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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. 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.

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

    PubMed

    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

    2016-09-01

    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

  4. Expression of DAI by an oncolytic vaccinia virus boosts the immunogenicity of the virus and enhances antitumor immunity

    PubMed Central

    Hirvinen, Mari; Capasso, Cristian; Guse, Kilian; Garofalo, Mariangela; Vitale, Andrea; Ahonen, Marko; Kuryk, Lukasz; Vähä-Koskela, Markus; Hemminki, Akseli; Fortino, Vittorio; Greco, Dario; Cerullo, Vincenzo

    2016-01-01

    In oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. Vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. However, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI) to boost the innate immune system and to activate adaptive immune cells in the tumor. We showed that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways. Treatment with DAI-armed VV resulted in significant reduction in the size of syngeneic melanoma tumors in mice. When the mice were rechallenged with the same tumor, DAI-VV-treated mice completely rejected growth of the new tumor, which indicates immunity established against the tumor. We also showed enhanced control of growth of human melanoma tumors and elevated levels of human T-cells in DAI-VV-treated mice humanized with human peripheral blood mononuclear cells. We conclude that expression of DAI by an oncolytic VV is a promising way to amplify the vaccine potency of an oncolytic vaccinia virus to trigger the innate—and eventually the long-lasting adaptive immunity against cancer. PMID:27626058

  5. Expression of DAI by an oncolytic vaccinia virus boosts the immunogenicity of the virus and enhances antitumor immunity.

    PubMed

    Hirvinen, Mari; Capasso, Cristian; Guse, Kilian; Garofalo, Mariangela; Vitale, Andrea; Ahonen, Marko; Kuryk, Lukasz; Vähä-Koskela, Markus; Hemminki, Akseli; Fortino, Vittorio; Greco, Dario; Cerullo, Vincenzo

    2016-01-01

    In oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. Vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. However, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI) to boost the innate immune system and to activate adaptive immune cells in the tumor. We showed that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways. Treatment with DAI-armed VV resulted in significant reduction in the size of syngeneic melanoma tumors in mice. When the mice were rechallenged with the same tumor, DAI-VV-treated mice completely rejected growth of the new tumor, which indicates immunity established against the tumor. We also showed enhanced control of growth of human melanoma tumors and elevated levels of human T-cells in DAI-VV-treated mice humanized with human peripheral blood mononuclear cells. We conclude that expression of DAI by an oncolytic VV is a promising way to amplify the vaccine potency of an oncolytic vaccinia virus to trigger the innate-and eventually the long-lasting adaptive immunity against cancer. PMID:27626058

  6. 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.

  7. Single-particle characterization of oncolytic vaccinia virus by flow virometry.

    PubMed

    Tang, Vera A; Renner, Tyler M; Varette, Oliver; Le Boeuf, Fabrice; Wang, Jiahu; Diallo, Jean-Simon; Bell, John C; Langlois, Marc-André

    2016-09-30

    Vaccinia virus (VV) is an oncolytic virus that is currently being evaluated as a promising cancer vaccine in several phase I, II and III clinical trials. Although several quality control tests are performed on each new batch of virus, these do not routinely include a systematic characterization of virus particle homogeneity, or relate the infectious titer to the total number of submicron sized particles (SSPs) present in the sample. SSPs are comprised of infectious virus and non-infectious viral particles, but also cell contaminants derived from the virus isolation procedures, such as cellular vesicles and debris. Here we have employed flow virometry (FV) analysis and sorting to isolate and characterize distinct SSP populations in therapeutic oncolytic VV preparations. We show that VV preparations contain SSPs heterogeneous in size and include large numbers of non-infectious VV particles. Furthermore, we used FV to illustrate how VV has a propensity to aggregate over time and under various handling and storage procedures. Accordingly, we find that together the infectious titer, the total number of SSPs, the number of viral genomes and the level of particle aggregation in a sample constitute useful parameters that greatly facilitate inter-sample assessment of physical quality, and also provides a means to monitor sample deterioration over time. Additionally, we have successfully employed FV sorting to further isolate virus from other particles by identifying a lipophilic dye that preferentially stains VV over other SSPs in the sample. Overall, we demonstrate that FV is a fast and effective tool that can be used to perform quality, and consistency control assessments of oncolytic VV vaccine preparations.

  8. Single-particle characterization of oncolytic vaccinia virus by flow virometry.

    PubMed

    Tang, Vera A; Renner, Tyler M; Varette, Oliver; Le Boeuf, Fabrice; Wang, Jiahu; Diallo, Jean-Simon; Bell, John C; Langlois, Marc-André

    2016-09-30

    Vaccinia virus (VV) is an oncolytic virus that is currently being evaluated as a promising cancer vaccine in several phase I, II and III clinical trials. Although several quality control tests are performed on each new batch of virus, these do not routinely include a systematic characterization of virus particle homogeneity, or relate the infectious titer to the total number of submicron sized particles (SSPs) present in the sample. SSPs are comprised of infectious virus and non-infectious viral particles, but also cell contaminants derived from the virus isolation procedures, such as cellular vesicles and debris. Here we have employed flow virometry (FV) analysis and sorting to isolate and characterize distinct SSP populations in therapeutic oncolytic VV preparations. We show that VV preparations contain SSPs heterogeneous in size and include large numbers of non-infectious VV particles. Furthermore, we used FV to illustrate how VV has a propensity to aggregate over time and under various handling and storage procedures. Accordingly, we find that together the infectious titer, the total number of SSPs, the number of viral genomes and the level of particle aggregation in a sample constitute useful parameters that greatly facilitate inter-sample assessment of physical quality, and also provides a means to monitor sample deterioration over time. Additionally, we have successfully employed FV sorting to further isolate virus from other particles by identifying a lipophilic dye that preferentially stains VV over other SSPs in the sample. Overall, we demonstrate that FV is a fast and effective tool that can be used to perform quality, and consistency control assessments of oncolytic VV vaccine preparations. PMID:27614781

  9. 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

  10. Photodynamic therapy augments the efficacy of oncolytic vaccinia virus against primary and metastatic tumours in mice

    PubMed Central

    Gil, M; Bieniasz, M; Seshadri, M; Fisher, D; Ciesielski, M J; Chen, Y; Pandey, R K; Kozbor, D

    2011-01-01

    Background: Therapies targeted towards the tumour vasculature can be exploited for the purpose of improving the systemic delivery of oncolytic viruses to tumours. Photodynamic therapy (PDT) is a clinically approved treatment for cancer that is known to induce potent effects on tumour vasculature. In this study, we examined the activity of PDT in combination with oncolytic vaccinia virus (OVV) against primary and metastatic tumours in mice. Methods: The effect of 2-[1-hexyloxyethyl-]-2-devinyl pyropheophorbide-a (HPPH)-sensitised-PDT on the efficacy of oncolytic virotherapy was investigated against subcutaneously implanted syngeneic murine NXS2 neuroblastoma and human FaDu head and neck squamous cell carcinoma xenografts in nude mice. Treatment efficacy was evaluated by monitoring tumour growth and survival. The effects of combination treatment on vascular function were examined using magnetic resonance imaging (MRI) and immunohistochemistry, whereas viral replication in tumour cells was analysed by a standard plaque assay. Normal tissue phototoxicity following PDT-OV treatment was studied using the mouse foot response assay. Results: Combination of PDT with OVV resulted in inhibition of primary and metastatic tumour growth compared with either monotherapy. PDT-induced vascular disruption resulted in higher intratumoural viral titres compared with the untreated tumours. Five days after delivery of OVV, there was a loss of blood flow to the interior of tumour that was associated with infiltration of neutrophils. Administration of OVV did not result in any additional photodynamic damage to normal mouse foot tissue. Conclusion: These results provide evidence into the usefulness of PDT as a means of enhancing intratumoural replication and therapeutic efficacy of OV. PMID:21989183

  11. A Novel Recombinant Vaccinia Virus Expressing the Human Norepinephrine Transporter Retains Oncolytic Potential and Facilitates Deep-Tissue Imaging

    PubMed Central

    Chen, Nanhai; Zhang, Qian; Yu, Yong A; Stritzker, Jochen; Brader, Peter; Schirbel, Andreas; Samnick, Samuel; Serganova, Inna; Blasberg, Ronald; Fong, Yuman; Szalay, Aladar A

    2009-01-01

    Noninvasive and repetitive monitoring of a virus in target tissues and/or specific organs of the body is highly desirable for the development of safe and efficient cancer virotherapeutics. We have previously shown that the oncolytic vaccinia virus GLV-1h68 can target and eradicate human tumors in mice and that its therapeutic effects can be monitored by using optical imaging. Here, we report on the development of a derivative of GLV-1h68, a novel recombinant vaccinia virus (VACV) GLV-1h99, which was constructed to carry the human norepinephrine transporter gene (hNET) under the VACV synthetic early promoter placed at the F14.5L locus for deep-tissue imaging. The hNET protein was expressed at high levels on the membranes of cells infected with this virus. Expression of the hNET protein did not negatively affect virus replication, cytolytic activity in cell culture, or in vivo virotherpeutic efficacy. GLV-1h99–mediated expression of the hNET protein in infected cells resulted in specific uptake of the radiotracer [131I]-meta-iodobenzylguanidine (MIBG). In mice, GLV-1h99–infected tumors were readily imaged by [124I]-MIBG positron emission tomography. To our knowledge, GLV-1h99 is the first oncolytic virus expressing the hNET protein that can efficiently eliminate tumors and simultaneously allow deep-tissue imaging of infected tumors. PMID:19287510

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

    PubMed

    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.

  13. 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

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

    PubMed

    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. Phase 1 study of intratumoral Pexa-Vec (JX-594), an oncolytic and immunotherapeutic vaccinia virus, in pediatric cancer patients.

    PubMed

    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

    2015-03-01

    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 10(6) or 10(7) 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.

  16. Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

    PubMed Central

    2011-01-01

    Introduction Oncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS) cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153. Methods GLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET) utilizing carrier-free 124I radiotracer. Results GLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P < 0.001). In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P < 0.001). Finally, intratumoral injection of GLV-1h153 facilitated imaging of virus replication in tumors via 124I-PET. Conclusion Insertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy. PMID:21453532

  17. Oncolytic virus therapy for cancer.

    PubMed

    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.

  18. 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

  19. 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

  20. Intratumoral INF-γ triggers an antiviral state in GL261 tumor cells: a major hurdle to overcome for oncolytic vaccinia virus therapy of cancer

    PubMed Central

    Kober, Christina; Weibel, Stephanie; Rohn, Susanne; Kirscher, Lorenz; Szalay, Aladar A

    2015-01-01

    Oncolytic vaccinia virus (VACV) therapy is an alternative treatment option for glioblastoma multiforme. Here, we used a comparison of different tumor locations and different immunologic and genetic backgrounds to determine the replication efficacy and oncolytic potential of the VACV LIVP 1.1.1, an attenuated wild-type isolate of the Lister strain, in murine GL261 glioma models. With this approach, we expected to identify microenvironmental factors, which may be decisive for failure or success of oncolytic VACV therapy. We found that GL261 glioma cells implanted subcutaneously or orthotopically into Balb/c athymic, C57BL/6 athymic, or C57BL/6 wild-type mice formed individual tumors that respond to oncolytic VACV therapy with different outcomes. Surprisingly, only Balb/c athymic mice with subcutaneous tumors supported viral replication. We identified intratumoral IFN-γ expression levels that upregulate MHCII expression on GL261 cells in C57BL/6 wild-type mice associated with a non-permissive status of the tumor cells. Moreover, this IFN-γ-induced tumor cell phenotype was reversible. PMID:27119106

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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. PMID:27057460

  2. A vaccinia virus renaissance

    PubMed Central

    Verardi, Paulo H.; Titong, Allison; Hagen, Caitlin J.

    2012-01-01

    In 1796, Edward Jenner introduced the concept of vaccination with cowpox virus, an Orthopoxvirus within the family Poxviridae that elicits cross protective immunity against related orthopoxviruses, including smallpox virus (variola virus). Over time, vaccinia virus (VACV) replaced cowpox virus as the smallpox vaccine, and vaccination efforts eventually led to the successful global eradication of smallpox in 1979. VACV has many characteristics that make it an excellent vaccine and that were crucial for the successful eradication of smallpox, including (1) its exceptional thermal stability (a very important but uncommon characteristic in live vaccines), (2) its ability to elicit strong humoral and cell-mediated immune responses, (3) the fact that it is easy to propagate, and (4) that it is not oncogenic, given that VACV replication occurs exclusively within the host cell cytoplasm and there is no evidence that the viral genome integrates into the host genome. Since the eradication of smallpox, VACV has experienced a renaissance of interest as a viral vector for the development of recombinant vaccines, immunotherapies, and oncolytic therapies, as well as the development of next-generation smallpox vaccines. This revival is mainly due to the successful use and extensive characterization of VACV as a vaccine during the smallpox eradication campaign, along with the ability to genetically manipulate its large dsDNA genome while retaining infectivity and immunogenicity, its wide mammalian host range, and its natural tropism for tumor cells that allows its use as an oncolytic vector. This review provides an overview of new uses of VACV that are currently being explored for the development of vaccines, immunotherapeutics, and oncolytic virotherapies. PMID:22777090

  3. Combined expression of miR-34a and Smac mediated by oncolytic vaccinia virus synergistically promote anti-tumor effects in Multiple Myeloma.

    PubMed

    Lei, Wen; Wang, Shibing; Yang, Chunmei; Huang, Xianbo; Chen, Zhenzhen; He, Wei; Shen, Jianping; Liu, Xinyuan; Qian, Wenbin

    2016-01-01

    Despite great progress made in the treatment of multiple myeloma (MM), it is still incurable. Promising phase II clinical results have been reported recently for oncolytic vaccinia virus (OVV) clinic therapeutics. One reason for this has focused on the critical therapeutic importance of the immune response raised by these viruses. However, few studies have performed their applications as an optimal delivery system for therapeutic gene, especially miRNA in MM. In this study, we constructed two novel OVVs (TK deletion) that express anti-tumor genes, miR-34a and Smac, respectively, in MM cell lines and xenograft model. The results demonstrated that the novel OVV can effectively infect MM cell lines, and forcefully enhance the exogenous gene (miR-34a or Smac) expression. Furthermore, utilization of VV-miR-34a combined with VV-Smac synergistically inhibited tumor growth and induced apoptosis in vitro and in vivo. The underlying mechanism is proposed that blocking of Bcl-2 by VV-miR-34a increases the release of cytochrome c from mitochondria and then synergistically amplifies the antitumor effects of Smac-induced cell apoptosis. Our study is the first to utilize OVV as the vector for miR-34a or Smac expression to treat MM, and lays the groundwork for future clinical therapy for MM. PMID:27552933

  4. Combined expression of miR-34a and Smac mediated by oncolytic vaccinia virus synergistically promote anti-tumor effects in Multiple Myeloma

    PubMed Central

    Lei, Wen; Wang, Shibing; Yang, Chunmei; Huang, Xianbo; Chen, Zhenzhen; He, Wei; Shen, Jianping; Liu, Xinyuan; Qian, Wenbin

    2016-01-01

    Despite great progress made in the treatment of multiple myeloma (MM), it is still incurable. Promising phase II clinical results have been reported recently for oncolytic vaccinia virus (OVV) clinic therapeutics. One reason for this has focused on the critical therapeutic importance of the immune response raised by these viruses. However, few studies have performed their applications as an optimal delivery system for therapeutic gene, especially miRNA in MM. In this study, we constructed two novel OVVs (TK deletion) that express anti-tumor genes, miR-34a and Smac, respectively, in MM cell lines and xenograft model. The results demonstrated that the novel OVV can effectively infect MM cell lines, and forcefully enhance the exogenous gene (miR-34a or Smac) expression. Furthermore, utilization of VV-miR-34a combined with VV-Smac synergistically inhibited tumor growth and induced apoptosis in vitro and in vivo. The underlying mechanism is proposed that blocking of Bcl-2 by VV-miR-34a increases the release of cytochrome c from mitochondria and then synergistically amplifies the antitumor effects of Smac-induced cell apoptosis. Our study is the first to utilize OVV as the vector for miR-34a or Smac expression to treat MM, and lays the groundwork for future clinical therapy for MM. PMID:27552933

  5. An Oncolytic Vaccinia Virus Expressing the Human Sodium Iodine Symporter Prolongs Survival and Facilitates SPECT/CT Imaging in an Orthotopic Model of Malignant Pleural Mesothelioma

    PubMed Central

    Belin, Laurence J.; Ady, Justin W.; Lewis, Christina; Marano, Drew; Gholami, Sepideh; Mojica, Kelly; Eveno, Clarisse; Longo, Valerie; Zanzonico, Pat B.; Chen, Nanhai G.; Szalay, Aladar A.; Fong, Yuman

    2014-01-01

    Background The purpose of this original work is to examine the ability of an oncolytic vaccinia virus expressing the human sodium iodine transporter (hNIS) to provide real time monitoring of viral therapy and effective treatment of malignant pleural mesothelioma (MPM). Methods Infectivity and cytotoxic effect of GLV-1h153 on mesothelioma cell lines of all histologic subtypes was assayed in vitro. Viral replication was examined by standard viral plaque assay. Orthotopic MPM xenografts were generated in athymic nude mice and treated with intrapleural GLV-1h153 and assessed for effect on tumor burden and survival. Orthotopic tumors were also imaged on SPECT/CT after 131I administration. Results GLV-1h153 infected and killed all cell lines in a time and concentration dependent manner. Viral replication demonstrated over a 2.5 log increase in titer over 4 days. Intrapleural treatment of orthotopic MPM xenografts resulted in a significant reduction in tumor burden one week after treatment and an improvement in survival. Infection of orthotopic xenografts was both therapeutic and facilitated monitoring by 131I-SPECT/CT via expression of hNIS in infected tissue. Conclusions Our results suggest GLV-1h153 is a promising therapeutic agent for MPM and warrants further investigation. PMID:23890748

  6. Oncolytic viruses: finally delivering

    PubMed Central

    Seymour, Leonard W; Fisher, Kerry D

    2016-01-01

    Oncolytic viruses can be found at the confluence of virology, genetic engineering and pharmacology where versatile platforms for molecularly targeted anticancer agents can be designed and optimised. Oncolytic viruses offer several important advantages over traditional approaches, including the following. (1) Amplification of the active agent (infectious virus particles) within the tumour. This avoids unnecessary exposure to normal tissues experienced during delivery of traditional stoichiometric chemotherapy and maximises the therapeutic index. (2) The active cell-killing mechanisms, often independent of programmed death mechanisms, should decrease the emergence of acquired drug resistance. (3) Lytic death of cancer cells provides a pro-inflammatory microenvironment and the potential for induction of an anticancer vaccine response. (4) Tumour-selective expression and secretion of encoded anticancer biologics, providing a new realm of potent and cost-effective-targeted therapeutics. PMID:26766734

  7. Group 2 vaccinia virus, Brazil.

    PubMed

    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; Abrahão, Jônatas Santos

    2012-12-01

    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.

  8. The Addition of Recombinant Vaccinia HER2/neu to Oncolytic Vaccinia-GMCSF Given into the Tumor Microenvironment Overcomes MDSC-Mediated Immune Escape and Systemic Anergy

    PubMed Central

    de Vries, Christiaan R.; Monken, Claude E.; Lattime, Edmund C.

    2015-01-01

    Effective immunotherapeutic strategies require the ability to generate a systemic antigen-specific response capable of impacting both primary and metastatic disease. We have built on our oncolytic vaccinia GM-CSF strategy by adding recombinant tumor antigen to increase the response in the tumor microenvironment and systemically. In the present study, orthotopic growth of a syngeneic HER2/neu-overexpressing mammary carcinoma in FVB/N mice (NBT1) was associated with increased Gr1+CD11b+ myeloid derived suppressor cells (MDSCs) both systemically and in the tumor microenvironment. This MDSC population had inhibitory effects on the HER2/neu specific Th1 immune response. VVneu and VVGMCSF are recombinant oncolytic vaccinia viruses that encode HER2/neu and GM-CSF, respectively. Naïve FVB mice vaccinated with combined VVneu and VVGMCSF given systemically developed systemic HER2/neu-specific immunity. NBT1 bearing mice became anergic to systemic immunization with combined VVneu and VVGMCSF. Intratumoral VVGMCSF failed to result in systemic antitumor immunity until combined with intratumoral VVneu. Infection/transfection of the tumor microenvironment with combined VVGMCSF and VVneu resulted in development of systemic tumor-specific immunity, reduction in splenic and tumor MDSC, and therapeutic efficacy against tumor. These studies demonstrate the enhanced efficacy of oncolytic vaccinia virus recombinants encoding combined tumor antigen and GM-CSF in modulating the microenvironment of MDSC-rich tumors. PMID:25633483

  9. 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

  10. Ex vivo infection of live tissue with oncolytic viruses.

    PubMed

    Diallo, Jean-Simon; Roy, Dominic; Abdelbary, Hesham; De Silva, Naomi; Bell, John C

    2011-06-25

    Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells(1). Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway(2-5). One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging(6,7). This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy(8). To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection(9). It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

  11. 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.

  12. Attenuation of Vaccinia Virus.

    PubMed

    Yakubitskiy, S N; Kolosova, I V; Maksyutov, R A; Shchelkunov, S N

    2015-01-01

    Since 1980, in the post-smallpox vaccination era the human population has become increasingly susceptible compared to a generation ago to not only the variola (smallpox) virus, but also other zoonotic orthopoxviruses. The need for safer vaccines against orthopoxviruses is even greater now. The Lister vaccine strain (LIVP) of vaccinia virus was used as a parental virus for generating a recombinant 1421ABJCN clone defective in five virulence genes encoding hemagglutinin (A56R), the IFN-γ-binding protein (B8R), thymidine kinase (J2R), the complement-binding protein (C3L), and the Bcl-2-like inhibitor of apoptosis (N1L). We found that disruption of these loci does not affect replication in mammalian cell cultures. The isogenic recombinant strain 1421ABJCN exhibits a reduced inflammatory response and attenuated neurovirulence relative to LIVP. Virus titers of 1421ABJCN were 3 lg lower versus the parent VACV LIVP when administered by the intracerebral route in new-born mice. In a subcutaneous mouse model, 1421ABJCN displayed levels of VACV-neutralizing antibodies comparable to those of LIVP and conferred protective immunity against lethal challenge by the ectromelia virus. The VACV mutant holds promise as a safe live vaccine strain for preventing smallpox and other orthopoxvirus infections. PMID:26798498

  13. Oncolytic and immunotherapeutic vaccinia induces antibody-mediated complement-dependent cancer cell lysis in humans.

    PubMed

    Kim, Mi Kyung; Breitbach, Caroline J; Moon, Anne; Heo, Jeong; Lee, Yu Kyoung; Cho, Mong; Lee, Jun Woo; Kim, Seong-Geun; Kang, Dae Hwan; Bell, John C; Park, Byeong Ho; Kirn, David H; Hwang, Tae-Ho

    2013-05-15

    Oncolytic viruses cause direct cytolysis and cancer-specific immunity in preclinical models. The goal of this study was to demonstrate induction of functional anticancer immunity that can lyse target cancer cells in humans. Pexa-Vec (pexastimogene devacirepvec; JX-594) is a targeted oncolytic and immunotherapeutic vaccinia virus engineered to express human granulocyte-macrophage colony-stimulating factor (GM-CSF). Pexa-Vec demonstrated replication, GM-CSF expression, and tumor responses in previous phase 1 trials. We now evaluated whether Pexa-Vec induced functional anticancer immunity both in the rabbit VX2 tumor model and in patients with diverse solid tumor types in phase 1. Antibody-mediated complement-dependent cancer cell cytotoxicity (CDC) was induced by intravenous Pexa-Vec in rabbits; transfer of serum from Pexa-Vec-treated animals to tumor-bearing animals resulted in tumor necrosis and improved survival. In patients with diverse tumor types treated on a phase 1 trial, CDC developed within 4 to 8 weeks in most patients; normal cells were resistant to the cytotoxic effects. T lymphocyte activation in patients was evidenced by antibody class switching. We determined that patients with the longest survival duration had the highest CDC activity, and identified candidate target tumor cell antigens. Thus, we demonstrated that Pexa-Vec induced polyclonal antibody-mediated CDC against multiple tumor antigens both in rabbits and in patients with diverse solid tumor types.

