence of vaccinia virus on materials.

    PubMed

    Wood, J P; Choi, Y W; Wendling, M Q; Rogers, J V; Chappie, D J

    2013-11-01

    Smallpox is caused by the variola virus, and ranks as one of the most serious diseases that could originate from a biological weapon. However, limited data exist on the persistence of variola and related viruses on materials (that may act as fomites), under controlled environmental conditions. To fill these data gaps, we determined the persistence of the vaccinia virus (an established surrogate for the variola virus) as a function of temperature, relative humidity and material. Experiments were conducted with vaccinia virus in a freeze-dried form, using four materials under four sets of environmental conditions. After elapsed times ranging from 1 to 56 days, the virus was extracted from small coupons and quantified via plaque-forming units (PFU). The vaccinia virus was most persistent at low temperature and low relative humidity, with greater than 10(4) PFU recovered from glass, galvanized steel and painted cinder block at 56 days (equivalent to only a c. 2 log reduction). Thus, vaccinia virus may persist from weeks to months, depending on the material and environmental conditions. This study may aid those responsible for infection control to make informed decisions regarding the need for environmental decontamination following the release of an agent such as variola.

  16. Oncolytic virus therapy using genetically engineered herpes simplex viruses.

    PubMed

    Todo, Tomoki

    2008-01-01

    Genetically engineered, conditionally replicating herpes simplex viruses type 1 (HSV-1) are promising therapeutic agents for cancer. They can replicate in situ, spread, and exhibit oncolytic activity via a direct cytocidal effect. In addition, oncolytic HSV-1 can transfer and express foreign genes in host cells. The phase I clinical study with G207, a double-mutated HSV-1, in recurrent malignant glioma patients has shown that oncolytic HSV-1 can be safely administered into human brains. The therapeutic benefits of oncolytic HSV-1 depend on the extent of both intratumoral viral replication and induction of host antitumor immune responses. We develop new-generation oncolytic HSV-1 by enhancing these properties while retaining the safety features. G47delta was created from G207 by introducing another genetic mutation. Compared with G207, G47delta showed 1) better stimulation of human antitumor immune cells, 2) better growth properties leading to higher virus yields and increased cytopathic effect in vitro, 3) better antitumor efficacy in both immuno-competent and -incompetent animals, and 4) preserved safety in the brain of HSV-1-sensitive mice. Preparation is under way for a clinical trial using G47delta in progressive glioblastoma patients. G47delta is also suited as a backbone vector for expressing foreign molecules. Using bacterial artificial chromosome and two DNA recombinases, we have created an "armed" oncolytic HSV-1 generation system that allows insertion of transgene(s) into the genome of G47delta in a rapid and accurate manner. We found that expression of immunostimulatory molecules can significantly enhance the antitumor efficacy of G47delta. Based on these advances, we anticipate that oncolytic virus therapy using oncolytic HSV-1 will soon be established as an important modality of cancer treatment.

  17. Retargeting Strategies for Oncolytic Herpes Simplex Viruses.

    PubMed

    Campadelli-Fiume, Gabriella; Petrovic, Biljana; Leoni, Valerio; Gianni, Tatiana; Avitabile, Elisa; Casiraghi, Costanza; Gatta, Valentina

    2016-02-26

    Most of the oncolytic herpes simplex viruses (HSVs) exhibit a high safety profile achieved through attenuation. They carry defects in virulence proteins that antagonize host cell response to the virus, including innate response, apoptosis, authophagy, and depend on tumor cell proliferation. They grow robustly in cancer cells, provided that these are deficient in host cell responses, which is often the case. To overcome the attenuation limits, a strategy is to render the virus highly cancer-specific, e.g., by retargeting their tropism to cancer-specific receptors, and detargeting from natural receptors. The target we selected is HER-2, overexpressed in breast, ovarian and other cancers. Entry of wt-HSV requires the essential glycoproteins gD, gH/gL and gB. Here, we reviewed that oncolytic HSV retargeting was achieved through modifications in gD: the addition of a single-chain antibody (scFv) to HER-2 coupled with appropriate deletions to remove part of the natural receptors' binding sites. Recently, we showed that also gH/gL can be a retargeting tool. The insertion of an scFv to HER-2 at the gH N-terminus, coupled with deletions in gD, led to a recombinant capable to use HER-2 as the sole receptor. The retargeted oncolytic HSVs can be administered systemically by means of carrier cells-forcedly-infected mesenchymal stem cells. Altogether, the retargeted oncolytic HSVs are highly cancer-specific and their replication is not dependent on intrinsic defects of the tumor cells. They might be further modified to express immunomodulatory molecules.

  18. Retargeting Strategies for Oncolytic Herpes Simplex Viruses

    PubMed Central

    Campadelli-Fiume, Gabriella; Petrovic, Biljana; Leoni, Valerio; Gianni, Tatiana; Avitabile, Elisa; Casiraghi, Costanza; Gatta, Valentina

    2016-01-01

    Most of the oncolytic herpes simplex viruses (HSVs) exhibit a high safety profile achieved through attenuation. They carry defects in virulence proteins that antagonize host cell response to the virus, including innate response, apoptosis, authophagy, and depend on tumor cell proliferation. They grow robustly in cancer cells, provided that these are deficient in host cell responses, which is often the case. To overcome the attenuation limits, a strategy is to render the virus highly cancer-specific, e.g., by retargeting their tropism to cancer-specific receptors, and detargeting from natural receptors. The target we selected is HER-2, overexpressed in breast, ovarian and other cancers. Entry of wt-HSV requires the essential glycoproteins gD, gH/gL and gB. Here, we reviewed that oncolytic HSV retargeting was achieved through modifications in gD: the addition of a single-chain antibody (scFv) to HER-2 coupled with appropriate deletions to remove part of the natural receptors’ binding sites. Recently, we showed that also gH/gL can be a retargeting tool. The insertion of an scFv to HER-2 at the gH N-terminus, coupled with deletions in gD, led to a recombinant capable to use HER-2 as the sole receptor. The retargeted oncolytic HSVs can be administered systemically by means of carrier cells-forcedly-infected mesenchymal stem cells. Altogether, the retargeted oncolytic HSVs are highly cancer-specific and their replication is not dependent on intrinsic defects of the tumor cells. They might be further modified to express immunomodulatory molecules. PMID:26927159

  19. ODE models for oncolytic virus dynamics

    PubMed Central

    Komarova, Natalia L.; Wodarz, Dominik

    2010-01-01

    Replicating oncolytic viruses are able to infect and lyse cancer cells and spread through the tumor, while leaving normal cells largely unharmed. This makes them potentially useful in cancer therapy, and a variety of viruses have shown promising results in clinical trials. Nevertheless, consistent success remains elusive and the correlates of success have been the subject of investigation, both from an experimental and a mathematical point of view. Mathematical modeling of oncolytic virus therapy is often limited by the fact that the predicted dynamics depend strongly on particular mathematical terms in the model, the nature of which remain uncertain. We aim to address this issue in the context of ODE modeling, by formulating a general computational framework that is independent of particular mathematical expressions. By analyzing this framework, we find some new insights into the conditions for successful virus therapy. We find that depending on our assumptions about the virus spread, there can be two distinct types of dynamics. In models of the first type (the “fast spread” models), we predict that the viruses can eliminate the tumor if the viral replication rate is sufficiently high. The second type of models is characterized by a suboptimal spread (the “slow spread” models). For such models, the simulated treatment may fail, even for very high viral replication rates. Our methodology can be used to study the dynamics of many biological systems, and thus has implications beyond the study of virus therapy of cancers. PMID:20085772

  20. The ex vivo purge of cancer cells using oncolytic viruses: recent advances and clinical implications.

    PubMed

    Tsang, Jovian J; Atkins, Harold L

    2015-01-01

    Hematological malignancies are treated with intensive high-dose chemotherapy, with or without radiation. This is followed by hematopoietic stem cell (HSC) transplantation (HSCT) to rescue or reconstitute hematopoiesis damaged by the anticancer therapy. Autologous HSC grafts may contain cancer cells and purging could further improve treatment outcomes. Similarly, allogeneic HSCT may be improved by selectively purging alloreactive effector cells from the graft rather than wholesale immune cell depletion. Viral agents that selectively replicate in specific cell populations are being studied in experimental models of cancer and immunological diseases and have potential applications in the context of HSC graft engineering. This review describes preclinical studies involving oncolytic virus strains of adenovirus, herpes simplex virus type 1, myxoma virus, and reovirus as ex vivo purging agents for HSC grafts, as well as in vitro and in vivo experimental studies using oncolytic coxsackievirus, measles virus, parvovirus, vaccinia virus, and vesicular stomatitis virus to eradicate hematopoietic malignancies. Alternative ex vivo oncolytic virus strategies are also outlined that aim to reduce the risk of relapse following autologous HSCT and mitigate morbidity and mortality due to graft-versus-host disease in allogeneic HSCT.

  1. The ex vivo purge of cancer cells using oncolytic viruses: recent advances and clinical implications

    PubMed Central

    Tsang, Jovian J; Atkins, Harold L

    2015-01-01

    Hematological malignancies are treated with intensive high-dose chemotherapy, with or without radiation. This is followed by hematopoietic stem cell (HSC) transplantation (HSCT) to rescue or reconstitute hematopoiesis damaged by the anticancer therapy. Autologous HSC grafts may contain cancer cells and purging could further improve treatment outcomes. Similarly, allogeneic HSCT may be improved by selectively purging alloreactive effector cells from the graft rather than wholesale immune cell depletion. Viral agents that selectively replicate in specific cell populations are being studied in experimental models of cancer and immunological diseases and have potential applications in the context of HSC graft engineering. This review describes preclinical studies involving oncolytic virus strains of adenovirus, herpes simplex virus type 1, myxoma virus, and reovirus as ex vivo purging agents for HSC grafts, as well as in vitro and in vivo experimental studies using oncolytic coxsackievirus, measles virus, parvovirus, vaccinia virus, and vesicular stomatitis virus to eradicate hematopoietic malignancies. Alternative ex vivo oncolytic virus strategies are also outlined that aim to reduce the risk of relapse following autologous HSCT and mitigate morbidity and mortality due to graft-versus-host disease in allogeneic HSCT. PMID:27512666

  2. Oncolytic virotherapy in veterinary medicine: current status and future prospects for canine patients.

    PubMed

    Patil, Sandeep S; Gentschev, Ivaylo; Nolte, Ingo; Ogilvie, Gregory; Szalay, Aladar A

    2012-01-04

    Oncolytic viruses refer to those that are able to eliminate malignancies by direct targeting and lysis of cancer cells, leaving non-cancerous tissues unharmed. Several oncolytic viruses including adenovirus strains, canine distemper virus and vaccinia virus strains have been used for canine cancer therapy in preclinical studies. However, in contrast to human studies, clinical trials with oncolytic viruses for canine cancer patients have not been reported. An 'ideal' virus has yet to be identified. This review is focused on the prospective use of oncolytic viruses in the treatment of canine tumors - a knowledge that will undoubtedly contribute to the development of oncolytic viral agents for canine cancer therapy in the future.

  3. Trial Watch-Oncolytic viruses and cancer therapy.

    PubMed

    Pol, Jonathan; Buqué, Aitziber; Aranda, Fernando; Bloy, Norma; Cremer, Isabelle; Eggermont, Alexander; Erbs, Philippe; Fucikova, Jitka; Galon, Jérôme; Limacher, Jean-Marc; Preville, Xavier; Sautès-Fridman, Catherine; Spisek, Radek; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2016-02-01

    Oncolytic virotherapy relies on the administration of non-pathogenic viral strains that selectively infect and kill malignant cells while favoring the elicitation of a therapeutically relevant tumor-targeting immune response. During the past few years, great efforts have been dedicated to the development of oncolytic viruses with improved specificity and potency. Such an intense wave of investigation has culminated this year in the regulatory approval by the US Food and Drug Administration (FDA) of a genetically engineered oncolytic viral strain for use in melanoma patients. Here, we summarize recent preclinical and clinical advances in oncolytic virotherapy.

  4. Myxoma and Vaccinia Viruses Bind Differentially to Human Leukocytes

    PubMed Central

    Chan, Winnie M.; Bartee, Eric C.; Moreb, Jan S.; Dower, Ken; Connor, John H.

    2013-01-01

    Myxoma virus (MYXV) and vaccinia virus (VACV), two distinct members of the family Poxviridae, are both currently being developed as oncolytic virotherapeutic agents. Recent studies have demonstrated that ex vivo treatment with MYXV can selectively recognize and kill contaminating cancerous cells from autologous bone marrow transplants without perturbing the engraftment of normal CD34+ hematopoietic stem and progenitor cells. However, the mechanism(s) by which MYXV specifically recognizes and eliminates the cancer cells in the autografts is not understood. While little is known about the cellular attachment factor(s) exploited by MYXV for entry into any target cells, VACV has been shown to utilize cell surface glycosaminoglycans such as heparan sulfate (HS), the extracellular matrix protein laminin, and/or integrin β1. We have constructed MYXV and VACV virions tagged with the Venus fluorescent protein and compared their characteristics of binding to various human cancer cell lines as well as to primary human leukocytes. We report that the binding of MYXV or VACV to some adherent cell lines could be partially inhibited by heparin, but laminin blocked only VACV binding. In contrast to cultured fibroblasts, the binding of MYXV and VACV to a wide spectrum of primary human leukocytes could not be competed by either HS or laminin. Additionally, MYXV and VACV exhibited very different binding characteristics against certain select human leukocytes, suggesting that the two poxviruses utilize different cell surface determinants for the attachment to these cells. These results indicate that VACV and MYXV can exhibit very different oncolytic tropisms against some cancerous human leukocytes. PMID:23388707

  5. Myxoma and vaccinia viruses bind differentially to human leukocytes.

    PubMed

    Chan, Winnie M; Bartee, Eric C; Moreb, Jan S; Dower, Ken; Connor, John H; McFadden, Grant

    2013-04-01

    Myxoma virus (MYXV) and vaccinia virus (VACV), two distinct members of the family Poxviridae, are both currently being developed as oncolytic virotherapeutic agents. Recent studies have demonstrated that ex vivo treatment with MYXV can selectively recognize and kill contaminating cancerous cells from autologous bone marrow transplants without perturbing the engraftment of normal CD34(+) hematopoietic stem and progenitor cells. However, the mechanism(s) by which MYXV specifically recognizes and eliminates the cancer cells in the autografts is not understood. While little is known about the cellular attachment factor(s) exploited by MYXV for entry into any target cells, VACV has been shown to utilize cell surface glycosaminoglycans such as heparan sulfate (HS), the extracellular matrix protein laminin, and/or integrin β1. We have constructed MYXV and VACV virions tagged with the Venus fluorescent protein and compared their characteristics of binding to various human cancer cell lines as well as to primary human leukocytes. We report that the binding of MYXV or VACV to some adherent cell lines could be partially inhibited by heparin, but laminin blocked only VACV binding. In contrast to cultured fibroblasts, the binding of MYXV and VACV to a wide spectrum of primary human leukocytes could not be competed by either HS or laminin. Additionally, MYXV and VACV exhibited very different binding characteristics against certain select human leukocytes, suggesting that the two poxviruses utilize different cell surface determinants for the attachment to these cells. These results indicate that VACV and MYXV can exhibit very different oncolytic tropisms against some cancerous human leukocytes.

  6. Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Mackett, Michael; Moss, Bernard

    1983-04-01

    Potential live vaccines against hepatitis B virus have been produced. The coding sequence for hepatitis B virus surface antigen (HBsAg) has been inserted into the vaccinia virus genome under control of vaccinia virus early promoters. Cells infected with these vaccinia virus recombinants synthesize and excrete HBsAg and vaccinated rabbits rapidly produce antibodies to HBsAg.

  7. Susceptibility of Vaccinia Virus to Chemical Disinfectants

    PubMed Central

    de Oliveira, Tércia Moreira Ludolfo; Rehfeld, Izabelle Silva; Coelho Guedes, Maria Isabel Maldonado; Ferreira, Jaqueline Maria Siqueira; Kroon, Erna Geessien; Lobato, Zélia Inês Portela

    2011-01-01

    Vaccinia virus (VACV) is the cause of bovine vaccinia (BV), an emerging zoonotic disease that affects dairy cows and milkers. Some chemical disinfectants have been used on farms affected by BV to disinfect cow teats and milkers' hands. To date, there is no information about the efficacy of disinfectants against VACV. Therefore, this study aimed to assess the virucidal activity of some active disinfectants commonly used in the field. Sodium hypochlorite, quaternary ammonium combined with chlorhexidine, and quaternary ammonium combined with glutaraldehyde were effective in inactivating the virus at all concentrations tested. Iodine and quaternary ammonium as the only active component were partially effective. The presence of bovine feces as organic matter and light decreased the effectiveness of sodium hypochlorite. These results show that an appropriated disinfection and asepsis of teats and hands may be helpful in the control and prevention of BV and other infections with VACV. PMID:21734141

  8. Retargeting of Viruses to Generate Oncolytic Agents

    PubMed Central

    Verheije, M. H.; Rottier, P. J. M.

    2012-01-01

    Oncolytic virus therapy is based on the ability of viruses to effectively infect and kill tumor cells without destroying the normal tissues. While some viruses seem to have a natural preference for tumor cells, most viruses require the modification of their tropism to specifically enter and replicate in such cells. This review aims to describe the transductional targeting strategies currently employed to specifically redirect viruses towards surface receptors on tumor cells. Three major strategies can be distinguished; they involve (i) the incorporation of new targeting specificity into a viral surface protein, (ii) the incorporation of a scaffold into a viral surface protein to allow the attachment of targeting moieties, and (iii) the use of bispecific adapters to mediate targeting of a virus to a specified moiety on a tumor cell. Of each strategy key features, advantages and limitations are discussed and examples are given. Because of their potential to cause sustained, multiround infection—a desirable characteristic for eradicating tumors—particular attention is given to viruses engineered to become self-targeted by the genomic expression of a bispecific adapter protein. PMID:22312365

  9. Therapeutic potential of oncolytic Newcastle disease virus: a critical review

    PubMed Central

    Tayeb, Shay; Zakay-Rones, Zichria; Panet, Amos

    2015-01-01

    Newcastle disease virus (NDV) features a natural preference for replication in many tumor cells compared with normal cells. The observed antitumor effect of NDV appears to be a result of both selective killing of tumor cells and induction of immune responses. Genetic manipulations to change viral tropism and arming the virus with genes encoding for cytokines improved the oncolytic capacity of NDV. Several intracellular proteins in tumor cells, including antiapoptotic proteins (Livin) and oncogenic proteins (H-Ras), are relevant for the oncolytic activity of NDV. Defects in the interferon system, found in some tumor cells, also contribute to the oncolytic selectivity of NDV. Notwithstanding, NDV displays effective oncolytic activity in many tumor types, despite having intact interferon signaling. Taken together, several cellular systems appear to dictate the selective oncolytic activity of NDV. Some barriers, such as neutralizing antibodies elicited during NDV treatment and the extracellular matrix in tumor tissue appear to interfere with spread of NDV and reduce oncolysis. To further understand the oncolytic activity of NDV, we compared two NDV strains, ie, an attenuated virus (NDV-HUJ) and a pathogenic virus (NDV-MTH-68/H). Significant differences in amino acid sequence were noted in several viral proteins, including the fusion precursor (F0) glycoprotein, an important determinant of replication and pathogenicity. However, no difference in the oncolytic activity of the two strains was noted using human tumor tissues maintained as organ cultures or in mouse tumor models. To optimize virotherapy in clinical trials, we describe here a unique organ culture methodology, using a biopsy taken from a patient’s tumor before treatment for ex vivo infection with NDV to determine the oncolytic potential on an individual basis. In conclusion, oncolytic NDV is an excellent candidate for cancer therapy, but more knowledge is needed to ensure success in clinical trials. PMID

  10. "Armed" oncolytic herpes simplex viruses for brain tumor therapy.

    PubMed

    Todo, Tomoki

    2008-01-01

    Genetically engineered, conditionally replicating herpes simplex viruses type 1 (HSV-1) are promising therapeutic agents for brain tumors and other solid cancers. They can replicate in situ, spread and exhibit oncolytic activity via a direct cytocidal effect. One of the advantages of HSV-1 is the capacity to incorporate large and/or multiple transgenes within the viral genome. Oncolytic HSV-1 can therefore be "armed" to add certain functions. Recently, the field of armed oncolytic HSV-1 has drastically advanced, due to development of recombinant HSV-1 generation systems that utilize bacterial artificial chromosome and multiple DNA recombinases. Because antitumor immunity is induced in the course of oncolytic activities of HSV-1, transgenes encoding immunomodulatory molecules have been most frequently used for arming. Other armed oncolytic HSV-1 include those that express antiangiogenic factors, fusogenic membrane glycoproteins, suicide gene products, and proapoptotic proteins. Provided that the transgene product does not interfere with viral replication, such arming of oncolytic HSV-1 results in augmentation of antitumor efficacy. Immediate-early viral promoters are often used to control the arming transgenes, but strict-late viral promoters have been shown useful to restrict the expression in the late stage of viral replication when desirable. Some armed oncolytic HSV-1 have been created for the purpose of noninvasive in vivo imaging of viral infection and replication. Development of a wide variety of armed oncolytic HSV-1 will lead to an establishment of a new genre of therapy for brain tumors as well as other cancers.

  11. Attenuated oncolytic Measles Virus strains as cancer therapeutics

    PubMed Central

    Msaouel, P.; Iankov, I.D.; Dispenzieri, A.; Galanis, E.

    2011-01-01

    Attenuated measles virus vaccine strains have emerged as a promising oncolytic vector platform, having shown significant anti-tumor activity against a broad range of malignant neoplasms. Measles virus strains derived from the attenuated Edmonston-B (MV-Edm) vaccine lineage have been shown to selectively infect, replicate in and lyse cancer cells while causing minimal cytopathic effect on normal tissues. This review summarizes the preclinical data that led to the rapid clinical translation of oncolytic measles vaccine strains and provides an overview of early clinical data using this oncolytic platform. Furthermore, novel approaches currently under development to further enhance the oncolytic efficacy of MV-Edm strains, including strategies to circumvent immunity or modulate immune system responses, combinatorial approaches with standard treatment modalities, virus retargeting as well as strategies for in vivo monitoring of viral replication are discussed. PMID:21740361

  12. Oncolytic Viruses: Therapeutics With an Identity Crisis.

    PubMed

    Breitbach, Caroline J; Lichty, Brian D; Bell, John C

    2016-07-01

    Oncolytic viruses (OV) are replicating viral therapeutics for the treatment of cancer and have been in laboratory development for about twenty years. Recently, the FDA approved Imlygic, a herpes virus based therapeutic for the treatment of melanoma and thus OVs have entered a new era where they are a weapon in the armament of the oncologist. OVs are unique therapeutics with multiple mechanisms of therapeutic activity. The exact path for their development and eventual uptake by pharmaceutical companies is somewhat clouded by an uncertain identity. Are they vaccines, tumour lysing therapeutics, inducers of innate immunity, gene therapy vectors, anti-vascular agents or all of the above? Should they be developed as stand-alone loco-regional therapeutics, systemically delivered tumour hunters or immune modulators best tested as combination therapeutics? We summarize data here supporting the idea, depending upon the virus, that OVs can be any or all of these things. Pursuing a "one-size fits all" approach is counter-productive to their clinical development and instead as a field we should build on the strengths of individual virus platforms.

  13. Brazilian Vaccinia Viruses and Their Origins

    PubMed Central

    Trindade, Giliane S.; Emerson, Ginny L.; Carroll, Darin S.; Kroon, Erna G.

    2007-01-01

    Although the World Health Organization (WHO) declared global smallpox eradicated in 1980, concerns over emergent poxvirus infections have increased. Most poxvirus infections are zoonotic; exploring their genetic diversity will illuminate the genetic and evolutionary aspects of poxvirus infections, ecology, and epidemiology. In recent decades, several strains of the orthopoxvirus vaccinia virus (VACV) have been isolated throughout Brazil, including many genetically distinct isolates within the same outbreak. To further investigate the diversity and origins of these viruses, we analyzed molecular data from 8 Brazilian VACV isolates and compared several genes involved in virus structure and pathogenicity. Genetic variation among isolates suggests that ancestral Brazilian VACVs existed before the beginning of the WHO smallpox eradication vaccination campaigns and that these viruses continue to circulate. PMID:18214166

  14. Myxoma virus oncolytic efficiency can be enhanced through chemical or genetic disruption of the actin cytoskeleton.

    PubMed

    Irwin, Chad R; Favis, Nicole A; Agopsowicz, Kate C; Hitt, Mary M; Evans, David H

    2013-01-01

    Myxoma virus (MYXV) is one of many animal viruses that exhibit oncolytic properties in transformed human cells. Compared to orthopoxviruses like vaccinia (VACV), MYXV spreads inefficiently, which could compromise its use in treating tumors and their associated metastases. The VACV F11 protein promotes virus exit and rapid spread by inhibiting Rho signalling, which results in a disruption of cortical actin. We have previously shown that although MYXV lacks an F11 homolog, the F11L gene can be introduced into MYXV promoting the spread of this Leporipoxvirus in natural host cells. Here we show that the F11-encoding (F11L(+)) MYXV strain replicates to higher levels in a number of human cancer cells. We also show that F11L(+) MYXV induces better tumor control and prolonged survival of mice bearing MDA-MB-231 cancer cells. Furthermore, we show that this virus also spreads more efficiently from the site of growth in one injected tumor, to a second untreated tumor. While we focused mostly on the use of a modified MYXV we were able to show that the effects of F11 on MYXV growth in cancer cells could be mimicked through the use of pharmacological inhibition or siRNA-mediated silencing of key regulators of cortical actin (RhoA, RhoC, mDia1, or LIMK2). These data suggest that it may be possible to increase the oncolytic efficacy of wild-type MYXV using chemical inhibitors of RhoA/C or their downstream targets. Furthermore, since all viruses must overcome barriers to exit posed by structures like cortical actin, these findings suggest that the oncolytic activity of other viruses may be enhanced through similar strategies.

  15. Myxoma Virus Oncolytic Efficiency Can Be Enhanced Through Chemical or Genetic Disruption of the Actin Cytoskeleton

    PubMed Central

    Irwin, Chad R.; Favis, Nicole A.; Agopsowicz, Kate C.; Hitt, Mary M.; Evans, David H.

    2013-01-01

    Myxoma virus (MYXV) is one of many animal viruses that exhibit oncolytic properties in transformed human cells. Compared to orthopoxviruses like vaccinia (VACV), MYXV spreads inefficiently, which could compromise its use in treating tumors and their associated metastases. The VACV F11 protein promotes virus exit and rapid spread by inhibiting Rho signalling, which results in a disruption of cortical actin. We have previously shown that although MYXV lacks an F11 homolog, the F11L gene can be introduced into MYXV promoting the spread of this Leporipoxvirus in natural host cells. Here we show that the F11-encoding (F11L+) MYXV strain replicates to higher levels in a number of human cancer cells. We also show that F11L+ MYXV induces better tumor control and prolonged survival of mice bearing MDA-MB-231 cancer cells. Furthermore, we show that this virus also spreads more efficiently from the site of growth in one injected tumor, to a second untreated tumor. While we focused mostly on the use of a modified MYXV we were able to show that the effects of F11 on MYXV growth in cancer cells could be mimicked through the use of pharmacological inhibition or siRNA-mediated silencing of key regulators of cortical actin (RhoA, RhoC, mDia1, or LIMK2). These data suggest that it may be possible to increase the oncolytic efficacy of wild-type MYXV using chemical inhibitors of RhoA/C or their downstream targets. Furthermore, since all viruses must overcome barriers to exit posed by structures like cortical actin, these findings suggest that the oncolytic activity of other viruses may be enhanced through similar strategies. PMID:24391902

  16. Oncolytic viruses as therapeutic cancer vaccines

    PubMed Central

    2013-01-01

    Oncolytic viruses (OVs) are tumor-selective, multi-mechanistic antitumor agents. They kill infected cancer and associated endothelial cells via direct oncolysis, and uninfected cells via tumor vasculature targeting and bystander effect. Multimodal immunogenic cell death (ICD) together with autophagy often induced by OVs not only presents potent danger signals to dendritic cells but also efficiently cross-present tumor-associated antigens from cancer cells to dendritic cells to T cells to induce adaptive antitumor immunity. With this favorable immune backdrop, genetic engineering of OVs and rational combinations further potentiate OVs as cancer vaccines. OVs armed with GM-CSF (such as T-VEC and Pexa-Vec) or other immunostimulatory genes, induce potent anti-tumor immunity in both animal models and human patients. Combination with other immunotherapy regimens improve overall therapeutic efficacy. Coadministration with a HDAC inhibitor inhibits innate immunity transiently to promote infection and spread of OVs, and significantly enhances anti-tumor immunity and improves the therapeutic index. Local administration or OV mediated-expression of ligands for Toll-like receptors can rescue the function of tumor-infiltrating CD8+ T cells inhibited by the immunosuppressive tumor microenvironment and thus enhances the antitumor effect. Combination with cyclophosphamide further induces ICD, depletes Treg, and thus potentiates antitumor immunity. In summary, OVs properly armed or in rational combinations are potent therapeutic cancer vaccines. PMID:24020520

  17. Oncolytic Seneca Valley Virus: past perspectives and future directions

    PubMed Central

    Burke, Michael J

    2016-01-01

    Seneca Valley Virus isolate 001 (SVV-001) is an oncolytic RNA virus of the Picornaviridae family. It is also the first picornavirus discovered of the novel genus Senecavirus. SVV-001 replicates through an RNA intermediate, bypassing a DNA phase, and is unable to integrate into the host genome. SVV-001 was originally discovered as a contaminant in the cell culture of fetal retinoblasts and has since been identified as a potent oncolytic virus against tumors of neuroendocrine origin. SVV-001 has a number of features that make it an attractive oncolytic virus, namely, its ability to target and penetrate solid tumors via intravenous administration, inability for insertional mutagenesis, and being a self-replicating RNA virus with selective tropism for cancer cells. SVV-001 has been studied in both pediatric and adult early phase studies reporting safety and some clinical efficacy, albeit primarily in adult tumors. This review summarizes the current knowledge of SVV-001 and what its future as an oncolytic virus may hold. PMID:27660749

  18. Oncolytic Seneca Valley Virus: past perspectives and future directions.

    PubMed

    Burke, Michael J

    2016-01-01

    Seneca Valley Virus isolate 001 (SVV-001) is an oncolytic RNA virus of the Picornaviridae family. It is also the first picornavirus discovered of the novel genus Senecavirus. SVV-001 replicates through an RNA intermediate, bypassing a DNA phase, and is unable to integrate into the host genome. SVV-001 was originally discovered as a contaminant in the cell culture of fetal retinoblasts and has since been identified as a potent oncolytic virus against tumors of neuroendocrine origin. SVV-001 has a number of features that make it an attractive oncolytic virus, namely, its ability to target and penetrate solid tumors via intravenous administration, inability for insertional mutagenesis, and being a self-replicating RNA virus with selective tropism for cancer cells. SVV-001 has been studied in both pediatric and adult early phase studies reporting safety and some clinical efficacy, albeit primarily in adult tumors. This review summarizes the current knowledge of SVV-001 and what its future as an oncolytic virus may hold.

  19. Active immunotherapy: oncolytic virus therapy using HSV-1.

    PubMed

    Todo, Tomoki

    2012-01-01

    Conditionally replicating herpes simplex viruses Type 1 (HSV-1) are promising therapeutic agents for glioma. They can replicate in situ, spread and exhibit oncolytic activity via a direct cytocidal effect. In addition, specific antitumor immunity is effectively induced in the course of oncolytic activities. G47Δ is a genetically engineered HSV-1 with triple mutations that realized augmented viral replication in tumor cells, strong induction of antitumor immunity and enhanced safety in normal tissues. A clinical trial of G47Δ in patients with recurrent glioblastoma has started in 2009. One of the advantages of HSV-1 is its capacity to incorporate large and/or multiple transgenes within the viral genome. In preclinical studies, "arming" of an oncolytic HSV-1 with transgenes encoding immunomodulatory molecules, such as interleukin 12, has been shown to greatly augment the efficacy of oncolytic HSV-1 therapy. Oncolytic virus therapy using HSV-1 may be a useful treatment for glioma that can also function as an efficient in situ tumor vaccination.

  20. Vaccinia Virus: A Tool for Research and Vaccine Development

    NASA Astrophysics Data System (ADS)

    Moss, Bernard

    1991-06-01

    Vaccinia virus is no longer needed for smallpox immunization, but now serves as a useful vector for expressing genes within the cytoplasm of eukaryotic cells. As a research tool, recombinant vaccinia viruses are used to synthesize biologically active proteins and analyze structure-function relations, determine the targets of humoral- and cell-mediated immunity, and investigate the immune responses needed for protection against specific infectious diseases. When more data on safety and efficacy are available, recombinant vaccinia and related poxviruses may be candidates for live vaccines and for cancer immunotherapy.

  1. Aptamer-facilitated Protection of Oncolytic Virus from Neutralizing Antibodies

    PubMed Central

    Muharemagic, Darija; Zamay, Anna; Ghobadloo, Shahrokh M; Evgin, Laura; Savitskaya, Anna; Bell, John C; Berezovski, Maxim V

    2014-01-01

    Oncolytic viruses promise to significantly improve current cancer treatments through their tumor-selective replication and multimodal attack against cancer cells. However, one of the biggest setbacks for oncolytic virus therapy is the intravenous delivery of the virus, as it can be cleared from the bloodstream by neutralizing antibodies before it reaches the tumor cells. We have selected DNA aptamers against an oncolytic virus, vesicular stomatitis virus, using a competitive binding approach, as well as against the antigen binding fragment (Fab) of antivesicular stomatitis virus polyclonal antibodies, in order to shield the virus from nAbs and enhance its in vivo survival. We used flow cytometry to identify these aptamers and evaluated their efficiency to shield vesicular stomatitis virus in a cell-based plaque forming assay. These oligonucleotides were then modified to obtain multivalent binders, which led to a decrease of viral aggregation, an increase in its infectivity and an increase in its stability in serum. The aptamers were also incubated in nondiluted serum, showing their effectiveness under conditions mimicking those in vivo. With this approach, we were able to increase viral infectivity by more than 70% in the presence of neutralizing antibodies. Thus, this method has the potential to enhance the delivery of vesicular stomatitis virus through the bloodstream without compromising the patient's immune system. PMID:24892725

  2. Big Data Offers Novel Insights for Oncolytic Virus Immunotherapy

    PubMed Central

    Swift, Stephanie L.; Stojdl, David F.

    2016-01-01

    Large-scale assays, such as microarrays, next-generation sequencing and various “omics” technologies, have explored multiple aspects of the immune response following virus infection, often from a public health perspective. Yet a lack of similar data exists for monitoring immune engagement during oncolytic virus immunotherapy (OVIT) in the cancer setting. Tracking immune signatures at the tumour site can create a snapshot or longitudinally analyse immune cell activation, infiltration and functionality within global populations or individual cells. Mapping immune changes over the course of oncolytic biotherapy—from initial infection to tumour stabilisation/regression through to long-term cure or escape/relapse—has the potential to generate important therapeutic insights around virus-host interactions. Further, correlating such immune signatures with specific tumour outcomes has significant value for guiding the development of novel oncolytic virus immunotherapy strategies. Here, we provide insights for OVIT from large-scale analyses of immune populations in the infection, vaccination and immunotherapy setting. We analyse several approaches to manipulating immune engagement during OVIT. We further explore immunocentric changes in the tumour tissue following immunotherapy, and compile several immune signatures of therapeutic success. Ultimately, we highlight clinically relevant large-scale approaches with the potential to strengthen future oncolytic strategies to optimally engage the immune system. PMID:26861383

  3. Big Data Offers Novel Insights for Oncolytic Virus Immunotherapy.

    PubMed

    Swift, Stephanie L; Stojdl, David F

    2016-02-05

    Large-scale assays, such as microarrays, next-generation sequencing and various "omics" technologies, have explored multiple aspects of the immune response following virus infection, often from a public health perspective. Yet a lack of similar data exists for monitoring immune engagement during oncolytic virus immunotherapy (OVIT) in the cancer setting. Tracking immune signatures at the tumour site can create a snapshot or longitudinally analyse immune cell activation, infiltration and functionality within global populations or individual cells. Mapping immune changes over the course of oncolytic biotherapy-from initial infection to tumour stabilisation/regression through to long-term cure or escape/relapse-has the potential to generate important therapeutic insights around virus-host interactions. Further, correlating such immune signatures with specific tumour outcomes has significant value for guiding the development of novel oncolytic virus immunotherapy strategies. Here, we provide insights for OVIT from large-scale analyses of immune populations in the infection, vaccination and immunotherapy setting. We analyse several approaches to manipulating immune engagement during OVIT. We further explore immunocentric changes in the tumour tissue following immunotherapy, and compile several immune signatures of therapeutic success. Ultimately, we highlight clinically relevant large-scale approaches with the potential to strengthen future oncolytic strategies to optimally engage the immune system.

  4. Complete Genome Sequence of the Oncolytic Sendai virus Strain Moscow

    PubMed Central

    Zainutdinov, Sergei S.; Tikunov, Artem Y.; Matveeva, Olga V.

    2016-01-01

    We report here the complete genome sequence of Sendai virus Moscow strain. Anecdotal evidence for the efficacy of oncolytic virotherapy exists for this strain. The RNA genome of the Moscow strain is 15,384 nucleotides in length and differs from the nearest strain, BB1, by 18 nucleotides and 11 amino acids. PMID:27516510

  5. Computational modeling approaches to the dynamics of oncolytic viruses

    PubMed Central

    Wodarz, Dominik

    2016-01-01

    Replicating oncolytic viruses represent a promising treatment approach against cancer, specifically targeting the tumor cells. Significant progress has been made through experimental and clinical studies. Besides these approaches, however, mathematical models can be useful when analyzing the dynamics of virus spread through tumors, because the interactions between a growing tumor and a replicating virus are complex and nonlinear, making them difficult to understand by experimentation alone. Mathematical models have provided significant biological insight into the field of virus dynamics, and similar approaches can be adopted to study oncolytic viruses. The review discusses this approach and highlights some of the challenges that need to be overcome in order to build mathematical and computation models that are clinically predictive. PMID:27001049

  6. Herpes Simplex Virus Oncolytic Therapy for Pediatric Malignancies

    PubMed Central

    Friedman, Gregory K; Pressey, Joseph G; Reddy, Alyssa T; Markert, James M; Gillespie, G Yancey

    2009-01-01

    Despite improving survival rates for children with cancer, a subset of patients exist with disease resistant to traditional therapies such as surgery, chemotherapy, and radiation. These patients require newer, targeted treatments used alone or in combination with more traditional approaches. Oncolytic herpes simplex virus (HSV) is one of these newer therapies that offer promise for several difficult to treat pediatric malignancies. The potential benefit of HSV therapy in pediatric solid tumors including brain tumors, neuroblastomas, and sarcomas is reviewed along with the many challenges that need to be addressed prior to moving oncolytic HSV therapy from the laboratory to the beside in the pediatric population. PMID:19367259

  7. Herpes simplex virus oncolytic therapy for pediatric malignancies.

    PubMed

    Friedman, Gregory K; Pressey, Joseph G; Reddy, Alyssa T; Markert, James M; Gillespie, G Yancey

    2009-07-01

    Despite improving survival rates for children with cancer, a subset of patients exist with disease resistant to traditional therapies such as surgery, chemotherapy, and radiation. These patients require newer, targeted treatments used alone or in combination with more traditional approaches. Oncolytic herpes simplex virus (HSV) is one of these newer therapies that offer promise for several difficult to treat pediatric malignancies. The potential benefit of HSV therapy in pediatric solid tumors including brain tumors, neuroblastomas, and sarcomas is reviewed along with the many challenges that need to be addressed prior to moving oncolytic HSV therapy from the laboratory to the beside in the pediatric population.

  8. Vaccinia Virus Vaccines: Past, Present and Future

    PubMed Central

    Jacobs, Bertram L.; Langland, Jeffrey O.; Kibler, Karen V.; Denzler, Karen L.; White, Stacy D.; Holechek, Susan A.; Wong, Shukmei; Huynh, Trung; Baskin, Carole R.

    2009-01-01

    Vaccinia virus (VACV) has been used more extensively for human immunization than any other vaccine. For almost two centuries, VACV was employed to provide cross-protection against variola virus, the causative agent of smallpox, until the disease was eradicated in the late 1970s. Since that time, continued research on VACV has produced a number of modified vaccines with improved safety profiles. Attenuation has been achieved through several strategies, including sequential passage in an alternative host, deletion of specific genes or genetic engineering of viral genes encoding immunomodulatory proteins. Some highly attenuated third- and fourth-generation VACV vaccines are now being considered for stockpiling against a possible re-introduction of smallpox through bioterrorism. Researchers have also taken advantage of the ability of the VACV genome to accommodate additional genetic material to produce novel vaccines against a wide variety of infectious agents, including a recombinant VACV encoding the rabies virus glycoprotein that is administered orally to wild animals. This review provides an in-depth examination of these successive generations of VACV vaccines, focusing on how the understanding of poxviral replication and viral gene function permits the deliberate modification of VACV immunogenicity and virulence. PMID:19563829

  9. Safety mechanism assisted by the repressor of tetracycline (SMART) vaccinia virus vectors for vaccines and therapeutics.

    PubMed

    Grigg, Patricia; Titong, Allison; Jones, Leslie A; Yilma, Tilahun D; Verardi, Paulo H

    2013-09-17

    Replication-competent viruses, such as Vaccinia virus (VACV), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. However, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. VACVs constitutively expressing interferon-γ (IFN-γ) replicate in cell culture indistinguishably from control viruses; however, they replicate in vivo to low or undetectable levels, and are rapidly cleared even in immunodeficient animals. In an effort to develop safe and highly effective replication-competent VACV vectors, we established a system to inducibly express IFN-γ. Our SMART (safety mechanism assisted by the repressor of tetracycline) vectors are designed to express the tetracycline repressor under a constitutive VACV promoter and IFN-γ under engineered tetracycline-inducible promoters. Immunodeficient SCID mice inoculated with VACVs not expressing IFN-γ demonstrated severe weight loss, whereas those given VACVs expressing IFN-γ under constitutive VACV promoters showed no signs of infection. Most importantly, mice inoculated with a VACV expressing the IFN-γ gene under an inducible promoter remained healthy in the presence of doxycycline, but exhibited severe weight loss in the absence of doxycycline. In this study, we developed a safety mechanism for VACV based on the conditional expression of IFN-γ under a tightly controlled tetracycline-inducible VACV promoter for use in vaccines and oncolytic cancer therapies.

  10. Zika virus has oncolytic activity against glioblastoma stem cells.

    PubMed

    Zhu, Zhe; Gorman, Matthew J; McKenzie, Lisa D; Chai, Jiani N; Hubert, Christopher G; Prager, Briana C; Fernandez, Estefania; Richner, Justin M; Zhang, Rong; Shan, Chao; Tycksen, Eric; Wang, Xiuxing; Shi, Pei-Yong; Diamond, Michael S; Rich, Jeremy N; Chheda, Milan G

    2017-10-02

    Glioblastoma is a highly lethal brain cancer that frequently recurs in proximity to the original resection cavity. We explored the use of oncolytic virus therapy against glioblastoma with Zika virus (ZIKV), a flavivirus that induces cell death and differentiation of neural precursor cells in the developing fetus. ZIKV preferentially infected and killed glioblastoma stem cells (GSCs) relative to differentiated tumor progeny or normal neuronal cells. The effects against GSCs were not a general property of neurotropic flaviviruses, as West Nile virus indiscriminately killed both tumor and normal neural cells. ZIKV potently depleted patient-derived GSCs grown in culture and in organoids. Moreover, mice with glioblastoma survived substantially longer and at greater rates when the tumor was inoculated with a mouse-adapted strain of ZIKV. Our results suggest that ZIKV is an oncolytic virus that can preferentially target GSCs; thus, genetically modified strains that further optimize safety could have therapeutic efficacy for adult glioblastoma patients. © 2017 Zhu et al.

  11. Genetic Modification of Oncolytic Newcastle Disease Virus for Cancer Therapy.

    PubMed

    Cheng, Xing; Wang, Weijia; Xu, Qi; Harper, James; Carroll, Danielle; Galinski, Mark S; Suzich, JoAnn; Jin, Hong

    2016-06-01

    Clinical development of a mesogenic strain of Newcastle disease virus (NDV) as an oncolytic agent for cancer therapy has been hampered by its select agent status due to its pathogenicity in avian species. Using reverse genetics, we have generated a lead candidate oncolytic NDV based on the mesogenic NDV-73T strain that is no longer classified as a select agent for clinical development. This recombinant NDV has a modification at the fusion protein (F) cleavage site to reduce the efficiency of F protein cleavage and an insertion of a 198-nucleotide sequence into the HN-L intergenic region, resulting in reduced viral gene expression and replication in avian cells but not in mammalian cells. In mammalian cells, except for viral polymerase (L) gene expression, viral gene expression is not negatively impacted or increased by the HN-L intergenic insertion. Furthermore, the virus can be engineered to express a foreign gene while still retaining the ability to grow to high titers in cell culture. The recombinant NDV selectively replicates in and kills tumor cells and is able to drive potent tumor growth inhibition following intratumoral or intravenous administration in a mouse tumor model. The candidate is well positioned for clinical development as an oncolytic virus. Avian paramyxovirus type 1, NDV, has been an attractive oncolytic agent for cancer virotherapy. However, this virus can cause epidemic disease in poultry, and concerns about the potential environmental and economic impact of an NDV outbreak have precluded its clinical development. Here we describe generation and characterization of a highly potent oncolytic NDV variant that is unlikely to cause Newcastle disease in its avian host, representing an essential step toward moving NDV forward as an oncolytic agent. Several attenuation mechanisms have been genetically engineered into the recombinant NDV that reduce chicken pathogenicity to a level that is acceptable worldwide without impacting viral production in

  12. Oncolytic virotherapy for oral squamous cell carcinoma using replication-competent viruses.

    PubMed

    Saito, Kengo; Shirasawa, Hiroshi; Isegawa, Naohisa; Shiiba, Masashi; Uzawa, Katsuhiro; Tanzawa, Hideki

    2009-12-01

    Oncolytic virotherapy utilizes viruses that can selectively destroy cancer cells without harming normal tissues. Clinical trials of oncolytic viruses show that most oncolytic agents are well tolerated and safe. The virotherapeutic agents currently in use have limited potency when administered alone; however, combination therapy using virotherapeutic agents and conventional anticancer agents, such as chemotherapeutics, radiation, and gene therapy, exhibits encouraging levels of efficacy. Advances in recombinant DNA technology have allowed the development of viruses that are tumor-selective and armed with transgenes, increasing the application potential and efficacy of this novel anticancer therapy. Here, we review the development of oncolytic viruses and the clinical trials of oncolytic virotherapy for oral cancers. We discuss current issues and perspectives of this evolving anticancer therapy, highlighting the potential applications of a unique, naturally occurring oncolytic virus, Sindbis virus.

  13. Cryo-electron tomography of vaccinia virus

    PubMed Central

    Cyrklaff, Marek; Risco, Cristina; Fernández, Jose Jesús; Jiménez, Maria Victoria; Estéban, Mariano; Baumeister, Wolfgang; Carrascosa, José L.

    2005-01-01

    The combination of cryo-microscopy and electron tomographic reconstruction has allowed us to determine the structure of one of the more complex viruses, intracellular mature vaccinia virus, at a resolution of 4–6 nm. The tomographic reconstruction allows us to dissect the different structural components of the viral particle, avoiding projection artifacts derived from previous microscopic observations. A surface-rendering representation revealed brick-shaped viral particles with slightly rounded edges and dimensions of ≈360 × 270 × 250 nm. The outer layer was consistent with a lipid membrane (5–6 nm thick), below which usually two lateral bodies were found, built up by a heterogeneous material without apparent ordering or repetitive features. The internal core presented an inner cavity with electron dense coils of presumptive DNA–protein complexes, together with areas of very low density. The core was surrounded by two layers comprising an overall thickness of ≈18–19 nm; the inner layer was consistent with a lipid membrane. The outer layer was discontinuous, formed by a periodic palisade built by the side interaction of T-shaped protein spikes that were anchored in the lower membrane and were arranged into small hexagonal crystallites. It was possible to detect a few pore-like structures that communicated the inner side of the core with the region outside the layer built by the T-shaped spike palisade. PMID:15699328

  14. Modelling Spread of Oncolytic Viruses in Heterogeneous Cell Populations

    NASA Astrophysics Data System (ADS)

    Ellis, Michael; Dobrovolny, Hana

    2014-03-01

    One of the most promising areas in current cancer research and treatment is the use of viruses to attack cancer cells. A number of oncolytic viruses have been identified to date that possess the ability to destroy or neutralize cancer cells while inflicting minimal damage upon healthy cells. Formulation of predictive models that correctly describe the evolution of infected tumor systems is critical to the successful application of oncolytic virus therapy. A number of different models have been proposed for analysis of the oncolytic virus-infected tumor system, with approaches ranging from traditional coupled differential equations such as the Lotka-Volterra predator-prey models, to contemporary modeling frameworks based on neural networks and cellular automata. Existing models are focused on tumor cells and the effects of virus infection, and offer the potential for improvement by including effects upon normal cells. We have recently extended the traditional framework to a 2-cell model addressing the full cellular system including tumor cells, normal cells, and the impacts of viral infection upon both populations. Analysis of the new framework reveals complex interaction between the populations and potential inability to simultaneously eliminate the virus and tumor populations.

  15. A Fusogenic Oncolytic Herpes Simplex Virus for Therapy of Advanced Ovarian Cancer

    DTIC Science & Technology

    2007-06-01

    AD_________________ Award Number: DAMD17-03-1-0434 TITLE: A Fusogenic Oncolytic Herpes Simplex ...TITLE AND SUBTITLE 5a. CONTRACT NUMBER A Fusogenic Oncolytic Herpes Simplex Virus for Therapy of Advanced Ovarian Cancer 5b. GRANT NUMBER...oncolytic herpes simplex virus (HSV) can significantly enhance the anti-tumor effect of the virus. Three specific aims have been proposed and they are: 1

  16. Myxoma and vaccinia viruses exploit different mechanisms to enter and infect human cancer cells

    SciTech Connect

    Villa, Nancy Y.; Bartee, Eric; Mohamed, Mohamed R.; Rahman, Masmudur M.; Barrett, John W.; McFadden, Grant

    2010-06-05

    Myxoma (MYXV) and vaccinia (VACV) viruses have recently emerged as potential oncolytic agents that can infect and kill different human cancer cells. Although both are structurally similar, it is unknown whether the pathway(s) used by these poxviruses to enter and cause oncolysis in cancer cells are mechanistically similar. Here, we compared the entry of MYXV and VACV-WR into various human cancer cells and observed significant differences: 1 - low-pH treatment accelerates fusion-mediated entry of VACV but not MYXV, 2 - the tyrosine kinase inhibitor genistein inhibits entry of VACV, but not MYXV, 3 - knockdown of PAK1 revealed that it is required for a late stage event downstream of MYXV entry into cancer cells, whereas PAK1 is required for VACV entry into the same target cells. These results suggest that VACV and MYXV exploit different mechanisms to enter into human cancer cells, thus providing some rationale for their divergent cancer cell tropisms.

  17. Myxoma and vaccinia viruses exploit different mechanisms to enter and infect human cancer cells.

    PubMed

    Villa, Nancy Y; Bartee, Eric; Mohamed, Mohamed R; Rahman, Masmudur M; Barrett, John W; McFadden, Grant

    2010-06-05

    Myxoma (MYXV) and vaccinia (VACV) viruses have recently emerged as potential oncolytic agents that can infect and kill different human cancer cells. Although both are structurally similar, it is unknown whether the pathway(s) used by these poxviruses to enter and cause oncolysis in cancer cells are mechanistically similar. Here, we compared the entry of MYXV and VACV-WR into various human cancer cells and observed significant differences: 1--low-pH treatment accelerates fusion-mediated entry of VACV but not MYXV, 2--the tyrosine kinase inhibitor genistein inhibits entry of VACV, but not MYXV, 3--knockdown of PAK1 revealed that it is required for a late stage event downstream of MYXV entry into cancer cells, whereas PAK1 is required for VACV entry into the same target cells. These results suggest that VACV and MYXV exploit different mechanisms to enter into human cancer cells, thus providing some rationale for their divergent cancer cell tropisms.

  18. Zoonotic Vaccinia Virus Infection in Brazil: Clinical Description and Implications for Health Professionals▿

    PubMed Central

    de Souza Trindade, Giliane; Drumond, Betania Paiva; Guedes, Maria Isabel Maldonado Coelho; Leite, Juliana Almeida; Mota, Bruno Eduardo Fernandes; Campos, Marco Antônio; da Fonseca, Flávio Guimarães; Nogueira, Maurício Lacerda; Lobato, Zélia Inês Portela; Bonjardim, Cláudio Antônio; Ferreira, Paulo César Peregrino; Kroon, Erna Geessien

    2007-01-01

    Bovine vaccinia virus outbreaks have been occurring in different regions of Brazil. We report here the time course of natural human infection by vaccinia virus and describe important clinical and epidemiological aspects of this zoonotic infection. The diagnosis of vaccinia virus infection was based on clinical, serological, and molecular procedures. PMID:17287326

  19. Detection of Vaccinia Virus in Dairy Cattle Serum Samples from 2009, Uruguay

    PubMed Central

    Franco-Luiz, Ana Paula Moreira; Oliveira, Danilo Bretas; Pereira, Alexandre Fagundes; Gasparini, Mirela Cristina Soares; Bonjardim, Cláudio Antônio; Ferreira, Paulo César Peregrino; Trindade, Giliane de Souza; Puentes, Rodrigo; Furtado, Agustin; Abrahão, Jônatas Santos

    2016-01-01

    We detected orthopoxvirus in 28 of 125 serum samples collected during 2009 from cattle in Uruguay. Two samples were PCR-positive for vaccinia virus and had sequences similar to those for vaccinia virus associated with outbreaks in Brazil. Autochthonous circulation of vaccinia virus in Uruguay and other South American countries cannot be ruled out. PMID:27869601

  20. Antitumor efficacy of vaccinia virus-modified tumor cell vaccine

    SciTech Connect

    Ito, T.; Wang, D.Q.; Maru, M.; Nakajima, K.; Kato, S.; Kurimura, T.; Wakamiya, N. )

    1990-11-01

    The antitumor efficacies of vaccinia virus-modified tumor cell vaccines were examined in murine syngeneic MH134 and X5563 tumor cells. UV-inactivated vaccinia virus was inoculated i.p. into C3H/HeN mice that had received whole body X-irradiation at 150 rads. After 3 weeks, the vaccines were administered i.p. 3 times at weekly intervals. One week after the last injection, mice were challenged i.p. with various doses of syngeneic MH134 or X5563 viable tumor cells. Four methods were used for preparing tumor cell vaccines: X-ray irradiation; fixation with paraformaldehyde for 1 h or 3 months; and purification of the membrane fraction. All four vaccines were effective, but the former two vaccines were the most effective. A mixture of the membrane fraction of untreated tumor cells and UV-inactivated vaccinia virus also had an antitumor effect. These results indicate that vaccine with the complete cell structure is the most effective. The membrane fraction of UV-inactivated vaccinia virus-absorbed tumor cells was also effective. UV-inactivated vaccinia virus can react with not only intact tumor cells but also the purified membrane fraction of tumor cells and augment antitumor activity.

  1. Susceptibility of different leukocyte cell types to Vaccinia virus infection

    PubMed Central

    Sánchez-Puig, Juana M; Sánchez, Laura; Roy, Garbiñe; Blasco, Rafael

    2004-01-01

    Background Vaccinia virus, the prototype member of the family Poxviridae, was used extensively in the past as the Smallpox vaccine, and is currently considered as a candidate vector for new recombinant vaccines. Vaccinia virus has a wide host range, and is known to infect cultures of a variety of cell lines of mammalian origin. However, little is known about the virus tropism in human leukocyte populations. We report here that various cell types within leukocyte populations have widely different susceptibility to infection with vaccinia virus. Results We have investigated the ability of vaccinia virus to infect human PBLs by using virus recombinants expressing green fluorescent protein (GFP), and monoclonal antibodies specific for PBL subpopulations. Flow cytometry allowed the identification of infected cells within the PBL mixture 1–5 hours after infection. Antibody labeling revealed that different cell populations had very different infection rates. Monocytes showed the highest percentage of infected cells, followed by B lymphocytes and NK cells. In contrast to those cell types, the rate of infection of T lymphocytes was low. Comparison of vaccinia virus strains WR and MVA showed that both strains infected efficiently the monocyte population, although producing different expression levels. Our results suggest that MVA was less efficient than WR in infecting NK cells and B lymphocytes. Overall, both WR and MVA consistently showed a strong preference for the infection of non-T cells. Conclusions When infecting fresh human PBL preparations, vaccinia virus showed a strong bias towards the infection of monocytes, followed by B lymphocytes and NK cells. In contrast, very poor infection of T lymphocytes was detected. These finding may have important implications both in our understanding of poxvirus pathogenesis and in the development of improved smallpox vaccines. PMID:15555076

  2. Measles to the Rescue: A Review of Oncolytic Measles Virus

    PubMed Central

    Aref, Sarah; Bailey, Katharine; Fielding, Adele

    2016-01-01

    Oncolytic virotherapeutic agents are likely to become serious contenders in cancer treatment. The vaccine strain of measles virus is an agent with an impressive range of oncolytic activity in pre-clinical trials with increasing evidence of safety and efficacy in early clinical trials. This paramyxovirus vaccine has a proven safety record and is amenable to careful genetic modification in the laboratory. Overexpression of the measles virus (MV) receptor CD46 in many tumour cells may direct the virus to preferentially enter transformed cells and there is increasing awareness of the importance of nectin-4 and signaling lymphocytic activation molecule (SLAM) in oncolysis. Successful attempts to retarget MV by inserting genes for tumour-specific ligands to antigens such as carcinoembryonic antigen (CEA), CD20, CD38, and by engineering the virus to express synthetic microRNA targeting sequences, and “blinding” the virus to the natural viral receptors are exciting measures to increase viral specificity and enhance the oncolytic effect. Sodium iodine symporter (NIS) can also be expressed by MV, which enables in vivo tracking of MV infection. Radiovirotherapy using MV-NIS, chemo-virotherapy to convert prodrugs to their toxic metabolites, and immune-virotherapy including incorporating antibodies against immune checkpoint inhibitors can also increase the oncolytic potential. Anti-viral host immune responses are a recognized barrier to the success of MV, and approaches such as transporting MV to the tumour sites by carrier cells, are showing promise. MV Clinical trials are producing encouraging preliminary results in ovarian cancer, myeloma and cutaneous non-Hodgkin lymphoma, and the outcome of currently open trials in glioblastoma multiforme, mesothelioma and squamous cell carcinoma are eagerly anticipated. PMID:27782084

  3. Oncolytic Measles Virus Strains as Novel Anticancer Agents

    PubMed Central

    Msaouel, Pavlos; Opyrchal, Mateusz; Domingo Musibay, Evidio; Galanis, Evanthia

    2013-01-01

    Introduction Replication-competent oncolytic measles virus (MV) strains preferentially infect and destroy a wide variety of cancer tissues. Clinical translation of engineered attenuated MV vaccine derivatives is demonstrating the therapeutic potential and negligible pathogenicity of these strains in humans. Areas covered The present review summarizes the mechanisms of MV tumor selectivity and cytopathic activity as well as the current data on the oncolytic efficacy and preclinical testing of MV strains. Investigational strategies to reprogram MV selectivity, escape antiviral immunity and modulate the immune system to enhance viral delivery and tumor oncolysis are also discussed. Expert Opinion Clinical viral kinetic data derived from non-invasive monitoring of reporter transgene expression will guide future protocols to enhance oncolytic MV efficacy. Anti-measles immunity is a major challenge of measles-based therapeutics and various strategies are being investigated to modulate immunity. These include the combination of MV therapy with immunosuppressive drugs such as cyclophosphamide, the use of cell carriers and the introduction of immunomodulatory transgenes and wild-type virulence genes. Available MV retargeting technologies can address safety considerations that may arise as more potent oncolytic MV vectors are being developed. PMID:23289598

  4. Vaccinia virus vectors: new strategies for producing recombinant vaccines.

    PubMed Central

    Hruby, D E

    1990-01-01

    The development and continued refinement of techniques for the efficient insertion and expression of heterologous DNA sequences from within the genomic context of infectious vaccinia virus recombinants are among the most promising current approaches towards effective immunoprophylaxis against a variety of protozoan, viral, and bacterial human pathogens. Because of its medical relevance, this area is the subject of intense research interest and has evolved rapidly during the past several years. This review (i) provides an updated overview of the technology that exists for assembling recombinant vaccinia virus strains, (ii) discusses the advantages and disadvantages of these approaches, (iii) outlines the areas of outgoing research directed towards overcoming the limitations of current techniques, and (iv) provides some insight (i.e., speculation) about probable future refinements in the use of vaccinia virus as a vector. PMID:2187593

  5. Vaccinia virus as a subhelper for AAV replication and packaging.

    PubMed

    Moore, Andrea R; Dong, Biao; Chen, Lingxia; Xiao, Weidong

    2015-01-01

    Adeno-associated virus (AAV) has been widely used as a gene therapy vector to treat a variety of disorders. While these vectors are increasingly popular and successful in the clinic, there is still much to learn about the viruses. Understanding the biology of these viruses is essential in engineering better vectors and generating vectors more efficiently for large-scale use. AAV requires a helper for production and replication making this aspect of the viral life cycle crucial. Vaccinia virus (VV) has been widely cited as a helper virus for AAV. However, to date, there are no detailed analyses of its helper function. Here, the helper role of VV was studied in detail. In contrast to common belief, we demonstrated that VV was not a sufficient helper virus for AAV replication. Vaccinia failed to produce rAAV and activate AAV promoters. While this virus could not support rAAV production, Vaccinia could initiate AAV replication and packaging when AAV promoter activation is not necessary. This activity is due to the ability of Vaccinia-driven Rep78 to transcribe in the cytoplasm and subsequently translate in the nucleus and undergo typical functions in the AAV life cycle. As such, VV is subhelper for AAV compared to complete helper functions of adenovirus.