  14. GMCSF-armed vaccinia virus induces an antitumor immune response.

    PubMed

    Parviainen, Suvi; Ahonen, Marko; Diaconu, Iulia; Kipar, Anja; Siurala, Mikko; Vähä-Koskela, Markus; Kanerva, Anna; Cerullo, Vincenzo; Hemminki, Akseli

    2015-03-01

    Oncolytic Western Reserve strain vaccinia virus selective for epidermal growth factor receptor pathway mutations and tumor-associated hypermetabolism was armed with human granulocyte-macrophage colony-stimulating factor (GMCSF) and a tdTomato fluorophore. As the assessment of immunological responses to human transgenes is challenging in the most commonly used animal models, we used immunocompetent Syrian golden hamsters, known to be sensitive to human GMCSF and semipermissive to vaccinia virus. Efficacy was initially tested in vitro on various human and hamster cell lines and oncolytic potency of transgene-carrying viruses was similar to unarmed virus. The hGMCSF-encoding virus was able to completely eradicate subcutaneous pancreatic tumors in hamsters, and to fully protect the animals from subsequent rechallenge with the same tumor. Induction of specific antitumor immunity was also shown by ex vivo co-culture experiments with hamster splenocytes. In addition, histological examination revealed increased infiltration of neutrophils and macrophages in GMCSF-virus-treated tumors. These findings help clarify the mechanism of action of GMCSF-armed vaccinia viruses undergoing clinical trials.

  15. Trial Watch:: Oncolytic viruses for cancer therapy.

    PubMed

    Pol, Jonathan; Bloy, Norma; Obrist, Florine; Eggermont, Alexander; Galon, Jérôme; Cremer, Isabelle; Erbs, Philippe; Limacher, Jean-Marc; Preville, Xavier; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2014-01-01

    Oncolytic viruses are natural or genetically modified viral species that selectively infect and kill neoplastic cells. Such an innate or exogenously conferred specificity has generated considerable interest around the possibility to employ oncolytic viruses as highly targeted agents that would mediate cancer cell-autonomous anticancer effects. Accumulating evidence, however, suggests that the therapeutic potential of oncolytic virotherapy is not a simple consequence of the cytopathic effect, but strongly relies on the induction of an endogenous immune response against transformed cells. In line with this notion, superior anticancer effects are being observed when oncolytic viruses are engineered to express (or co-administered with) immunostimulatory molecules. Although multiple studies have shown that oncolytic viruses are well tolerated by cancer patients, the full-blown therapeutic potential of oncolytic virotherapy, especially when implemented in the absence of immunostimulatory interventions, remains unclear. Here, we cover the latest advances in this active area of translational investigation, summarizing high-impact studies that have been published during the last 12 months and discussing clinical trials that have been initiated in the same period to assess the therapeutic potential of oncolytic virotherapy in oncological indications. PMID:25097804

  16. Vaccinia Virus Induces Programmed Necrosis in Ovarian Cancer Cells

    PubMed Central

    Whilding, Lynsey M; Archibald, Kyra M; Kulbe, Hagen; Balkwill, Frances R; Öberg, Daniel; McNeish, Iain A

    2013-01-01

    The mechanisms by which oncolytic vaccinia virus induces tumor cell death are poorly understood. We have evaluated cell death pathways following infection of ovarian cancer cells with both wild-type and thymidine kinase-deleted (dTK) Lister strain vaccinia. We show that death does not rely upon classical apoptosis despite the appearances of some limited apoptotic features, including phosphatidylserine externalization and appearance of sub-G1 DNA populations. Vaccinia infection induces marked lipidation of LC3 proteins, but there is no general activation of the autophagic process and cell death does not rely upon autophagy induction. We show that vaccinia induces necrotic morphology on transmission electron microscopy, accompanied by marked by reductions in intracellular adenosine triphosphate, altered mitochondrial metabolism, and release of high mobility group box 1 (HMGB1) protein. This necrotic cell death appears regulated, as infection induces formation of a receptor interacting protein (RIP1)/caspase-8 complex. In addition, pharmacological inhibition of both RIP1 and substrates downstream of RIP1, including MLKL, significantly attenuate cell death. Blockade of TNF-α, however, does not alter virus efficacy, suggesting that necrosis does not result from autocrine cytokine release. Overall, these results show that, in ovarian cancer cells, vaccinia virus causes necrotic cell death that is mediated through a programmed series of events. PMID:23985697

  17. Vaccinia virus-mediated melanin production allows MR and optoacoustic deep tissue imaging and laser-induced thermotherapy of cancer.

    PubMed

    Stritzker, Jochen; Kirscher, Lorenz; Scadeng, Miriam; Deliolanis, Nikolaos C; Morscher, Stefan; Symvoulidis, Panagiotis; Schaefer, Karin; Zhang, Qian; Buckel, Lisa; Hess, Michael; Donat, Ulrike; Bradley, William G; Ntziachristos, Vasilis; Szalay, Aladar A

    2013-02-26

    We reported earlier the delivery of antiangiogenic single chain antibodies by using oncolytic vaccinia virus strains to enhance their therapeutic efficacy. Here, we provide evidence that gene-evoked production of melanin can be used as a therapeutic and diagnostic mediator, as exemplified by insertion of only one or two genes into the genome of an oncolytic vaccinia virus strain. We found that produced melanin is an excellent reporter for optical imaging without addition of substrate. Melanin production also facilitated deep tissue optoacoustic imaging as well as MRI. In addition, melanin was shown to be a suitable target for laser-induced thermotherapy and enhanced oncolytic viral therapy. In conclusion, melanin as a mediator for thermotherapy and reporter for different imaging modalities may soon become a versatile alternative to replace fluorescent proteins also in other biological systems. After ongoing extensive preclinical studies, melanin overproducing oncolytic virus strains might be used in clinical trials in patients with cancer.

  18. 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.

  19. Vaccine Therapy, Oncolytic Viruses, and Gliomas.

    PubMed

    Desjardins, Annick; Vlahovic, Gordana; Friedman, Henry S

    2016-03-01

    After years of active research and refinement, vaccine therapy and oncolytic viruses are becoming part of the arsenal in the treatment of gliomas. In contrast to standard treatment with radiation therapy and chemotherapy, vaccines are more specific to the patient and the tumor. The majority of ongoing vaccine trials are investigating peptide, heat shock protein, and dendritic cell vaccines. The immunosuppression triggered by the tumor itself and by its treatment is a major obstacle to vaccine and oncolytic virus therapy. Thus, combination therapy with different agents that affect the immune system will probably be necessary. PMID:26984213

  20. 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

  1. 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.

  2. Preparation of cell cultures and vaccinia virus stocks.

    PubMed

    Earl, P L; Cooper, N; Wyatt, L S; Moss, B; Carroll, M W

    2001-05-01

    This unit describes the maintenance of cell lines used with vaccinia virus, both in monolayer cultures and in suspension. The suspended cell culture is then used in the preparation of vaccinia virus stocks. The preparation of chick embryo fibroblasts (CEF) is also presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Additionally, support protocols are presented for the titration of standard and MVA vaccinia virus stocks.

  3. Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent

    PubMed Central

    Kirscher, Lorenz; Deán-Ben, Xosé Luis; Scadeng, Miriam; Zaremba, Angelika; Zhang, Qian; Kober, Christina; Fehm, Thomas Felix; Razansky, Daniel; Ntziachristos, Vasilis; Stritzker, Jochen; Szalay, Aladar A.

    2015-01-01

    We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system. PMID:26199644

  4. Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent.

    PubMed

    Kirscher, Lorenz; Deán-Ben, Xosé Luis; Scadeng, Miriam; Zaremba, Angelika; Zhang, Qian; Kober, Christina; Fehm, Thomas Felix; Razansky, Daniel; Ntziachristos, Vasilis; Stritzker, Jochen; Szalay, Aladar A

    2015-01-01

    We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

  5. High-throughput screening to enhance oncolytic virus immunotherapy.

    PubMed

    Allan, K J; Stojdl, David F; Swift, S L

    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

  6. 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

  7. Features of the Antitumor Effect of Vaccinia Virus Lister Strain.

    PubMed

    Zonov, Evgeniy; Kochneva, Galina; Yunusova, Anastasiya; Grazhdantseva, Antonina; Richter, Vladimir; Ryabchikova, Elena

    2016-01-01

    Oncolytic abilities of vaccinia virus (VACV) served as a basis for the development of various recombinants for treating cancer; however, "natural" oncolytic properties of the virus are not examined in detail. Our study was conducted to know how the genetically unmodified L-IVP strain of VACV produces its antitumor effect. Human A431 carcinoma xenografts in nude mice and murine Ehrlich carcinoma in C57Bl mice were used as targets for VACV, which was injected intratumorally. A set of virological methods, immunohistochemistry, light and electron microscopy was used in the study. We found that in mice bearing A431 carcinoma, the L-IVP strain was observed in visceral organs within two weeks, but rapidly disappeared from the blood. The L-IVP strain caused decrease of sizes in both tumors, however, in different ways. Direct cell destruction by replicating virus plays a main role in regression of A431 carcinoma xenografts, while in Ehrlich carcinoma, which poorly supported VACV replication, the virus induced decrease of mitoses by pushing tumor cells into S-phase of cell cycle. Our study showed that genetically unmodified VACV possesses at least two mechanisms of antitumor effect: direct destruction of tumor cells and suppression of mitoses in tumor cells. PMID:26771631

  8. Features of the Antitumor Effect of Vaccinia Virus Lister Strain.

    PubMed

    Zonov, Evgeniy; Kochneva, Galina; Yunusova, Anastasiya; Grazhdantseva, Antonina; Richter, Vladimir; Ryabchikova, Elena

    2016-01-12

    Oncolytic abilities of vaccinia virus (VACV) served as a basis for the development of various recombinants for treating cancer; however, "natural" oncolytic properties of the virus are not examined in detail. Our study was conducted to know how the genetically unmodified L-IVP strain of VACV produces its antitumor effect. Human A431 carcinoma xenografts in nude mice and murine Ehrlich carcinoma in C57Bl mice were used as targets for VACV, which was injected intratumorally. A set of virological methods, immunohistochemistry, light and electron microscopy was used in the study. We found that in mice bearing A431 carcinoma, the L-IVP strain was observed in visceral organs within two weeks, but rapidly disappeared from the blood. The L-IVP strain caused decrease of sizes in both tumors, however, in different ways. Direct cell destruction by replicating virus plays a main role in regression of A431 carcinoma xenografts, while in Ehrlich carcinoma, which poorly supported VACV replication, the virus induced decrease of mitoses by pushing tumor cells into S-phase of cell cycle. Our study showed that genetically unmodified VACV possesses at least two mechanisms of antitumor effect: direct destruction of tumor cells and suppression of mitoses in tumor cells.

  9. 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

  10. Preparation of Cell Cultures and Vaccinia Virus Stocks.

    PubMed

    Cotter, Catherine A; Earl, Patricia L; Wyatt, Linda S; Moss, Bernard

    2015-11-01

    The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included.

  11. CD40 ligand and tdTomato-armed vaccinia virus for induction of antitumor immune response and tumor imaging.

    PubMed

    Parviainen, S; Ahonen, M; Diaconu, I; Hirvinen, M; Karttunen, Å; Vähä-Koskela, M; Hemminki, A; Cerullo, V

    2014-02-01

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

  12. 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

  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. 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.

  15. Oncolytic measles virus retargeting by ligand display.

    PubMed

    Msaouel, Pavlos; Iankov, Ianko D; Allen, Cory; Russell, Stephen J; Galanis, Evanthia

    2012-01-01

    Despite significant advances in recent years, treatment of metastatic malignancies remains a significant challenge. There is an urgent need for development of novel therapeutic approaches. Virotherapy approaches have considerable potential, and among them measles virus (MV) vaccine strains have emerged as a promising oncolytic platform. Retargeted MV strains deriving from the Edmonston vaccine lineage (MV-Edm) have shown comparable antitumor efficacy to unmodified strains against receptor expressing tumor cells with improved therapeutic index. Here, we describe the construction, rescue, amplification, and titration of fully retargeted MV-Edm derivatives displaying tumor specific receptor binding ligands on the viral surface in combination with H protein CD46 and SLAM entry ablating mutations.

  16. An update on the vaccinia virus genome.

    PubMed

    Johnson, G P; Goebel, S J; Paoletti, E

    1993-10-01

    This communication is intended as a single source update of the initial report (Goebel et al., 1990a,b) which described the complete DNA sequence of the vaccinia virus genome. We have integrated published information as well as unpublished data. Our understanding of the complexities of the genetic functional organization of poxviruses is increasing at a remarkable rate. While some previously unknown identities have since been elucidated, the fact that the majority of vaccinia-encoded gene products still lack assigned functions lends excitement to the immediate future of poxvirus research.

  17. 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

  18. 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

  19. The complete DNA sequence of vaccinia virus.

    PubMed

    Goebel, S J; Johnson, G P; Perkus, M E; Davis, S W; Winslow, J P; Paoletti, E

    1990-11-01

    The complete DNA sequence of the genome of vaccinia virus has been determined. The genome consisted of 191,636 bp with a base composition of 66.6% A + T. We have identified 198 "major" protein-coding regions and 65 overlapping "minor" regions, for a total of 263 potential genes. Genes encoded by the virus were located by examination of DNA sequence characteristics and compared with existing vaccinia virus mapping analyses, sequence data, and transcription data. These genes were found to be compactly organized along the genome with relatively few regions of noncoding sequences. Whereas several similarities to proteins of known function were discerned, the function of the majority of proteins encoded by these open reading frames is as yet undetermined.

  20. 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. PMID:27407036

  1. 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.

  2. 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.

  3. Oncolytic Sendai virus-based virotherapy for cancer: recent advances

    PubMed Central

    Saga, Kotaro; Kaneda, Yasufumi

    2015-01-01

    Many drugs have been developed and optimized for the treatment of cancer; however, it is difficult to completely cure cancer with anticancer drugs alone. Therefore, the development of new therapeutic technologies, in addition to new anticancer drugs, is necessary for more effective oncotherapy. Oncolytic viruses are one potential new anticancer strategy. Various oncolytic viruses have been developed for safe and effective oncotherapy. Recently, Sendai virus-based oncotherapy has been reported by several groups, and attention has been drawn to its unique anticancer mechanisms, which are different from those of the conventional oncolytic viruses that kill cancer cells by cancer cell-selective replication. Here, we introduce Sendai virus-based virotherapy and its anticancer mechanisms. PMID:27512677

  4. Experimental therapies: gene therapies and oncolytic viruses.

    PubMed

    Hulou, M Maher; Cho, Choi-Fong; Chiocca, E Antonio; Bjerkvig, Rolf

    2016-01-01

    Glioblastoma is the most common and aggressive primary brain tumor in adults. Over the past three decades, the overall survival time has only improved by a few months, therefore novel alternative treatment modalities are needed to improve clinical management strategies. Such strategies should ultimately extend patient survival. At present, the extensive insight into the molecular biology of gliomas, as well as into genetic engineering techniques, has led to better decision processes when it comes to modifying the genome to accommodate suicide genes, cytokine genes, and tumor suppressor genes that may kill cancer cells, and boost the host defensive immune system against neoantigenic cytoplasmic and nuclear targets. Both nonreplicative viral vectors and replicating oncolytic viruses have been developed for brain cancer treatment. Stem cells, microRNAs, nanoparticles, and viruses have also been designed. These have been armed with transgenes or peptides, and have been used both in laboratory-based experiments as well as in clinical trials, with the aim of improving selective killing of malignant glioma cells while sparing normal brain tissue. This chapter reviews the current status of gene therapies for malignant gliomas and highlights the most promising viral and cell-based strategies under development. PMID:26948355

  5. Reemergence of vaccinia virus during Zoonotic outbreak, Pará State, Brazil.

    PubMed

    de Assis, Felipe L; Vinhote, Wagner M; Barbosa, José D; de Oliveira, Cairo H S; de Oliveira, Carlos M G; Campos, Karinny F; Silva, Natália S; Trindade, Giliane de Souza

    2013-12-01

    In 2010, vaccinia virus caused an outbreak of bovine vaccinia that affected dairy cattle and rural workers in Pará State, Brazil. Genetic analyses identified the virus as distinct from BeAn58058 vaccinia virus (identified in 1960s) and from smallpox vaccine virus strains. These findings suggest spread of autochthonous group 1 vaccinia virus in this region.

  6. Outbreak of severe zoonotic vaccinia virus infection, Southeastern Brazil.

    PubMed

    Abrahão, Jônatas Santos; Campos, Rafael Kroon; Trindade, Giliane de Souza; Guimarães da Fonseca, Flávio; Ferreira, Paulo César Peregrino; Kroon, Erna Geessien

    2015-04-01

    In 2010, a vaccinia virus isolate caused an atypically severe outbreak that affected humans and cattle in Brazil. Of 26 rural workers affected, 12 were hospitalized. Our data raise questions about the risk factors related to the increasing number and severity of vaccinia virus infections.

  7. Complete Genome Sequence of Vaccinia Virus Strain L-IVP

    PubMed Central

    Shvalov, Alexander N.; Sivolobova, Galina F.; Kuligina, Elena V.

    2016-01-01

    Most of the live vaccine doses of vaccinia virus donated to the Intensified Smallpox Eradication Programme after 1971 were prepared using the L-IVP strain. A mixture of three clones of the L-IVP strain was sequenced using MySEQ. Consensus sequence similarity with the vaccinia virus Lister strain is 99.5%. PMID:27174282

  8. Complete Genome Sequence of Vaccinia Virus Strain L-IVP.

    PubMed

    Shvalov, Alexander N; Sivolobova, Galina F; Kuligina, Elena V; Kochneva, Galina V

    2016-01-01

    Most of the live vaccine doses of vaccinia virus donated to the Intensified Smallpox Eradication Programme after 1971 were prepared using the L-IVP strain. A mixture of three clones of the L-IVP strain was sequenced using MySEQ. Consensus sequence similarity with the vaccinia virus Lister strain is 99.5%. PMID:27174282

  9. Plasmodium knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Godson, G. Nigel; Nussenzweig, Victor; Nussenzweig, Ruth S.; Barnwell, John; Moss, Bernard

    1984-04-01

    The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Cells infected with the recombinant virus synthesized polypeptides of 53,000 to 56,000 daltons that reacted with monoclonal antibody against the repeating epitope of the malaria protein. Furthermore, rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as live malaria vaccines.

  10. 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.

  11. 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. PMID:27660749

  12. 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

  13. 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.

  14. 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

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

    PubMed

    Swift, Stephanie L; Stojdl, David F

    2016-02-01

    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

  16. 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.

  17. Functional hyper-IL-6 from vaccinia virus-colonized tumors triggers platelet formation and helps to alleviate toxicity of mitomycin C enhanced virus therapy

    PubMed Central

    2012-01-01

    Background Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic. Methods Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects. Results We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia. Conclusion Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects. PMID:22236378

  18. Structure of vaccinia virus early promoters.

    PubMed

    Davison, A J; Moss, B

    1989-12-20

    Functional elements of a vaccinia virus early promoter were characterized by making a complete set of single nucleotide substitutions, as well as more complex mutations, and assaying their effects on gene expression. Synthetic oligonucleotides, based primarily on the sequence of the 7.5-kD early promoter, were inserted into a plasmid vector containing the lacZ gene of Escherichia coli flanked by sequences from the thymidine kinase (TK) gene of vaccinia virus. The lacZ gene, under control of the synthetic promoter, was introduced into the vaccinia virus genome at the TK locus by homologous recombination, and each of the 331 different recombinant viruses thus obtained was assayed for beta-galactosidase expression. The relative amounts and precise 5' ends of lacZ mRNAs specified by a subset of the recombinants were determined by primer extension. Many promoters were tested for their ability to direct specific transcription in vitro. A generally good correlation was noted between measurements of promoter strength estimated by beta-galactosidase expression, primer extension of in vivo mRNA and transcription in vitro. A relatively simple picture emerged from the analysis. The early promoter consists of a 16 base-pair critical region, in which most single nucleotide substitutions have a major effect on expression, separated by 11 base-pairs of a less critical T-rich sequence from a seven base-pair region within which initiation with a purine usually occurs. For the critical region of the 7.5-kD promoter, AAAAgTaGAAAataTA, any substitution of an upper-case nucleotide reduced expression, usually drastically, whereas certain substitutions of lower-case nucleotides maintained or significantly enhanced expression. On the basis of this analysis, the wide range of activities of natural promoters could be attributed to the presence of one or more non-optimal nucleotides in the critical region. Moreover, single nucleotide substitutions in such promoters had the predicted enhancing

  19. 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.

  20. Construction of chimeric vaccinia viruses by molecular cloning and packaging.

    PubMed Central

    Scheiflinger, F; Dorner, F; Falkner, F G

    1992-01-01

    Foreign DNA was inserted into unique restriction endonuclease cleavage sites (Sma I or Not I) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. The modified vaccinia virus DNA was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. In contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and families of chimeras with multiple inserts were obtained. The different genomic configurations of chimeric viruses provide a broader basis for screening of optimal viruses. In addition to packaging in avian cells, a second packaging procedure for vaccinia DNA, based on the abortive infection of mammalian cells with the fowlpox helper virus, was developed. This procedure permits simultaneous packaging and host-range selection for the packaged virus. The cloning/packaging procedure allows the direct insertion of foreign DNA without the need for plasmids having flanking regions homologous to viral nonessential regions and is independent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% of the total vaccinia virus yield consisted of chimeras. The procedure is, therefore, a useful tool in molecular virology. Images PMID:1438247

  1. A vaccinia virus renaissance: new vaccine and immunotherapeutic uses after smallpox eradication.

    PubMed

    Verardi, Paulo H; Titong, Allison; Hagen, Caitlin J

    2012-07-01

    In 1796, Edward Jenner introduced the concept of vaccination with cowpox virus, an Orthopoxvirus within the family Poxviridae that elicits cross protective immunity against related orthopoxviruses, including smallpox virus (variola virus). Over time, vaccinia virus (VACV) replaced cowpox virus as the smallpox vaccine, and vaccination efforts eventually led to the successful global eradication of smallpox in 1979. VACV has many characteristics that make it an excellent vaccine and that were crucial for the successful eradication of smallpox, including (1) its exceptional thermal stability (a very important but uncommon characteristic in live vaccines), (2) its ability to elicit strong humoral and cell-mediated immune responses, (3) the fact that it is easy to propagate, and (4) that it is not oncogenic, given that VACV replication occurs exclusively within the host cell cytoplasm and there is no evidence that the viral genome integrates into the host genome. Since the eradication of smallpox, VACV has experienced a renaissance of interest as a viral vector for the development of recombinant vaccines, immunotherapies, and oncolytic therapies, as well as the development of next-generation smallpox vaccines. This revival is mainly due to the successful use and extensive characterization of VACV as a vaccine during the smallpox eradication campaign, along with the ability to genetically manipulate its large dsDNA genome while retaining infectivity and immunogenicity, its wide mammalian host range, and its natural tropism for tumor cells that allows its use as an oncolytic vector. This review provides an overview of new uses of VACV that are currently being explored for the development of vaccines, immunotherapeutics, and oncolytic virotherapies. PMID:22777090

  2. A vaccinia virus renaissance: new vaccine and immunotherapeutic uses after smallpox eradication.

    PubMed

    Verardi, Paulo H; Titong, Allison; Hagen, Caitlin J

    2012-07-01

    In 1796, Edward Jenner introduced the concept of vaccination with cowpox virus, an Orthopoxvirus within the family Poxviridae that elicits cross protective immunity against related orthopoxviruses, including smallpox virus (variola virus). Over time, vaccinia virus (VACV) replaced cowpox virus as the smallpox vaccine, and vaccination efforts eventually led to the successful global eradication of smallpox in 1979. VACV has many characteristics that make it an excellent vaccine and that were crucial for the successful eradication of smallpox, including (1) its exceptional thermal stability (a very important but uncommon characteristic in live vaccines), (2) its ability to elicit strong humoral and cell-mediated immune responses, (3) the fact that it is easy to propagate, and (4) that it is not oncogenic, given that VACV replication occurs exclusively within the host cell cytoplasm and there is no evidence that the viral genome integrates into the host genome. Since the eradication of smallpox, VACV has experienced a renaissance of interest as a viral vector for the development of recombinant vaccines, immunotherapies, and oncolytic therapies, as well as the development of next-generation smallpox vaccines. This revival is mainly due to the successful use and extensive characterization of VACV as a vaccine during the smallpox eradication campaign, along with the ability to genetically manipulate its large dsDNA genome while retaining infectivity and immunogenicity, its wide mammalian host range, and its natural tropism for tumor cells that allows its use as an oncolytic vector. This review provides an overview of new uses of VACV that are currently being explored for the development of vaccines, immunotherapeutics, and oncolytic virotherapies.

  3. Comparison of the genetic maps of variola and vaccinia viruses.

    PubMed

    Shchelkunov, S N; Resenchuk, S M; Totmenin, A V; Blinov, V M; Marennikova, S S; Sandakhchiev, L S

    1993-08-01

    The complete genetic map of the variola major virus strain India-1967 is built basing on the sequence data. The suggested map is compared with the maps of the sequenced genomic regions of Copenhagen and Western Reserve strains of vaccinia virus and Harvey strain of variola major virus. The principle differences revealed in the genomic organization of these viruses are discussed.

  4. 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.

  5. Chimpanzee/human mAbs to vaccinia virus B5 protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus.

    PubMed

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

    2006-02-01

    Chimpanzee Fabs against the B5 envelope glycoprotein of vaccinia virus were isolated and converted into complete mAbs with human gamma 1 heavy chain constant regions. The two mAbs (8AH8AL and 8AH7AL) displayed high binding affinities to B5 (Kd of 0.2 and 0.7 nM). The mAb 8AH8AL inhibited the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro, protected mice from subsequent intranasal challenge with virulent vaccinia virus, protected mice when administered 2 days after challenge, and provided significantly greater protection than that afforded by a previously isolated rat anti-B5 mAb (19C2) or by vaccinia immune globulin. The mAb bound to a conformational epitope between amino acids 20 and 130 of B5. These chimpanzee/human anti-B5 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.

  6. Highly immunogenic variant of attenuated vaccinia virus.

    PubMed

    Yakubitskyi, S N; Kolosova, I V; Maksyutov, R A; Shchelkunov, S N

    2016-01-01

    The LIVPΔ6 strain of vaccinia virus (VACV) was created by genetic engineering on the basis of previously obtained attenuated 1421ABJCN strain by target deletion of the A35R gene encoding an inhibitor of antigen presentation by the major histocompatibility complex class II. 1421ABJCN is the LIVP strain of VACV with five inactivated virulence genes encoding hemagglutinin (A56R), γ-interferon-binding protein (B8R), thymidine kinase (J2R), complement-binding protein (C3L), and Bcl2-like inhibitor of apoptosis (N1L). The highly immunogenic LIVPΔ6 strain could be an efficient fourth-generation attenuated vaccine against smallpox and other orthopoxvirus infections. PMID:27025484

  7. 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

  8. 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.

  9. 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

  10. 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

  11. Use of vaccinia virus to express biopharmaceutical products.

    PubMed

    Hruby, D E; Thomas, G

    1987-04-01

    Recent technological advancements have fostered the continued development of vaccinia virus as an efficient eukaryotic cloning and expression vector system. Genetically engineered vaccinia virus strains have been constructed for use (i) as recombinant vaccines for the prophylaxis of infectious disease, (ii) in producing significant quantities of biologically active polypeptide factors or enzymes, and (iii) as basic research tools with which to investigate primary structure-function relationships between proteins and their catalytic activities. This review examines the basic vaccinia vector system, its advantages and limitations, and current areas of research. As a specific example of the power and utility of this approach, attention is focused on the application of this technology to the field of neurobiology, specifically the use of recombinant vaccinia to study the expression, processing, and transport of cellular neuropeptides.

  12. 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.

  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. History of oncolytic viruses: genesis to genetic engineering.

    PubMed

    Kelly, Elizabeth; Russell, Stephen J

    2007-04-01

    Since the turn of the nineteenth century, when their existence was first recognized, viruses have attracted considerable interest as possible agents of tumor destruction. Early case reports emphasized regression of cancers during naturally acquired virus infections, providing the basis for clinical trials where body fluids containing human or animal viruses were used to transmit infections to cancer patients. Most often the viruses were arrested by the host immune system and failed to impact tumor growth, but sometimes, in immunosuppressed patients, infection persisted and tumors regressed, although morbidity as a result of the infection of normal tissues was unacceptable. With the advent of rodent models and new methods for virus propagation, there were numerous attempts through the 1950s and 1960s to force the evolution of viruses with greater tumor specificity, but success was limited and many researchers abandoned the field. Technology employing reverse genetics later brought about a renewal of interest in virotherapy that allowed the generation of more potent, tumor-specific oncolytics. Here, examination of early oncolytic virotherapy before genetic engineering serves to highlight tremendous advances, yet also hints at ways to penetrate host immune defenses, a significant remaining challenge in modern virotherapy research.

  15. 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.

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

    PubMed Central

    2012-01-01

    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. PMID:22216938

  17. Identification of genetically modified Maraba virus as an oncolytic rhabdovirus.

    PubMed

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

    2010-08-01

    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.

  18. 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

  19. Modification of HSV-1 to an oncolytic virus.

    PubMed

    Nakashima, Hiroshi; Chiocca, E Antonio

    2014-01-01

    Cancer-permissive viruses or oncolytic viruses consist of either genetically engineered or naturally occurring strains that possess relatively selective replicative and/or infection abilities for cancer vs. normal cells (Chiocca, Nat Rev Cancer 2: 938-950, 2002). They can also be armed with additional anticancer cDNAs (e.g., cytokines, prodrug-activating, anti-angiogenesis genes, and others) to extend therapeutic effects (Kaur et al., Curr Gene Ther 9: 341-355, 2009). Herpes simplex virus type 1 (HSV-1) possesses several advantages as an oncolytic virus such as a rapid lytic cycle and a large capacity for insertion of heterologous DNA sequences (Wade-Martins et al., Nat Biotechnol, 19: 1067-1070, 2001). However, the technical nuances of genetic manipulation of the HSV-1 genome may still be relatively challenging. Here, we describe a system that has been durable and consistent in providing the ability to generate multiple recombinant HSV-1. The HsvQuik technology utilizes an HSV-1 genome cloned in a bacterial artificial chromosome to recombine heterologous cDNAs in a relatively rapid and reliable manner (Terada et al., Gene Ther 13: 705-714, 2006). PMID:24671680

  20. 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.

  1. 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

  2. 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.

  3. A recombinant vaccinia virus expressing human carcinoembryonic antigen (CEA).

    PubMed

    Kaufman, H; Schlom, J; Kantor, J

    1991-07-30

    Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein expressed on most gastrointestinal carcinomas. A 2.4-kb cDNA clone, containing the complete coding sequence, was isolated from a human colon tumor cell library and inserted into a vaccinia virus genome. This newly developed construct was characterized by Southern blotting, DNA hybridization studies, and polymerase chain reaction analysis. The CEA gene was stably integrated into the vaccinia virus thymidine kinase gene. The recombinant was efficiently replicated upon serial passages in cell cultures and in animals. The recombinant virus expresses on the surface of infected cells a protein product recognized by a monoclonal antibody (COL-I) directed against CEA. Immunization of mice with the vaccinia construct elicited a humoral immune response against CEA. Pilot studies also showed that administration of the recombinant CEA vaccinia construct was able to greatly reduce the growth in mice of a syngeneic murine colon adenocarcinoma which had been transduced with the human CEA gene. The use of this new recombinant CEA vaccinia construct may thus provide an approach in the specific active immunotherapy of human GI cancer and other CEA expressing carcinoma types.

  4. 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

  5. Mechanistic insights into the oncolytic activity of vesicular stomatitis virus in cancer immunotherapy

    PubMed Central

    Simovic, Boris; Walsh, Scott R; Wan, Yonghong

    2015-01-01

    Immunotherapy and oncolytic virotherapy have both shown anticancer efficacy in the clinic as monotherapies but the greatest promise lies in therapies that combine these approaches. Vesicular stomatitis virus is a prominent oncolytic virus with several features that promise synergy between oncolytic virotherapy and immunotherapy. This review will address the cytotoxicity of vesicular stomatitis virus in transformed cells and what this means for antitumor immunity and the virus’ immunogenicity, as well as how it facilitates the breaking of tolerance within the tumor, and finally, we will outline how these features can be incorporated into the rational design of new treatment strategies in combination with immunotherapy. PMID:27512679

  6. Preclinical Mouse Models for Analysis of the Therapeutic Potential of Engineered Oncolytic Herpes Viruses.

    PubMed

    Speranza, Maria-Carmela; Kasai, Kazue; Lawler, Sean E

    2016-01-01

    After more than two decades of research and development, oncolytic herpes viruses (oHSVs) are moving into the spotlight due to recent encouraging clinical trial data. oHSV and other oncolytic viruses function through direct oncolytic cancer cell-killing mechanisms and by stimulating antitumor immunity. As further viruses are developed and optimized for the treatment of various types of cancer, appropriate predictive preclinical models will be of great utility. This review will discuss existing data in this area, focusing on the mouse tumor models that are commonly used. PMID:27034396

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

    PubMed Central

    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. PMID:27512683

  8. 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.

  9. 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.

  10. Early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization.

    PubMed

    Vennema, H; de Groot, R J; Harbour, D A; Dalderup, M; Gruffydd-Jones, T; Horzinek, M C; Spaan, W J

    1990-03-01

    The gene encoding the fusogenic spike protein of the coronavirus causing feline infectious peritonitis was recombined into the genome of vaccinia virus. The recombinant induced spike-protein-specific, in vitro neutralizing antibodies in mice. When kittens were immunized with the recombinant, low titers of neutralizing antibodies were obtained. After challenge with feline infectious peritonitis virus, these animals succumbed earlier than did the control group immunized with wild-type vaccinia virus (early death syndrome).

  11. Integrin β1 mediates vaccinia virus entry through activation of PI3K/Akt signaling.

    PubMed

    Izmailyan, Roza; Hsao, Jye-Chian; Chung, Che-Sheng; Chen, Chein-Hung; Hsu, Paul Wei-Che; Liao, Chung-Lin; Chang, Wen

    2012-06-01

    Vaccinia virus has a broad range of infectivity in many cell lines and animals. Although it is known that the vaccinia mature virus binds to cell surface glycosaminoglycans and extracellular matrix proteins, whether additional cellular receptors are required for virus entry remains unclear. Our previous studies showed that the vaccinia mature virus enters through lipid rafts, suggesting the involvement of raft-associated cellular proteins. Here we demonstrate that one lipid raft-associated protein, integrin β1, is important for vaccinia mature virus entry into HeLa cells. Vaccinia virus associates with integrin β1 in lipid rafts on the cell surface, and the knockdown of integrin β1 in HeLa cells reduces vaccinia mature virus entry. Additionally, vaccinia mature virus infection is reduced in a mouse cell line, GD25, that is deficient in integrin β1 expression. Vaccinia mature virus infection triggers the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, and the treatment of cells with inhibitors to block P13K activation reduces virus entry in an integrin β1-dependent manner, suggesting that integrin β1-mediates PI3K/Akt activation induced by vaccinia virus and that this signaling pathway is essential for virus endocytosis. The inhibition of integrin β1-mediated cell adhesion results in a reduction of vaccinia virus entry and the disruption of focal adhesion and PI3K/Akt activation. In summary, our results show that the binding of vaccinia mature virus to cells mimics the outside-in activation process of integrin functions to facilitate vaccinia virus entry into HeLa cells.

  12. From Crescent to Mature Virion: Vaccinia Virus Assembly and Maturation

    PubMed Central

    Liu, Liang; Cooper, Tamara; Howley, Paul M.; Hayball, John D.

    2014-01-01

    Vaccinia virus (VACV) has achieved unprecedented success as a live viral vaccine for smallpox which mitigated eradication of the disease. Vaccinia virus has a complex virion morphology and recent advances have been made to answer some of the key outstanding questions, in particular, the origin and biogenesis of the virion membrane, the transformation from immature virion (IV) to mature virus (MV), and the role of several novel genes, which were previously uncharacterized, but have now been shown to be essential for VACV virion formation. This new knowledge will undoubtedly contribute to the rational design of safe, immunogenic vaccine candidates, or effective antivirals in the future. This review endeavors to provide an update on our current knowledge of the VACV maturation processes with a specific focus on the initiation of VACV replication through to the formation of mature virions. PMID:25296112

  13. Expression of functional Bunyamwera virus L protein by recombinant vaccinia viruses.

    PubMed

    Jin, H; Elliott, R M

    1991-08-01

    A cDNA containing the complete coding sequence of the Bunyamwera virus (family Bunyaviridae) L genome segment has been constructed and cloned into two recombinant vaccinia virus expression systems. In the first, the L gene is under control of vaccinia virus P7.5 promoter; in the second, the L gene is under control of the bacteriophage T7 phi 10 promoter, and expression of the L gene requires coinfection with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase. Both systems express a protein which is the same size as the Bunyamwera virus L protein and is recognized by a monospecific L antiserum. The expressed L protein was shown to be functional in synthesizing Bunyamwera virus RNA in a nucleocapsid transfection assay: recombinant vaccinia virus-infected cells were transfected with purified Bunyamwera virus nucleocapsids, and subsequently, total cellular RNA was analyzed by Northern (RNA) blotting. No Bunyamwera virus RNA was detected in control transfections, but in cells which had previously been infected with recombinant vaccinia viruses expressing the L protein, both positive- and negative-sense Bunyamwera virus S segment RNA was detected. The suitability of this system to delineate functional domains within the Bunyamwera virus L protein is discussed.

  14. 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

  15. 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.

  16. [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. PMID:26319388

  17. 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

  18. 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. PMID:27088104

  19. Targeted delivery of a suicide gene to human colorectal tumors by a conditionally replicating vaccinia virus.

    PubMed

    Foloppe, J; Kintz, J; Futin, N; Findeli, A; Cordier, P; Schlesinger, Y; Hoffmann, C; Tosch, C; Balloul, J-M; Erbs, P

    2008-10-01

    We have generated a thymidine kinase gene-deleted vaccinia virus (VV) (Copenhagen strain) that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. Intratumoral inoculation of this thymidine kinase gene-deleted VV encoding FCU1 (VV-FCU1) in the presence of systemically administered prodrug 5-fluorocytosine (5-FC) produced statistically significant reductions in the growth of subcutaneous human colon cancer in nude mice compared with thymidine kinase gene-deleted VV treatments or with control 5-fluorouracil alone. A limitation of prodrug therapies has often been the requirement for the direct injection of the virus into relatively large, accessible tumors. Here we demonstrate vector targeting of tumors growing subcutaneously following systemic administration of VV-FCU1. More importantly we also demonstrate that the systemic injection of VV-FCU1 in nude mice bearing orthotopic liver metastasis of a human colon cancer, with concomitant administration of 5-FC, leads to substantial tumor growth retardation. In conclusion, the insertion of the fusion FCU1 suicide gene potentiates the oncolytic efficiency of the thymidine kinase gene-deleted VV and represents a potentially efficient means for gene therapy of distant metastasis from colon and other cancers. PMID:18480846

  20. Phosphorylation of vaccinia virus core proteins during transcription in vitro.

    PubMed Central

    Moussatche, N; Keller, S J

    1991-01-01

    The phosphorylation of vaccinia virus core proteins has been studied in vitro during viral transcription. The incorporation of [gamma-32P]ATP into protein is linear for the first 2 min of the reaction, whereas incorporation of [3H]UTP into RNA lags for 1 to 2 min before linear synthesis. At least 12 different proteins are phosphorylated on autoradiograms of acrylamide gels, and the majority of label is associated with low-molecular-weight proteins. If the transcription reaction is reduced by dropping the pH to 7 from its optimal of 8.5, two proteins (70 and 80 kDa) are no longer phosphorylated. RNA isolated from the pH 7 transcription reaction hybridized primarily to the vaccinia virus HindIII DNA fragments D to F, whereas the transcripts synthesized at pH 8.5 hybridized to almost all of the HindIII-digested vaccinia virus DNA fragments. The differences between the pH 7.0 and 8.5 transcription reactions in phosphorylation and transcription could be eliminated by preincubating the viral cores with 2 mM ATP. In sum, the results suggest that the phosphorylation of the 70- and 80-kDa peptides may contribute to the regulation of early transcription. Images PMID:2016772

  1. 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.

  2. Oncolytic viruses: From bench to bedside with a focus on safety.

    PubMed

    Buijs, Pascal R A; Verhagen, Judith H E; van Eijck, Casper H J; van den Hoogen, Bernadette G

    2015-01-01

    Oncolytic viruses are a relatively new class of anti-cancer immunotherapy agents. Several viruses have undergone evaluation in clinical trials in the last decades, and the first agent is about to be approved to be used as a novel cancer therapy modality. In the current review, an overview is presented on recent (pre)clinical developments in the field of oncolytic viruses that have previously been or currently are being evaluated in clinical trials. Special attention is given to possible safety issues like toxicity, environmental shedding, mutation and reversion to wildtype virus. PMID:25996182

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

    PubMed

    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.

  4. Cultivation of vaccinia virus in sheep kidney cell cultures.

    PubMed

    SUBRAMANYAM, P; DIVAKARAN, S; VINODRAJ, P

    1961-01-01

    Attempts to find a suitable tissue for the preparation of cell monolayers for the cultivation of vaccinia virus and for the titration of this virus and its antibodies resulted in the use of sheep kidneys procured from freshly slaughtered healthy young sheep. The cultures are easy and economical to prepare and support the multiplication of the virus well. They can be used for the titration of the virus and its antibody and their sensitivity to virus is comparable to that of the chorio-allantoic membranes of chicken embryos. Preliminary trials indicate that the sheep kidney cell culture virus can be freeze-dried without suffering a significant loss in titre. Studies are in progress to determine the efficacy of a vaccine prepared from sheep kidney cell cultures.

  5. 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

  6. 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.

  7. Targeting Pediatric Cancer Stem Cells with Oncolytic Virotherapy

    PubMed Central

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

    2013-01-01

    Cancer stem cells (CSC), 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 CSC are thought to be responsible for tumorigenesis, 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 CSC by utilizing mechanisms of cell killing that differ from conventional therapies. Moreover, oncolytic viruses have the ability to target specific features of CSC 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 (HSV-1), adenovirus, reovirus, Seneca Valley virus, vaccinia virus, Newcastle disease virus, myxoma virus, vesicular stomatitis virus) to target and kill both CSC and tumor cells in pediatric tumors. We highlight advantages and limitations of each virus and potential ways next-generation engineered viruses may target resilient CSC. PMID:22430386

  8. 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.

  9. Protective efficacy of Modified Vaccinia virus Ankara in preclinical studies.

    PubMed

    Volz, Asisa; Sutter, Gerd

    2013-09-01

    Modified Vaccinia virus Ankara (MVA) is a tissue culture-derived, highly attenuated strain of vaccinia virus (VACV) exhibiting characteristic defective replication in cells from mammalian hosts. In the 1960s MVA was originally generated as a candidate virus for safer vaccination against smallpox. Now, MVA is widely used in experimental vaccine development targeting important infectious diseases and cancer. Versatile technologies for genetic engineering, large-scale production, and quality control facilitate R&D of recombinant and non-recombinant MVA vaccines matching today's requirements for new biomedical products. Such vaccines are attractive candidates for delivering antigens from pathogens against which no, or no effective vaccine is available, including emerging infections caused by highly pathogenic influenza viruses, chikungunya virus, West Nile virus or zoonotic orthopoxviruses. Other directions are seeking valuable vaccines against highly complex diseases such as AIDS, malaria, and tuberculosis. Here, we highlight examples of MVA candidate vaccines against infectious diseases, and review the efforts made to assess both the efficacy of vaccination and immune correlates of protection in preclinical studies.

  10. 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

  11. Oncolytic virus expressing RANTES and IL-15 enhances function of CAR-modified T cells in solid tumors

    PubMed Central

    Nishio, Nobuhiro; Dotti, Gianpietro

    2015-01-01

    We improved the migration and survival of chimeric antigen receptor (CAR)-modified T cells in solid tumors by combining CAR-T cells with an armed oncolytic virus. Local delivery of the chemokine RANTES and the cytokine IL-15 by the oncolytic virus enhanced the trafficking and persistence of the CAR-T cells, resulting in improved antitumor effects. PMID:25949885

  12. Clinical development of Modified Vaccinia virus Ankara vaccines.

    PubMed

    Gilbert, Sarah C

    2013-09-01

    The smallpox vaccine Vaccinia was successfully used to eradicate smallpox, but although very effective, it was a very reactogenic vaccine and responsible for the deaths of one or two people per million vaccinated. Modified Vaccinia virus Ankara (MVA) is a replication-deficient and attenuated derivative, also used in the smallpox eradication campaign and now being developed as a recombinant viral vector to produce vaccines against infectious diseases and cancer. Many clinical trials of these new vaccines have been conducted, and the findings of these trials are reviewed here. The safety of MVA is now well documented, immunogenicity is influenced by the dose and vaccination regimen, and information on the efficacy of MVA-vectored vaccines is now beginning to accumulate.

  13. Expression of the structural proteins of dengue 2 virus and yellow fever virus by recombinant vaccinia viruses.

    PubMed

    Hahn, Y S; Lenches, E M; Galler, R; Rice, C M; Dalrymple, J; Strauss, J H

    1990-01-01

    Vaccinia virus recombinants were constructed which contained cDNA sequences encoding the structural region of dengue 2 virus (PR159/S1 strain) or yellow fever virus (17D strain). The flavivirus cDNA sequences were expressed under the control of the vaccinia 7.5k early/late promotor. Cultured cells infected with these recombinants expressed immunologically reactive flavivirus structural proteins, precursor prM and E. These proteins appeared to be cleaved and glycosylated properly since they comigrated with the authentic proteins from dengue 2 virus- and yellow fever virus-infected cells. Mice immunized with the dengue/vaccinia recombinant showed a dengue-specific immune response that included low levels of neutralizing antibodies. Immunization of mice with the yellow fever/vaccinia recombinant was less effective at inducing an immune response to yellow fever virus and in only some of the mice were low titers of neutralizing antibodies produced.

  14. Exploring Reovirus Plasticity for Improving Its Use as Oncolytic Virus

    PubMed Central

    Kemp, Vera; Hoeben, Rob C.; van den Wollenberg, Diana J. M.