  6. Oncolytic herpes viruses, chemotherapeutics, and other cancer drugs

    PubMed Central

    Braidwood, Lynne; Graham, Sheila V; Graham, Alex; Conner, Joe

    2013-01-01

    Oncolytic viruses are emerging as a potential new way of treating cancers. They are selectively replication-competent viruses that propagate only in actively dividing tumor cells but not in normal cells and, as a result, destroy the tumor cells by consequence of lytic infection. At least six different oncolytic herpes simplex viruses (oHSVs) have undergone clinical trials worldwide to date, and they have demonstrated an excellent safety profile and intimations of efficacy. The first pivotal Phase III trial with an oHSV, talimogene laherparepvec (T-Vec [OncoVexGM-CSF]), is almost complete, with extremely positive early results reported. Intuitively, therapeutically beneficial interactions between oHSV and chemotherapeutic and targeted therapeutic drugs would be limited as the virus requires actively dividing cells for maximum replication efficiency and most anticancer agents are cytotoxic or cytostatic. However, combinations of such agents display a range of responses, with antagonistic, additive, or, perhaps most surprisingly, synergistic enhancement of antitumor activity. When synergistic interactions in cancer cell killing are observed, chemotherapy dose reductions that achieve the same overall efficacy may be possible, resulting in a valuable reduction of adverse side effects. Therefore, the combination of an oHSV with “standard-of-care” drugs makes a logical and reasonable approach to improved therapy, and the addition of a targeted oncolytic therapy with “standard-of-care” drugs merits further investigation, both preclinically and in the clinic. Numerous publications report such studies of oncolytic HSV in combination with other drugs, and we review their findings here. Viral interactions with cellular hosts are complex and frequently involve intracellular signaling networks, thus creating diverse opportunities for synergistic or additive combinations with many anticancer drugs. We discuss potential mechanisms that may lead to synergistic interactions

  7. Oncolytic viruses & their specific targeting to tumour cells

    PubMed Central

    Singh, Prafull K.; Doley, Juwar; Kumar, G. Ravi; Sahoo, A.P.; Tiwari, Ashok K.

    2012-01-01

    Cancer is one of the major causes of death worldwide. In spite of achieving significant successes in medical sciences in the past few decades, the number of deaths due to cancer remains unchecked. The conventional chemotherapy and radiotherapy have limited therapeutic index and a plethora of treatment related side effects. This situation has provided an impetus for search of novel therapeutic strategies that can selectively destroy the tumour cells, leaving the normal cells unharmed. Viral oncotherapy is such a promising treatment modality that offers unique opportunity for tumour targeting. Numerous viruses with inherent anti-cancer activity have been identified and are in different phases of clinical trials. In the era of modern biotechnology and with better understanding of cancer biology and virology, it has become feasible to engineer the oncolytic viruses (OVs) to increase their tumour selectivity and enhance their oncolytic activity. In this review, the mechanisms by which oncolytic viruses kill the tumour cells have been discussed as also the development made in virotherapy for cancer treatment with emphasis on their tumour specific targeting. PMID:23168697

  8. Oncolytic Virus Therapy of Glioblastoma Multiforme – Concepts and Candidates

    PubMed Central

    Wollmann, Guido; Ozduman, Koray; van den Pol, Anthony N.

    2012-01-01

    Twenty years of oncolytic virus (OV) development have created a field that is driven by the potential promise of lasting impact on our cancer treatment repertoire. With the field constantly expanding – over 20 viruses have been recognized as potential OVs – new virus candidates continue to emerge even as established viruses reach clinical trials. They all share the defining commonalities of selective replication in tumors, subsequent tumor cell lysis, and dispersion within the tumor. Members from diverse virus classes with distinctly different biologies and host species have been identified. Of these viruses, 15 have been tested on human glioblastoma multiforme (GBM). So far, 20 clinical trials have been conducted or initiated using attenuated strains of 7 different oncolytic viruses against GBM. In this review, we present an overview of viruses that have been developed or considered for GBM treatment. We outline the principles of tumor targeting and selective viral replication, which include mechanisms of tumor-selective binding, and molecular elements usurping cellular biosynthetic machinery in transformed cells. Results from clinical trials have clearly established the proof of concept and have confirmed the general safety of OV application in the brain. The moderate clinical efficacy has not yet matched the promising preclinical lab results; next-generation OVs that are either “armed” with therapeutic genes or that are embedded in a multimodality treatment regimen should enhance the clinical results. PMID:22290260

  9. Deletion of the vaccinia virus F13L gene results in a highly attenuated virus that mounts a protective immune response against subsequent vaccinia virus challenge.

    PubMed

    Vliegen, Inge; Yang, Guang; Hruby, Dennis; Jordan, Robert; Neyts, Johan

    2012-01-01

    Vaccinia virus F13L encodes the envelope protein p37, which is the target of the anti-pox virus drug ST-246 (Yang et al., 2005) and that is required for production of extracellular vaccinia virus. The F13L (p37)-deleted (and ST-246 resistant) vaccinia virus recombinant (Vac-ΔF13L) produced smaller plaques than the wild-type vaccinia (Western Reserve vaccinia). In addition, Vac-ΔF13L proved, when inoculated either intravenously or intracutaneously in both immunocompetent and immunodeficient (athymic nude or SCID) mice, to be severely attenuated. Intravenous or intracutaneous inoculation of immunocompetent mice with the ΔF13L virus efficiently protected against a subsequent intravenous, intracutaneous or intranasal challenge with vaccinia WR (Western Reserve). This was corroborated by the observation that Vac-ΔF13L induced a humoral immune response against vaccinia following either intravenous or intracutaneous challenge. In conclusion, F13L-deleted vaccinia virus may have the potential to be developed as a smallpox vaccine. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. UNIT 14A.4 Generation of Recombinant Vaccinia Viruses

    PubMed Central

    Earl, Patricia L.; Moss, Bernard; Wyatt, Linda S.

    2016-01-01

    This unit describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector or PCR fragment to generate a recombinant virus. Selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses are described. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented. This unit first describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector or PCR fragment to generate a recombinant virus (see Basic Protocol 1). Also presented are selection and screening methods used to isolate recombinant viruses (see Basic Protocol 2) and a method for the amplification of recombinant viruses (see Basic Protocol 3). Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented (see Basic Protocol 4). HeLa S3 cells are recommended for large-scale growth of vaccinia virus. BS-C-1 cells may be used for xanthine-guanine phosphoribosyltransferase (XGPRT) and plaque size selection, fluorescent protein screening, transfection and determination of virus titer (UNIT 14A.3). For thymidine kinase (TK) selection, HuTK− 143B cells are used. With MVA, all steps are carried out in CEF or BHK-21 cells (UNIT 14A.3). CAUTION Proceed carefully and follow biosafety level 2 (BL-2) practices when working with standard vaccinia virus (see UNIT 14A.3 for safety precautions). [*Copy Editor: The original CPMB unit referenced CPMB Unit 16.15 for safety. The chapter editor asked that the authors include some of the safety information in the revised units – CPMB 16.16 and 16.17 – which are now CPMC Unit 14A.3 and 14A.4. As a result, the authors changed the safety citation here to “Unit 14A.3”, which doesn’t have nearly as much information as the original CPMB Unit 16.15. Perhaps the

  11. Detection of Vaccinia Virus DNA, but not Infectious Virus, in the Blood of Smallpox Vaccine Recipients

    PubMed Central

    Cohen, Jeffrey I.; Hohman, Patricia; Preuss, Jeanne C.; Li, Li; Fischer, Steven H.; Fedorko, Daniel P.

    2007-01-01

    The authors of a recent study suggested that the duration of deferral for blood donations by smallpox vaccinees should be extended, based on detection of vaccinia virus DNA in 5 blood samples by PCR and the potential for viremia. We found that 4 of 202 blood specimens (from 3 of 27 smallpox vaccinees) were positive for vaccinia virus DNA by PCR; none were positive for virus by culture. Throat swabs were negative by PCR and culture. Thus, while some blood specimens contained vaccinia virus DNA, infectious virus was not detected. Current guidelines for deferral of blood donation in vaccinees seem appropriate. PMID:17493714

  12. Oncolytic Viruses in the Treatment of Bladder Cancer

    PubMed Central

    Potts, Kyle G.; Hitt, Mary M.; Moore, Ronald B.

    2012-01-01

    Bladder carcinoma is the second most common malignancy of the urinary tract. Up to 85% of patients with bladder cancer are diagnosed with a tumor that is limited to the bladder mucosa (Ta, T1, and CIS). These stages are commonly termed as non-muscle-invasive bladder cancer (NMIBC). Although the treatment of NMIBC has greatly improved in recent years, there is a need for additional therapies when patients fail bacillus Calmette-Guérin (BCG) and chemotherapeutic agents. We propose that bladder cancer may be an ideal target for oncolytic viruses engineered to selectively replicate in and lyse tumor cells leaving normal cells unharmed. In support of this hypothesis, here we review current treatment strategies for bladder cancer and their shortcomings, as well as recent advancements in oncolytic viral therapy demonstrating encouraging safety profiles and antitumor activity. PMID:22899907

  13. Exploiting diversity: genetic approaches to creating highly potent and efficacious oncolytic viruses.

    PubMed

    Bauzon, Maxine; Hermiston, Terry W

    2008-08-01

    Oncolytic viruses possess several key attributes that make them a highly attractive treatment for cancer. They exhibit clinically validated synergy with chemotherapy and an ability to selectively destroy tumor cells to the exclusion of normal cells. Oncolytic viruses can replicate and, therefore, amplify their dose in a tumor-dependent manner. In addition, they can be genetically manipulated to include additional therapeutic factors to create a multimodal anticancer agent. These characteristics lead to the expectation that oncolytic viruses will serve as an additional tool in the treatment repertoire of clinical oncologists. In their clinical development to date, these agents were safe and well tolerated, but lacked efficacy as monotherapies. In this review, three genetic-based methods to increase the potency and efficacy of oncolytic viruses, in which human adenovirus is utilized as an example of a prototype oncolytic virus, are discussed.

  14. Cell carriers for oncolytic viruses: Fed Ex for cancer therapy.

    PubMed

    Willmon, Candice; Harrington, Kevin; Kottke, Timothy; Prestwich, Robin; Melcher, Alan; Vile, Richard

    2009-10-01

    Oncolytic viruses delivered directly into the circulation face many hazards that impede their localization to, and infection of, metastatic tumors. Such barriers to systemic delivery could be overcome if couriers, which confer both protection, and tumor localization, to their viral cargoes, could be found. Several preclincal studies have shown that viruses can be loaded into, or onto, different types of cells without losing the biological activity of either virus or cell carrier. Importantly, such loading can significantly protect the viruses from immune-mediated virus-neutralizing activities, including antiviral antibody. Moreover, an impressive portfolio of cellular vehicles, which have some degree of tropism for tumor cells themselves, or for the biological properties associated with the tumor stroma, is already available. Therefore, it will soon be possible to initiate clinical protocols to test the hypopthesis that cell-mediated delivery can permit efficient shipping of oncolytic viruses from the loading bay (the production laboratory) directly to the tumor in immune-competent patients with metastatic disease.

  15. Detection of Vaccinia Virus in Urban Domestic Cats, Brazil

    PubMed Central

    Costa, Galileu Barbosa; Miranda, Júlia Bahia; Almeida, Gregório Guilherme; Silva de Oliveira, Jaqueline; Pinheiro, Mariana Siqueira; Gonçalves, Stefanne Aparecida; Pimenta dos Reis, Jenner Karlisson; Gonçalves, Ricardo; Ferreira, Paulo César Peregrino; Bonjardim, Cláudio Antônio; Abrahão, Jônatas Santos; Kroon, Erna Geessien

    2017-01-01

    We investigated possible vaccinia virus (VACV) in urban house cats in Brazil. Serum samples from 6 cats were positive for VACV by PCR, indicating likely VACV circulation among house cats in urban areas of Brazil. This finding highlights the importance of epidemiologic surveillance to avoid outbreaks among urban human populations. PMID:28098542

  16. [Enhancement of epidermal regeneration by recombinant vaccinia virus growth factor].

    PubMed

    Petrov, V S; Cheshenko, I O; Omigov, V V; Azaev, M Sh; Krendel'shchikov, A V; Ovechkina, L G; Cheshenko, N V; Malygin, E G

    1998-01-01

    Examining the specific activity has showed that recombinant vaccinia virus growth factor binds to appropriate receptors on the A-431 cell surface and prompts the healing acceleration of degree III burns in rats. This recombinant factor did not demonstrate pyrogenicity or toxicogenicity in tests on rabbits, guinea-pits, noninbred albino mice.

  17. Identification of Genetically Modified Maraba Virus as an Oncolytic Rhabdovirus

    PubMed Central

    Brun, Jan; McManus, Dan; Lefebvre, Charles; Hu, Kang; Falls, Theresa; Atkins, Harold; Bell, John C; McCart, J. Andrea; Mahoney, Douglas; Stojdl, David F

    2010-01-01

    To expand our current array of safe and potent oncolytic viruses, we screened a variety of wild-type (WT) rhabdoviruses against a panel of tumor cell lines. Our screen identified a number of viruses with varying degrees of killing activity. Maraba virus was the most potent of these strains. We built a recombinant system for the Maraba virus platform, engineered a series of attenuating mutations to expand its therapeutic index, and tested their potency in vitro and in vivo. A double mutant (MG1) strain containing both G protein (Q242R) and M protein (L123W) mutations attenuated Maraba virus in normal diploid cell lines, yet appeared to be hypervirulent in cancer cells. This selective attenuation was mediated through interferon (IFN)-dependent and -independent mechanisms. Finally, the Maraba MG1 strain had a 100-fold greater maximum tolerable dose (MTD) than WT Maraba in vivo and resulted in durable cures when systemically administered in syngeneic and xenograft models. In summary, we report a potent new oncolytic rhabdovirus platform with unique tumor-selective attenuating mutations. PMID:20551913

  18. Integrating the biological characteristics of oncolytic viruses and immune cells can optimize therapeutic benefits of cell-based delivery.

    PubMed

    Thorne, S H; Contag, C H

    2008-05-01

    Despite significant advances in the development of tumor-selective agents, strategies for effective delivery of these agents across biological barriers to cells within the tumor microenvironment has been limiting. One tactical approach to overcoming biological barriers is to use cells as delivery vehicles, and a variety of different cell types have been investigated with a range of agents. In addition to transporting agents with targeted delivery, cells can also produce their own tumoricidal effect, conceal a payload from an immune response, amplify a selective agent at the target site and facilitate an antitumor immune response. We have reported a therapeutic combination consisting of cytokine induced killer cells and an oncolytic vaccinia virus with many of these features that led to therapeutic synergy in animal models of human cancer. The synergy was due to the interaction of the two agents to enhance the antitumor benefits of each individual component. As both of these agents display broad tumor-targeting potential and possess unique tumor killing mechanisms, together they were able to recognize and destroy a far greater number of malignant cells within the heterogeneous tumor than either agent alone. Effective cancer therapy will require recognition and elimination of the root of the disease, the cancer stem cell, and the combination of CIK cells and oncolytic vaccinia viruses has this potential. To create effective tumor-selective agents the viruses are modified to take advantage of the unique biology of the cancer cell. Similarly, if we are to develop targeted therapies that are sufficiently multifaceted to eliminate cancer cells at all stages of disease, we should integrate the virus into the unique biology of the cell delivery vehicle.

  19. Hydroxyurea-resistant vaccinia virus: overproduction of ribonucleotide reductase

    SciTech Connect

    Slabaugh, M.B.; Mathews, C.K.

    1986-11-01

    Repeated passage of vaccinia virus in increasing concentrations of hydroxyurea followed by plaque purification resulted in the isolation of variants capable of growth in 5 mM hydroxyurea, a drug concentration which inhibited the reproduction of wild-type vaccinia virus 1000-fold. Analyses of viral protein synthesis by using (/sup 35/S)methionine pulse-labeling at intervals throughout the infection cycle revealed that all isolates overproduced a 34,000-molecular-weight (MW) early polypeptide. Measurement of ribonucleoside-diphosphate reductase activity after infection indicated that 4- to 10-fold more activity was induced by hydroxyurea-resistant viruses than by the wild-type virus. A two-step partial purification resulted in a substantial enrichment for the 34,000-MW protein from extracts of wild-type and hydroxyurea-resistant-virus-infected, but not mock-infected, cells. In the presence of the drug, the isolates incorporated (/sup 3/H)thymidine into DNA earlier and a rate substantially greater than that of the wild type, although the onset of DNA synthesis was delayed in both cases. The drug resistance trait was markedly unstable in all isolates. In the absence of selective pressure, plaque-purified isolated readily segregated progeny that displayed a wide range of resistance phenotypes. The results of this study indicate that vaccinia virus encodes a subunit of ribonucleotide reductase which is 34,000-MW early protein whose overproduction confers hydroxyurea resistance on reproducing viruses.

  20. Targeting Prostate Cancer for Gene Therapy Utilizing Lentivirus and Oncolytic VSV Virus

    DTIC Science & Technology

    2010-04-01

    specific fashion. Ad ditionally, mutated form of Vesicular Stomatitis Virus (VSV), an oncolytic virus capable of replicating in interferon (IFN) response...Our results indicated that direct injection of VSV (AV3) intra-prostaticaly lead to selective infection, replication, and overall i ncrease i n ap...ully re plication-competent a nd r apidly s pread through a nd ki ll cancerous cells. Vesicular Stomatitis Virus (VSV) is an oncolytic virus which

  1. CRISPR-Cas9 as a Powerful Tool for Efficient Creation of Oncolytic Viruses.

    PubMed

    Yuan, Ming; Webb, Eika; Lemoine, Nicholas Robert; Wang, Yaohe

    2016-03-07

    The development of oncolytic viruses has led to an emerging new class of cancer therapeutics. Although the safety profile has been encouraging, the transition of oncolytic viruses to the clinical setting has been a slow process due to modifications. Therefore, a new generation of more potent oncolytic viruses needs to be exploited, following our better understanding of the complex interactions between the tumor, its microenvironment, the virus, and the host immune response. The conventional method for creation of tumor-targeted oncolytic viruses is based on homologous recombination. However, the creation of new mutant oncolytic viruses with large genomes remains a challenge due to the multi-step process and low efficiency of homologous recombination. The CRISPR-associated endonuclease Cas9 has hugely advanced the potential to edit the genomes of various organisms due to the ability of Cas9 to target a specific genomic site by a single guide RNA. In this review, we discuss the CRISPR-Cas9 system as an efficient viral editing method for the creation of new oncolytic viruses, as well as its potential future applications in the development of oncolytic viruses. Further, this review discusses the potential of off-target effects as well as CRISPR-Cas9 as a tool for basic research into viral biology.

  2. CRISPR-Cas9 as a Powerful Tool for Efficient Creation of Oncolytic Viruses

    PubMed Central

    Yuan, Ming; Webb, Eika; Lemoine, Nicholas Robert; Wang, Yaohe

    2016-01-01

    The development of oncolytic viruses has led to an emerging new class of cancer therapeutics. Although the safety profile has been encouraging, the transition of oncolytic viruses to the clinical setting has been a slow process due to modifications. Therefore, a new generation of more potent oncolytic viruses needs to be exploited, following our better understanding of the complex interactions between the tumor, its microenvironment, the virus, and the host immune response. The conventional method for creation of tumor-targeted oncolytic viruses is based on homologous recombination. However, the creation of new mutant oncolytic viruses with large genomes remains a challenge due to the multi-step process and low efficiency of homologous recombination. The CRISPR-associated endonuclease Cas9 has hugely advanced the potential to edit the genomes of various organisms due to the ability of Cas9 to target a specific genomic site by a single guide RNA. In this review, we discuss the CRISPR-Cas9 system as an efficient viral editing method for the creation of new oncolytic viruses, as well as its potential future applications in the development of oncolytic viruses. Further, this review discusses the potential of off-target effects as well as CRISPR-Cas9 as a tool for basic research into viral biology. PMID:26959050

  3. Detection of vaccinia virus DNA, but not infectious virus, in the blood of smallpox vaccine recipients.

    PubMed

    Cohen, Jeffrey I; Hohman, Patricia; Preuss, Jeanne C; Li, Li; Fischer, Steven H; Fedorko, Daniel P

    2007-06-06

    The authors of a recent study [Savona MR, Dela Cruz WP, Jones MS, Thornton JA, Xia D, Hadfield TL, et al. Detection of vaccinia DNA in the blood following smallpox vaccination. JAMA 2006; 295:1898-1900] suggested that the duration of deferral for blood donations by smallpox vaccinees should be extended, based on detection of vaccinia virus DNA in five blood samples by polymerase chain reaction (PCR) and the potential for viremia. We found that 4 of 202 blood specimens (from 3 of 27 smallpox vaccinees) were positive for vaccinia virus DNA by PCR; none were positive for virus by culture. Throat swabs were negative by PCR and culture. Thus, while some blood specimens contained vaccinia virus DNA, infectious virus was not detected. Current guidelines for deferral of blood donation in vaccinees seem appropriate.

  4. Evaluation of Therapeutic Interventions for Vaccinia Virus Keratitis

    PubMed Central

    Altmann, Sharon; Murphy, Christopher J.; Patnaikuni, Ravi; Takla, Teresa; Toomey, Megan; Nesbit, Brittany; McIntyre, Kimberly; Covert, Jill; Dubielzig, Richard; Leatherberry, Gary; Adkins, Elizabeth; Kodihalli, Shantha

    2011-01-01

    Background. Vaccinia virus keratitis (VACVK) is a complication of smallpox vaccination that can result in blindness. There are no Food and Drug Administration–approved treatments for VACVK, and vaccinia immunoglobulin (VIG) is contraindicated in isolated VACVK. We used a rabbit model of infection to compare several therapeutic options for VACVK. Methods. Rabbit eyes were infected with 105 plaque-forming units of the Dryvax strain of vaccinia virus and scored daily for 28 days using a modified MacDonald-Shadduck scoring system. Animals were treated for 10 days after the onset of keratitis with albumin, VIG, prednisolone acetate, trifluridine, or combinations thereof. Ocular viral titers and vaccinia-specific antibody titers were determined by plaque assay and enzyme-linked immunosorbent assay, respectively. Results. Treatment with intravenous VIG neither exacerbated nor ameliorated VACVK. Topical prednisolone acetate interfered with viral clearance, and ocular disease rebounded in prednisolone-treated groups. The most effective treatment was topical trifluridine alone. Conclusions. We conclude that (1) VIG did not negatively affect the treatment of isolated keratitis, (2) topical corticosteroids should not be used for treating VACVK, and (3) treatment with topical trifluridine, with or without intravenous VIG, is the preferred therapeutic regimen for treating VACVK. PMID:21278209

  5. Advances in the design and development of oncolytic measles viruses

    PubMed Central

    Hutzen, Brian; Raffel, Corey; Studebaker, Adam W

    2015-01-01

    A successful oncolytic virus is one that selectively propagates and destroys cancerous tissue without causing excessive damage to the normal surrounding tissue. Oncolytic measles virus (MV) is one such virus that exhibits this characteristic and thus has rapidly emerged as a potentially useful anticancer modality. Derivatives of the Edmonston MV vaccine strain possess a remarkable safety record in humans. Promising results in preclinical animal models and evidence of biological activity in early phase trials contribute to the enthusiasm. Genetic modifications have enabled MV to evolve from a vaccine agent to a potential anticancer therapy. Specifically, alterations of the MV genome have led to improved tumor selectivity and delivery, therapeutic potency, and immune system modulation. In this article, we will review the advancements that have been made in the design and development of MV that have led to its use as a cancer therapy. In addition, we will discuss the evidence supporting its use, as well as the challenges associated with MV as a potential cancer therapeutic. PMID:27512675

  6. Oncolytic virotherapy using herpes simplex virus: how far have we come?

    PubMed

    Sokolowski, Nicolas As; Rizos, Helen; Diefenbach, Russell J

    2015-01-01

    Oncolytic virotherapy exploits the properties of human viruses to naturally cytolysis of cancer cells. The human pathogen herpes simplex virus (HSV) has proven particularly amenable for use in oncolytic virotherapy. The relative safety of HSV coupled with extensive knowledge on how HSV interacts with the host has provided a platform for manipulating HSV to enhance the targeting and killing of human cancer cells. This has culminated in the approval of talimogene laherparepvec for the treatment of melanoma. This review focuses on the development of HSV as an oncolytic virus and where the field is likely to head in the future.

  7. Oncolytic Herpes Simplex Virus Vectors Fully Retargeted to Tumor-Associated Antigens.

    PubMed

    Uchida, Hiroaki; Hamada, Hirofumi; Nakano, Kenji; Kwon, Heechung; Tahara, Hideaki; Cohen, Justus B; Glorioso, Joseph C

    2017-02-05

    Oncolytic virotherapy is a novel therapeutic modality for malignant diseases that exploits selective viral replication in cancer cells. Herpes simplex virus (HSV) is a promising agent for oncolytic virotherapy due to its broad cell tropism and the identification of mutations that favor its replication in tumor over normal cells. However, these attenuating mutations also tend to limit the potency of current oncolytic HSV vectors that have entered clinical studies. As an alternative, vector retargeting to novel entry receptors has the potential to achieve tumor specificity at the stage of virus entry, eliminating the need for replication-attenuating mutations. Here we summarize the molecular mechanism of HSV entry and recent advances in the development of fully retargeted HSV vectors for oncolytic virotherapy. Retargeted HSV vectors offer an attractive platform for the creation of a new generation of oncolytic HSV with improved efficacy and specificity.

  8. Bovine vaccinia: Inactivated Vaccinia virus vaccine induces protection in murine model.

    PubMed

    Matos, Ana Carolina Diniz; Guedes, Maria Isabel Maldonado Coelho; Rehfeld, Izabelle Silva; Costa, Erica Azevedo; Costa, Aristoteles Gomes; Silva, Natalia Lopes da; Lage, Andrey Pereira; Lobato, Zélia Inês Portela

    2017-05-01

    Bovine vaccinia (BV), caused by Vaccinia virus (VACV), is a zoonosis characterized by exanthematous lesions on the teats of dairy cows and the milkers' hands. Since 1999, due to the occurrence of many BV outbreaks in dairy farms across all Brazilian regions, there is a need to improve the control and prevention measures of the disease. Vaccination is one of the major tools to prevent viral diseases, and it could be an alternative for BV prevention. The main objective of this study was the development of vaccine formulations against BV using the inactivated VACV strain GP2 as antigen combined with different adjuvants. Potency tests were performed in mice, which were vaccinated with two doses at a 21-day interval, and then challenged with the vaccine homologous virus. VACV strain GP2 inactivated by beta-propiolactone (BPL) in association with adjuvants was effective in inducing a humoral immune response against VACV, as measured by neutralizing antibody (NA) titers, and was variable depending on the adjuvant used in each vaccine formulation. The vaccine formulation containing aluminum hydroxide (AH) associated with saponin as adjuvant induced the production of high NA titers in all vaccinated mice, giving 100% protection in Balb/c murine model after challenge with homologous virus. Copyright © 2017. Published by Elsevier B.V.

  9. Studies on the serological relationships between avian pox, sheep pox, goat pox and vaccinia viruses

    PubMed Central

    Uppal, P. K.; Nilakantan, P. R.

    1970-01-01

    By using neutralization, complement fixation and immunogel-diffusion tests, it has been demonstrated that cross-reactions occur between various avian pox viruses and between sheep pox and goat pox viruses. No such reactions were demonstrated between avian pox viruses and vaccinia virus or between avian pox and sheep pox and goat pox viruses. Furthermore, no serological relationship was demonstrable between vaccinia virus and sheep pox and goat pox viruses. PMID:4989854

  10. Oncolytic Viruses and Their Application to Cancer Immunotherapy

    PubMed Central

    Chiocca, EA; Rabkin, SD

    2015-01-01

    Oncolytic viruses (OVs) selectively replicate in and kill cancer cells, and spread within the tumor, while not harming normal tissue. In addition to this direct oncolytic activity, OVs are also very effective at inducing immune responses to themselves and to the infected tumor cells. OVs encompass a broad diversity of DNA and RNA viruses that are naturally cancer-selective or can be genetically-engineered. OVs provide a diverse platform for immunotherapy; they act as in situ vaccines, and can be armed with immune modulatory transgenes or combined with other immunotherapies. However, the interactions of OVs with the immune system may affect therapeutic outcomes in opposing fashions: negatively by limiting virus replication and/or spread, or positively by inducing antitumor immune responses. Many aspects of the OV-tumor/host interaction are important in delineating the effectiveness of therapy; they include: (i) innate immune responses and the degree of inflammation induced, (ii) types of virus-induced cell death, (iii) inherent tumor physiology, such as infiltrating and resident immune cells, vascularity/hypoxia, lymphatics, and stromal architecture, and (iv) tumor cell phenotype, including alterations in IFN signaling, oncogenic pathways, cell surface immune markers (MHC, co-stimulatory, NK receptors), and the expression of immunosuppressive factors. Recent clinical trials with a variety of OVs, especially those expressing GM-CSF, have demonstrated efficacy and induction of antitumor immune responses in the absence of significant toxicity. Manipulating the balance between anti-virus and antitumor responses, often involving overlapping immune pathways, will be critical to the clinical success of OVs. PMID:24764576

  11. Protein Composition of the Vaccinia Virus Mature Virion

    SciTech Connect

    Resch, Wolfgang; Hixson, Kim K.; Moore, Ronald J.; Lipton, Mary S.; Moss, Bernard

    2007-02-05

    The protein content of vaccinia virus mature virions, purified by rate zonal and isopycnic centrifugation and solubilized by SDS or a solution of urea and thiourea, was determined by the accurate mass and time tag technology which uses both tandem mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry to detect tryptic peptides separated by high-resolution liquid chromatography. Eighty vaccinia virus-encoded proteins representing 37% of the 218 genes annotated in the complete genome sequence were detected in at least three analyses. Ten proteins accounted for approximately 80% of the mass, while the least abundant proteins made up 1% or less of the mass. Thirteen identified proteins were not previously reported as components of virions. On the other hand, 8 previously described virion proteins were not detected here, presumably due to technical reasons including small size and hydrophobicity. In addition to vaccinia virus-encoded proteins, 24 host proteins omitting isoforms were detected. The most abundant of these were cytoskeletal proteins, heat shock proteins, and proteins involved in translation.

  12. Oncolytic viruses on the cusp of success?: proceedings of the 9th International Conference on Oncolytic Virus Therapeutics

    PubMed Central

    Peters, Cole; Nigim, Fares; Chiocca, E Antonio; Rabkin, Samuel D

    2016-01-01

    Boston, Massachusetts, was the site of the 9th International Conference on Oncolytic Virus Therapeutics held 13–16 June 2015. An overarching theme of the meeting was the continued development of combinatorial treatment regimens to bolster the therapeutic potential of oncolytic viruses (OVs). Several talks focused on combining OVs with immune checkpoint inhibitors in a wide array of tumors, signaling an experimental and thematic shift toward driving immune activation to clear a tumor versus relying on direct viral oncolysis. An important aspect of the meeting was the variety of ongoing OV clinical trials. Topics ranged from basic virology to clinical trials and from academic research to intellectual property and biotechnology. There was much excitement due to the US Food and Drug Administration’s recent consideration of talimogene laherparepvec (T-VEC) for the treatment of advanced melanoma (T-VEC was approved in October, following the conference). Here, we summarize the meeting’s primary themes, which reflect the current state of the field.

  13. Oncolytic viruses and their application to cancer immunotherapy.

    PubMed

    Chiocca, E Antonio; Rabkin, Samuel D

    2014-04-01

    Oncolytic viruses (OV) selectively replicate and kill cancer cells and spread within the tumor, while not harming normal tissue. In addition to this direct oncolytic activity, OVs are also very effective at inducing immune responses to themselves and to the infected tumor cells. OVs encompass a broad diversity of DNA and RNA viruses that are naturally cancer selective or can be genetically engineered. OVs provide a diverse platform for immunotherapy; they act as in situ vaccines and can be armed with immunomodulatory transgenes or combined with other immunotherapies. However, the interactions of OVs with the immune system may affect therapeutic outcomes in opposing fashions: negatively by limiting virus replication and/or spread, or positively by inducing antitumor immune responses. Many aspects of the OV-tumor/host interaction are important in delineating the effectiveness of therapy: (i) innate immune responses and the degree of inflammation induced; (ii) types of virus-induced cell death; (iii) inherent tumor physiology, such as infiltrating and resident immune cells, vascularity/hypoxia, lymphatics, and stromal architecture; and (iv) tumor cell phenotype, including alterations in IFN signaling, oncogenic pathways, cell surface immune markers [MHC, costimulatory, and natural killer (NK) receptors], and the expression of immunosuppressive factors. Recent clinical trials with a variety of OVs, especially those expressing granulocyte macrophage colony-stimulating factor (GM-CSF), have demonstrated efficacy and induction of antitumor immune responses in the absence of significant toxicity. Manipulating the balance between antivirus and antitumor responses, often involving overlapping immune pathways, will be critical to the clinical success of OVs.

  14. Characterization of chimpanzee/human monoclonal antibodies to vaccinia virus A33 glycoprotein and its variola virus homolog in vitro and in a vaccinia virus mouse protection model.

    PubMed

    Chen, Zhaochun; Earl, Patricia; Americo, Jeffrey; Damon, Inger; Smith, Scott K; Yu, Fujuan; Sebrell, Andrew; Emerson, Suzanne; Cohen, Gary; Eisenberg, Roselyn J; Gorshkova, Inna; Schuck, Peter; Satterfield, William; Moss, Bernard; Purcell, Robert

    2007-09-01

    Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human gamma 1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (K(d) of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.

  15. Structure of intracellular mature vaccinia virus observed by cryoelectron microscopy.

    PubMed Central

    Dubochet, J; Adrian, M; Richter, K; Garces, J; Wittek, R

    1994-01-01

    Intracellular mature vaccinia virus, also called intracellular naked virus, and its core envelope have been observed in their native, unfixed, unstained, hydrated states by cryoelectron microscopy of vitrified samples. The virion appears as a smooth rounded rectangle of ca. 350 by 270 nm. The core seems homogeneous and is surrounded by a 30-nm-thick surface domain delimited by membranes. We show that surface tubules and most likely also the characteristic dumbbell-shaped core with the lateral bodies which are generally observed in negatively stained or conventionally embedded samples are preparation artifacts. Images PMID:8107253

  16. Efficacy and Safety/Toxicity Study of Recombinant Vaccinia Virus JX-594 in Two Immunocompetent Animal Models of Glioma

    PubMed Central

    Lun, XueQing; Chan, Jennifer; Zhou, Hongyuan; Sun, Beichen; Kelly, John JP; Stechishin, Owen Owen; Bell, John C; Parato, Kelley; Hu, Kang; Vaillant, Dominique; Wang, Jiahu; Liu, Ta-Chiang; Breitbach, Caroline; Kirn, David; Senger, Donna L; Forsyth, Peter A

    2010-01-01

    The purpose of this study was to investigate the oncolytic potential of the recombinant, granulocyte macrophage colony-stimulating factor (GM-CSF)-expressing vaccinia virus (VV) JX-594 in experimental malignant glioma (MGs) in vitro and in immunocompetent rodent models. We have found that JX-594 killed all MG cell lines tested in vitro. Intratumoral (i.t.) administration of JX-594 significantly inhibited tumor growth and prolonged survival in rats-bearing RG2 intracranial (i.c.) tumors and mice-bearing GL261 brain tumors. Combination therapy with JX-594 and rapamycin significantly increased viral replication and further prolonged survival in both immunocompetent i.c. MG models with several animals considered “cured” (three out of seven rats >120 days, terminated experiment). JX-594 infected and killed brain tumor-initiating cells (BTICs) from patient samples grown ex vivo, and did so more efficiently than other oncolytic viruses MYXV, Reovirus type-3, and VSVΔM51. Additional safety/toxicity studies in nontumor-bearing rodents treated with a supratherapeutic dose of JX-594 demonstrated GM-CSF-dependent inflammation and necrosis. These results suggest that i.c. administered JX-594 triggers a predictable GM-CSF-mediated inflammation in murine models. Before proceeding to clinical trials, JX-594 should be evaluated in the brains of nonhuman primates and optimized for the viral doses, delivery routes as well as the combination agents (e.g., mTOR inhibitors). PMID:20808290

  17. Efficacy and safety/toxicity study of recombinant vaccinia virus JX-594 in two immunocompetent animal models of glioma.

    PubMed

    Lun, XueQing; Chan, Jennifer; Zhou, Hongyuan; Sun, Beichen; Kelly, John J P; Stechishin, Owen Owen; Bell, John C; Parato, Kelley; Hu, Kang; Vaillant, Dominique; Wang, Jiahu; Liu, Ta-Chiang; Breitbach, Caroline; Kirn, David; Senger, Donna L; Forsyth, Peter A

    2010-11-01

    The purpose of this study was to investigate the oncolytic potential of the recombinant, granulocyte macrophage colony-stimulating factor (GM-CSF)-expressing vaccinia virus (VV) JX-594 in experimental malignant glioma (MGs) in vitro and in immunocompetent rodent models. We have found that JX-594 killed all MG cell lines tested in vitro. Intratumoral (i.t.) administration of JX-594 significantly inhibited tumor growth and prolonged survival in rats-bearing RG2 intracranial (i.c.) tumors and mice-bearing GL261 brain tumors. Combination therapy with JX-594 and rapamycin significantly increased viral replication and further prolonged survival in both immunocompetent i.c. MG models with several animals considered "cured" (three out of seven rats >120 days, terminated experiment). JX-594 infected and killed brain tumor-initiating cells (BTICs) from patient samples grown ex vivo, and did so more efficiently than other oncolytic viruses MYXV, Reovirus type-3, and VSV(ΔM51). Additional safety/toxicity studies in nontumor-bearing rodents treated with a supratherapeutic dose of JX-594 demonstrated GM-CSF-dependent inflammation and necrosis. These results suggest that i.c. administered JX-594 triggers a predictable GM-CSF-mediated inflammation in murine models. Before proceeding to clinical trials, JX-594 should be evaluated in the brains of nonhuman primates and optimized for the viral doses, delivery routes as well as the combination agents (e.g., mTOR inhibitors).

  18. A New Inhibitor of Apoptosis from Vaccinia Virus and Eukaryotes

    PubMed Central

    Gubser, Caroline; Bergamaschi, Daniele; Hollinshead, Michael; Lu, Xin; van Kuppeveld, Frank J. M; Smith, Geoffrey L

    2007-01-01

    A new apoptosis inhibitor is described from vaccinia virus, camelpox virus, and eukaryotic cells. The inhibitor is a hydrophobic, multiple transmembrane protein that is resident in the Golgi and is named GAAP (Golgi anti-apoptotic protein). Stable expression of both viral GAAP (v-GAAP) and human GAAP (h-GAAP), which is expressed in all human tissues tested, inhibited apoptosis induced by intrinsic and extrinsic apoptotic stimuli. Conversely, knockout of h-GAAP by siRNA induced cell death by apoptosis. v-GAAP and h-GAAP display overlapping functions as shown by the ability of v-GAAP to complement for the loss of h-GAAP. Lastly, deletion of the v-GAAP gene from vaccinia virus did not affect virus replication in cell culture, but affected virus virulence in a murine infection model. This study identifies a new regulator of cell death that is highly conserved in evolution from plants to insects, amphibians, mammals, and poxviruses. PMID:17319741

  19. Changing Faces in Virology: The Dutch Shift from Oncogenic to Oncolytic Viruses

    PubMed Central

    Belcaid, Zineb; Lamfers, Martine L.M.; van Beusechem, Victor W.

    2014-01-01

    Abstract Viruses have two opposing faces. On the one hand, they can cause harm and disease. A virus may manifest directly as a contagious disease with a clinical pathology of varying significance. A viral infection can also have delayed consequences, and in rare cases may cause cellular transformation and cancer. On the other hand, viruses may provide hope: hope for an efficacious treatment of serious disease. Examples of the latter are the use of viruses as a vaccine, as transfer vector for therapeutic genes in a gene therapy setting, or, more directly, as therapeutic anticancer agent in an oncolytic-virus therapy setting. Already there is evidence for antitumor activity of oncolytic viruses. The antitumor efficacy seems linked to their capacity to induce a tumor-directed immune response. Here, we will provide an overview on the development of oncolytic viruses and their clinical evaluation from the Dutch perspective. PMID:25141764

  20. Changing faces in virology: the dutch shift from oncogenic to oncolytic viruses.

    PubMed

    Belcaid, Zineb; Lamfers, Martine L M; van Beusechem, Victor W; Hoeben, Rob C

    2014-10-01

    Viruses have two opposing faces. On the one hand, they can cause harm and disease. A virus may manifest directly as a contagious disease with a clinical pathology of varying significance. A viral infection can also have delayed consequences, and in rare cases may cause cellular transformation and cancer. On the other hand, viruses may provide hope: hope for an efficacious treatment of serious disease. Examples of the latter are the use of viruses as a vaccine, as transfer vector for therapeutic genes in a gene therapy setting, or, more directly, as therapeutic anticancer agent in an oncolytic-virus therapy setting. Already there is evidence for antitumor activity of oncolytic viruses. The antitumor efficacy seems linked to their capacity to induce a tumor-directed immune response. Here, we will provide an overview on the development of oncolytic viruses and their clinical evaluation from the Dutch perspective.

  1. [Oncolytic viruses as a new way of treatment of neoplastic diseases].

    PubMed

    Kukla, Urszula; Chronowska, Justyna; Łabuzek, Krzysztof; Okopień, Bogusław

    2015-08-01

    Despite the unceasing progression in chemotherapy, radiotherapy and surgery, neoplasms are still the second, after cardiovascular diseases, cause of death in the world. The creation of oncolytic viruses gives hope for increase of anticancer therapy effectiveness. Oncolytic viruses are the type of viruses that selectively infect and cause the lyse of tumor cells excluding normal cells. This mechanism allows to avoid the consequences of the possible replication of the virus, which having entered to the organism, replicates in organism's cells by using the DNA of host cells. The development of genetic engineering and molecular biology has enabled the creation of this kind of genetically modified viruses, which deprive them of their virulence. Currently, there are many clinical trials in progress including the use of oncolytic viruses in head and neck squamous cell carcinoma, thyroid cancer, colorectal cancer, liver cancer, melanoma and glioblastoma multiforme treatment. There are parallel studies in animals using the subsequent viruses. Oncolytic viruses treatment is generally well tolerated, without significant side effects. It is worth to point out that this method combined with chemotherapy and radiotherapy allows to reduce the use of therapeutic doses, which significantly reduces the toxicity of conventional treatment. Further clinical studies evaluating the efficacy and safety of oncolytic viruses will develop more effective and better tolerated therapeutic protocols in the future.

  2. Moving oncolytic viruses into the clinic: clinical-grade production, purification, and characterization of diverse oncolytic viruses.

    PubMed

    Ungerechts, Guy; Bossow, Sascha; Leuchs, Barbara; Holm, Per S; Rommelaere, Jean; Coffey, Matt; Coffin, Rob; Bell, John; Nettelbeck, Dirk M

    2016-01-01

    Oncolytic viruses (OVs) are unique anticancer agents based on their pleotropic modes of action, which include, besides viral tumor cell lysis, activation of antitumor immunity. A panel of diverse viruses, often genetically engineered, has advanced to clinical investigation, including phase 3 studies. This diversity of virotherapeutics not only offers interesting opportunities for the implementation of different therapeutic regimens but also poses challenges for clinical translation. Thus, manufacturing processes and regulatory approval paths need to be established for each OV individually. This review provides an overview of clinical-grade manufacturing procedures for OVs using six virus families as examples, and key challenges are discussed individually. For example, different virus features with respect to particle size, presence/absence of an envelope, and host species imply specific requirements for measures to ensure sterility, for handling, and for determination of appropriate animal models for toxicity testing, respectively. On the other hand, optimization of serum-free culture conditions, increasing virus yields, development of scalable purification strategies, and formulations guaranteeing long-term stability are challenges common to several if not all OVs. In light of the recent marketing approval of the first OV in the Western world, strategies for further upscaling OV manufacturing and optimizing product characterization will receive increasing attention.

  3. Moving oncolytic viruses into the clinic: clinical-grade production, purification, and characterization of diverse oncolytic viruses

    PubMed Central

    Ungerechts, Guy; Bossow, Sascha; Leuchs, Barbara; Holm, Per S; Rommelaere, Jean; Coffey, Matt; Coffin, Rob; Bell, John; Nettelbeck, Dirk M

    2016-01-01

    Oncolytic viruses (OVs) are unique anticancer agents based on their pleotropic modes of action, which include, besides viral tumor cell lysis, activation of antitumor immunity. A panel of diverse viruses, often genetically engineered, has advanced to clinical investigation, including phase 3 studies. This diversity of virotherapeutics not only offers interesting opportunities for the implementation of different therapeutic regimens but also poses challenges for clinical translation. Thus, manufacturing processes and regulatory approval paths need to be established for each OV individually. This review provides an overview of clinical-grade manufacturing procedures for OVs using six virus families as examples, and key challenges are discussed individually. For example, different virus features with respect to particle size, presence/absence of an envelope, and host species imply specific requirements for measures to ensure sterility, for handling, and for determination of appropriate animal models for toxicity testing, respectively. On the other hand, optimization of serum-free culture conditions, increasing virus yields, development of scalable purification strategies, and formulations guaranteeing long-term stability are challenges common to several if not all OVs. In light of the recent marketing approval of the first OV in the Western world, strategies for further upscaling OV manufacturing and optimizing product characterization will receive increasing attention. PMID:27088104

  4. Optimal Control Model of Tumor Treatment with Oncolytic Virus and MEK Inhibitor

    PubMed Central

    Jia, Chen; Chen, Ying

    2016-01-01

    Tumors are a serious threat to human health. The oncolytic virus is a kind of tumor killer virus which can infect and lyse cancer cells and spread through the tumor, while leaving normal cells largely unharmed. Mathematical models can help us to understand the tumor-virus dynamics and find better treatment strategies. This paper gives a new mathematical model of tumor therapy with oncolytic virus and MEK inhibitor. Stable analysis was given. Because mitogen-activated protein kinase (MEK) can not only lead to greater oncolytic virus infection into cancer cells, but also limit the replication of the virus, in order to provide the best dosage of MEK inhibitors and balance the positive and negative effect of the inhibitors, we put forward an optimal control problem of the inhibitor. The optimal strategies are given by theory and simulation. PMID:28097139

  5. Recombinant vaccinia virus/Venezuelan equine encephalitis (VEE) virus protects mice from peripheral VEE virus challenge.

    PubMed

    Kinney, R M; Esposito, J J; Mathews, J H; Johnson, B J; Roehrig, J T; Barrett, A D; Trent, D W

    1988-12-01

    Mice immunized with recombinant vaccinia virus (VACC) expressing Venezuelan equine encephalitis (VEE) virus capsid protein and glycoproteins E1 and E2 or with attenuated VEE TC-83 virus vaccine developed VEE-specific neutralizing antibody and survived intraperitoneal challenge with virulent VEE virus strains including Trinidad donkey (subtype 1AB), P676 (subtype 1C), 3880 (subtype 1D), and Everglades (subtype 2). However, unlike immunization with TC-83 virus, immunization with the recombinant VACC/VEE virus did not protect mice from intranasal challenge with VEE Trinidad donkey virus. These results suggest that recombinant VACC/VEE virus is a vaccine candidate for equines and humans at risk of mosquito-transmitted VEE disease but not for laboratory workers at risk of accidental exposure to aerosol infection with VEE virus.

  6. Vaccinia Virus Requires Glutamine but Not Glucose for Efficient Replication

    PubMed Central

    Fontaine, Krystal A.; Camarda, Roman

    2014-01-01

    ABSTRACT Viruses require host cell metabolism to provide the necessary energy and biosynthetic precursors for successful viral replication. Vaccinia virus (VACV) is a member of the Poxviridae family, and its use as a vaccine enabled the eradication of variola virus, the etiologic agent of smallpox. A global metabolic screen of VACV-infected primary human foreskin fibroblasts suggested that glutamine metabolism is altered during infection. Glutamine and glucose represent the two main carbon sources for mammalian cells. Depriving VACV-infected cells of exogenous glutamine led to a substantial decrease in infectious virus production, whereas starving infected cells of exogenous glucose had no significant impact on replication. Viral yield in glutamine-deprived cells or in cells treated with an inhibitor of glutaminolysis, the pathway of glutamine catabolism, could be rescued by the addition of multiple tricarboxylic acid (TCA) cycle intermediates. Thus, VACV infection induces a metabolic alteration to fully rely on glutamine to anaplerotically maintain the TCA cycle. VACV protein synthesis, but not viral transcription, was decreased in glutamine-deprived cells, which corresponded with a dramatic reduction in all VACV morphogenetic intermediates. This study reveals the unique carbon utilization program implemented during poxvirus infection and provides a potential metabolic pathway to target viral replication. IMPORTANCE Viruses are dependent on the metabolic machinery of the host cell to supply the energy and molecular building blocks needed for critical processes including genome replication, viral protein synthesis, and membrane production. This study investigates how vaccinia virus (VACV) infection alters global cellular metabolism, providing the first metabolomic analysis for a member of the poxvirus family. Unlike most viruses examined to date, VACV does not activate glycolysis, and exogenous glucose is not required for maximal virus production. Instead, VACV

  7. Development of a selective biopharmaceutical from Herpes simplex virus type 1 glycoproteins E and I for blocking antibody mediated neutralization of oncolytic viruses.

    PubMed

    Bucurescu, Septimiu

    2010-12-01

    Future cancer therapies will be molecular cures. They will correct, block or destroy cancer cells by targeting molecular changes that lead to carcinogenesis. Destroying cancer cells can be done using oncolytic viruses. By blocking antibody mediated neutralization of oncolytic viruses, Herpes simplex virus type 1 glycoproteins E and I could be used in the adjuvant treatment of cancer for improving the chances of oncolytic viruses to kill cancer cells in vivo.

  8. Modified Vaccinia Ankara Virus Vaccination Provides Long-Term Protection against Nasal Rabbitpox Virus Challenge.

    PubMed

    Jones, Dorothy I; McGee, Charles E; Sample, Christopher J; Sempowski, Gregory D; Pickup, David J; Staats, Herman F

    2016-07-01

    Modified vaccinia Ankara virus (MVA) is a smallpox vaccine candidate. This study was performed to determine if MVA vaccination provides long-term protection against rabbitpox virus (RPXV) challenge, an animal model of smallpox. Two doses of MVA provided 100% protection against a lethal intranasal RPXV challenge administered 9 months after vaccination.

  9. Modified Vaccinia Ankara Virus Vaccination Provides Long-Term Protection against Nasal Rabbitpox Virus Challenge

    PubMed Central

    Jones, Dorothy I.; McGee, Charles E.; Sample, Christopher J.; Sempowski, Gregory D.; Pickup, David J.

    2016-01-01

    Modified vaccinia Ankara virus (MVA) is a smallpox vaccine candidate. This study was performed to determine if MVA vaccination provides long-term protection against rabbitpox virus (RPXV) challenge, an animal model of smallpox. Two doses of MVA provided 100% protection against a lethal intranasal RPXV challenge administered 9 months after vaccination. PMID:27146001

  10. Deoxyadenosine reverses hydroxyurea inhibition of vaccinia virus growth.

    PubMed Central

    Slabaugh, M B; Howell, M L; Wang, Y; Mathews, C K

    1991-01-01

    Hydroxyurea, an inhibitor of ribonucleotide reductase, blocks replication of vaccinia virus. However, when medium containing hydroxyurea and dialyzed serum was supplemented with deoxyadenosine, the block to viral reproduction was circumvented, provided that an inhibitor of adenosine deaminase was also present. Deoxyguanosine, deoxycytidine, and deoxythymidine were ineffective alone and did not augment the deoxyadenosine effect. In fact, increasing concentrations of deoxyguanosine and deoxythymidine, but not deoxycytidine, eliminated the deoxyadenosine rescue, an effect that was reversed by the addition of low concentrations of deoxycytidine. These results suggested that the inhibition of viral replication by hydroxyurea was primarily due to a deficiency of dATP. Deoxyribonucleoside triphosphate pools in vaccinia virus-infected cells were measured at the height of viral DNA synthesis after a synchronous infection. With 0.5 mM hydroxyurea, the dATP pool was greater than 90% depleted, the dCTP and dGTP pools were 40 to 50% reduced, and the dTTP pool was increased. Assay of ribonucleotide reductase activity in intact virus-infected cells suggested that hydroxyurea may differentially affect reduction of the various substrates of the enzyme. PMID:2016760

  11. Targeting pediatric cancer stem cells with oncolytic virotherapy.

    PubMed

    Friedman, Gregory K; Cassady, Kevin A; Beierle, Elizabeth A; Markert, James M; Gillespie, G Yancey

    2012-04-01

    Cancer stem cells (CSCs), also termed "cancer-initiating cells" or "cancer progenitor cells," which have the ability to self-renew, proliferate, and maintain the neoplastic clone, have recently been discovered in a wide variety of pediatric tumors. These CSCs are thought to be responsible for tumorigenesis and tumor maintenance, aggressiveness, and recurrence due to inherent resistance to current treatment modalities such as chemotherapy and radiation. Oncolytic virotherapy offers a novel, targeted approach for eradicating pediatric CSCs using mechanisms of cell killing that differ from conventional therapies. Moreover, oncolytic viruses have the ability to target specific features of CSCs such as cell-surface proteins, transcription factors, and the CSC microenvironment. Through genetic engineering, a wide variety of foreign genes may be expressed by oncolytic viruses to augment the oncolytic effect. We review the current data regarding the ability of several types of oncolytic viruses (herpes simplex virus-1, adenovirus, reovirus, Seneca Valley virus, vaccinia virus, Newcastle disease virus, myxoma virus, vesicular stomatitis virus) to target and kill both CSCs and tumor cells in pediatric tumors. We highlight advantages and limitations of each virus and potential ways in which next-generation engineered viruses may target resilient CSCs.

  12. Fusogenic oncolytic herpes simplex viruses as a potent and personalized cancer vaccine.

    PubMed

    Li, Qi-Xiang; Liu, Guohong; Zhang, Xiaoliu

    2012-07-01

    The recent FDA approval of Sipuleucel-T for the treatment of prostate cancer represents an important milestone of cancer immunotherapy, which, for the first time, validates the concept of bringing true clinical benefit to cancer patients by stimulating patients' own anti-tumor immunity. Among the different experimental cancer immunotherapies, oncolytic virotherapy may represent a low-cost yet potent and personalized cancer vaccine for the treatment of solid tumors. This review describes the constructions of several human herpes simplex virus (HSV)-derived oncolytic viruses as candidate cancer vaccines, which induce specific and potent anti-tumor immunity in pre-clinical models, and thus resulting in stronger overall anti-tumor efficacy as compared to oncolytic effect alone. This article also describes the approaches to enhance the antitumor immunity of oncolytic HSVs, and in particular, the key role played by integrating membrane-fusion activity into these viruses. Additionally, this article reviews the potential effect of certain chemotherapeutic agents (e.g. cyclophosphamide) in boosting antitumor immunity induced by oncolytic HSV, and the mechanisms behind it. In summary, all the preclinical and clinical data have suggested that HSV-based oncolytic virotherapies could likely be developed as a new generation of cancer vaccines for the treatment of solid tumors.

  13. Genome Scale Patterns of Recombination between Coinfecting Vaccinia Viruses

    PubMed Central

    Qin, Li

    2014-01-01

    ABSTRACT Recombination plays a critical role in virus evolution. It helps avoid genetic decline and creates novel phenotypes. This promotes survival, and genome sequencing suggests that recombination has facilitated the evolution of human pathogens, including orthopoxviruses such as variola virus. Recombination can also be used to map genes, but although recombinant poxviruses are easily produced in culture, classical attempts to map the vaccinia virus (VACV) genome this way met with little success. We have sequenced recombinants formed when VACV strains TianTan and Dryvax are crossed under different conditions. These were a single round of growth in coinfected cells, five rounds of sequential passage, or recombinants obtained using leporipoxvirus-mediated DNA reactivation. Our studies showed that recombinants contain a patchwork of DNA, with the number of exchanges increasing with passage. Further passage also selected for TianTan DNA and correlated with increased plaque size. The recombinants produced through a single round of coinfection contain a disproportionate number of short conversion tracks (<1 kbp) and exhibited 1 exchange per 12 kbp, close to the ∼1 per 8 kbp in the literature. One by-product of this study was that rare mutations were also detected; VACV replication produces ∼1 × 10−8 mutation per nucleotide copied per cycle of replication and ∼1 large (21 kbp) deletion per 70 rounds of passage. Viruses produced using DNA reactivation appeared no different from recombinants produced using ordinary methods. An attractive feature of this approach is that when it is combined with selection for a particular phenotype, it provides a way of mapping and dissecting more complex virus traits. IMPORTANCE When two closely related viruses coinfect the same cell, they can swap genetic information through a process called recombination. Recombination produces new viruses bearing different combinations of genes, and it plays an important role in virus

  14. Initial characterization of Vaccinia Virus B4 suggests a role in virus spread

    SciTech Connect

    Burles, Kristin; Irwin, Chad R.; Burton, Robyn-Lee; Schriewer, Jill; Evans, David H.; Buller, R. Mark; Barry, Michele

    2014-05-15

    Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a natural mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus. - Highlights: • B4R-null viruses show reduced plaque size, and decreased ability to spread. • B4R-null viruses formed mature and extracellular virions; and rearranged actin. • Virus devoid of EVM154, the B4R homolog, was attenuated during infection. • Initial characterization suggests that B4 may play a role in virus spread. • Unidentified mediators of actin tail formation may exist in vaccinia virus.

  15. Ultrasound-mediated oncolytic virus delivery and uptake for increased therapeutic efficacy: state of art

    PubMed Central

    Nande, Rounak; Howard, Candace M; Claudio, Pier Paolo

    2015-01-01

    The field of ultrasound (US) has changed significantly from medical imaging and diagnosis to treatment strategies. US contrast agents or microbubbles (MB) are currently being used as potential carriers for chemodrugs, small molecules, nucleic acids, small interfering ribonucleic acid, proteins, adenoviruses, and oncolytic viruses. Oncolytic viruses can selectively replicate within and destroy a cancer cell, thus making them a powerful therapeutic in treating late-stage or metastatic cancer. These viruses have been shown to have robust activity in clinical trials when injected directly into tumor nodules. However limitations in oncolytic virus’ effectiveness and its delivery approach have warranted exploration of ultrasound-mediated delivery. Gene therapy bearing adenoviruses or oncolytic viruses can be coupled with MBs and injected intravenously. Following application of US energy to the target region, the MBs cavitate, and the resulting shock wave enhances drug, gene, or adenovirus uptake. Though the underlying mechanism is yet to be fully understood, there is evidence to suggest that mechanical pore formation of cellular membranes allows for the temporary uptake of drugs. This delivery method circumvents the limitations due to stimulation of the immune system that prevented intravenous administration of viruses. This review provides insight into this intriguing new frontier on the delivery of oncolytic viruses to tumor sites. PMID:27512682

  16. Low-Resolution Structure of Vaccinia Virus DNA Replication Machinery

    PubMed Central

    Sèle, Céleste; Gabel, Frank; Gutsche, Irina; Ivanov, Ivan; Burmeister, Wim P.