    2015-01-01

    Reoviruses are non-enveloped viruses with a segmented double stranded RNA genome. In humans, they are not associated with serious disease. Human reoviruses exhibit an inherent preference to replicate in tumor cells, which makes them ideally suited for use in oncolytic virotherapies. Their use as anti-cancer agent has been evaluated in several clinical trials, which revealed that intra-tumoral and systemic delivery of reoviruses are well tolerated. Despite evidence of anti-tumor effects, the efficacy of reovirus in anti-cancer monotherapy needs to be further enhanced. The opportunity to treat both the primary tumor as well as metastases makes systemic delivery a preferred administration route. Several pre-clinical studies have been conducted to address the various hurdles connected to systemic delivery of reoviruses. The majority of those studies have been done in tumor-bearing immune-deficient murine models. This thwarts studies on the impact of the contribution of the immune system to the tumor cell eradication. This review focuses on key aspects of the reovirus/host-cell interactions and the methods that are available to modify the virus to alter these interactions. These aspects are discussed with a focus on improving the reovirus’ antitumor efficacy. PMID:26712782

  15. 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

  16. Oncolytic viral therapy: targeting cancer stem cells

    PubMed Central

    Smith, Tyrel T; Roth, Justin C; Friedman, Gregory K; Gillespie, G Yancey

    2014-01-01

    Cancer stem cells (CSCs) are defined as rare populations of tumor-initiating cancer cells that are capable of both self-renewal and differentiation. Extensive research is currently underway to develop therapeutics that target CSCs for cancer therapy, due to their critical role in tumorigenesis, as well as their resistance to chemotherapy and radiotherapy. To this end, oncolytic viruses targeting unique CSC markers, signaling pathways, or the pro-tumor CSC niche offer promising potential as CSCs-destroying agents/therapeutics. We provide a summary of existing knowledge on the biology of CSCs, including their markers and their niche thought to comprise the tumor microenvironment, and then we provide a critical analysis of the potential for targeting CSCs with oncolytic viruses, including herpes simplex virus-1, adenovirus, measles virus, reovirus, and vaccinia virus. Specifically, we review current literature regarding first-generation oncolytic viruses with their innate ability to replicate in CSCs, as well as second-generation viruses engineered to enhance the oncolytic effect and CSC-targeting through transgene expression. PMID:24834430

  17. Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants.

    PubMed Central

    Riviere, M; Tartaglia, J; Perkus, M E; Norton, E K; Bongermino, C M; Lacoste, F; Duret, C; Desmettre, P; Paoletti, E

    1992-01-01

    Glycoproteins gp50, gII, and gIII of pseudorabies virus (PRV) were expressed either individually or in combination by vaccinia virus recombinants. In vitro analysis by immunoprecipitation and immunofluorescence demonstrated the expression of a gII protein of approximately 120 kDa that was proteolytically processed to the gIIb (67- to 74-kDa) and gIIc (58-kDa) mature protein species similar to those observed in PRV-infected cells. Additionally, the proper expression of the 90-kDa gIII and 50-kDa gp50 was observed. All three of these PRV-derived glycoproteins were detectable on the surface of vaccinia virus-PRV recombinant-infected cells. In vivo, mice were protected against a virulent PRV challenge after immunization with the PRV glycoprotein-expressing vaccinia virus recombinants. The coexpression of gII and gIII by a single vaccinia virus recombinant resulted in a significantly reduced vaccination dose required to protect mice against PRV challenge. Inoculation of piglets with the various vaccinia virus-PRV glycoprotein recombinants also resulted in protection against virulent PRV challenge as measured by weight gain. The simultaneous expression of gII and gp50 in swine resulted in a significantly enhanced level of protection as evaluated by weight evolution following challenge with live PRV. Images PMID:1316458

  18. A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate.

    PubMed

    Chung, C S; Hsiao, J C; Chang, Y S; Chang, W

    1998-02-01

    Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.

  19. Immune control strategies for vaccinia virus-related laboratory-acquired infections.

    PubMed

    Wei, Qiang; Jiang, Meng Nan; Han, Jun; Wang, Zi Jun

    2014-02-01

    While presenting biological characteristics of vaccinia virus and laboratory-acquired infections during related research processes, this paper focuses on benefits and risks of vaccinia virus immunization in relation to laboratory-acquired infections, describes characteristics and the adaptation of vaccinia virus vaccine, analyses the role vaccinia virus immunization plays in the prevention and control of laboratory-acquired infections, and finally proposes solutions and countermeasures to further promote and implement immune control strategies. The problem related to immune strategy and laboratory- acquired infections which is being raised, analyzed and explored plays an active and instructive role in vaccinia virus related researches and laboratory- acquired infections, and also helps to recommend and develop relevant immune strategy for future vaccine control of such infections.

  20. Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity.

    PubMed

    Smith, Geoffrey L; Benfield, Camilla T O; Maluquer de Motes, Carlos; Mazzon, Michela; Ember, Stuart W J; Ferguson, Brian J; Sumner, Rebecca P

    2013-11-01

    Virus infection of mammalian cells is sensed by pattern recognition receptors and leads to an innate immune response that restricts virus replication and induces adaptive immunity. In response, viruses have evolved many countermeasures that enable them to replicate and be transmitted to new hosts, despite the host innate immune response. Poxviruses, such as vaccinia virus (VACV), have large DNA genomes and encode many proteins that are dedicated to host immune evasion. Some of these proteins are secreted from the infected cell, where they bind and neutralize complement factors, interferons, cytokines and chemokines. Other VACV proteins function inside cells to inhibit apoptosis or signalling pathways that lead to the production of interferons and pro-inflammatory cytokines and chemokines. In this review, these VACV immunomodulatory proteins are described and the potential to create more immunogenic VACV strains by manipulation of the gene encoding these proteins is discussed.

  1. 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. PMID:27146001

  2. 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.

  3. Nucleotide sequence of the vaccinia virus hemagglutinin gene.

    PubMed

    Shida, H

    1986-04-30

    Vaccinia virus hemagglutinin (HA) is expressed at late time of infection cycle, and it is nonessential for virus growth. Location of the HA structural gene was determined by hybrid-arrested and hybrid-selected translation methods at the right terminus of the HindIII A fragment. The position of the HA gene was confirmed by the production of the complete HA protein in the cells transfected with the plasmid containing that region. Examination of this nucleotide sequence revealed the positions of cleavage sites for a number of restriction endonucleases. The deduced amino acid sequence revealed that the HA protein is a member of typical surface membrane glycoproteins. Comparison of the nucleotide sequence upstream of the HA coding region with corresponding region of other late genes suggested the existence of the consensus decanucleotides TTCATTTa/tGT between 34 to 18 bp upstream to the initiation codon followed by a cluster of A or T, a unique feature of the late genes of vaccinia virus. These results in conjunction with the ease of isolating HA- mutants provide a basis for a new site suitable for inserting foreign genes.

  4. Evolution of and Evolutionary Relationships between Extant Vaccinia Virus Strains

    PubMed Central

    Qin, Li; Favis, Nicole; Famulski, Jakub

    2014-01-01

    ABSTRACT Although vaccinia virus (VACV) was once used as a vaccine to eradicate smallpox on a worldwide scale, the biological origins of VACV are uncertain, as are the historical relationships between the different strains once used as smallpox vaccines. Here, we sequenced additional VACV strains that either represent relatively pristine examples of old vaccines (e.g., Dryvax, Lister, and Tashkent) or have been subjected to additional laboratory passage (e.g., IHD-W and WR). These genome sequences were compared with those previously reported for other VACVs as well as other orthopoxviruses. These extant VACVs do not always cluster in simple phylogenetic trees that are aligned with the known historical relationships between these strains. Rather, the pattern of deletions suggests that all existing strains likely come from a complex stock of viruses that has been passaged, distributed, and randomly sampled over time, thus obscuring simple historical or geographic links. We examined surviving nonclonal vaccine stocks, like Dryvax, which continue to harbor larger and now rare variants, including one that we have designated “clone DPP25.” DPP25 encodes genes not found in most VACV strains, including an ankyrin-F-box protein, a homolog of the variola virus (Bangladesh) B18R gene which we show can be deleted without affecting virulence in mice. We propose a simple common mechanism by which recombination of a larger and hypothetical DPP25-like ancestral strain, combined with selection for retention of critically important genes near the terminal inverted repeat boundaries (vaccinia virus growth factor gene and an interferon alpha/beta receptor homolog), could produce all known VACV variants. IMPORTANCE Smallpox was eradicated by using a combination of intensive disease surveillance and vaccination using vaccinia virus (VACV). Interestingly, little is known about the historical relationships between different strains of VACV and how these viruses may have evolved from a

  5. Vaccinia virus recombinants expressing an 11-kilodalton beta-galactosidase fusion protein incorporate active beta-galactosidase in virus particles.

    PubMed

    Huang, C; Samsonoff, W A; Grzelecki, A

    1988-10-01

    Recombinant plasmids in which vaccinia virus transcriptional regulatory sequences were fused to the Escherichia coli lacZ gene were constructed for insertion of the lacZ gene into the vaccinia virus genome. beta-Galactosidase (beta-gal) was found in some purified recombinant vaccinia virions. By enzyme activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and microscopic techniques, the evidence suggested that beta-gal accounted for 5% of the total protein in the virion. These recombinant viruses were constructed so that a portion of the coding sequences of a late vaccinia virus structural polypeptide was fused to the amino terminus of beta-gal to produce the fusion protein. Removal of the coding sequences resulted in the complete loss of beta-gal activity. This demonstrated that a vaccinia virus DNA segment from a late structural gene is responsible for the incorporation of beta-gal into the virion.

  6. Simultaneous expression of the Lassa virus N and GPC genes from a single recombinant vaccinia virus.

    PubMed

    Morrison, H G; Goldsmith, C S; Regnery, H L; Auperin, D D

    1991-03-01

    A new transfer vector was constructed that directs the insertion of two heterologous genes into the vaccinia virus thymidine kinase (TK) gene during a single recombination event. This vector, pDAVAC2, contains bidirectional vaccinia P7.5 early/late promoter elements and two unique cloning sites. cDNA clones containing the complete coding sequences for the Lassa virus (Josiah strain) nucleoprotein (N) and glycoprotein (GPC) genes were inserted into the vaccinia TK gene using this transfer vector. The recombinant virus, V-LSGN-II, expressed proteins in cell culture that appeared to be authentic with respect to electrophoretic mobility, glycosylation, and post-translational cleavage. Indirect immunofluorescence (IFA) of recombinant virus-infected cells demonstrated both the bright granular and diffuse patterns of staining characteristic of the Lassa nucleoprotein and glycoprotein, respectively. Electron-dense inclusion bodies typical of arenavirus-infected cells were observed by electron microscopy in V-LSN and V-LSGN-II-infected cells, but not in V-LSGPC-infected cells. Mice inoculated with V-LSGN-II by intraperitoneal injection developed serum antibodies that reacted with authentic Lassa proteins in immunofluorescence and radioimmune precipitation assays. This recombinant virus represents an additional candidate for a Lassa fever vaccine and demonstrates the feasibility of expressing any two genes of interest in a single recombinant vaccinia virus through the use of the transfer vector pDAVAC2.

  7. 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.

  8. THE FATE OF VACCINIA VIRUS ON CULTIVATION IN VITRO WITH KUPFFER CELLS (RETICULO-ENDOTHELIAL CELLS)

    PubMed Central

    Beard, Joseph W.; Rous, Peyton

    1938-01-01

    The pathogenic activity of vaccinia virus is in large part suppressed when it is mixed with living Kupffer cells or clasmatocytes in the test-tube and injected intradermally. Vaccinia increases in quantity when introduced into cultures of Kupffer cells in vitro, and survives in immediate association with these elements. No antiviral principle is elaborated by them under such conditions. PMID:19870763

  9. 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.

  10. Semliki Forest virus and Sindbis virus, but not vaccinia virus, require glycolysis for optimal replication.

    PubMed

    Findlay, James S; Ulaeto, David

    2015-09-01

    Viruses are obligate intracellular pathogens which rely on the cell's machinery to produce the energy and macromolecules required for replication. Infection is associated with a modified metabolic profile and one pathway which can be modified is glycolysis. In this study, we investigated if the glycolysis pathway is required for alphavirus replication. Pre-treatment of Vero cells with three different glycolysis inhibitors (2-deoxyglucose, lonidamine and oxamate) resulted in a significant reduction (but not abrogation) of Semliki Forest virus and Sindbis virus replication, but not of the unrelated virus, vaccinia virus. Reduced virus yield was not associated with any significant cytotoxic effect and delayed treatment up to 3 h post-infection still resulted in a significant reduction. This suggested that glycolysis is required for optimal replication of alphaviruses by supporting post-entry life cycle steps.

  11. 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

  12. Oncolytic viruses as immunotherapy: progress and remaining challenges.

    PubMed

    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

  13. 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

  14. 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.

  15. Oncolytic Newcastle Disease Virus as Cutting Edge between Tumor and Host

    PubMed Central

    Fournier, Philippe; Schirrmacher, Volker

    2013-01-01

    Oncolytic viruses (OVs) replicate selectively in tumor cells and exert anti-tumor cytotoxic activity. Among them, Newcastle Disease Virus (NDV), a bird RNA virus of the paramyxovirus family, appears outstanding. Its anti-tumor effect is based on: (i) oncolytic activity and (ii) immunostimulation. Together these activities facilitate the induction of post-oncolytic adaptive immunity. We will present milestones during the last 60 years of clinical evaluation of this virus. Two main strategies of clinical application were followed using the virus (i) as a virotherapeutic agent, which is applied systemically or (ii) as an immunostimulatory agent combined with tumor cells for vaccination of cancer patients. More recently, a third strategy evolved. It combines the strategies (i) and (ii) and includes also dendritic cells (DCs). The first step involves systemic application of NDV to condition the patient. The second step involves intradermal application of a special DC vaccine pulsed with viral oncolysate. This strategy, called NDV/DC, combines anti-cancer activity (oncolytic virotherapy) and immune-stimulatory properties (oncolytic immunotherapy) with the high potential of DCs (DC therapy) to prime naive T cells. The aim of such treatment is to first prepare the cancer-bearing host for immunocompetence and then to instruct the patient’s immune system with information about tumor-associated antigens (TAAs) of its own tumor together with danger signals derived from virus infection. This multimodal concept should optimize the generation of strong polyclonal T cell reactivity targeted against the patient’s TAAs and lead to the establishment of a long-lasting memory T cell repertoire. PMID:24833054

  16. 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

  17. Mitophagy switches cell death from apoptosis to necrosis in NSCLC cells treated with oncolytic measles virus.

    PubMed

    Xia, Mao; Meng, Gang; Jiang, Aiqin; Chen, Aiping; Dahlhaus, Meike; Gonzalez, Patrick; Beltinger, Christian; Wei, Jiwu

    2014-06-15

    Although apoptotic phenomena have been observed in malignant cells infected by measles virus vaccine strain Edmonston B (MV-Edm), the precise oncolytic mechanisms are poorly defined. In this study we found that MV-Edm induced autophagy and sequestosome 1-mediated mitophagy leading to decreased cytochrome c release, which blocked the pro-apoptotic cascade in non-small cell lung cancer cells (NSCLCs). The decrease of apoptosis by mitophagy favored viral replication. Persistent viral replication sustained by autophagy ultimately resulted in necrotic cell death due to ATP depletion. Importantly, when autophagy was impaired in NSCLCs MV-Edm-induced cell death was significantly abrogated despite of increased apoptosis. Taken together, our results define a novel oncolytic mechanism by which mitophagy switches cell death from apoptosis to more efficient necrosis in NSCLCs following MV-Edm infection. This provides a foundation for future improvement of oncolytic virotherapy or antiviral therapy.

  18. 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

  19. 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.

  20. 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. PMID:27672294

  1. 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

  2. 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.

  3. Transcriptional mapping of the DNA polymerase gene of vaccinia virus

    SciTech Connect

    Traktman, P.; Sridhar, P.; Condit, R.C.; Roberts, B.E.

    1984-01-01

    Vaccinia virus DNA polymerase, a single-subunit enzyme of 110,000 molecular weight, is induced early after infection. Genetic analysis suggests that the gene encoding the enzyme maps within a 15-kilobase HindIII fragment located 45 kilobases from the left-hand end of the genome. The authors identified the in vitro translation product with these propeties and mapped the transcript by hybrid selection, RNA filter hybridization, and S1 nuclease mapping. Two mRNAs from this region, 3.4 and 3.9 kilobases in size, could be translated in vitro to yield a 110K polypeptide. The two RNAs shared a common 5' terminus and had staggered 3' ends. Sequences mapping entirely within the gene were shown to be biologically active in rescuing mutants with temperature-sensitive or drug-resistant polymerase activity to the wild-type phenotype.

  4. High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara.

    PubMed

    Hebben, Matthias; Brants, Jan; Birck, Catherine; Samama, Jean-Pierre; Wasylyk, Bohdan; Spehner, Danièle; Pradeau, Karine; Domi, Arban; Moss, Bernard; Schultz, Patrick; Drillien, Robert

    2007-12-01

    Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of beta-galactosidase, about 15 mg protein could be produced from 10(8) BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production. PMID:17892951

  5. Easy and efficient protocols for working with recombinant vaccinia virus MVA.

    PubMed

    Kremer, Melanie; Volz, Asisa; Kreijtz, Joost H C M; Fux, Robert; Lehmann, Michael H; Sutter, Gerd

    2012-01-01

    Modified vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient strain of vaccinia virus that is increasingly used as vector for expression of recombinant genes in the research laboratory and in biomedicine for vaccine development. Major benefits of MVA include the clear safety advantage compared to conventional vaccinia viruses, the longstanding experience in the genetic engineering of the virus, and the availability of established procedures for virus production at an industrial scale. MVA vectors can be handled under biosafety level 1 conditions, and a multitude of recombinant MVA vaccines has proven to be immunogenic and protective when delivering various heterologous antigens in animals and humans. In this chapter we provide convenient state-of-the-art protocols for generation, amplification, and purification of recombinant MVA viruses. Importantly, we include methodology for rigid quality control to obtain best possible vector viruses for further investigations including clinical evaluation. PMID:22688761

  6. 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. PMID:20211576

  7. N-Myc expression enhances the oncolytic effects of vesicular stomatitis virus in human neuroblastoma cells.

    PubMed

    Corredor, Juan C; Redding, Nicole; Bloté, Karen; Robbins, Stephen M; Senger, Donna L; Bell, John C; Beaudry, Paul

    2016-01-01

    N-myc oncogene amplification is associated but not present in all cases of high-risk neuroblastoma (NB). Since oncogene expression could often modulate sensitivity to oncolytic viruses, we wanted to examine if N-myc expression status would determine virotherapy efficacy to high-risk NB. We showed that induction of exogenous N-myc in a non-N-myc-amplified cell line background (TET-21N) increased susceptibility to oncolytic vesicular stomatitis virus (mutant VSVΔM51) and alleviated the type I IFN-induced antiviral state. Cells with basal N-myc, on the other hand, were less susceptible to virus-induced oncolysis and established a robust IFN-mediated antiviral state. The same effects were also observed in NB cell lines with and without N-myc amplification. Microarray analysis showed that N-myc overexpression in TET-21N cells downregulated IFN-stimulated genes (ISGs) with known antiviral functions. Furthermore, virus infection caused significant changes in global gene expression in TET-21N cells overexpressing N-myc. Such changes involved ISGs with various functions. Therefore, the present study showed that augmented susceptibility to VSVΔM51 by N-myc at least involves downregulation of ISGs with antiviral functions and alleviation of the IFN-stimulated antiviral state. Our studies suggest the potential utility of N-myc amplification/overexpression as a predictive biomarker of virotherapy response for high-risk NB using IFN-sensitive oncolytic viruses. PMID:27626059

  8. N-Myc expression enhances the oncolytic effects of vesicular stomatitis virus in human neuroblastoma cells

    PubMed Central

    Corredor, Juan C; Redding, Nicole; Bloté, Karen; Robbins, Stephen M; Senger, Donna L; Bell, John C; Beaudry, Paul

    2016-01-01

    N-myc oncogene amplification is associated but not present in all cases of high-risk neuroblastoma (NB). Since oncogene expression could often modulate sensitivity to oncolytic viruses, we wanted to examine if N-myc expression status would determine virotherapy efficacy to high-risk NB. We showed that induction of exogenous N-myc in a non-N-myc-amplified cell line background (TET-21N) increased susceptibility to oncolytic vesicular stomatitis virus (mutant VSVΔM51) and alleviated the type I IFN-induced antiviral state. Cells with basal N-myc, on the other hand, were less susceptible to virus-induced oncolysis and established a robust IFN-mediated antiviral state. The same effects were also observed in NB cell lines with and without N-myc amplification. Microarray analysis showed that N-myc overexpression in TET-21N cells downregulated IFN-stimulated genes (ISGs) with known antiviral functions. Furthermore, virus infection caused significant changes in global gene expression in TET-21N cells overexpressing N-myc. Such changes involved ISGs with various functions. Therefore, the present study showed that augmented susceptibility to VSVΔM51 by N-myc at least involves downregulation of ISGs with antiviral functions and alleviation of the IFN-stimulated antiviral state. Our studies suggest the potential utility of N-myc amplification/overexpression as a predictive biomarker of virotherapy response for high-risk NB using IFN-sensitive oncolytic viruses. PMID:27626059

  9. N-Myc expression enhances the oncolytic effects of vesicular stomatitis virus in human neuroblastoma cells

    PubMed Central

    Corredor, Juan C; Redding, Nicole; Bloté, Karen; Robbins, Stephen M; Senger, Donna L; Bell, John C; Beaudry, Paul

    2016-01-01

    N-myc oncogene amplification is associated but not present in all cases of high-risk neuroblastoma (NB). Since oncogene expression could often modulate sensitivity to oncolytic viruses, we wanted to examine if N-myc expression status would determine virotherapy efficacy to high-risk NB. We showed that induction of exogenous N-myc in a non-N-myc-amplified cell line background (TET-21N) increased susceptibility to oncolytic vesicular stomatitis virus (mutant VSVΔM51) and alleviated the type I IFN-induced antiviral state. Cells with basal N-myc, on the other hand, were less susceptible to virus-induced oncolysis and established a robust IFN-mediated antiviral state. The same effects were also observed in NB cell lines with and without N-myc amplification. Microarray analysis showed that N-myc overexpression in TET-21N cells downregulated IFN-stimulated genes (ISGs) with known antiviral functions. Furthermore, virus infection caused significant changes in global gene expression in TET-21N cells overexpressing N-myc. Such changes involved ISGs with various functions. Therefore, the present study showed that augmented susceptibility to VSVΔM51 by N-myc at least involves downregulation of ISGs with antiviral functions and alleviation of the IFN-stimulated antiviral state. Our studies suggest the potential utility of N-myc amplification/overexpression as a predictive biomarker of virotherapy response for high-risk NB using IFN-sensitive oncolytic viruses.

  10. Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

    PubMed

    Artois, M; Charlton, K M; Tolson, N D; Casey, G A; Knowles, M K; Campbell, J B

    1990-10-01

    Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies. PMID:2249183

  11. Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

    PubMed Central

    Artois, M; Charlton, K M; Tolson, N D; Casey, G A; Knowles, M K; Campbell, J B

    1990-01-01

    Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies. PMID:2249183

  12. Vaccinia virus leads to ATG12–ATG3 conjugation and deficiency in autophagosome formation.

    PubMed

    Moloughney, Joseph G; Monken, Claude E; Tao, Hanlin; Zhang, Haiyan; Thomas, Janice D; Lattime, Edmund C; Jin, Shengkan

    2011-12-01

    The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12–ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia

  13. Expression of the synthetic gene for human angiogenin in recombinant vaccinia virus

    SciTech Connect

    Netesova, N.A.; Petrov, V.S.; Cheshenko, N.V.

    1995-08-01

    The gene for angiogenin was cloned into vaccinia virus genome. The recombinant virus expressing angiogenin was obtained. The level of protein synthesis directed by the recombinant virus was analyzed by immunoblotting using monoclonal antibodies against human angiogenin. 15 refs., 2 figs.

  14. 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

  15. 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.

  16. Locally Produced IL-10 Limits Cutaneous Vaccinia Virus Spread.

    PubMed

    Cush, Stephanie S; Reynoso, Glennys V; Kamenyeva, Olena; Bennink, Jack R; Yewdell, Jonathan W; Hickman, Heather D

    2016-03-01

    Skin infection with the poxvirus vaccinia (VV) elicits a powerful, inflammatory cellular response that clears virus infection in a coordinated, spatially organized manner. Given the high concentration of pro-inflammatory effectors at areas of viral infection, it is unclear how tissue pathology is limited while virus-infected cells are being eliminated. To better understand the spatial dynamics of the anti-inflammatory response to a cutaneous viral infection, we first screened cytokine mRNA expression levels after epicutaneous (ec.) VV infection and found a large increase the anti-inflammatory cytokine IL-10. Ex vivo analyses revealed that T cells in the skin were the primary IL-10-producing cells. To understand the distribution of IL-10-producing T cells in vivo, we performed multiphoton intravital microscopy (MPM) of VV-infected mice, assessing the location and dynamic behavior of IL-10 producing cells. Although virus-specific T cells were distributed throughout areas of the inflamed skin lacking overt virus-infection, IL-10+ cells closely associated with large keratinocytic foci of virus replication where they exhibited similar motility patterns to bulk antigen-specific CD8+ T cells. Paradoxically, neutralizing secreted IL-10 in vivo with an anti-IL-10 antibody increased viral lesion size and viral replication. Additional analyses demonstrated that IL-10 antibody administration decreased recruitment of CCR2+ inflammatory monocytes, which were important for reducing viral burden in the infected skin. Based upon these findings, we conclude that spatially concentrated IL-10 production limits cutaneous viral replication and dissemination, likely through modulation of the innate immune repertoire at the site of viral growth.

  17. 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.

  18. 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

  19. Intrafamilial transmission of Vaccinia virus during a bovine Vaccinia outbreak in Brazil: a new insight in viral transmission chain.

    PubMed

    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

    2014-06-01

    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.

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

    PubMed

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

    2014-11-01

    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. PMID:25450361

  1. 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.

  2. 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. PMID:12243138

  3. Redundancy complicates the definition of essential genes for vaccinia virus.

    PubMed

    Dobson, Bianca M; Tscharke, David C

    2015-11-01

    Vaccinia virus (VACV) genes are characterized as either essential or non-essential for growth in culture. It seems intuitively obvious that if a gene can be deleted without imparting a growth defect in vitro it does not have a function related to basic replication or spread. However, this interpretation relies on the untested assumption that there is no redundancy across the genes that have roles in growth in cell culture. First, we provide a comprehensive summary of the literature that describes the essential genes of VACV. Next, we looked for interactions between large blocks of non-essential genes located at the ends of the genome by investigating sets of VACVs with large deletions at the genomic termini. Viruses with deletions at either end of the genome behaved as expected, exhibiting only mild or host-range defects. In contrast, combining deletions at both ends of the genome for the VACV Western Reserve (WR) strain caused a devastating growth defect on all cell lines tested. Unexpectedly, we found that the well-studied VACV growth factor homologue encoded by C11R has a role in growth in vitro that is exposed when 42 genes are absent from the left end of the VACV WR genome. These results demonstrate that some non-essential genes contribute to basic viral growth, but redundancy means these functions are not revealed by single-gene-deletion mutants.

  4. Interactions of the vaccinia virus A19 protein.

    PubMed

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

    2013-10-01

    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.

  5. Enhanced expression of the Epstein-Barr virus latent membrane protein by a recombinant vaccinia virus.

    PubMed

    Stewart, J P; Hampson, I N; Heinrich, H W; Mackett, M; Arrand, J R

    1989-05-01

    The complete coding sequence of the Epstein-Barr virus strain B95-8 latent membrane protein (LMP) was cloned using a Raji cell cDNA library and genomic B95-8 DNA. The clone was characterized by sequencing and then used to make a recombinant vaccinia virus. This virus (VLMP) was shown to express a relatively high level of LMP in an authentic fashion. Antisera raised in rabbits against VLMP were shown to react with B95-8 LMP as well as cross-reacting with a 50K cellular protein.

  6. 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. PMID:20951746

  7. Initial characterization of vaccinia virus B4 suggests a role in virus spread.

    PubMed

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

    2014-05-01

    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.

  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. 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. PMID:27579298

  10. Characteristics of oncolytic vesicular stomatitis virus displaying tumor-targeting ligands.

    PubMed

    Ammayappan, Arun; Peng, Kah-Whye; Russell, Stephen J

    2013-12-01

    We sought proof of principle that tumor-targeting ligands can be displayed on the surface of vesicular stomatitis virus (VSV) by engineering its glycoprotein. Here, we successfully rescued VSVs displaying tumor vasculature-targeting ligands. By using a rational approach, we investigated various feasible insertion sites on the G protein of VSV (VSV-G) for display of tumor vasculature-targeting ligands, cyclic RGD (cRGD) and echistatin. We found seven sites on VSV-G that tolerated insertion of the 9-residue cRGD peptide, two of which could tolerate insertion of the 49-amino acid echistatin domain. All of the ligand-displaying viruses replicated as well as the parental virus. In vitro studies demonstrated that the VSV-echistatin viruses specifically bound to targeted integrins. Since the low-density lipoprotein receptor (LDLR) was recently identified as a major receptor for VSV, we investigated the entry of ligand-displaying viruses after masking LDLR. The experiment showed that the modified viruses can enter the cell independently of LDLR, whereas entry of unmodified virus is significantly blocked by a specific monoclonal antibody against LDLR. Both parental and ligand-displaying viruses displayed equal oncolytic efficacies in a syngeneic mouse myeloma model. We further demonstrated that single-chain antibody fragments against tumor-specific antigens can be inserted at the N terminus of the G protein and that corresponding replication-competent VSVs can be rescued efficiently. Overall, we demonstrated that functional tumor-targeting ligands can be displayed on replication-competent VSVs without perturbing viral growth and oncolytic efficacy. This study provides a rational foundation for the future development of fully retargeted oncolytic VSVs.

  11. Oncolytic vesicular stomatitis virus expressing interferon-γ has enhanced therapeutic activity

    PubMed Central

    Bourgeois-Daigneault, Marie-Claude; Roy, Dominic Guy; Falls, Theresa; Twumasi-Boateng, Kwame; St-Germain, Lauren Elizabeth; Marguerie, Monique; Garcia, Vanessa; Selman, Mohammed; Jennings, Victoria Ann; Pettigrew, Jessica; Amos, Sally; Diallo, Jean-Simon; Nelson, Brad; Bell, John Cameron

    2016-01-01

    Oncolytic viruses are known to stimulate the antitumor immune response by specifically replicating in tumor cells. This is believed to be an important aspect of the durable responses observed in some patients and the field is rapidly moving toward immunotherapy. As a further means to engage the immune system, we engineered a virus, vesicular stomatitis virus (VSV), to encode the proinflammatory cytokine interferon-γ. We used the 4T1 mammary adenocarcinoma as well as other murine tumor models to characterize immune responses in tumor-bearing animals generated by treatment with our viruses. The interferon-γ-encoding virus demonstrated greater activation of dendritic cells and drove a more profound secretion of proinflammatory cytokines compared to the parental virus. From a therapeutic point of view, the interferon-γ virus slowed tumor growth, minimized lung tumors, and prolonged survival in several murine tumor models. The improved efficacy was lost in immunocompromized animals; hence the mechanism appears to be T-cell-mediated. Taken together, these results demonstrate the ability of oncolytic viruses to act as immune stimulators to drive antitumor immunity as well as their potential for targeted gene therapy. PMID:27119116

  12. Treatment of medulloblastoma with oncolytic measles viruses expressing the angiogenesis inhibitors endostatin and angiostatin

    PubMed Central

    2014-01-01

    Background Medulloblastoma is the most common type of pediatric brain tumor. Although numerous factors influence patient survival rates, more than 30% of all cases will ultimately be refractory to conventional therapies. Current standards of care are also associated with significant morbidities, giving impetus for the development of new treatments. We have previously shown that oncolytic measles virotherapy is effective against medulloblastoma, leading to significant prolongation of survival and even cures in mouse xenograft models of localized and metastatic disease. Because medulloblastomas are known to be highly vascularized tumors, we reasoned that the addition of angiogenesis inhibitors could further enhance the efficacy of oncolytic measles virotherapy. Toward this end, we have engineered an oncolytic measles virus that express a fusion protein of endostatin and angiostatin, two endogenous and potent inhibitors of angiogenesis. Methods Oncolytic measles viruses encoding human and mouse variants of a secretable endostatin/angiostatin fusion protein were designed and rescued according to established protocols. These viruses, known as MV-hE:A and MV-mE:A respectively, were then evaluated for their anti-angiogenic potential and efficacy against medulloblastoma cell lines and orthotopic mouse models of localized disease. Results Medulloblastoma cells infected by MV-E:A readily secrete endostatin and angiostatin prior to lysis. The inclusion of the endostatin/angiostatin gene did not negatively impact the measles virus’ cytotoxicity against medulloblastoma cells or alter its growth kinetics. Conditioned media obtained from these infected cells was capable of inhibiting multiple angiogenic factors in vitro, significantly reducing endothelial cell tube formation, viability and migration compared to conditioned media derived from cells infected by a control measles virus. Mice that were given a single intratumoral injection of MV-E:A likewise showed reduced numbers

  13. 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.

  14. Recombinant Immunomodulating Lentogenic or Mesogenic Oncolytic Newcastle Disease Virus for Treatment of Pancreatic Adenocarcinoma

    PubMed Central

    Buijs, Pascal; van Nieuwkoop, Stefan; Vaes, Vincent; Fouchier, Ron; van Eijck, Casper; van den Hoogen, Bernadette

    2015-01-01

    Oncolytic Newcastle disease virus (NDV) might be a promising new therapeutic agent for the treatment of pancreatic cancer. We evaluated recombinant NDVs (rNDVs) expressing interferon (rNDV-hIFNβ-F0) or an IFN antagonistic protein (rNDV-NS1-F0), as well as rNDV with increased virulence (rNDV-F3aa) for oncolytic efficacy in human pancreatic adenocarcinoma cells. Expression of additional proteins did not hamper virus replication or cytotoxic effects on itself. However, expression of interferon, but not NS1, resulted in loss of multicycle replication. Conversely, increasing the virulence (rNDV-F3aa) resulted in enhanced replication of the virus. Type I interferon was produced in high amounts by all tumor cells inoculated with rNDV-hIFNβ-F0, while inoculation with rNDV-NS1-F0 resulted in a complete block of interferon production in most cells. Inoculation of human pancreatic adenocarcinoma cells with rNDV-F3aa caused markedly more cytotoxicity compared to rNDV-F0, while inoculation with rNDV-hIFNβ-F0 and rNDV-NS1-F0 induced cytotoxic effects comparable to those induced by the parental rNDV-F0. Evaluation in vivo using mice bearing subcutaneous pancreatic cancer xenografts revealed that only intratumoral injection with rNDV-F3aa resulted in regression of tumors. We conclude that although lentogenic rNDVs harboring proteins that modulate the type I interferon pathway proteins do have an oncolytic effect, a more virulent mesogenic rNDV might be needed to improve oncolytic efficacy. PMID:26110582

  15. 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.

  16. CTLA-4 and PD-L1 Checkpoint Blockade Enhances Oncolytic Measles Virus Therapy

    PubMed Central

    Engeland, Christine E; Grossardt, Christian; Veinalde, Rūta; Bossow, Sascha; Lutz, Diana; Kaufmann, Johanna K; Shevchenko, Ivan; Umansky, Viktor; Nettelbeck, Dirk M; Weichert, Wilko; Jäger, Dirk; von Kalle, Christof; Ungerechts, Guy

    2014-01-01

    We hypothesized that the combination of oncolytic virotherapy with immune checkpoint modulators would reduce tumor burden by direct cell lysis and stimulate antitumor immunity. In this study, we have generated attenuated Measles virus (MV) vectors encoding antibodies against CTLA-4 and PD-L1 (MV-aCTLA-4 and MV-aPD-L1). We characterized the vectors in terms of growth kinetics, antibody expression, and cytotoxicity in vitro. Immunotherapeutic effects were assessed in a newly established, fully immunocompetent murine model of malignant melanoma, B16-CD20. Analyses of tumor-infiltrating lymphocytes and restimulation experiments indicated a favorable immune profile after MV-mediated checkpoint modulation. Therapeutic benefits in terms of delayed tumor progression and prolonged median overall survival were observed for animals treated with vectors encoding anti-CTLA-4 and anti-PD-L1, respectively. Combining systemic administration of antibodies with MV treatment also improved therapeutic outcome. In vivo oncolytic efficacy against human tumors was studied in melanoma xenografts. MV-aCTLA-4 and MV-aPD-L1 were equally efficient as parental MV in this model, with high rates of complete tumor remission (> 80%). Furthermore, we could demonstrate lysis of tumor cells and transgene expression in primary tissue from melanoma patients. The current results suggest rapid translation of combining immune checkpoint modulation with oncolytic viruses into clinical application. PMID:25156126

  17. CTLA-4 and PD-L1 checkpoint blockade enhances oncolytic measles virus therapy.

    PubMed

    Engeland, Christine E; Grossardt, Christian; Veinalde, Rūta; Bossow, Sascha; Lutz, Diana; Kaufmann, Johanna K; Shevchenko, Ivan; Umansky, Viktor; Nettelbeck, Dirk M; Weichert, Wilko; Jäger, Dirk; von Kalle, Christof; Ungerechts, Guy

    2014-11-01

    We hypothesized that the combination of oncolytic virotherapy with immune checkpoint modulators would reduce tumor burden by direct cell lysis and stimulate antitumor immunity. In this study, we have generated attenuated Measles virus (MV) vectors encoding antibodies against CTLA-4 and PD-L1 (MV-aCTLA-4 and MV-aPD-L1). We characterized the vectors in terms of growth kinetics, antibody expression, and cytotoxicity in vitro. Immunotherapeutic effects were assessed in a newly established, fully immunocompetent murine model of malignant melanoma, B16-CD20. Analyses of tumor-infiltrating lymphocytes and restimulation experiments indicated a favorable immune profile after MV-mediated checkpoint modulation. Therapeutic benefits in terms of delayed tumor progression and prolonged median overall survival were observed for animals treated with vectors encoding anti-CTLA-4 and anti-PD-L1, respectively. Combining systemic administration of antibodies with MV treatment also improved therapeutic outcome. In vivo oncolytic efficacy against human tumors was studied in melanoma xenografts. MV-aCTLA-4 and MV-aPD-L1 were equally efficient as parental MV in this model, with high rates of complete tumor remission (> 80%). Furthermore, we could demonstrate lysis of tumor cells and transgene expression in primary tissue from melanoma patients. The current results suggest rapid translation of combining immune checkpoint modulation with oncolytic viruses into clinical application. PMID:25156126

  18. Can vaccinia virus be replaced by MVA virus for testing virucidal activity of chemical disinfectants?

    PubMed Central

    2010-01-01

    Background Vaccinia virus strain Lister Elstree (VACV) is a test virus in the DVV/RKI guidelines as representative of the stable enveloped viruses. Since the potential risk of laboratory-acquired infections with VACV persists and since the adverse effects of vaccination with VACV are described, the replacement of VACV by the modified vaccinia Ankara strain (MVA) was studied by testing the activity of different chemical biocides in three German laboratories. Methods The inactivating properties of different chemical biocides (peracetic acid, aldehydes and alcohols) were tested in a quantitative suspension test according to the DVV/RKI guideline. All tests were performed with a protein load of 10% fetal calf serum with both viruses in parallel using different concentrations and contact times. Residual virus was determined by endpoint dilution method. Results The chemical biocides exhibited similar virucidal activity against VACV and MVA. In three cases intra-laboratory differences were determined between VACV and MVA - 40% (v/v) ethanol and 30% (v/v) isopropanol are more active against MVA, whereas MVA seems more stable than VACV when testing with 0.05% glutardialdehyde. Test accuracy across the three participating laboratories was high. Remarkably inter-laboratory differences in the reduction factor were only observed in two cases. Conclusions Our data provide valuable information for the replacement of VACV by MVA for testing chemical biocides and disinfectants. Because MVA does not replicate in humans this would eliminate the potential risk of inadvertent inoculation with vaccinia virus and disease in non-vaccinated laboratory workers. PMID:20573218

  19. 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.

  20. Engineered measles virus as a novel oncolytic viral therapy system for hepatocellular carcinoma.

    PubMed

    Blechacz, Boris; Splinter, Patrick L; Greiner, Suzanne; Myers, Rae; Peng, Kah-Whye; Federspiel, Mark J; Russell, Stephen J; LaRusso, Nicholas F

    2006-12-01

    The oncolytic measles virus Edmonston strain (MV-Edm), a nonpathogenic virus targeting cells expressing abundant CD46, selectively destroys neoplastic tissue. Clinical development of MV-Edm would benefit from noninvasive monitoring strategies to determine the speed and extent of the spread of the virus in treated patients and the location of virus-infected cells. We evaluated recombinant MV-Edm expressing carcinoembryonic antigen (CEA) or the human sodium iodide symporter (hNIS) for oncolytic potential in hepatocellular carcinoma (HCC) and efficiency in tracking viruses in vivo by noninvasive monitoring. CD46 expression in human HCC and primary hepatocytes was assessed by flow cytometry and immunohistochemistry. Infectivity, syncytium formation, and cytotoxicity of recombinant MV-Edm in HCC cell lines were evaluated by fluorescence microscopy, crystal violet staining, and the MTS assay. Transgene expression in HCC cell lines after infection with recombinant MV-Edm in vitro and in vivo was assessed by CEA concentration, 125I-uptake, and 123I-imaging studies. Toxicology studies were performed in Ifnar(KO)xCD46 transgenic mice. The CD46 receptor was highly expressed in HCC compared to nonmalignant hepatic tissue. Recombinant MV-Edm efficiently infected HCC cell lines, resulting in extensive syncytium formation followed by cell death. Transduction of HCC cell lines and subcutaneous HCC xenografts with recombinant MV-Edm resulted in high-level expression of transgenes in vitro and in vivo. MV-Edm was nontoxic in susceptible mice. Intratumoral and intravenous therapy with recombinant MV-Edm resulted in inhibition of tumor growth and prolongation of survival with complete tumor regression in up to one third of animals. In conclusion, engineered MV-Edm may be a potent and novel cancer gene therapy system for HCC. MV-Edm expressing CEA or hNIS elicited oncolytic effects in human HCC cell lines in vitro and in vivo, enabling the spread of the virus to be monitored in a

  1. 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

  2. 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. PMID:26645902

  3. A tetrazolium-based colorimetric assay for titration of neutralizing antibodies against vaccinia virus.

    PubMed

    Ifrah, M; Stienlauf, S; Shoresh, M; Katz, E

    1998-01-01

    A colorimetric assay for titration of neutralizing antibodies against vaccinia virus was developed. The test is based on the ability of live cells in culture to reduce the yellow tetrazolium salt MTT (thiazolyl-blue), to its blue formazan derivative. Antisera from individuals vaccinated with vaccinia virus against smallpox were serially diluted, incubated with 100 plaque-forming units (PFU) of vaccinia virus for 1 hour at 37 degrees C, and then transferred to a 96-well plate containing monolayers of B-SC-1 cells. After incubation for 3 to 4 days at 37 degrees C, when more than 80% of the control infected cultures exhibited high degree of cytopathogenic effect, MTT was added. The absorbance of the formazan formed and extracted by dimethylsulfoxide was read at 492 nm by an automatic microplate spectrophotometer. A good correlation was found between the results obtained using this newly developed method and those of the plaque-reduction assay.

  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. Biochemical and biophysical properties of a putative hub protein expressed by vaccinia virus.

    PubMed

    Kay, Nicole E; Bainbridge, Travis W; Condit, Richard C; Bubb, Michael R; Judd, Reuben E; Venkatakrishnan, Balasubramanian; McKenna, Robert; D'Costa, Susan M

    2013-04-19

    H5 is a constitutively expressed, phosphorylated vaccinia virus protein that has been implicated in viral DNA replication, post-replicative gene expression, and virus assembly. For the purpose of understanding the role of H5 in vaccinia biology, we have characterized its biochemical and biophysical properties. Previously, we have demonstrated that H5 is associated with an endoribonucleolytic activity. In this study, we have shown that this cleavage results in a 3'-OH end suitable for polyadenylation of the nascent transcript, corroborating a role for H5 in vaccinia transcription termination. Furthermore, we have shown that H5 is intrinsically disordered, with an elongated rod-shaped structure that preferentially binds double-stranded nucleic acids in a sequence nonspecific manner. The dynamic phosphorylation status of H5 influences this structure and has implications for the role of H5 in multiple processes during virus replication. PMID:23476017

  6. Biochemical and Biophysical Properties of a Putative Hub Protein Expressed by Vaccinia Virus*

    PubMed Central

    Kay, Nicole E.; Bainbridge, Travis W.; Condit, Richard C.; Bubb, Michael R.; Judd, Reuben E.; Venkatakrishnan, Balasubramanian; McKenna, Robert; D'Costa, Susan M.

    2013-01-01

    H5 is a constitutively expressed, phosphorylated vaccinia virus protein that has been implicated in viral DNA replication, post-replicative gene expression, and virus assembly. For the purpose of understanding the role of H5 in vaccinia biology, we have characterized its biochemical and biophysical properties. Previously, we have demonstrated that H5 is associated with an endoribonucleolytic activity. In this study, we have shown that this cleavage results in a 3′-OH end suitable for polyadenylation of the nascent transcript, corroborating a role for H5 in vaccinia transcription termination. Furthermore, we have shown that H5 is intrinsically disordered, with an elongated rod-shaped structure that preferentially binds double-stranded nucleic acids in a sequence nonspecific manner. The dynamic phosphorylation status of H5 influences this structure and has implications for the role of H5 in multiple processes during virus replication. PMID:23476017

  7. Vaccinia Virus Telomeres: Interaction with the Viral I1, I6, and K4 Proteins

    PubMed Central

    DeMasi, Joseph; Du, Shan; Lennon, David; Traktman, Paula

    2001-01-01

    The 192-kb linear DNA genome of vaccinia virus has covalently closed hairpin termini that are extremely AT rich and contain 12 extrahelical bases. Vaccinia virus telomeres have previously been implicated in the initiation of viral genome replication; therefore, we sought to determine whether the telomeres form specific protein-DNA complexes. Using an electrophoretic mobility shift assay, we found that extracts prepared from virions and from the cytoplasm of infected cells contain telomere binding activity. Four shifted complexes were detected using hairpin probes representing the viral termini, two of which represent an interaction with the “flip” isoform and two with the “flop” isoform. All of the specificity for protein binding lies within the terminal 65-bp hairpin sequence. Viral hairpins lacking extrahelical bases cannot form the shifted complexes, suggesting that DNA structure is crucial for complex formation. Using an affinity purification protocol, we purified the proteins responsible for hairpin-protein complex formation. The vaccinia virus I1 protein was identified as being necessary and sufficient for the formation of the upper doublet of shifted complexes, and the vaccinia virus I6 protein was shown to form the lower doublet of shifted complexes. Competition and challenge experiments confirmed that the previously uncharacterized I6 protein binds tightly and with great specificity to the hairpin form of the viral telomeric sequence. Incubation of viral hairpins with extracts from infected cells also generates a smaller DNA fragment that is likely to reflect specific nicking at the apex of the hairpin; we show that the vaccinia virus K4 protein is necessary and sufficient for this reaction. We hypothesize that these telomere binding proteins may play a role in the initiation of vaccinia virus genome replication and/or genome encapsidation. PMID:11581377

  8. Expression of the F and HN glycoproteins of human parainfluenza virus type 3 by recombinant vaccinia viruses: contributions of the individual proteins to host immunity.

    PubMed

    Spriggs, M K; Murphy, B R; Prince, G A; Olmsted, R A; Collins, P L

    1987-11-01

    cDNA clones containing the complete coding sequences for the human parainfluenza virus type 3 (PIV3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes were inserted into the thymidine kinase gene of vaccinia virus (WR strain) under the control of the P7.5 early-late vaccinia virus promotor. The recombinant vaccinia viruses, designated vaccinia-F and vaccinia-HN, expressed glycoproteins in cell culture that appeared to be authentic with respect to glycosylation, disulfide linkage, electrophoretic mobility, cell surface expression, and, in the case of the HN protein, biological activity. Cotton rats inoculated intradermally with vaccinia-HN developed serum neutralizing antibody titers equal to that induced by respiratory tract infection with PIV3, whereas animals receiving vaccinia-F had threefold lower neutralizing antibody titers. A single immunization with either recombinant vaccinia virus induced nearly complete resistance in the lower respiratory tract of these animals. With regard to protection in the upper respiratory tract, animals immunized with vaccinia-HN or vaccinia-F exhibited reductions in PIV3 replication of greater than 3,000-fold and 6-fold, respectively. This large difference (greater than 500-fold) in reduction of PIV3 replication in the upper respiratory tract was in contrast to the relatively modest difference (3-fold) in serum neutralizing antibody titers induced by vaccinia-HN versus vaccinia-F. This dissociation between the level of neutralizing antibodies and protection suggested that immunity to PIV3 is complex, and that immune mechanisms other than serum neutralizing antibodies make important contributions to resistance to infection. Overall, under these experimental conditions, vaccinia-HN induced a substantially more protective immune response than did vaccinia-F.