    2013-01-01

    Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages. PMID:23175373

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

    PubMed Central

    Tong, Jessica G; Valdes, Yudith Ramos; Barrett, John W; Bell, John C; Stojdl, David; McFadden, Grant; McCart, J Andrea; DiMattia, Gabriel E; Shepherd, Trevor G

    2015-01-01

    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. PMID:27119108

  18. Vaccinia virus strain differences in cell attachment and entry

    SciTech Connect

    Bengali, Zain; Townsley, Alan C.; Moss, Bernard

    2009-06-20

    Vaccinia virus (VACV) strain WR can enter cells by a low pH endosomal pathway or direct fusion with the plasma membrane at neutral pH. Here, we compared attachment and entry of five VACV strains in six cell lines and discovered two major patterns. Only WR exhibited pH 5-enhanced rate of entry following neutral pH adsorption to cells, which correlated with sensitivity to bafilomycin A1, an inhibitor of endosomal acidification. Entry of IHD-J, Copenhagen and Elstree strains were neither accelerated by pH 5 treatment nor prevented by bafilomycin A1. Entry of the Wyeth strain, although not augmented by pH 5, was inhibited by bafilomycin A1. WR and Wyeth were both relatively resistant to the negative effects of heparin on entry, whereas the other strains were extremely sensitive due to inhibition of cell binding. The relative sensitivities of individual vaccinia virus strains to heparin correlated inversely with their abilities to bind to and enter glycosaminoglycan-deficient sog9 cells but not other cell lines tested. These results suggested that that IHD-J, Copenhagen and Elstree have a more limited ability than WR and Wyeth to use the low pH endosomal pathway and are more dependent on binding to glycosaminoglycans for cell attachment.

  19. Analysis of variola and vaccinia virus neutralization assays for smallpox vaccines.

    PubMed

    Hughes, Christine M; Newman, Frances K; Davidson, Whitni B; Olson, Victoria A; Smith, Scott K; Holman, Robert C; Yan, Lihan; Frey, Sharon E; Belshe, Robert B; Karem, Kevin L; Damon, Inger K

    2012-07-01

    Possible smallpox reemergence drives research for third-generation vaccines that effectively neutralize variola virus. A comparison of neutralization assays using different substrates, variola and vaccinia (Dryvax and modified vaccinia Ankara [MVA]), showed significantly different 90% neutralization titers; Dryvax underestimated while MVA overestimated variola neutralization. Third-generation vaccines may rely upon neutralization as a correlate of protection.

  20. Natural Vaccinia Virus Infection: Diagnosis, Isolation, and Characterization.

    PubMed

    Geessien Kroon, Erna; Santos Abrahão, Jônatas; de Souza Trindade, Giliane; Pereira Oliveira, Graziele; Moreira Franco Luiz, Ana Paula; Barbosa Costa, Galileu; Teixeira Lima, Mauricio; Silva Calixto, Rafael; de Oliveira, Danilo Bretas; Drumond, Betânia Paiva

    2016-08-12

    Natural infections of Vaccinia virus (VACV)-the prototype species of the Orthopoxvirus genus, from the family Poxviridae and subfamily Chordopoxvirinae-cause an occupational emergent zoonotic disease that is primarily associated with the handling of infected dairy cattle. In humans, VACV infection is characterized by skin lesions, primarily on the hands, and accompanied by systemic symptoms such as fever, myalgia, headache, and lymphadenopathy. The diagnosis of VACV is usually performed according to the methods described for other orthopoxviruses. This unit describes the methods utilized to obtain clinical samples, the serological and molecular techniques used for diagnosis, and the isolation methods and techniques used for molecular and biological characterization of the viruses. © 2016 by John Wiley & Sons, Inc.

  1. Immune Modulation in Primary Vaccinia virus Zoonotic Human Infections

    PubMed Central

    Gomes, Juliana Assis Silva; de Araújo, Fernanda Fortes; Trindade, Giliane de Souza; Quinan, Bárbara Resende; Drumond, Betânia Paiva; Ferreira, Jaqueline Maria Siqueira; Mota, Bruno Eduardo Fernandes; Nogueira, Maurício Lacerda; Kroon, Erna Geessien; Abrahão, Jônatas Santos; Côrrea-Oliveira, Rodrigo; da Fonseca, Flávio Guimarães

    2012-01-01

    In 2010, the WHO celebrated the 30th anniversary of the smallpox eradication. Ironically, infections caused by viruses related to smallpox are being increasingly reported worldwide, including Monkeypox, Cowpox, and Vaccinia virus (VACV). Little is known about the human immunological responses elicited during acute infections caused by orthopoxviruses. We have followed VACV zoonotic outbreaks taking place in Brazil and analyzed cellular immune responses in patients acutely infected by VACV. Results indicated that these patients show a biased immune modulation when compared to noninfected controls. Amounts of B cells are low and less activated in infected patients. Although present, T CD4+ cells are also less activated when compared to noninfected individuals, and so are monocytes/macrophages. Similar results were obtained when Balb/C mice were experimentally infected with a VACV sample isolated during the zoonotic outbreaks. Taking together, the data suggest that zoonotic VACVs modulate specific immune cell compartments during an acute infection in humans. PMID:22229039

  2. Domain Organization of Vaccinia Virus Helicase-Primase D5

    PubMed Central

    Hutin, Stephanie; Ling, Wai Li; Round, Adam; Effantin, Gregory; Reich, Stefan; Iseni, Frédéric; Tarbouriech, Nicolas; Schoehn, Guy

    2016-01-01

    ABSTRACT Poxviridae are viruses with a large linear double-stranded DNA genome coding for up to 250 open reading frames and a fully cytoplasmic replication. The double-stranded DNA genome is covalently circularized at both ends. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) and in the Phycodnaviridae. We are studying the machinery which replicates this peculiar genome structure. From our work with vaccinia virus, we give first insights into the overall structure and function of the essential poxvirus virus helicase-primase D5 and show that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has ATPase and, more generally, nucleoside triphosphatase activities that are indistinguishable from the activities of full-length D5 and that are independent of the nature of the base. In addition, hexameric helicase domains bind tightly to single- and double-stranded DNA. Still, the monomeric D5 helicase construct truncated within the D5N domain leads to a well-defined structure, but it does not have ATPase or DNA-binding activity. This shows that the full D5N domain has to be present for hexamerization. This allowed us to assign a function to the D5N domain which is present not only in D5 but also in other viruses of the nucleocytoplasmic large DNA virus (NCLDV) clade. The primase domain and the helicase domain were structurally analyzed via a combination of small-angle X-ray scattering and, when appropriate, electron microscopy, leading to consistent low-resolution models of the different proteins. IMPORTANCE Since the beginning of the 1980s, research on the vaccinia virus replication mechanism has basically stalled due to the absence of structural information. As a result, this important class of pathogens is less well understood than most other viruses. This lack of information concerns in general viruses of the NCLDV clade, which use a superfamily 3 helicase

  3. Oncolytic viruses: emerging options for the treatment of breast cancer.

    PubMed

    Suryawanshi, Yogesh R; Zhang, Tiantian; Essani, Karim

    2017-03-01

    Breast cancer (BC) is the most common type of cancer among women and is the second most common cause of cancer-related deaths, following lung cancer. Severe toxicity associated with a long-term use of BC chemo- and radiotherapy makes it essential to look for newer therapeutics. Additionally, molecular heterogeneity at both intratumoral and intertumoral levels among BC subtypes is known to result in a differential response to standard therapeutics. Oncolytic viruses (OVs) have emerged as one of the most promising treatment options for BC. Many preclinical and clinical studies have shown that OVs are effective in treating BC, both as a single therapeutic agent and as a part of combination therapies. Combination therapies involving multimodal therapeutics including OVs are becoming popular as they allow to achieve the synergistic therapeutic effects, while minimizing the associated toxicities. Here, we review the OVs for BC therapy in preclinical studies and in clinical trials, both as a monotherapy and as part of a combination therapy. We also briefly discuss the potential therapeutic targets for BC, as these are likely to be critical for the development of new OVs.

  4. Expression of Herpes Simplex Virus 1 Glycoprotein B by a Recombinant Vaccinia Virus and Protection of Mice against Lethal Herpes Simplex Virus 1 Infection

    NASA Astrophysics Data System (ADS)

    Cantin, Edouard M.; Eberle, Richard; Baldick, Joseph L.; Moss, Bernard; Willey, Dru E.; Notkins, Abner L.; Openshaw, Harry

    1987-08-01

    The herpes simplex virus 1 (HSV-1) strain F gene encoding glycoprotein gB was isolated and modified at the 5' end by in vitro oligonucleotide-directed mutagenesis. The modified gB gene was inserted into the vaccinia virus genome and expressed under the control of a vaccinia virus promoter. The mature gB glycoprotein produced by the vaccinia virus recombinant was glycosylated, was expressed at the cell surface, and was indistinguishable from authentic HSV-1 gB in terms of electrophoretic mobility. Mice immunized intradermally with the recombinant vaccinia virus produced gB-specific neutralizing antibodies and were resistant to a lethal HSV-1 challenge.

  5. Oncolytic virotherapy.

    PubMed

    Russell, Stephen J; Peng, Kah-Whye; Bell, John C

    2012-07-10

    Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancers. Recent advances include preclinical proof of feasibility for a single-shot virotherapy cure, identification of drugs that accelerate intratumoral virus propagation, strategies to maximize the immunotherapeutic action of oncolytic viruses and clinical confirmation of a critical viremic threshold for vascular delivery and intratumoral virus replication. The primary clinical milestone has been completion of accrual in a phase 3 trial of intratumoral herpes simplex virus therapy using talimogene laherparepvec for metastatic melanoma. Key challenges for the field are to select 'winners' from a burgeoning number of oncolytic platforms and engineered derivatives, to transiently suppress but then unleash the power of the immune system to maximize both virus spread and anticancer immunity, to develop more meaningful preclinical virotherapy models and to manufacture viruses with orders-of-magnitude higher yields than is currently possible.

  6. [Oncolytic viruses for genetic therapy of gastrointestinal tumors].

    PubMed

    Bitzer, M; Lauer, U M

    2003-07-01

    Gastroenterological oncology requires new strategies with new mechanisms of action and without cross-resistance to currently available treatment regimes. Virotherapy which is based on the employment of replication-competent viral vectors exhibiting strong oncolytic properties is such an approach currently under preclinical/clinical investigation. Techniques of molecular virology are required for further improvement of current vectors, particularly with respect to oncolytic activity, tumour selectivity, tumour spread capacity, and safety.

  7. Therapy of Experimental Nerve Sheath Tumors Using Oncolytic Viruses

    DTIC Science & Technology

    2005-06-01

    SUPPLEMENTARY NOTES 14. ABSTRACT Abstract follows. 15. SUBJECT TERMS Oncolytic HSV, angiogenesis, MPNST , mouse model 16. SECURITY CLASSIFICATION OF: 17...reliable tumor models for malignant peripheral nerve sheath tumors ( MPNST ). Several existing and novel oncolytic HSV vectors will then be tested on these...from G47A increases cytotoxicity in vitro to human endothelial cells and murine Nfl" MPNST cell lines. Inhibition of MPNST M2 tumor growth in vivo was

  8. The Lipid Raft-Associated Protein CD98 Is Required for Vaccinia Virus Endocytosis

    PubMed Central

    Schroeder, Nina; Chung, Che-Sheng; Chen, Chein-Hung; Liao, Chung-Lin

    2012-01-01

    Mature vaccinia virus (vaccinia MV) infects a broad range of animals in vivo and cell cultures in vitro; however, the cellular receptors that determine vaccinia MV tropism and entry pathways are poorly characterized. Here, we performed quantitative proteomic analyses of lipid raft-associated proteins upon vaccinia MV entry into HeLa cells. We found that a type II membrane glycoprotein, CD98, is enriched in lipid rafts upon vaccinia MV infection compared to mock-infected HeLa cells. The knockdown of CD98 expression in HeLa cells significantly reduced vaccinia MV entry. Furthermore, CD98 knockout (KO) mouse embryonic fibroblasts (MEFs) also exhibited reduced vaccinia MV infectivity without affecting MV attachment to cells, suggesting a role for CD98 in the postbinding step of virus entry. Further characterization with inhibitors and dominant negative proteins that block different endocytic pathways revealed that vaccinia MV entry into MEFs occurs through a clathrin-independent, caveolin-independent, dynamin-dependent, fluid-phase endocytic pathway, implying that CD98 plays a specific role in the vaccinia MV endocytic pathway. Infections of wild-type and CD98 KO MEF cells with different strains of vaccinia MV provided further evidence that CD98 plays a specific role in MV endocytosis but not in plasma membrane fusion. Finally, different CD98-C69 chimeric proteins were expressed in CD98 KO MEFs, but none were able to reconstitute MV infectivity, suggesting that the overall structure of the CD98 protein is required for vaccinia MV endocytosis. PMID:22345471

  9. Recombinant modified vaccinia virus Ankara-based malaria vaccines.

    PubMed

    Sebastian, Sarah; Gilbert, Sarah C

    2016-01-01

    A safe and effective malaria vaccine is a crucial part of the roadmap to malaria elimination/eradication by the year 2050. Viral-vectored vaccines based on adenoviruses and modified vaccinia virus Ankara (MVA) expressing malaria immunogens are currently being used in heterologous prime-boost regimes in clinical trials for induction of strong antigen-specific T-cell responses and high-titer antibodies. Recombinant MVA is a safe and well-tolerated attenuated vector that has consistently shown significant boosting potential. Advances have been made in large-scale MVA manufacture as high-yield producer cell lines and high-throughput purification processes have recently been developed. This review describes the use of MVA as malaria vaccine vector in both preclinical and clinical studies in the past 5 years.

  10. Oncolytic virotherapy.

    PubMed

    Cervantes-García, Daniel; Ortiz-López, Rocío; Mayek-Pérez, Netzahualcoyotl; Rojas-Martínez, Augusto

    2008-01-01

    Current oncolytic virotherapy strategies are based in the accumulated understanding of the common molecular mechanisms displayed during cell transformation and viral infection, like cell cycle and apoptosis deregulations. Oncolytic virotherapy aims to achieve a strong cytolytic effect, highly restricted to transformed cells. Here, we describe the oncolytic virotherapy defined as the use of viruses like antitumor agents (wild and gene-modified oncolytic viruses) and the developed strategies to increase antitumor efficacy and safety. In addition, we discuss the advances and challenges concerning the use virotherapy in animal models and clinical trials. Some clinical trials of virotherapy have demonstrated promising results, particularly when combined with standard antineoplastic therapies. These preliminary accomplishments are opening the field for more research in several aspects, like vector modifications, pharmacodynamics, biosafety, new clinical applications, etc.

  11. [Analysis on status and characteristics of laboratory-acquired vaccinia virus infections cases].

    PubMed

    Wei, Qiang; Lu, Xuan-cheng; Wu, Gui-zhen

    2013-02-01

    By analyzing the status and characteristics of vaccinia virus laboratory-acquired infections in the bibliographical information, this paper provides relevant recommendations and measures for prevention and control of vaccinia virus laboratory-acquired infections in China. Choosing PubMed, Embase, Biosis and SCIE, SSCI, CPCI-S as well as CPCI-SSH covered by Web of Science as the data source, indexing the bibliography of vaccinia virus laboratory-acquired infections, this paper analyzes the information on whether to vaccinate, the occurrence time of symptoms, diseasedparts, symptom characteristics and the disease-causing reasons. The outcome shows that 52. 9% of the cases never get vaccinated, 82.4% engaged in vaccinia virus related researches never get vaccinated in 10 years, 52. 9% get infected by the accidental needlestick in hands during the process of handling animal experiments, 70. 6% of infections occur in the hands and having symptoms after being exposed with an average of 5. 1 days. Although it is still controversial that whether or not to be vaccinated before carrying out vaccinia virus related works, it should be important aspects of prevention and control of vaccinia virus laboratory-acquired infections with the strict compliance with the operating requirements of the biosafety, by strengthening personal protection and timely taking emergency measures when unforeseen circumstances occur, as well as providing the research background information to doctors.

  12. Use of an Oncolytic Virus Secreting GM-CSF as Combined Oncolytic and Immunotherapy for Treatment of Colorectal and Hepatic Adenocarcinomas

    PubMed Central

    Malhotra, Sandeep; Kim, Teresa; Zager, Jonathan; Bennett, Joseph; Ebright, Michael; D’Angelica, Michael; Fong, Yuman

    2007-01-01

    Oncolytic cancer therapy using herpes simplex viruses (HSV) that have direct tumoricidal effects and cancer immunotherapy using the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) have each been effective in preclinical testing. NV1034 is a multi-mutated oncolytic HSV carrying the gene for murine GM-CSF that attempts to combine these two anticancer strategies. The purpose of this study was to compare NV1034 to NV1023, the parent HSV mutants lacking GM-CSF, in order to determine if such combined oncolytic and immunotherapy using a single vector has advantages over oncolytic therapy alone. In vitro, expression GM-CSF did not alter the infectivity, in vitro cytotoxicity, or replication of NV1034 compared to the non-cytokine secreting control. Tumors infected with NV1034 produced GM-CSF in picogram quantities. In vivo efficacy of the viruses against murine colorectal carcinoma CT26 and murine hepatoma Hepa l–6 was then tested in subcutaneous tumors in syngeneic Balb/c and C57 L/J mice respectively. In these immune competent models, NV1034 or NV1023 each demonstrated potent antitumor activity. Treatment with NV1034 had significantly better antitumor effect compared to treatment with NV1023. Furthermore, in mice depleted of CD4+ and CD8+ T-lymphocytes, there was no difference in the antitumor efficacy of these viruses. Viral vectors combining oncolytic and immunotherapy are promising agents in treatment of colorectal carcinoma and hepatoma. PMID:17383529

  13. Construction of Poxviruses as Cloning Vectors: Insertion of the Thymidine Kinase Gene from Herpes Simplex Virus into the DNA of Infectious Vaccinia Virus

    NASA Astrophysics Data System (ADS)

    Panicali, Dennis; Paoletti, Enzo

    1982-08-01

    We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by contransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.

  14. Vaccinia virus infection & temporal analysis of virus gene expression: Part 3.

    PubMed

    Yen, Judy; Golan, Ron; Rubins, Kathleen

    2009-04-13

    The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (approximately 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

  15. Vaccinia virus infection & temporal analysis of virus gene expression: part 1.

    PubMed

    Yen, Judy; Golan, Ron; Rubins, Kathleen

    2009-04-08

    The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (approximately 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

  16. Vaccinia virus infection & temporal analysis of virus gene expression: Part 2.

    PubMed

    Yen, Judy; Golan, Ron; Rubins, Kathleen

    2009-04-10

    The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (approximately 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

  17. Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly▿

    PubMed Central

    Deng, Liang; Dai, Peihong; Ciro, Anthony; Smee, Donald F.; Djaballah, Hakim; Shuman, Stewart

    2007-01-01

    The bioterror threat of a smallpox outbreak in an unvaccinated population has mobilized efforts to develop new antipoxviral agents. By screening a library of known drugs, we identified 13 compounds that inhibited vaccinia virus replication at noncytotoxic doses. The anticancer drug mitoxantrone is unique among the inhibitors identified in that it has no apparent impact on viral gene expression. Rather, it blocks processing of viral structural proteins and assembly of mature progeny virions. The isolation of mitoxantrone-resistant vaccinia strains underscores that a viral protein is the likely target of the drug. Whole-genome sequencing of mitoxantrone-resistant viruses pinpointed missense mutations in the N-terminal domain of vaccinia DNA ligase. Despite its favorable activity in cell culture, mitoxantrone administered intraperitoneally at the maximum tolerated dose failed to protect mice against a lethal intranasal infection with vaccinia virus. PMID:17928345

  18. Oncolytic viruses as immunotherapy: progress and remaining challenges

    PubMed Central

    Aurelian, Laure

    2016-01-01

    Oncolytic viruses (OVs) comprise an emerging cancer therapeutic modality whose activity involves both direct tumor cell lysis and the induction of immunogenic cell death (ICD). Cellular proteins released from the OV-lysed tumor cells, known as damage-associated molecular patterns and tumor-associated antigens, activate dendritic cells and elicit adaptive antitumor immunity. Interaction with the innate immune system and the development of long-lasting immune memory also contribute to OV-induced cell death. The degree to which the ICD component contributes to the clinical efficacy of OV therapy is still unclear. Modulation of a range of immune interactions may be beneficial or detrimental in nature and the interactions depend on the specific tumor, the site and extent of the disease, the immunosuppressive tumor microenvironment, the OV platform, the dose, time, and delivery conditions, as well as individual patient responses. To enhance the contribution of ICD, OVs have been engineered to express immunostimulatory genes and strategies have been developed to combine OV therapy with chemo- and immune-based therapeutic regimens. However, these approaches carry the risk that they may also be tolerogenic depending on their levels and the presence of other cytokines, their direct antiviral effects, and the timing and conditions of their expression. The contribution of autophagy to adaptive immunity, the ability of the OVs to kill cancer stem cells, and the patient’s baseline immune status are additional considerations. This review focuses on the complex and as yet poorly understood balancing act that dictates the outcome of OV therapy. We summarize current understanding of the OVs’ function in eliciting antitumor immunity and its relationship to therapeutic efficacy. Also discussed are the criteria involved in restraining antiviral immune responses and minimizing pathology while promoting antitumor immunity to override immune tolerance. PMID:27226725

  19. Vaccinia virus infection after sexual contact with a military smallpox vaccinee -Washington, 2010.

    PubMed

    2010-07-02

    On March 1, 2010, the Washington State Department of Health (WADOH) notified Public Health - Seattle & King County (PHSKC) of a suspected case of contact transmission of vaccinia virus from sexual contact with a member of the military who had been vaccinated against smallpox. Vaccinia virus infection after sexual contact has been reported previously (1-4). Despite the patient's exposure history and clinical presentation, the diagnosis initially was not considered by the patient's physician, who ordered laboratory testing for several common sexually transmitted infections. The patient was seen by a second physician and referred to an infectious disease specialist, who obtained a swab sample of a genital lesion for laboratory testing for vaccinia virus. Vaccinia virus was confirmed by the Washington State Public Health Laboratory (WAPHL) and the CDC Poxvirus Laboratory. The patient resided in a household with an immunosuppressed renal transplant recipient. Appropriate contact precautions were recommended to the patient. No additional cases of contact transmission were reported. This report describes the patient's clinical course and the associated epidemiologic investigation. Health-care providers caring for U.S. military personnel or their contacts should consider vaccinia virus infection in the differential diagnosis of clinically compatible genital lesions. Contact precautions should be emphasized to all persons who are vaccinated, as well as their contacts with unexplained lesions that might represent vaccinia infection from contact transmission.

  20. Myxoma virus suppresses proliferation of activated T lymphocytes yet permits oncolytic virus transfer to cancer cells

    PubMed Central

    Villa, Nancy Y.; Wasserfall, Clive H.; Meacham, Amy M.; Wise, Elizabeth; Chan, Winnie; Wingard, John R.; McFadden, Grant

    2015-01-01

    Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain hematologic malignancies, but the risk of graft-versus-host disease (GVHD) is a major limitation for wider application. Ideally, strategies to improve allo-HCT would involve suppression of T lymphocytes that drive GVHD while sparing those that mediate graft-versus-malignancy (GVM). Recently, using a xenograft model, we serendipitously discovered that myxoma virus (MYXV) prevented GVHD while permitting GVM. In this study, we show that MYXV binds to resting, primary human T lymphocytes but will only proceed into active virus infection after the T cells receive activation signals. MYXV-infected T lymphocytes exhibited impaired proliferation after activation with reduced expression of interferon-γ, interleukin-2 (IL-2), and soluble IL-2Rα, but did not affect expression of IL-4 and IL-10. MYXV suppressed T-cell proliferation in 2 patterns (full vs partial) depending on the donor. In terms of GVM, we show that MYXV-infected activated human T lymphocytes effectively deliver live oncolytic virus to human multiple myeloma cells, thus augmenting GVM by transfer of active oncolytic virus to residual cancer cells. Given this dual capacity of reducing GVHD plus increasing the antineoplastic effectiveness of GVM, ex vivo virotherapy with MYXV may be a promising clinical adjunct to allo-HCT regimens. PMID:25904246

  1. Myxoma virus suppresses proliferation of activated T lymphocytes yet permits oncolytic virus transfer to cancer cells.

    PubMed

    Villa, Nancy Y; Wasserfall, Clive H; Meacham, Amy M; Wise, Elizabeth; Chan, Winnie; Wingard, John R; McFadden, Grant; Cogle, Christopher R

    2015-06-11

    Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain hematologic malignancies, but the risk of graft-versus-host disease (GVHD) is a major limitation for wider application. Ideally, strategies to improve allo-HCT would involve suppression of T lymphocytes that drive GVHD while sparing those that mediate graft-versus-malignancy (GVM). Recently, using a xenograft model, we serendipitously discovered that myxoma virus (MYXV) prevented GVHD while permitting GVM. In this study, we show that MYXV binds to resting, primary human T lymphocytes but will only proceed into active virus infection after the T cells receive activation signals. MYXV-infected T lymphocytes exhibited impaired proliferation after activation with reduced expression of interferon-γ, interleukin-2 (IL-2), and soluble IL-2Rα, but did not affect expression of IL-4 and IL-10. MYXV suppressed T-cell proliferation in 2 patterns (full vs partial) depending on the donor. In terms of GVM, we show that MYXV-infected activated human T lymphocytes effectively deliver live oncolytic virus to human multiple myeloma cells, thus augmenting GVM by transfer of active oncolytic virus to residual cancer cells. Given this dual capacity of reducing GVHD plus increasing the antineoplastic effectiveness of GVM, ex vivo virotherapy with MYXV may be a promising clinical adjunct to allo-HCT regimens.

  2. ONCOLYTIC HERPES SIMPLEX VIRUS 1 (HSV-1) VECTORS: INCREASING TREATMENT EFFICACY AND RANGE THROUGH STRATEGIC VIRUS DESIGN

    PubMed Central

    Carson, J.; Haddad, D.; Bressman, M.; Fong, Y.

    2012-01-01

    SUMMARY Viruses have long been considered potential anticancer treatments. Wild-type viruses have been tested as anticancer agents in clinical trials since the 1960s. The possibility of viral oncolysis as an alternate cancer therapy was transformed by the emergence of modern genetic engineering. The herpes simplex virus (HSV) family offers particular advantages for use as a viral oncolytic. The engineered vectors that make up oncolytic HSVs (oHSVs) have demonstrated remarkable safety in clinical trials, with some evidence of efficacy. The past decade has seen a focus on increasing the efficacy of oncolytic vectors by adding exogenous transgenes to enhance tumor destruction. The current paper describes the various strategies for engineering HSV for increased cancer tissue specificity and efficacy. Presented are the rationale, preclinical data and clinical data where available. This is meant to illustrate a basic framework for the development of a novel therapy meant to exploit the viral life cycle for the killing of cancer. PMID:22287818

  3. Neuroblastoma cell lines contain pluripotent tumor initiating cells that are susceptible to a targeted oncolytic virus.

    PubMed

    Mahller, Yonatan Y; Williams, Jon P; Baird, William H; Mitton, Bryan; Grossheim, Jonathan; Saeki, Yoshinaga; Cancelas, Jose A; Ratner, Nancy; Cripe, Timothy P

    2009-01-01

    Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers. Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice. These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

  4. Construction of recombinant Newcastle disease virus Italien strain for oncolytic virotherapy of tumors.

    PubMed

    Wei, Ding; Sun, Na; Nan, Gang; Wang, Yuan; Liu, Hong-Qi; Peeters, Ben; Chen, Zhi-Nan; Bian, Huijie

    2012-07-01

    Newcastle disease virus (NDV) is a naturally oncolytic virus that has been shown to be safe and effective for cancer therapy. Tumor virotherapy using NDV emerged in the 1950s and has advanced more recently by the increased availability of reverse genetics technology. In this study, we constructed a reverse genetics system based on the virulent and oncolytic NDV Italien strain, and generated two recombinant NDVs carrying a gene encoding either enhanced green fluorescent protein or firefly luciferase. We evaluated the replication and antitumor characteristics of these viruses in vitro and in vivo. Our data showed that the insertion of exogenous reporter genes did not affect NDV replication and sensitivity to type I interferon. The recombinant NDVs kept the property of tumor-selective replication both in vitro and in vivo and strongly induced syncytium formation leading to cell death. Moreover, the recombinant NDVs significantly prolonged the survival of tumor-bearing athymic mice (p=0.017) and suppressed the loss of body weight after intratumoral injection. Taken together, our study provides a novel platform to develop recombinant oncolytic viruses based on the NDV Italien strain and shows the efficiency of recombinant NDV Italien for oncolytic virotherapy of tumors.

  5. The immunoregulatory properties of oncolytic myxoma virus and their implications in therapeutics.

    PubMed

    Liu, Jia; Wennier, Sonia; McFadden, Grant

    2010-12-01

    Myxoma virus (MYXV) is a poxvirus with a strict rabbit-specific host-tropism for pathogenesis. The immunoregulatory factors encoded by MYXV can suppress some functions of immune effectors from other species. We review their mechanisms of action, implications in therapeutics and the potential to improve MYXV as an oncolytic agent in humans.

  6. Non-plaque-forming virions of Modified Vaccinia virus Ankara express viral genes.

    PubMed

    Lülf, Anna-Theresa; Freudenstein, Astrid; Marr, Lisa; Sutter, Gerd; Volz, Asisa

    2016-12-01

    In cell culture infections with vaccinia virus the number of counted virus particles is substantially higher than the number of plaques obtained by titration. We found that standard vaccine preparations of recombinant Modified Vaccinia virus Ankara produce only about 20-30% plaque-forming virions in fully permissive cell cultures. To evaluate the biological activity of the non-plaque-forming particles, we generated recombinant viruses expressing fluorescent reporter proteins under transcriptional control of specific viral early and late promoters. Live cell imaging and automated counting by fluorescent microscopy indicated that virtually all virus particles can enter cells and switch on viral gene expression. Although most of the non-plaque-forming infections are arrested at the level of viral early gene expression, we detected activation of late viral transcription in 10-20% of single infected cells. Thus, non-plaque-forming particles are biologically active, and likely contribute to the immunogenicity of vaccinia virus vaccines.

  7. Recombinant vaccinia/Venezuelan equine encephalitis (VEE) virus expresses VEE structural proteins.

    PubMed

    Kinney, R M; Esposito, J J; Johnson, B J; Roehrig, J T; Mathews, J H; Barrett, A D; Trent, D W

    1988-12-01

    cDNA molecules encoding the structural proteins of the virulent Trinidad donkey and the TC-83 vaccine strains of Venezuelan equine encephalitis (VEE) virus were inserted under control of the vaccinia virus 7.5K promoter into the thymidine kinase gene of vaccinia virus. Synthesis of the capsid protein and glycoproteins E2 and E1 of VEE virus was demonstrated by immunoblotting of lysates of CV-1 cells infected with recombinant vaccinia/VEE viruses. VEE glycoproteins were detected in recombinant virus-infected cells by fluorescent antibody (FA) analysis performed with a panel of VEE-specific monoclonal antibodies. Seven E2-specific epitopes and two of four E1-specific epitopes were demonstrated by FA.

  8. Enhanced efficacy of cidofovir combined with vaccinia immune globulin in treating progressive cutaneous vaccinia virus infections in immunosuppressed hairless mice.

    PubMed

    Smee, Donald F; Dagley, Ashley; Downs, Brittney; Hagloch, Joseph; Tarbet, E Bart

    2015-01-01

    The treatment of progressive vaccinia in individuals has involved antiviral drugs, such as cidofovir (CDV), brincidofovir, and/or tecovirimat, combined with vaccinia immune globulin (VIG). VIG is costly, and its supply is limited, so sparing the use of VIG during treatment is an important objective. VIG sparing was modeled in immunosuppressed mice by maximizing the treatment benefits of CDV combined with VIG to determine the effective treatments that delayed the time to death, reduced cutaneous lesion severity, and/or decreased tissue viral titers. SKH-1 hairless mice immunosuppressed with cyclophosphamide and hairless SCID mice (SHO strain) were infected cutaneously with vaccinia virus. Monotherapy, dual combinations (CDV plus VIG), or triple therapy (topical CDV, parenteral CDV, and VIG) were initiated 2 days postinfection and were given every 3 to 4 days through day 11. The efficacy assessment included survival rate, cutaneous lesion severity, and viral titers. Delays in the time to death and the reduction in lesion severity occurred in the following order of efficacy: triple therapy had greater efficacy than double combinations (CDV plus VIG or topical plus parenteral CDV), which had greater efficacy than VIG alone. Parenteral administration of CDV or VIG was necessary to suppress virus titers in internal organs (liver, lung, and spleen). The skin viral titers were significantly reduced by triple therapy only. The greatest efficacy was achieved by triple therapy. In humans, this regimen should translate to a faster cure rate, thus sparing the amount of VIG used for treatment.

  9. Enhanced Efficacy of Cidofovir Combined with Vaccinia Immune Globulin in Treating Progressive Cutaneous Vaccinia Virus Infections in Immunosuppressed Hairless Mice

    PubMed Central

    Dagley, Ashley; Downs, Brittney; Hagloch, Joseph; Tarbet, E. Bart

    2014-01-01

    The treatment of progressive vaccinia in individuals has involved antiviral drugs, such as cidofovir (CDV), brincidofovir, and/or tecovirimat, combined with vaccinia immune globulin (VIG). VIG is costly, and its supply is limited, so sparing the use of VIG during treatment is an important objective. VIG sparing was modeled in immunosuppressed mice by maximizing the treatment benefits of CDV combined with VIG to determine the effective treatments that delayed the time to death, reduced cutaneous lesion severity, and/or decreased tissue viral titers. SKH-1 hairless mice immunosuppressed with cyclophosphamide and hairless SCID mice (SHO strain) were infected cutaneously with vaccinia virus. Monotherapy, dual combinations (CDV plus VIG), or triple therapy (topical CDV, parenteral CDV, and VIG) were initiated 2 days postinfection and were given every 3 to 4 days through day 11. The efficacy assessment included survival rate, cutaneous lesion severity, and viral titers. Delays in the time to death and the reduction in lesion severity occurred in the following order of efficacy: triple therapy had greater efficacy than double combinations (CDV plus VIG or topical plus parenteral CDV), which had greater efficacy than VIG alone. Parenteral administration of CDV or VIG was necessary to suppress virus titers in internal organs (liver, lung, and spleen). The skin viral titers were significantly reduced by triple therapy only. The greatest efficacy was achieved by triple therapy. In humans, this regimen should translate to a faster cure rate, thus sparing the amount of VIG used for treatment. PMID:25385098

  10. Live-Cell Imaging of Vaccinia Virus Recombination.

    PubMed

    Paszkowski, Patrick; Noyce, Ryan S; Evans, David H

    2016-08-01

    Recombination between co-infecting poxviruses provides an important mechanism for generating the genetic diversity that underpins evolution. However, poxviruses replicate in membrane-bound cytoplasmic structures known as factories or virosomes. These are enclosed structures that could impede DNA mixing between co-infecting viruses, and mixing would seem to be essential for this process. We hypothesize that virosome fusion events would be a prerequisite for recombination between co-infecting poxviruses, and this requirement could delay or limit viral recombination. We have engineered vaccinia virus (VACV) to express overlapping portions of mCherry fluorescent protein fused to a cro DNA-binding element. In cells also expressing an EGFP-cro fusion protein, this permits live tracking of virus DNA and genetic recombination using confocal microscopy. Our studies show that different types of recombination events exhibit different timing patterns, depending upon the relative locations of the recombining elements. Recombination between partly duplicated sequences is detected soon after post-replicative genes are expressed, as long as the reporter gene sequences are located in cis within an infecting genome. The same kinetics are also observed when the recombining elements are divided between VACV and transfected DNA. In contrast, recombination is delayed when the recombining sequences are located on different co-infecting viruses, and mature recombinants aren't detected until well after late gene expression is well established. The delay supports the hypothesis that factories impede inter-viral recombination, but even after factories merge there remain further constraints limiting virus DNA mixing and recombinant gene assembly. This delay could be related to the continued presence of ER-derived membranes within the fused virosomes, membranes that may once have wrapped individual factories.

  11. Live-Cell Imaging of Vaccinia Virus Recombination

    PubMed Central

    Paszkowski, Patrick; Noyce, Ryan S.; Evans, David H.

    2016-01-01

    Recombination between co-infecting poxviruses provides an important mechanism for generating the genetic diversity that underpins evolution. However, poxviruses replicate in membrane-bound cytoplasmic structures known as factories or virosomes. These are enclosed structures that could impede DNA mixing between co-infecting viruses, and mixing would seem to be essential for this process. We hypothesize that virosome fusion events would be a prerequisite for recombination between co-infecting poxviruses, and this requirement could delay or limit viral recombination. We have engineered vaccinia virus (VACV) to express overlapping portions of mCherry fluorescent protein fused to a cro DNA-binding element. In cells also expressing an EGFP-cro fusion protein, this permits live tracking of virus DNA and genetic recombination using confocal microscopy. Our studies show that different types of recombination events exhibit different timing patterns, depending upon the relative locations of the recombining elements. Recombination between partly duplicated sequences is detected soon after post-replicative genes are expressed, as long as the reporter gene sequences are located in cis within an infecting genome. The same kinetics are also observed when the recombining elements are divided between VACV and transfected DNA. In contrast, recombination is delayed when the recombining sequences are located on different co-infecting viruses, and mature recombinants aren’t detected until well after late gene expression is well established. The delay supports the hypothesis that factories impede inter-viral recombination, but even after factories merge there remain further constraints limiting virus DNA mixing and recombinant gene assembly. This delay could be related to the continued presence of ER-derived membranes within the fused virosomes, membranes that may once have wrapped individual factories. PMID:27525721

  12. Intrafamilial Transmission of Vaccinia virus during a Bovine Vaccinia Outbreak in Brazil: A New Insight in Viral Transmission Chain

    PubMed Central

    Pereira Oliveira, Graziele; Tavares Silva Fernandes, André; Lopes de Assis, Felipe; Augusto Alves, Pedro; Moreira Franco Luiz, Ana Paula; Barcelos Figueiredo, Leandra; Costa de Almeida, Cláudia Maria; Pires Ferreira Travassos, Carlos Eurico; de Souza Trindade, Giliane; Santos Abrahão, Jônatas; Geessien Kroon, Erna

    2014-01-01

    Bovine vaccinia (BV) is an emerging zoonosis caused by the Vaccinia virus (VACV), genus Orthopoxvirus (OPV), Poxviridae family. In general, human cases are related to direct contact with sick cattle but there is a lack of information about human-to-human transmission of VACV during BV outbreaks. In this study, we epidemiologically and molecularly show a case of VACV transmission between humans in São Francisco de Itabapoana County, Rio de Janeiro state. Our group collected samples from the patients, a 49-year-old patient and his son. Our results showed that patients had developed anti-OPV IgG or IgM antibodies and presented neutralizing antibodies against OPV. The VACV isolates displayed high identity (99.9%) and were grouped in the same phylogenetic tree branch. Our data indicate that human-to-human VACV transmission occurred during a BV outbreak, raising new questions about the risk factors of the VACV transmission chain. PMID:24615135

  13. Oncolytic viruses against cancer stem cells: A promising approach for gastrointestinal cancer.

    PubMed

    Huang, Fang; Wang, Bin-Rong; Wu, Ye-Qing; Wang, Fan-Chao; Zhang, Jian; Wang, Yi-Gang

    2016-09-21

    Gastrointestinal cancer has been one of the five most commonly diagnosed and leading causes of cancer mortality over the past few decades. Great progress in traditional therapies has been made, which prolonged survival in patients with early cancer, yet tumor relapse and drug resistance still occurred, which is explained by the cancer stem cell (CSC) theory. Oncolytic virotherapy has attracted increasing interest in cancer because of its ability to infect and lyse CSCs. This paper reviews the basic knowledge, CSC markers and therapeutics of gastrointestinal cancer (liver, gastric, colon and pancreatic cancer), as well as research advances and possible molecular mechanisms of various oncolytic viruses against gastrointestinal CSCs. This paper also summarizes the existing obstacles to oncolytic virotherapy and proposes several alternative suggestions to overcome the therapeutic limitations.

  14. Oncolytic viruses against cancer stem cells: A promising approach for gastrointestinal cancer

    PubMed Central

    Huang, Fang; Wang, Bin-Rong; Wu, Ye-Qing; Wang, Fan-Chao; Zhang, Jian; Wang, Yi-Gang

    2016-01-01

    Gastrointestinal cancer has been one of the five most commonly diagnosed and leading causes of cancer mortality over the past few decades. Great progress in traditional therapies has been made, which prolonged survival in patients with early cancer, yet tumor relapse and drug resistance still occurred, which is explained by the cancer stem cell (CSC) theory. Oncolytic virotherapy has attracted increasing interest in cancer because of its ability to infect and lyse CSCs. This paper reviews the basic knowledge, CSC markers and therapeutics of gastrointestinal cancer (liver, gastric, colon and pancreatic cancer), as well as research advances and possible molecular mechanisms of various oncolytic viruses against gastrointestinal CSCs. This paper also summarizes the existing obstacles to oncolytic virotherapy and proposes several alternative suggestions to overcome the therapeutic limitations. PMID:27672294

  15. Non-coding RNAs and heme oxygenase-1 in vaccinia virus infection

    SciTech Connect

    Meseda, Clement A.; Srinivasan, Kumar; Wise, Jasen; Catalano, Jennifer; Yamada, Kenneth M.; Dhawan, Subhash

    2014-11-07

    Highlights: • Heme oxygenase-1 (HO-1) induction inhibited vaccinia virus infection of macrophages. • Reduced infectivity inversely correlated with increased expression of non-coding RNAs. • The regulation of HO-1 and ncRNAs suggests a novel host defense response against vaccinia virus infection. - Abstract: Small nuclear RNAs (snRNAs) are <200 nucleotide non-coding uridylate-rich RNAs. Although the functions of many snRNAs remain undetermined, a population of snRNAs is produced during the early phase of infection of cells by vaccinia virus. In the present study, we demonstrate a direct correlation between expression of the cytoprotective enzyme heme oxygenase-1 (HO-1), suppression of selective snRNA expression, and inhibition of vaccinia virus infection of macrophages. Hemin induced HO-1 expression, completely reversed virus-induced host snRNA expression, and suppressed vaccinia virus infection. This involvement of specific virus-induced snRNAs and associated gene clusters suggests a novel HO-1-dependent host-defense pathway in poxvirus infection.

  16. Vaccination of vampire bats using recombinant vaccinia-rabies virus.

    PubMed

    Aguilar-Setién, Alvaro; Leon, Yolanda Campos; Tesoro, Emiliano Cruz; Kretschmer, Roberto; Brochier, Bernard; Pastoret, Paul-Pierre

    2002-07-01

    Adult vampire bats (Desmodus rotundus) were vaccinated by intramuscular, scarification, oral, or aerosol routes (n = 8 in each group) using a vaccinia-rabies glycoprotein recombinant virus. Sera were obtained before and 30 days after vaccination. All animals were then challenged intramuscularly with a lethal dose of rabies virus. Neutralizing antirabies antibodies were measured by rapid fluorescent focus inhibition test (RFFIT). Seroconversion was observed with each of the routes employed, but some aerosol and orally vaccinated animals failed to seroconvert. The highest antibody titers were observed in animals vaccinated by intramuscular and scarification routes. All animals vaccinated by intramuscular, scarification, and oral routes survived the viral challenge, but one of eight vampire bats receiving aerosol vaccination succumbed to the challenge. Of 31 surviving vaccinated and challenged animals, nine lacked detectable antirabies antibodies by RFFIT (five orally and four aerosol immunized animals). In contrast, nine of 10 non-vaccinated control bats succumbed to viral challenge. The surviving control bat had antiviral antibodies 90 days after viral challenge. These results suggest that the recombinant vaccine is an adequate and safe immunogen for bats by all routes tested.

  17. Pediatric cancer gone viral. Part I: strategies for utilizing oncolytic herpes simplex virus-1 in children

    PubMed Central

    Cripe, Timothy P; Chen, Chun-Yu; Denton, Nicholas L; Haworth, Kellie B; Hutzen, Brian; Leddon, Jennifer L; Streby, Keri A; Wang, Pin-Yi; Markert, James M; Waters, Alicia M; Gillespie, George Yancey; Beierle, Elizabeth A; Friedman, Gregory K

    2015-01-01

    Progress for improving outcomes in pediatric patients with solid tumors remains slow. In addition, currently available therapies are fraught with numerous side effects, often causing significant life-long morbidity for long-term survivors. The use of viruses to kill tumor cells based on their increased vulnerability to infection is gaining traction, with several viruses moving through early and advanced phase clinical testing. The prospect of increased efficacy and decreased toxicity with these agents is thus attractive for pediatric cancer. In part I of this two-part review, we focus on strategies for utilizing oncolytic engineered herpes simplex virus (HSV) to target pediatric malignancies. We discuss mechanisms of action, routes of delivery, and the role of preexisting immunity on antitumor efficacy. Challenges to maximizing oncolytic HSV in children are examined, and we highlight how these may be overcome through various arming strategies. We review the preclinical and clinical evidence demonstrating safety of a variety of oncolytic HSVs. In Part II, we focus on the antitumor efficacy of oncolytic HSV in pediatric tumor types, pediatric clinical advances made to date, and future prospects for utilizing HSV in pediatric patients with solid tumors. PMID:26436135

  18. Pediatric cancer gone viral. Part I: strategies for utilizing oncolytic herpes simplex virus-1 in children.

    PubMed

    Cripe, Timothy P; Chen, Chun-Yu; Denton, Nicholas L; Haworth, Kellie B; Hutzen, Brian; Leddon, Jennifer L; Streby, Keri A; Wang, Pin-Yi; Markert, James M; Waters, Alicia M; Gillespie, George Yancey; Beierle, Elizabeth A; Friedman, Gregory K

    Progress for improving outcomes in pediatric patients with solid tumors remains slow. In addition, currently available therapies are fraught with numerous side effects, often causing significant life-long morbidity for long-term survivors. The use of viruses to kill tumor cells based on their increased vulnerability to infection is gaining traction, with several viruses moving through early and advanced phase clinical testing. The prospect of increased efficacy and decreased toxicity with these agents is thus attractive for pediatric cancer. In part I of this two-part review, we focus on strategies for utilizing oncolytic engineered herpes simplex virus (HSV) to target pediatric malignancies. We discuss mechanisms of action, routes of delivery, and the role of preexisting immunity on antitumor efficacy. Challenges to maximizing oncolytic HSV in children are examined, and we highlight how these may be overcome through various arming strategies. We review the preclinical and clinical evidence demonstrating safety of a variety of oncolytic HSVs. In Part II, we focus on the antitumor efficacy of oncolytic HSV in pediatric tumor types, pediatric clinical advances made to date, and future prospects for utilizing HSV in pediatric patients with solid tumors.

  19. Effect of Repeat Dosing of Engineered Oncolytic Herpes Simplex Virus on Preclinical Models of Rhabdomyosarcoma.

    PubMed

    Waters, Alicia M; Stafman, Laura L; Garner, Evan F; Mruthyunjayappa, Smitha; Stewart, Jerry E; Friedman, Gregory K; Coleman, Jennifer M; Markert, James M; Gillespie, G Yancey; Beierle, Elizabeth A

    2016-10-01

    Rhabdomyosarcoma (RMS), a tumor of skeletal muscle origin, is the most common sarcoma of childhood. Despite multidrug chemotherapy regimens, surgical intervention, and radiation treatment, outcomes remain poor, especially in advanced disease, and novel therapies are needed for the treatment of these aggressive malignancies. Genetically engineered oncolytic viruses, such as herpes simplex virus-1 (HSV), are currently being explored as treatments for pediatric tumors. M002, an oncolytic HSV, has both copies of the γ134.5 gene deleted, enabling replication in tumor cells but thwarting infection of normal, postmitotic cells. We hypothesized that M002 would infect human RMS tumor cells and lead to decreased tumor cell survival in vitro and impede tumor growth in vivo. In the current study, we demonstrated that M002 could infect, replicate in, and decrease cell survival in both embryonal (ERMS) and alveolar rhabdomyosarcoma (ARMS) cells. Additionally, M002 reduced xenograft tumor growth and increased animal survival in both ARMS and ERMS. Most importantly, we showed for the first time that repeated dosing of oncolytic virus coupled with low-dose radiation provided improved tumor response in RMS. These findings provide support for the clinical investigation of oncolytic HSV in pediatric RMS.

  20. Systemic therapy of spontaneous prostate cancer in transgenic mice with oncolytic herpes simplex viruses.

    PubMed

    Varghese, Susan; Rabkin, Samuel D; Nielsen, G Petur; MacGarvey, Usha; Liu, Renbin; Martuza, Robert L

    2007-10-01

    Oncolytic viruses are an innovative therapeutic strategy for cancer, wherein viral replication and cytotoxicity are selective for tumor cells. Here we show the efficacy of systemically administered oncolytic viruses for the treatment of spontaneously arising tumors, specifically the use of oncolytic herpes simplex viruses (HSV) administered i.v. to treat spontaneously developing primary and metastatic prostate cancer in the transgenic TRAMP mouse, which recapitulates human prostate cancer progression. Four administrations of systemically delivered NV1023 virus, an HSV-1/HSV-2 oncolytic recombinant, to TRAMP mice at 12 or 18 weeks of age (presence of prostate adenocarcinoma or metastatic disease, respectively) inhibited primary tumor growth and metastases to lymph nodes. Expression of interleukin 12 (IL-12) from NV1042 virus, a derivative of NV1023, was additionally effective, significantly reducing the frequency of development of prostate cancer and lung metastases, even when the mice were treated after the onset of metastasis at 18 weeks of age. NV1042-infected cells, as detected by 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining for Lac Z expressed by the virus, were present in prostate tumors 1 week after the final virus injection and viral DNA was detected at 2 weeks after final virus injection by real-time PCR in primary and metastatic tumors but not in liver or blood. No toxicity was observed in any of the treated mice. The efficacy of the IL-12-expressing NV1042 virus in this aggressive prostate cancer model using a clinically relevant treatment paradigm merits its consideration for clinical studies.

  1. Orf virus encodes a homolog of the vaccinia virus interferon-resistance gene E3L.

    PubMed

    McInnes, C J; Wood, A R; Mercer, A A

    1998-01-01

    A homolog of the vaccinia virus (VAC) interferon resistance gene E3L has been discovered in orf virus strain NZ-2, a parapoxvirus that infects sheep, goats and humans. The gene is located 20 kb from the left terminus of the orf virus genome and is transcribed towards this terminus. RNase protection studies have been used to define the limits of the gene and Northern analysis revealed that it is expressed early in infection. The predicted amino acid sequence of the orf virus protein shares 31% identity (57% similarity) with the VAC E3L protein. Four of the six residues identified as being essential to dsRNA binding in the vaccinia virus protein are conserved in the orf virus protein whilst the other two amino acid changes are conservative substitutions. The orf virus gene has been sequenced in two other orf virus strains which vary markedly in their ability to produce experimental lesions in vivo. Their predicted protein sequences vary by less than 3% from the NZ-2 protein. The recombinant orf virus protein, expressed as a fusion protein in E. coli, bound double-stranded (ds)RNA but not dsDNA, single-stranded (ss)DNA or ssRNA . This is the first demonstration of a VAC E3L-like gene encoded by a parapoxvirus.

  2. Interactions of the Vaccinia Virus A19 Protein

    PubMed Central

    Satheshkumar, P. S.; Olano, L. Renee; Hammer, Carl H.; Zhao, Ming

    2013-01-01

    The A19 protein of vaccinia virus (VACV) is conserved among chordopoxviruses, expressed late in infection, packaged in the virus core, and required for a late step in morphogenesis. Multiple-sequence alignments of A19 homologs indicated conservation of a series of lysines and arginines, which could represent a nuclear localization or nucleic acid binding motif, and a pair of CXXC motifs that suggested a zinc finger or redox active sites. The importance of the CXXC motif was confirmed by cysteine-to-serine substitutions, which rendered the altered protein unable to trans-complement infectivity of a null mutant. Nevertheless, the cysteines were not required for function of the poxvirus-specific redox pathway. Epitope-tagged A19 proteins were detected in the nucleus and cytoplasm in both infected and uninfected cells, but this distribution was unaffected by alanine substitutions of the arginine residues, which only partially reduced the ability of the mutated protein to trans-complement infectivity. Viral proteins specifically associated with affinity-purified A19 were identified by mass spectrometry as components of the transcription complex, including RNA polymerase subunits, RAP94 (RNA polymerase-associated protein 94), early transcription factors, capping enzyme, and nucleoside triphosphate phosphohydrolase I, and two core proteins required for morphogenesis. Further studies suggested that the interaction of A19 with the RNA polymerase did not require RAP94 or other intermediate or late viral proteins but was reduced by mutation of cysteines in the putative zinc finger domain. Although A19 was not required for incorporation of the transcription complex in virus particles, the transcriptional activity of A19-deficient virus particles was severely reduced. PMID:23885084

  3. Mathematical modeling of tumor therapy with oncolytic viruses: effects of parametric heterogeneity on cell dynamics

    PubMed Central

    Karev, Georgy P; Novozhilov, Artem S; Koonin, Eugene V

    2006-01-01

    Background: One of the mechanisms that ensure cancer robustness is tumor heterogeneity, and its effects on tumor cells dynamics have to be taken into account when studying cancer progression. There is no unifying theoretical framework in mathematical modeling of carcinogenesis that would account for parametric heterogeneity. Results: Here we formulate a modeling approach that naturally takes stock of inherent cancer cell heterogeneity and illustrate it with a model of interaction between a tumor and an oncolytic virus. We show that several phenomena that are absent in homogeneous models, such as cancer recurrence, tumor dormancy, and others, appear in heterogeneous setting. We also demonstrate that, within the applied modeling framework, to overcome the adverse effect of tumor cell heterogeneity on the outcome of cancer treatment, a heterogeneous population of an oncolytic virus must be used. Heterogeneity in parameters of the model, such as tumor cell susceptibility to virus infection and the ability of an oncolytic virus to infect tumor cells, can lead to complex, irregular evolution of the tumor. Thus, quasi-chaotic behavior of the tumor-virus system can be caused not only by random perturbations but also by the heterogeneity of the tumor and the virus. Conclusion: The modeling approach described here reveals the importance of tumor cell and virus heterogeneity for the outcome of cancer therapy. It should be straightforward to apply these techniques to mathematical modeling of other types of anticancer therapy. Reviewers: Leonid Hanin (nominated by Arcady Mushegian), Natalia Komarova (nominated by Orly Alter), and David Krakauer. PMID:17018145

  4. A novel, polymer-coated oncolytic measles virus overcomes immune suppression and induces robust antitumor activity

    PubMed Central

    Nosaki, Kaname; Hamada, Katsuyuki; Takashima, Yuto; Sagara, Miyako; Matsumura, Yumiko; Miyamoto, Shohei; Hijikata, Yasuki; Okazaki, Toshihiko; Nakanishi, Yoichi; Tani, Kenzaburo

    2016-01-01

    Although various therapies are available to treat cancers, including surgery, chemotherapy, and radiotherapy, cancer has been the leading cause of death in Japan for the last 30 years, and new therapeutic modalities are urgently needed. As a new modality, there has recently been great interest in oncolytic virotherapy, with measles virus being a candidate virus expected to show strong antitumor effects. The efficacy of virotherapy, however, was strongly limited by the host immune response in previous clinical trials. To enhance and prolong the antitumor activity of virotherapy, we combined the use of two newly developed tools: the genetically engineered measles virus (MV-NPL) and the multilayer virus-coating method of layer-by-layer deposition of ionic polymers. We compared the oncolytic effects of this polymer-coated MV-NPL with the naked MV-NPL, both in vitro and in vivo. In the presence of anti-MV neutralizing antibodies, the polymer-coated virus showed more enhanced oncolytic activity than did the naked MV-NPL in vitro. We also examined antitumor activities in virus-treated mice. Complement-dependent cytotoxicity and antitumor activities were higher in mice treated with polymer-coated MV-NPL than in mice treated with the naked virus. This novel, polymer-coated MV-NPL is promising for clinical cancer therapy in the future. PMID:27847861

  5. A novel, polymer-coated oncolytic measles virus overcomes immune suppression and induces robust antitumor activity.

    PubMed

    Nosaki, Kaname; Hamada, Katsuyuki; Takashima, Yuto; Sagara, Miyako; Matsumura, Yumiko; Miyamoto, Shohei; Hijikata, Yasuki; Okazaki, Toshihiko; Nakanishi, Yoichi; Tani, Kenzaburo

    2016-01-01

    Although various therapies are available to treat cancers, including surgery, chemotherapy, and radiotherapy, cancer has been the leading cause of death in Japan for the last 30 years, and new therapeutic modalities are urgently needed. As a new modality, there has recently been great interest in oncolytic virotherapy, with measles virus being a candidate virus expected to show strong antitumor effects. The efficacy of virotherapy, however, was strongly limited by the host immune response in previous clinical trials. To enhance and prolong the antitumor activity of virotherapy, we combined the use of two newly developed tools: the genetically engineered measles virus (MV-NPL) and the multilayer virus-coating method of layer-by-layer deposition of ionic polymers. We compared the oncolytic effects of this polymer-coated MV-NPL with the naked MV-NPL, both in vitro and in vivo. In the presence of anti-MV neutralizing antibodies, the polymer-coated virus showed more enhanced oncolytic activity than did the naked MV-NPL in vitro. We also examined antitumor activities in virus-treated mice. Complement-dependent cytotoxicity and antitumor activities were higher in mice treated with polymer-coated MV-NPL than in mice treated with the naked virus. This novel, polymer-coated MV-NPL is promising for clinical cancer therapy in the future.

  6. Assessment of a vaccinia virus vectored multi-epitope live vaccine candidate for Plasmodium falciparum.

    PubMed

    Dong, W; Li, M; Bi, H; Li, Y; Wu, J; Qu, L

    2001-01-01

    We constructed a live recombinant vaccinia virus vaccine candidate containing a synthesised hybrid gene termed 'HGFSP' encoding circumsporozoite protein (CSP), major merozoite surface antigen-1(MSA1), major merozoite surface antigen-2 (MSA2), and ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum, interleukin-1 (IL-1) and tetanus toxin (TT) epitopes. Anti-recombinant vaccinia virus rabbit sera and IgG were tested in inhibition experiments in vitro. Results showed that the recombinant vaccinia virus had some capability to inhibit the growth of P. falciparum in vitro. The sera of rabbits, rats, and mice immunised with recombinant virus showed obvious IL-2 activity 4-6 weeks after immunisation. The interferon (IFN) level of sera from these animals 6 weeks after immunisation was significantly higher than before immunisation. These results indicate that the recombinant vaccinia virus can stimulate cell mediated responses (Th1 cell response) in immunised animals, and has the capability to inhibit multiplication of in vitro cultured P. falciparum. Thus this recombinant vaccinia virus is an appropriate vaccine candidate for further evaluation in Aotus monkey or human clinical trails.