  9. 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. PMID:26719429

  10. Treatment of malignant effusion by oncolytic virotherapy in an experimental subcutaneous xenograft model of lung cancer

    PubMed Central

    2013-01-01

    Background Malignant pleural effusion (MPE) is associated with advanced stages of lung cancer and is mainly dependent on invasion of the pleura and expression of vascular endothelial growth factor (VEGF) by cancer cells. As MPE indicates an incurable disease with limited palliative treatment options and poor outcome, there is an urgent need for new and efficient treatment options. Methods In this study, we used subcutaneously generated PC14PE6 lung adenocarcinoma xenografts in athymic mice that developed subcutaneous malignant effusions (ME) which mimic pleural effusions of the orthotopic model. Using this approach monitoring of therapeutic intervention was facilitated by direct observation of subcutaneous ME formation without the need of sacrificing mice or special imaging equipment as in case of MPE. Further, we tested oncolytic virotherapy using Vaccinia virus as a novel treatment modality against ME in this subcutaneous PC14PE6 xenograft model of advanced lung adenocarcinoma. Results We demonstrated significant therapeutic efficacy of Vaccinia virus treatment of both advanced lung adenocarcinoma and tumor-associated ME. We attribute the efficacy to the virus-mediated reduction of tumor cell-derived VEGF levels in tumors, decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of the blood vessel-invading tumor cells. Moreover, we showed that the use of oncolytic Vaccinia virus encoding for a single-chain antibody (scAb) against VEGF (GLAF-1) significantly enhanced mono-therapy of oncolytic treatment. Conclusions Here, we demonstrate for the first time that oncolytic virotherapy using tumor-specific Vaccinia virus represents a novel and promising treatment modality for therapy of ME associated with advanced lung cancer. PMID:23635329

  11. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.

    PubMed Central

    Janknecht, R; de Martynoff, G; Lou, J; Hipskind, R A; Nordheim, A; Stunnenberg, H G

    1991-01-01

    Vaccinia virus has been used as a vector to express foreign genes for the production of functional and posttranslationally modified proteins. A procedure is described here that allows the rapid native purification of vaccinia-expressed proteins fused to an amino-terminal tag of six histidines. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+.nitrilotriacetic acid (Ni2+.NTA)-agarose and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. In the case of the human serum response factor (SRF), a transcription factor involved in the regulation of the c-fos protooncogene, the vaccinia-expressed histidine-tagged SRF (SRF-6His) could be purified solely by this step to greater than 95% purity. SRF-6His was shown to resemble authentic SRF by functional criteria: it was transported to the nucleus, bound specifically the c-fos serum response element, interacted with the p62TCF protein to form a ternary complex, and stimulated in vitro transcription from the serum response element. Thus, the combination of vaccinia virus expression and affinity purification by Ni2+.NTA chromatography promises to be useful for the production of proteins in a functional and posttranslationally modified form. Images PMID:1924358

  12. 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.

  13. Relationship between RNA polymerase II and efficiency of vaccinia virus replication

    SciTech Connect

    Wilton, S.; Dales, S.

    1989-04-01

    It is clear from previous studies that host transcriptase or RNA polymerase II (pol II) has a role in poxvirus replication. To elucidate the participation of this enzyme further, in this study the authors examined several parameters related to pol II during the cycle of vaccinia virus infection in L-strain fibroblasts, HeLa cells, and L/sub 6/H/sub 9/ rat myoblasts. Nucleocytoplasmic transposition of pol II into virus factories and virions was assessed by immunofluorescence and immunoblotting by using anti-pol II immunoglobulin G. RNA polymerase activities were compared in nuclear extracts containing cured enzyme preparations. Rates of translation into cellular or viral polypeptides were ascertained by labeling with (/sup 35/S)methionine. In L and HeLa cells, which produced vaccinia virus more abundantly, the rate of RNA polymerase and translation in controls and following infection were higher than in myoblasts. The data on synthesis and virus formation could be correlated with observations on transmigration of pol II, which was more efficient and complete in L and HeLa cells. The stimulus for pol II to leave the nucleus required the expression of both early and late viral functions. On the basis of current and past information, the authors suggest that mobilization of pol II depends on the efficiency of vaccinia virus replication and furthermore that control over vaccinia virus production by the host is related to the content or availability (or both) of pol II in different cell types.

  14. 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

    2016-02-01

    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. PMID:26861381

  15. Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

    PubMed

    Taylor, J; Pincus, S; Tartaglia, J; Richardson, C; Alkhatib, G; Briedis, D; Appel, M; Norton, E; Paoletti, E

    1991-08-01

    cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge. PMID:1830113

  16. Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

    PubMed Central

    Taylor, J; Pincus, S; Tartaglia, J; Richardson, C; Alkhatib, G; Briedis, D; Appel, M; Norton, E; Paoletti, E

    1991-01-01

    cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge. Images PMID:1830113

  17. 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.

  18. Cell Carriage, Delivery, and Selective Replication of an Oncolytic Virus in Tumor in Patients

    PubMed Central

    Adair, Robert A.; Roulstone, Victoria; Scott, Karen J.; Morgan, Ruth; Nuovo, Gerard J.; Fuller, Martin; Beirne, Deborah; West, Emma J.; Jennings, Victoria A.; Rose, Ailsa; Kyula, Joan; Fraser, Sheila; Dave, Rajiv; Anthoney, David A.; Merrick, Alison; Prestwich, Robin; Aldouri, Amer; Donnelly, Oliver; Pandha, Hardev; Coffey, Matt; Selby, Peter; Vile, Richard; Toogood, Giles; Harrington, Kevin; Melcher, Alan A.

    2013-01-01

    Oncolytic viruses, which preferentially lyse cancer cells and stimulate an antitumor immune response, represent a promising approach to the treatment of cancer. However, how they evade the antiviral immune response and their selective delivery to, and replication in, tumor over normal tissue has not been investigated in humans. Here,we treated patients with a single cycle of intravenous reovirus before planned surgery to resect colorectal cancer metastases in the liver. Tracking the viral genome in the circulation showed that reovirus could be detected in plasma and blood mononuclear, granulocyte, and platelet cell compartments after infusion. Despite the presence of neutralizing antibodies before viral infusion in all patients, replication-competent reovirus that retained cytotoxicity was recovered from blood cells but not plasma, suggesting that transport by cells could protect virus for potential delivery to tumors. Analysis of surgical specimens demonstrated greater, preferential expression of reovirus protein in malignant cells compared to either tumor stroma or surrounding normal liver tissue. There was evidence of viral factories within tumor, and recovery of replicating virus from tumor (but not normal liver)was achieved in all four patients from whom fresh tissue was available. Hence, reovirus could be protected from neutralizing antibodies after systemic administration by immune cell carriage, which delivered reovirus to tumor.These findings suggest new preclinical and clinical scheduling and treatment combination strategies to enhance in vivo immune evasion and effective intravenous delivery of oncolytic viruses to patients in vivo. PMID:22700953

  19. Cell carriage, delivery, and selective replication of an oncolytic virus in tumor in patients.

    PubMed

    Adair, Robert A; Roulstone, Victoria; Scott, Karen J; Morgan, Ruth; Nuovo, Gerard J; Fuller, Martin; Beirne, Deborah; West, Emma J; Jennings, Victoria A; Rose, Ailsa; Kyula, Joan; Fraser, Sheila; Dave, Rajiv; Anthoney, David A; Merrick, Alison; Prestwich, Robin; Aldouri, Amer; Donnelly, Oliver; Pandha, Hardev; Coffey, Matt; Selby, Peter; Vile, Richard; Toogood, Giles; Harrington, Kevin; Melcher, Alan A

    2012-06-13

    Oncolytic viruses, which preferentially lyse cancer cells and stimulate an antitumor immune response, represent a promising approach to the treatment of cancer. However, how they evade the antiviral immune response and their selective delivery to, and replication in, tumor over normal tissue has not been investigated in humans. Here, we treated patients with a single cycle of intravenous reovirus before planned surgery to resect colorectal cancer metastases in the liver. Tracking the viral genome in the circulation showed that reovirus could be detected in plasma and blood mononuclear, granulocyte, and platelet cell compartments after infusion. Despite the presence of neutralizing antibodies before viral infusion in all patients, replication-competent reovirus that retained cytotoxicity was recovered from blood cells but not plasma, suggesting that transport by cells could protect virus for potential delivery to tumors. Analysis of surgical specimens demonstrated greater, preferential expression of reovirus protein in malignant cells compared to either tumor stroma or surrounding normal liver tissue. There was evidence of viral factories within tumor, and recovery of replicating virus from tumor (but not normal liver) was achieved in all four patients from whom fresh tissue was available. Hence, reovirus could be protected from neutralizing antibodies after systemic administration by immune cell carriage, which delivered reovirus to tumor. These findings suggest new preclinical and clinical scheduling and treatment combination strategies to enhance in vivo immune evasion and effective intravenous delivery of oncolytic viruses to patients in vivo. PMID:22700953

  20. 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

  1. In vitro inhibition of protein synthesis by purified cores from vaccinia virus.

    PubMed Central

    Ben-Hamida, F; Beaud, G

    1978-01-01

    The mechanism of the shutoff of cellular protein synthesis in vaccinia virus-infected cells has been investigated by using in vitro systems. Purified vaccinia cores cause inhibition of endogenous mRNA translation in nonpreincubated reticulocyte lysates and Ehrlich ascites tumor cell-free systems. Translation of viral mRNA from turnip yellow mosaic virus is also impaired in wheat germ cell-free extracts. The block induced by vaccinia cores in protein synthesis is not due to a decrease in the availability of mRNA but rather to an alteration of the cellular translational machinery. No nucleolytic activity able of digesting mRNA could be detected in purified vaccinia cores with three sensitive tests. There is a lack of inhibition in the poly(Phe)-poly(U) system, which bypasses the normal initiation process. An almost complete disaggregation of polyribosomes in the reticulocyte lysate appears when vaccinia cores are present. These results indicate that mRNA translation in a cell-free system is affected predominantly at the level of polypeptide chain initiation. PMID:272632

  2. The role of vaccinia termination factor and cis-acting elements in vaccinia virus early gene transcription termination.

    PubMed

    Tate, Jessica; Gollnick, Paul

    2015-11-01

    Vaccinia virus early gene transcription termination requires the virion form of the viral RNA polymerase (vRNAP), Nucleoside Triphosphate Phosphohydrolase I (NPHI), ATP, the vaccinia termination factor (VTF), and a U5NU termination signal in the nascent transcript. VTF, also the viral mRNA capping enzyme, binds U5NU, and NPHI hydrolyzes ATP to release the transcript. NPHI can release transcripts independent of VTF and U5NU if vRNAP is not actively elongating. However, VTF and U5NU are required for transcript release from an elongating vRNAP, suggesting that the function of VTF and U5NU may be to stall the polymerase. Here we demonstrate that VTF inhibits transcription elongation by enhancing vRNAP pausing. Hence VTF provides the connection between the termination signal in the RNA transcript and viral RNA polymerase to initiate transcription termination. We also provide evidence that a second cis-acting element downstream of U5NU influences the location and efficiency of early gene transcription termination.

  3. Shaping the tumor microenvironment with Modified Vaccinia Virus Ankara and TLR9 ligand

    PubMed Central

    Préville, Xavier; Rittner, Karola; Fend, Laetitia

    2015-01-01

    Our preclinical data demonstrate that an intravenous injection of Modified Vaccinia virus Ankara induces CD8+ lymphocytes to infiltrate organs to control the growth of orthotopic renal carcinoma upon combination with a toll-like receptor 9 agonist. Such shaping of the tumor microenvironment could constitute the basis of more effective clinical protocols of tumor immunotherapy. PMID:26155396

  4. 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.

  5. 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

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26436134

  7. New vaccinia virus promoter as a potential candidate for future vaccines.

    PubMed

    Di Pilato, Mauro; Mejías-Pérez, Ernesto; Gómez, Carmen Elena; Perdiguero, Beatriz; Sorzano, Carlos Oscar S; Esteban, Mariano

    2013-12-01

    Here we describe the design and strength of a new synthetic late-early optimized (LEO) vaccinia virus (VACV) promoter used as a transcriptional regulator of GFP expression during modified vaccinia Ankara infection. In contrast to the described synthetic VACV promoter (pS), LEO induced significantly higher levels of GFP expression in vitro within the first hour after infection, which correlated with an enhancement in the GFP-specific CD8 T-cell response detected in vivo, demonstrating its potential use in future vaccines.

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

    PubMed Central

    Thippeshappa, Rajesh; Tian, Baoping; Cleveland, Brad; Guo, Wenjin; Polacino, Patricia

    2015-01-01

    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 × 108 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. PMID:26718849

  9. The Immunological Relationship of the Vaccinia and Pig Pox Viruses demonstrated by Gel Diffusion

    PubMed Central

    Datt, N. S.; Orlans, E. S.

    1958-01-01

    The Ouchterlony double diffusion method in agar gel has been used to study the antigens of the vaccinia and pig pox viruses and their corresponding antibodies. The existence of a common antigenic constituent in the two viruses has been demonstrated. The sensitivity of the method was found to be adequate for sera giving complement fixation titres of 1/80. The complications arising from the presence of antibodies to heterologous (host) antigens are illustrated. ImagesFIG. 6FIG. 7 PMID:13513144

  10. Relationship between RNA polymerase II and efficiency of vaccinia virus replication.

    PubMed Central

    Wilton, S; Dales, S

    1989-01-01

    It is clear from previous studies that host transcriptase or RNA polymerase II (pol II) has a role in poxvirus replication. To elucidate the participation of this enzyme further, in this study we examined several parameters related to pol II during the cycle of vaccinia virus infection in L-strain fibroblasts, HeLa cells, and L6H9 rat myoblasts. Nucleocytoplasmic transposition of pol II into virus factories and virions was assessed by immunofluorescence and immunoblotting by using anti-pol II immunoglobulin G. RNA polymerase activities were compared in nuclear extracts containing crude enzyme preparations. Rates of translation into cellular or viral polypeptides were ascertained by labeling with [35S]methionine. In L and HeLa cells, which produced vaccinia virus more abundantly, the rates of RNA polymerase and translation in controls and following infection were higher than in myoblasts. The data on synthesis and virus formation could be correlated with observations on transmigration of pol II, which was more efficient and complete in L and HeLa cells. The stimulus for pol II to leave the nucleus required the expression of both early and late viral functions. On the basis of current and past information, we suggest that mobilization of pol II depends on the efficiency of vaccina virus replication and furthermore that control over vaccinia virus production by the host is related to the content or availability (or both) of pol II in different cell types. Images PMID:2648021

  11. The vaccinia virus-encoded Bcl-2 homologues do not act as direct Bax inhibitors.

    PubMed

    Postigo, Antonio; Way, Michael

    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.

  12. Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors.

    PubMed

    Falkner, F G; Moss, B

    1988-06-01

    Mycophenolic acid, an inhibitor of purine metabolism, was shown to block the replication of vaccinia virus in normal cell lines. This observation led to the development of a dominant one-step plaque selection system, based on expression of the Escherichia coli gpt gene, for the isolation of recombinant vaccinia viruses. Synthesis of xanthine-guanine phosphoribosyltransferase enabled only the recombinant viruses to form large plaques in a selective medium containing mycophenolic acid, xanthine, and hypoxanthine. To utilize the selection system efficiently, we constructed a series of plasmids that contain the E. coli gpt gene and allow insertion of foreign genes into multiple unique restriction endonuclease sites in all three reading frames between the translation initiation codon of a strong late promoter and synthetic translation termination sequences. The selection-expression cassette is flanked by vaccinia virus DNA that directs homologous recombination into the virus genome. The new vectors allow high-level expression of complete or partial open reading frames and rapid construction of recombinant viruses by facilitating the cloning steps and by simplifying their isolation. The system was tested by cloning the E. coli beta-galactosidase gene; in 24 h, this enzyme accounted for approximately 3.5% of the total infected-cell protein.

  13. Ty virus-like particles, DNA vaccines and Modified Vaccinia Virus Ankara; comparisons and combinations.

    PubMed

    Gilbert, S C; Schneider, J; Plebanski, M; Hannan, C M; Blanchard, T J; Smith, G L; Hill, A V

    1999-03-01

    Three types of vaccine, all expressing the same antigen from Plasmodium berghei, or a CD8+ T cell epitope from that antigen, were compared for their ability to induce CD8+ T cell responses in mice. Higher levels of lysis and numbers of IFN-gamma secreting T cells were primed with Ty virus-like particles and Modified Vaccinia Virus Ankara (MVA) than with DNA vaccines, but none of the vaccines were able to protect immunised mice from infectious challenge even after repeated doses. However, when the immune response was primed with one type of vaccine (Ty-VLPs or DNA) and boosted with another (MVA) complete protection against infection was achieved. Protection correlated with very high levels of IFN-gamma secreting T cells and lysis. This method of vaccination uses delivery systems and routes that can be used in humans and could provide a generally applicable regime for the induction of high levels of CD8+ T cells.

  14. Oncolytic herpes simplex virus-based strategies: toward a breakthrough in glioblastoma therapy

    PubMed Central

    Ning, Jianfang; Wakimoto, Hiroaki

    2014-01-01

    Oncolytic viruses (OV) are a class of antitumor agents that selectively kill tumor cells while sparing normal cells. Oncolytic herpes simplex virus (oHSV) has been investigated in clinical trials for patients with the malignant brain tumor glioblastoma for more than a decade. These clinical studies have shown the safety of oHSV administration to the human brain, however, therapeutic efficacy of oHSV as a single treatment remains unsatisfactory. Factors that could hamper the anti-glioblastoma efficacy of oHSV include: attenuated potency of oHSV due to deletion or mutation of viral genes involved in virulence, restricting viral replication and spread within the tumor; suboptimal oHSV delivery associated with intratumoral injection; virus infection-induced inflammatory and cellular immune responses which could inhibit oHSV replication and promote its clearance; lack of effective incorporation of oHSV into standard-of-care, and poor knowledge about the ability of oHSV to target glioblastoma stem cells (GSCs). In an attempt to address these issues, recent research efforts have been directed at: (1) design of new engineered viruses to enhance potency, (2) better understanding of the role of the cellular immunity elicited by oHSV infection of tumors, (3) combinatorial strategies with different antitumor agents with a mechanistic rationale, (4) “armed” viruses expressing therapeutic transgenes, (5) use of GSC-derived models in oHSV evaluation, and (6) combinations of these. In this review, we will describe the current status of oHSV clinical trials for glioblastoma, and discuss recent research advances and future directions toward successful oHSV-based therapy of glioblastoma. PMID:24999342

  15. Tumour-specific triple-regulated oncolytic herpes virus to target glioma

    PubMed Central

    Delwar, Zahid M.; Liu, Guoyu; Kuo, Yvonne; Lee, Cleo; Bu, Luke; Rennie, Paul S.; Jia, William W.

    2016-01-01

    Oncolytic herpes simplex virus type 1 (oHSV-1) therapy is an emerging treatment modality that selectively destroys cancer. Here we report use of a glioma specific HSV-1 amplicon virus (SU4-124 HSV-1) to selectively target tumour cells. To achieve transcriptional regulation of the SU4-124 HSV-1 virus, the promoter for the essential HSV-1 gene ICP4 was replaced with a tumour specific survivin promoter. Translational regulation was achieved by incorporating 5 copies of microRNA 124 target sequences into the 3′UTR of the ICP4 gene. Additionally, a 5′UTR of rat fibroblast growth factor -2 was added in front of the viral ICP4 gene open reading frame. Our results confirmed enhanced expression of survivin and eIF4E in different glioma cells and increased micro-RNA124 expression in normal human and mouse brain tissue. SU4-124 HSV-1 had an increased ICP4 expression and virus replication in different glioma cells compared to normal neuronal cells. SU4-124 HSV-1 exerted a strong antitumour effect against a panel of glioma cell lines. Intracranial injection of SU4-124 HSV-1 did not reveal any sign of toxicity on day 15 after the injection. Moreover, a significantly enhanced antitumour effect with the intratumourally injected SU4-124 HSV-1 virus was demonstrated in mice bearing human glioma U87 tumours, whereas viral DNA was almost undetectable in normal organs. Our study indicates that incorporation of multiple cancer-specific regulators in an HSV-1 system significantly enhances both cancer specificity and oncolytic activity. PMID:27070093

  16. Protection of mice from respiratory Sendai virus infections by recombinant vaccinia viruses.

    PubMed

    Takao, S I; Kiyotani, K; Sakaguchi, T; Fujii, Y; Seno, M; Yoshida, T

    1997-01-01

    Mechanisms of protection of mice from Sendai virus, which is exclusively pneumotropic and causes a typical respiratory disease, by immunization with recombinant vaccinia viruses (RVVs) were investigated. Although the RVV carrying a hemagglutinin-neuraminidase gene of Sendai virus (Vac-HN) propagated in the noses and lungs of mice by either intranasal (i.n.) or intraperitoneal (i.p.) inoculation, no vaccinia virus antigens were detected in the mucosal layer of upper and lower airways of the i.p.-inoculated mice. The mice immunized i.n. with Vac-HN or Vac-F (the RVV carrying a fusion protein gene of Sendai virus) demonstrated the strong resistance to Sendai virus challenge both in the lung and in the nose, whereas the i.p.-immunized mice showed almost no resistance in the nose but showed a partial resistance in the lung. Titration of Sendai virus-specific antibodies in the nasal wash (NW), bronchoalveolar lavage (BAL), and serum collected from the Vac-F-immunized mice showed that the NW from the i.n.-immunized mice contained immunoglobulin A (IgA) antibodies but no IgG and the BAL from the mice contained both IgA and IgG antibodies. On the other hand, neither IgA nor IgG antibodies were detected in the NW from the i.p.-immunized mice and only IgG antibodies were detected in the BAL, although both i.n.- and i.p.-immunized mice exhibited similar levels of serum IgG, IgA, and neutralizing antibodies. The resistance to Sendai virus in the noses of i.n.-immunized mice could be abrogated by the intranasal instillation of anti-mouse IgA but not of anti-IgG antiserum, while the resistance in the lung was not significantly abrogated by such treatments. These results demonstrate that IgA is a major mediator for the immunity against Sendai virus induced by the RVVs and IgG is a supplementary one, especially in the lung, and that the RVV should be intranasally inoculated to induce an efficient mucosal immunity even if it has a pantropic nature.

  17. Characterization of a molluscum contagiosum virus homolog of the vaccinia virus p37K major envelope antigen.

    PubMed

    Blake, N W; Porter, C D; Archard, L C

    1991-07-01

    We present the first nucleotide sequence data for molluscum contagiosum virus (MCV), an unclassified poxvirus. A 2,276-bp XhoI fragment from a near left-terminal fragment of MCV subtype I (MCVI) and a 1,920-bp XhoI fragment from the corresponding locus of MCV subtype II (MCVII) were sequenced and analyzed for open reading frames (ORFs). A large, complete ORF of 1,167 bp was present in both fragments. The putative polypeptide has a calculated molecular mass of 43 kDa (p43K protein) and was shown to have a high degree of homology to the vaccinia virus p37K major envelope antigen (40% amino acid identity and 22% conservative changes). The nucleotide content of the MCV fragments sequenced was 66% G or C. The codon usage within the gene for p43K reflected this high G + C content, with position 3 of codons being predominantly G or C (82 and 87% for MCVI and MCVII, respectively). The MCV p43K-encoding gene has motifs immediately upstream which are similar to those required for vaccinia virus late gene expression. The location and direction of transcription of the MCV p43K-encoding gene were equivalent to those of the vaccinia virus p37K gene, revealing similarity in genetic organization between MCV and vaccinia virus. Another, incomplete ORF was identified downstream of the p43K-encoding gene in both MCVI and MCVII. The sequence immediately upstream of this ORF overlapped the termination codon of the p43K-encoding gene and contained a motif which had homology to the derived consensus sequence for vaccinia virus early gene promoters.