  7. Nigericin is a potent inhibitor of the early stage of vaccinia virus replication.

    PubMed

    Myskiw, Chad; Piper, Jessica; Huzarewich, Rhiannon; Booth, Tim F; Cao, Jingxin; He, Runtao

    2010-12-01

    Poxviruses remain a significant public health concern due to their potential use as bioterrorist agents and the spread of animal borne poxviruses, such as monkeypox virus, to humans. Thus, the identification of small molecule inhibitors of poxvirus replication is warranted. Vaccinia virus is the prototypic member of the Orthopoxvirus genus, which also includes variola and monkeypox virus. In this study, we demonstrate that the carboxylic ionophore nigericin is a potent inhibitor of vaccinia virus replication in several human cell lines. In HeLa cells, we found that the 50% inhibitory concentration of nigericin against vaccinia virus was 7.9 nM, with a selectivity index of 1038. We present data demonstrating that nigericin targets vaccinia virus replication at a post-entry stage. While nigericin moderately inhibits both early vaccinia gene transcription and translation, viral DNA replication and intermediate and late gene expression are severely compromised in the presence of nigericin. Our results demonstrate that nigericin has the potential to be further developed into an effective antiviral to treat poxvirus infections. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

  8. Human CD4+ T Cell Epitopes from Vaccinia Virus Induced by Vaccination or Infection

    PubMed Central

    Calvo-Calle, J. Mauricio; Strug, Iwona; Nastke, Maria-Dorothea; Baker, Stephen P; Stern, Lawrence J

    2007-01-01

    Despite the importance of vaccinia virus in basic and applied immunology, our knowledge of the human immune response directed against this virus is very limited. CD4+ T cell responses are an important component of immunity induced by current vaccinia-based vaccines, and likely will be required for new subunit vaccine approaches, but to date vaccinia-specific CD4+ T cell responses have been poorly characterized, and CD4+ T cell epitopes have been reported only recently. Classical approaches used to identify T cell epitopes are not practical for large genomes like vaccinia. We developed and validated a highly efficient computational approach that combines prediction of class II MHC-peptide binding activity with prediction of antigen processing and presentation. Using this approach and screening only 36 peptides, we identified 25 epitopes recognized by T cells from vaccinia-immune individuals. Although the predictions were made for HLA-DR1, eight of the peptides were recognized by donors of multiple haplotypes. T cell responses were observed in samples of peripheral blood obtained many years after primary vaccination, and were amplified after booster immunization. Peptides recognized by multiple donors are highly conserved across the poxvirus family, including variola, the causative agent of smallpox, and may be useful in development of a new generation of smallpox vaccines and in the analysis of the immune response elicited to vaccinia virus. Moreover, the epitope identification approach developed here should find application to other large-genome pathogens. PMID:17937498

  9. Targeting Prostate Cancer for Gene Therapy Utilizing Lentivirus and Oncolytic VSV Virus

    DTIC Science & Technology

    2009-04-01

    Prostate cancer is the most commonly diagnosed non- skin carcinoma, and one of the leading causes of cancerrelated deaths in North American men. Presently...primary and metastatic cancer cells while sparing normal cells. Vesicular Stomatitis Virus (VSV) is an oncolytic virus which is able to replicate in...capable of selectively infecting and killing malignant prostate cells while sparing normal cells. This cancer-specific cell death was not due to

  10. Use of oncolytic viruses for the eradication of drug-resistant cancer cells

    PubMed Central

    Wodarz, Dominik

    2008-01-01

    Targeted therapy using small-molecule inhibitors is a promising new therapy approach against cancer, but drug-resistant mutants present an obstacle to success. Oncolytic virus therapy, where viruses replicate specifically in cancer cells and kill them, is another promising therapy approach against cancer. While encouraging results have been observed in clinical trials, consistent success has not been possible so far. Based on a computational framework, I report that even if oncolytic virus therapy fails to eradicate a cancer, it can have the potential to eradicate the sub-population of drug-resistant cancer cells. Once this has occurred, targeted drug therapy can be used to induce cancer remission. For this to work, a drug resistance mutation must confer a certain fitness cost to the cell, as has been documented in the literature. The reason for this finding is that in the presence of a shared virus, the faster growing (drug-sensitive) cell population produces an amount of virus that is too much for the slower growing (drug-resistant) cell population to survive. This is derived from a population dynamic principle known as apparent competition. Therefore, a sequential combination of oncolytic virus and targeted therapies can overcome major weaknesses of either approach alone. PMID:18664430

  11. Comparison of methods for detection of vaccinia virus in patient specimens.

    PubMed

    Fedorko, Daniel P; Preuss, Jeanne C; Fahle, Gary A; Li, Li; Fischer, Steven H; Hohman, Patricia; Cohen, Jeffrey I

    2005-09-01

    We analyzed a shell vial culture assay (SVA), real-time PCR, and a direct fluorescent antibody assay (DFA) for rapid detection of vaccinia virus from vaccination sites of Dryvax vaccine recipients. Of 47 samples assayed, 100% were positive by PCR, 89% were positive by SVA, and 40% were positive by DFA. DFA was limited by the need for adequate numbers of cells, with 32% of samples inadequate for interpretation. DFA performed better with specimens from patients who had not previously received the vaccine. PCR was positive for longer times postvaccination than was SVA. Infectious virus could be recovered after 45 min of acetone fixation of shell vial coverslips. Commercially available polyclonal antibodies cross-reacted with other orthopoxviruses and herpes simplex 1, but commercially available monoclonal antibodies were specific for vaccinia virus. In summary, PCR was the most sensitive test for detecting vaccinia virus in clinical specimens, while the DFA was the most rapid but the least sensitive test.

  12. Antitumor activity and immune responses induced by a recombinant carcinoembryonic antigen-vaccinia virus vaccine.

    PubMed

    Kantor, J; Irvine, K; Abrams, S; Kaufman, H; DiPietro, J; Schlom, J

    1992-07-15

    Human carcinoembryonic antigen (CEA) is a 180-kd glycoprotein expressed in human colorectal, gastric, pancreatic, breast, and non-small-cell lung carcinomas. Previous studies have demonstrated enhanced immune responses to other antigens presented with vaccinia virus proteins via a recombinant vaccinia virus construct. In addition, we have developed a recombinant CEA-vaccinia virus construct, designated rV(WR)-CEA, and have demonstrated humoral anti-CEA responses in mice after immunization with that virus. The goals of this study were (a) to construct a recombinant CEA-vaccinia vaccine in a less virulent vaccinia strain that is potentially safe and effective for treatment of patients whose tumors express CEA and (b) to evaluate the ability of the recombinant CEA-vaccinia vaccine to prevent and reverse tumor growth in mice and to elicit cell-mediated and humoral anti-CEA immune responses. Using the New York City strain of vaccinia virus, which is used in smallpox vaccination and is more attenuated for humans than rV(WR), we derived a recombinant CEA-vaccinia construct, designated rV(NYC)-CEA. The ability of this construct to induce antitumor immunity was evaluated in mice receiving subcutaneous injections of murine colon adenocarcinoma cells expressing the human CEA gene. Administration of rV(NYC)-CEA in mice induced strong anti-CEA antibody responses, as well as CEA-specific cell-mediated responses, including delayed-type hypersensitivity, lymphoproliferative, and cytotoxic responses. Vaccination of mice with the rV(NYC)-CEA rendered them resistant to the growth of subsequently transplanted CEA-expressing tumors. Moreover, when mice were vaccinated 7 days after tumor cell injection, tumor growth was either greatly reduced or eliminated. No toxic effects were observed in any of the mice. These studies demonstrate that antitumor activity can be induced with the use of a recombinant CEA-vaccinia virus construct derived from an attenuated vaccinia strain, and they

  13. Oncolytic viral purging of leukemic hematopoietic stem and progenitor cells with Myxoma virus

    PubMed Central

    Rahman, Masmudur M.; Madlambayan, Gerard J.; Cogle, Christopher R.; McFadden, Grant

    2010-01-01

    High-dose chemotherapy and radiation followed by autologous blood and marrow transplantation (ABMT) has been extensively used for the treatment of certain cancers that are refractory to standard therapeutic regimes. However, a major challenge with ABMT for patients with hematologic malignancies is disease relapse, mainly due to either contamination with cancerous hematopoietic stem and progenitor cells (HSPCs) within the autograft or the persistence of residual therapy-resistant disease niches within the patient. Oncolytic viruses represent a promising therapeutic approach to prevent cancer relapse by eliminating tumor-initiating cells that contaminate the autograft. Here we summarize an ex vivo “purging” strategy with oncolytic myxoma virus (MYXV) to remove cancer-initiating cells from patient autografts prior to transplantation. MYXV, a novel oncolytic poxvirus with potent anti-cancer properties in a variety of in vivo tumor models, can specifically eliminate cancerous stem and progenitor cells from samples obtained from acute myelogenous leukemia (AML) patients, while sparing normal CD34+ hematopoietic stem and progenitor cells capable of rescuing hematopoiesis following high dose conditioning. We propose that a broader subset of patients with intractable hematologic malignancies who have failed standard therapy could become eligible for ABMT when the treatment schema is coupled with ex vivo oncolytic therapy. PMID:20211576

  14. Oncolytic viral purging of leukemic hematopoietic stem and progenitor cells with Myxoma virus.

    PubMed

    Rahman, Masmudur M; Madlambayan, Gerard J; Cogle, Christopher R; McFadden, Grant

    2010-01-01

    High-dose chemotherapy and radiation followed by autologous blood and marrow transplantation (ABMT) has been used for the treatment of certain cancers that are refractory to standard therapeutic regimes. However, a major challenge with ABMT for patients with hematologic malignancies is disease relapse, mainly due to either contamination with cancerous hematopoietic stem and progenitor cells (HSPCs) within the autograft or the persistence of residual therapy-resistant disease niches within the patient. Oncolytic viruses represent a promising therapeutic approach to prevent cancer relapse by eliminating tumor-initiating cells that contaminate the autograft. Here we summarize an ex vivo "purging" strategy with oncolytic Myxoma virus (MYXV) to remove cancer-initiating cells from patient autografts prior to transplantation. MYXV, a novel oncolytic poxvirus with potent anti-cancer properties in a variety of in vivo tumor models, can specifically eliminate cancerous stem and progenitor cells from samples obtained from acute myelogenous leukemia (AML) patients, while sparing normal CD34+ hematopoietic stem and progenitor cells capable of rescuing hematopoiesis following high dose conditioning. We propose that a broader subset of patients with intractable hematologic malignancies who have failed standard therapy could become eligible for ABMT when the treatment schema is coupled with ex vivo oncolytic therapy.

  15. Perfusion Pressure Is a Critical Determinant of the Intratumoral Extravasation of Oncolytic Viruses

    PubMed Central

    Miller, Amber; Nace, Rebecca; Ayala-Breton C, Camilo; Steele, Michael; Bailey, Kent; Peng, Kah Whye; Russell, Stephen J

    2016-01-01

    Antitumor efficacy of oncolytic virotherapy is determined by the density and distribution of infectious centers within the tumor, which may be heavily influenced by the permeability and blood flow in tumor microvessels. Here, we investigated whether systemic perfusion pressure, a key driver of tumor blood flow, could influence the intratumoral extravasation of systemically administered oncolytic vesicular stomatitis virus (VSV) in myeloma tumor-bearing mice. Exercise was used to increase mean arterial pressure, and general anesthesia to decrease it. A recombinant VSV expressing the sodium iodide symporter (NIS), which concentrates radiotracers at sites of infection, was administered intravenously to exercising or anesthetized mice, and nuclear NIS reporter gene imaging was used to noninvasively track the density and spatial distribution of intratumoral infectious centers. Anesthesia resulted in decreased intratumoral infection density, while exercise increased the density and uniformity of infectious centers. Perfusion state also had a significant impact on the antitumor efficacy of the VSV therapy. In conclusion, quantitative dynamic radiohistologic imaging was used to noninvasively interrogate delivery of oncolytic virotherapy, highlighting the critical importance of perfusion pressure as a driver of intratumoral delivery and efficacy of oncolytic viruses. PMID:26647825

  16. Vaccinia Virus Recombinants: Expression of VSV Genes and Protective Immunization of Mice and Cattle

    NASA Astrophysics Data System (ADS)

    Mackett, M.; Yilma, T.; Rose, J. K.; Moss, B.

    1985-01-01

    Vesicular stomatitis virus (VSV) causes a contagious disease of horses, cattle, and pigs. When DNA copies of messenger RNA's for the G or N proteins of VSV were linked to a vaccinia virus promoter and inserted into the vaccinia genome, the recombinants retained infectivity and synthesized VSV polypeptides. After intradermal vaccination with live recombinant virus expressing the G protein, mice produced VSV-neutralizing antibodies and were protected against lethal encephalitis upon intravenous challenge with VSV. In cattle, the degree of protection against intradermalingually injected VSV was correlated with the level of neutralizing antibody produced following vaccination.

  17. Spatial and temporal epithelial ovarian cancer cell heterogeneity impacts Maraba virus oncolytic potential.

    PubMed

    Tong, Jessica G; Valdes, Yudith Ramos; Sivapragasam, Milani; Barrett, John W; Bell, John C; Stojdl, David; DiMattia, Gabriel E; Shepherd, Trevor G

    2017-08-30

    Epithelial ovarian cancer exhibits extensive interpatient and intratumoral heterogeneity, which can hinder successful treatment strategies. Herein, we investigated the efficacy of an emerging oncolytic, Maraba virus (MRBV), in an in vitro model of ovarian tumour heterogeneity. Four ovarian high-grade serous cancer (HGSC) cell lines were isolated and established from a single patient at four points during disease progression. Limiting-dilution subcloning generated seven additional subclone lines to assess intratumoral heterogeneity. MRBV entry and oncolytic efficacy were assessed among all 11 cell lines. Low-density receptor (LDLR) expression, conditioned media treatments and co-cultures were performed to determine factors impacting MRBV oncolysis. Temporal and intratumoral heterogeneity identified two subpopulations of cells: one that was highly sensitive to MRBV, and another set which exhibited 1000-fold reduced susceptibility to MRBV-mediated oncolysis. We explored both intracellular and extracellular mechanisms influencing sensitivity to MRBV and identified that LDLR can partially mediate MRBV infection. LDLR expression, however, was not the singular determinant of sensitivity to MRBV among the HGSC cell lines and subclones. We verified that there were no apparent extracellular factors, such as type I interferon responses, contributing to MRBV resistance. However, direct cell-cell contact by co-culture of MRBV-resistant subclones with sensitive cells restored virus infection and oncolytic killing of mixed population. Our data is the first to demonstrate differential efficacy of an oncolytic virus in the context of both spatial and temporal heterogeneity of HGSC cells and to evaluate whether it will constitute a barrier to effective viral oncolytic therapy.

  18. Vaccinia Virus Recombinant Expressing Herpes Simplex Virus Type 1 Glycoprotein D Prevents Latent Herpes in Mice

    NASA Astrophysics Data System (ADS)

    Cremer, Kenneth J.; Mackett, Michael; Wohlenberg, Charles; Notkins, Abner Louis; Moss, Bernard

    1985-05-01

    In humans, herpes simplex virus causes a primary infection and then often a latent ganglionic infection that persists for life. Because these latent infections can recur periodically, vaccines are needed that can protect against both primary and latent herpes simplex infections. Infectious vaccinia virus recombinants that contain the herpes simplex virus type 1 (HSV-1) glycoprotein D gene under control of defined early or late vaccinia virus promoters were constructed. Tissue culture cells infected with these recombinant viruses synthesized a glycosylated protein that had the same mass (60,000 daltons) as the glycoprotein D produced by HSV-1. Immunization of mice with one of these recombinant viruses by intradermal, subcutaneous, or intraperitoneal routes resulted in the production of antibodies that neutralized HSV-1 and protected the mice against subsequent lethal challenge with HSV-1 or HSV-2. Immunization with the recombinant virus also protected the majority of the mice against the development of a latent HSV-1 infection of the trigeminal ganglia. This is the first demonstration that a genetically engineered vaccine can prevent the development of latency.

  19. 4th European Seminars in Virology on Oncogenic and Oncolytic Viruses, in Bertinoro (Bologna), Italy.

    PubMed

    Reale, Alberto; Messa, Lorenzo; Vitiello, Adriana; Loregian, Arianna; Palù, Giorgio

    2017-10-01

    The 4th European Seminars in Virology (EuSeV), which was focused on oncogenic and oncolytic viruses, was held in Bertinoro (Bologna), Italy, from June 10 to 12, 2016. This article summarizes the plenary lectures and aims to illustrate the main topics discussed at 4th EuSeV, which brought together knowledge and expertise in the field of oncogenic and oncolytic viruses from all over the world. The meeting was divided in two parts, "Mechanisms of Viral Oncogenesis" and "Viral Oncolysis and Immunotherapy," which were both focused on dissecting the complex and multi-factorial interplay between cancer and human viruses and on exploring new anti-cancer strategies. J. Cell. Physiol. 232: 2641-2648, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. Directed Evolution Generates a Novel Oncolytic Virus for the Treatment of Colon Cancer

    PubMed Central

    Kuhn, Irene; Harden, Paul; Bauzon, Maxine; Chartier, Cecile; Nye, Julie; Thorne, Steve; Reid, Tony; Ni, Shaoheng; Lieber, Andre; Fisher, Kerry; Seymour, Len; Rubanyi, Gabor M.; Harkins, Richard N.; Hermiston, Terry W.

    2008-01-01

    Background Viral-mediated oncolysis is a novel cancer therapeutic approach with the potential to be more effective and less toxic than current therapies due to the agents selective growth and amplification in tumor cells. To date, these agents have been highly safe in patients but have generally fallen short of their expected therapeutic value as monotherapies. Consequently, new approaches to generating highly potent oncolytic viruses are needed. To address this need, we developed a new method that we term “Directed Evolution” for creating highly potent oncolytic viruses. Methodology/Principal Findings Taking the “Directed Evolution” approach, viral diversity was increased by pooling an array of serotypes, then passaging the pools under conditions that invite recombination between serotypes. These highly diverse viral pools were then placed under stringent directed selection to generate and identify highly potent agents. ColoAd1, a complex Ad3/Ad11p chimeric virus, was the initial oncolytic virus derived by this novel methodology. ColoAd1, the first described non-Ad5-based oncolytic Ad, is 2–3 logs more potent and selective than the parent serotypes or the most clinically advanced oncolytic Ad, ONYX-015, in vitro. ColoAd1's efficacy was further tested in vivo in a colon cancer liver metastasis xenograft model following intravenous injection and its ex vivo selectivity was demonstrated on surgically-derived human colorectal tumor tissues. Lastly, we demonstrated the ability to arm ColoAd1 with an exogenous gene establishing the potential to impact the treatment of cancer on multiple levels from a single agent. Conclusions/Significance Using the “Directed Evolution” methodology, we have generated ColoAd1, a novel chimeric oncolytic virus. In vitro, this virus demonstrated a >2 log increase in both potency and selectivity when compared to ONYX-015 on colon cancer cells. These results were further supported by in vivo and ex vivo studies. Furthermore

  1. Animal Movement and Establishment of Vaccinia Virus Cantagalo Strain in Amazon Biome, Brazil

    PubMed Central

    Quixabeira-Santos, Jociane Cristina; Medaglia, Maria Luiza G.; Pescador, Caroline A.

    2011-01-01

    To understand the emergence of vaccinia virus Cantagalo strain in the Amazon biome of Brazil, during 2008–2010 we conducted a molecular and epidemiologic survey of poxvirus outbreaks. Data indicate that animal movement was the major cause of virus dissemination within Rondônia State, leading to the establishment and spread of this pathogen. PMID:21470472

  2. Effects of poliovirus 2A(pro) on vaccinia virus gene expression.

    PubMed

    Feduchi, E; Aldabe, R; Novoa, I; Carrasco, L

    1995-12-15

    The effects of transient expression of poliovirus 2A(pro) on p220 cleavage in COS cells have been analyzed. When 2A(pro) was cloned in plasmid pTM1 and transiently expressed in COS cells, efficient cleavage of p220 occurred after infection of these cells with a recombinant vaccinia virus bearing phage T7 RNA polymerase. High numbers of COS cells were transfected with pTM1-2A, as judged by p220 cleavage, thereby allowing an analysis of the effects of poliovirus 2A(pro) on vaccinia virus gene expression. A 40-50% cleavage of p220 by transfected poliovirus 2A(pro) was observed ten hours post infection and cleavage was almost complete (80-90%) 20-25 hours post infection with vaccinia virus. Profound inhibition of vaccinia virus protein synthesis was detectable ten hours post infection and was maximal 20-25 hours post infection. This inhibition resulted from neither a blockade of transcription of vaccinia virus nor a lack of translatability of the mRNAs present in cells that synthesize poliovirus 2A(pro). Addition of ara-C inhibited the replication of vaccinia virus and allowed the continued synthesis of cellular proteins. Under these conditions, 2A(pro) is expressed and blocks cellular translation. Finally, p220 cleavage by 2A(pro) did not inhibit the translation of a mRNA encoding poliovirus protein 2C, as directed by the 5' leader sequences of encephalomiocarditis virus. Therefore, these findings show a correlation between p220 cleavage and inhibition of translation from newly made mRNAs. Our results are discussed in the light of present knowledge of p220 function, and new approaches are considered that might provide further insights into the function(s) of initiation factor eIF-4F.

  3. Immunization with vaccinia virus induces polyfunctional and phenotypically distinctive CD8+ T cell responses

    PubMed Central

    Precopio, Melissa L.; Betts, Michael R.; Parrino, Janie; Price, David A.; Gostick, Emma; Ambrozak, David R.; Asher, Tedi E.; Douek, Daniel C.; Harari, Alexandre; Pantaleo, Giuseppe; Bailer, Robert; Graham, Barney S.; Roederer, Mario; Koup, Richard A.

    2007-01-01

    Vaccinia virus immunization provides lifelong protection against smallpox, but the mechanisms of this exquisite protection are unknown. We used polychromatic flow cytometry to characterize the functional and phenotypic profile of CD8+ T cells induced by vaccinia virus immunization in a comparative vaccine trial of modified vaccinia virus Ankara (MVA) versus Dryvax immunization in which protection was assessed against subsequent Dryvax challenge. Vaccinia virus–specific CD8+ T cells induced by both MVA and Dryvax were highly polyfunctional; they degranulated and produced interferon γ, interleukin 2, macrophage inflammatory protein 1β, and tumor necrosis factor α after antigenic stimulation. Responding CD8+ T cells exhibited an unusual phenotype (CD45RO−CD27intermediate). The unique phenotype and high degree of polyfunctionality induced by vaccinia virus also extended to inserted HIV gene products of recombinant NYVAC. This quality of the CD8+ T cell response may be at least partially responsible for the profound efficacy of these vaccines in protection against smallpox and serves as a benchmark against which other vaccines can be evaluated. PMID:17535971

  4. Horizontal study of vaccinia virus infections in an endemic area: epidemiologic, phylogenetic and economic aspects.

    PubMed

    Assis, Felipe L; Franco-Luiz, Ana Paula M; Paim, Luis M; Oliveira, Graziele P; Pereira, Alexandre F; de Almeida, Gabriel M F; Figueiredo, Leandra B; Tanus, Adriano; Trindade, Giliane S; Ferreira, Paulo P; Kroon, Erna G; Abrahão, Jônatas S

    2015-11-01

    Vaccinia virus (VACV), the etiological agent of bovine vaccinia (BV), is widespread in Brazil and present in most of the milk-producing regions. We conducted a horizontal study of BV in Bahia, a state of Brazil in which the production of milk is increasing. During 2011, human and bovine clinical samples were collected during outbreaks for BV diagnosis, virus isolation and molecular analysis. We collected data for epidemiological inferences. Vaccinia virus was detected in 87.7% of the analyzed outbreaks, highlighting the effective circulation of VACV in Bahia. The molecular data showed the spreading of group 1 Brazilian VACV to Bahia. We observed a seasonal profile of BV, with its peak in the drier and cooler season. Manual milking was observed in 96 % of the visited properties, showing its importance to viral spread in herds. Under-notification of BV, ineffective animal trade surveillance, and bad milking practices have contributed to the spread of VACV in Brazil.

  5. The vaccinia virus E6 protein influences virion protein localization during virus assembly

    PubMed Central

    Condit, Richard C.; Moussatche, Nissin

    2015-01-01

    Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulate in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a “pre-nucleocapsid”, and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. PMID:25863879

  6. Use of a recombinant vaccinia virus expressing interferon gamma for post-exposure protection against vaccinia and ectromelia viruses.

    PubMed

    Holechek, Susan A; Denzler, Karen L; Heck, Michael C; Schriewer, Jill; Buller, R Mark; Legrand, Fatema A; Verardi, Paulo H; Jones, Leslie A; Yilma, Tilahun; Jacobs, Bertram L

    2013-01-01

    Post-exposure vaccination with vaccinia virus (VACV) has been suggested to be effective in minimizing death if administered within four days of smallpox exposure. While there is anecdotal evidence for efficacy of post-exposure vaccination this has not been definitively studied in humans. In this study, we analyzed post-exposure prophylaxis using several attenuated recombinant VACV in a mouse model. A recombinant VACV expressing murine interferon gamma (IFN-γ) was most effective for post-exposure protection of mice infected with VACV and ectromelia virus (ECTV). Untreated animals infected with VACV exhibited severe weight loss and morbidity leading to 100% mortality by 8 to 10 days post-infection. Animals treated one day post-infection had milder symptoms, decreased weight loss and morbidity, and 100% survival. Treatment on days 2 or 3 post-infection resulted in 40% and 20% survival, respectively. Similar results were seen in ECTV-infected mice. Despite the differences in survival rates in the VACV model, the viral load was similar in both treated and untreated mice while treated mice displayed a high level of IFN-γ in the serum. These results suggest that protection provided by IFN-γ expressed by VACV may be mediated by its immunoregulatory activities rather than its antiviral effects. These results highlight the importance of IFN-γ as a modulator of the immune response for post-exposure prophylaxis and could be used potentially as another post-exposure prophylaxis tool to prevent morbidity following infection with smallpox and other orthopoxviruses.

  7. Oncolytic virotherapy for pediatric malignancies: future prospects.

    PubMed

    Waters, Alicia M; Friedman, Gregory K; Ring, Eric K; Beierle, Elizabeth A

    2016-01-01

    Pediatric solid tumors remain a major health concern, with nearly 16,000 children diagnosed each year. Of those, ~2,000 succumb to their disease, and survivors often suffer from lifelong disability secondary to toxic effects of current treatments. Countless multimodality treatment regimens are being explored to make advances against this deadly disease. One targeted treatment approach is oncolytic virotherapy. Conditionally replicating viruses can infect tumor cells while leaving normal cells unharmed. Four viruses have been advanced to pediatric clinical trials, including herpes simplex virus-1, Seneca Valley virus, reovirus, and vaccinia virus. In this review, we discuss the mechanism of action of each virus, pediatric preclinical studies conducted to date, past and ongoing pediatric clinical trials, and potential future direction for these novel viral therapeutics.

  8. Oncolytic virotherapy for pediatric malignancies: future prospects

    PubMed Central

    Waters, Alicia M; Friedman, Gregory K; Ring, Eric K; Beierle, Elizabeth A

    2016-01-01

    Pediatric solid tumors remain a major health concern, with nearly 16,000 children diagnosed each year. Of those, ~2,000 succumb to their disease, and survivors often suffer from lifelong disability secondary to toxic effects of current treatments. Countless multimodality treatment regimens are being explored to make advances against this deadly disease. One targeted treatment approach is oncolytic virotherapy. Conditionally replicating viruses can infect tumor cells while leaving normal cells unharmed. Four viruses have been advanced to pediatric clinical trials, including herpes simplex virus-1, Seneca Valley virus, reovirus, and vaccinia virus. In this review, we discuss the mechanism of action of each virus, pediatric preclinical studies conducted to date, past and ongoing pediatric clinical trials, and potential future direction for these novel viral therapeutics. PMID:27579298

  9. TRAF2 Facilitates Vaccinia Virus Replication by Promoting Rapid Virus Entry

    PubMed Central

    Haga, Ismar R.; Pechenick Jowers, Tali; Griffiths, Samantha J.; Haas, Juergen

    2014-01-01

    ABSTRACT Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) is a pivotal intracellular mediator of signaling pathways downstream of TNFR1 and -2 with known pro- and antiviral effects. We investigated its role in the replication of the prototype poxvirus vaccinia virus (VACV). Loss of TRAF2 expression, either through small interfering RNA treatment of HeLa cells or through genetic knockout in murine embryonic fibroblasts (MEFs), led to significant reductions in VACV growth following low-multiplicity infection. In single-cycle infections, there was delayed production of both early and late VACV proteins as well as accelerated virus-induced alterations to cell morphology, indicating that TRAF2 influences early stages of virus replication. Consistent with an early role, uncoating assays showed normal virus attachment but delayed virus entry in the absence of TRAF2. Although alterations to c-Jun N-terminal kinase (JNK) signaling were apparent in VACV-infected TRAF2−/− MEFs, treatment of wild-type cells with a JNK inhibitor did not affect virus entry. Instead, treatment with an inhibitor of endosomal acidification greatly reduced virus entry into TRAF2−/− MEFs, suggesting that VACV is reliant on the endosomal route of entry in the absence of TRAF2. Thus, TRAF2 is a proviral factor for VACV that plays a role in promoting efficient viral entry, most likely via the plasma membrane. IMPORTANCE Tumor necrosis factor receptor-associated factors (TRAFs) are key facilitators of intracellular signaling with roles in innate and adaptive immunity and stress responses. We have discovered that TRAF2 is a proviral factor in vaccinia virus replication in both HeLa cells and mouse embryonic fibroblasts and that its influence is exercised through promotion of efficient virus entry. PMID:24429366

  10. Cellular factors promoting resistance to effective treatment of glioma with oncolytic myxoma virus.

    PubMed

    Zemp, Franz J; McKenzie, Brienne A; Lun, Xueqing; Reilly, Karlyne M; McFadden, Grant; Yong, V Wee; Forsyth, Peter A

    2014-12-15

    Oncolytic virus therapy is being evaluated in clinical trials for human glioma. While it is widely assumed that the immune response of the patient to the virus infection limits the utility of the therapy, investigations into the specific cell type(s) involved in this response have been performed using nonspecific pharmacologic inhibitors or allogeneic models with compromised immunity. To identify the immune cells that participate in clearing an oncolytic infection in glioma, we used flow cytometry and immunohistochemistry to immunophenotype an orthotopic glioma model in immunocompetent mice after Myxoma virus (MYXV) administration. These studies revealed a large resident microglia and macrophage population in untreated tumors, and robust monocyte, T-, and NK cell infiltration 3 days after MYXV infection. To determine the role on the clinical utility of MYXV therapy for glioma, we used a combination of knockout mouse strains and specific immunocyte ablation techniques. Collectively, our experiments identify an important role for tumor-resident myeloid cells and overlapping roles for recruited NK and T cells in the clearance and efficacy of oncolytic MYXV from gliomas. Using a cyclophosphamide regimen to achieve lymphoablation prior and during MYXV treatment, we prevented treatment-induced peripheral immunocyte recruitment and, surprisingly, largely ablated the tumor-resident macrophage population. Virotherapy of cyclophosphamide-treated animals resulted in sustained viral infection within the glioma as well as a substantial survival advantage. This study demonstrates that resistance to MYXV virotherapy in syngeneic glioma models involves a multifaceted cellular immune response that can be overcome with cyclophosphamide-mediated lymphoablation.

  11. Aurintricarboxylic Acid Inhibits the Early Stage of Vaccinia Virus Replication by Targeting both Cellular and Viral Factors▿

    PubMed Central

    Myskiw, Chad; Deschambault, Yvon; Jefferies, Kristel; He, Runtao; Cao, Jingxin

    2007-01-01

    Aurintricarboxylic acid (ATA) has been shown to inhibit the replication of viruses from several different families, including human immunodeficiency virus, vesicular stomatitis virus, and the coronavirus causing severe acute respiratory syndrome. This study characterizes the inhibitory effect of ATA on vaccinia virus replication in HeLa, Huh7, and AD293 cells. Vaccinia virus replication is significantly abrogated upon ATA treatment, which is associated with the inhibition of early viral gene transcription. This inhibitory effect may be attributed to two findings. First, ATA blocks the phosphorylation of extracellular signal-regulated kinase 1/2, an event shown to be essential for vaccinia virus replication. Second, ATA inhibits the phosphatase activity of the viral enzyme H1L, which is required to initiate viral transcription. Thus, ATA inhibits vaccinia virus replication by targeting both cellular and viral factors essential for the early stage of replication. PMID:17192307

  12. Mechanism of Poly (A) Synthesis by Vaccinia Virus

    PubMed Central

    Sheldon, Robert; Kates, Joseph

    1974-01-01

    Data are presented which indicate that vaccinia DNA does not contain poly(dT) sequences the size of poly(A) sequences (50 to 200 nucleotides in length) found in vaccinia RNA. A hybridization experiment and polyacrylamide gel electrophoresis and DEAE-Sephadex chromatography of pyrimidine tracts show that poly(dT) sequences can account for no more than 0.1% of vaccinia DNA. Ultraviolet irradiation (which causes thymine dimer formation) and phleomycin (which binds to thymidine) both inhibit RNA synthesis but not poly(A) synthesis by vaccinia cores. These data are consistent with a nontranscriptive mechanism for vaccinia poly(A) synthesis. Both trypsin and 50 C heat treatment inhibit RNA synthesis more than poly(A) synthesis by cores, suggesting that separate enzymes may be involved in these syntheses. When the rate of core RNA synthesis is reduced by lowering the UTP and GTP concentrations, the size of the poly(A) sequences increase. These and other data suggest that transcription is involved in the termination of poly(A) synthesis in cores. This might be due to the displacement of growing poly(A) chains by recently completed RNA 3′ termini which have not yet acquired poly(A) sequences. PMID:4847326

  13. Mechanism of poly(A) synthesis by vaccinia virus.

    PubMed

    Sheldon, R; Kates, J

    1974-08-01

    Data are presented which indicate that vaccinia DNA does not contain poly(dT) sequences the size of poly(A) sequences (50 to 200 nucleotides in length) found in vaccinia RNA. A hybridization experiment and polyacrylamide gel electrophoresis and DEAE-Sephadex chromatography of pyrimidine tracts show that poly(dT) sequences can account for no more than 0.1% of vaccinia DNA. Ultraviolet irradiation (which causes thymine dimer formation) and phleomycin (which binds to thymidine) both inhibit RNA synthesis but not poly(A) synthesis by vaccinia cores. These data are consistent with a nontranscriptive mechanism for vaccinia poly(A) synthesis. Both trypsin and 50 C heat treatment inhibit RNA synthesis more than poly(A) synthesis by cores, suggesting that separate enzymes may be involved in these syntheses. When the rate of core RNA synthesis is reduced by lowering the UTP and GTP concentrations, the size of the poly(A) sequences increase. These and other data suggest that transcription is involved in the termination of poly(A) synthesis in cores. This might be due to the displacement of growing poly(A) chains by recently completed RNA 3' termini which have not yet acquired poly(A) sequences.

  14. The vaccinia virus E6 protein influences virion protein localization during virus assembly

    SciTech Connect

    Condit, Richard C. Moussatche, Nissin

    2015-08-15

    Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulate in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a “pre-nucleocapsid”, and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. - Highlights: • Mutation of E6 disrupts association of viral membranes with viral core proteins • Mutation of E6 does not perturb viral membrane biosynthesis • Mutation of E6 does not perturb localization of viral transcription enzymes • Mutation of E6 causes mis-localization and aggregation of viral core proteins • Vaccinia assembly uses three subassemblies: membranes, viroplasm, prenucleocapsid.

  15. Generation and evaluation of a recombinant modified vaccinia virus Ankara vaccine for rabies.

    PubMed

    Weyer, Jacqueline; Rupprecht, Charles E; Mans, Janet; Viljoen, Gerrit J; Nel, Louis H

    2007-05-22

    Modified vaccinia virus Ankara (MVA) has become a vaccine vector of choice for recombinant vaccine development. A MVA-based rabies vaccine would be advantageous for use as a vaccine for dogs (and wildlife), particularly if it proves innocuous and efficacious by the oral route. Here, the generation and immunological testing of a recombinant MVA expressing a rabies virus glycoprotein gene is described. In a murine model, higher dosages of recombinant MVA were needed to induce equivocal immune responses as with Vaccinia Copenhagen or Vaccinia Western Reserve recombinants, when administered by a parenteral route. The MVA recombinant was not immunogenic or efficacious when administered per os in naïve mice. The ability of the recombinant MVA to induce anamnestic responses in dogs and raccoons was also investigated. Recombinant MVA boosted humoral immune responses in these animals when administered peripherally, but not when administered orally.

  16. [The construction of attenuated Tiantan recombinant vaccinia virus vector with IFN-gamma receptor gene deletion].

    PubMed

    Huang, Wei; Liu, Ying; Duan, Dan-li; Li, Hai-shan; Liu, Yong; Hong, Kun-Xue; Zhu, Jia-hong; Shao, Yi-ming

    2004-03-01

    B8R gene encodes a secreted protein with homology to IFN-gamma receptor, which neutralizes the antiviral and immunological regulation activities of IFN-gamma. To improve the safety of vaccinia virus vector, an attenuated recombinant vaccinia virus with the B8R gene deletion from Tiantan vaccine strain (VTT) was constructed. The transfer vectors were generated by joining B8R left flank, B8R right flank, vv promoter, LacZ, multicloning site and pBRSK fragments. The recombinant viruses VTTdeltaB8RLacZ (VTT with B8R deletion and LacZ insertion) were constructed by homologous recombination. The B8R deletion mutants were confirmed by dot blot with B8R gene probe and PCR amplification. The replication ability of VTTdeltaB8RLacZ strain in vitro was similar to that of the VTT. The skin lesions formed by VTTdeltaB8RLacZ (10(6) pfu) were significantly smaller and healed faster than those formed by VTT when injected intradermally to the rabbits,and no visible ulceration occurred. Meanwhile LacZ in VTKgpedeltaB8RLacZ was expressed stably. The attenuated vector with B8R gene deletion improves the safety of recombinant vaccinia virus vaccine B8R locus may be used as a new site for insertion of foreign genes in vaccinia virus vector.

  17. Vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis

    PubMed Central

    Wasilenko, Shawn T.; Stewart, Tara L.; Meyers, Adrienne F. A.; Barry, Michele

    2003-01-01

    To circumvent apoptotic death, many viruses encode Bcl-2 homologous proteins that function at the mitochondria. Vaccinia virus, the prototypic member of the Poxviridae family, does not encode a Bcl-2 homolog but inhibits the mitochondrial arm of the apoptotic cascade by an unknown mechanism. We now report that F1L, a previously unidentified protein in vaccinia virus, is responsible for the inhibition of apoptosis. Cells infected with vaccinia virus are resistant to staurosporine-mediated cleavage of poly(ADP-ribose) polymerase, caspases 3 and 9, and release of cytochrome c. In contrast, a vaccinia virus deletion mutant, VV811, was unable to inhibit apoptosis; however, the antiapoptotic function was restored by expression of the F1L ORF, which is absent in VV811. Although F1L displays no homology to members of the Bcl-2 family, it localizes to the mitochondria through a C-terminal hydrophobic domain. We show that expression of F1L interferes with apoptosis by inhibiting the loss of the inner mitochondrial membrane potential and the release of cytochrome c. PMID:14610284

  18. Selective replication of oncolytic virus M1 results in a bystander killing effect that is potentiated by Smac mimetics.

    PubMed

    Cai, Jing; Lin, Yuan; Zhang, Haipeng; Liang, Jiankai; Tan, Yaqian; Cavenee, Webster K; Yan, Guangmei

    2017-06-27

    Oncolytic virotherapy is a treatment modality that uses native or genetically modified viruses that selectively replicate in and kill tumor cells. Viruses represent a type of pathogen-associated molecular pattern and thereby induce the up-regulation of dozens of cytokines via activating the host innate immune system. Second mitochondria-derived activator of caspases (Smac) mimetic compounds (SMCs), which antagonize the function of inhibitor of apoptosis proteins (IAPs) and induce apoptosis, sensitize tumor cells to multiple cytokines. Therefore, we sought to determine whether SMCs sensitize tumor cells to cytokines induced by the oncolytic M1 virus, thus enhancing a bystander killing effect. Here, we report that SMCs potentiate the oncolytic effect of M1 in vitro, in vivo, and ex vivo. This strengthened oncolytic efficacy resulted from the enhanced bystander killing effect caused by the M1 virus via cytokine induction. Through a microarray analysis and subsequent validation using recombinant cytokines, we identified IL-8, IL-1A, and TRAIL as the key cytokines in the bystander killing effect. Furthermore, SMCs increased the replication of M1, and the accumulation of virus protein induced irreversible endoplasmic reticulum stress- and c-Jun N-terminal kinase-mediated apoptosis. Nevertheless, the combined treatment with M1 and SMCs had little effect on normal and human primary cells. Because SMCs selectively and significantly enhance the bystander killing effect and the replication of oncolytic virus M1 specifically in cancer cells, this combined treatment may represent a promising therapeutic strategy.

  19. Using clinically approved cyclophosphamide regimens to control the humoral immune response to oncolytic viruses

    PubMed Central

    Peng, K-W; Myers, R; Greenslade, A; Mader, E; Greiner, S; Federspiel, MJ; Dispenzieri, A; Russell, SJ

    2013-01-01

    Oncolytic viruses can be neutralized in the bloodstream by antiviral antibodies whose titers increase progressively with each exposure, resulting in faster virus inactivation and further reductions in efficacy with each successive dose. A single dose of cyclophosphamide (CPA) at 370 mg m−2 was not sufficient to control the primary antiviral immune responses in mice, squirrel monkeys and humans. We therefore tested clinically approved multidose CPA regimens, which are known to kill proliferating lymphocytes, to determine if more intensive CPA therapy can more effectively suppress antiviral antibody responses during virotherapy. In virus-susceptible mice, primary antibody responses to intravenously (i.v.) administered oncolytic measles virus (MV) or vesicular stomatitis virus (VSV) were partially or completely suppressed, respectively, by oral (1 mg × 8 days) or systemic (3 mg × 4 days) CPA regimens initiated 1 day before virus. When MV- or VSV-immune mice were re-challenged with the respective viruses and concurrently treated with four daily systemic doses of CPA, their anamnestic antibody responses were completely suppressed and antiviral antibody titers fell significantly below pre-booster levels. We conclude that the CPA regimen of four daily doses at 370 mg m−2 should be evaluated clinically with i.v. virotherapy to control the antiviral antibody response and facilitate effective repeat dosing. PMID:22476202

  20. Using clinically approved cyclophosphamide regimens to control the humoral immune response to oncolytic viruses.

    PubMed

    Peng, K-W; Myers, R; Greenslade, A; Mader, E; Greiner, S; Federspiel, M J; Dispenzieri, A; Russell, S J

    2013-03-01

    Oncolytic viruses can be neutralized in the bloodstream by antiviral antibodies whose titers increase progressively with each exposure, resulting in faster virus inactivation and further reductions in efficacy with each successive dose. A single dose of cyclophosphamide (CPA) at 370 mg m(-2) was not sufficient to control the primary antiviral immune responses in mice, squirrel monkeys and humans. We therefore tested clinically approved multidose CPA regimens, which are known to kill proliferating lymphocytes, to determine if more intensive CPA therapy can more effectively suppress antiviral antibody responses during virotherapy. In virus-susceptible mice, primary antibody responses to intravenously (i.v.) administered oncolytic measles virus (MV) or vesicular stomatitis virus (VSV) were partially or completely suppressed, respectively, by oral (1 mg × 8 days) or systemic (3 mg × 4 days) CPA regimens initiated 1 day before virus. When MV- or VSV-immune mice were re-challenged with the respective viruses and concurrently treated with four daily systemic doses of CPA, their anamnestic antibody responses were completely suppressed and antiviral antibody titers fell significantly below pre-booster levels. We conclude that the CPA regimen of four daily doses at 370 mg m(-2) should be evaluated clinically with i.v. virotherapy to control the antiviral antibody response and facilitate effective repeat dosing.

  1. Transient fasting enhances replication of oncolytic herpes simplex virus in glioblastoma.

    PubMed

    Esaki, Shinichi; Rabkin, Samuel D; Martuza, Robert L; Wakimoto, Hiroaki

    2016-01-01

    Short-term nutritional restriction (fasting) has been shown to enhance the efficacy of chemotherapy by sensitizing cancer cells and protecting normal cells in a variety of cancer models, including glioblastoma (GBM). Cancer cells, unlike normal cells, respond to fasting by promoting oncogenic signaling and protein synthesis. We hypothesized that fasting would increase the replication of oncolytic herpes simplex virus (oHSV) in GBM. Patient-derived GBM cell lines were fasted by growth in glucose and fetal calf serum restricted culture medium. "Transient fasting", 24-hour fasting followed by 24-hour recovery in complete medium, increased late virus gene expression and G47Δ yields about 2-fold in GBM cells, but not in human astrocytes, and enhanced G47Δ killing of GBM cells. Mechanistically, "transient fasting" suppressed phosphorylation of the subunit of eukaryotic initiation factor 2α (eIF2α) and c-Jun N-terminal kinases (JNK) in GBM cells, but not in astrocytes. Pharmacological inhibition of JNK also increased G47Δ yield. In vivo, transient fasting (48-hour food restriction and 24-hour recovery) doubled luciferase activity after intratumoral G47Δ-US11fluc injection into orthotopic GBM xenografts. Thus, "transient fasting" increases G47Δ replication and oncolytic activity in human GBM cells. These results suggest that "transient fasting" may be effectively combined to enhance oncolytic HSV therapy of GBM.

  2. Oncolytic herpes simplex virus vectors and chemotherapy: are combinatorial strategies more effective for cancer?

    PubMed Central

    Kanai, Ryuichi; Wakimoto, Hiroaki; Cheema, Tooba; Rabkin, Samuel D

    2010-01-01

    Despite aggressive treatments, including chemotherapy and radiotherapy, cancers often recur owing to resistance to conventional therapies. Oncolytic viruses such as oncolytic herpes simplex virus (oHSV) represent an exciting biological approach to cancer therapy. A range of viral mutations has been engineered into HSV to engender oncolytic activity. While oHSV as a single agent has been tested in a number of cancer clinical trials, preclinical studies have demonstrated enhanced efficacy when it is combined with cytotoxic anticancer drugs. Among the strategies that will be discussed in this article are combinations with standard-of-care chemotherapeutics, expression of prodrug-activating enzymes to enhance chemotherapy and small-molecule inhibitors. The combination of oHSV and chemotherapy can achieve much more efficient cancer cell killing than either single agent alone, often through synergistic interactions. This can be clinically important not just for improving efficacy but also for permitting lower and less toxic chemotherapeutic doses. The viral mutations in an oHSV vector often determine the favorability of its interactions with chemotherapy, just as different cancer cells, due to genetic alterations, vary in their response to chemotherapy. As chemotherapeutics are often the standard of care, combining them with an investigational new drug, such as oHSV, is clinically easier than combining multiple novel agents. As has become clear for most cancer therapies, multimodal treatments are usually more effective. In this article, we will discuss the recent progress of these combinatorial strategies between virotherapy and chemotherapy and future directions. PMID:20373873

  3. Secondary and Tertiary Transmission of Vaccinia Virus from US Military Service Member

    PubMed Central

    Hidalgo, Christina M.; Sullivan-Frohm, Ann; Schulte, Cynthia; Davis, Stephen; Kelly-Cirino, Cassandra; Egan, Christina; Wilkins, Kimberly; Emerson, Ginny L.; Noyes, Kimberly; Blog, Debra

    2011-01-01

    During February and March 2010, the New York State Department of Health investigated secondary and tertiary vaccinia contact transmission from a military vaccinee to 4 close contacts. Identification of these cases underscores the need for strict adherence to postvaccination infection control guidance to avoid transmission of the live virus. PMID:21470470

  4. DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome.

    PubMed Central

    Jones, E V; Puckett, C; Moss, B

    1987-01-01

    Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A. Images PMID:3033308

  5. The vaccinia virus K7 protein promotes histone methylation associated with heterochromatin formation

    PubMed Central

    Noyce, Ryan S.; Shenouda, Mira; Umer, Brittany

    2017-01-01

    It has been well established that many vaccinia virus proteins suppress host antiviral pathways by targeting the transcription of antiviral proteins, thus evading the host innate immune system. However, whether viral proteins have an effect on the host’s overall cellular transcription is less understood. In this study we investigated the regulation of heterochromatin during vaccinia virus infection. Heterochromatin is a highly condensed form of chromatin that is less transcriptionally active and characterized by methylation of histone proteins. We examined the change in methylation of two histone proteins, H3 and H4, which are major markers of heterochromatin, during the course of viral infection. Using immunofluorescence microscopy and flow cytometry we were able to track the overall change in the methylated levels of H3K9 and H4K20. Our results suggest that there is significant increase in methylation of H3K9 and H4K20 during Orthopoxviruses infection compared to mock-infected cells. However, this effect was not seen when we infected cells with Leporipoxviruses. We further screened several vaccinia virus single and multi-gene deletion mutant and identified the vaccinia virus gene K7R as a contributor to the increase in cellular histone methylation during infection. PMID:28257484

  6. One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

    PubMed Central

    Nitsche, Andreas; Kurth, Andreas; Dunkhorst, Anna; Pänke, Oliver; Sielaff, Hendrik; Junge, Wolfgang; Muth, Doreen; Scheller, Frieder; Stöcklein, Walter; Dahmen, Claudia; Pauli, Georg; Kage, Andreas

    2007-01-01

    Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method. PMID:17697378

  7. A Strategy for Cultivation of Retargeted Oncolytic Herpes Simplex Viruses in Non-cancer Cells.

    PubMed

    Leoni, Valerio; Gatta, Valentina; Casiraghi, Costanza; Nicosia, Alfredo; Petrovic, Biljana; Campadelli-Fiume, Gabriella

    2017-05-15

    The oncolytic herpes simplex virus (HSV) that has been approved for clinical practice and those HSVs in clinical trials are attenuated viruses, often with the neurovirulence gene γ134.5 and additional genes deleted. One strategy to engineer nonattenuated oncolytic HSVs consists of retargeting the viral tropism to a cancer-specific receptor of choice, exemplified by HER2 (human epidermal growth factor receptor 2), which is present in breast, ovary, and other cancers, and in detargeting from the natural receptors. Because the HER2-retargeted HSVs strictly depend on this receptor for infection, the viruses employed in preclinical studies were cultivated in HER2-positive cancer cells. The production of clinical-grade viruses destined for humans should avoid the use of cancer cells. Here, we engineered the R-213 recombinant, by insertion of a 20-amino-acid (aa) short peptide (named GCN4) in the gH of R-LM113; this recombinant was retargeted to HER2 through insertion in gD of a single-chain antibody (scFv) to HER2. Next, we generated a Vero cell line expressing an artificial receptor (GCN4R) whose N terminus consists of an scFv to GCN4 and therefore is capable of interacting with GCN4 present in gH of R-213. R-213 replicated as well as R-LM113 in SK-OV-3 cells, implying that addition of the GCN4 peptide was not detrimental to gH. R-213 grew to relatively high titers in Vero-GCN4R cells, efficiently spread from cell to cell, and killed both Vero-GCN4R and SK-OV-3 cells, as expected for an oncolytic virus. Altogether, Vero-GCN4R cells represent an efficient system for cultivation of retargeted oncolytic HSVs in non-cancer cells.IMPORTANCE There is growing interest in viruses as oncolytic agents, which can be administered in combination with immunotherapeutic compounds, including immune checkpoint inhibitors. The oncolytic HSV approved for clinical practice and those in clinical trials are attenuated viruses. An alternative to attenuation is a cancer specificity achieved

  8. Modified Vaccinia Virus Ankara: History, Value in Basic Research, and Current Perspectives for Vaccine Development.

    PubMed

    Volz, A; Sutter, G

    2017-01-01

    Safety tested Modified Vaccinia virus Ankara (MVA) is licensed as third-generation vaccine against smallpox and serves as a potent vector system for development of new candidate vaccines against infectious diseases and cancer. Historically, MVA was developed by serial tissue culture passage in primary chicken cells of vaccinia virus strain Ankara, and clinically used to avoid the undesirable side effects of conventional smallpox vaccination. Adapted to growth in avian cells MVA lost the ability to replicate in mammalian hosts and lacks many of the genes orthopoxviruses use to conquer their host (cell) environment. As a biologically well-characterized mutant virus, MVA facilitates fundamental research to elucidate the functions of poxvirus host-interaction factors. As extremely safe viral vectors MVA vaccines have been found immunogenic and protective in various preclinical infection models. Multiple recombinant MVA currently undergo clinical testing for vaccination against human immunodeficiency viruses, Mycobacterium tuberculosis or Plasmodium falciparum. The versatility of the MVA vector vaccine platform is readily demonstrated by the swift development of experimental vaccines for immunization against emerging infections such as the Middle East Respiratory Syndrome. Recent advances include promising results from the clinical testing of recombinant MVA-producing antigens of highly pathogenic avian influenza virus H5N1 or Ebola virus. This review summarizes our current knowledge about MVA as a unique strain of vaccinia virus, and discusses the prospects of exploiting this virus as research tool in poxvirus biology or as safe viral vector vaccine to challenge existing and future bottlenecks in vaccinology.

  9. Turning killer into cure -- the story of oncolytic herpes simplex viruses.

    PubMed

    Zhang, Shaun Xiaoliu

    2015-11-01

    Viruses have the intrinsic capability to kill host cells. Even when the initial infection consists of only a few viruses, they can reproduce themselves in large quantities within a short time and quickly spread to nearby cells, causing substantial tissue damage. These same infectious properties become desirable if they can be converted into killer agents with specificity for malignant cells. Cancer virotherapy is doing exactly that by modifying viruses in ways that allow them to replicate in malignant cells but not in normal cells. Although relatively young, the field has seen significant progress in recent years. For example, the most recent phase III trial data on a herpes simplex virus (HSV)-based oncolytic virus (T-VEC) show substantial improvement in objective and durable responses over the control arm in melanoma patients, prompting speculation that a virotherapy may receive FDA approval for clinical use in the very near future. This review focuses on HSV-based oncolytic viruses, from their early history to their most recent development, with discussion of promising directions for further improvement.

  10. The potential application of a transcriptionally regulated oncolytic herpes simplex virus for human cancer therapy

    PubMed Central

    Miao, L; Fraefel, C; Sia, K C; Newman, J P; Mohamed-Bashir, S A; Ng, W H; Lam, P Y P

    2014-01-01

    Background: Emerging studies have shown the potential benefit of arming oncolytic viruses with therapeutic genes. However, most of these therapeutic genes are placed under the regulation of ubiquitous viral promoters. Our goal is to generate a safer yet potent oncolytic herpes simplex virus type-1 (HSV-1) for cancer therapy. Methods: Using bacterial artificial chromosome (BAC) recombineering, a cell cycle-regulatable luciferase transgene cassette was replaced with the infected cell protein 6 (ICP6) coding region (encoded for UL39 or large subunit of ribonucleotide reductase) of the HSV-1 genome. These recombinant viruses, YE-PC8, were further tested for its proliferation-dependent luciferase gene expression. Results: The ability of YE-PC8 to confer proliferation-dependent transgene expression was demonstrated by injecting similar amount of viruses into the tumour-bearing region of the brain and the contralateral normal brain parenchyma of the same mouse. The results showed enhanced levels of luciferase activities in the tumour region but not in the normal brain parenchyma. Similar findings were observed in YE-PC8-infected short-term human brain patient-derived glioma cells compared with normal human astrocytes. intratumoural injection of YE-PC8 viruses resulted in 77% and 80% of tumour regression in human glioma and human hepatocellular carcinoma xenografts, respectively. Conclusion: YE-PC8 viruses confer tumour selectivity in proliferating cells and may be developed further as a feasible approach to treat human cancers. PMID:24196790

  11. Angiogenesis inhibition using an oncolytic herpes simplex virus expressing endostatin in a murine lung cancer model.

    PubMed

    Goodwin, Jonathan M; Schmitt, Anthony D; McGinn, Christopher M; Fuchs, Bryan C; Kuruppu, Darshini; Tanabe, Kenneth K; Lanuti, Michael

    2012-03-01

    Herpes-mediated viral oncolysis alone is not sufficient to completely eradicate tumors. In this study we used a replication conditional, endostatin-expressing herpes simplex virus-1 mutant (HSV-Endo) in a murine lung cancer model. We hypothesized that the anti-angiogenic action of endostatin would improve upon the oncolytic effect of HSV-1. HSV-Endo was evaluated in a pulmonary metastases and orthotopic flank model, where there was significantly less tumor burden and reduced microvessel density compared to a control virus. Endostatin expression appears to improve the anti-tumor effect of HSV-1 in a lung cancer model.

  12. Sensitization with vaccinia virus encoding H5N1 hemagglutinin restores immune potential against H5N1 influenza virus

    PubMed Central

    Yasui, Fumihiko; Itoh, Yasushi; Ikejiri, Ai; Kitabatake, Masahiro; Sakaguchi, Nobuo; Munekata, Keisuke; Shichinohe, Shintaro; Hayashi, Yukiko; Ishigaki, Hirohito; Nakayama, Misako; Sakoda, Yoshihiro; Kida, Hiroshi; Ogasawara, Kazumasa; Kohara, Michinori

    2016-01-01

    H5N1 highly pathogenic avian influenza (H5N1 HPAI) virus causes elevated mortality compared with seasonal influenza viruses like H1N1 pandemic influenza (H1N1 pdm) virus. We identified a mechanism associated with the severe symptoms seen with H5N1 HPAI virus infection. H5N1 HPAI virus infection induced a decrease of dendritic cell number in the splenic extrafollicular T-cell zone and impaired formation of the outer layers of B-cell follicles, resulting in insufficient levels of antibody production after infection. However, in animals vaccinated with a live recombinant vaccinia virus expressing the H5 hemagglutinin, infection with H5N1 HPAI virus induced parafollicular dendritic cell accumulation and efficient antibody production. These results indicate that a recombinant vaccinia encoding H5 hemagglutinin gene does not impair dendritic cell recruitment and can be a useful vaccine candidate. PMID:27892498

  13. Complex Dynamics of Virus Spread from Low Infection Multiplicities: Implications for the Spread of Oncolytic Viruses

    PubMed Central

    Rodriguez-Brenes, Ignacio A.; Hofacre, Andrew; Fan, Hung; Wodarz, Dominik

    2017-01-01

    While virus growth dynamics have been well-characterized in several infections, data are typically collected once the virus population becomes easily detectable. Earlier dynamics, however, remain less understood. We recently reported unusual early dynamics in an experimental system using adenovirus infection of human embryonic kidney (293) cells. Under identical experimental conditions, inoculation at low infection multiplicities resulted in either robust spread, or in limited spread that eventually stalled, with both outcomes occurring with approximately equal frequencies. The reasons underlying these observations have not been understood. Here, we present further experimental data showing that inhibition of interferon-induced antiviral states in cells results in a significant increase in the percentage of robust infections that are observed, implicating a race between virus replication and the spread of the anti-viral state as a central mechanism. Analysis of a variety of computational models, however, reveals that this alone cannot explain the simultaneous occurrence of both viral growth outcomes under identical conditions, and that additional biological mechanisms have to be invoked to explain the data. One such mechanism is the ability of the virus to overcome the antiviral state through multiple infection of cells. If this is included in the model, two outcomes of viral spread are found to be simultaneously stable, depending on initial conditions. In stochastic versions of such models, the system can go by chance to either state from identical initial conditions, with the relative frequency of the outcomes depending on the strength of the interferon-based anti-viral response, consistent with the experiments. This demonstrates considerable complexity during the early phase of the infection that can influence the ability of a virus to become successfully established. Implications for the initial dynamics of oncolytic virus spread through tumors are discussed

  14. Complex Dynamics of Virus Spread from Low Infection Multiplicities: Implications for the Spread of Oncolytic Viruses.

    PubMed

    Rodriguez-Brenes, Ignacio A; Hofacre, Andrew; Fan, Hung; Wodarz, Dominik

    2017-01-01

    While virus growth dynamics have been well-characterized in several infections, data are typically collected once the virus population becomes easily detectable. Earlier dynamics, however, remain less understood. We recently reported unusual early dynamics in an experimental system using adenovirus infection of human embryonic kidney (293) cells. Under identical experimental conditions, inoculation at low infection multiplicities resulted in either robust spread, or in limited spread that eventually stalled, with both outcomes occurring with approximately equal frequencies. The reasons underlying these observations have not been understood. Here, we present further experimental data showing that inhibition of interferon-induced antiviral states in cells results in a significant increase in the percentage of robust infections that are observed, implicating a race between virus replication and the spread of the anti-viral state as a central mechanism. Analysis of a variety of computational models, however, reveals that this alone cannot explain the simultaneous occurrence of both viral growth outcomes under identical conditions, and that additional biological mechanisms have to be invoked to explain the data. One such mechanism is the ability of the virus to overcome the antiviral state through multiple infection of cells. If this is included in the model, two outcomes of viral spread are found to be simultaneously stable, depending on initial conditions. In stochastic versions of such models, the system can go by chance to either state from identical initial conditions, with the relative frequency of the outcomes depending on the strength of the interferon-based anti-viral response, consistent with the experiments. This demonstrates considerable complexity during the early phase of the infection that can influence the ability of a virus to become successfully established. Implications for the initial dynamics of oncolytic virus spread through tumors are discussed.

  15. Molecular Pathways: Mechanism of Action for Talimogene Laherparepvec, a New Oncolytic Virus Immunotherapy.

    PubMed

    Kohlhapp, Frederick J; Kaufman, Howard L

    2016-03-01

    Oncolytic viruses are native or engineered viruses that preferentially replicate in and lyse cancer cells. Selective tumor cell replication is thought to depend on infection of neoplastic cells, which harbor low levels of protein kinase R (PKR) and dysfunctional type I IFN signaling elements. These changes allow more efficient viral replication, and with selected deletion of specific viral genes, replication in normal cells with activated PKR may not be possible. Direct tumor cell lysis, release of soluble tumor antigens, and danger-associated molecular factors are all thought to help prime and promote tumor-specific immunity. Talimogene laherparepvec (T-VEC) is a genetically modified herpes simplex virus, type I and is the first oncolytic virus to demonstrate a clinical benefit in patients with melanoma. T-VEC has also been evaluated for the treatment of head and neck cancer, pancreatic cancer, and likely other types of cancer will be targeted in the near future. T-VEC has been modified for improved safety, tumor-selective replication, and induction of host immunity by deletion of several viral genes and expression of human granulocyte-macrophage colony stimulating factor. Although the mechanism of action for T-VEC is incompletely understood, the safety profile of T-VEC and ability to promote immune responses suggest future combination studies with other immunotherapy approaches including checkpoint blockade through PD-1, PD-L1, and CTLA-4 to be a high priority for clinical development. Oncolytic viruses also represent unique regulatory and biosafety challenges but offer a potential new class of agents for the treatment of cancer.

  16. Mutations in the glycoprotein of vesicular stomatitis virus affect cytopathogenicity: potential for oncolytic virotherapy.

    PubMed

    Janelle, Valérie; Brassard, Frédérick; Lapierre, Pascal; Lamarre, Alain; Poliquin, Laurent

    2011-07-01

    Vesicular stomatitis virus (VSV) has been widely used to characterize cellular processes, viral resistance, and cytopathogenicity. Recently, VSV has also been used for oncolytic virotherapy due to its capacity to selectively lyse tumor cells. Mutants of the matrix (M) protein of VSV have generally been preferred to the wild-type virus for oncolysis because of their ability to induce type I interferon (IFN) despite causing weaker cytopathic effects. However, due to the large variability of tumor types, it is quite clear that various approaches and combinations of multiple oncolytic viruses will be needed to effectively treat most cancers. With this in mind, our work focused on characterizing the cytopathogenic profiles of four replicative envelope glycoprotein (G) VSV mutants. In contrast to the prototypic M mutant, VSV G mutants are as efficient as wild-type virus at inhibiting cellular transcription and host protein translation. Despite being highly cytopathic, the mutant G(6R) triggers type I interferon secretion as efficiently as the M mutant. Importantly, most VSV G mutants are more effective at killing B16 and MC57 tumor cells in vitro than the M mutant or wild-type virus through apoptosis induction. Taken together, our results demonstrate that VSV G mutants retain the high cytopathogenicity of wild-type VSV, with G(6R) inducing type I IFN secretion at levels similar to that of the M mutant. VSV G protein mutants could therefore prove to be highly valuable for the development of novel oncolytic virotherapy strategies that are both safe and efficient for the treatment of various types of cancer.

  17. A Vaccinia Virus Recombinant Transcribing an Alphavirus Replicon and Expressing Alphavirus Structural Proteins Leads to Packaging of Alphavirus Infectious Single Cycle Particles

    PubMed Central

    Sánchez-Puig, Juana M.; Lorenzo, María M.; Blasco, Rafael

    2013-01-01

    Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses. PMID:24130722

  18. A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

    PubMed

    Sánchez-Puig, Juana M; Lorenzo, María M; Blasco, Rafael

    2013-01-01

    Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.

  19. Vaccinia viruses: vaccines against smallpox and vectors against infectious diseases and tumors

    PubMed Central

    Walsh, Stephen R; Dolin, Raphael

    2011-01-01

    Less than 200 years after its introduction, widespread use of vaccinia virus (VACV) as a smallpox vaccine has eradicated variola virus. Along with the remarkable success of the vaccination program, frequent and sometimes severe adverse reactions to VACV were encountered. After eradication, VACV has been reserved for select populations who might be at significant risk for orthopoxvirus infections. Events over the past decade have renewed concerns over the potential use of variola virus as a biological weapon. Accordingly, interest in VACV and attenuated derivatives has increased, both as vaccines against smallpox and as vectors for other vaccines. This article will focus on new developments in the field of orthopoxvirus immunization and will highlight recent advances in the use of vaccinia viruses as vectors for infectious diseases and malignancies. PMID:21854314

  20. Contact transmission of vaccinia virus from smallpox vaccinees in the United States, 2003-2011.

    PubMed

    Wertheimer, Ellen R; Olive, Denise S; Brundage, John F; Clark, Leslie L

    2012-02-01

    Since 2002, approximately 40,000 US civilians and 2.1 million military personnel have been vaccinated against smallpox. The vaccine contains live vaccinia virus that can be transferred through physical contact. This report summarizes numbers, rates, and characteristics of contact vaccinia cases that presented between December 2002 and March 2011. Cases were identified from reports in adverse event reporting systems and peer-reviewed literature. One hundred fifteen cases of vaccinia transmission through contact were identified (5.4 per 100,000 vaccinees); 52 reports (45%) noted laboratory confirmation. Three-quarters of vaccinees, but fewer than 8% of contact vaccinia cases, were described as military members. Most cases were household or intimate contacts (n=86, 75%) or wrestling partners (n=18, 16%) of vaccinees. Nearly all cases manifested mild, local skin reactions; of 14 hospitalized cases, one was life-threatening. Vaccinia transmission from vaccinees is relatively infrequent. Continued attention to both vaccinee education and screening for contraindications to vaccination is appropriate.

  1. Oral Immunization with Recombinant Vaccinia Virus Prime and Intramuscular Protein Boost Provides Protection against Intrarectal Simian-Human Immunodeficiency Virus Challenge in Macaques.

    PubMed

    Thippeshappa, Rajesh; Tian, Baoping; Cleveland, Brad; Guo, Wenjin; Polacino, Patricia; Hu, Shiu-Lok

    2015-12-30

    Human immunodeficiency virus type 1 (HIV-1) acquisition occurs predominantly through mucosal transmission. We hypothesized that greater mucosal immune responses and protective efficacy against mucosal HIV-1 infection may be achieved by prime-boost immunization at mucosal sites. We used a macaque model to determine the safety, immunogenicity, and protective efficacy of orally delivered, replication-competent but attenuated recombinant vaccinia viruses expressing full-length HIV-1 SF162 envelope (Env) or simian immunodeficiency virus (SIV) Gag-Pol proteins. We examined the dose and route that are suitable for oral immunization with recombinant vaccinia viruses. We showed that sublingual inoculation of two vaccinia virus-naive pigtailed macaques with 5 × 10(8) PFU of recombinant vaccinia viruses was safe. However, sublingual inoculation with a higher dose or tonsillar inoculation resulted in secondary oral lesions, indicating the need to optimize the dose and route for oral immunization with replication-competent vaccinia virus vectors. Oral priming alone elicited antibody responses to vaccinia virus and to the SF162 Env protein. Intramuscular immunization with the SF162 gp120 protein at either 20 or 21 weeks postpriming resulted in a significant boost in antibody responses in both systemic and mucosal compartments. Furthermore, we showed that immune responses induced by recombinant vaccinia virus priming and intramuscular protein boosting provided protection against intrarectal challenge with the simian-human immunodeficiency virus SHIV-SF162-P4.

  2. Systemic therapy with oncolytic myxoma virus cures established residual multiple myeloma in mice

    PubMed Central

    Bartee, Eric; Bartee, Mee Y; Bogen, Bjarne; Yu, Xue-Zhong

    2016-01-01

    Multiple myeloma is an incurable malignancy of plasma B-cells. Traditional chemotherapeutic regimes often induce initial tumor regression; however, virtually all patients eventually succumb to relapse caused by either reintroduction of disease during autologous transplant or expansion of chemotherapy resistant minimal residual disease. It has been previously demonstrated that an oncolytic virus known as myxoma can completely prevent myeloma relapse caused by reintroduction of malignant cells during autologous transplant. The ability of this virus to treat established residual disease in vivo, however, remained unknown. Here we demonstrate that intravenous administration of myxoma virus into mice bearing disseminated myeloma results in the elimination of 70–90% of malignant cells within 24 hours. This rapid debulking was dependent on direct contact of myxoma virus with residual myeloma and did not occur through destruction of the hematopoietic bone marrow niche. Importantly, systemic myxoma therapy also induced potent antimyeloma CD8+ T cell responses which localized to the bone marrow and were capable of completely eradicating established myeloma in some animals. These results demonstrate that oncolytic myxoma virus is not only effective at preventing relapse caused by reinfusion of tumor cells during stem cell transplant, but is also potentially curative for patients bearing established minimal residual disease. PMID:27933316

  3. Modulation of the Myxoma Virus Plaque Phenotype by Vaccinia Virus Protein F11

    PubMed Central

    Irwin, Chad R.

    2012-01-01

    Vaccinia virus (VACV) produces large plaques consisting of a rapidly expanding ring of infected cells surrounding a lytic core, whereas myxoma virus (MYXV) produces small plaques that resemble a focus of transformed cells. This is odd, because bioinformatics suggests that MYXV carries homologs of nearly all of the genes regulating Orthopoxvirus attachment, entry, and exit. So why does MYXV produce foci? One notable difference is that MYXV-infected cells produce few of the actin microfilaments that promote VACV exit and spread. This suggested that although MYXV carries homologs of the required genes (A33R, A34R, A36R, and B5R), they are dysfunctional. To test this, we produced MYXV recombinants expressing these genes, but we could not enhance actin projectile formation even in cells expressing all four VACV proteins. Another notable difference between these viruses is that MYXV lacks a homolog of the F11L gene. F11 inhibits the RhoA-mDia signaling that maintains the integrity of the cortical actin layer. We constructed an MYXV strain encoding F11L and observed that, unlike wild-type MYXV, the recombinant virus disrupted actin stress fibers and produced plaques up to 4-fold larger than those of controls, and these plaques expanded ∼6-fold faster. These viruses also grew to higher titers in multistep growth conditions, produced higher levels of actin projectiles, and promoted infected cell movement, although neither process was to the extent of that observed in VACV-infected cells. Thus, one reason for why MYXV produces small plaques is that it cannot spread via actin filaments, although the reason for this deficiency remains obscure. A second reason is that leporipoxviruses lack vaccinia's capacity to disrupt cortical actin. PMID:22514354

  4. Modulation of the myxoma virus plaque phenotype by vaccinia virus protein F11.

    PubMed

    Irwin, Chad R; Evans, David H

    2012-07-01

    Vaccinia virus (VACV) produces large plaques consisting of a rapidly expanding ring of infected cells surrounding a lytic core, whereas myxoma virus (MYXV) produces small plaques that resemble a focus of transformed cells. This is odd, because bioinformatics suggests that MYXV carries homologs of nearly all of the genes regulating Orthopoxvirus attachment, entry, and exit. So why does MYXV produce foci? One notable difference is that MYXV-infected cells produce few of the actin microfilaments that promote VACV exit and spread. This suggested that although MYXV carries homologs of the required genes (A33R, A34R, A36R, and B5R), they are dysfunctional. To test this, we produced MYXV recombinants expressing these genes, but we could not enhance actin projectile formation even in cells expressing all four VACV proteins. Another notable difference between these viruses is that MYXV lacks a homolog of the F11L gene. F11 inhibits the RhoA-mDia signaling that maintains the integrity of the cortical actin layer. We constructed an MYXV strain encoding F11L and observed that, unlike wild-type MYXV, the recombinant virus disrupted actin stress fibers and produced plaques up to 4-fold larger than those of controls, and these plaques expanded ∼6-fold faster. These viruses also grew to higher titers in multistep growth conditions, produced higher levels of actin projectiles, and promoted infected cell movement, although neither process was to the extent of that observed in VACV-infected cells. Thus, one reason for why MYXV produces small plaques is that it cannot spread via actin filaments, although the reason for this deficiency remains obscure. A second reason is that leporipoxviruses lack vaccinia's capacity to disrupt cortical actin.

  5. Targeting of vaccinia virus using biotin-avidin viral coating and biotinylated antibodies.

    PubMed

    Purow, Benjamin; Staveley-O'Carroll, Kevin

    2005-01-01

    To test a general method for altering the tropism of viral vectors, we conjugated targeting antibody to the surface of recombinant vaccinia virus with a biotin-avidin-biotin linker and assessed the resulting infectivity in target cells and controls. We biotinylated a vaccinia viral vector and used avidin to crosslink the biotinylated viral surface to a biotinylated antibody specific for a molecule on the surface of a target cell. In an in vitro model system, we coated a recombinant vaccinia construct containing the E. coli beta-galactosidase gene with antibody to the murine class I MHC molecule Db. Target cells were B78H1 murine melanoma cells transduced with either the Db gene or, as a control, the Kb gene. Infectivity was assessed by staining target cells with x-gal to demonstrate expression of virally delivered beta-galactosidase. This technique was also assessed in a second system with vaccinia/beta-gal targeted to the murine B7.2 molecule. The infectivity of the resulting construct was assessed for murine SA1 fibrosarcoma cells transfected with the B7.2 gene and for wild-type, B7.2-negative SA1. Experiments were repeated in each system with similar results. This strategy demonstrated antibody-mediated viral targeting in both the B78H1 and the SA1 models. Importantly, addition of the targeting coat diminished the infectivity of the modified vaccinia for control cells but preserved infectivity for targeted cells. In the B78H1 system, Db-targeted vaccinia consistently had 2- to 3-fold greater infectivity for B78H1Db than B78H1Kb. Increasing the number of avidin molecules used per virion in the synthesis of the viral coat led to greater selectivity but decreased overall infectivity. In the SA1 system, B7.2-targeted vaccinia demonstrated completely ablated infectivity for control SA1 cells, but maintained infectivity for target SA1/B7.2 cells. Recombinant viral vectors such as vaccinia may be coated with biotin/avidin and linked to biotinylated antibodies to

  6. Targeting the Vaccinia Virus L1 Protein to the Cell Surface Enhances Production of Neutralizing Antibodies

    DTIC Science & Technology

    2008-04-28

    vaccine can protect mice and non-humanprimates from lethal challengewith VACV ormon- keypox virus, respectively [21–23]. Fogg et al. demonstrated...23. 24] Fogg C, Lustig S, Whitbeck JC, Eisenberg RJ, Cohen GH, Moss B. Protective immunity to vaccinia virus induced by vaccination with multiple...Allison TJ, Fogg C, Moss B, Garboczi DN. The 1.51-Angstrom structure of the orthopoxvirus L1 protein, a target of potent neutralizing anti- bodies

  7. To Infection and Beyond: The Multi-Pronged Anti-Cancer Mechanisms of Oncolytic Viruses.

    PubMed

    Cassady, Kevin A; Haworth, Kellie B; Jackson, Josh; Markert, James M; Cripe, Timothy P

    2016-02-04

    Over the past 1-2 decades we have witnessed a resurgence of efforts to therapeutically exploit the attributes of lytic viruses to infect and kill tumor cells while sparing normal cells. We now appreciate that the utility of viruses for treating cancer extends far beyond lytic cell death. Viruses are also capable of eliciting humoral and cellular innate and adaptive immune responses that may be directed not only at virus-infected cells but also at uninfected cancer cells. Here we review our current understanding of this bystander effect, and divide the mechanisms into lytic, cytokine, innate cellular, and adaptive phases. Knowing the key pathways and molecular players during virus infection in the context of the cancer microenvironment will be critical to devise strategies to maximize the therapeutic effects of oncolytic viroimmunotherapy.

  8. Protection against Friend retrovirus-induced leukemia by recombinant vaccinia viruses expressing the gag gene.

    PubMed Central

    Miyazawa, M; Nishio, J; Chesebro, B

    1992-01-01

    High sequence variability in the envelope gene of human immunodeficiency virus has provoked interest in nonenvelope antigens as potential immunogens against retrovirus infection. However, the role of core protein antigens encoded by the gag gene in protective immunity against retroviruses is unclear. By using recombinant vaccinia viruses expressing the Friend murine leukemia helper virus (F-MuLV) gag gene, we could prime CD4+ T-helper cells and protectively immunize susceptible strains of mice against Friend retrovirus infection. Recovery from leukemic splenomegaly developed more slowly after immunization with vaccinia virus-F-MuLV gag than with vaccinia virus-F-MuLV env; however, genetic nonresponders to the envelope protein could be partially protected with Gag vaccines. Class switching of F-MuLV-neutralizing antibodies from immunoglobulin M to immunoglobulin G after challenge with Friend virus complex was facilitated in mice immunized with the Gag antigen. Sequential deletion of the gag gene revealed that the major protective epitope was located on the N-terminal hydrophobic protein p15. Images PMID:1534853

  9. The Vaccinia Virus-Encoded Bcl-2 Homologues Do Not Act as Direct Bax Inhibitors

    PubMed Central

    Postigo, Antonio

    2012-01-01

    Many viruses, including members of several poxvirus genera, encode inhibitors that block apoptosis by simultaneously binding the proapoptotic Bcl-2 proteins Bak and Bax. The Orthopoxvirus vaccinia virus encodes the Bcl-2-like F1 protein, which sequesters Bak but not Bax. However, N1, a potent virulence factor, is reported to be antiapoptotic and to interact with Bax. Here we investigated whether vaccinia virus inhibits Bak/Bax-dependent apoptosis via the cooperative action of F1 and N1. We found that Western Reserve (WR) and ΔN1L viruses inhibited drug- and infection-induced apoptosis equally. Meanwhile, infections with ΔF1L or ΔN1L/F1L virus resulted in similar levels of Bax activation and apoptosis. Outside the context of infection, N1 did not block drug- or Bax-induced cell death or interact with Bax. In addition to F1 and N1, vaccinia virus encodes further structural homologs of Bcl-2 proteins that are conserved in orthopoxviruses, including A46, A52, B14, C1, C6, C16/B22, K7, and N2. However, we found that these do not associate with Bax or inhibit drug-induced cell death. Based on our findings that N1 is not an antiapoptotic protein, we propose that the F1 orthologs represent the only orthopoxvirus Bcl-2 homolog to directly inhibit the Bak/Bax checkpoint. PMID:22013032

  10. Exponential enhancement of oncolytic vesicular stomatitis virus potency by vector-mediated suppression of inflammatory responses in vivo.

    PubMed

    Altomonte, Jennifer; Wu, Lan; Chen, Li; Meseck, Marcia; Ebert, Oliver; García-Sastre, Adolfo; Fallon, John; Woo, Savio L C

    2008-01-01

    Oncolytic virotherapy is a promising strategy for treatment of malignancy, although its effectiveness is hampered by host antiviral inflammatory responses. The efficacy of treatment of oncolytic vesicular stomatitis virus (VSV) in rats bearing multifocal hepatocellular carcinoma (HCC) can be substantially elevated by antibody-mediated depletion of natural killer (NK) cells. In order to test the hypothesis that the oncotyic potency of VSV can be exponentially elevated by evasion of inflammatory responses in vivo, we constructed a recombinant VSV vector expressing equine herpes virus-1 glycoprotein G, which is a broad-spectrum viral chemokine binding protein (rVSV-gG). Infusion of rVSV-gG via the hepatic artery into immune-competent rats bearing syngeneic and multifocal HCC in their livers, resulted in a reduction of NK and NKT cells in the tumors and a 1-log enhancement in intratumoral virus titer in comparison with a reference rVSV vector. The treatment led to increased tumor necrosis and substantially prolonged animal survival without toxicities. These results indicate that rVSV-gG has the potential to be developed as an effective and safe oncolytic agent to treat patients with advanced HCC. Furthermore, the novel concept that oncolytic potency can be substantially enhanced by vector-mediated suppression of host antiviral inflammatory responses could have general applicability in the field of oncolytic virotherapy for cancer.

  11. Clinical testing of engineered oncolytic measles virus strains in the treatment of cancer: An overview

    PubMed Central

    Msaouel, Pavlos; Dispenzieri, Angela; Galanis, Evanthia

    2009-01-01

    Viruses have adapted through millennia of evolution to effectively invade and lyse cells through diverse mechanisms. Strains of the attenuated measles virus Edmonston (MV-Edm) vaccine lineage can preferentially infect and destroy cancerous cells while sparing the surrounding tissues. This specificity is predominantly due to overexpression of the measles virus receptor CD46 in tumor cells. To facilitate in vivo monitoring of viral gene expression and replication, these oncolytic strains have been engineered to either express soluble marker peptides, such as the human carcinoembryonic antigen (CEA; MV-CEA virus), or genes that facilitate imaging and therapy, such as the human thyroidal sodium iodide symporter (NIS) gene (MV-NIS). Preclinical efficacy and safety data for engineered oncolytic MV-Edm derivatives that led to their clinical translation are discussed in this review, and an overview of the early experience in three ongoing clinical trials of patients with ovarian cancer, glioblastoma multiforme and multiple myeloma is provided. The information obtained from these ongoing trials will guide the future clinical application and further development of MV strains as anticancer agents. PMID:19169959

  12. Clinical trials involving the oncolytic virus, reovirus: ready for prime time?

    PubMed

    Black, Allison J; Morris, Don G

    2012-09-01

    The use of oncolytic viruses as a potential cancer therapeutic has been studied extensively over the past 15 years and is now in Phase III human clinical testing. One of the most promising of the viruses is the nonattenuated reovirus type-3 Dearing (RT3D; Reolysin(®), Oncolytics Biotech Inc., AB, Canada). The virus is a laboratory strain of a ubiquitous common environmental virus commonly infecting the respiratory and GI tracts of humans without major sequelae. The Phase I/II clinical trial conducted by Karapanagiotou et al. involved dose escalation of Reolysin to 3 × 10(10) tissue culture infectious dose 50 (TCID(50)) daily for 5 days in combination with paclitaxel (175 mg/m(2)) and carboplatin (area under the curve 5) given on day 1 every 3 weeks. Maximum tolerated dose was not reached in the dose-escalation phase and was only limited by manufacturing concentration limitation. Efficacy was suggested in this heavily pretreated head and neck cancer predominate patient population with a 26.9% response rate (seven out of 26 evaluable patients) of the 34 patients intended to treat. Although this was not a randomized trial, the fact that many of the patients (83%) had already received a platinum agent and subsequently progressed and then responded is of interest.

  13. Chemokine (C-C Motif) Receptor 1 Is Required for Efficient Recruitment of Neutrophils during Respiratory Infection with Modified Vaccinia Virus Ankara

    PubMed Central

    Price, Philip J. R.; Luckow, Bruno; Torres-Domínguez, Lino E.; Brandmüller, Christine; Zorn, Julia; Kirschning, Carsten J.; Sutter, Gerd

    2014-01-01

    ABSTRACT Modified vaccinia virus Ankara (MVA) serves as a versatile platform in vaccine development. This highly attenuated orthopoxvirus, which cannot replicate in mammalian cells, triggers strong innate immune responses, including cell migration. Previously, we have shown that induction of chemokine (C-C motif) ligand 2 (CCL2) by MVA is necessary for the recruitment of monocytes and T cells, but not neutrophils, to the lung. Here, we identified neutrophil-attracting chemokines produced by MVA-infected primary murine lung fibroblasts and murine bone marrow-derived macrophages. We demonstrate that MVA, but not vaccinia virus (VACV) strain WR, induces chemokine expression, which is independent of Toll-like receptor 2 (TLR2) signaling. Additionally, we show that both chemokine (C-C motif) receptor 1 (CCR1) and chemokine (C-X-C motif) receptor 2 (CXCR2) are involved in MVA-induced neutrophil chemotaxis in vitro. Finally, intranasal infection of Ccr1−/− mice with MVA, as well as application of the CCR1 antagonist J-113863, revealed a role for CCR1 in leukocyte recruitment, including neutrophils, into the lung. IMPORTANCE Rapid attraction of leukocytes to the site of inoculation is unique to MVA in comparison to other VACV strains. The findings here extend current knowledge about the regulation of MVA-induced leukocyte migration, particularly regarding neutrophils, which could potentially be exploited to improve other VACV strains currently in development as oncolytic viruses and viral vectors. Additionally, the data presented here indicate that the inflammatory response may vary depending on the cell type infected by MVA, highlighting the importance of the site of vaccine application. Moreover, the rapid recruitment of neutrophils and other leukocytes can directly contribute to the induction of adaptive immune responses elicited by MVA inoculation. Thus, a better understanding of leukocyte migration upon MVA infection is particularly relevant for further

  14. Society for Assisted Reproductive Technology position statement on donor suitability of recipients of smallpox vaccine (vaccinia virus).

    PubMed

    2006-11-01

    Although there is presently no definitive evidence linking vaccinia virus transmission through reproductive cells, SART/ASRM accordingly recommends that ART practitioners consider deferring donors who have recently received smallpox vaccine or contracted symptomatic vaccinia virus infection through close contact with a vaccine recipient (until after the vaccine or infectious scab has spontaneously separated). Good donor practice further suggests that donors who are not in good health, including those with recent complications from smallpox vaccine, should be similarly deferred.

  15. Society for assisted reproductive technology position statement on donor suitability of recipients of smallpox vaccine (vaccinia virus).

    PubMed

    2004-09-01

    Although there is presently no definitive evidence linking vaccinia virus transmission through reproductive cells, SART/ASRM accordingly recommends that assisted reproductive technology (ART) practitioners consider deferring donors who have recently received smallpox vaccine or contracted symptomatic vaccinia virus infection through close contact with a vaccine recipient (until after the vaccine or infectious scab has spontaneously separated). Good donor practice further suggests that donors who are not in good health, including those with recent complications from smallpox vaccine, should be similarly deferred.

  16. Society for assisted reproductive technology position statement on donor suitability of recipients of smallpox vaccine (vaccinia virus).

    PubMed

    2004-04-01

    Although there is presently no definitive evidence linking vaccinia virus transmission through reproductive cells, SART/ASRM accordingly recommends that assisted reproductive technology (ART) practitioners consider deferring donors who have recently received smallpox vaccine or contracted symptomatic vaccinia virus infection through close contact with a vaccine recipient (until after the vaccine or infectious scab has spontaneously separated). Good donor practice further suggests that donors who are not in good health, including those with recent complications from smallpox vaccine, should be similarly deferred.

  17. Biological Activity of an Intravenous Preparation of Human Vaccinia Immune Globulin in Mouse Models of Vaccinia Virus Infection

    PubMed Central

    Shearer, Jeffry D.; Siemann, Linda; Gerkovich, Mary; House, Robert V.

    2005-01-01

    The biological activity of a new intravenous (i.v.) preparation of human vaccinia immune globulin (VIGIV) was evaluated in two mouse models of vaccinia virus (VV) infection. In a mouse tail lesion model, female CD-1 mice were inoculated i.v. with 7 × 104 PFU of VV to produce >10 lesions per tail 8 days later. In a mouse lethality model, female severe combined immunodeficient (SCID) mice were inoculated i.v. with 3 × 104 PFU of VV to produce 100% mortality within 45 days. The ability of VIGIV to reduce tail lesion formation in CD-1 mice and mortality in SCID mice was determined by (i) pretreatment of a lethal VV dose with VIGIV prior to i.v. inoculation into SCID mice and (ii) i.v. administration of VIGIV to CD-1 and SCID mice the day before and up to 8 days after VV infection. VIGIV reduced the proportion of CD-1 mice with >10 tail lesions in a dose-related manner when VIGIV was given 1 day before and up to 1 day after VV inoculation. The pretreatment of VV with VIGIV prolonged survival and decreased mortality. VIGIV (100 and 400 mg/kg) prolonged survival when given up to 4 days after VV inoculation, and the 400-mg/kg dose reduced the mortality rate by 80% when given the day before or immediately after VV inoculation. The biological activity of VIGIV was demonstrated in both the immunocompetent and immunocompromised murine models. The timing of treatment relative to VV inoculation appeared to be important for the demonstration of VIGIV's biological activity. PMID:15980330

  18. Cellular factors promoting resistance to effective treatment of glioma with oncolytic Myxoma virus

    PubMed Central

    Zemp, Franz J.; McKenzie, Brienne A.; Lun, Xueqing; Reilly, Karlyne M.; McFadden, Grant; Yong, V. Wee; Forsyth, Peter A.

    2014-01-01

    Oncolytic virus therapy is being evaluated in clinical trials for human glioma. While it is widely assumed that the patient's immune response to the virus infection limits the therapy's utility, investigations into the specific cell type(s) involved in this response have been performed using non-specific pharmacological inhibitors or allogeneic models with compromised immunity. To identify the immune cells that participate in clearing an oncolytic infection in glioma, we used flow cytometry and immunohistochemistry to immunophenotype an orthotopic glioma model in immunocompetent mice after Myxoma virus (MYXV) administration. These studies revealed a large resident microglia and macrophage population in untreated tumours, and robust monocyte, T and NK cell infiltration 3 days following MYXV infection. To determine the role on the clinical utility of MYXV therapy for glioma, we used a combination of knockout mouse strains and specific immunocyte ablation techniques. Collectively, our experiments identify an important role for tumour-resident myeloid cells and overlapping roles for recruited NK and T cells in the clearance and efficacy of oncolytic MYXV from gliomas. Using a cyclophosphamide regimen to achieve lymphoablation prior and during MYXV treatment, we prevented treatment-induced peripheral immunocyte recruitment and, surprisingly, largely ablated the tumour-resident macrophage population. Virotherapy of CPA-treated animals resulted in sustained viral infection within the glioma as well as a substantial survival advantage. This study demonstrates that resistance to MYXV virotherapy in syngeneic glioma models involves a multi-faceted cellular immune response that can be overcome with CPA-mediated lymphoablation. PMID:25336188

  19. Sensitivity to ultraviolet radiation of Lassa, vaccinia, and Ebola viruses dried on surfaces.

    PubMed

    Sagripanti, Jose-Luis; Lytle, C David

    2011-03-01

    Germicidal UV (also known as UVC) provides a means to decontaminate infected environments as well as a measure of viral sensitivity to sunlight. The present study determined UVC inactivation slopes (and derived D(37) values) of viruses dried onto nonporous (glass) surfaces. The data obtained indicate that the UV resistance of Lassa virus is higher than that of Ebola virus. The UV sensitivity of vaccinia virus (a surrogate for variola virus) appeared intermediate between that of the two virulent viruses studied. In addition, the three viruses dried on surfaces showed a relatively small but significant population of virions (from 3 to 10 % of virus in the inoculum) that appeared substantially more protected by their environment from the effect of UV than the majority of virions tested. The findings reported in this study should assist in estimating the threat posed by the persistence of virus in environments contaminated during epidemics or after an accidental or intentional release.

  20. Targeting vaccinia virus-expressed secretory beta subunit of human chorionic gonadotropin to the cell surface induces antibodies.

    PubMed Central

    Srinivasan, J; Singh, O; Chakrabarti, S; Talwar, G P

    1995-01-01

    We carried out experiments designed to study the effect of a protein's localization on its immunogenicity. A novel cell-surface protein was generated from a small, glycosylated secretory protein. The DNA sequence encoding the entire precursor of the human chorionic gonadotropin beta (beta hCG) subunit was fused in the correct reading frame to the DNA sequence encoding the transmembrane and cytoplasmic domains of vesicular stomatitis virus glycoprotein. This chimeric gene was introduced into the vaccinia virus genome to generate a recombinant virus. The recombinant virus, when used to infect animal cells, expressed a 135-amino-acid beta hCG subunit anchored in cellular membranes by the 48 carboxy-terminal amino acids of vesicular stomatitis virus glycoprotein. The immunogenicity of this recombinant virus with respect to its ability to generate anti-hCG antibodies was compared with that of a second recombinant vaccinia virus expressing the native secretory form of beta hCG. All animals immunized with the vaccinia virus expressing beta hCG on the cell surface elicited high titers of anti-hCG antibodies. Even after a single immunization with the recombinant vaccinia virus, the anti-hCG antibody titers persisted for a long period of time (more than 6 months). None of the animals immunized with vaccinia virus expressing the native secretory form of beta hCG showed any hCG-specific antibody response. PMID:7591154

  1. Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response.

    PubMed

    Paran, Nir; Lustig, Shlomo; Zvi, Anat; Erez, Noam; Israely, Tomer; Melamed, Sharon; Politi, Boaz; Ben-Nathan, David; Schneider, Paula; Lachmi, Batel; Israeli, Ofir; Stein, Dana; Levin, Reuven; Olshevsky, Udy

    2013-07-10

    Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104-120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.

  2. Dominant negative selection of vaccinia virus using a thymidine kinase/thymidylate kinase fusion gene and the prodrug azidothymidine

    SciTech Connect

    Holzer, Georg W. . E-mail: falknef@baxter.com

    2005-07-05

    The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3'-azido-2',3'-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes.

  3. Pediatric cancer gone viral. Part II: potential clinical application of oncolytic herpes simplex virus-1 in children.

    PubMed

    Friedman, Gregory K; Beierle, Elizabeth A; Gillespie, George Yancey; Markert, James M; Waters, Alicia M; Chen, Chun-Yu; Denton, Nicholas L; Haworth, Kellie B; Hutzen, Brian; Leddon, Jennifer L; Streby, Keri A; Wang, Pin-Yi; Cripe, Timothy P

    Oncolytic engineered herpes simplex viruses (HSVs) possess many biologic and functional attributes that support their use in clinical trials in children with solid tumors. Tumor cells, in an effort to escape regulatory mechanisms that would impair their growth and progression, have removed many mechanisms that would have protected them from virus infection and eventual virus-mediated destruction. Viruses engineered to exploit this weakness, like mutant HSV, can be safely employed as tumor cell killers, since normal cells retain these antiviral strategies. Many preclinical studies and early phase trials in adults demonstrated that oncolytic HSV can be safely used and are highly effective in killing tumor cells that comprise pediatric malignancies, without generating the toxic side effects of nondiscriminatory chemotherapy or radiation therapy. A variety of engineered viruses have been developed and tested in numerous preclinical models of pediatric cancers and initial trials in patients are underway. In Part II of this review series, we examine the preclinical evidence to support the further advancement of oncolytic HSV in the pediatric population. We discuss clinical advances made to date in this emerging era of oncolytic virotherapy.

  4. Oncolytic virotherapy.

    PubMed

    Sze, Daniel Y; Reid, Tony R; Rose, Steven C

    2013-08-01

    Oncolytic virotherapy is an emerging technology that uses engineered viruses to treat malignancies. Viruses can be designed with biological specificity to infect cancerous cells preferentially, and to replicate in these cells exclusively. Malignant cells may be killed directly by overwhelming viral infection and lysis, which releases additional viral particles to infect neighboring cells and distant metastases. Viral infections may also activate the immune system, unmask stealthy tumor antigens, and aid the immune system to recognize and attack neoplasms. Delivery of live virus particles is potentially complex, and may require the expertise of the interventional community.

  5. Linked Tumor-Selective Virus Replication and Transgene Expression from E3-Containing Oncolytic Adenoviruses†

    PubMed Central

    Zhu, Mingzhu; Bristol, J. Andrew; Xie, Yuefeng; Mina, Mervat; Ji, Hong; Forry-Schaudies, Suzanne; Ennist, David L.

    2005-01-01

    Historically, the adenoviral E3 region was found to be nonessential for viral replication in vitro. In addition, adenoviruses whose genome was more than approximately 105% the size of the native genome were inefficiently packaged. These profound observations were used experimentally to insert transgenes into the adenoviral backbone. More recently, however, the reintroduction of the E3 region into oncolytic adenoviruses has been found to positively influence antitumor efficacy in preclinical models and clinical trials. In the studies reported here, the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA sequence has been substituted for the E3-gp19 gene in oncolytic adenoviruses that otherwise retained the E3 region. Five viruses that differed slightly in the method of transgene insertion were generated and compared to Ar6pAE2fGmF (E2F/GM/ΔE3), a previously described E3-deleted oncolytic adenovirus encoding GM-CSF. In all of the viruses, the human E2F-1 promoter regulated E1A expression and GM-CSF expression was under the control of the adenoviral E3 promoter and the packaging signal was relocated immediately upstream from the right terminal repeat. The E3-gp19-deleted viruses had similar cytolytic properties, as measured in vitro by cytotoxicity assays, but differed markedly in their capacity to express and secrete GM-CSF. Ar15pAE2fGmF (E2F/GM/E3b), the virus that produced the highest levels of GM-CSF and retained the native GM-CSF leader sequence, was selected for further analysis. The E2F/GM/E3b and E2F/GM/ΔE3 viruses exhibited similar cytotoxic activity and GM-CSF production in several tumor cell lines in vitro. However, when compared in vivo in nude mouse xenograft tumor models, E2F/GM/E3b spread through tumors to a greater extent, resulted in higher peak GM-CSF and total exposure levels in both tumor and serum, and was more efficacious than the E3-deleted virus. Using the matched WI-38 (parental) and WI-38-VA13 (simian virus 40 large T antigen

  6. Analysis of vaccinia virus temperature-sensitive I7L mutants reveals two potential functional domains

    PubMed Central

    Moerdyk, Megan J; Byrd, Chelsea M; Hruby, Dennis E

    2006-01-01

    As an approach to initiating a structure-function analysis of the vaccinia virus I7L core protein proteinase, a collection of conditional-lethal mutants in which the mutation had been mapped to the I7L locus were subjected to genomic sequencing and phenotypic analyses. Mutations in six vaccinia virus I7L temperature sensitive mutants fall into two groups: changes at three positions at the N-terminal end between amino acids 29 and 37 and two different substitutions at amino acid 344, near the catalytic domain. Regardless of the position of the mutation, mutants at the non-permissive temperature failed to cleave core protein precursors and had their development arrested prior to core condensation. Thus it appears that the two clusters of mutations may affect two different functional domains required for proteinase activity. PMID:16945137

  7. Use of Reporter Genes in the Generation of Vaccinia Virus-Derived Vectors

    PubMed Central

    Al Ali, Sally; Baldanta, Sara; Fernández-Escobar, Mercedes; Guerra, Susana

    2016-01-01

    Vaccinia virus (VACV) is one of the most extensively-studied viruses of the Poxviridae family. It is easy to genetically modify, so it has become a key tool for many applications. In this context, reporter genes facilitate the study of the role of foreign genes introduced into the genome of VACV. In this review, we describe the type of reporter genes that have been used to generate reporter-expressing VACV and the applications of the recombinant viruses obtained. Reporter-expressing VACV are currently employed in basic and immunology research, in the development of vaccines and cancer treatment. PMID:27213433

  8. Towards Predictive Computational Models of Oncolytic Virus Therapy: Basis for Experimental Validation and Model Selection

    PubMed Central

    Wodarz, Dominik; Komarova, Natalia

    2009-01-01

    Oncolytic viruses are viruses that specifically infect cancer cells and kill them, while leaving healthy cells largely intact. Their ability to spread through the tumor makes them an attractive therapy approach. While promising results have been observed in clinical trials, solid success remains elusive since we lack understanding of the basic principles that govern the dynamical interactions between the virus and the cancer. In this respect, computational models can help experimental research at optimizing treatment regimes. Although preliminary mathematical work has been performed, this suffers from the fact that individual models are largely arbitrary and based on biologically uncertain assumptions. Here, we present a general framework to study the dynamics of oncolytic viruses that is independent of uncertain and arbitrary mathematical formulations. We find two categories of dynamics, depending on the assumptions about spatial constraints that govern that spread of the virus from cell to cell. If infected cells are mixed among uninfected cells, there exists a viral replication rate threshold beyond which tumor control is the only outcome. On the other hand, if infected cells are clustered together (e.g. in a solid tumor), then we observe more complicated dynamics in which the outcome of therapy might go either way, depending on the initial number of cells and viruses. We fit our models to previously published experimental data and discuss aspects of model validation, selection, and experimental design. This framework can be used as a basis for model selection and validation in the context of future, more detailed experimental studies. It can further serve as the basis for future, more complex models that take into account other clinically relevant factors such as immune responses. PMID:19180240

  9. Vaccinia Virus Induces Rapid Necrosis in Keratinocytes by a STAT3-Dependent Mechanism

    PubMed Central

    He, Yong; Fisher, Robert; Chowdhury, Soma; Sultana, Ishrat; Pereira, Claudia P.; Bray, Mike; Reed, Jennifer L.

    2014-01-01

    Rationale Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens. Methods To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin. Results Mice treated topically with a STAT3 inhibitor (Stattic) developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3. Conclusions Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus. PMID:25419841

  10. Rescue of vaccinia virus lacking the E3L gene by mutants of E3L.

    PubMed Central

    Chang, H W; Uribe, L H; Jacobs, B L

    1995-01-01

    Vaccinia virus with the E3L gene deleted was able to replicate in RK-13 but not HeLa cells. This host range phenotype could be complemented by an E3L gene expressed transiently from a plasmid. Analysis of mutants of E3L indicates that the ability to complement deletion of E3L correlates with the ability of mutated proteins to bind double-stranded RNA but not with their ability to migrate to the nucleus. PMID:7666567

  11. Development of an oncolytic Herpes Simplex Virus using a tumor-specific HIF-responsive promoter

    PubMed Central

    Longo, Sharon L.; Griffith, Christopher; Glass, Aaron; Shillitoe, Edward J.; Post, Dawn E.

    2010-01-01

    We exploited the differential activation of hypoxia-inducible factor (HIF)-dependent gene expression in tumors versus normal tissue for the design of a targeted oncolytic Herpes simplex virus type-1 (HSV-1). A gene that is essential for viral replication, ICP4, was placed under the regulation of a HIF-responsive promoter and then introduced into the thymidine kinase locus (UL23) of HSV d120 which contains partial deletions in the two endogenous ICP4 genes. Recombinant HIF-HSV were isolated and their derivation from d120 was verified by expression of a truncated, nonfunctional form of ICP4 protein. Disruption of the UL23 locus was confirmed by loss of thymidine kinase expression and resistance to acyclovir. Unexpectedly, HIF-HSV expressed ICP4 and induced tumor cell lysis at similar levels under normoxia and hypoxia. The lack of HIF-dependent ICP4 transgene expression by HIF-HSV was due to two factors that have not previously been reported- reversion of the ICP4 gene region to its wild-type configuration and increased HIF-transcriptional activity under normoxia when cells were infected with any strain of HSV-1. The findings that an oncolytic HSV-1 is genetically unstable and can activate a tumor-related promoter in a non-specific manner have important implications for any proposed use of this virus in cancer therapy. PMID:20930860

  12. Retargeting Oncolytic Vesicular Stomatitis Virus to Human T-Cell Lymphotropic Virus Type 1-Associated Adult T-Cell Leukemia.

    PubMed

    Betancourt, Dillon; Ramos, Juan Carlos; Barber, Glen N

    2015-12-01

    Adult T cell leukemia/lymphoma (ATL) is an aggressive cancer of CD4/CD25(+) T lymphocytes, the etiological agent of which is human T-cell lymphotropic virus type 1 (HTLV-1). ATL is highly refractory to current therapies, making the development of new treatments a high priority. Oncolytic viruses such as vesicular stomatitis virus (VSV) are being considered as anticancer agents since they readily infect transformed cells compared to normal cells, the former appearing to exhibit defective innate immune responses. Here, we have evaluated the efficacy and safety of a recombinant VSV that has been retargeted to specifically infect and replicate in transformed CD4(+) cells. This was achieved by replacing the single VSV glycoprotein (G) with human immunodeficiency virus type 1 (HIV-1) gp160 to create a hybrid fusion protein, gp160G. The resultant virus, VSV-gp160G, was found to only target cells expressing CD4 and retained robust oncolytic activity against HTLV-1 actuated ATL cells. VSV-gp160G was further noted to be highly attenuated and did not replicate efficiently in or induce significant cell death of primary CD4(+) T cells. Accordingly, VSV-gp160G did not elicit any evidence of neurotoxicity even in severely immunocompromised animals such as NOD/Shi-scid, IL-2Rγ-c-null (NSG) mice. Importantly, VSV-gp160G effectively exerted potent oncolytic activity in patient-derived ATL transplanted into NSG mice and facilitated a significant survival benefit. Our data indicate that VSV-gp160G exerts potent oncolytic efficacy against CD4(+) malignant cells and either alone or in conjunction with established therapies may provide an effective treatment in patients displaying ATL. Adult T cell leukemia (ATL) is a serious form of cancer with a high mortality rate. HTLV-1 infection is the etiological agent of ATL and, unfortunately, most patients succumb to the disease within a few years. Current treatment options have failed to significantly improve survival rate. In this study, we

  13. Retargeting Oncolytic Vesicular Stomatitis Virus to Human T-Cell Lymphotropic Virus Type 1-Associated Adult T-Cell Leukemia

    PubMed Central

    Betancourt, Dillon; Ramos, Juan Carlos

    2015-01-01

    ABSTRACT Adult T cell leukemia/lymphoma (ATL) is an aggressive cancer of CD4/CD25+ T lymphocytes, the etiological agent of which is human T-cell lymphotropic virus type 1 (HTLV-1). ATL is highly refractory to current therapies, making the development of new treatments a high priority. Oncolytic viruses such as vesicular stomatitis virus (VSV) are being considered as anticancer agents since they readily infect transformed cells compared to normal cells, the former appearing to exhibit defective innate immune responses. Here, we have evaluated the efficacy and safety of a recombinant VSV that has been retargeted to specifically infect and replicate in transformed CD4+ cells. This was achieved by replacing the single VSV glycoprotein (G) with human immunodeficiency virus type 1 (HIV-1) gp160 to create a hybrid fusion protein, gp160G. The resultant virus, VSV-gp160G, was found to only target cells expressing CD4 and retained robust oncolytic activity against HTLV-1 actuated ATL cells. VSV-gp160G was further noted to be highly attenuated and did not replicate efficiently in or induce significant cell death of primary CD4+ T cells. Accordingly, VSV-gp160G did not elicit any evidence of neurotoxicity even in severely immunocompromised animals such as NOD/Shi-scid, IL-2Rγ-c-null (NSG) mice. Importantly, VSV-gp160G effectively exerted potent oncolytic activity in patient-derived ATL transplanted into NSG mice and facilitated a significant survival benefit. Our data indicate that VSV-gp160G exerts potent oncolytic efficacy against CD4+ malignant cells and either alone or in conjunction with established therapies may provide an effective treatment in patients displaying ATL. IMPORTANCE Adult T cell leukemia (ATL) is a serious form of cancer with a high mortality rate. HTLV-1 infection is the etiological agent of ATL and, unfortunately, most patients succumb to the disease within a few years. Current treatment options have failed to significantly improve survival rate. In

  14. High CD46 receptor density determines preferential killing of tumor cells by oncolytic measles virus.

    PubMed

    Anderson, Bambi D; Nakamura, Takafumi; Russell, Stephen J; Peng, Kah-Whye

    2004-07-15

    Live attenuated Edmonston B strain of measles virus (MV-Edm) is a potent and specific oncolytic agent, but the mechanism underlying its tumor selectivity is unknown. The virus causes cytopathic effects (CPEs) of extensive syncytial formation in tumor cells but minimal damage or cell killing in normal cells. The CPE is dependent on expression of viral proteins and the presence of CD46, the major cellular receptor of MV-Edm. Using a virally encoded soluble marker peptide to provide a quantitative readout of the level of viral gene expression, we determined that tumor cells and normal cells expressed comparable levels of viral proteins. CD46 mediates virus attachment, entry, and virus-induced cell-to-cell fusion. Using engineered cells expressing a range of CD46 densities, we determined that whereas virus entry increased progressively with CD46 density, cell fusion was minimal at low receptor densities but increased dramatically above a threshold density of CD46 receptors. It is well established that tumor cells express abundant CD46 receptors on their surfaces compared with their normal counterparts. Thus, at low CD46 densities typical of normal cells, infection occurs, but intercellular fusion is negligible. At higher densities typical of tumor cells, infection leads to extensive cell fusion. Intercellular fusion also results in enhancement of viral gene expression through recruitment of neighboring uninfected cells into the syncytium, further amplifying the CPE. Discrimination between high and low CD46 receptor density provides a compelling basis for the oncolytic specificity of MV-Edm and establishes MV-Edm as a promising CD46-targeted cancer therapeutic agent.

  15. Vaccinia virus p37 interacts with host proteins associated with LE-derived transport vesicle biogenesis.

    PubMed

    Chen, Yali; Honeychurch, Kady M; Yang, Guang; Byrd, Chelsea M; Harver, Chris; Hruby, Dennis E; Jordan, Robert

    2009-04-28

    Proteins associated with the late endosome (LE) appear to play a central role in the envelopment of a number of taxonomically diverse viruses. How viral proteins interact with LE-associated proteins to facilitate envelopment is not well understood. LE-derived transport vesicles form through the interaction of Rab9 GTPase with cargo proteins, and TIP47, a Rab9-specific effector protein. Vaccinia virus (VV) induces a wrapping complex derived from intracellular host membranes to envelope intracellular mature virus particles producing egress-competent forms of virus. We show that VV p37 protein associates with TIP47-, Rab9-, and CI-MPR-containing membranes. Mutation of a di-aromatic motif in p37 blocks association with TIP47 and inhibits plaque formation. ST-246, a specific inhibitor of p37 function, inhibits these interactions and also blocks wrapped virus particle formation. Vaccinia virus expressing p37 variants with reduced ST-246 susceptibility associates with Rab9 and co-localizes with CI-MPR in the presence and absence of compound. These results suggest that p37 localizes to the LE and interacts with proteins associated with LE-derived transport vesicle biogenesis to facilitate assembly of extracellular forms of virus.

  16. Vaccinia virus p37 interacts with host proteins associated with LE-derived transport vesicle biogenesis

    PubMed Central

    Chen, Yali; Honeychurch, Kady M; Yang, Guang; Byrd, Chelsea M; Harver, Chris; Hruby, Dennis E; Jordan, Robert

    2009-01-01

    Background Proteins associated with the late endosome (LE) appear to play a central role in the envelopment of a number of taxonomically diverse viruses. How viral proteins interact with LE-associated proteins to facilitate envelopment is not well understood. LE-derived transport vesicles form through the interaction of Rab9 GTPase with cargo proteins, and TIP47, a Rab9-specific effector protein. Vaccinia virus (VV) induces a wrapping complex derived from intracellular host membranes to envelope intracellular mature virus particles producing egress-competent forms of virus. Results We show that VV p37 protein associates with TIP47-, Rab9-, and CI-MPR-containing membranes. Mutation of a di-aromatic motif in p37 blocks association with TIP47 and inhibits plaque formation. ST-246, a specific inhibitor of p37 function, inhibits these interactions and also blocks wrapped virus particle formation. Vaccinia virus expressing p37 variants with reduced ST-246 susceptibility associates with Rab9 and co-localizes with CI-MPR in the presence and absence of compound. Conclusion These results suggest that p37 localizes to the LE and interacts with proteins associated with LE-derived transport vesicle biogenesis to facilitate assembly of extracellular forms of virus. PMID:19400954

  17. Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

    PubMed Central

    Ink, B S; Gilbert, C S; Evan, G I

    1995-01-01

    The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family. PMID:7815529

  18. Combination of Paclitaxel and MG1 oncolytic virus as a successful strategy for breast cancer treatment.

    PubMed

    Bourgeois-Daigneault, Marie-Claude; St-Germain, Lauren Elizabeth; Roy, Dominic Guy; Pelin, Adrian; Aitken, Amelia Sadie; Arulanandam, Rozanne; Falls, Theresa; Garcia, Vanessa; Diallo, Jean-Simon; Bell, John Cameron

    2016-08-08

    Breast cancer is the most common malignant disease amongst Western women. The lack of treatment options for patients with chemotherapy-resistant or recurrent cancers is pushing the field toward the rapid development of novel therapies. The use of oncolytic viruses is a promising approach for the treatment of disseminated diseases like breast cancer, with the first candidate recently approved by the Food and Drug Administration for use in patients. In this report, we demonstrate the compatibility of oncolytic virotherapy and chemotherapy using various murine breast cancer models. This one-two punch has been explored in the past by several groups with different viruses and drugs and was shown to be a successful approach. Our strategy is to combine Paclitaxel, one of the most common drugs used to treat patients with breast cancer, and the oncolytic Rhabdovirus Maraba-MG1, a clinical trial candidate in a study currently recruiting patients with late-stage metastatic cancer. We used the EMT6, 4 T1 and E0771 murine breast cancer models to evaluate in vitro and in vivo the effects of co-treatment with MG1 and Paclitaxel. Treatment-induced cytotoxicity was assessed and plaque assays, flow cytometry, microscopy and immunocytochemistry analysis were performed to quantify virus production and transgene expression. Orthotopically implanted tumors were measured during and after treatment to evaluate efficacy and Kaplan-Meier survival curves were generated. Our data demonstrate not only the compatibility of the treatments, but also their synergistic cytopathic activity. With Paclitaxel, EMT6 and 4 T1 tumors demonstrated increased virus production both in vitro and in vivo. Our results also show that Paclitaxel does not impair the safety profile of the virus treatment. Importantly, when combined, MG1 and the drug controlled tumor growth and prolonged survival. The combination of MG1 and Paclitaxel improved efficacy in all of the breast cancer models we tested and thus is a

  19. Vaccinia virus protein C4 inhibits NF-κB activation and promotes virus virulence

    PubMed Central

    Ember, Stuart W. J.; Ren, Hongwei; Ferguson, Brian J.

    2012-01-01

    Vaccinia virus (VACV) strain Western Reserve protein C4 has been characterized and its function and contribution to virus virulence assessed. Bioinformatic analysis showed that C4 is conserved in six orthopoxvirus species and shares 43 % amino acid identity with VACV protein C16, a known virulence factor. A recombinant VACV expressing a C-terminally tagged version of C4 showed that, like C16, this 37 kDa protein is expressed early during infection and localizes to both the cytoplasm and the nucleus. Functional assays using a firefly luciferase reporter plasmid under the control of a nuclear factor kappa B (NF-κB)-dependent promoter demonstrated that C4 inhibits NF-κB activation at, or downstream of, the inhibitor of kappa kinase (IKK) complex. Consistent with this, C4 inhibited interleukin-1β-induced translocation of p65 into the nucleus. A VACV lacking the C4L gene (vΔC4) showed no significant differences from wild-type virus in growth kinetics or spread in cell culture, but had reduced virulence in a murine intranasal model of infection. vΔC4-infected mice exhibited fewer symptoms, lost less weight and recovered 7 days earlier than animals infected with control viruses expressing C4. Furthermore, bronchoalveolar lavage fluid from vΔC4-infected mice had increased cell numbers at day 5 post-infection, which correlated with reduced lung virus titres from this time onward. C4 represents the ninth VACV protein to inhibit NF-κB activation and remarkably, in every case examined, loss of each protein individually caused an alteration in virus virulence, despite the presence of other NF-κB inhibitors. PMID:22791606

  20. Oncolytic Virotherapy for Hematological Malignancies

    PubMed Central

    Bais, Swarna; Bartee, Eric; Rahman, Masmudur M.; McFadden, Grant; Cogle, Christopher R.

    2012-01-01

    Hematological malignancies such as leukemias, lymphomas, multiple myeloma (MM), and the myelodysplastic syndromes (MDSs) primarily affect adults and are difficult to treat. For high-risk disease, hematopoietic stem cell transplant (HCT) can be used. However, in the setting of autologous HCT, relapse due to contamination of the autograft with cancer cells remains a major challenge. Ex vivo manipulations of the autograft to purge cancer cells using chemotherapies and toxins have been attempted. Because these past strategies lack specificity for malignant cells and often impair the normal hematopoietic stem and progenitor cells, prior efforts to ex vivo purge autografts have resulted in prolonged cytopenias and graft failure. The ideal ex vivo purging agent would selectively target the contaminating cancer cells while spare normal stem and progenitor cells and would be applied quickly without toxicities to the recipient. One agent which meets these criteria is oncolytic viruses. This paper details experimental progress with reovirus, myxoma virus, measles virus, vesicular stomatitis virus, coxsackievirus, and vaccinia virus as well as requirements for translation of these results to the clinic. PMID:22312362

  1. Targeting tumor vasculature through oncolytic virotherapy: recent advances.

    PubMed

    Toro Bejarano, Marcela; Merchan, Jaime R

    2015-01-01

    The oncolytic virotherapy field has made significant advances in the last decade, with a rapidly increasing number of early- and late-stage clinical trials, some of them showing safety and promising therapeutic efficacy. Targeting tumor vasculature by oncolytic viruses (OVs) is an attractive strategy that offers several advantages over nontargeted viruses, including improved tumor viral entry, direct antivascular effects, and enhanced antitumor efficacy. Current understanding of the biological mechanisms of tumor neovascularization, novel vascular targets, and mechanisms of resistance has allowed the development of oncolytic viral vectors designed to target tumor neovessels. While some OVs (such as vaccinia and vesicular stomatitis virus) can intrinsically target tumor vasculature and induce vascular disruption, the majority of reported vascular-targeted viruses are the result of genetic manipulation of their viral genomes. Such strategies include transcriptional or transductional endothelial targeting, "armed" viruses able to downregulate angiogenic factors, or to express antiangiogenic molecules. The above strategies have shown preclinical safety and improved antitumor efficacy, either alone, or in combination with standard or targeted agents. This review focuses on the recent efforts toward the development of vascular-targeted OVs for cancer treatment and provides a translational/clinical perspective into the future development of new generation biological agents for human cancers.

  2. Oncolytic herpes simplex virus expressing yeast cytosine deaminase: relationship between viral replication, transgene expression, prodrug bioactivation.

    PubMed

    Yamada, S; Kuroda, T; Fuchs, B C; He, X; Supko, J G; Schmitt, A; McGinn, C M; Lanuti, M; Tanabe, K K

    2012-03-01

    Yeast cytosine deaminase (yCD) is a well-characterized prodrug/enzyme system that converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and has been combined with oncolytic viruses. However, in vivo studies of the interactions between 5-FC bioactivation and viral replication have not been previously reported, nor have the kinetics of transgene expression and the pharmacokinetics of 5-FC and 5-FU. We constructed a replication-conditional Herpes simplex virus 1 (HSV-1) expressing yCD and examined cytotoxicity when 5-FC was initiated at different times after viral infection, and observed that earlier 5-FC administration led to greater cytotoxicity than later 5-FC administration in vitro and in vivo. In animal models, 12 days of 5-FC administration was superior to 6 days, but dosing beyond 12 days did not further enhance efficacy. Consistent with the dosing-schedule results, both viral genomic DNA copy number and viral titers were observed to peak on Day 3 after viral injection and gradually decrease thereafter. The virus is replication-conditional and was detected in tumors for as long as 2 weeks after viral injection. The maximum relative extent of yCD conversion of 5-FC to 5-FU in tumors was observed on Day 6 after viral injection and it decreased progressively thereafter. The observation that 5-FU generation within tumors did not lead to appreciable levels of systemic 5-FU (<10 ng ml⁻¹) is important and has not been previously reported. The approaches used in these studies of the relationship between the viral replication kinetics, transgene expression, prodrug administration and anti-tumor efficacy are useful in the design of clinical trials of armed, oncolytic viruses.

  3. Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein.

    PubMed

    Volz, Asisa; Kupke, Alexandra; Song, Fei; Jany, Sylvia; Fux, Robert; Shams-Eldin, Hosam; Schmidt, Jörg; Becker, Christin; Eickmann, Markus; Becker, Stephan; Sutter, Gerd

    2015-08-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8(+) T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine.

  4. Host range, growth property, and virulence of the smallpox vaccine: Vaccinia virus Tian Tan strain

    SciTech Connect

    Fang Qing; Yang Lin; Zhu Weijun; Liu Li; Wang Haibo; Yu Wenbo; Xiao Genfu; Tien Po; Zhang Linqi; Chen Zhiwei . E-mail: zchen@adarc.org

    2005-05-10

    Vaccinia Tian Tan (VTT) was used as a vaccine against smallpox in China for millions of people before 1980, yet the biological characteristics of the virus remain unclear. We have characterized VTT with respect to its host cell range, growth properties in vitro, and virulence in vivo. We found that 11 of the 12 mammalian cell lines studied are permissive to VTT infection whereas one, CHO-K1, is non-permissive. Using electron microscopy and sequence analysis, we found that the restriction of VTT replication in CHO-K1 is at a step before viral maturation probably due to the loss of the V025 gene. Moreover, VTT is significantly less virulent than vaccinia WR but remains neurovirulent in mice and causes significant body weight loss after intranasal inoculation. Our data demonstrate the need for further attenuation of VTT to serve either as a safer smallpox vaccine or as a live vaccine vector for other pathogens.

  5. Serologic and Molecular Evidence of Vaccinia Virus Circulation among Small Mammals from Different Biomes, Brazil

    PubMed Central

    Miranda, Júlia B.; Borges, Iara A.; Campos, Samantha P.S.; Vieira, Flávia N.; de Ázara, Tatiana M.F.; Marques, Fernanda A.; Costa, Galileu B.; Luis, Ana Paula M.F.; de Oliveira, Jaqueline S.; Ferreira, Paulo César P.; Bonjardim, Cláudio Antônio; da Silva, Silvio L.M.; Eiras, Álvaro E.; Abrahão, Jônatas S.; Kroon, Erna G.; Drumond, Betânia P.; Paglia, Adriano P.