  18. Production and characterisation of a monoclonal antibody to human papillomavirus type 16 using recombinant vaccinia virus.

    PubMed

    McLean, C S; Churcher, M J; Meinke, J; Smith, G L; Higgins, G; Stanley, M; Minson, A C

    1990-06-01

    A monoclonal antibody was raised against the major capsid protein L1 of human papillomavirus type 16, using a recombinant vaccinia virus that expresses the L1 protein, as a target for screening. This antibody, designated CAMVIR-1, reacted with a 56 kilodalton protein in cells infected with L1-vaccinia virus, and the protein was present in a predominantly nuclear location. The antibody also detects the HPV-16 L1 antigen in formalin fixed, paraffin wax embedded biopsy specimens and on routine cervical smears. The antibody reacts strongly and consistently with biopsy specimens containing HPV-16 or HPV-33, but very weak reactions were occasionally observed with biopsy specimens or smears containing HPV-6 or HPV-11. The potential advantages of using a vaccinia recombinant are (i) the target protein is synthesised in a eukoryotic cell so that its "processing" and location are normal; (ii) cells infected with vaccinia recombinants can be subjected to various fixing procedures similar to those used for routine clinical material. This greatly increases the probability that an identified antibody will be useful in a clinical setting.

  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. PMID:21104283

  20. A vaccinia virus double recombinant expressing the F and H genes of rinderpest virus protects cattle against rinderpest and causes no pock lesions.

    PubMed Central

    Giavedoni, L; Jones, L; Mebus, C; Yilma, T

    1991-01-01

    Rinderpest is a highly contagious viral disease of ruminants with greater than 95% morbidity and mortality. We have constructed an infectious vaccinia virus recombinant that expresses both the fusion (F) gene and the hemagglutinin (H) gene of rinderpest virus. The Wyeth strain of vaccinia virus was used for the construction of the recombinant. Cattle vaccinated with the recombinant virus were 100% protected from challenge inoculation with greater than 1000 times the lethal dose of rinderpest virus. No transmission of recombinant vaccinia virus from vaccinated animals to contact animals was observed. The lyophilized form of vaccinia virus is thermostable and allows circumvention of the logistical problems associated with the distribution and administration of vaccines in the arid and hot regions of Asia and Africa. The insertional inactivation of both the thymidine kinase and the hemagglutinin genes of vaccinia virus led to increased attenuation of the virus; this was manifested by the lack of detectable pock lesions in vaccinated animals. This approach may have wide application in the development of safe and efficacious recombinant vaccines for humans and animals. This becomes quite relevant with the concern of the use of vaccinia virus in a population with high incidence of the human immunodeficiency virus. Images PMID:1896447

  1. Glycosylated and nonglycosylated complement control protein of the lister strain of vaccinia virus.

    PubMed

    Meseda, Clement A; Kuhn, Jordan; Atukorale, Vajini; Campbell, Joseph; Weir, Jerry P

    2014-09-01

    The vaccinia virus complement control protein (VCP) is a secreted viral protein that binds the C3b and C4b complement components and inhibits the classic and alternative complement pathways. Previously, we reported that an attenuated smallpox vaccine, LC16m8, which was derived from the Lister strain of vaccinia virus (VV-Lister), expressed a glycosylated form of VCP, whereas published sequence data at that time indicated that the VV-Lister VCP has no motif for N-linked glycosylation. We were interested in determining whether the glycosylation of VCP impairs its biological activity, possibly contributing to the attenuation of LC16m8, and the likely origin of the glycosylated VCP. Expression analysis indicated that VV-Lister contains substrains expressing glycosylated VCP and substrains expressing nonglycosylated VCP. Other strains of smallpox vaccine, as well as laboratory strains of vaccinia virus, all expressed nonglycosylated VCP. Individual Lister virus clones expressing either the glycosylated VCP or the nonglycosylated species were isolated, and partially purified VCP from the isolates were found to be functional equivalents in binding human C3b and C4b complement proteins and inhibiting hemolysis and in immunogenicity. Recombinant vaccinia viruses expressing FLAG-tagged glycosylated VCP (FLAG-VCPg) and nonglycosylated VCP (FLAG-VCP) were constructed based on the Western Reserve strain. Purified FLAG-VCP and FLAG-VCPg bind human C3b and C4b and blocked complement-mediated hemolysis. Our data suggest that glycosylation did not affect the biological activity of VCP and thus may not have contributed to the attenuation of LC16m8. In addition, the LC16m8 virus likely originated from a substrain of VV-Lister that expresses glycosylated VCP.

  2. Incomplete but Infectious Vaccinia Virions Are Produced in the Absence of Oncolysis in Feline SCCF1 Cells

    PubMed Central

    Parviainen, Suvi; Autio, Karoliina; Vähä-Koskela, Markus; Guse, Kilian; Pesonen, Sari; Rosol, Thomas J.; Zhao, Fang; Hemminki, Akseli

    2015-01-01

    Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus. PMID:25799430

  3. Incomplete but infectious vaccinia virions are produced in the absence of oncolysis in feline SCCF1 cells.

    PubMed

    Parviainen, Suvi; Autio, Karoliina; Vähä-Koskela, Markus; Guse, Kilian; Pesonen, Sari; Rosol, Thomas J; Zhao, Fang; Hemminki, Akseli

    2015-01-01

    Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus.

  4. Mutagenesis of the palmitoylation site in vaccinia virus envelope glycoprotein B5.

    PubMed

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

    2012-04-01

    The outer envelope of vaccinia virus extracellular virions is derived from intracellular membranes that, at late times in infection, are enriched in several virus-encoded proteins. Although palmitoylation is common in vaccinia virus envelope proteins, little is known about the role of palmitoylation in the biogenesis of the enveloped virus. We have studied the palmitoylation of B5, a 42 kDa type I transmembrane glycoprotein comprising a large ectodomain and a short (17 aa) cytoplasmic tail. Mutation of two cysteine residues located in the cytoplasmic tail in close proximity to the transmembrane domain abrogated palmitoylation of the protein. Virus mutants expressing non-palmitoylated versions of B5 and/or lacking most of the cytoplasmic tail were isolated and characterized. Cell-to-cell virus transmission and extracellular virus formation were only slightly affected by those mutations. Notably, B5 versions lacking palmitate showed decreased interactions with proteins A33 and F13, but were still incorporated into the virus envelope. Expression of mutated B5 by transfection into uninfected cells showed that both the cytoplasmic tail and palmitate have a role in the intracellular transport of B5. These results indicate that the C-terminal portion of protein B5, while involved in protein transport and in protein-protein interactions, is broadly dispensable for the formation and egress of infectious extracellular virus and for virus transmission.

  5. 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.

  6. Targeting tumor vasculature through oncolytic virotherapy: recent advances

    PubMed Central

    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. PMID:27512680

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

    PubMed

    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

  9. 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.

  10. Hepatitis B virus large surface protein is not secreted but is immunogenic when selectively expressed by recombinant vaccinia virus.

    PubMed Central

    Cheng, K C; Smith, G L; Moss, B

    1986-01-01

    The envelope region of the hepatitis B virus (HBV) genome contains an open reading frame that begins upstream of the major surface protein gene. The two minor proteins that are initiated within this pre-s segment are immunogenic and may be involved in virus attachment to hepatocytes. We have constructed a recombinant vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus promoter. Cells infected with the recombinant virus synthesized HBV polypeptides of 39 and 42 kilodaltons, corresponding to the unglycosylated and glycosylated forms of LS, respectively. The presence of pre-s epitopes in the 39- and 42-kilodalton polypeptides was demonstrated by binding of antibody prepared against a synthetic peptide. Synthesis of the 42-kilodalton species was specifically inhibited by tunicamycin, suggesting that it is N-glycosylated. Despite apparent glycosylation, LS was not secreted into the medium of infected cells. Nevertheless, rabbits vaccinated with the purified recombinant virus made antibodies that recognized s and pre-s epitopes. Antibody to the NH2 terminus of LS appeared before or simultaneously with antibody that bound to the major surface protein. The additional immunogenicity provided by expression of LS may be advantageous for the development of an HBV vaccine. Images PMID:2430108

  11. Expression of African swine fever virus envelope protein j13L inhibits vaccinia virus morphogenesis.

    PubMed

    Jacobs, S C; Dixon, L K; Brookes, S M; Smith, G L

    1998-05-01

    The African swine fever virus (ASFV) strain Malawi LIL20/1 open reading frame (ORF) j13L was expressed in vaccinia virus (VV) from a strong synthetic late promoter as either a complete ORF (vSJ1) or lacking codons 1-31 (vSJ2). Each recombinant VV produced a small plaque which rapidly reverted to a normal size upon passage. The yield of infectious virus from a single cycle infection with vSJ1 or vSJ2 was reduced 50- to 100-fold compared to wild-type (wt) and a revertant virus (vSJ5) in which the j13L ORF was removed and the VV thymidine kinase gene restored. PCR analysis of nine spontaneous large plaque revertant viruses, recovered after passage of vSJ1 in BSC-40 cells, showed that six had lost the j13L ORF and the co-inserted beta-galactosidase gene. Three viruses retained the j13L and beta-galactosidase genes, but in each case the j13L protein was not expressed due to a different single base deletion near the 5' end of the j13L coding region which introduced a stop codon a short distance downstream. The formation of intracellular mature virus (IMV) and extracellular enveloped virus was reduced 50- to 75-fold in cells infected with vSJ1 compared to wt VV and revertant vSJ5. Electron microscopy showed aberrant IMV precursor structures in vSJ1-infected cells, and immunoelectron microscopy demonstrated that these structures contained j13L protein. These results indicate that expression of the j13L protein is toxic for VV replication due to interference with VV morphogenesis prior to IMV formation.

  12. 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

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

    PubMed Central

    Yamada, Suguru; Kuroda, Toshihiko; Fuchs, Bryan C.; He, Xiaoying; Supko, Jeffrey G.; Schmitt, Anthony; McGinn, Christopher M.; Lanuti, Michael; Tanabe, Kenneth K.

    2011-01-01

    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 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. Twelve days of 5-FC administration was superior to 6 days in animal models, 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) 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. PMID:22076044

  14. Host range restriction of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff of protein synthesis.

    PubMed

    Drillien, R; Spehner, D; Kirn, A

    1978-12-01

    Chinese hamster ovary cells were found to be nonpermissive for vaccinia virus. Although early virus-induced events occurred in these cells (RNA and polypeptide synthesis), subsequent events appeared to be prevented by a very rapid and nonselective shutoff of protein synthesis. Within less than 2 h after infection, both host and viral protein syntheses were arrested. At low multiplicities of infection, inhibition of RNA synthesis with cordycepin resulted in failure of the virus to block protein synthesis. Moreover, infection of the cells in the presence of cycloheximide prevented the immediate onset of shutoff after reversal of cycloheximide. Inactivation of virus particles by UV irradiation also impaired the capacity of the virus to inhibit protein synthesis. These results suggested that an early vaccinia virus-coded product was implicated in the shutoff of protein synthesis. Either the nonpermissive Chinese hamster ovary cells were more sensitive to this inhibition than permissive cells, or a regulatory control of the vaccinia shutoff function was defective.

  15. 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

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

    PubMed

    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

  17. 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

  18. Modified Vaccinia Virus Ankara Encoding Influenza Virus Hemagglutinin Induces Heterosubtypic Immunity in Macaques

    PubMed Central

    Florek, Nicholas W.; Weinfurter, Jason T.; Jegaskanda, Sinthujan; Brewoo, Joseph N.; Powell, Tim D.; Young, Ginger R.; Das, Subash C.; Hatta, Masato; Broman, Karl W.; Hungnes, Olav; Dudman, Susanne G.; Kawaoka, Yoshihiro; Kent, Stephen J.; Stinchcomb, Dan T.

    2014-01-01

    ABSTRACT Current influenza virus vaccines primarily aim to induce neutralizing antibodies (NAbs). Modified vaccinia virus Ankara (MVA) is a safe and well-characterized vector for inducing both antibody and cellular immunity. We evaluated the immunogenicity and protective efficacy of MVA encoding influenza virus hemagglutinin (HA) and/or nucleoprotein (NP) in cynomolgus macaques. Animals were given 2 doses of MVA-based vaccines 4 weeks apart and were challenged with a 2009 pandemic H1N1 isolate (H1N1pdm) 8 weeks after the last vaccination. MVA-based vaccines encoding HA induced potent serum antibody responses against homologous H1 or H5 HAs but did not stimulate strong T cell responses prior to challenge. However, animals that received MVA encoding influenza virus HA and/or NP had high frequencies of virus-specific CD4+ and CD8+ T cell responses within the first 7 days of H1N1pdm infection, while animals vaccinated with MVA encoding irrelevant antigens did not. We detected little or no H1N1pdm replication in animals that received vaccines encoding H1 (homologous) HA, while a vaccine encoding NP from an H5N1 isolate afforded no protection. Surprisingly, H1N1pdm viral shedding was reduced in animals vaccinated with MVA encoding HA and NP from an H5N1 isolate. This reduced shedding was associated with cross-reactive antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC) effector functions. Our results suggest that ADCC plays a role in cross-protective immunity against influenza. Vaccines optimized to stimulate cross-reactive antibodies with ADCC function may provide an important measure of protection against emerging influenza viruses when NAbs are ineffective. IMPORTANCE Current influenza vaccines are designed to elicit neutralizing antibodies (NAbs). Vaccine-induced NAbs typically are effective but highly specific for particular virus strains. Consequently, current vaccines are poorly suited for preventing the spread of newly emerging

  19. Characterization of a New Vaccinia virus Isolate Reveals the C23L Gene as a Putative Genetic Marker for Autochthonous Group 1 Brazilian Vaccinia virus

    PubMed Central

    Oliveira, Danilo B.; Franco-Luiz, Ana P. M.; Campos, Rafael K.; Guedes, Maria I. M.; Fonseca, Flávio G.; Trindade, Giliane S.; Drumond, Betânia P.; Kroon, Erna G.; Abrahão, Jônatas S.

    2012-01-01

    Since 1999, several Vaccinia virus (VACV) isolates, the etiological agents of bovine vaccinia (BV), have been frequently isolated and characterized with various biological and molecular methods. The results from these approaches have grouped these VACV isolates into two different clusters. This dichotomy has elicited debates surrounding the origin of the Brazilian VACV and its epidemiological significance. To ascertain vital information to settle these debates, we and other research groups have made efforts to identify molecular markers to discriminate VACV from other viruses of the genus Orthopoxvirus (OPV) and other VACV-BR groups. In this way, some genes have been identified as useful markers to discriminate between the VACV-BR groups. However, new markers are needed to infer ancestry and to correlate each sample or group with its unique epidemiological and biological features. The aims of this work were to characterize a new VACV isolate (VACV DMTV-2005) molecularly and biologically using conserved and non-conserved gene analyses for phylogenetic inference and to search for new genes that would elucidate the VACV-BR dichotomy. The VACV DMTV-2005 isolate reported in this study is biologically and phylogenetically clustered with other strains of Group 1 VACV-BR, the most prevalent VACV group that was isolated during the bovine vaccinia outbreaks in Brazil. Sequence analysis of C23L, the gene that encodes for the CC-chemokine-binding protein, revealed a ten-nucleotide deletion, which is a new Group 1 Brazilian VACV genetic marker. This deletion in the C23L open reading frame produces a premature stop-codon that is shared by all Group 1 VACV-BR strains and may also reflect the VACV-BR dichotomy; the deletion can also be considered to be a putative genetic marker for non-virulent Brazilian VACV isolates and may be used for the detection and molecular characterization of new isolates. PMID:23189200

  20. Characterization of a new Vaccinia virus isolate reveals the C23L gene as a putative genetic marker for autochthonous Group 1 Brazilian Vaccinia virus.

    PubMed

    Assis, Felipe L; Almeida, Gabriel M F; Oliveira, Danilo B; Franco-Luiz, Ana P M; Campos, Rafael K; Guedes, Maria I M; Fonseca, Flávio G; Trindade, Giliane S; Drumond, Betânia P; Kroon, Erna G; Abrahão, Jônatas S

    2012-01-01

    Since 1999, several Vaccinia virus (VACV) isolates, the etiological agents of bovine vaccinia (BV), have been frequently isolated and characterized with various biological and molecular methods. The results from these approaches have grouped these VACV isolates into two different clusters. This dichotomy has elicited debates surrounding the origin of the Brazilian VACV and its epidemiological significance. To ascertain vital information to settle these debates, we and other research groups have made efforts to identify molecular markers to discriminate VACV from other viruses of the genus Orthopoxvirus (OPV) and other VACV-BR groups. In this way, some genes have been identified as useful markers to discriminate between the VACV-BR groups. However, new markers are needed to infer ancestry and to correlate each sample or group with its unique epidemiological and biological features. The aims of this work were to characterize a new VACV isolate (VACV DMTV-2005) molecularly and biologically using conserved and non-conserved gene analyses for phylogenetic inference and to search for new genes that would elucidate the VACV-BR dichotomy. The VACV DMTV-2005 isolate reported in this study is biologically and phylogenetically clustered with other strains of Group 1 VACV-BR, the most prevalent VACV group that was isolated during the bovine vaccinia outbreaks in Brazil. Sequence analysis of C23L, the gene that encodes for the CC-chemokine-binding protein, revealed a ten-nucleotide deletion, which is a new Group 1 Brazilian VACV genetic marker. This deletion in the C23L open reading frame produces a premature stop-codon that is shared by all Group 1 VACV-BR strains and may also reflect the VACV-BR dichotomy; the deletion can also be considered to be a putative genetic marker for non-virulent Brazilian VACV isolates and may be used for the detection and molecular characterization of new isolates.

  1. 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

  2. Clinical development of reovirus for cancer therapy: An oncolytic virus with immune-mediated antitumor activity

    PubMed Central

    Gong, Jun; Sachdev, Esha; Mita, Alain C; Mita, Monica M

    2016-01-01

    Reovirus is a double-stranded RNA virus with demonstrated oncolysis or preferential replication in cancer cells. The oncolytic properties of reovirus appear to be dependent, in part, on activated Ras signaling. In addition, Ras-transformation promotes reovirus oncolysis by affecting several steps of the viral life cycle. Reovirus-mediated immune responses can present barriers to tumor targeting, serve protective functions against reovirus systemic toxicity, and contribute to therapeutic efficacy through antitumor immune-mediated effects via innate and adaptive responses. Preclinical studies have demonstrated the broad anticancer activity of wild-type, unmodified type 3 Dearing strain reovirus (Reolysin®) across a spectrum of malignancies. The development of reovirus as an anticancer agent and available clinical data reported from 22 clinical trials will be reviewed. PMID:27019795

  3. Sequence and transcriptional analysis of the vaccinia virus HindIII I fragment.

    PubMed

    Schmitt, J F; Stunnenberg, H G

    1988-06-01

    The complete sequence of the vaccinia virus HindIII I fragment, which is composed of 6,498 base pairs, encodes six complete and two incomplete open reading frames (ORFs). Computer analysis revealed an amino acid sequence homology between ORF I 4 and the large subunit of the ribonucleotide reductase complex. The two small polypeptides derived from ORFs I 2 and I 5, with molecular weights of 8,500 and 8,700, respectively, have a very high hydrophobic amino acid sequence composition. S1 analysis revealed that ORF I 4 is expressed at early stages of infection, ORFs I 1, I 2, I 5, and I 7 are expressed in the late phase of infection, and ORF I 3 is constitutively expressed. Screening a vaccinia virus genomic library revealed a large vaccinia virus insert overlapping the HindIII I and O fragments which contains a previously undetected HindIII P fragment of approximately 300 base pairs. S1 analysis revealed an early (O1) and a late (O2) start site of transcription initiation located within the HindIII O fragment.

  4. 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.

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

    PubMed

    Ember, Stuart W J; Ren, Hongwei; Ferguson, Brian J; Smith, Geoffrey L

    2012-10-01

    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

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

    PubMed

    Ember, Stuart W J; Ren, Hongwei; Ferguson, Brian J; Smith, Geoffrey L

    2012-10-01

    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.

  7. Non-essential genes in the vaccinia virus HindIII K fragment: a gene related to serine protease inhibitors and a gene related to the 37K vaccinia virus major envelope antigen.

    PubMed

    Boursnell, M E; Foulds, I J; Campbell, J I; Binns, M M

    1988-12-01

    The complete nucleotide sequence of a cloned copy of the HindIII K fragment of the WR strain of vaccinia virus has been determined. Eight open reading frames (ORFs) have been identified, on the basis of size and codon usage. The predicted amino acid sequences of the putative genes have been compared to the Protein Identification Resource and to published vaccinia virus sequences. One gene, predicted to encode a 42.2K protein, is highly related to the family of serine protease inhibitors. It shows approximately 25% identity to human antithrombin III and 19% identity to the cowpox virus 38K protein gene which is also related to serine protease inhibitors. The product of another gene shows a similar high level of identity to the 37K vaccinia virus major envelope antigen. The existence of viable deletion mutants and recombinants containing foreign DNA inserted into both these genes indicates that they are non-essential.

  8. Determination of the promoter region of an early vaccinia virus gene encoding thymidine kinase.

    PubMed

    Weir, J P; Moss, B

    1987-05-01

    Nine recombinant vaccinia viruses that contain overlapping segments of the putative promoter region of the vaccinia virus thymidine kinase (TK) gene linked to DNA coding for the prokaryotic enzyme chloramphenicol acetyltransferase (CAT) were constructed. In each case, the RNA start site and 5 bp of DNA downstream were retained. No significant difference in CAT expression occurred as the deletion was extended from 352 to 32 bp before the RNA start site. Deletion of a further 10 bp, however, led to complete cessation of early promoter activity. Primer extension analysis of the 5' ends of the transcripts verified that the natural TK RNA start site was still used when only 32 bp of upstream DNA remained. Loss of early promoter activity was previously found when deletions were extended from 31 to 24 bp before the RNA start site of another vaccinia gene that is expressed constitutively throughout infection (M.A. Cochran, C. Puckett, and B. Moss, 1985, Proc. Natl. Acad. Sci. USA 82, 19-23). Sequence similarities in the promoter regions of these two genes were noted.

  9. Induction of neutralizing antibodies by varicella-zoster virus gpII glycoprotein expressed from recombinant vaccinia virus.

    PubMed

    Massaer, M; Haumont, M; Place, M; Bollen, A; Jacobs, P

    1993-03-01

    The gpII glycoprotein of varicella-zoster virus (VZV) was produced in CV1 cells via vaccinia virus recombinants. Two different DNA constructs were expressed: the first one encodes the complete gpII protein (gpII s+a+) and the second a truncated species lacking the membrane anchorage domain (gpII s+a-). To achieve expression both coding sequences had to be engineered at the 5' end by substituting the unusually short (24 bp) natural signal sequence by a more conventional one encoding 29 amino acids. Recombinant gpII proteins were detected in vaccinia virus-infected cells by ELISA and immunoprecipitation. Both forms of recombinant gpII proteins were produced as glycosylated single-chain molecules of respectively 110K and 90K. Upon reduction these were only partially converted into subunits. A rabbit infected with the vaccinia virus recombinant expressing the complete gpII produced antibodies which recognized VZV antigens and neutralized VZV infectivity in vitro, independent of complement.

  10. 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.

  11. Bovine respiratory syncytial virus nucleocapsid protein: mRNA sequence analysis and expression from recombinant vaccinia virus vectors.