    2017-01-01

    Vaccinia virus (VACV) is a zoonotic agent that causes a disease called bovine vaccinia, which is detected mainly in milking cattle and humans in close contact with these animals. Even though many aspects of VACV infection have been described, much is still unknown about its circulation in the environment and its natural hosts/reservoirs. To investigate the presence of Orthopoxvirus antibodies or VACV DNA, we captured small rodents and marsupials in 3 areas of Minas Gerais state, Brazil, and tested their samples in a laboratory. A total of 336 animals were tested; positivity ranged from 18.1% to 25.5% in the 3 studied regions located in different biomes, including the Atlantic Forest and the Cerrado. Analysis of nucleotide sequences indicated co-circulation of VACV groups I and II. Our findings reinforce the possible role played by rodents and marsupials in VACV maintenance and its transmission chain. PMID:28518030

  6. Host range, growth property, and virulence of the smallpox vaccine: vaccinia virus Tian Tan strain.

    PubMed

    Fang, Qing; Yang, Lin; Zhu, Weijun; Liu, Li; Wang, Haibo; Yu, Wenbo; Xiao, Genfu; Tien, Po; Zhang, Linqi; Chen, Zhiwei

    2005-05-10

    Vaccinia Tian Tan (VTT) was used as a vaccine against smallpox in China for millions of people before 1980, yet the biological characteristics of the virus remain unclear. We have characterized VTT with respect to its host cell range, growth properties in vitro, and virulence in vivo. We found that 11 of the 12 mammalian cell lines studied are permissive to VTT infection whereas one, CHO-K1, is non-permissive. Using electron microscopy and sequence analysis, we found that the restriction of VTT replication in CHO-K1 is at a step before viral maturation probably due to the loss of the V025 gene. Moreover, VTT is significantly less virulent than vaccinia WR but remains neurovirulent in mice and causes significant body weight loss after intranasal inoculation. Our data demonstrate the need for further attenuation of VTT to serve either as a safer smallpox vaccine or as a live vaccine vector for other pathogens.

  7. Oncolytic herpes simplex virus kills stem-like tumor-initiating colon cancer cells

    PubMed Central

    Warner, Susanne G; Haddad, Dana; Au, Joyce; Carson, Joshua S; O’Leary, Michael P; Lewis, Christina; Monette, Sebastien; Fong, Yuman

    2016-01-01

    Stem-like tumor-initiating cells (TICs) are implicated in cancer progression and recurrence, and can be identified by sphere-formation and tumorigenicity assays. Oncolytic viruses infect, replicate in, and kill a variety of cancer cells. In this study, we seek proof of principle that TICs are susceptible to viral infection. HCT8 human colon cancer cells were subjected to serum-free culture to generate TIC tumorspheres. Parent cells and TICs were infected with HSV-1 subtype NV1066. Cytotoxicity, viral replication, and Akt1 expression were assessed. TIC tumorigenicity was confirmed and NV1066 efficacy was assessed in vivo. NV1066 infection was highly cytotoxic to both parent HCT8 cells and TICs. In both populations, cell-kill of >80% was achieved within 3 days of infection at a multiplicity of infection (MOI) of 1.0. However, the parent cells required 2-log greater viral replication to achieve the same cytotoxicity. TICs overexpressed Akt1 in vitro and formed flank tumors from as little as 100 cells, growing earlier, faster, larger, and with greater histologic atypia than tumors from parent cells. Treatment of TIC-induced tumors with NV1066 yielded tumor regression and slowed tumor growth. We conclude that colon TICs are selected for by serum-free culture, overexpress Akt1, and are susceptible to oncolytic viral infection. PMID:27347556

  8. Analysis of genetically engineered oncolytic herpes simplex viruses in human prostate cancer organotypic cultures.

    PubMed

    Passer, B J; Wu, C-l; Wu, S; Rabkin, S D; Martuza, R L

    2009-12-01

    Oncolytic herpes simplex viruses type 1 (oHSVs) such as G47Delta and G207 are genetically engineered for selective replication competence in cancer cells. Several factors can influence the overall effectiveness of oHSV tropism, including HSV-1 receptor expression, extracellular matrix milieu and cellular permissiveness. We have taken advantage of human prostate organ cultures derived from radical prostatectomies to investigate oHSV tropism. In this study, we show that both G47Delta and G207 specifically replicate in epithelial cells of the prostatic glands but not in the surrounding stroma. In contrast, both the epithelial and stromal cell compartments were readily infected by wild-type HSV-1. Analysis of oHSV replication in prostate surgical specimens 3 days post infection showed that G47Delta generated approximately 30-fold more viral progeny than did G207. This correlated with the enhanced expression of G47Delta-derived glycoprotein gB protein levels as compared with G207. In benign prostate tissues, G207 and G47Delta titers were notably reduced, whereas strain F titers were maintained at similar levels compared with prostate cancer specimens. Overall, our results show that these oncolytic herpes vectors show both target specificity and replication competence in human prostate cancer specimens and point to the utility of using human prostate organ cultures in assessing oHSV tropism and cellular specificity.

  9. Preclinical evaluation of engineered oncolytic herpes simplex virus for the treatment of pediatric solid tumors.

    PubMed

    Megison, Michael L; Gillory, Lauren A; Stewart, Jerry E; Nabers, Hugh C; Mroczek-Musulman, Elizabeth; Waters, Alicia M; Coleman, Jennifer M; Kelly, Virginia; Markert, James M; Gillespie, G Yancey; Friedman, Gregory K; Beierle, Elizabeth A

    2014-01-01

    Recently, investigators showed that mice with syngeneic murine gliomas that were treated with a neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ134.5 gene, enabling replication in tumor cells but precluding infection of normal cells. Previous studies have shown antitumor effects of other oHSV against a number of adult tumors including hepatocellular carcinoma and renal cell carcinoma. The purpose of the current study was to investigate the oncolytic potential of M002 against difficult to treat pediatric liver and kidney tumors. We showed that the oHSV, M002, infected, replicated, and decreased cell survival in hepatoblastoma, malignant rhabdoid kidney tumor, and renal sarcoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly increased survival and decreased tumor growth. Finally, these studies showed that the primary entry protein for oHSV, CD111 (nectin-1) was present in human hepatoblastoma and malignant rhabdoid kidney tumor specimens. We concluded that M002 effectively targeted these rare aggressive tumor types and that M002 may have potential for use in children with unresponsive or relapsed pediatric solid tumors.

  10. Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus.

    PubMed

    Schlehofer, J R; Ehrbar, M; zur Hausen, H

    1986-07-15

    The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens ("initiators") as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. As reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.

  11. Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

    SciTech Connect

    Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

    1986-07-15

    The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. As reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.

  12. Dynamic monitoring of membrane nanotubes formation induced by vaccinia virus on a high throughput microfluidic chip

    PubMed Central

    Xiao, Min; Xu, Na; Wang, Cheng; Pang, Dai-Wen; Zhang, Zhi-Ling

    2017-01-01

    Membrane nanotubes (MNTs) are physical connections for intercellular communication and induced by various viruses. However, the formation of vaccinia virus (VACV)-induced MNTs has never been studied. In this report, VACV-induced MNTs formation process was monitored on a microfluidic chip equipped with a series of side chambers, which protected MNTs from fluidic shear stress. MNTs were formed between susceptible cells and be facilitated by VACV infection through three patterns. The formed MNTs varied with cell migration and virus concentration. The length of MNTs was positively correlated with the distance of cell migration. With increasing virus titer, the peak value of the ratio of MNT-carried cell appeared earlier. The immunofluorescence assay indicated that the rearrangement of actin fibers induced by VACV infection may lead to the formation of MNTs. This study presents evidence for the formation of MNTs induced by virus and helps us to understand the relationship between pathogens and MNTs. PMID:28317863

  13. Dynamic monitoring of membrane nanotubes formation induced by vaccinia virus on a high throughput microfluidic chip

    NASA Astrophysics Data System (ADS)

    Xiao, Min; Xu, Na; Wang, Cheng; Pang, Dai-Wen; Zhang, Zhi-Ling

    2017-03-01

    Membrane nanotubes (MNTs) are physical connections for intercellular communication and induced by various viruses. However, the formation of vaccinia virus (VACV)-induced MNTs has never been studied. In this report, VACV-induced MNTs formation process was monitored on a microfluidic chip equipped with a series of side chambers, which protected MNTs from fluidic shear stress. MNTs were formed between susceptible cells and be facilitated by VACV infection through three patterns. The formed MNTs varied with cell migration and virus concentration. The length of MNTs was positively correlated with the distance of cell migration. With increasing virus titer, the peak value of the ratio of MNT-carried cell appeared earlier. The immunofluorescence assay indicated that the rearrangement of actin fibers induced by VACV infection may lead to the formation of MNTs. This study presents evidence for the formation of MNTs induced by virus and helps us to understand the relationship between pathogens and MNTs.

  14. Elements in the Development of a Production Process for Modified Vaccinia Virus Ankara.

    PubMed

    Jordan, Ingo; Lohr, Verena; Genzel, Yvonne; Reichl, Udo; Sandig, Volker

    2013-11-01

    The production of several viral vaccines depends on chicken embryo fibroblasts or embryonated chicken eggs. To replace this logistically demanding substrate, we created continuous anatine suspension cell lines (CR and CR.pIX), developed chemically-defined media, and established production processes for different vaccine viruses. One of the processes investigated in greater detail was developed for modified vaccinia virus Ankara (MVA). MVA is highly attenuated for human recipients and an efficient vector for reactogenic expression of foreign genes. Because direct cell-to-cell spread is one important mechanism for vaccinia virus replication, cultivation of MVA in bioreactors is facilitated if cell aggregates are induced after infection. This dependency may be the mechanism behind our observation that a novel viral genotype (MVA-CR) accumulates with serial passage in suspension cultures. Sequencing of a major part of the genomic DNA of the new strain revealed point mutations in three genes. We hypothesize that these changes confer an advantage because they may allow a greater fraction of MVA-CR viruses to escape the host cells for infection of distant targets. Production and purification of MVA-based vaccines may be simplified by this combination of designed avian cell line, chemically defined media and the novel virus strain.

  15. Vaccinia virus interactions with the cell membrane studied by new chromatic vesicle and cell sensor assays.

    PubMed

    Orynbayeva, Z; Kolusheva, S; Groysman, N; Gavrielov, N; Lobel, L; Jelinek, R

    2007-02-01

    The potential danger of cross-species viral infection points to the significance of understanding the contributions of nonspecific membrane interactions with the viral envelope compared to receptor-mediated uptake as a factor in virus internalization and infection. We present a detailed investigation of the interactions of vaccinia virus particles with lipid bilayers and with epithelial cell membranes using newly developed chromatic biomimetic membrane assays. This analytical platform comprises vesicular particles containing lipids interspersed within reporter polymer units that emit intense fluorescence following viral interactions with the lipid domains. The chromatic vesicles were employed as membrane models in cell-free solutions and were also incorporated into the membranes of epithelial cells, thereby functioning as localized membrane sensors on the cell surface. These experiments provide important insight into membrane interactions with and fusion of virions and the kinetic profiles of these processes. In particular, the data emphasize the significance of cholesterol/sphingomyelin domains (lipid rafts) as a crucial factor promoting bilayer insertion of the viral particles. Our analysis of virus interactions with polymer-labeled living cells exposed the significant role of the epidermal growth factor receptor in vaccinia virus infectivity; however, the data also demonstrated the existence of additional non-receptor-mediated mechanisms contributing to attachment of the virus to the cell surface and its internalization.

  16. Oncolytic Activity of Avian Influenza Virus in Human Pancreatic Ductal Adenocarcinoma Cell Lines

    PubMed Central

    Pizzuto, Matteo S.; Silic-Benussi, Micol; Pavone, Silvia; Ciminale, Vincenzo; Capua, Ilaria

    2014-01-01

    ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is the most lethal form of human cancer, with dismal survival rates due to late-stage diagnoses and a lack of efficacious therapies. Building on the observation that avian influenza A viruses (IAVs) have a tropism for the pancreas in vivo, the present study was aimed at testing the efficacy of IAVs as oncolytic agents for killing human PDA cell lines. Receptor characterization confirmed that human PDA cell lines express the alpha-2,3- and the alpha-2,6-linked glycan receptor for avian and human IAVs, respectively. PDA cell lines were sensitive to infection by human and avian IAV isolates, which is consistent with this finding. Growth kinetic experiments showed preferential virus replication in PDA cells over that in a nontransformed pancreatic ductal cell line. Finally, at early time points posttreatment, infection with IAVs caused higher levels of apoptosis in PDA cells than gemcitabine and cisplatin, which are the cornerstone of current therapies for PDA. In the BxPC-3 PDA cell line, apoptosis resulted from the engagement of the intrinsic mitochondrial pathway. Importantly, IAVs did not induce apoptosis in nontransformed pancreatic ductal HPDE6 cells. Using a model based on the growth of a PDA cell line as a xenograft in SCID mice, we also show that a slightly pathogenic avian IAV significantly inhibited tumor growth following intratumoral injection. Taken together, these results are the first to suggest that IAVs may hold promise as future agents of oncolytic virotherapy against pancreatic ductal adenocarcinomas. IMPORTANCE Despite intensive studies aimed at designing new therapeutic approaches, PDA still retains the most dismal prognosis among human cancers. In the present study, we provide the first evidence indicating that avian IAVs of low pathogenicity display a tropism for human PDA cells, resulting in viral RNA replication and a potent induction of apoptosis in vitro and antitumor effects in vivo. These

  17. Suitability of vaccinia virus and bovine viral diarrhea virus (BVDV) for determining activities of three commonly-used alcohol-based hand rubs against enveloped viruses

    PubMed Central

    Kampf, Günter; Steinmann, Jochen; Rabenau, Holger

    2007-01-01

    Background A procedure for including activity against enveloped viruses in the post-contamination treatment of hands has been recommended, but so far no European standard is available to implement it. In 2004, the German Robert Koch-Institute (RKI) and the German Association for the Control of Virus Disease (DVV) suggested that vaccinia virus and bovine viral diarrhea virus (BVDV) should be used as test viruses in a quantitative suspension test to determine the activity of a disinfectant against all enveloped viruses. Methods We have studied the activities of three commonly-used alcohol-based hand rubs (hand rub A, based on 45% propan-2-ol, 30% propan-1-ol and 0.2% mecetronium etilsulfate; hand rub B, based on 80% ethanol; hand rub C, based on 95% ethanol) against vaccinia virus and BVDV, and in addition against four other clinically relevant enveloped viruses: herpes simplex virus (HSV) types 1 and 2, and human and avian influenza A virus. The hand rubs were challenged with different organic loads at exposure time of 15, 30 and 60 s. According to the guidelines of both BGA/RKI and DVV, and EN 14476:2005, the reduction of infectivity of each test virus was measured on appropriate cell lines using a quantitative suspension test. Results All three alcohol-based hand rubs reduced the infectivity of vaccinia virus and BVDV by ≥ 4 log10-steps within 15 s, irrespective of the type of organic load. Similar reductions of infectivity were seen against the other four enveloped viruses within 15 s in the presence of different types of organic load. Conclusion Commonly used alcohol-based hand rubs with a total alcohol concentration ≥ 75% can be assumed to be active against clinically relevant enveloped viruses if they effectively reduce the infectivities of vaccinia virus and BVDV in a quantitative suspension test. PMID:17291338

  18. Suitability of vaccinia virus and bovine viral diarrhea virus (BVDV) for determining activities of three commonly-used alcohol-based hand rubs against enveloped viruses.

    PubMed

    Kampf, Günter; Steinmann, Jochen; Rabenau, Holger

    2007-02-09

    A procedure for including activity against enveloped viruses in the post-contamination treatment of hands has been recommended, but so far no European standard is available to implement it. In 2004, the German Robert Koch-Institute (RKI) and the German Association for the Control of Virus Disease (DVV) suggested that vaccinia virus and bovine viral diarrhea virus (BVDV) should be used as test viruses in a quantitative suspension test to determine the activity of a disinfectant against all enveloped viruses. We have studied the activities of three commonly-used alcohol-based hand rubs (hand rub A, based on 45% propan-2-ol, 30% propan-1-ol and 0.2% mecetronium etilsulfate; hand rub B, based on 80% ethanol; hand rub C, based on 95% ethanol) against vaccinia virus and BVDV, and in addition against four other clinically relevant enveloped viruses: herpes simplex virus (HSV) types 1 and 2, and human and avian influenza A virus. The hand rubs were challenged with different organic loads at exposure time of 15, 30 and 60 s. According to the guidelines of both BGA/RKI and DVV, and EN 14476:2005, the reduction of infectivity of each test virus was measured on appropriate cell lines using a quantitative suspension test. All three alcohol-based hand rubs reduced the infectivity of vaccinia virus and BVDV by > or = 4 log10-steps within 15 s, irrespective of the type of organic load. Similar reductions of infectivity were seen against the other four enveloped viruses within 15 s in the presence of different types of organic load. Commonly used alcohol-based hand rubs with a total alcohol concentration > or = 75% can be assumed to be active against clinically relevant enveloped viruses if they effectively reduce the infectivities of vaccinia virus and BVDV in a quantitative suspension test.

  19. Complex spatial dynamics of oncolytic viruses in vitro: mathematical and experimental approaches.

    PubMed

    Wodarz, Dominik; Hofacre, Andrew; Lau, John W; Sun, Zhiying; Fan, Hung; Komarova, Natalia L

    2012-01-01

    Oncolytic viruses replicate selectively in tumor cells and can serve as targeted treatment agents. While promising results have been observed in clinical trials, consistent success of therapy remains elusive. The dynamics of virus spread through tumor cell populations has been studied both experimentally and computationally. However, a basic understanding of the principles underlying virus spread in spatially structured target cell populations has yet to be obtained. This paper studies such dynamics, using a newly constructed recombinant adenovirus type-5 (Ad5) that expresses enhanced jellyfish green fluorescent protein (EGFP), AdEGFPuci, and grows on human 293 embryonic kidney epithelial cells, allowing us to track cell numbers and spatial patterns over time. The cells are arranged in a two-dimensional setting and allow virus spread to occur only to target cells within the local neighborhood. Despite the simplicity of the setup, complex dynamics are observed. Experiments gave rise to three spatial patterns that we call "hollow ring structure", "filled ring structure", and "disperse pattern". An agent-based, stochastic computational model is used to simulate and interpret the experiments. The model can reproduce the experimentally observed patterns, and identifies key parameters that determine which pattern of virus growth arises. The model is further used to study the long-term outcome of the dynamics for the different growth patterns, and to investigate conditions under which the virus population eliminates the target cells. We find that both the filled ring structure and disperse pattern of initial expansion are indicative of treatment failure, where target cells persist in the long run. The hollow ring structure is associated with either target cell extinction or low-level persistence, both of which can be viewed as treatment success. Interestingly, it is found that equilibrium properties of ordinary differential equations describing the dynamics in local

  20. Genome Sequence of WAU86/88-1, a New Variant of Vaccinia Virus Lister Strain from Poland

    PubMed Central

    Mavian, Carla; López-Bueno, Alberto

    2014-01-01

    The poxviruses Warsaw Agricultural University 86 (WAU86) and 88-1 (WAU88-1) were isolated in 1986 to 1988 from separate outbreaks in laboratory mice in Poland and described as ectromelia virus isolates. The genome sequences of these poxviruses reveal that they are almost identical and represent a novel variant of the vaccinia virus Lister strain. PMID:24407630

  1. Chimeric antigen receptor–engineered T cells as oncolytic virus carriers

    PubMed Central

    VanSeggelen, Heather; Tantalo, Daniela GM; Afsahi, Arya; Hammill, Joanne A; Bramson, Jonathan L

    2015-01-01

    The use of engineered T cells in adoptive transfer therapies has shown significant promise in treating hematological cancers. However, successes treating solid tumors are much less prevalent. Oncolytic viruses (OVs) have the capacity to induce specific lysis of tumor cells and indirectly impact tumor growth via vascular shutdown. These viruses bear natural abilities to associate with lymphocytes upon systemic administration, but therapeutic doses must be very high in order to evade antibodies and other components of the immune system. As T cells readily circulate through the body, using these cells to deliver OVs directly to tumors may provide an ideal combination. Our studies demonstrate that loading chimeric antigen receptor–engineered T cells with low doses of virus does not impact receptor expression or function in either murine or human T cells. Engineered T cells can deposit virus onto a variety of tumor targets, which can enhance the tumoricidal activity of the combination treatment. This concept appears to be broadly applicable, as we observed similar results using murine or human T cells, loaded with either RNA or DNA viruses. Overall, loading of engineered T cells with OVs represents a novel combination therapy that may increase the efficacy of both treatments. PMID:27119109

  2. Vesicular stomatitis virus as a flexible platform for oncolytic virotherapy against cancer.

    PubMed

    Hastie, Eric; Grdzelishvili, Valery Z

    2012-12-01

    Oncolytic virus (OV) therapy is an emerging anti-cancer approach that utilizes viruses to preferentially infect and kill cancer cells, while not harming healthy cells. Vesicular stomatitis virus (VSV) is a prototypic non-segmented, negative-strand RNA virus with inherent OV qualities. Antiviral responses induced by type I interferon pathways are believed to be impaired in most cancer cells, making them more susceptible to VSV than normal cells. Several other factors make VSV a promising OV candidate for clinical use, including its well-studied biology, a small, easily manipulated genome, relative independence of a receptor or cell cycle, cytoplasmic replication without risk of host-cell transformation, and lack of pre-existing immunity in humans. Moreover, various VSV-based recombinant viruses have been engineered via reverse genetics to improve oncoselectivity, safety, oncotoxicity and stimulation of tumour-specific immunity. Alternative delivery methods are also being studied to minimize premature immune clearance of VSV. OV treatment as a monotherapy is being explored, although many studies have employed VSV in combination with radiotherapy, chemotherapy or other OVs. Preclinical studies with various cancers have demonstrated that VSV is a promising OV; as a result, a human clinical trial using VSV is currently in progress.

  3. Enhanced lysis by bispecific oncolytic measles viruses simultaneously using HER2/neu or EpCAM as target receptors

    PubMed Central

    Hanauer, Jan RH; Gottschlich, Lisa; Riehl, Dennis; Rusch, Tillmann; Koch, Vivian; Friedrich, Katrin; Hutzler, Stefan; Prüfer, Steffen; Friedel, Thorsten; Hanschmann, Kay-Martin; Münch, Robert C; Jost, Christian; Plückthun, Andreas; Cichutek, Klaus; Buchholz, Christian J; Mühlebach, Michael D

    2016-01-01

    To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass. PMID:27119117

  4. Naturally Existing Oncolytic Virus M1 Is Nonpathogenic for the Nonhuman Primates After Multiple Rounds of Repeated Intravenous Injections.

    PubMed

    Zhang, Haipeng; Lin, Yuan; Li, Kai; Liang, Jiankai; Xiao, Xiao; Cai, Jing; Tan, Yaqian; Xing, Fan; Mai, Jialuo; Li, Yuan; Chen, Wenli; Sheng, Longxiang; Gu, Jiayu; Zhu, Wenbo; Yin, Wei; Qiu, Pengxin; Su, Xingwen; Lu, Bingzheng; Tian, Xuyan; Liu, Jinhui; Lu, Wanjun; Dou, Yunling; Huang, Yijun; Hu, Bing; Kang, Zhuang; Gao, Guangping; Mao, Zixu; Cheng, Shi-Yuan; Lu, Ling; Bai, Xue-Tao; Gong, Shoufang; Yan, Guangmei; Hu, Jun

    2016-09-01

    Cancers figure among the leading causes of morbidity and mortality worldwide. The number of new cases is expected to rise by about 70% over the next 2 decades. Development of novel therapeutic agents is urgently needed for clinical cancer therapy. Alphavirus M1 is a Getah-like virus isolated from China with a genome of positive single-strand RNA. We have previously identified that alphavirus M1 is a naturally existing oncolytic virus with significant anticancer activity against different kinds of cancer (e.g., liver cancer, bladder cancer, and colon cancer). To support the incoming clinical trial of intravenous administration of alphavirus M1 to cancer patients, we assessed the safety of M1 in adult nonhuman primates. We previously presented the genome sequencing data of the cynomolgus macaques (Macaca fascicularis), which was demonstrated as an ideal animal species for virus infection study. Therefore, we chose cynomolgus macaques of either sex for the present safety study of oncolytic virus M1. In the first round of administration, five experimental macaques were intravenously injected with six times of oncolytic virus M1 (1 × 10(9) pfu/dose) in 1 week, compared with five vehicle-injected control animals. The last two rounds of injections were further completed in the following months in the same way as the first round. Body weight, temperature, complete blood count, clinical biochemistries, cytokine profiles, lymphocytes subsets, neutralizing antibody, and clinical symptoms were closely monitored at different time points. Magnetic resonance imaging was also performed to assess the possibility of encephalitis or arthritis. As a result, no clinical, biochemical, immunological, or medical imaging or other pathological evidence of toxicity was found during the whole process of the study. Our results in cynomolgus macaques suggested the safety of intravenous administration of oncolytic virus M1 in cancer patients in the future.

  5. Molecular cloning, encoding sequence, and expression of vaccinia virus nucleic acid-dependent nucleoside triphosphatase gene.

    PubMed Central

    Rodriguez, J F; Kahn, J S; Esteban, M

    1986-01-01

    A rabbit poxvirus genomic library contained within the expression vector lambda gt11 was screened with polyclonal antiserum prepared against vaccinia virus nucleic acid-dependent nucleoside triphosphatase (NTPase)-I enzyme. Five positive phage clones containing from 0.72- to 2.5-kilobase-pair (kbp) inserts expressed a beta-galactosidase fusion protein that was reactive by immunoblotting with the NTPase-I antibody. Hybridization analysis allowed the location of this gene within the vaccinia HindIIID restriction fragment. From the known nucleotide sequence of the 16-kbp vaccinia HindIIID fragment, we identified a region that contains a 1896-base open reading frame coding for a 631-amino acid protein. Analysis of the complete sequence revealed a highly basic protein, with hydrophilic COOH and NH2 termini, various hydrophobic domains, and no significant homology to other known proteins. Translational studies demonstrate that NTPase-I belongs to a late class of viral genes. This protein is highly conserved among Orthopoxviruses. Images PMID:3025846

  6. Pharmacological Modulation of Anti-Tumor Immunity Induced by Oncolytic Viruses

    PubMed Central

    Forbes, Nicole E.; Krishnan, Ramya; Diallo, Jean-Simon

    2014-01-01

    Oncolytic viruses (OVs) not only kill cancer cells by direct lysis but also generate a significant anti-tumor immune response that allows for prolonged cancer control and in some cases cures. How to best stimulate this effect is a subject of intense investigation in the OV field. While pharmacological manipulation of the cellular innate anti-viral immune response has been shown by several groups to improve viral oncolysis and spread, it is increasingly clear that pharmacological agents can also impact the anti-tumor immune response generated by OVs and related tumor vaccination strategies. This review covers recent progress in using pharmacological agents to improve the activity of OVs and their ability to generate robust anti-tumor immune responses. PMID:25101247

  7. Adeno-Associated Virus Enhances Wild-Type and Oncolytic Adenovirus Spread

    PubMed Central

    Laborda, Eduardo; Puig-Saus, Cristina; Cascalló, Manel; Chillón, Miguel

    2013-01-01

    Abstract The contamination of adenovirus (Ad) stocks with adeno-associated viruses (AAV) is usually unnoticed, and it has been associated with lower Ad yields upon large-scale production. During Ad propagation, AAV contamination needs to be detected routinely by polymerase chain reaction without symptomatic suspicion. In this study, we describe that the coinfection of either Ad wild type 5 or oncolytic Ad with AAV results in a large-plaque phenotype associated with an accelerated release of Ad from coinfected cells. This accelerated release was accompanied with the expected decrease in Ad yields in two out of three cell lines tested. Despite this lower Ad yield, coinfection with AAV accelerated cell death and enhanced the cytotoxicity mediated by Ad propagation. Intratumoral coinjection of Ad and AAV in two xenograft tumor models improved antitumor activity and mouse survival. Therefore, we conclude that accidental or intentional AAV coinfection has important implications for Ad-mediated virotherapy. PMID:24020980

  8. Efficient cleavage of p220 by poliovirus 2Apro expression in mammalian cells: effects on vaccinia virus.

    PubMed

    Aldabe, R; Feduchi, E; Novoa, I; Carrasco, L

    1995-10-24

    Poliovirus protease 2A cleaves p220, a component of initiation factor eIF-4F. Polyclonal antibodies that recognize p220 and the cleaved products from different species have been raised. Transfection of several cell lines with poliovirus 2Apro cloned in different plasmids leads to efficient cleavage of p220 upon infection with VT7, a recombinant vaccinia virus that expresses the T7 RNA polymerase. Under these conditions vaccinia virus protein synthesis is severely inhibited, while expression of poliovirus protein 2C from a similar plasmid has no effect. These results show by the first time the effects of p220 cleavage on vaccinia virus translation in the infected cells.

  9. Treatment strategies for combining immunostimulatory oncolytic virus therapeutics with dendritic cell injections.

    PubMed

    Wares, Joanna R; Crivelli, Joseph J; Yun, Chae-Ok; Choi, Il-Kyu; Gevertz, Jana L; Kim, Peter S

    2015-12-01

    Oncolytic viruses (OVs) are used to treat cancer, as they selectively replicate inside of and lyse tumor cells. The efficacy of this process is limited and new OVs are being designed to mediate tumor cell release of cytokines and co-stimulatory molecules, which attract cytotoxic T cells to target tumor cells, thus increasing the tumor-killing effects of OVs. To further promote treatment efficacy, OVs can be combined with other treatments, such as was done by Huang et al., who showed that combining OV injections with dendritic cell (DC) injections was a more effective treatment than either treatment alone. To further investigate this combination, we built a mathematical model consisting of a system of ordinary differential equations and fit the model to the hierarchical data provided from Huang et al. We used the model to determine the effect of varying doses of OV and DC injections and to test alternative treatment strategies. We found that the DC dose given in Huang et al. was near a bifurcation point and that a slightly larger dose could cause complete eradication of the tumor. Further, the model results suggest that it is more effective to treat a tumor with immunostimulatory oncolytic viruses first and then follow-up with a sequence of DCs than to alternate OV and DC injections. This protocol, which was not considered in the experiments of Huang et al., allows the infection to initially thrive before the immune response is enhanced. Taken together, our work shows how the ordering, temporal spacing, and dosage of OV and DC can be chosen to maximize efficacy and to potentially eliminate tumors altogether.

  10. Vaccinia Virus LC16m8∆ as a Vaccine Vector for Clinical Applications

    PubMed Central

    Kidokoro, Minoru; Shida, Hisatoshi

    2014-01-01

    The LC16m8 strain of vaccinia virus, the active ingredient in the Japanese smallpox vaccine, was derived from the Lister/Elstree strain. LC16m8 is replication-competent and has been administered to over 100,000 infants and 3,000 adults with no serious adverse reactions. Despite this outstanding safety profile, the occurrence of spontaneously-generated large plaque-forming virulent LC16m8 revertants following passage in cell culture is a major drawback. We identified the gene responsible for the reversion and deleted the gene (B5R) from LC16m8 to derive LC16m8Δ. LC16m8∆ is non-pathogenic in immunodeficient severe combined immunodeficiency (SCID) mice, genetically-stable and does not reverse to a large-plaque phenotype upon passage in cell culture, even under conditions in which most LC16m8 populations are replaced by revertants. Moreover, LC16m8∆ is >500-fold more effective than the non-replicating vaccinia virus (VV), Modified Vaccinia Ankara (MVA), at inducing murine immune responses against pathogenic VV. LC16m8∆, which expresses the SIV gag gene, also induced anti-Gag CD8+ T-cells more efficiently than MVA and another non-replicating VV, Dairen I minute-pock variants (DIs). Moreover, LC16m8∆ expressing HIV-1 Env in combination with a Sendai virus vector induced the production of anti-Env antibodies and CD8+ T-cells. Thus, the safety and efficacy of LC16m8∆ mean that it represents an outstanding platform for the development of human vaccine vectors. PMID:26344890

  11. Nucleotide sequence of a cluster of early and late genes in a conserved segment of the vaccinia virus genome.

    PubMed Central

    Plucienniczak, A; Schroeder, E; Zettlmeissl, G; Streeck, R E

    1985-01-01

    The nucleotide sequence of a 7.6 kb vaccinia DNA segment from a genomic region conserved among different orthopox virus has been determined. This segment contains a tight cluster of 12 partly overlapping open reading frames most of which can be correlated with previously identified early and late proteins and mRNAs. Regulatory signals used by vaccinia virus have been studied. Presumptive promoter regions are rich in A, T and carry the consensus sequences TATA and AATAA spaced at 20-24 base pairs. Tandem repeats of a CTATTC consensus sequence are proposed to be involved in the termination of early transcription. PMID:2987815

  12. Attenuated vaccinia virus-circumsporozoite protein recombinants confer protection against rodent malaria.

    PubMed Central

    Lanar, D E; Tine, J A; de Taisne, C; Seguin, M C; Cox, W I; Winslow, J P; Ware, L A; Kauffman, E B; Gordon, D; Ballou, W R; Paoletti, E; Sadoff, J C

    1996-01-01

    NYVAC-based vaccinia virus recombinants expressing the circumsporozoite protein (CSP) were evaluated in the Plasmodium berghei rodent malaria model system. Immunization of mice with a NYVAC-based CSP recombinant elicited a high level of protection (60 to 100%). Protection did not correlate with CS repeat-specific antibody responses and was abrogated by in vivo CD8+ T-cell depletion. Protection was not enhanced by modification of the subcellular localization of CSP. These results suggest the potential of poxvirus-based vectors for the development of vaccine candidates for human malaria. PMID:8613376

  13. Treatment of Vaccinia and Cowpox Virus Infections in Mice with CMX001 and ST-246.

    PubMed

    Quenelle, Debra C; Kern, Earl R

    2010-12-01

    Although a large number of compounds have been identified with antiviral activity against orthopoxviruses in tissue culture systems, it is highly preferred that these compounds have activity in vivo before they can be seriously considered for further development. One of the most commonly used animal models for the confirmation of this activity has been the use of mice infected with either vaccinia or cowpox viruses. These model systems have the advantage that they are relatively inexpensive, readily available and do not require any special containment facilities; therefore, relatively large numbers of compounds can be evaluated in vivo for their activity. The two antiviral agents that have progressed from preclinical studies to human safety trials for the treatment of orthopoxvirus infections are the cidofovir analog, CMX001, and an inhibitor of extracellular virus formation, ST-246. These compounds are the ones most likely to be used in the event of a bioterror attack. The purpose of this communication is to review the advantages and disadvantages of using mice infected with vaccinia and cowpox virus as surrogate models for human orthopoxvirus infections and to summarize the activity of CMX001 and ST-246 in these model infections.

  14. Treatment of Vaccinia and Cowpox Virus Infections in Mice with CMX001 and ST-246

    PubMed Central

    Quenelle, Debra C.; Kern, Earl R.

    2010-01-01

    Although a large number of compounds have been identified with antiviral activity against orthopoxviruses in tissue culture systems, it is highly preferred that these compounds have activity in vivo before they can be seriously considered for further development. One of the most commonly used animal models for the confirmation of this activity has been the use of mice infected with either vaccinia or cowpox viruses. These model systems have the advantage that they are relatively inexpensive, readily available and do not require any special containment facilities; therefore, relatively large numbers of compounds can be evaluated in vivo for their activity. The two antiviral agents that have progressed from preclinical studies to human safety trials for the treatment of orthopoxvirus infections are the cidofovir analog, CMX001, and an inhibitor of extracellular virus formation, ST-246. These compounds are the ones most likely to be used in the event of a bioterror attack. The purpose of this communication is to review the advantages and disadvantages of using mice infected with vaccinia and cowpox virus as surrogate models for human orthopoxvirus infections and to summarize the activity of CMX001 and ST-246 in these model infections. PMID:21994637

  15. Vaccinia virus detection in dairy products made with milk from experimentally infected cows.

    PubMed

    de Oliveira, T M L; Guedes, M I M C; Rehfeld, I S; Matos, A C D; Rivetti Júnior, A V; da Cunha, A F; Cerqueira, M M O P; Abrahão, J S; Lobato, Z I P

    2017-06-26

    Vaccinia virus (VACV) is the agent of bovine vaccinia (BV), an emerging zoonosis that causes exanthematic lesions on the teats of dairy cows and on the hands of milkers. The virus has been detected in the milk of naturally infected cows. The objective of this study was to investigate and quantify VACV DNA as well as the presence of infectious virus particles in samples of cheese curd, cheese whey and pasteurized milk produced using milk from cows experimentally inoculated with VACV-GP2, a Brazilian isolate of VACV (VACV-BR). VACV DNA was detected in samples of cheese and pasteurized milk at different time points, even after the resolution of the typical lesions caused by VACV, which occurred after 22 days post-infection (dpi), on average. Moreover, it was possible to detect infectious viral particles in cheese samples on alternate days until 27 dpi. The presence of both VACV DNA and infectious viral particles in cheese samples throughout the clinical course of BV and even after the disappearance of the typical clinical signs of disease draws attention to the risk associated with consumption of the cheese. Furthermore, VACV-contaminated milk and cheese may represent an occupational risk to cheesemakers who often manipulate milk and cheese curd without wearing gloves. © 2017 Blackwell Verlag GmbH.

  16. A novel high-throughput vaccinia virus neutralization assay and preexisting immunity in populations from different geographic regions in China.

    PubMed

    Liu, Qiang; Huang, Weijin; Nie, Jianhui; Zhu, Rong; Gao, Dongying; Song, Aijing; Meng, Shufang; Xu, Xuemei; Wang, Youchun

    2012-01-01

    Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China. A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30-55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin. A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui provinces of China.

  17. A Novel High-Throughput Vaccinia Virus Neutralization Assay and Preexisting Immunity in Populations from Different Geographic Regions in China

    PubMed Central

    Liu, Qiang; Huang, Weijin; Nie, Jianhui; Zhu, Rong; Gao, Dongying; Song, Aijing; Meng, Shufang; Xu, Xuemei; Wang, Youchun

    2012-01-01

    Background Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China. Methodology/Principal Findings A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30–55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin. Conclusion A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui

  18. Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses.

    PubMed Central

    Ralston, R; Thudium, K; Berger, K; Kuo, C; Gervase, B; Hall, J; Selby, M; Kuo, G; Houghton, M; Choo, Q L

    1993-01-01

    We constructed recombinant vaccinia virus vectors for expression of the structural region of hepatitis C virus (HCV). Infection of mammalian cells with a vector (vv/HCV1-906) encoding C-E1-E2-NS2 generated major protein species of 22 kDa (C), 33 to 35 kDa (E1), and 70 to 72 kDa (E2), as observed previously with other mammalian expression systems. The bulk of the E1 and E2 expressed by vv/HCV1-906 was found integrated into endoplasmic reticulum membranes as core-glycosylated species, suggesting that these E1 and E2 species represent intracellular forms of the HCV envelope proteins. HCV E1 and E2 formed E1-E2 complexes which were precipitated by either anti-E1 or anti-E2 serum and which sedimented at approximately 15 S on glycerol density gradients. No evidence of intermolecular disulfide bonding between E1 and E2 was detected. E1 and E2 were copurified to approximately 90% purity by mild detergent extraction followed by chromatography on Galanthus nivalus lectin-agarose and DEAE-Fractogel. Immunization of chimpanzees with purified E1-E2 generated high titers of anti-E1 and anti-E2 antibodies. Further studies, to be reported separately, demonstrated that purified E1-E2 complexes were recognized at high frequency by HCV+ human sera (D. Y. Chien, Q.-L. Choo, R. Ralston, R. Spaete, M. Tong, M. Houghton, and G. Kuo, Lancet, in press) and generated protective immunity in chimpanzees (Q.-L. Choo, G. Kuo, R. Ralston, A. Weiner, D. Chien, G. Van Nest, J. Han, K. Berger, K. Thudium, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Mass, L. B. Cummins, E. Muchmore, and M. Houghton, submitted for publication), suggesting that these purified HCV envelope proteins display native HCV epitopes. Images PMID:8411378

  19. Resistance to Human Respiratory Syncytial Virus (RSV) Infection Induced by Immunization of Cotton Rats with a Recombinant Vaccinia Virus Expressing the RSV G Glycoprotein

    NASA Astrophysics Data System (ADS)

    Elango, Narayanasamy; Prince, Gregory A.; Murphy, Brian R.; Venkatesan, Sundararajan; Chanock, Robert M.; Moss, Bernard

    1986-03-01

    A cDNA copy of the G glycoprotein gene of human respiratory syncytial virus (RSV) was placed under control of a vaccinia virus promoter and inserted into the thymidine kinase locus of the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and expressed a 93-kDa protein that migrated with the authentic RSV G glycoprotein upon polyacrylamide gel electrophoresis. Glycosylation of the expressed protein and transport to the cell surface were demonstrated in the absence of other RSV proteins. Cotton rats that were inoculated intradermally with the infectious recombinant virus produced serum antibody to the G glycoprotein that neutralized RSV in vitro. Furthermore, the vaccinated animals were resistant to lower respiratory tract infection upon intranasal inoculation with RSV and had reduced titers of RSV in the nose.

  20. RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

    PubMed Central

    Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K.; James, John; Prescott, Alan; Haga, Ismar R.; Beard, Philippa M.

    2015-01-01

    Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. PMID:25462347

  1. Inhibition of Vaccinia virus entry by a broad spectrum antiviral peptide

    SciTech Connect

    Altmann, S.E.; Jones, J.C.; Schultz-Cherry, S.; Brandt, C.R.

    2009-06-05

    Concerns about the possible use of Variola virus, the causative agent of smallpox, as a weapon for bioterrorism have led to renewed efforts to identify new antivirals against orthopoxviruses. We identified a peptide, EB, which inhibited infection by Vaccinia virus with an EC{sub 50} of 15 muM. A control peptide, EBX, identical in composition to EB but differing in sequence, was inactive (EC{sub 50} > 200 muM), indicating sequence specificity. The inhibition was reversed upon removal of the peptide, and EB treatment had no effect on the physical integrity of virus particles as determined by electron microscopy. Viral adsorption was unaffected by the presence of EB, and the addition of EB post-entry had no effect on viral titers or on early gene expression. The addition of EB post-adsorption resulted in the inhibition of beta-galactosidase expression from an early viral promoter with an EC{sub 50} of 45 muM. A significant reduction in virus entry was detected in the presence of the peptide when the number of viral cores released into the cytoplasm was quantified. Electron microscopy indicated that 88% of the virions remained on the surface of cells in the presence of EB, compared to 37% in the control (p < 0.001). EB also blocked fusion-from-within, suggesting that virus infection is inhibited at the fusion step. Analysis of EB derivatives suggested that peptide length may be important for the activity of EB. The EB peptide is, to our knowledge, the first known small molecule inhibitor of Vaccinia virus entry.

  2. Prostate-Specific and Tumor-Specific Targeting of an Oncolytic HSV-1 Amplicon/Helper Virus for Prostate Cancer Treatment

    DTIC Science & Technology

    2009-11-01

    Targeting of an Oncolytic HSV - 1 Amplicon/Helper Virus for Prostate Cancer Treatment PRINCIPAL INVESTIGATOR: Cleo Lee CONTRACTING...5a. CONTRACT NUMBER Prostate-Specific and Tumor-Specific Targeting of an Oncolytic HSV - 1 Amplicon/Helper Virus for Prostate Cancer Treatment...untranslated region (3’UTR) of a herpes simplex virus- 1 ( HSV - 1 ) essential viral gene, ICP4, to create CMV-ICP4-143T and CMV-ICP4-145T amplicon viruses. Our

  3. Intravenously injected Newcastle disease virus in non-human primates is safe to use for oncolytic virotherapy.

    PubMed

    Buijs, P R A; van Amerongen, G; van Nieuwkoop, S; Bestebroer, T M; van Run, P R W A; Kuiken, T; Fouchier, R A M; van Eijck, C H J; van den Hoogen, B G

    2014-11-01

    Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic potential. Detailed preclinical information regarding the safety of oncolytic NDV is scarce. In this study, we evaluated the toxicity, biodistribution and shedding of intravenously injected oncolytic NDVs in non-human primates (Macaca fascicularis). Two animals were injected with escalating doses of a non-recombinant vaccine strain, a recombinant lentogenic strain or a recombinant mesogenic strain. To study transmission, naive animals were co-housed with the injected animals. Injection with NDV did not lead to severe illness in the animals or abnormalities in hematologic or biochemistry measurements. Injected animals shed low amounts of virus, but this did not lead to seroconversion of the contact animals. Postmortem evaluation demonstrated no pathological changes or evidence of virus replication. This study demonstrates that NDV generated in embryonated chicken eggs is safe for intravenous administration to non-human primates. In addition, our study confirmed results from a previous report that naïve primate and human sera are able to neutralize egg-generated NDV. We discuss the implications of these results for our study and the use of NDV for virotherapy.

  4. Oral Vaccination With Vaccinia Virus Expressing the Tick Antigen Subolesin Inhibits Tick Feeding and Transmission of Borrelia burgdorferi Vaccination

    PubMed Central

    Bensaci, Mekki; Bhattacharya, Debaditya; Clark, Roger; Hu, Linden T.

    2014-01-01

    Immunization with the Ixodes scapularis protein, subolesin, has previously been shown to protect hosts against tick infestation and to decrease acquisition of Anaplsma marginale and Babesia bigemina. Here we report the efficacy of subolesin expressed from Vaccinia virus for use as an orally delivered reservoir–targeted vaccine for prevention of tick infestation and acquisition/transmission of Borrelia burgdorferi to its tick and mouse hosts. We cloned subolesin into Vaccinia virus and showed that it is expressed from mammalian cells infected with the recombinant virus in vitro. We then vaccinated mice by oral gavage. A single dose of the vaccine was sufficient for mice to generate antibody response to subolesin. Vaccination with the subolesin expressing Vaccinia virus inhibited tick infestation by 52% compared to control vaccination with Vaccinia virus and reduced uptake of B. burgdorferi among the surviving ticks that fed to repletion by 34%. There was a reduction in transmission of B. burgdorferi to uninfected vaccinated mice of 40% compared to controls. These results suggest that subolesin has potential as a component of a reservoir targeted vaccine to decrease B. burgdorferi, Babesia and Anaplasma species infections in their natural hosts. PMID:22864146

  5. Prevention of EBV lymphoma development by oncolytic myxoma virus in a murine xenograft model of post-transplant lymphoproliferative disease

    SciTech Connect

    Kim, Manbok; Rahman, Masmudur M.; Cogle, Christopher R.

    2015-07-10

    Epstein–Barr virus (EBV) has been associated with a variety of epithelial and hematologic malignancies, including B-, T- and NK cell-lymphomas, Hodgkin's disease (HD), post-transplant lymphoproliferative diseases (LPDs), nasopharyngeal and gastric carcinomas, smooth muscle tumors, and HIV-associated lymphomas. Currently, treatment options for EBV-associated malignancies are limited. We have previously shown that myxoma virus specifically targets various human solid tumors and leukemia cells in a variety of animal models, while sparing normal human or murine tissues. Since transplant recipients of bone marrow or solid organs often develop EBV-associated post-transplant LPDs and lymphoma, myxoma virus may be of utility to prevent EBV-associated malignancies in immunocompromised transplant patients where treatment options are frequently limited. In this report, we demonstrate the safety and efficacy of myxoma virus purging as a prophylactic strategy for preventing post-transplant EBV-transformed human lymphomas, using a highly immunosuppressed mouse xenotransplantation model. This provides support for developing myxoma virus as a potential oncolytic therapy for preventing EBV-associated LPDs following transplantation of bone marrow or solid organ allografts. - Highlights: • Myxoma virus effectively infects and purges EBV lymphoma cells in vivo. • Oncolytic myxoma virus effectively eradicates oncogenic EBV tumorigenesis. • Ex vivo pre-treatment of myxoma virus can be effective as a preventive treatment modality for post-transplant lymphoproliferative diseases.

  6. Review of Vaccinia Virus and Baculovirus Viability Versus Virucides

    DTIC Science & Technology

    2008-03-01

    164, pp 45-63. Shapiro, M. Use of Optical Brighteners as Radiation Protectants for Gypsy Moth (Lepidoptera: Lymantriidae) Nuclear Polyhedrosis Virus. J...Econ. Entomol. 1992, 85, pp 1682-1686. Shapiro, M. The Effects of Cations on the Activity of the Gypsy Moth (Lepidoptera: Lymantriidae) Nuclear...described from coddling moth larvae (Cydia pomonella L.) collected in Mexican apple and pear orchards (Tanada, 1964). The viral occlusions are formed in the

  7. Structure and Assembly of Intracellular Mature Vaccinia Virus: Thin-Section Analyses

    PubMed Central

    Griffiths, Gareth; Roos, Norbert; Schleich, Sybille; Locker, Jacomine Krijnse

    2001-01-01

    In the preceding study (see accompanying paper), we showed by a variety of different techniques that intracellular mature vaccinia virus (vaccinia IMV) is unexpectedly complex in its structural organization and that this complexity also extends to the underlying viral core, which is highly folded. With that analysis as a foundation, we now present different thin-section electron microscopy approaches for analyzing the IMV and the processes by which it is assembled in infected HeLa cells. We focus on conventional epoxy resin thin sections as well as cryosections to describe key intermediates in the assembly process. We took advantage of streptolysin O's ability to selectively permeabilize the plasma membrane of infected cells to improve membrane contrast, and we used antibodies against bone fide integral membrane proteins of the virus to unequivocally identify membrane profiles in thin sections. All of the images presented here can be rationalized with respect to the model put forward for the assembly of the IMV in the accompanying paper. PMID:11602745

  8. Microbiota is an essential element for mice to initiate a protective immunity against Vaccinia virus.

    PubMed

    Lima, Maurício T; Andrade, Ana C S P; Oliveira, Graziele P; Calixto, Rafael S; Oliveira, Danilo B; Souza, Éricka L S; Trindade, Giliane S; Nicoli, Jacques R; Kroon, Erna G; Martins, Flaviano S; Abrahão, Jônatas S

    2016-02-01

    The gastrointestinal tract of vertebrates harbors one of the most complex ecosystems known in microbial ecology and this indigenous microbiota almost always has a profound influence on host-parasite relationships, which can enhance or reduce the pathology of the infection. In this context, the impact of the microbiota during the infection of several viral groups remains poorly studied, including the family Poxviridae. Vaccinia virus (VACV) is a member of this family and is the causative agent of bovine vaccinia, responsible for outbreaks that affect bovines and humans. To determine the influence of the microbiota in the development of the disease caused by VACV, a comparative study using a murine model was performed. Germ-free and conventional, 6- to 7-week-old Swiss NIH mice were infected by tail scarification and intranasally with VACV. Moreover, immunosuppression and microbiota reposition were performed, to establish the interactions among the host's immune system, microbiota and VACV. The data demonstrate that the microbiota is essential for the effective immune response of mice against VACV in intranasal inoculation and to control the virus at the primary site of infection. Furthermore, this study is the first to show that Swiss conventional mice are refractory to the intranasal infection of VACV. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Development of a vaccinia virus based reservoir-targeted vaccine against Yersinia pestis

    PubMed Central

    Bhattacharya, Debaditya; Mecsas, Joan; Hu, Linden T.

    2010-01-01

    Yersinia pestis, the causative organism of plague, is a zoonotic organism with a worldwide distribution. Although the last plague epidemic occurred in early 1900s, human cases continue to occur due to contact with infected wild animals. In this study, we have developed a reservoir-targeted vaccine against Y. pestis, to interrupt transmission of disease in wild animals as a potential strategy for decreasing human disease. A vaccinia virus delivery system was used to express the F1 capsular protein and the LcrV type III secretion component of Y. pestis as a fusion protein. Here we show that a single dose of this vaccine administered orally, generates a dose-dependent antibody response in mice. Antibody titers peak by 3 weeks after administration and remain elevated for a minimum of 45 weeks. Vaccination provided up to 100% protection against challenge with Y. pestis administered by intranasal challenge at 10 times the lethal dose with protection lasting a minimum of 45 weeks. An orally available, vaccinia virus expressed vaccine against Y. pestis may be a suitable vaccine for a reservoir targeted strategy for the prevention of enzootic plague. PMID:20875494

  10. Evaluation of Imiquimod for Topical Treatment of Vaccinia Virus Cutaneous Infections in Immunosuppressed Hairless Mice

    PubMed Central

    Tarbet, E. Bart; Larson, Deanna; Anderson, Bentley J.; Bailey, Kevin W.; Wong, Min-Hui; Smee, Donald F.

    2011-01-01

    Imiquimod is an immune response modifier prescribed as a topical medication for a number of viral and neoplastic conditions. We evaluated the antiviral activity of imiquimod against vaccinia virus (WR strain) cutaneous infections in immunosuppressed (with cyclophosphamide) hairless mice when administered after virus exposure. Primary lesions progressed in severity, satellite lesions developed, and infection eventually killed the mice. Once daily topical treatment with 1% imiquimod cream for three, four, or five days were compared to twice daily topical treatment with 1% cidofovir cream for seven days. Survival time of mice in all treated groups was significantly prolonged compared to placebo controls. The mean day of death for the placebo group, three-day imiquimod, four day imiquimod, five-day imiquimod, and cidofovir groups were 15.5, 20.0, 20.5, 19.5, and 20.5 days post-infection, respectively. All treatment groups showed significant reductions in primary lesion size and in the number of satellite lesions. The cidofovir and 4-day imiquimod treatments delayed the appearance of lung virus titers by 3 and 6 days, respectively, although cutaneous lesion and snout virus titers were not as affected by treatment. Benefits in survival and lesion reduction were observed when imiquimod treatment was delayed from 24, 48 and 72 hours post-infection. However, increasing the treatment dose of imiquimod from 1% to 5% led to a significant decrease in antiviral efficacy. These results demonstrate the protective effects of topically administered imiquimod against a disseminated vaccinia virus infection in this mouse model. PMID:21439326

  11. Construction and Characterization of an Infectious Vaccinia Virus Recombinant That Expresses the Influenza Hemagglutinin Gene and Induces Resistance to Influenza Virus Infection in Hamsters

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Murphy, Brian R.; Moss, Bernard

    1983-12-01

    A DNA copy of the influenza virus hemagglutinin gene, derived from influenza virus A/Jap/305/57 (H2N2) was inserted into the genome of vaccinia virus under the control of an early vaccinia virus promoter. Tissue culture cells infected with the purified recombinant virus synthesized influenza hemagglutinin, which was glycosylated and transported to the cell surface where it could be cleaved with trypsin into HA1 and HA2 subunits. Rabbits and hamsters inoculated intradermally with recombinant virus produced circulating antibodies that inhibited hemagglutination by influenza virus. Furthermore, vaccinated hamsters achieved levels of antibody similar to those obtained upon primary infection with influenza virus and were protected against respiratory infection with the A/Jap/305/57 influenza virus.

  12. Oncolytic reovirus induces intracellular redistribution of Ras to promote apoptosis and progeny virus release.

    PubMed

    Garant, K A; Shmulevitz, M; Pan, L; Daigle, R M; Ahn, D-G; Gujar, S A; Lee, P W K

    2016-02-11

    Reovirus is a naturally oncolytic virus that preferentially replicates in Ras-transformed cells and is currently undergoing clinical trials as a cancer therapeutic. Ras transformation promotes reovirus oncolysis by enhancing virion disassembly during entry, viral progeny production, and virus release through apoptosis; however, the mechanism behind the latter is not well understood. Here, we show that reovirus alters the intracellular location of oncogenic Ras to induce apoptosis of H-RasV12-transformed fibroblasts. Reovirus infection decreases Ras palmitoylation levels and causes accumulation of Ras in the Golgi through Golgi fragmentation. With the Golgi being the site of Ras palmitoylation, treatment of target cells with the palmitoylation inhibitor, 2-bromopalmitate (2BP), prompts a greater accumulation of H-RasV12 in the Golgi, and a dose-dependent increase in progeny virus release and subsequent spread. Conversely, tethering H-RasV12 to the plasma membrane (thereby preventing its movement to the Golgi) allows for efficient virus production, but results in basal levels of reovirus-induced cell death. Analysis of Ras downstream signaling reveals that cells expressing cycling H-RasV12 have elevated levels of phosphorylated JNK (c-Jun N-terminal kinase), and that Ras retained at the Golgi body by 2BP increases activation of the MEKK1/MKK4/JNK signaling pathway to promote cell death. Collectively, our data suggest that reovirus induces Golgi fragmentation of target cells, and the subsequent accumulation of oncogenic Ras in the Golgi body initiates apoptotic signaling events required for virus release and spread.

  13. Vaccinia virus and Cowpox virus are not susceptible to the interferon-induced antiviral protein MxA.

    PubMed

    Lorenzo, María M; Sanchez-Puig, Juana M; Blasco, Rafael

    2017-01-01

    MxA protein is expressed in response to type I and type III Interferon and constitute an important antiviral factor with broad antiviral activity to diverse RNA viruses. In addition, some studies expand the range of MxA antiviral activity to include particular DNA viruses like Monkeypox virus (MPXV) and African Swine Fever virus (ASFV). However, a broad profile of activity of MxA to large DNA viruses has not been established to date. Here, we investigated if some well characterized DNA viruses belonging to the Poxviridae family are sensitive to human MxA. A cell line inducibly expressing MxA to inhibitory levels showed no anti-Vaccinia virus (VACV) virus activity, indicating either lack of susceptibility of the virus, or the existence of viral factors capable of counteracting MxA inhibition. To determine if VACV resistance to MxA was due to a virus-encoded anti-MxA activity, we performed coinfections of VACV and the MxA-sensitive Vesicular Stomatitis virus (VSV), and show that VACV does not protect VSV from MxA inhibition in trans. Those results were extended to several VACV strains and two CPXV strains, thus confirming that those Orthopoxviruses do not block MxA action. Overall, these results point to a lack of susceptibility of the Poxviridae to MxA antiviral activity.

  14. A recombinant vaccinia virus containing the papilloma E2 protein promotes tumor regression by stimulating macrophage antibody-dependent cytotoxicity.

    PubMed

    Rosales, C; Graham, V V; Rosas, G A; Merchant, H; Rosales, R

    2000-09-01

    Human papillomavirus infection is associated with cervical cancer. The E6 and E7 papillomavirus proteins are normally required for the maintenance of the malignant phenotype. Expression of these proteins in infected cells is negatively regulated by the binding of the papilloma E2 protein to the long terminal control region of the papilloma virus genome. The E2 protein can also promote cell arrest and apoptosis in HeLa cells. Therefore, it is clear that this protein has the potential of inhibiting the malignant phenotype. Because, anticancer vaccines based in vaccinia viruses have recently been shown to be an effective way to treat and to eradicate tumors, a recombinant vaccinia virus expressing the E2 gene of bovine papilloma virus (Modified Vaccinia Ankara, MVA E2) was created, to explore further the antitumor potential of the E2 protein. A series of rabbits, containing the VX2 transplantable papilloma carcinoma, were treated with MVA E2. An impressive tumor regression, up to a complete disappearance of tumor, was observed in most animals (80%). In contrast, very little or no regression was detected if the normal vaccinia virus was used. Lymphocytes isolated from MVA E2-treated rabbits did not show cytotoxic activity against tumor cells. However, in these animals a humoral immune response against tumor cells was observed. These antitumor antibodies were capable of activating macrophages to destroy tumor cells efficiently. These data indicate that injecting the MVA E2 recombinant vaccinia virus directly into the tumor results in a robust and long-lasting tumor regression. Data also suggest that antitumor antibodies are responsible, at least in part, for eliminating tumors by activating macrophage antibody-dependent cytotoxicity.

  15. Comparison of the replication characteristics of vaccinia virus strains Guang 9 and Tian Tan in vivo and in vitro.

    PubMed

    Zhu, Rong; Liu, Qiang; Huang, Weijin; Yu, Yongxin; Wang, Youchun

    2014-10-01

    Vaccinia virus is widely used as a vector in the development of recombinant vaccines. Vaccinia virus strain Guang 9 (VG9), which was derived from vaccinia virus strain Tian Tan (VTT) by successive plaque-cloning purification, was more attenuated than VTT. In this study, the host cell range and the growth and replication of VG9 were compared with those of VTT. The results showed that both VG9 and VTT could infect permissive cells (Vero, TK-143 and CEF) and semipermissive cells PK (15) and induced a visible cytopathic effect (CPE). Both strains could infect nonpermissive CHO-K1 cells but neither was able to reproduce. The replicative ability of VG9 was a little lower than that of VTT. Additionally, recombinant vaccinia viruses containing a firefly luciferase gene (VG9-L and VTT-L) were constructed, and their expression in vitro and replication and spread in vivo were compared. The expression ability of VG9-L was lower than that of VTT-L. Whole-animal imaging data indicated that VG9-L could reproduce quickly and express the exogenous protein at the site of inoculation, regardless of whether the intramuscular, intracutaneous, subcutaneous or celiac inoculation route was used. VG9-L was better in its ability to express a foreign protein than VTT-L, but the time during which expression occurred was shorter. There was no dissemination of virus in mice inoculated with either strain. In summary, this study demonstrates the possibility of using VG9 for the production of smallpox vaccines or the construction of recombinant vaccinia virus vaccines.

  16. Dogs and Opossums Positive for Vaccinia Virus during Outbreak Affecting Cattle and Humans, São Paulo State, Brazil.

    PubMed

    Peres, Marina G; Barros, Claudenice B; Appolinário, Camila M; Antunes, João M A P; Mioni, Mateus S R; Bacchiega, Thais S; Allendorf, Susan D; Vicente, Acácia F; Fonseca, Clóvis R; Megid, Jane

    2016-02-01

    During a vaccinia virus (VACV) outbreak in São Paulo State, Brazil, blood samples were collected from cows, humans, other domestic animals, and wild mammals. Samples from 3 dogs and 3 opossums were positive for VACV by PCR. Results of gene sequencing yielded major questions regarding other mammalian species acting as reservoirs of VACV.

  17. Dogs and Opossums Positive for Vaccinia Virus during Outbreak Affecting Cattle and Humans, São Paulo State, Brazil

    PubMed Central

    Peres, Marina G.; Barros, Claudenice B.; Appolinário, Camila M.; Antunes, João M.A.P.; Mioni, Mateus S.R.; Bacchiega, Thais S.; Allendorf, Susan D.; Vicente, Acácia F.; Fonseca, Clóvis R.

    2016-01-01

    During a vaccinia virus (VACV) outbreak in São Paulo State, Brazil, blood samples were collected from cows, humans, other domestic animals, and wild mammals. Samples from 3 dogs and 3 opossums were positive for VACV by PCR. Results of gene sequencing yielded major questions regarding other mammalian species acting as reservoirs of VACV. PMID:26812352

  18. Deletion of the Vaccinia Virus N2L Gene Encoding an Inhibitor of IRF3 Improves the Immunogenicity of Modified Vaccinia Virus Ankara Expressing HIV-1 Antigens

    PubMed Central

    García-Arriaza, Juan; Gómez, Carmen E.; Sorzano, Carlos Óscar S.

    2014-01-01

    ABSTRACT A modified vaccinia virus Ankara poxvirus vector expressing the HIV-1 Env, Gag, Pol, and Nef antigens from clade B (MVA-B) is currently being tested in clinical trials. To improve its immunogenicity, we have generated and characterized the immune profile of MVA-B containing a deletion of the vaccinia viral gene N2L, which codes for an inhibitor of IRF3 (MVA-B ΔN2L). Deletion of N2L had no effect on virus growth kinetics or on the expression of HIV-1 antigens; hence, the N2 protein is not essential for MVA replication. The innate immune responses triggered by MVA-B ΔN2L revealed an increase in beta interferon, proinflammatory cytokines, and chemokines. Mouse prime-boost protocols showed that MVA-B ΔN2L improves the magnitude and polyfunctionality of HIV-1-specific CD4+ and CD8+ T cell adaptive and memory immune responses, with most of the HIV-1 responses mediated by CD8+ T cells. In the memory phase, HIV-1-specific CD8+ T cells with an effector phenotype were predominant and in a higher percentage with MVA-B ΔN2L than with MVA-B. In both immunization groups, CD4+ and CD8+ T cell responses were directed mainly against Env. Furthermore, MVA-B ΔN2L in the memory phase enhanced levels of antibody against Env. For the vector immune responses, MVA-B ΔN2L induced a greater magnitude and polyfunctionality of VACV-specific CD8+ T memory cells than MVA-B, with an effector phenotype. These results revealed the immunomodulatory role of N2L, whose deletion enhanced the innate immunity and improved the magnitude and quality of HIV-1-specific T cell adaptive and memory immune responses. These findings are relevant for the optimization of poxvirus vectors as vaccines. IMPORTANCE On the basis of the limited efficacy of the RV144 phase III clinical trial, new optimized poxvirus vectors as vaccines against HIV/AIDS are needed. Here we have generated and characterized a new HIV/AIDS vaccine candidate on the basis of the poxvirus MVA vector expressing HIV-1 Env, Gag, Pol

  19. Dendritic Cells in Oncolytic Virus-Based Anti-Cancer Therapy

    PubMed Central

    Kim, Youra; Clements, Derek R.; Sterea, Andra M.; Jang, Hyun Woo; Gujar, Shashi A.; Lee, Patrick W. K.

    2015-01-01

    Dendritic cells (DCs) are specialized antigen-presenting cells that have a notable role in the initiation and regulation of innate and adaptive immune responses. In the context of cancer, appropriately activated DCs can induce anti-tumor immunity by activating innate immune cells and tumor-specific lymphocytes that target cancer cells. However, the tumor microenvironment (TME) imposes different mechanisms that facilitate the impairment of DC functions, such as inefficient antigen presentation or polarization into immunosuppressive DCs. These tumor-associated DCs thus fail to initiate tumor-specific immunity, and indirectly support tumor progression. Hence, there is increasing interest in identifying interventions that can overturn DC impairment within the TME. Many reports thus far have studied oncolytic viruses (OVs), viruses that preferentially target and kill cancer cells, for their capacity to enhance DC-mediated anti-tumor effects. Herein, we describe the general characteristics of DCs, focusing on their role in innate and adaptive immunity in the context of the TME. We also examine how DC-OV interaction affects DC recruitment, OV delivery, and anti-tumor immunity activation. Understanding these roles of DCs in the TME and OV infection is critical in devising strategies to further harness the anti-tumor effects of both DCs and OVs, ultimately enhancing the efficacy of OV-based oncotherapy. PMID:26690204

  20. The E6 protein from vaccinia virus is required for the formation of immature virions

    SciTech Connect

    Boyd, Olga; Turner, Peter C.; Moyer, Richard W.; Condit, Richard C.; Moussatche, Nissin

    2010-04-10

    An IPTG-inducible mutant in the E6R gene of vaccinia virus was used to study the role of the E6 virion core protein in viral replication. In the absence of the inducer, the mutant exhibited a normal pattern DNA replication, concatemer resolution and late gene expression, but it showed an inhibition of virion structural protein processing it failed to produce infectious particles. Electron microscopic analysis showed that in the absence of IPTG viral morphogenesis was arrested before IV formation: crescents, aberrant or empty IV-like structures, and large aggregated virosomes were observed throughout the cytoplasm. The addition of IPTG to release a 12-h block showed that virus infectious particles could be formed in the absence of de novo DNA synthesis. Our observations show that in the absence of E6 the association of viroplasm with viral membrane crescents is impaired.

  1. Expression of the highly conserved vaccinia virus E6 protein is required for virion morphogenesis

    SciTech Connect

    Resch, Wolfgang; Weisberg, Andrea S.; Moss, Bernard

    2009-04-10

    The vaccinia virus E6R gene (VACVWR062) is conserved in all members of the poxvirus family and encodes a protein associated with the mature virion. We confirmed this association and provided evidence for an internal location. An inducible mutant that conditionally expresses E6 was constructed. In the absence of inducer, plaque formation and virus production were severely inhibited in several cell lines, whereas some replication occurred in others. This difference could be due to variation in the stringency of repression, since we could not isolate a stable deletion mutant even in the more 'permissive' cells. Under non-permissive conditions, viral late proteins were synthesized but processing of core proteins was inefficient, indicative of an assembly block. Transmission electron microscopy of sections of cells infected with the mutant in the absence of inducer revealed morphogenetic defects with crescents and empty immature virions adjacent to dense inclusions of viroplasm. Mature virions were infrequent and cores appeared to have lucent centers.

  2. Characterization of an attenuated TE3L-deficient vaccinia virus Tian Tan strain.

    PubMed

    Wang, Yuhang; Kan, Shifu; Du, Shouwen; Qi, Yanxin; Wang, Jinhui; Liu, Liming; Ji, Huifan; He, Dongyun; Wu, Na; Li, Chang; Chi, Baorong; Li, Xiao; Jin, Ningyi

    2012-12-01

    An attenuated vaccinia virus (VACV), TE3L(-)VTT, was evaluated for virulence and safety to determine its potential use as a vaccine or as a recombinant virus vector to express foreign genes. The virulence of TE3L(-)VTT was compared with that of the wild-type VTT both in vivo and in vitro. The humoral and cellular immune responses were detected in a mouse model to test the vaccine efficacy of the TE3L mutant. The results suggested that deletion of the TE3L gene decreased the virulence and neurovirulence significantly in mice and rabbit models, yet retained the immunogenicity. Thus, the deletion of TE3L improved the safety of the VTT vector; this approach may yield a valuable resource for studies of recombinant VACV-vectored vaccines.

  3. A36-dependent Actin Filament Nucleation Promotes Release of Vaccinia Virus

    PubMed Central

    Horsington, Jacquelyn; Lynn, Helena; Turnbull, Lynne; Cheng, Delfine; Braet, Filip; Diefenbach, Russell J.; Whitchurch, Cynthia B.; Karupiah, Guna; Newsome, Timothy P.

    2013-01-01

    Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36YdF virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36YdF infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36YdF extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5P189S mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin

  4. Elongation properties of vaccinia virus RNA polymerase: pausing, slippage, 3' end addition, and termination site choice.

    PubMed

    Deng, L; Shuman, S

    1997-12-16

    We have analyzed the elongation properties of vaccinia virus RNA polymerase during a single round of transcription in vitro. RNA-labeled ternary complexes were halted at a unique template position located upstream of a T-run (TTTTTTTTT) in the nontemplate strand; this element encodes an RNA signal for factor-dependent transcription termination at distal sites on the template. The halted ternary complexes were purified and allowed to resume elongation under a variety of conditions. We found that the T-run constituted a strong elongation block, even at high nucleotide concentrations. The principal sites of pausing were at a C position situated two nucleotides upstream of the first T in the T-run and at the first three to four T positions within the T-run. There was relatively little pausing at the five downstream Ts. Intrinsic pausing was exacerbated at suboptimal nucleotide concentrations. Ternary complexes arrested by the T-run at 10 microM NTPs rapidly traversed the T-run when the NTP pool was increased to 1 mM. Limiting GTP (1 microM) resulted in polymerase stuttering at the 3' margin of the T-run, immediately prior to a templated G position; this generated a ladder of slippage synthesis products. We found that vaccinia ternary complexes remained intact after elongating to the very end of a linear DNA template and that such complexes catalyzed the addition of extra nucleotides to the 3' end of the RNA chain. The 3' end addition required much higher concentrations of NTPs than did templated chain elongation. Finally, we report that factor-dependent transcription termination by vaccinia RNA polymerase downstream of the T-run was affected by nucleotide concentration. Limiting UTP caused the polymerase to terminate at sites closer to the UUUUUNU termination signal. This is consistent with the kinetic coupling model for factor-dependent termination.

  5. Vaccinia virus-induced smallpox postvaccinal encephalitis in case of blood-brain barrier damage.

    PubMed

    Garcel, Aude; Fauquette, William; Dehouck, Marie-Pierre; Crance, Jean-Marc; Favier, Anne-Laure

    2012-02-08

    Smallpox vaccination is the only currently effective mean to combat the threat of variola virus used as a bioterrorism agent, although it is responsible for a rare but serious complication, the postvaccinal encephalitis (PVE). Development of safer vaccines therefore is a high priority as the PVE physiopathology is not well understood to date. If vaccinia virus (VACV) is responsible for PVE by central nervous system (CNS) dissemination, trans-migration of the VACV across the blood-brain barrier (BBB) would be supposed to be essential. Given the complexity of the pathogenesis of vaccinia neurovirulence, an in vitro BBB model was used to explore the mechanism of VACV to induce BBB permeability. Two VACV strains were studied, the neurovirulent Western Reserve strain (VACV-WR) and the vaccine reference Lister strain (VACV-List). A mouse model was also developed to study the ability of these two viral strains to propagate in the brain from the blood compartment, their neurovirulence and their neuropathogenesis. In vitro, the loss of permeability resulted from the tight-junctions disruption was induced by virus replication. The ability of VACV to release infectious particles at the abluminal side suggests the capacity of both VACV strains to migrate across the BBB from the blood to the CNS. In vivo, the virus replication in mice CNS was strain-dependent. The VACV-WR laboratory strain proved to be neuroinvasive and neurovirulent, whereas the VACV-List strain is safe in physiological conditions. Mice PVE was observed only with VACV-WR in the co-infection model, when BBB opening was obtained by lipopolysaccharide (LPS) treatment. This study suggests that VACV is able to cross the BBB but encephalitis occurs only in the presence of a co-infection by bacteria. So, a model of co-infection, mimicked by LPS treatment, could have important implication towards the assessment of neurovirulence of new vaccines.

  6. Emergence of foreign H-2-like cytotoxicity and transplantation targets on vaccinia and Moloney virus-infected Meth. A tumour cells.

    PubMed

    Matossian-Rogers, A; Garrido, F; Festenstein, H

    1977-01-01

    Using cytotoxic effector cells of different anti-H-2 specificities, new cell-mediated lympholysis targets, normally undetected on Meth. A tumour cells, were shown after passage with vaccinia or Moloney virus. The H-2d CML targets on Meth. A cells recognized by B10.BR anti-B10.D2 effector cells were presented only after simultaneous vaccinia virus passage, while passage with Moloney virus caused the emergence of H-2b targets. Small but significant killing of vaccinia virus-passaged Meth.A was also obtained by anti-H-2k effector cells. These results are discussed in relation to in vivo experiments: retardation of tumour growth was noted in mice which had received several injections of vaccinia or Moloney virus, showing that the new CML targets were probably acting as transplantation targets.

  7. Single-Virus Fusion Experiments Reveal Proton Influx into Vaccinia Virions and Hemifusion Lag Times

    PubMed Central

    Schmidt, Florian I.; Kuhn, Phillip; Robinson, Tom; Mercer, Jason; Dittrich, Petra S.

    2013-01-01

    Recent studies have revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. We developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which we analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concentrations of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addition, we found that EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiological pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further investigations into a potential VACV viroporin are justified. Our findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements. PMID:23870263

  8. Inactivation of vaccinia virus by natural sunlight and by artificial UVB radiation.

    PubMed

    Sagripanti, Jose-Luis; Voss, Luzie; Marschall, Hans-Juergen; Lytle, Carl David

    2013-01-01

    This study determined the sensitivity of vaccinia virus, an orthopox virus commonly used as a surrogate for variola virus (etiological agent of smallpox), exposed to UVB radiation emitted by a solar simulator, or to direct natural sunlight. The data obtained indicate that: (1) the virucidal effect of natural sunlight can be mimicked adequately by an artificial light source with similar spectral characteristics in the UVB, (2) viral sensitivity to UVB or to solar radiation can be correlated with experimental data previously obtained with UVC, (3) the correlation factor between virus inactivation by solar radiation (measured at 300 ± 5 nm) and by UVC (254 nm) is between 33 and 60, and (4) the sensitivity of viruses either dry on glass surfaces or in liquid suspension is similar when in the presence of similar amounts of cellular debris and growth media. The findings reported in this study should assist in estimating the threat posed by the persistence of virus during epidemics or after an accidental or intentional release.

  9. Assessing the variability of Brazilian Vaccinia virus isolates from a horse exanthematic lesion: coinfection with distinct viruses.

    PubMed

    Campos, Rafael K; Brum, Mário C S; Nogueira, Carlos E W; Drumond, Betânia P; Alves, Pedro A; Siqueira-Lima, Larissa; Assis, Felipe L; Trindade, Giliane S; Bonjardim, Cláudio A; Ferreira, Paulo C; Weiblen, Rudi; Flores, Eduardo F; Kroon, Erna G; Abrahão, Jônatas S

    2011-02-01

    During the last bovine vaccinia (BV) outbreaks, several Vaccinia virus (VACV) strains were isolated and characterised, revealing significant polymorphisms between strains, even within conserved genes. Although the epidemiology of VACV has been studied in BV outbreaks, there is little data about the circulation of the Brazilian VACV isolates. This study describes the genetic and biological characterisation of two VACV isolates, Pelotas 1 virus (P1V) and Pelotas 2 virus (P2V), which were obtained concomitantly from a horse affected by severe cutaneous disease. Despite being isolated from the same exanthematic clinical sample, P1V and P2V showed differences in their plaque phenotype and in one-step growth curves. Moreover, P1V and P2V presented distinct virulence profiles in a BALB/c mouse model, as observed with other Brazilian VACV isolates. Sequencing and phylogenetic analysis of four different genes demonstrated that the isolates are segregated in different VACV clusters. Our results raise interesting questions about the diversity of VACV isolates in Brazil.

  10. Discovery of naturally processed and HLA-presented class I peptides from vaccinia virus infection using mass spectrometry for vaccine development.

    PubMed

    Johnson, Kenneth L; Ovsyannikova, Inna G; Mason, Christopher J; Bergen, H Robert; Poland, Gregory A

    2009-12-10

    An important approach for developing a safer smallpox vaccine is to identify naturally processed immunogenic vaccinia-derived peptides rather than live whole vaccinia virus. We used two-dimensional liquid chromatography coupled to mass spectrometry to identify 116 vaccinia peptides, encoded by 61 open reading frames, from a B-cell line (homozygous for HLA class I A*0201, B*1501, and C*03) after infection with vaccinia virus (Dryvax). Importantly, 68 of these peptides are conserved in variola, providing insight into the peptides that induce protection against smallpox. Twenty-one of these 68 conserved peptides were 11 amino acids long or longer, outside of the range of most predictive algorithms. Thus, direct identification of naturally processed and presented HLA peptides gives important information not provided by current computational methods for identifying potential vaccinia epitopes.

  11. Crystallization and preliminary X-ray diffraction analysis of vaccinia virus H1L phosphatase

    PubMed Central

    Roces, Laura; Knowles, Phillip P.; Fox, Gavin; Juanhuix, Jordi; Scaplehorn, Nicki; Way, Michael; McDonald, Neil Q.

    2008-01-01

    The cysteine-based protein phosphatase H1L was the first reported dual-specificity protein phosphatase. H1L is encapsidated within the vaccinia virus and is required for successful host infection and for the production of viable vaccinia progeny. H1L has therefore been proposed as a target candidate for antiviral compounds. Recombinant H1L has been expressed in a catalytically inactive form using an Escherichia coli host, leading to purification and crystallization by the microbatch method. The crystals diffract to 2.1 Å resolution using synchrotron radiation. These crystals belong to space group P422, with unit-cell parameters a = b = 98.31, c = 169.15 Å, and are likely to contain four molecules in the asymmetric unit. A sulfur SAD data set was collected to 2.8 Å resolution on beamline BM14 at the ESRF to facilitate structure determination. Attempts to derivatize these crystals with xenon gas changed the space group to I422, with unit-cell parameters a = b = 63.28, c = 169.68 Å and a single molecule in the asymmetric unit. The relationship between these two crystal forms is discussed. PMID:18323605

  12. Short communication: Survival of Vaccinia virus in inoculated cheeses during 60-day ripening.

    PubMed

    Rehfeld, Izabelle S; Fraiha, Ana Luiza S; Matos, Ana Carolina D; Guedes, Maria Isabel M C; Costa, Erica A; de Souza, Marcelo R; Cavalcante, Luigi F L; Lobato, Zélia I P

    2017-09-01

    Bovine vaccinia is a neglected zoonosis caused by Vaccinia virus (VACV) and has a major economic and public health effect in Brazil. Previous studies showed infectious VACV particles in milk from either experimentally or naturally infected cows and in fresh cheeses prepared with experimentally contaminated milk. Ripening is a process that leads to major changes in the physical and chemical characteristics of cheese, reducing contamination by spoilage, pathogenic microorganisms, or both. However, it is not known if VACV infectious particles persist after the ripening process. To investigate this issue, viral infectivity at different ripening times was studied in cheeses manufactured with milk experimentally contaminated with VACV strain Guarani P2 (GP2). Cheeses were analyzed at 1, 7, 14, 21, 45, and 60 d of ripening at 25°C. Viral DNA was quantified by real-time PCR, and VACV isolation and titration were performed in Vero cells. The whole experiment was repeated 4 times. Analysis of the mean viral DNA quantification and infectivity indicated a reduction of approximately 2 logs along the ripening process; however, infectious viral particles (1.7 × 10(2) pfu/mL) could still be recovered at d 60 of ripening. These findings indicate that the ripening process reduces VACV infectivity, but it was not able to inactivate completely the viral particles after 60 d. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  13. Clinical, hematological and biochemical parameters of dairy cows experimentally infected with Vaccinia virus.

    PubMed

    Rehfeld, Izabelle S; Guedes, Maria Isabel M C; Matos, Ana Carolina D; de Oliveira, Tércia M L; Rivetti, Anselmo V; Moura, Ana Carolina J; Paes, Paulo Ricardo O; do Lago, Luiz Alberto; Kroon, Erna G; Lobato, Zélia Inês P

    2013-10-01

    Vaccinia virus (VACV) is the etiological agent of bovine vaccinia (BV), an important zoonosis that affects dairy cattle. There are many aspects of the disease that remain unknown, and aiming to answer some of these questions, the clinical, hematological, and biochemical parameters of VACV experimentally infected cows were evaluated. In the first part of the study, lactating cows were infected with VACV-GP2 strain. In the second part, animals previously infected with VACV-GP2 were divided into two treatment groups: Group 1, immunosuppressed cows; and Group 2, re-infected cows. In this study, BV could be experimentally reproduced, with similar lesions as observed in natural infections. Moreover, a short incubation period and local lymphadenopathy were also observed. VACV could be detected by PCR and isolated from scabs taken from teat lesions of all inoculated and re-inoculated animals. Lymphocytosis and neutrophilia were observed in all animals from the first part of the experiment, and lymphopenia and relative neutrophilia were observed in the immunosuppressed animals. Detection of viral DNA in oral mucosa lesions suggests that viral reactivation might occur in immunosuppressed animals. Moreover, clinical disease with teat lesions may occur in previously VACV-infected cows under the experimental conditions of the present study.

  14. Targeting CXCL12/CXCR4 signaling with oncolytic virotherapy disrupts tumor vasculature and inhibits breast cancer metastases.

    PubMed

    Gil, Margaret; Seshadri, Mukund; Komorowski, Marcin P; Abrams, Scott I; Kozbor, Danuta

    2013-04-02

    Oncolytic viruses hold promise for the treatment of cancer, but their interaction with the tumor microenvironment needs to be elucidated for optimal tumor cell killing. Because the CXCR4 receptor for the stromal cell-derived factor-1 (SDF-1/CXCL12) chemokine is one of the key stimuli involved in signaling interactions between tumor cells and their stromal microenvironment, we used oncolytic virotherapy with a CXCR4 antagonist to target the CXCL12/CXCR4 signaling axis in a triple-negative 4T1 breast carcinoma in syngeneic mice. We show here that CXCR4 antagonist expression from an oncolytic vaccinia virus delivered intravenously to mice with orthotopic tumors attains higher intratumoral concentration than its soluble counterpart and exhibits increased efficacy over that mediated by oncolysis alone. A systemic delivery of the armed virus after resection of the primary tumor was efficacious in inhibiting the development of spontaneous metastasis and increased overall tumor-free survival. Inhibition of tumor growth with the armed virus was associated with destruction of tumor vasculature, reductions in expression of CXCL12 and VEGF, and decrease in intratumoral numbers of bone marrow-derived endothelial and myeloid cells. These changes led to induction of antitumor antibody responses and resistance to tumor rechallenge. Engineering an oncolytic virus armed with a CXCR4 antagonist represents an innovative strategy that targets multiple elements within the tumor microenvironment. As such, this approach could have a significant therapeutic impact against primary and metastatic breast cancer.

  15. Association of Vaccinia Virus Fusion Regulatory Proteins with the Multicomponent Entry/Fusion Complex▿

    PubMed Central

    Wagenaar, Timothy R.; Moss, Bernard

    2007-01-01

    The proteins encoded by the A56R and K2L genes of vaccinia virus form a heterodimer (A56/K2) and have a fusion regulatory role as deletion or mutation of either causes infected cells to form large syncytia spontaneously. Here, we showed that syncytia formation is dependent on proteins of the recently described entry fusion complex (EFC), which are also required for virus-cell fusion and low-pH-triggered cell-cell fusion. This finding led us to consider that A56/K2 might prevent fusion by direct or indirect interaction with the EFC. To test this hypothesis, we made a panel of recombinant vaccinia viruses that have a tandem affinity purification tag attached to A56, K2, or the A28 EFC protein. Interaction between A56/K2 and the EFC was demonstrated by their copurification from detergent-treated lysates of infected cells and identification by mass spectrometry or Western blotting. In addition, a purified soluble transmembrane-deleted form of A56/K2 was shown to interact with the EFC. Tagged A56 did not interact with the EFC in the absence of K2, nor did tagged K2 interact with the EFC in the absence of A56. The finding that both A56 and K2 are required for efficient binding to the EFC fits well with prior experiments showing that mutation of either A56 or K2 results in spontaneous fusion of infected cells. Because A56 and K2 are located on the surface of infected cells, they are in position to interact with the EFC of released progeny virions and prevent back-fusion and syncytia formation. PMID:17409143

  16. Association of vaccinia virus fusion regulatory proteins with the multicomponent entry/fusion complex.

    PubMed

    Wagenaar, Timothy R; Moss, Bernard

    2007-06-01

    The proteins encoded by the A56R and K2L genes of vaccinia virus form a heterodimer (A56/K2) and have a fusion regulatory role as deletion or mutation of either causes infected cells to form large syncytia spontaneously. Here, we showed that syncytia formation is dependent on proteins of the recently described entry fusion complex (EFC), which are also required for virus-cell fusion and low-pH-triggered cell-cell fusion. This finding led us to consider that A56/K2 might prevent fusion by direct or indirect interaction with the EFC. To test this hypothesis, we made a panel of recombinant vaccinia viruses that have a tandem affinity purification tag attached to A56, K2, or the A28 EFC protein. Interaction between A56/K2 and the EFC was demonstrated by their copurification from detergent-treated lysates of infected cells and identification by mass spectrometry or Western blotting. In addition, a purified soluble transmembrane-deleted form of A56/K2 was shown to interact with the EFC. Tagged A56 did not interact with the EFC in the absence of K2, nor did tagged K2 interact with the EFC in the absence of A56. The finding that both A56 and K2 are required for efficient binding to the EFC fits well with prior experiments showing that mutation of either A56 or K2 results in spontaneous fusion of infected cells. Because A56 and K2 are located on the surface of infected cells, they are in position to interact with the EFC of released progeny virions and prevent back-fusion and syncytia formation.

  17. Potential effect of prior raccoonpox virus infection in raccoons on vaccinia-based rabies immunization

    PubMed Central

    Root, J Jeffrey; McLean, Robert G; Slate, Dennis; MacCarthy, Kathleen A; Osorio, Jorge E

    2008-01-01

    Background The USDA, Wildlife Services cooperative oral rabies vaccination (ORV) program uses a live vaccinia virus-vectored (genus Orthopoxvirus) vaccine, Raboral V-RG® (V-RG), to vaccinate specific wildlife species against rabies virus in several regions of the U.S. Several naturally occurring orthopoxviruses have been found in North America, including one isolated from asymptomatic raccoons (Procyon lotor). The effect of naturally occurring antibodies to orthopoxviruses on successful V-RG vaccination in raccoons is the focus of this study. Results Overall, raccoons pre-immunized (n = 10) with a recombinant raccoonpox virus vaccine (RCN-F1) responded to vaccination with V-RG with lower rabies virus neutralizing antibody (VNA) titers than those which were not pre-immunized (n = 10) and some failed to seroconvert for rabies VNA to detectable levels. Conclusion These results suggest that the success of some ORV campaigns may be hindered where raccoonpox virus or possibly other orthopoxvirus antibodies are common in wildlife species targeted for ORV. If these areas are identified, different vaccination strategies may be warranted. PMID:18834520

  18. Novel biomarkers of resistance of pancreatic cancer cells to oncolytic vesicular stomatitis virus

    PubMed Central

    Steuerwald, Nury; Grdzelishvili, Valery Z.

    2016-01-01

    Vesicular stomatitis virus (VSV) based recombinant viruses (such as VSV-ΔM51) are effective oncolytic viruses (OVs) against a majority of pancreatic ductal adenocarcinoma (PDAC) cell lines. However, some PDAC cell lines are highly resistant to VSV-ΔM51. We recently showed that treatment of VSV-resistant PDAC cells with ruxolitinib (JAK1/2 inhibitor) or TPCA-1 (IKK-β inhibitor) breaks their resistance to VSV-ΔM51. Here we compared the global effect of ruxolitinib or TPCA-1 treatment on cellular gene expression in PDAC cell lines highly resistant to VSV-ΔM51. Our study identified a distinct subset of 22 interferon-stimulated genes (ISGs) downregulated by both ruxolitinib and TPCA-1. Further RNA and protein analyses demonstrated that 4 of these genes (MX1, EPSTI1, XAF1, and GBP1) are constitutively co-expressed in VSV-resistant, but not in VSV-permissive PDACs, thus serving as potential biomarkers to predict OV therapy success. Moreover, shRNA-mediated knockdown of one of such ISG, MX1, showed a positive effect on VSV-ΔM51 replication in resistant PDAC cells, suggesting that at least some of the identified ISGs contribute to resistance of PDACs to VSV-ΔM51. As certain oncogene and tumor suppressor gene variants are often associated with increased tropism of OVs to cancer cells, we also analyzed genomic DNA in a set of PDAC cell lines for frequently occurring cancer associated mutations. While no clear correlation was found between such mutations and resistance of PDACs to VSV-ΔM51, the analysis generated valuable genotypic data for future studies. PMID:27533247

  19. Cross-protective immunity against multiple influenza virus subtypes by a novel modified vaccinia Ankara (MVA) vectored vaccine in mice.

    PubMed

    Brewoo, Joseph N; Powell, Tim D; Jones, Jeremy C; Gundlach, Nancy A; Young, Ginger R; Chu, Haiyan; Das, Subash C; Partidos, Charalambos D; Stinchcomb, Dan T; Osorio, Jorge E

    2013-04-03

    Development of an influenza vaccine that provides cross-protective immunity remains a challenge. Candidate vaccines based on a recombinant modified vaccinia Ankara (MVA) viral vector expressing antigens from influenza (MVA/Flu) viruses were constructed. A vaccine candidate, designated MVA/HA1/C13L/NP, that expresses the hemagglutinin from pandemic H1N1 (A/California/04/09) and the nucleoprotein (NP) from highly pathogenic H5N1 (A/Vietnam/1203/04) fused to a secretory signal sequence from vaccinia virus was highly protective. The vaccine elicited strong antibody titers to homologous H1N1 viruses while cross-reactive antibodies to heterologous viruses were not detectable. In mice, this MVA/HA1/C13L/NP vaccine conferred complete protection against lethal challenge with A/Vietnam/1203/04 (H5N1), A/Norway/3487-2/09 (pandemic H1N1) or A/Influenza/Puerto Rico/8/34 (seasonal H1N1) and partial protection (57.1%) against challenge with seasonal H3N2 virus (A/Aichi/68). The protective efficacy of the vaccine was not affected by pre-existing immunity to vaccinia. Our findings highlight MVA as suitable vector to express multiple influenza antigens that could afford broad cross-protective immunity against multiple subtypes of influenza virus.

  20. Cellular DNA ligase I is recruited to cytoplasmic vaccinia virus factories and masks the role of the vaccinia ligase in viral DNA replication.

    PubMed

    Paran, Nir; De Silva, Frank S; Senkevich, Tatiana G; Moss, Bernard

    2009-12-17

    Vaccinia virus (VACV) encodes DNA polymerase and additional proteins that enable cytoplasmic replication. We confirmed the ability of VACV DNA ligase mutants to replicate and tested the hypothesis that cellular ligases compensate for loss of viral gene expression. RNA silencing of human DNA ligase I expression and a small molecule inhibitor of human DNA ligase I [corrected] severely reduced replication of viral DNA in cells infected with VACV ligase-deficient mutants, indicating that the cellular enzyme plays a complementary role. Replication of ligase-deficient VACV was greatly reduced and delayed in resting primary cells, correlating with initial low levels of ligase I and subsequent viral induction and localization of ligase I in virus factories. These studies indicate that DNA ligation is essential for poxvirus replication and explain the ability of ligase deletion mutants to replicate in dividing cells but exhibit decreased pathogenicity in mice. Encoding its own ligase might allow VACV to "jump-start" DNA synthesis.

  1. Comparison of host cell gene expression in cowpox, monkeypox or vaccinia virus-infected cells reveals virus-specific regulation of immune response genes

    PubMed Central

    2013-01-01

    Background Animal-borne orthopoxviruses, like monkeypox, vaccinia and the closely related cowpox virus, are all capable of causing zoonotic infections in humans, representing a potential threat to human health. The disease caused by each virus differs in terms of symptoms and severity, but little is yet know about the reasons for these varying phenotypes. They may be explained by the unique repertoire of immune and host cell modulating factors encoded by each virus. In this study, we analysed the specific modulation of the host cell’s gene expression profile by cowpox, monkeypox and vaccinia virus infection. We aimed to identify mechanisms that are either common to orthopoxvirus infection or specific to certain orthopoxvirus species, allowing a more detailed description of differences in virus-host cell interactions between individual orthopoxviruses. To this end, we analysed changes in host cell gene expression of HeLa cells in response to infection with cowpox, monkeypox and vaccinia virus, using whole-genome gene expression microarrays, and compared these to each other and to non-infected cells. Results Despite a dominating non-responsiveness of cellular transcription towards orthopoxvirus infection, we could identify several clusters of infection-modulated genes. These clusters are either commonly regulated by orthopoxvirus infection or are uniquely regulated by infection with a specific orthopoxvirus, with major differences being observed in immune response genes. Most noticeable was an induction of genes involved in leukocyte migration and activation in cowpox and monkeypox virus-infected cells, which was not observed following vaccinia virus infection. Conclusion Despite their close genetic relationship, the expression profiles induced by infection with different orthopoxviruses vary significantly. It may be speculated that these differences at the cellular level contribute to the individual characteristics of cowpox, monkeypox and vaccinia virus

  2. Viruses as nanomedicine for cancer

    PubMed Central

    Badrinath, Narayanasamy; Heo, Jeong; Yoo, So Young

    2016-01-01

    Oncolytic virotherapy, a type of nanomedicine in which oncolytic viruses (OVs) are used to selectively infect and lyse cancer cells, is an emerging field in cancer therapy. Some OVs exhibit a specific tropism for cancer cells, whereas others require genetic modification to enhance their binding with and entry into cancer cells. OVs both kill tumor cells and induce the host’s immune response against tumor cells. Armed with antitumor cellular molecules, antibodies, and/or in combination with anticancer drugs, OVs can accelerate the lysis of cancer cells. Among the OVs, vaccinia virus has been the focus of preclinical and clinical research because of its many favorable properties. In this review, the basic mechanisms of action of OVs are presented, including their entry, survival, tumor lysis, and immune activation, and the latest research in vaccinia virus-based virotherapy and its status as an anticancer nanomedicine in prospective clinical trials are discussed. PMID:27703350

  3. Viruses as nanomedicine for cancer.

    PubMed

    Badrinath, Narayanasamy; Heo, Jeong; Yoo, So Young

    Oncolytic virotherapy, a type of nanomedicine in which oncolytic viruses (OVs) are used to selectively infect and lyse cancer cells, is an emerging field in cancer therapy. Some OVs exhibit a specific tropism for cancer cells, whereas others require genetic modification to enhance their binding with and entry into cancer cells. OVs both kill tumor cells and induce the host's immune response against tumor cells. Armed with antitumor cellular molecules, antibodies, and/or in combination with anticancer drugs, OVs can accelerate the lysis of cancer cells. Among the OVs, vaccinia virus has been the focus of preclinical and clinical research because of its many favorable properties. In this review, the basic mechanisms of action of OVs are presented, including their entry, survival, tumor lysis, and immune activation, and the latest research in vaccinia virus-based virotherapy and its status as an anticancer nanomedicine in prospective clinical trials are discussed.

  4. Induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express PreM and E glycoproteins of Japanese encephalitis virus.

    PubMed Central

    Yasuda, A; Kimura-Kuroda, J; Ogimoto, M; Miyamoto, M; Sata, T; Sato, T; Takamura, C; Kurata, T; Kojima, A; Yasui, K

    1990-01-01

    A cDNA clone representing the genome of structural proteins of Japanese encephalitis virus (JEV) was inserted into the thymidine kinase gene of vaccinia virus strains LC16mO and WR under the control of a strong early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. Indirect immunofluorescence and fluorescence-activated flow cytometric analysis revealed that the recombinant vaccinia viruses expressed JEV E protein on the membrane surface, as well as in the cytoplasm, of recombinant-infected cells. In addition, the E protein expressed from the JEV recombinants reacted to nine different characteristic monoclonal antibodies, some of which have hemagglutination-inhibiting and JEV-neutralizing activities. Radioimmunoprecipitation analysis demonstrated that two major proteins expressed in recombinant-infected cells were processed and glycosylated as the authentic PreM and E glycoproteins of JEV. Inoculation of rabbits with the infectious recombinant vaccinia virus resulted in rapid production of antiserum specific for the PreM and E glycoproteins of JEV. This antiserum had both hemagglutination-inhibiting and virus-neutralizing activities against JEV. Furthermore, mice vaccinated with the recombinant also produced JEV-neutralizing antibodies and were resistant to challenge with JEV. Images PMID:2159544

  5. Retrograde Transport from Early Endosomes to the trans-Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions

    PubMed Central

    Sivan, Gilad; Weisberg, Andrea S.; Americo, Jeffrey L.

    2016-01-01

    ABSTRACT The anterograde pathway, from the endoplasmic reticulum through the trans-Golgi network to the cell surface, is utilized by trans-membrane and secretory proteins. The retrograde pathway, which directs traffic in the opposite direction, is used following endocytosis of exogenous molecules and recycling of membrane proteins. Microbes exploit both routes: viruses typically use the anterograde pathway for envelope formation prior to exiting the cell, whereas ricin and Shiga-like toxins and some nonenveloped viruses use the retrograde pathway for cell entry. Mining a human genome-wide RNA interference (RNAi) screen revealed a need for multiple retrograde pathway components for cell-to-cell spread of vaccinia virus. We confirmed and extended these results while discovering that retrograde trafficking was required for virus egress rather than entry. Retro-2, a specific retrograde trafficking inhibitor of protein toxins, potently prevented spread of vaccinia virus as well as monkeypox virus, a human pathogen. Electron and confocal microscopy studies revealed that Retro-2 prevented wrapping of virions with an additional double-membrane envelope that enables microtubular transport, exocytosis, and actin polymerization. The viral B5 and F13 protein components of this membrane, which are required for wrapping, normally colocalize in the trans-Golgi network. However, only B5 traffics through the secretory pathway, suggesting that F13 uses another route to the trans-Golgi network. The retrograde route was demonstrated by finding that F13 was largely confined to early endosomes and failed to colocalize with B5 in the presence of Retro-2. Thus, vaccinia virus makes novel use of the retrograde transport system for formation of the viral wrapping membrane. IMPORTANCE Efficient cell-to-cell spread of vaccinia virus and other orthopoxviruses depends on the wrapping of infectious particles with a double membrane that enables microtubular transport, exocytosis, and actin

  6. Polycyclic N-benzamido imides with potent activity against vaccinia virus.

    PubMed

    Torres, Eva; Duque, María D; Camps, Pelayo; Naesens, Lieve; Calvet, Teresa; Font-Bardia, Mercè; Vázquez, Santiago

    2010-12-03

    The synthesis and antiviral activity of a series of novel polycyclic analogues of the orthopoxvirus egress inhibitor tecovirimat (ST-246) is presented. Several of these compounds display sub-micromolar activity against vaccinia virus, and were more potent than cidofovir (CDV). The more active compounds were about 10-fold more active than CDV, with minimum cytotoxic concentrations above 100 μM. Chemical manipulations of the two carbon-carbon double bonds present in the compounds were carried out to further explore the structure-activity relationships of these new polycyclic imides. Hydrogenation of the two carbon-carbon double bonds decreases antiviral activity, whereas either cyclopropanation or epoxidation of the double bonds fully eliminates the antiviral activity.

  7. Environmental Risk Assessment of Clinical Trials Involving Modified Vaccinia Virus Ankara (MVA)-Based Vectors

    PubMed Central

    Goossens, Martine; Pauwels, Katia; Willemarck, Nicolas; Breyer, Didier

    2013-01-01

    The modified vaccinia virus Ankara (MVA) strain, which has been developed as a vaccine against smallpox, is since the nineties widely tested in clinical trials as recombinant vector for vaccination or gene therapy applications. Although MVA is renowned for its safety, several biosafety aspects need to be considered when performing the risk assessment of a recombinant MVA (rMVA). This paper presents the biosafety issues and the main lessons learned from the evaluation of the clinical trials with rMVA performed in Belgium. Factors such as the specific characteristics of the rMVA, the inserted foreign sequences/transgene, its ability for reconversion, recombination and dissemination in the population and the environment are the main points of attention. Measures to prevent or manage identified risks are also discussed. PMID:24397528

  8. Reverse Genetics of SARS-Related Coronavirus Using Vaccinia Virus-Based Recombination

    PubMed Central

    Zevenhoven, Jessika C.; Weber, Friedemann; Züst, Roland; Kuri, Thomas; Dijkman, Ronald; Chang, Guohui; Siddell, Stuart G.; Snijder, Eric J.; Thiel, Volker; Davidson, Andrew D.

    2012-01-01

    Severe acute respiratory syndrome (SARS) is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV) that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virus-based SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime) as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV). Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs). In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E). Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs. PMID:22412934

  9. Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination.

    PubMed

    van den Worm, Sjoerd H E; Eriksson, Klara Kristin; Zevenhoven, Jessika C; Weber, Friedemann; Züst, Roland; Kuri, Thomas; Dijkman, Ronald; Chang, Guohui; Siddell, Stuart G; Snijder, Eric J; Thiel, Volker; Davidson, Andrew D

    2012-01-01

    Severe acute respiratory syndrome (SARS) is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV) that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virus-based SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime) as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV). Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs). In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E). Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs.

  10. siRNA targeting vaccinia virus double-stranded RNA binding protein [E3L] exerts potent antiviral effects.

    PubMed

    Dave, Rajnish S; McGettigan, James P; Qureshi, Tazeen; Schnell, Matthias J; Nunnari, Giuseppe; Pomerantz, Roger J

    2006-05-10

    The Vaccinia virus gene, E3L, encodes a double-stranded RNA [dsRNA]-binding protein. We hypothesized that, owing to the critical nature of dsRNA in triggering host innate antiviral responses, E3L-specific small-interfering RNAs [siRNAs] should be effective antiviral agents against pox viruses, for which Vaccinia virus is an appropriate surrogate. In this study, we have utilized two human cell types, namely, HeLa and 293T, one which responds to interferon [IFN]-beta and the other produces and responds to IFN-beta, respectively. The antiviral effects were equally robust in HeLa and 293T cells. However, in the case of 293T cells, several distinct features were observed, when IFN-beta is activated in these cells. Vaccinia virus replication was inhibited by 97% and 98% as compared to control infection in HeLa and 293T cells transfected with E3L-specific siRNAs, respectively. These studies demonstrate the utility of E3L-specific siRNAs as potent antiviral agents for small pox and related pox viruses.

  11. Cytotoxic T-cell responses in mice infected with influenza and vaccinia viruses vary in magnitude with H-2 genotype

    PubMed Central

    1978-01-01

    Secondary effector T-cell populations generated by cross-priming with heterologous influenza A viruses operate only in H-2K or H-2D compatible situations, when assayed on SV40-transformed target cells infected with a range of influenza A viruses. The H2-Kb allele is associated with a total failure in the generation of influenza-immune cytotoxic T cells, though this is not seen for the primary response to vaccinia virus. In both influenza and vaccinia development of effector T cells operating at H-2Db is greatly depressed in B10.A(2R) (kkkddb) and B10.A(4R) (kkbbbb), but not in B10 (bbbbbb), mice. However, there is no defect in viral antigen expression at either H-2Kk or H-2Db in B10.A(2R) target cells. This apparently reflects some inadequacy in the stimulator environment, as (A/J X B6) F1 T cells can be induced to respond at H-2Db when exposed to vaccinia virus in an irradiated B6 but not in a B10.A(4R) recipient. The present report, together with the accompanying paper by Zinkernagel and colleagues, records the first rigorous demonstration of both a nonresponder situation and a probable Ir-gene effect for conventional infectious viruses. Possible implications for the evolution of H-2 polymorphism and mechanisms of Ir gene function are discussed. PMID:100569

  12. Intratumoral modulation of the inducible co-stimulator ICOS by recombinant oncolytic virus promotes systemic anti-tumour immunity

    PubMed Central

    Zamarin, Dmitriy; Holmgaard, Rikke B.; Ricca, Jacob; Plitt, Tamar; Palese, Peter; Sharma, Padmanee; Merghoub, Taha; Wolchok, Jedd D.; Allison, James P.

    2017-01-01

    Emerging data suggest that locoregional cancer therapeutic approaches with oncolytic viruses can lead to systemic anti-tumour immunity, although the appropriate targets for intratumoral immunomodulation using this strategy are not known. Here we find that intratumoral therapy with Newcastle disease virus (NDV), in addition to the activation of innate immunity, upregulates the expression of T-cell co-stimulatory receptors, with the inducible co-stimulator (ICOS) being most notable. To explore ICOS as a direct target in the tumour, we engineered a recombinant NDV-expressing ICOS ligand (NDV-ICOSL). In the bilateral flank tumour models, intratumoral administration of NDV-ICOSL results in enhanced infiltration with activated T cells in both virus-injected and distant tumours, and leads to effective rejection of both tumours when used in combination with systemic CTLA-4 blockade. These findings highlight that intratumoral immunomodulation with an oncolytic virus expressing a rationally selected ligand can be an effective strategy to drive systemic efficacy of immune checkpoint blockade. PMID:28194010

  13. Gamma interferon-induced, nitric oxide-mediated inhibition of vaccinia virus replication.

    PubMed Central

    Harris, N; Buller, R M; Karupiah, G

    1995-01-01

    Gamma interferon (IFN-gamma)-induced nitric oxide synthase (iNOS) and nitric oxide (NO) production in the murine macrophage-like RAW 264.7 cells were previously shown to inhibit the replication of the poxviruses vaccinia virus (VV) and ectromelia virus and herpes simplex virus type 1. In the current study, we performed biochemical analyses to determine the stage in the viral life cycle blocked by IFN-gamma-induced NO. Antibodies specific for temporally expressed viral proteins, a VV-specific DNA probe, and transmission electron microscopy were used to show that the cytokine-induced NO inhibited late protein synthesis, DNA replication, and virus particle formation but not expression of the early proteins analyzed. Essentially similar results were obtained with hydroxyurea and cytosine arabinoside, inhibitors of DNA replication. Enzymatically active iNOS was detected in the lysates of IFN-gamma-treated but not in untreated RAW 264.7 cells. The IFN-gamma-treated RAW 264.7 cells which express iNOS not only were resistant to productive infection but also efficiently blocked the replication of VV in infected bystander cells of epithelial origin. This inhibition was arginine dependent, correlated with nitric production in cultures, and was reversible by the NOS inhibitor N omega-monomethyl-L-arginine. PMID:7529336

  14. Generation of an attenuated Tiantan vaccinia virus by deletion of the ribonucleotide reductase large subunit.

    PubMed

    Kan, Shifu; Jia, Peng; Sun, Lili; Hu, Ningning; Li, Chang; Lu, Huijun; Tian, Mingyao; Qi, Yanxin; Jin, Ningyi; Li, Xiao

    2014-09-01

    Attenuation of the virulence of vaccinia Tiantan virus (VTT) underlies the strategy adopted for mass vaccination campaigns. This strategy provides advantages of safety and efficacy over traditional vaccines and is aimed at minimization of adverse health effects. In this study, a mutant form of the virus, MVTT was derived from VTT by deletion of the ribonucleotide reductase large subunit (R1) (TI4L). Compared to wild-type parental (VTT) and revertant (VTT-rev) viruses, virulence of the mutant MVTT was reduced by 100-fold based on body weight reduction and by 3,200-fold based on determination of the intracranial 50% lethal infectious dose. However, the immunogenicity of MVTT was equivalent to that of the parental VTT. We also demonstrated that the TI4L gene is not required for efficient replication. These data support the conclusion that MVTT can be used as a replicating virus vector or as a platform for the development of vaccines against infectious diseases and for cancer therapy.

  15. Vaccinia virus Transmission through Experimentally Contaminated Milk Using a Murine Model.

    PubMed

    Rehfeld, Izabelle Silva; Guedes, Maria Isabel Maldonado Coelho; Fraiha, Ana Luiza Soares; Costa, Aristóteles Gomes; Matos, Ana Carolina Diniz; Fiúza, Aparecida Tatiane Lino; Lobato, Zélia Inês Portela

    2015-01-01

    Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects dairy cattle and humans. Previous studies have detected the presence of viable virus particles in bovine milk samples naturally and experimentally contaminated with VACV. However, it is not known whether milk contaminated with VACV could be a route of viral transmission. However, anti-Orthopoxvirus antibodies were detected in humans from BV endemic areas, whom had no contact with affected cows, which suggest that other VACV transmission routes are possible, such as consumption of contaminated milk and dairy products. Therefore, it is important to study the possibility of VACV transmission by contaminated milk. This study aimed to examine VACV transmission, pathogenesis and shedding in mice orally inoculated with experimentally contaminated milk. Thirty mice were orally inoculated with milk containing 107 PFU/ml of VACV, and ten mice were orally inoculated with uncontaminated milk. Clinical examinations were performed for 30 consecutive days, and fecal samples and oral swabs (OSs) were collected every other day. Mice were euthanized on predetermined days, and tissue and blood samples were collected. Nested-PCR, plaque reduction neutralization test (PRNT), viral isolation, histopathology, and immunohistochemistry (IHC) methods were performed on the collected samples. No clinical changes were observed in the animals. Viral DNA was detected in feces, blood, OSs and tissues, at least in one of the times tested. The lungs displayed moderate to severe interstitial lymphohistiocytic infiltrates, and only the heart, tonsils, tongue, and stomach did not show immunostaining at the IHC analysis. Neutralizing antibodies were detected at the 20th and 30th days post infection in 50% of infected mice. The results revealed that VACV contaminated milk could be a route of viral transmission in mice experimentally infected, showing systemic distribution and shedding through feces and oral mucosa, albeit

  16. From lesions to viral clones: biological and molecular diversity amongst autochthonous Brazilian vaccinia virus.

    PubMed

    Oliveira, Graziele; Assis, Felipe; Almeida, Gabriel; Albarnaz, Jonas; Lima, Maurício; Andrade, Ana Cláudia; Calixto, Rafael; Oliveira, Cairo; Diomedes Neto, José; Trindade, Giliane; Ferreira, Paulo César; Kroon, Erna Geessien; Abrahão, Jônatas

    2015-03-16

    Vaccinia virus (VACV) has had an important role for humanity because of its use during the smallpox eradication campaign. VACV is the etiologic agent of the bovine vaccinia (BV), an emerging zoonosis that has been associated with economic, social, veterinary and public health problems, mainly in Brazil and India. Despite the current and historical VACV importance, there is little information about its circulation, prevalence, origins and maintenance in the environment, natural reservoirs and diversity. Brazilian VACV (VACV-BR) are grouped into at least two groups based on genetic and biological diversity: group 1 (G1) and group 2 (G2). In this study, we went to the field and investigated VACV clonal diversity directly from exanthemous lesions, during BV outbreaks. Our results demonstrate that the G1 VACV-BR were more frequently isolated. Furthermore, we were able to co-detect the two variants (G1 and G2) in the same sample. Molecular and biological analysis corroborated previous reports and confirmed the co-circulation of two VACV-BR lineages. The detected G2 clones presented exclusive genetic and biological markers, distinct to reference isolates, including VACV-Western Reserve. Two clones presented a mosaic profile, with both G1 and G2 features based on the molecular analysis of A56R, A26L and C23L genes. Indeed, some SNPs and INDELs in A56R nucleotide sequences were observed among clones of the same virus population, maybe as a result of an increased mutation rate in a mixed population. These results provide information about the diversity profile in VACV populations, highlighting its importance to VACV evolution and maintenance in the environment.

  17. Vaccinia virus Transmission through Experimentally Contaminated Milk Using a Murine Model

    PubMed Central

    Rehfeld, Izabelle Silva; Guedes, Maria Isabel Maldonado Coelho; Fraiha, Ana Luiza Soares; Costa, Aristóteles Gomes; Matos, Ana Carolina Diniz; Fiúza, Aparecida Tatiane Lino; Lobato, Zélia Inês Portela

    2015-01-01

    Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects dairy cattle and humans. Previous studies have detected the presence of viable virus particles in bovine milk samples naturally and experimentally contaminated with VACV. However, it is not known whether milk contaminated with VACV could be a route of viral transmission. However, anti-Orthopoxvirus antibodies were detected in humans from BV endemic areas, whom had no contact with affected cows, which suggest that other VACV transmission routes are possible, such as consumption of contaminated milk and dairy products. Therefore, it is important to study the possibility of VACV transmission by contaminated milk. This study aimed to examine VACV transmission, pathogenesis and shedding in mice orally inoculated with experimentally contaminated milk. Thirty mice were orally inoculated with milk containing 107 PFU/ml of VACV, and ten mice were orally inoculated with uncontaminated milk. Clinical examinations were performed for 30 consecutive days, and fecal samples and oral swabs (OSs) were collected every other day. Mice were euthanized on predetermined days, and tissue and blood samples were collected. Nested-PCR, plaque reduction neutralization test (PRNT), viral isolation, histopathology, and immunohistochemistry (IHC) methods were performed on the collected samples. No clinical changes were observed in the animals. Viral DNA was detected in feces, blood, OSs and tissues, at least in one of the times tested. The lungs displayed moderate to severe interstitial lymphohistiocytic infiltrates, and only the heart, tonsils, tongue, and stomach did not show immunostaining at the IHC analysis. Neutralizing antibodies were detected at the 20th and 30th days post infection in 50% of infected mice. The results revealed that VACV contaminated milk could be a route of viral transmission in mice experimentally infected, showing systemic distribution and shedding through feces and oral mucosa, albeit

  18. From Lesions to Viral Clones: Biological and Molecular Diversity amongst Autochthonous Brazilian Vaccinia Virus

    PubMed Central

    Oliveira, Graziele; Assis, Felipe; Almeida, Gabriel; Albarnaz, Jonas; Lima, Maurício; Andrade, Ana Cláudia; Calixto, Rafael; Oliveira, Cairo; Neto, José Diomedes; Trindade, Giliane; Ferreira, Paulo César; Kroon, Erna Geessien; Abrahão, Jônatas

    2015-01-01

    Vaccinia virus (VACV) has had an important role for humanity because of its use during the smallpox eradication campaign. VACV is the etiologic agent of the bovine vaccinia (BV), an emerging zoonosis that has been associated with economic, social, veterinary and public health problems, mainly in Brazil and India. Despite the current and historical VACV importance, there is little information about its circulation, prevalence, origins and maintenance in the environment, natural reservoirs and diversity. Brazilian VACV (VACV-BR) are grouped into at least two groups based on genetic and biological diversity: group 1 (G1) and group 2 (G2). In this study, we went to the field and investigated VACV clonal diversity directly from exanthemous lesions, during BV outbreaks. Our results demonstrate that the G1 VACV-BR were more frequently isolated. Furthermore, we were able to co-detect the two variants (G1 and G2) in the same sample. Molecular and biological analysis corroborated previous reports and confirmed the co-circulation of two VACV-BR lineages. The detected G2 clones presented exclusive genetic and biological markers, distinct to reference isolates, including VACV-Western Reserve. Two clones presented a mosaic profile, with both G1 and G2 features based on the molecular analysis of A56R, A26L and C23L genes. Indeed, some SNPs and INDELs in A56R nucleotide sequences were observed among clones of the same virus population, maybe as a result of an increased mutation rate in a mixed population. These results provide information about the diversity profile in VACV populations, highlighting its importance to VACV evolution and maintenance in the environment. PMID:25785515

  19. IL-12 Is an Effective Adjuvant to Recombinant Vaccinia Virus-Based Tumor Vaccines

    PubMed Central

    Rao, Jay B.; Chamberlain, Ronald S.; Bronte, Vincenzo; Carroll, Miles W.; Irvine, Kari R.; Moss, Bernard; Rosenberg, Steven A.; Restifo, Nicholas P.

    2007-01-01

    A number of cytokines and costimulatory molecules involved in immune activation have recently been identified including IL-12, a heterodimeric cytokine that supports the development of cell-mediated immunity, and B7-1, a costimulatory molecule involved in the activation of T lymphocytes. We explored the use of these immunomodulants as molecularly defined adjuvants in the function of recombinant anticancer vaccines using a murine model adenocarcinoma, CT26, transduced with a model Ag, β-galactosidase (β-gal). Although IL-12 given alone to mice bearing tumors established for 3 days did not have consistent antitumor activity, a profound therapeutic effect was observed when IL-12 administration was combined with a recombinant vaccinia virus (rVV) encoding β-gal called VJS6. On the basis of the reported synergistic effects of IL-12 and the costimulatory molecule B7-1 (CD80) in vitro, we immunized mice with a double recombinant vaccinia encoding both the model tumor Ag the costimulatory molecule B7-1, designated B7-1β-gal rVV. The adjuvant administration of IL-12 after immunization with this virus significantly enhanced survival in tumor-bearing animals. T cell subset depletions demonstrated that the in vivo activity of IL-12 was largely independent of CD4+ T lymphocytes, whereas the in vivo activity of a B7-1 rVV required both CD4+ and CD8+ T cells to elicit maximal therapeutic effect. To our knowledge, this is the first description of B7-1 and IL-12 cooperation in vivo and represents a novel strategy to enhance the efficacy of recombinant anticancer vaccines. PMID:8617961

  20. Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.

    PubMed

    Marín-López, Alejandro; Ortego, Javier

    2016-01-01

    Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification.

  1. Expression and processing of human immunodeficiency virus type 1 gag and pol genes by cells infected with a recombinant vaccinia virus.

    PubMed Central

    Gowda, S D; Stein, B S; Steimer, K S; Engleman, E G

    1989-01-01

    Human cells infected with a recombinant vaccinia virus containing human immunodeficiency virus type 1 gag-pol genes produced large amounts of human immunodeficiency virus gag proteins beginning at 1 h and peaking at 48 h postinfection. We show that these polyproteins are processed accurately into mature forms and that the viral polymerase gene is encoded as a 160-kilodalton gag-pol fusion protein, most likely by translational frameshifting from the gag into the pol reading frame. Images PMID:2464705

  2. Immunogenicity of modified vaccinia virus Ankara expressing the hemagglutinin stalk domain of pandemic (H1N1) 2009 influenza virus.

    PubMed

    Di Mario, Giuseppina; Soprana, Elisa; Gubinelli, Francesco; Panigada, Maddalena; Facchini, Marzia; Fabiani, Concetta; Garulli, Bruno; Basileo, Michela; Cassone, Antonio; Siccardi, Antonio; Donatelli, Isabella; Castrucci, Maria R

    2017-03-01

    Vaccination offers protection against influenza, although current vaccines need to be reformulated each year. The development of a broadly protective influenza vaccine would guarantee the induction of heterosubtypic immunity also against emerging influenza viruses of a novel subtype. Vaccine candidates based on the stalk region of the hemagglutinin (HA) have the potential to induce broad and persistent protection against diverse influenza A viruses. Modified vaccinia virus Ankara (MVA) expressing a headless HA (hlHA) of A/California/4/09 (CA/09) virus was used as a vaccine to immunize C57BL/6 mice. Specific antibody and cell-mediated immune responses were determined, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus. Immunization of mice with CA/09-derived hlHA, vectored by MVA, was able to elicit influenza-specific broad cross-reactive antibodies and cell-mediated immune responses, but failed to induce neutralizing antibodies and did not protect mice against virus challenge. Although highly immunogenic, our vaccine was unable to induce a protective immunity against influenza. A misfolded and unstable conformation of the hlHA molecule may have affected its capacity of inducing neutralizing antiviral, conformational antibodies. Design of stable hlHA-based immunogens and their delivery by recombinant MVA-based vectors has the potential of improving this promising approach for a universal influenza vaccine.

  3. Overexpression of IL-1α in skin differentially modulates the immune response to scarification with vaccinia virus

    PubMed Central

    Tian, Tian; Liu, Luzheng; Freyschmidt, Eva-Jasmin; Murphy, George F.; Kupper, Thomas S.; Fuhlbrigge, Robert C.

    2010-01-01

    Transepidermal inoculation of vaccinia virus (VV), or scarification, has been used effectively for the induction of specific and long-lasting immunity to smallpox and is superior to other routes of immunization. Scarification of individuals with atopic skin disease or immune deficiency, however, can lead to persistent viral replication and cause significant morbidity and mortality. These effects of scarification presumably reflect the unique immunologic properties of skin and the immune cells resident in, or recruited to, the site of inoculation. To explore these phenomena, we utilized transgenic mice engineered to over-express interleukin-1α, a critical mediator of cutaneous inflammation, in the epidermis. Following scarification with vaccinia virus, both transgenic and wild-type mice develop local pox. At high doses of vaccinia virus, IL-1α transgenic mice recruited immune cells to the inoculation site more rapidly and demonstrated enhanced T-cell and humoral immune responses. At limiting doses, however, IL-1α transgenic mice could effectively control virus replication without formation of pox lesions or activation of a memory response. This study suggests IL-1 may have use as an adjuvant to enhance antiviral immunity and promote safer vaccination strategies; however, understanding the balance of IL-1 effects on innate and adaptive immune functions will be critical to achieve optimal results. PMID:18615110

  4. Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22.

    PubMed

    Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen; Ping, Yueh-Hsin; Chang, Wen

    2015-08-01

    Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal

  5. Isolation and identification of compounds from Kalanchoe pinnata having human alphaherpesvirus and vaccinia virus antiviral activity.

    PubMed

    Cryer, Matthew; Lane, Kyle; Greer, Mary; Cates, Rex; Burt, Scott; Andrus, Merritt; Zou, Jiping; Rogers, Paul; Hansen, Marc D H; Burgado, Jillybeth; Panayampalli, Subbian Satheshkumar; Day, Craig W; Smee, Donald F; Johnson, Brent F

    2017-12-01

    Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) is a succulent plant that is known for its traditional antivirus and antibacterial usage. This work examines two compounds identified from the K. pinnata plant for their antivirus activity against human alphaherpesvirus (HHV) 1 and 2 and vaccinia virus (VACV). Compounds KPB-100 and KPB-200 were isolated using HPLC and were identified using NMR and MS. Both compounds were tested in plaque reduction assay of HHV-2 wild type (WT) and VACV. Both compounds were then tested in virus spread inhibition and virus yield reduction (VYR) assays of VACV. KPB-100 was further tested in viral cytopathic effect (CPE) inhibition assay of HHV-2 TK-mutant and VYR assay of HHV-1 WT. KPB-100 and KPB-200 inhibited HHV-2 at IC50 values of 2.5 and 2.9 μg/mL, respectively, and VACV at IC50 values of 3.1 and 7.4 μg/mL, respectively, in plaque reduction assays. In virus spread inhibition assay of VACV KPB-100 and KPB-200 yielded IC50 values of 1.63 and 13.2 μg/mL, respectively, and KPB-100 showed a nearly 2-log reduction in virus in VYR assay of VACV at 20 μg/mL. Finally, KPB-100 inhibited HHV-2 TK- at an IC50 value of 4.5 μg/mL in CPE inhibition assay and HHV-1 at an IC90 of 3.0 μg/mL in VYR assay. Both compounds are promising targets for synthetic optimization and in vivo study. KPB-100 in particular showed strong inhibition of all viruses tested.

  6. A role for vaccinia virus protein C16 in reprogramming cellular energy metabolism

    PubMed Central

    Mazzon, Michela; Castro, Cecilia; Roberts, Lee D.; Griffin, Julian L.

    2015-01-01

    Vaccinia virus (VACV) is a large DNA virus that replicates in the cytoplasm and encodes about 200 proteins of which approximately 50 % may be non-essential for viral replication. These proteins enable VACV to suppress transcription and translation of cellular genes, to inhibit the innate immune response, to exploit microtubule- and actin-based transport for virus entry and spread, and to subvert cellular metabolism for the benefit of the virus. VACV strain WR protein C16 induces stabilization of the hypoxia-inducible transcription factor (HIF)-1α by binding to the cellular oxygen sensor prolylhydroxylase domain-containing protein (PHD)2. Stabilization of HIF-1α is induced by several virus groups, but the purpose and consequences are unclear. Here, 1H-NMR spectroscopy and liquid chromatography-mass spectrometry are used to investigate the metabolic alterations during VACV infection in HeLa and 2FTGH cells. The role of C16 in such alterations was examined by comparing infection to WT VACV (strain WR) and a derivative virus lacking gene C16L (vΔC16). Compared with uninfected cells, VACV infection caused increased nucleotide and glutamine metabolism. In addition, there were increased concentrations of glutamine derivatives in cells infected with WT VACV compared with vΔC16. This indicates that C16 contributes to enhanced glutamine metabolism and this may help preserve tricarboxylic acid cycle activity. These data show that VACV infection reprogrammes cellular energy metabolism towards increased synthesis of the metabolic precursors utilized during viral replication, and that C16 contributes to this anabolic reprogramming of the cell, probably via the stabilization of HIF-1α. PMID:25351724

  7. A role for vaccinia virus protein C16 in reprogramming cellular energy metabolism.

    PubMed

    Mazzon, Michela; Castro, Cecilia; Roberts, Lee D; Griffin, Julian L; Smith, Geoffrey L

    2015-02-01

    Vaccinia virus (VACV) is a large DNA virus that replicates in the cytoplasm and encodes about 200 proteins of which approximately 50 % may be non-essential for viral replication. These proteins enable VACV to suppress transcription and translation of cellular genes, to inhibit the innate immune response, to exploit microtubule- and actin-based transport for virus entry and spread, and to subvert cellular metabolism for the benefit of the virus. VACV strain WR protein C16 induces stabilization of the hypoxia-inducible transcription factor (HIF)-1α by binding to the cellular oxygen sensor prolylhydroxylase domain-containing protein (PHD)2. Stabilization of HIF-1α is induced by several virus groups, but the purpose and consequences are unclear. Here, (1)H-NMR spectroscopy and liquid chromatography-mass spectrometry are used to investigate the metabolic alterations during VACV infection in HeLa and 2FTGH cells. The role of C16 in such alterations was examined by comparing infection to WT VACV (strain WR) and a derivative virus lacking gene C16L (vΔC16). Compared with uninfected cells, VACV infection caused increased nucleotide and glutamine metabolism. In addition, there were increased concentrations of glutamine derivatives in cells infected with WT VACV compared with vΔC16. This indicates that C16 contributes to enhanced glutamine metabolism and this may help preserve tricarboxylic acid cycle activity. These data show that VACV infection reprogrammes cellular energy metabolism towards increased synthesis of the metabolic precursors utilized during viral replication, and that C16 contributes to this anabolic reprogramming of the cell, probably via the stabilization of HIF-1α.

  8. Myxoma virus sensitizes cancer cells to gemcitabine and is an effective oncolytic virotherapeutic in models of disseminated pancreatic cancer.

    PubMed

    Wennier, Sonia Tusell; Liu, Jia; Li, Shoudong; Rahman, Masmudur M; Mona, Mahmoud; McFadden, Grant

    2012-04-01

    Myxoma virus (MYXV) is a novel oncolytic virus that has been shown to replicate in pancreatic cancer cells, but its efficacy in animal models of pancreatic cancer has not been determined. The efficacy of MYXV as monotherapy or in combination with gemcitabine was evaluated in intraperitoneal dissemination (IPD) models of pancreatic cancer. The effects of an intact immune system on the efficacy of MYXV therapy was tested by comparing immunodeficient versus immunocompetent murine models and combination therapy with gemcitabine was also evaluated. In cell culture, MYXV replication was robust in a broad range of pancreatic cancer cells and also showed increased oncolysis in combination with gemcitabine. In animal models, MYXV treatment conferred survival benefits over control or gemcitabine-treated cohorts regardless of the cell line or animal model used. MYXV monotherapy was most effective in an immunocompetent IPD model, and resulted in 60% long-term survivors. In Pan02 engrafted immunocompetent IPD models, sequential treatment in which MYXV was administered first, followed by gemcitabine, was the most effective and resulted in 100% long-term survivors. MYXV is an effective oncolytic virus for pancreatic cancer and can be combined with gemcitabine to enhance survival, particularly in the presence of an intact host immune system.

  9. Sensitivity of human pleural mesothelioma to oncolytic measles virus depends on defects of the type I interferon response

    PubMed Central

    Achard, Carole; Boisgerault, Nicolas; Delaunay, Tiphaine; Roulois, David; Nedellec, Steven; Royer, Pierre-Joseph; Pain, Mallory; Combredet, Chantal; Mesel-Lemoine, Mariana; Cellerin, Laurent; Magnan, Antoine; Tangy, Frédéric; Grégoire, Marc; Fonteneau, Jean-François

    2015-01-01

    Attenuated measles virus (MV) is currently being evaluated as an oncolytic virus in clinical trials and could represent a new therapeutic approach for malignant pleural mesothelioma (MPM). Herein, we screened the sensitivity to MV infection and replication of twenty-two human MPM cell lines and some healthy primary cells. We show that MV replicates in fifteen of the twenty-two MPM cell lines. Despite overexpression of CD46 by a majority of MPM cell lines compared to healthy cells, we found that the sensitivity to MV replication did not correlate with this overexpression. We then evaluated the antiviral type I interferon (IFN) responses of MPM cell lines and healthy cells. We found that healthy cells and the seven insensitive MPM cell lines developed a type I IFN response in presence of the virus, thereby inhibiting replication. In contrast, eleven of the fifteen sensitive MPM cell lines were unable to develop a complete type I IFN response in presence of MV. Finally, we show that addition of type I IFN onto MV sensitive tumor cell lines inhibits replication. These results demonstrate that defects in type I IFN response are frequent in MPM and that MV takes advantage of these defects to exert oncolytic activity. PMID:26539644

  10. RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

    SciTech Connect

    Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K.; James, John; Prescott, Alan; Haga, Ismar R.; Beard, Philippa M.

    2015-01-15

    Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.

  11. Endoplasmic Reticulum-Golgi Intermediate Compartment Membranes and Vimentin Filaments Participate in Vaccinia Virus Assembly

    PubMed Central

    Risco, Cristina; Rodríguez, Juan R.; López-Iglesias, Carmen; Carrascosa, José L.; Esteban, Mariano; Rodríguez, Dolores

    2002-01-01

    Vaccinia virus (VV) has a complex morphogenetic pathway whose first steps are poorly characterized. We have studied the early phase of VV assembly, when viral factories and spherical immature viruses (IVs) form in the cytoplasm of the infected cell. After freeze-substitution numerous cellular elements are detected around assembling viruses: membranes, ribosomes, microtubules, filaments, and unidentified structures. A double membrane is clearly resolved in the VV envelope for the first time, and freeze fracture reveals groups of tubules interacting laterally on the surface of the viroplasm foci. These data strongly support the hypothesis of a cellular tubulovesicular compartment, related to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), as the origin of the first VV envelope. Moreover, the cytoskeletal vimentin intermediate filaments are found around viral factories and inside the viroplasm foci, where vimentin and the VV core protein p39 colocalize in the areas where crescents protrude. Confocal microscopy showed that ERGIC elements and vimentin filaments concentrate in the viral factories. We propose that modified cellular ERGIC membranes and vimentin intermediate filaments act coordinately in the construction of viral factories and the first VV form through a unique mechanism of viral morphogenesis from cellular elements. PMID:11799179

  12. Human CD4 T cell epitopes selective for Vaccinia versus Variola virus.

    PubMed

    Probst, Alicia; Besse, Aurore; Favry, Emmanuel; Imbert, Gilles; Tanchou, Valérie; Castelli, Florence Anne; Maillere, Bernard

    2013-04-01

    Due to the high degree of sequence identity between Orthopoxvirus species, the specific B and T cell responses raised against these viruses are largely cross-reactive and poorly selective. We therefore searched for CD4 T cell epitopes present in the conserved parts of the Vaccinia genome (VACV) but absent from Variola viruses (VARV), with a view to identifying immunogenic sequences selective for VACV. We identified three long peptide fragments from the B7R, B10R and E7R proteins by in silico comparisons of the poxvirus genomes, and evaluated the recognition of these fragments by VACV-specific T cell lines derived from healthy donors. For the 12 CD4 T cell epitopes identified, we assessed their binding to common HLA-DR allotypes and their capacity to induce peptide-specific CD4 T-cell lines. Four peptides from B7R and B10R displayed a broad binding specificity for HLA-DR molecules and induced multiple T cell lines from healthy donors. Besides their absence from VARV, the two B10R peptide sequences were mutated in the Cowpox virus and completely absent from the Monkeypox genome. This work contributes to the development of differential diagnosis of poxvirus infections. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Vaccinia virus A11 is required for membrane rupture and viral membrane assembly.

    PubMed

    Suarez, Cristina; Hoppe, Simone; Pénard, Esthel; Walther, Paul; Krijnse-Locker, Jacomine

    2017-10-01

    Although most enveloped viruses acquire their membrane from the host by budding or by a wrapping process, collective data argue that nucleocytoplasmic large DNA viruses (NCLDVs) may be an exception. The prototype member of NCLDVs, vaccinia virus (VACV) may induce rupture of endoplasmic-reticulum-derived membranes to build an open-membrane sphere that closes after DNA uptake. This unconventional membrane assembly pathway is also used by at least 3 other members of the NCLDVs. In this study, we identify the VACV gene product of A11, as required for membrane rupture, hence for VACV membrane assembly and virion formation. By electron tomography, in the absence of A11, the site of assembly formed by the viral scaffold protein D13 is surrounded by endoplasmic reticulum cisternae that are closed. We use scanning transmission electron microscopy-electron tomography to analyse large volumes of cells and demonstrate that in the absence of A11, no open membranes are detected. Given the pivotal role of D13 in initiating VACV membrane assembly, we also analyse viral membranes in the absence of D13 synthesis and show that this protein is not required for rupture. Finally, consistent with a role in rupture, we show that during wild-type infection, A11 localises predominantly to the small ruptured membranes, the precursors of VACV membrane assembly. These data provide strong evidence in favour of the unusual membrane biogenesis of VACV and are an important step towards understanding its molecular mechanism. © 2017 John Wiley & Sons Ltd.

  14. DNA and Modified Vaccinia Virus Ankara Vaccines Encoding Multiple Cytotoxic and Helper T-Lymphocyte Epitopes of Human Immunodeficiency Virus Type 1 (HIV-1) Are Safe but Weakly Immunogenic in HIV-1-Uninfected, Vaccinia Virus-Naive Adults

    PubMed Central

    Newman, Mark J.; deCamp, Allan; Hay, Christine Mhorag; De Rosa, Stephen C.; Noonan, Elizabeth; Livingston, Brian D.; Fuchs, Jonathan D.; Kalams, Spyros A.; Cassis-Ghavami, Farah L.

    2012-01-01

    We evaluated a DNA plasmid-vectored vaccine and a recombinant modified vaccinia virus Ankara vaccine (MVA-mBN32), each encoding cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) in a randomized, double-blinded, placebo-controlled trial in 36 HIV-1-uninfected adults using a heterologous prime-boost schedule. HIV-1-specific cellular immune responses, measured as interleukin-2 and/or gamma interferon production, were induced in 1 (4%) of 28 subjects after the first MVA-mBN32 immunization and in 3 (12%) of 25 subjects after the second MVA-mBN32 immunization. Among these responders, polyfunctional T-cell responses, including the production of tumor necrosis factor alpha and perforin, were detected. Vaccinia virus-specific antibodies were induced to the MVA vector in 27 (93%) of 29 and 26 (93%) of 28 subjects after the first and second immunizations with MVA-mBN32. These peptide-based vaccines were safe but were ineffective at inducing HIV-1-specific immune responses and induced much weaker responses than MVA vaccines expressing the entire open reading frames of HIV-1 proteins. PMID:22398243

  15. DNA and modified vaccinia virus Ankara vaccines encoding multiple cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) are safe but weakly immunogenic in HIV-1-uninfected, vaccinia virus-naive adults.

    PubMed

    Gorse, Geoffrey J; Newman, Mark J; deCamp, Allan; Hay, Christine Mhorag; De Rosa, Stephen C; Noonan, Elizabeth; Livingston, Brian D; Fuchs, Jonathan D; Kalams, Spyros A; Cassis-Ghavami, Farah L

    2012-05-01

    We evaluated a DNA plasmid-vectored vaccine and a recombinant modified vaccinia virus Ankara vaccine (MVA-mBN32), each encoding cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) in a randomized, double-blinded, placebo-controlled trial in 36 HIV-1-uninfected adults using a heterologous prime-boost schedule. HIV-1-specific cellular immune responses, measured as interleukin-2 and/or gamma interferon production, were induced in 1 (4%) of 28 subjects after the first MVA-mBN32 immunization and in 3 (12%) of 25 subjects after the second MVA-mBN32 immunization. Among these responders, polyfunctional T-cell responses, including the production of tumor necrosis factor alpha and perforin, were detected. Vaccinia virus-specific antibodies were induced to the MVA vector in 27 (93%) of 29 and 26 (93%) of 28 subjects after the first and second immunizations with MVA-mBN32. These peptide-based vaccines were safe but were ineffective at inducing HIV-1-specific immune responses and induced much weaker responses than MVA vaccines expressing the entire open reading frames of HIV-1 proteins.

  16. Vaccinia viruses with mutations in the E3L gene as potential replication-competent, attenuated vaccines: scarification vaccination.

    PubMed

    Jentarra, Garilyn M; Heck, Michael C; Youn, Jin Won; Kibler, Karen; Langland, Jeffrey O; Baskin, Carole R; Ananieva, Olga; Chang, Yung; Jacobs, Bertram L

    2008-06-02

    In this study, we evaluated the efficacy of vaccinia virus (VACV) containing mutations in the E3L virulence gene to protect mice against a lethal poxvirus challenge after vaccination by scarification. VACV strains with mutations in the E3L gene had significantly decreased pathogenicity, even in immune deficient mice, yet retained the ability to produce a potent Th1-dominated immune response in mice after vaccination by scarification, while protecting against challenge with wild type, pathogenic VACV. Initial experiments were done using the mouse-adapted, neurovirulent Western Reserve (WR) strain of vaccinia virus. Testing of the full E3L deletion mutation in the Copenhagen and NYCBH strains of VACV, which are more appropriate for use in humans, produced similar results. These results suggest that highly attenuated strains of VACV containing mutations in E3L have the potential for use as scarification administered vaccines.

  17. Vaccinia Viruses with Mutations in the E3L Gene as Potential Replication-Competent, Attenuated Vaccines: Scarification Vaccination

    PubMed Central

    Jentarra, Garilyn M.; Heck, Michael C.; Youn, Jin Won; Kibler, Karen; Langland, Jeffrey O.; Baskin, Carole R.; Ananieva, Olga; Chang, Yung; Jacobs, Bertram L.

    2008-01-01

    In this study, we evaluated the efficacy of vaccinia virus (VACV) containing mutations in the E3L virulence gene to protect mice against a lethal poxvirus challenge after vaccination by scarification. VACV strains with mutations in the E3L gene had significantly decreased pathogenicity, even in immune deficient mice, yet retained the ability to produce a potent Th1-dominated immune response in mice after vaccination by scarification, while protecting against challenge with wild type, pathogenic VACV. Initial experiments were done using the mouse-adapted, neurovirulent Western Reserve (WR) strain of vaccinia virus. Testing of the full E3L deletion mutation in the Copenhagen and NYCBH strains of VACV, which are more appropriate for use in humans, produced similar results. These results suggest that highly attenuated strains of VACV containing mutations in E3L have the potential for use as scarification administered vaccines. PMID:18455281

  18. Local Production of Tumor Necrosis Factor Encoded by Recombinant Vaccinia Virus is Effective in Controlling Viral Replication in vivo

    NASA Astrophysics Data System (ADS)

    Sambhi, Sharan K.; Kohonen-Corish, Maija R. J.; Ramshaw, Ian A.

    1991-05-01

    Tumor necrosis factor (TNF) has pleiotropic effects on a wide variety of cell types. In vitro studies have demonstrated that TNF has antiviral properties and is induced in response to viral infections. However, a role for TNF in the antiviral immune response of the host has yet to be demonstrated. Here we describe the construction of and studies using a recombinant vaccinia virus that encodes the gene for murine TNF-α. By comparing the replication of and immune responses elicited by the TNF-encoding virus to a similarly constructed control virus, we hoped to observe immunobiological effects of TNF in the host. The in vivo experiments with this recombinant virus demonstrate that the localized production of TNF-α during a viral infection leads to the rapid and efficient clearance of the virus in normal mice and attenuates the otherwise lethal pathogenicity of the virus in immunodeficient animals. This attenuation occurs early in the infection (by postinfection hour 24) and is not due to the enhancement of cellular or antibody responses by the vaccinia virus-encoded TNF. This evidence suggests that attenuation of the recombinant virus is due to a direct antiviral effect of TNF on cells at the site of infection. Therefore, these results support the suggestion that TNF produced by immune cells may be an important effector mechanism of viral clearance in vivo.

  19. Local production of tumor necrosis factor encoded by recombinant vaccinia virus is effective in controlling viral replication in vivo.

    PubMed Central

    Sambhi, S K; Kohonen-Corish, M R; Ramshaw, I A

    1991-01-01

    Tumor necrosis factor (TNF) has pleiotropic effects on a wide variety of cell types. In vitro studies have demonstrated that TNF has antiviral properties and is induced in response to viral infections. However, a role for TNF in the antiviral immune response of the host has yet to be demonstrated. Here we describe the construction of and studies using a recombinant vaccinia virus that encodes the gene for murine TNF-alpha. By comparing the replication of and immune responses elicited by the TNF-encoding virus to a similarly constructed control virus, we hoped to observe immunobiological effects of TNF in the host. The in vivo experiments with this recombinant virus demonstrate that the localized production of TNF-alpha during a viral infection leads to the rapid and efficient clearance of the virus in normal mice and attenuates the otherwise lethal pathogenicity of the virus in immunodeficient animals. This attenuation occurs early in the infection (by postinfection hour 24) and is not due to the enhancement of cellular or antibody responses by the vaccinia virus-encoded TNF. This evidence suggests that attenuation of the recombinant virus is due to a direct antiviral effect of TNF on cells at the site of infection. Therefore, these results support the suggestion that TNF produced by immune cells may be an important effector mechanism of viral clearance in vivo. Images PMID:2023951

  20. Cell-based delivery of oncolytic viruses: a new strategic alliance for a biological strike against cancer.

    PubMed

    Power, Anthony T; Bell, John C

    2007-04-01

    Recent years have seen tremendous advances in the development of exquisitely targeted replicating virotherapeutics that can safely destroy malignant cells. Despite this promise, clinical advancement of this powerful and unique approach has been hindered by vulnerability to host defenses and inefficient systemic delivery. However, it now appears that delivery of oncolytic viruses within carrier cells may offer one solution to this critical problem. In this review, we compare the advantages and limitations of the numerous cell lineages that have been investigated as delivery platforms for viral therapeutics, and discuss examples showing how combined cell-virus biotherapeutics can be used to achieve synergistic gains in antitumor activity. Finally, we highlight avenues for future preclinical research that might be taken in order to refine cell-virus biotherapeutics in preparation for human trials.

  1. Comparison of the Cowpox Virus and Vaccinia Virus Mature Virion Proteome: Analysis of the Species- and Strain-Specific Proteome

    PubMed Central

    Doellinger, Joerg; Schaade, Lars; Nitsche, Andreas

    2015-01-01

    Cowpox virus (CPXV) causes most zoonotic orthopoxvirus (OPV) infections in Europe and Northern as well as Central Asia. The virus has the broadest host range of OPV and is transmitted to humans from rodents and other wild or domestic animals. Increasing numbers of human CPXV infections in a population with declining immunity have raised concerns about the virus’ zoonotic potential. While there have been reports on the proteome of other human-pathogenic OPV, namely vaccinia virus (VACV) and monkeypox virus (MPXV), the protein composition of the CPXV mature virion (MV) is unknown. This study focused on the comparative analysis of the VACV and CPXV MV proteome by label-free single-run proteomics using nano liquid chromatography and high-resolution tandem mass spectrometry (nLC-MS/MS). The presented data reveal that the common VACV and CPXV MV proteome contains most of the known conserved and essential OPV proteins and is associated with cellular proteins known to be essential for viral replication. While the species-specific proteome could be linked mainly to less genetically-conserved gene products, the strain-specific protein abundance was found to be of high variance in proteins associated with entry, host-virus interaction and protein processing. PMID:26556597

  2. [Modified vaccinia virus ankara (MVA)--development as recombinant vaccine and prospects for use in veterinary medicine].

    PubMed

    Volz, Asisa; Fux, Robert; Langenmayer, Martin C; Sutter, Gerd

    2015-01-01

    Poxviruses as expression vectors are widely used in medical research for the development of recombinant vaccines and molecular therapies. Here we review recent accomplishments in vaccine research using recombinant modified vaccinia virus ankara (MVA). MVA is a highly attenuated vaccinia virus strain that originated from serial tissue culture passage in chicken embryo fibroblasts more than 40 years ago. Growth adaptation to avian host cells caused deletions and mutations in the viral genome affecting about 15% of the original genetic information. In consequence, MVA is replication-deficient in cells of mammalian origin and fails to produce many of the virulence factors encoded by conventional vaccinia virus. Because of its safety for the general environment MVA can be handled under conditions of biosafety level one. Non-replicating MVA can enter any target cell and activate its molecular life cycle to express all classes of viral and recombinant genes. Therefore, recombinant MVA have been established as an extremely safe and efficient vector system for vaccine development in medical research. By now, various recombinant MVA vaccines have been found safe and immunogenic when used for phase I/II clinical testing in humans, and suitable for industrial scale production following good practice of manufacturing. Thus, there is an obvious usefulness of recombinant MVA vaccines for novel prophylactic and therapeutic approaches also in veterinary medicine. Results from first studies in companion and farm animals are highly promising.

  3. Biosafety aspects of modified vaccinia virus Ankara (MVA)-based vectors used for gene therapy or vaccination.

    PubMed

    Verheust, Céline; Goossens, Martine; Pauwels, Katia; Breyer, Didier

    2012-03-30

    The modified vaccinia virus Ankara (MVA) strain is a highly attenuated strain of vaccinia virus that has been demonstrated to be safe for humans. MVA is widely considered as the vaccinia virus strain of choice for clinical investigation because of its high safety profile. It also represents an excellent candidate for use as vector system in recombinant vaccine development for gene delivery or vaccination against infectious diseases or tumours, even in immunocompromised individuals. The use of MVA and recombinant MVA vectors must comply with various regulatory requirements, particularly relating to the assessment of potential risks for human health and the environment. The purpose of the present paper is to highlight some biological characteristics of MVA and MVA-based recombinant vectors and to discuss these from a biosafety point of view in the context of the European regulatory framework for genetically modified organisms with emphasis on the assessment of potential risks associated with environmental release. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. [Current state of oncolytic virotherapy in Japan].

    PubMed

    Nakamori, Mikihito; Yamaue, Hiroki

    2013-05-01

    Oncolytic virotherapy is an emerging treatment strategy that uses replication-competent viruses to destroy cancers. Recent advances include preclinical proof of feasibility for a single-shot virotherapy cure, identification of drugs that accelerate intratumoral virus propagation, and strategies to maximize the immunotherapeutic action of oncolytic viruses. The primary clinical milestone has been completion of accrual in a phase 3 trial of intratumoral herpes simplex virus therapy using OncoVEX for metastatic melanoma. In Japan, clinical treatments such as oncolytic adenoviruses(OBP-301)for esophageal cancer and oncolytic herpes simplex viruses(G47b)for brain cancer have accelerated considerably. We hope that a steady stream of new oncolytic viruses will enter the clinical arena in our country.

  5. In Vivo Safety, Biodistribution and Antitumor Effects of uPAR Retargeted Oncolytic Measles Virus in Syngeneic Cancer Models

    PubMed Central

    Jing, Yuqi; Zaias, Julia; Duncan, Robert; Russell, Stephen J.; Merchan, Jaime R.

    2014-01-01

    The urokinase receptor (uPAR) is a clinically relevant target for novel biological therapies. We have previously rescued oncolytic measles viruses fully retargeted against human (MV-h-uPA) or murine (MV-m-uPA) uPAR. Here, we investigated the in vivo effects of systemic administration of MV-m-uPA in immunocompetent cancer models. MV-m-uPA induced in vitro cytotoxicity and replicated in a receptor dependent manner in murine mammary (4T1), and colon (MC-38 and CT-26) cancer cells. Intravenous administration of MV-m-uPA to 4T1 tumor bearing mice was not associated with significant clinical or laboratory toxicity. Higher MV-N RNA copy numbers were detected in primary tumors, and viable viral particles were recovered from tumor bearing tissues only. Non-tumor bearing organs did not show histological signs of viral induced toxicity. Serum anti-MV antibodies were detected at day 14 of treatment. Immunohistochemistry and immunofluorescence studies confirmed successful tumor targeting and demonstrated enhanced MV-m-uPA induced tumor cell apoptosis in treated, compared to control mice. Significant antitumor effects and prolonged survival were observed after systemic administration of MV-m-uPA in colon (CT-26) and mammary (4T1) cancer models. The above results demonstrate safety and feasibility of uPAR targeting by an oncolytic virus, and confirm significant antitumor effects in highly aggressive syngeneic immunocompetent cancer models. PMID:24430235

  6. Ethacrynic and alpha-lipoic acids inhibit vaccinia virus late gene expression.

    PubMed

    Spisakova, Martina; Cizek, Zdenek; Melkova, Zora

    2009-02-01

    Smallpox was declared eradicated in 1980. However recently, the need of agents effective against poxvirus infection has emerged again. In this paper, we report an original finding that two redox-modulating agents, the ethacrynic and alpha-lipoic acids (EA, LA), inhibit growth of vaccinia virus (VACV) in vitro. The effect of EA and LA was compared with those of beta-mercaptoethanol, DTT and ascorbic acid, but these agents increased VACV growth in HeLa G cells. The inhibitory effects of EA and LA on the growth of VACV were further confirmed in several cell lines of different embryonic origin, in epithelial cells, fibroblasts, macrophages and T-lymphocytes. Finally, we have analyzed the mechanism of action of the two agents. They both decreased expression of VACV late genes, as demonstrated by western blot analysis and activity of luciferase expressed under control of different VACV promoters. In contrast, they did not inhibit virus entry into the cell, expression of VACV early genes or VACV DNA synthesis. The results suggest new directions in development of drugs effective against poxvirus infection.

  7. Spectrum of Physical Properties Among the Virions of a Whole Population of Vaccinia Virus Particles

    PubMed Central

    Sharp, D. G.; McGuire, Peter M.

    1970-01-01

    Populations of virions released from cultures of L cells infected with vaccinia virus are composed of particles which differ substantially from each other in sedimentation rate and buoyant density. Clumps of two and three virions sediment enough faster than single particles so that fractions containing only singles and others with predominantly pairs can be isolated. The observed velocity range for single particles is much greater than that attributable to diffusion and convection in the centrifuge. Plaquing efficiency is three times higher in a small fraction of the slowest-moving virions than in the major part of the population, even though no size difference can be seen in the electron microscope. Isopycnic densities in potassium tartrate range from 1.15 to 1.23, enough to account for the observed range in velocities. Centrifugation was done in the BXIV zonal rotor at very low virion concentration (less than 108 per ml at any point in the spectrum). Virus count and state of aggregation were determined by electron microscopy. Images PMID:5438106

  8. Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication.

    PubMed Central

    Rempel, R E; Anderson, M K; Evans, E; Traktman, P

    1990-01-01

    Vaccinia virus mutants ts2 and ts25, members of the same complementation group, exhibit a temperature-dependent arrest at the stage of viral DNA replication. The lesions responsible for the mutant phenotypes have been localized to the far left region of the HindIII B genomic fragment by marker rescue studies. Hybrid selection analyses established that the DNA fragments positive for rescue represented the first open reading frame of the HindIII B fragment and encoded a 30-kilodalton protein. The gene is expressed early after infection as a rightwardly transcribed 1-kilobase-pair mRNA whose coordinates were determined by S1 nuclease mapping. To further the phenotypic analysis of the mutants, the accumulation of viral DNA sequences during permissive and nonpermissive infections was quantitated. The extent of the DNA- phenotype was shown to vary in different cell types. In mouse L cells at either high or low multiplicity of infection, nonpermissive DNA synthesis was less than 5% of that seen in permissive infections. This severe defect was mirrored by correspondingly low viral yields. In infections of BSC40 monkey cells, however, the deficiencies in both DNA synthesis and virus production were far less severe. For one mutant (ts2), the temperature sensitivity in BSC40 cells varied inversely with the multiplicity of infection. Images PMID:2296077

  9. Genomic identification of human vaccinia virus keratoconjunctivitis and its importance as a laboratory-acquired infection

    PubMed Central

    Motlagh, Zahra Movahedi; Mokhtari, Azam; Mahzounieh, Mohammadreza

    2016-01-01

    Context: Vaccinia virus (VACV) is a member of orthopoxvirus genus of the family Poxviridae. VACVs are enveloped, double-stranded DNA viruses. Several species of this family, for example, molluscum contagiosum, smallpox, deerpox, horsepox, rabbitpox, and VACVs may cause conjunctivitis. Aims: Given the high incidence of keratoconjunctivitis in Iran (approximately 3.6%–53.9%) and insufficient clinical diagnostic measures, laboratory tests for detection of its causes and determination of accurate keratoconjunctivitis/conjunctivitis prevalence due to different pathogens are essential. Settings and Design: In this research, conjunctival samples collected from 100 patients with keratoconjunctivitis signs were referred to an eye hospital of Iran. Subjects and Methods: After DNA extraction, polymerase chain reaction (PCR) was carried out for detection of VACV. PCR-positive products were further subjected to DNA sequencing. Statistical Analysis Used: The results were analyzed using Chi-square test. Results: In this study, 28% of the samples were positive and a statistically significant relationship obtained between working in medical or research laboratories and VACV prevalence (P < 0.05). Conclusions: This study showed a high rate of VACV keratoconjunctivitis, and therefore, further studies for its prevention and control are necessary. PMID:27958202

  10. Vaccinia Virus Inhibits T Cell Receptor–Dependent Responses by Human γδ T Cells

    PubMed Central

    Li, Haishan; Deetz, Carl O.; Zapata, Juan Carlos; Cairo, Cristiana; Hebbeler, Andrew M.; Propp, Nadia; Salvato, Maria S.; Shao, Yiming; Pauza, C. David

    2008-01-01

    Vaccinia virus (VV) is an effective vaccine and vector but has evolved multiple mechanisms for evading host immunity. We characterized the interactions of VV (TianTan and New York City Board of Health strains) with human γδ T cells because of the role they play in immune control of this virus. Exposure to VV failed to trigger proliferative responses in γδ T cells from unprimed individuals, but it was an unexpected finding that VV blocked responses to model antigens by the Vγ2Vδ2 T cell subset. Infectious or ultraviolet light–inactivated VV inhibited proliferative Vγ2Vδ2 T cell responses to phosphoantigens and tumor cells, prevented cytolysis of Daudi B cells, and reduced cytokine production. Inhibiting Vγ2Vδ2 T cells may be a mechanism for evading host immunity and increasing VV virulence. Increased VV replication or expression in the absence of γδ T cell responses might contribute to its potency as a vaccine against poxvirus and recombinant antigens. PMID:17152007

  11. Vaccinia virus inhibits T cell receptor-dependent responses by human gammadelta T cells.

    PubMed

    Li, Haishan; Deetz, Carl O; Zapata, Juan Carlos; Cairo, Cristiana; Hebbeler, Andrew M; Propp, Nadia; Salvato, Maria S; Shao, Yiming; Pauza, C David

    2007-01-01

    Vaccinia virus (VV) is an effective vaccine and vector but has evolved multiple mechanisms for evading host immunity. We characterized the interactions of VV (TianTan and New York City Board of Health strains) with human gammadelta T cells because of the role they play in immune control of this virus. Exposure to VV failed to trigger proliferative responses in gammadelta T cells from unprimed individuals, but it was an unexpected finding that VV blocked responses to model antigens by the Vgamma2Vdelta2 T cell subset. Infectious or ultraviolet light-inactivated VV inhibited proliferative Vgamma2Vdelta2 T cell responses to phosphoantigens and tumor cells, prevented cytolysis of Daudi B cells, and reduced cytokine production. Inhibiting Vgamma2Vdelta2 T cells may be a mechanism for evading host immunity and increasing VV virulence. Increased VV replication or expression in the absence of gammadelta T cell responses might contribute to its potency as a vaccine against poxvirus and recombinant antigens.

  12. Genomic identification of human vaccinia virus keratoconjunctivitis and its importance as a laboratory-acquired infection.

    PubMed

    Motlagh, Zahra Movahedi; Mokhtari, Azam; Mahzounieh, Mohammadreza

    2016-11-01

    Vaccinia virus (VACV) is a member of orthopoxvirus genus of the family Poxviridae. VACVs are enveloped, double-stranded DNA viruses. Several species of this family, for example, molluscum contagiosum, smallpox, deerpox, horsepox, rabbitpox, and VACVs may cause conjunctivitis. Given the high incidence of keratoconjunctivitis in Iran (approximately 3.6%-53.9%) and insufficient clinical diagnostic measures, laboratory tests for detection of its causes and determination of accurate keratoconjunctivitis/conjunctivitis prevalence due to different pathogens are essential. In this research, conjunctival samples collected from 100 patients with keratoconjunctivitis signs were referred to an eye hospital of Iran. After DNA extraction, polymerase chain reaction (PCR) was carried out for detection of VACV. PCR-positive products were further subjected to DNA sequencing. The results were analyzed using Chi-square test. In this study, 28% of the samples were positive and a statistically significant relationship obtained between working in medical or research laboratories and VACV prevalence (P < 0.05). This study showed a high rate of VACV keratoconjunctivitis, and therefore, further studies for its prevention and control are necessary.

  13. High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus.

    PubMed Central

    Stunnenberg, H G; Lange, H; Philipson, L; van Miltenburg, R T; van der Vliet, P C

    1988-01-01

    Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system. Images PMID:3362670

  14. IL-12 Expressing oncolytic herpes simplex virus promotes anti-tumor activity and immunologic control of metastatic ovarian cancer in mice.

    PubMed

    Thomas, Eric D; Meza-Perez, Selene; Bevis, Kerri S; Randall, Troy D; Gillespie, G Yancey; Langford, Catherine; Alvarez, Ronald D

    2016-10-27

    Despite advances in surgical aggressiveness and conventional chemotherapy, ovarian cancer remains the most lethal cause of gynecologic cancer mortality; consequently there is a need for new therapeutic agents and innovative treatment paradigms for the treatment of ovarian cancer. Several studies have demonstrated that ovarian cancer is an immunogenic disease and immunotherapy represents a promising and novel approach that has not been completely evaluated in ovarian cancer. Our objective was to evaluate the anti-tumor activity of an oncolytic herpes simplex virus "armed" with murine interleukin-12 and its ability to elicit tumor-specific immune responses. We evaluated the ability of interleukin-12-expressing and control oncolytic herpes simplex virus to kill murine and human ovarian cancer cell lines in vitro. We also administered interleukin-12-expressing oncolytic herpes simplex virus to the peritoneal cavity of mice that had developed spontaneous, metastatic ovarian cancer and determined overall survival and tumor burden at 95 days. We used flow cytometry to quantify the tumor antigen-specific CD8(+) T cell response in the omentum and peritoneal cavity. All ovarian cancer cell lines demonstrated susceptibility to oncolytic herpes simplex virus in vitro. Compared to controls, mice treated with interleukin-12-expressing oncolytic herpes simplex virus demonstrated a more robust tumor antigen-specific CD8(+) T-cell immune response in the omentum (471.6 cells vs 33.1 cells; p = 0.02) and peritoneal cavity (962.3 cells vs 179.5 cells; p = 0.05). Compared to controls, mice treated with interleukin-12-expressing oncolytic herpes simplex virus were more likely to control ovarian cancer metastases (81.2 % vs 18.2 %; p = 0.008) and had a significantly longer overall survival (p = 0.02). Finally, five of 6 mice treated with interleukin-12-expressing oHSV had no evidence of metastatic tumor when euthanized at 6 months, compared to two of 4 mice treated

  15. Increased expression in vivo and in vitro of foreign genes directed by A-type inclusion body hybrid promoters in recombinant vaccinia viruses.

    PubMed Central

    Funahashi, S; Itamura, S; Iinuma, H; Nerome, K; Sugimoto, M; Shida, H

    1991-01-01

    We constructed A-type inclusion body (ATI) hybrid promoters, that is, late ATI promoters followed by tandemly repeated early regions of the promoter for the 7.5-kDa protein (the 7.5-kDa promoter). The repetition of the whole early promoter sequence of the 7.5-kDa gene, including the upstream consensus sequence and initiation region, efficiently increased the early expression of the bacterial chloramphenicol acetyltransferase gene in recombinant vaccinia virus. Recombinant vaccinia virus could express influenza virus hemagglutinin via the hybrid promoter more efficiently, induced higher levels of neutralizing antibody and cytotoxic T lymphocytes, and consequently protected mice more efficiently against challenge with influenza virus than did recombinant vaccinia virus containing the widely used 7.5-kDa promoter. Images PMID:1654453

  16. Involvement of the Cellular Phosphatase DUSP1 in Vaccinia Virus Infection

    PubMed Central

    Cáceres, Ana; Perdiguero, Beatriz; Gómez, Carmen E.; Cepeda, Maria Victoria; Caelles, Carme; Sorzano, Carlos Oscar; Esteban, Mariano

    2013-01-01

    Poxviruses encode a large variety of proteins that mimic, block or enhance host cell signaling pathways on their own benefit. It has been reported that mitogen-activated protein kinases (MAPKs) are specifically upregulated during vaccinia virus (VACV) infection. Here, we have evaluated the role of the MAPK negative regulator dual specificity phosphatase 1 (DUSP1) in the infection of VACV. We demonstrated that DUSP1 expression is enhanced upon infection with the replicative WR virus and with the attenuated VACV viruses MVA and NYVAC. This upregulation is dependent on early viral gene expression. In the absence of DUSP1 in cultured cells, there is an increased activation of its molecular targets JNK and ERK and an enhanced WR replication. Moreover, DUSP1 knock-out (KO) mice are more susceptible to WR infection as a result of enhanced virus replication in the lungs. Significantly, MVA, which is known to produce non-permissive infections in most mammalian cell lines, is able to grow in DUSP1 KO immortalized murine embryo fibroblasts (MEFs). By confocal and electron microscopy assays, we showed that in the absence of DUSP1 MVA morphogenesis is similar as in permissive cell lines and demonstrated that DUSP1 is involved at the stage of transition between IVN and MV in VACV morphogenesis. In addition, we have observed that the secretion of pro-inflammatory cytokines at early times post-infection in KO mice infected with MVA and NYVAC is increased and that the adaptive immune response is enhanced in comparison with WT-infected mice. Altogether, these findings reveal that DUSP1 is involved in the replication and host range of VACV and in the regulation of host immune responses through the modulation of MAPKs. Thus, in this study we demonstrate that DUSP1 is actively involved in the antiviral host defense mechanism against a poxvirus infection. PMID:24244156

  17. First North American field release of a vaccinia-rabies glycoprotein recombinant virus.

    PubMed

    Hanlon, C A; Niezgoda, M; Hamir, A N; Schumacher, C; Koprowski, H; Rupprecht, C E

    1998-04-01

    Following nearly 10 yr of extensive laboratory evaluation, a vaccinia-rabies glycoprotein (V-RG) vaccine was the first recombinant virus to undergo limited North American field release on 20 August 1990. The free-ranging raccoon population on Parramore Island (Virginia, USA) was exposed to a high density (10 baits/ha) of vaccine-laden baits distributed on a 300 ha vaccination area. An annual total of 887 raccoons were live-trapped for sedation, physical examination and blood collection for rabies antibody determination; there was no evidence of adverse effects or lesions due to the vaccine. Age and sex distributions, mean body weights, and live-capture histories of raccoons from the vaccination and non-baited control areas were compared. There were no statistically significant differences in survivorship between the baited and non-baited areas, nor between rabies antibody-positive and antibody-negative raccoons from the vaccination area. There was no trend in field mortality that suggested an association with either tetracycline or sulfadimethoxine, used as biomakers, or with vaccine contact determined by antibody status. No gross or histopathologic lesions due to the vaccine were demonstrated among a subsample of live-trapped raccoons collected for gross necropsy, biomarker analysis, histopathologic examination, and V-RG virus isolation attempts. Recovery of V-RG virus was limited to the tonsils of two biomarker-positive, clinically healthy raccoons collected from the vaccination area for postmortem examination on days 2 and 4 following bait distribution. These data reinforce the extensive body of safety data on the V-RG virus and extend it to include field evaluation where vaccine is offered free-choice in abundance, in baits designed to attract free-ranging raccoons, in a relatively simple ecosystem.

  18. Vaccinia Virus Protein Synthesis Has a Low Requirement for the Intact Translation Initiation Factor eIF4F, the Cap-Binding Complex, within Infected Cells

    PubMed Central

    Mulder, Jacqueline; Robertson, Morwenna E. M.; Seamons, Rachael A.; Belsham, Graham J.

    1998-01-01

    The role of the cap-binding complex, eIF4F, in the translation of vaccinia virus mRNAs has been analyzed within infected cells. Plasmid DNAs, which express dicistronic mRNAs containing a picornavirus internal ribosome entry site, produced within vaccinia virus-infected cells both β-glucuronidase and a cell surface-targeted single-chain antibody (sFv). Cells expressing sFv were selected from nonexpressing cells, enabling analysis of protein synthesis specifically within the transfected cells. Coexpression of poliovirus 2A or foot-and-mouth disease virus Lb proteases, which cleaved translation initiation factor eIF4G, greatly inhibited cap-dependent protein (β-glucuronidase) synthesis. Under these conditions, internal ribosome entry site-directed expression of sFv continued and cell selection was maintained. Furthermore, vaccinia virus protein synthesis persisted in the selected cells containing cleaved eIF4G. Thus, late vaccinia virus protein synthesis has a low requirement for the intact cap-binding complex eIF4F. This may be attributed to the short unstructured 5′ noncoding regions of the vaccinia virus mRNAs, possibly aided by the presence of poly(A) at both 5′ and 3′ termini. PMID:9765426

  19. Vaccination with recombinant modified vaccinia virus Ankara prevents the onset of intestinal allergy in mice.

    PubMed

    Bohnen, C; Wangorsch, A; Schülke, S; Nakajima-Adachi, H; Hachimura, S; Burggraf, M; Süzer, Y; Schwantes, A; Sutter, G; Waibler, Z; Reese, G; Toda, M; Scheurer, S; Vieths, S

    2013-08-01

    Modified vaccinia virus Ankara (MVA)-encoding antigens are considered as safe vaccine candidates for various infectious diseases in humans. Here, we investigated the immune-modulating properties of MVA-encoding ovalbumin (MVA-OVA) on the allergen-specific immune response. The immune-modulating properties of MVA-OVA were investigated using GM-CSF-differentiated BMDCs from C57BL/6 mice. OVA expression upon MVA-OVA infection of BMDCs was monitored. Activation and maturation markers on viable MVA-OVA-infected mDCs were analyzed by flow cytometry. Secretion of INF-γ, IL-2, and IL-10 was determined in a co-culture of BMDCs infected with wtMVA or MVA-OVA and OVA-specific OT-I CD8(+) and OT-II CD4(+ ) T cells. BALB/c mice were vaccinated with wtMVA, MVA-OVA, or PBS, sensitized to OVA/alum and challenged with a diet containing chicken egg white. OVA-specific IgE, IgG1, and IgG2a and cytokine secretion from mesenteric lymph node (MLN) cells were analyzed. Body weight, body temperature, food uptake, intestinal inflammation, and health condition of mice were monitored. Infection with wtMVA and MVA-OVA induced comparable activation of mDCs. MVA-OVA-infected BMDCs expressed OVA and induced enhanced IFN-γ and IL-2 secretion from OVA-specific CD8(+ ) T cells in comparison with OVA, wtMVA, or OVA plus wtMVA. Prophylactic vaccination with MVA-OVA significantly repressed OVA-specific IgE, whereas OVA-specific IgG2a was induced. MVA-OVA vaccination suppressed TH 2 cytokine production in MLN cells and prevented the onset of allergic symptoms and inflammation in a mouse model of OVA-induced intestinal allergy. Modified vaccinia virus Ankara-ovalbumin (MVA-OVA) vaccination induces a strong OVA-specific TH 1- immune response, likely mediated by the induction of IFN-γ and IgG2a. Finally, MVA-based vaccines need to be evaluated for their therapeutic potential in established allergy models. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Adverse events post smallpox-vaccination: insights from tail scarification infection in mice with Vaccinia virus.

    PubMed

    Mota, Bruno E F; Gallardo-Romero, Nadia; Trindade, Giliane; Keckler, M Shannon; Karem, Kevin; Carroll, Darin; Campos, Marco A; Vieira, Leda Q; da Fonseca, Flávio G; Ferreira, Paulo C P; Bonjardim, Cláudio A; Damon, Inger K; Kroon, Erna G

    2011-04-15

    Adverse events upon smallpox vaccination with fully-replicative strains of Vaccinia virus (VACV) comprise an array of clinical manifestations that occur primarily in immunocompromised patients leading to significant host morbidity/mortality. The expansion of immune-suppressed populations and the possible release of Variola virus as a bioterrorist act have given rise to concerns over vaccination complications should more widespread vaccination be reinitiated. Our goal was to evaluate the components of the host immune system that are sufficient to prevent morbidity/mortality in a murine model of tail scarification, which mimics immunological and clinical features of smallpox vaccination in humans. Infection of C57BL/6 wild-type mice led to a strictly localized infection, with complete viral clearance by day 28 p.i. On the other hand, infection of T and B-cell deficient mice (Rag1(-/-)) produced a severe disease, with uncontrolled viral replication at the inoculation site and dissemination to internal organs. Infection of B-cell deficient animals (µMT) produced no mortality. However, viral clearance in µMT animals was delayed compared to WT animals, with detectable viral titers in tail and internal organs late in infection. Treatment of Rag1(-/-) with rabbit hyperimmune anti-vaccinia serum had a subtle effect on the morbidity/mortality of this strain, but it was effective in reduce viral titers in ovaries. Finally, NUDE athymic mice showed a similar outcome of infection as Rag1(-/-), and passive transfer of WT T cells to Rag1(-/-) animals proved fully effective in preventing morbidity/mortality. These results strongly suggest that both T and B cells are important in the immune response to primary VACV infection in mice, and that T-cells are required to control the infection at the inoculation site and providing help for B-cells to produce antibodies, which help to prevent viral dissemination. These insights might prove helpful to better identify individuals with

  1. Adverse Events Post Smallpox-Vaccination: Insights from Tail Scarification Infection in Mice with Vaccinia virus

    PubMed Central

    Mota, Bruno E. F.; Gallardo-Romero, Nadia; Trindade, Giliane; Keckler, M. Shannon; Karem, Kevin; Carroll, Darin; Campos, Marco A.; Vieira, Leda Q.; da Fonseca, Flávio G.; Ferreira, Paulo C. P.; Bonjardim, Cláudio A.; Damon, Inger K.; Kroon, Erna G.

    2011-01-01

    Adverse events upon smallpox vaccination with fully-replicative strains of Vaccinia virus (VACV) comprise an array of clinical manifestations that occur primarily in immunocompromised patients leading to significant host morbidity/mortality. The expansion of immune-suppressed populations and the possible release of Variola virus as a bioterrorist act have given rise to concerns over vaccination complications should more widespread vaccination be reinitiated. Our goal was to evaluate the components of the host immune system that are sufficient to prevent morbidity/mortality in a murine model of tail scarification, which mimics immunological and clinical features of smallpox vaccination in humans. Infection of C57BL/6 wild-type mice led to a strictly localized infection, with complete viral clearance by day 28 p.i. On the other hand, infection of T and B-cell deficient mice (Rag1−/−) produced a severe disease, with uncontrolled viral replication at the inoculation site and dissemination to internal organs. Infection of B-cell deficient animals (µMT) produced no mortality. However, viral clearance in µMT animals was delayed compared to WT animals, with detectable viral titers in tail and internal organs late in infection. Treatment of Rag1−/− with rabbit hyperimmune anti-vaccinia serum had a subtle effect on the morbidity/mortality of this strain, but it was effective in reduce viral titers in ovaries. Finally, NUDE athymic mice showed a similar outcome of infection as Rag1−/−, and passive transfer of WT T cells to Rag1−/− animals proved fully effective in preventing morbidity/mortality. These results strongly suggest that both T and B cells are important in the immune response to primary VACV infection in mice, and that T-cells are required to control the infection at the inoculation site and providing help for B-cells to produce antibodies, which help to prevent viral dissemination. These insights might prove helpful to better identify individuals

  2. Pathogenicity and immunogenicity of recombinant Tiantan Vaccinia Virus with deleted C12L and A53R genes.

    PubMed

    Dai, Kaifan; Liu, Ying; Liu, Mingjie; Xu, Jianqing; Huang, Wei; Huang, Xianggang; Liu, Lianxing; Wan, Yanmin; Hao, Yanling; Shao, Yiming

    2008-09-15

    Interest is increasing regarding replicating poxvirus as HIV vaccine vector. In China, the Tiantan Vaccinia Virus (TV) has been used most extensively in the battle of eradicating smallpox. Recently, TV was developing as vaccine vector to fight against infectious diseases such as human immunodeficiency virus (HIV). However, replicating vaccinia virus sometimes may pose serious post-vaccination complications, especially in immunosuppressed individuals. To develop a safer and more effective TV-based vector, we constructed C12L (vIL-18 binding protein) and A53R (vTNF receptor homolog) gene-deleted mutants which are based on parental TV and VTKgpe (TV expressing HIV gagpol and env gene), respectively. The pathogenicity and immunogenicity were also evaluated. Deleting these two immunomodulatory genes lessened the virulence of the parental virus in both mice and rabbit models. Notably, C12L deletion mutant attenuated the skin virulence of parental virus by as high as approximate 2 logs. Furthermore, VTKgpe with A53R and C12L gene deletion retains the high immunogenicity of the parental virus to elicit strong humoral and cellular responses to the HIV target genes despite the remarkable attenuation. These data suggest that deletion of the cytokine viroceptor gene is feasible to obtain a safer and replication-competent TV vector for vaccination and immunotherapy.

  3. Robust Intrapulmonary CD8 T Cell Responses and Protection with an Attenuated N1L Deleted Vaccinia Virus

    PubMed Central

    Mathew, Anuja; O'Bryan, Joel; Marshall, William; Kotwal, Girish J.; Terajima, Masanori; Green, Sharone; Rothman, Alan L.; Ennis, Francis A.

    2008-01-01

    Background Vaccinia viruses have been used as a model for viral disease and as a protective live vaccine. Methodology and Principal Findings We investigated the immunogenicity of an attenuated strain of vaccinia virus engineered to inactivate the N1L gene (vGK5). Using the intranasal route, this recombinant virus was 2 logs less virulent compared to the wildtype VACV-WR. Infection by the intranasal, intraperitoneal, and tail scarification routes resulted in the robust induction of cytolytic virus-specific CD8 T cells in the spleens and the lungs. VACV-specific antibodies were also detected in the sera of mice infected 3–5 months prior with the attenuated vGK5 virus. Finally, mice immunized with vGK5 were significantly protected when challenged with a lethal dose of VACV-WR. Conclusions These results indicate that the attenuated vGK5 virus protects against subsequent infection and suggest that the N1L protein limits the strength of the early antiviral CD8 T cell response following respiratory infection. PMID:18830408

  4. Herpes virus oncolytic therapy reverses tumor immune dysfunction and facilitates tumor antigen presentation.

    PubMed

    Benencia, Fabian; Courrèges, Maria C; Fraser, Nigel W; Coukos, George

    2008-08-01

    We have previously shown that intratumor administration of HSV-1716 (an ICP34.5 null mutant) resulted in significant reduction of tumor growth and a significant survival advantage in a murine model of ovarian cancer. Herewith we report that oncolytic HSV-1716 generates vaccination effects in the same model. Upon HSV-1716 infection, mouse ovarian tumor cells showed high levels of expression viral glycoproteins B and D and were highly phagocyted by dendritic cells (DCs). Interestingly, increased phagocytosis of tumor-infected cells by DCs was impaired by heparin, and anti-HSV glycoproteins B and D, indicating that viral infection enhances adhesive interactions between DCs and tumor apoptotic bodies. Moreover, HSV-1716 infected cells expressed high levels of heat shock proteins 70 and GRP94, molecules that have been reported to induce maturation of DCs, increase cross-presentation of antigens and promote antitumor immune response. After phagocytosis of tumor-infected cells, DCs acquired a mature status in vitro and in vivo, upregulated the expression of costimulatory molecule and increased migration towards MIP-3beta. Furthermore, HSV-1716 oncolytic treatment markedly reduced vascular endothelial growth factor (VEGF) levels in tumor-bearing animals thus abrogating tumor immunosuppressive milieu. These mechanisms may account for the highly enhanced antitumoral immune responses observed in HSV-1716 treated animals. Oncolytic treatment induced a significantly higher frequency of tumor-reactive IFNgamma producing cells, and induced a robust tumor infiltration by T cells. These results indicate that oncolytic therapy with HSV-1716 facilitates antitumor immune responses.

  5. A Fusogenic Oncolytic Herpes Simplex Virus for Therapy of Advanced Ovarian Cancer

    DTIC Science & Technology

    2006-06-01

    therapy of solid tumors such as ovarian cancer, two obstacles need to be overcome before the therapeutic potential of virotherapy could be fully... virotherapy could be fully materialized. Firstly, the potency of oncolytic HSVs needs to be improved. During the first two years of this funded

  6. Oncolytic polio virotherapy of cancer.

    PubMed

    Brown, Michael C; Dobrikova, Elena Y; Dobrikov, Mikhail I; Walton, Ross W; Gemberling, Sarah L; Nair, Smita K; Desjardins, Annick; Sampson, John H; Friedman, Henry S; Friedman, Allan H; Tyler, Douglas S; Bigner, Darell D; Gromeier, Matthias

    2014-11-01

    Recently, the century-old idea of targeting cancer with viruses (oncolytic viruses) has come of age, and promise has been documented in early stage and several late-stage clinical trials in a variety of cancers. Although originally prized for their direct tumor cytotoxicity (oncolytic virotherapy), recently, the proinflammatory and immunogenic effects of viral tumor infection (oncolytic immunotherapy) have come into focus. Indeed, a capacity for eliciting broad, sustained antineoplastic effects stemming from combined direct viral cytotoxicity, innate antiviral activation, stromal proinflammatory stimulation, and recruitment of adaptive immune effector responses is the greatest asset of oncolytic viruses. However, it also is the source for enormous mechanistic complexity that must be considered for successful clinical translation. Because of fundamentally different relationships with their hosts (malignant or not), diverse replication strategies, and distinct modes of tumor cytotoxicity/killing, oncolytic viruses should not be referred to collectively. These agents must be evaluated based on their individual merits. In this review, the authors highlight key mechanistic principles of cancer treatment with the polio:rhinovirus chimera PVSRIPO and their implications for oncolytic immunotherapy in the clinic.

  7. Oncolytic vaccine virus harbouring the IL-24 gene suppresses the growth of lung cancer by inducing apoptosis.

    PubMed

    Lv, Chunwei; Su, Qunshu; Liang, Yupei; Hu, Jinqing; Yuan, Sujing

    2016-07-15

    Lung cancer has an especially high incidence rate worldwide, and its resistance to cell death and chemotherapeutic drugs increases its intractability. The vaccinia virus has been shown to destroy neoplasm within a short time and disseminate rapidly and extensively as an enveloped virion throughout the circulatory system, and this virus has also demonstrated a strong ability to overexpress exogenous genes. Interleukin-24 (IL-24/mda-7) is an important cytokine that belongs to the activating caspase family and facilitates the inhibition of STAT3 when a cell enters the apoptosis pathway. In this study, we constructed a cancer-targeted vaccinia virus carrying the IL-24 gene knocked in the region of the viral thymidine kinase (TK) gene (VV-IL-24). Our results showed that VV-IL-24 efficiently infected and destroyed lung cancer cells via caspase-dependent apoptosis and decreased the expression of STAT3. In vivo, VV-IL-24 expressed IL-24 at a high level in the transplanted tumour, reduced STAT3 activity, and eventually led to apoptosis. In conclusion, we demonstrated that vv-IL-24 has the potential for use as a new human lung cancer treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Genomic sequence and virulence of clonal isolates of vaccinia virus Tiantan, the Chinese smallpox vaccine strain.

    PubMed

    Zhang, Qicheng; Tian, Meijuan; Feng, Yi; Zhao, Kai; Xu, Jing; Liu, Ying; Shao, Yiming

    2013-01-01

    Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.

  9. A Loss of Function Analysis of Host Factors Influencing Vaccinia virus Replication by RNA Interference

    PubMed Central

    Gonzalez, Orland; Haga, Ismar R.; Pechenick Jowers, Tali; Reynolds, Danielle K.; Wildenhain, Jan; Tekotte, Hille; Auer, Manfred; Tyers, Mike; Ghazal, Peter; Zimmer, Ralf; Haas, Jürgen

    2014-01-01

    Vaccinia virus (VACV) is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA) screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF) influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics. PMID:24901222

  10. Choindroitinase ABC I-mediated enhancement of oncolytic virus spread and anti tumor efficacy: a mathematical model.