    PubMed

    Amann, V L; Lerch, R A; Anderson, K; Wertz, G W

    1992-04-01

    The nucleotide sequence of the mRNA encoding the nucleocapsid (N) protein of bovine respiratory syncytial (BRS) virus, strain 391-2, was determined. Recombinant vectors containing a cDNA of the complete N gene were constructed, and expression of the N protein in eukaryotic cells was demonstrated using two different vector systems. The BRS virus N mRNA was 1197 nucleotides in length, exclusive of poly(A), and had a single major open reading frame that encoded a polypeptide of 391 amino acids with a calculated M(r) of 42.6K. The nucleotide and amino acid sequences of the BRS virus N gene were compared to those of human respiratory syncytial (HRS) virus strains A2 and 18537, and to BRS virus strain A51908. The level of nucleic acid identity between the N mRNA of BRS virus 391-2 and both HRS virus subtypes was 80 to 81%, whereas the identity between the two BRS virus strains was 97%. A 93 to 94% level of identity was observed between the deduced amino acid sequences of the N protein of BRS virus 391-2 and the corresponding sequences of the two HRS virus strains. The two BRS virus N proteins differed in amino acid sequence at only three positions. Recombinant BRS virus N protein was expressed using two different vector systems: in cells from a plasmid using the vaccinia virus/T7 polymerase expression system or from a recombinant vaccinia virus. N proteins synthesized by the two vector systems migrated with an electrophoretic mobility identical to that of authentic BRS virus N protein, and were precipitated by anti-BRS virus antibodies.

  12. 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.

  13. 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.

  14. RNA capping by the vaccinia virus guanylyltransferase. Structure of enzyme-guanylate intermediate.

    PubMed

    Roth, M J; Hurwitz, J

    1984-11-10

    GTP:RNA guanylyltransferase isolated from vaccinia virus catalyzes the transfer of GMP from GTP to the 5' terminus of RNA via an enzyme-guanylate intermediate. Incubation of the purified vaccinia RNA guanylyltransferase with [alpha- 32P]GTP and MgCl2 yields [32P]GMP covalently linked to the Mr = 95,000 subunit. The bond involves the phosphate moiety of GMP and the Ne-amino group of lysine. This was verified by treatment of the isolated 95-kDa subunit-[32P]GMP complex with sodium periodate, followed by methylamine-catalyzed beta-elimination. The product was then hydrolyzed by alkali producing 32P-labeled lysine (Ne-P)phosphate.

  15. Targeted release of oncolytic measles virus by blood outgrowth endothelial cells in situ inhibits orthotopic gliomas.

    PubMed

    Wei, J; Wahl, J; Nakamura, T; Stiller, D; Mertens, T; Debatin, K-M; Beltinger, C

    2007-11-01

    Malignant gliomas remain largely incurable despite intensive efforts to develop novel therapies. Replicating oncolytic viruses have shown great promise, among them attenuated measles viruses of the Edmonston B strain (MV-Edm). However, host immune response and the infiltrative nature of gliomas limit their efficacy. We show that human blood outgrowth endothelial cells (BOECs), readily expandable from peripheral blood, are easily infected by MV-Edm and allow replication of MV-Edm while surviving long enough after infection to serve as vehicles for MV-Edm (BOEC/MV-Edm). After intravenous and peritumoral injection, BOEC/MV-Edm deliver the viruses selectively to irradiated orthotopic U87 gliomas in mice. At the tumor, MV-Edm produced by the BOECs infect glioma cells. Subsequent spread from tumor cell to tumor cell leads to focal infection and cytopathic effects that decrease tumor size and, in the case of peritumoral injection, prolong survival of mice. Since MV-Edm within BOECs are not readily neutralized and because BOEC/MV-Edm search and destroy glioma cells, BOEC/MV-Edm constitute a promising novel approach for glioma therapy.

  16. Protection against lethal vaccinia virus challenge in HLA-A2 transgenic mice by immunization with a single CD8+ T-cell peptide epitope of vaccinia and variola viruses.

    PubMed

    Snyder, James T; Belyakov, Igor M; Dzutsev, Amiran; Lemonnier, François; Berzofsky, Jay A

    2004-07-01

    CD8(+) T lymphocytes have been shown to be involved in controlling poxvirus infection, but no protective cytotoxic T-lymphocyte (CTL) epitopes are defined for variola virus, the causative agent of smallpox, or for vaccinia virus. Of several peptides in vaccinia virus predicted to bind HLA-A2.1, three, VETFsm(498-506), A26L(6-14), and HRP2(74-82), were found to bind HLA-A2.1. Splenocytes from HLA-A2.1 transgenic mice immunized with vaccinia virus responded only to HRP2(74-82) at 1 week and to all three epitopes by ex vivo enzyme-linked immunosorbent spot (ELISPOT) assay at 4 weeks postimmunization. To determine if these epitopes could elicit a protective CD8(+) T-cell response, we challenged peptide-immunized HLA-A2.1 transgenic mice intranasally with a lethal dose of the WR strain of vaccinia virus. HRP2(74-82) peptide-immunized mice recovered from infection, while naïve mice died. Depletion of CD8(+) T cells eliminated protection. Protection of HHD-2 mice, lacking mouse class I major histocompatibility complex molecules, implicates CTLs restricted by human HLA-A2.1 as mediators of protection. These results suggest that HRP2(74-82), which is shared between vaccinia and variola viruses, may be a CD8(+) T-cell epitope of vaccinia virus that will provide cross-protection against smallpox in HLA-A2.1-positive individuals, representing almost half the population.

  17. Genome Sequence of Vaccinia virus Strain Lister-Butantan, a Lister Vaccine Variant Used during a Smallpox Eradication Campaign in Brazil

    PubMed Central

    Assis, Felipe; Trindade, Giliane; Drumond, Betânia; Frace, Mike; Sammons, Scott; Emerson, Ginny; Li, Yu; Carroll, Darin; Batra, Dhwani; Kroon, Erna

    2016-01-01

    Here, we report the 187.8-kb genome sequence of Vaccinia virus Lister-Butantan, which was used in Brazil during the WHO smallpox eradication campaign. Its genome showed an average similarity of 98.18% with the original Lister isolate, highlighting the low divergence among related Vaccinia virus vaccine strains, even after several passages in animals and cell culture. PMID:27340056

  18. Genome Sequence of Vaccinia virus Strain Lister-Butantan, a Lister Vaccine Variant Used during a Smallpox Eradication Campaign in Brazil.

    PubMed

    Assis, Felipe; Trindade, Giliane; Drumond, Betânia; Frace, Mike; Sammons, Scott; Emerson, Ginny; Li, Yu; Carroll, Darin; Batra, Dhwani; Abrahão, Jonatas; Kroon, Erna

    2016-01-01

    Here, we report the 187.8-kb genome sequence of Vaccinia virus Lister-Butantan, which was used in Brazil during the WHO smallpox eradication campaign. Its genome showed an average similarity of 98.18% with the original Lister isolate, highlighting the low divergence among related Vaccinia virus vaccine strains, even after several passages in animals and cell culture. PMID:27340056

  19. 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.

  20. Non-replicating recombinant vaccinia virus expressing CD80 to enhance T-cell stimulation.

    PubMed

    Zajac, Paul

    2009-01-01

    The following method describes the generation of a recombinant vaccinia virus expressing a costimulatory molecule (human CD80 or B7.1).The procedure first requires the cloning, by classical methods not described here, of the gene of interest, e.g. CD80, into a vaccinia shuttle plasmid under the control of a virus-specific promoter enabling a transcription during the early phase of infection. Flanking the insert, the plasmid contains viral sequences and a selection maker needed for the insertion into the viral genome. The successive plaque isolation of recombinant virus on cell monolayer described here is based on the transient "gpt" selection system which enables other insertions in different loci of the same virus. Finally, after verification amplification and titration of the recombinant vector, replication will be impaired by a psoralen-UV treatment in order to produce a non-replicating virus. Expression and function of inserts, following infection of cells, are verified by specific phenotypic and functional assays.

  1. Elements in the Development of a Production Process for Modified Vaccinia Virus Ankara

    PubMed Central

    Jordan, Ingo; Lohr, Verena; Genzel, Yvonne; Reichl, Udo; Sandig, Volker

    2013-01-01

    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.

  2. Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

    PubMed Central

    Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

    2015-01-01

    ABSTRACT 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. IMPORTANCE 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

  3. Molecular network, pathway, and functional analysis of time-dependent gene changes associated with pancreatic cancer susceptibility to oncolytic vaccinia virotherapy

    PubMed Central

    Haddad, Dana; Socci, Nicholas; Chen, Chun-Hao; Chen, Nanhai G; Zhang, Qian; Carpenter, Susanne G; Mittra, Arjun; Szalay, Aladar A; Fong, Yuman

    2016-01-01

    .01). By 24 hours, prominent pathways included P53- and Myc-induced apoptotic processes, pancreatic adenocarcinoma signaling, and phosphoinositide 3-kinase/v-akt murine thymoma vial oncogene homolog 1 (PI3/AKT) pathways. Conclusions: Our study reveals the ability to assess time-dependent changes in gene expression patterns in pancreatic cancer cells associated with infection and susceptibility to vaccinia viruses. This suggests that molecular assays may be useful to develop safer and more efficacious oncolyticvirotherapies and support the idea that these treatments may target pathways implicated in pancreatic cancer resistance to conventional therapies. PMID:27119120

  4. 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.

  5. An improved high pressure freezing and freeze substitution method to preserve the labile vaccinia virus nucleocapsid.

    PubMed

    Jesus, Desyree Murta; Moussatche, Nissin; Condit, Richard C

    2016-07-01

    In recent years, high pressure freezing and freeze substitution have been widely used for electron microscopy to reveal viral and cellular structures that are difficult to preserve. Vaccinia virus, a member of the Poxviridae family, presents one of the most complex viral structures. The classical view of vaccinia virus structure consists of an envelope surrounding a biconcave core, with a lateral body in each concavity of the core. This classical view was challenged by Peters and Muller (1963), who demonstrated the presence of a folded tubular structure inside the virus core and stated the difficulty in visualizing this structure, possibly because it is labile and cannot be preserved by conventional sample preparation. Therefore, this tubular structure, now called the nucleocapsid, has been mostly neglected over the years. Earlier studies were able to preserve the nucleocapsid, but with low efficiency. In this study, we report the protocol (and troubleshooting) that resulted in preservation of the highest numbers of nucleocapsids in several independent preparations. Using this protocol, we were able to demonstrate an interdependence between the formation of the virus core wall and the nucleocapsid, leading to the hypothesis that an interaction exists between the major protein constituents of these compartments, A3 (core wall) and L4 (nucleocapsid). Our results show that high pressure freezing and freeze substitution can be used in more in-depth studies concerning the nucleocapsid structure and function.

  6. Granzyme B Inhibits Vaccinia Virus Production through Proteolytic Cleavage of Eukaryotic Initiation Factor 4 Gamma 3

    PubMed Central

    Marcet-Palacios, Marcelo; Duggan, Brenda Lee; Shostak, Irene; Barry, Michele; Geskes, Tracy; Wilkins, John A.; Yanagiya, Akiko; Sonenberg, Nahum; Bleackley, R. Chris

    2011-01-01

    Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD1408S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery. PMID:22194691

  7. 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

  8. Molecular cloning, encoding sequence, and expression of vaccinia virus nucleic acid-dependent nucleoside triphosphatase gene.

    PubMed

    Rodriguez, J F; Kahn, J S; Esteban, M

    1986-12-01

    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.

  9. RNAi screening reveals proteasome- and Cullin3-dependent stages in vaccinia virus infection.

    PubMed

    Mercer, Jason; Snijder, Berend; Sacher, Raphael; Burkard, Christine; Bleck, Christopher Karl Ernst; Stahlberg, Henning; Pelkmans, Lucas; Helenius, Ari

    2012-10-25

    A two-step, automated, high-throughput RNAi silencing screen was used to identify host cell factors required during vaccinia virus infection. Validation and analysis of clustered hits revealed previously unknown processes during virus entry, including a mechanism for genome uncoating. Viral core proteins were found to be already ubiquitinated during virus assembly. After entering the cytosol of an uninfected cell, the viral DNA was released from the core through the activity of the cell's proteasomes. Next, a Cullin3-based ubiquitin ligase mediated a further round of ubiquitination and proteasome action. This was needed in order to initiate viral DNA replication. The results accentuate the value of large-scale RNAi screens in providing directions for detailed cell biological investigation of complex pathways. The list of cell functions required during poxvirus infection will, moreover, provide a resource for future virus-host cell interaction studies and for the discovery of antivirals. PMID:23084750

  10. CD8+ T-cell Immune Evasion Enables Oncolytic Virus Immunotherapy

    PubMed Central

    Pourchet, Aldo; Fuhrmann, Steven R.; Pilones, Karsten A.; Demaria, Sandra; Frey, Alan B.; Mulvey, Matthew; Mohr, Ian

    2016-01-01

    Although counteracting innate defenses allows oncolytic viruses (OVs) to better replicate and spread within tumors, CD8+ T-cells restrict their capacity to trigger systemic anti-tumor immune responses. Herpes simplex virus-1 (HSV-1) evades CD8+ T-cells by producing ICP47, which limits immune recognition of infected cells by inhibiting the transporter associated with antigen processing (TAP). Surprisingly, removing ICP47 was assumed to benefit OV immuno-therapy, but the impact of inhibiting TAP remains unknown because human HSV-1 ICP47 is not effective in rodents. Here, we engineer an HSV-1 OV to produce bovine herpesvirus UL49.5, which unlike ICP47, antagonizes rodent and human TAP. Significantly, UL49.5-expressing OVs showed superior efficacy treating bladder and breast cancer in murine models that was dependent upon CD8+ T-cells. Besides injected subcutaneous tumors, UL49.5-OV reduced untreated, contralateral tumor size and metastases. These findings establish TAP inhibitor-armed OVs that evade CD8+ T-cells as an immunotherapy strategy to elicit potent local and systemic anti-tumor responses. PMID:27077112

  11. 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

  12. 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.

  13. Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant.

    PubMed

    Kotwal, G J; Moss, B

    1988-12-01

    The principal objectives of this study were to analyze the structure and coding potential of a long segment of DNA missing from a previously isolated (B. Moss, E. Winters, and J. A. Cooper (1981) J. Virol. 40, 387-395) attenuated variant of vaccinia virus strain WR and to examine the precise changes in the genome accompanying the deletion. The sequences of a 14.5-kbp region located at the left end of the standard vaccinia virus genome, extending from within the inverted terminal repetition (ITR) of the HindIII C fragment to the end of the HindIII N fragment, and of a 3-kbp segment from a corresponding region of the variant genome were determined. A comparison of these sequences revealed that the variant contained a deletion of 12 kbp and an insertion of 2.1 kbp. The origin of the inserted DNA was traced to the HindIII B region by using oligonucleotide probes indicating that a transposition of unique DNA located adjacent to the right ITR had occurred. Structural analysis indicated no extensive homologies, nucleotide substitutions, additions, or deletions at the boundaries of the transposed DNA. Examination of the right end of the variant genome indicated that a copy of the transposed DNA was still present and, therefore, the length of the ITR had been increased by 2.1 kbp. The variant genome could have formed by a mechanism that resulted in the replacement of a 22-kbp left-terminal fragment with a 12-kbp right-terminal fragment. The DNA missing from the variant and contained within the standard vaccinia virus WR genome contains 17 contiguous open reading frames (ORFs), all of which are directed leftward and apparently not required for replication in cultured cells. One deleted ORF has a 60% sequence similarity to another gene encoding a 42,000-Da protein present within the ITR suggesting that duplications have previously occurred during the evolution of vaccinia virus. Another deleted ORF has a 39% sequence similarity to a complement 4b binding protein. The

  14. Membrane-bound complement regulatory activity is decreased on vaccinia virus-infected cells.

    PubMed Central

    Baranyi, L; Okada, N; Baranji, K; Takizawa, H; Okada, H

    1994-01-01

    Decay accelerating factor (DAF), membrane cofactor protein (MCP), complement receptor 1 and mouse Crry are cell surface-bound complement regulatory proteins capable of inhibiting C3 convertase activity on cell membranes, and therefore provide a substantial protection from attack by homologous complement activated either by the classical or by the alternative pathway. Decrease in complement regulatory activity might lead to spontaneous complement deposition and subsequent cell injury. MoAb 5I2 can inhibit the complement regulatory activity of molecules on rat cells, resulting in deposition of homologous complement. The antigen recognized by 5I2 MoAb in rats is homologous to mouse Crry. Fifteen to 20 h after infection with vaccinia virus, in vitro cultured KDH-8 rat hepatoma cells show a strong decrease in expression of Crry-like antigen, and proved to be sensitive to complement deposition when 1:5 diluted normal rat serum was added to the culture medium as a source of complement. Addition of complement to the cultured KDH-8 cells infected with a very low dose of vaccinia virus (1 plaque-forming unit (PFU)/1000 cells) substantially reduced spreading of virus infection in the cell culture, while inactivation of complement by heat or zymosan treatment abrogated the protective effect. PMID:7923872

  15. Modified Vaccinia virus Ankara: innate immune activation and induction of cellular signalling.

    PubMed

    Price, Philip J R; Torres-Domínguez, Lino E; Brandmüller, Christine; Sutter, Gerd; Lehmann, Michael H

    2013-09-01

    Attenuated poxviruses are currently under development as vaccine vectors against a number of diseases including, influenza, HIV, malaria and tuberculosis. Modified Vaccinia virus Ankara (MVA) is an attenuated, replication deficient vaccinia virus (VACV) strain which, similar to replication competent VACV, is highly immunogenic. The lack of productive viral replication further improves the safety profile of MVA as a vector, minimizing the potential for reversion to virulent forms particularly if used in immunocompromised individuals. Despite its inability to replicate in most mammalian cells, MVA still efficiently expresses viral and recombinant genes making it a potent antigen delivery platform. Moreover, due to the loss of various immunomodulatory factors MVA infection leads to rapid local immune responses, fulfilling a requirement of an adjuvant. In this review we take a look at the immunostimulatory properties of MVA, paying particular attention to the signalling of the innate immune system in response to MVA and VACV infection. Understanding the cellular and molecular mechanisms modulated by VACV will help in the future design and engineering of new vaccines and may provide insight into previously unknown mechanisms of dominant virus-host interactions.

  16. DNA sequence analysis of the Hind III M fragment from Chinese vaccine strain of vaccinia virus.

    PubMed

    Liu, V J; Jin, Q; Jin, D Y; Hou, Y D

    1989-01-01

    The complete DNA sequence of the Hind III M fragment of vaccinia virus (VV) Tian Tan strain genome was determined by the dideoxynucleotide chain termination method. Three open reading frames (ORFs) were identified in the complementary strand of the sequence, comprised of 2218bp. Among them, ORF K1 initiates its transcription at -45 of the Hind III K fragment. The deduced peptide encoded by K1 contains 284 amino acids with a calculated molecular weight of 32.48 KDa. Its sequence is homologous to the host range protein of VV Copenhagen strain; the variation is only 2.46% at the amino acid level. ORF M2 could encode a peptide of 21.94 KDa with 196 amino acids. This gene was shown to be homologous to that of the 23 KDa peptide of herpes simplex virus type I. A non-coding region of 204bp located between K1 and M2 is rich in palindromic structures. ORF M1 extends its 3' terminus into the Hind III N fragment. Within the M fragment, M1 can only encode 212 amino acids. The major part of ORF M1 is very similar to the M portion of a possible alpha-amanitin resistance gene isolated from VV-WR strain. This work provides a molecular foundation in the construction of a new insertion vector for the preparation of a recombinant vaccinia virus to be used as a polyvalent live vaccine.

  17. 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.

  18. Vaccinia virus recombinant expressing an 87-kilodalton polyprotein that is sufficient to form astrovirus-like particles.

    PubMed

    Dalton, Rosa M; Pastrana, Esperanza P; Sánchez-Fauquier, Alicia

    2003-08-01

    Human astrovirus is an important cause of acute gastroenteritis. We have generated, for the first time, a vaccinia virus recombinant expressing the astrovirus 87-kDa structural polyprotein. The results demonstrate that this expression results in the formation of virus-like particles in the absence of other astrovirus proteins and genomic RNA. The purified trypsin-activated virus-like particles strongly resemble the complete astrovirus particles.

  19. Encapsulated Stem Cells Loaded With Hyaluronidase-expressing Oncolytic Virus for Brain Tumor Therapy

    PubMed Central

    Martinez-Quintanilla, Jordi; He, Derek; Wakimoto, Hiroaki; Alemany, Ramon; Shah, Khalid

    2015-01-01

    Despite the proven safety of oncolytic viruses (OV) in clinical trials for glioblastoma (GBM), their efficacy has been hindered by suboptimal spreading within the tumor. We show that hyaluronan or hyaluronic acid (HA), an important component of extracellular matrix (ECM), is highly expressed in a majority of tumor xenografts established from patient-derived GBM lines that present both invasive and nodular phenotypes. Intratumoral injection of a conditionally replicating adenovirus expressing soluble hyaluronidase (ICOVIR17) into nodular GBM, mediated HA degradation and enhanced viral spread, resulting in a significant antitumor effect and mice survival. In an effort to translate OV-based therapeutics into clinical settings, we encapsulated human adipose-derived mesenchymal stem cells (MSC) loaded with ICOVIR17 in biocompatible synthetic extracellular matrix (sECM) and tested their efficacy in a clinically relevant mouse model of GBM resection. Compared with direct injection of ICOVIR17, sECM-MSC loaded with ICOVIR17 resulted in a significant decrease in tumor regrowth and increased mice survival. This is the first report of its kind revealing the expression of HA in GBM and the role of OV-mediated HA targeting in clinically relevant mouse model of GBM resection and thus has clinical implications. PMID:25352242

  20. Targeting CXCL12/CXCR4 signaling with oncolytic virotherapy disrupts tumor vasculature and inhibits breast cancer metastases

    PubMed Central

    Gil, Margaret; Seshadri, Mukund; Komorowski, Marcin P.; Abrams, Scott I.; Kozbor, Danuta

    2013-01-01

    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. PMID:23509246

  1. Crystallization and preliminary X-ray diffraction analysis of vaccinia virus H1L phosphatase

    SciTech Connect

    Roces, Laura; Knowles, Phillip P.; Fox, Gavin; Juanhuix, Jordi; Scaplehorn, Nicki; Way, Michael; McDonald, Neil Q.

    2008-03-01

    A catalytically inactive mutant of the dual-specificity phosphatase H1L from vaccinia virus was expressed recombinantly, purified and crystallized by the microbatch method. The crystals belong to the tetragonal space group P422 and diffraction data were collected to 2.1 Å resolution using a synchrotron-radiation source. Attempts to derivatize these crystals with xenon gas lead to a space-group change to I422 with a smaller unit cell and a diffraction limit of 3.0 Å. 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.

  2. Intracellular forms of pox viruses as shown by the electron microscope (Vaccinia, Ectromelia, Molluscum Contagiosum).

    PubMed

    GAYLORD, W H; MELNICK, J L

    1953-08-01

    The intracellular development of three pox viruses has been studied with the electron microscope using thin sections of infected tissue. Cells infected with vaccinia, ectromelia, and molluscum contagiosum viruses all form developmental bodies preliminary to the production of mature virus. Developmental bodies, believed to be virus precursors, are round to oval, slightly larger than mature virus particles, less dense to electrons, and have a more varied morphology. It is suggested as a working hypothesis that the process of maturation of a virus particle takes place as follows. In the earliest form the developmental bodies appear as hollow spheres, imbedded in a very dense cytoplasmic mass constituting an inclusion body, or in a less dense matrix near the nucleus in cells without typical inclusion bodies. The spheres become filled with a homogeneous material of low electron density. A small, dense granule appears in each developmental body and grows in size at the expense of the low density material. Following growth of the granule, particles are found with the dimensions of mature virus and having complex internal structure resembling bars or dumbells. Mature virus is ovoid and very dense to electrons. An "empty" interior may be found within its thick walls.

  3. Combination effect of oncolytic adenovirus therapy and herpes simplex virus thymidine kinase/ganciclovir in hepatic carcinoma animal models

    PubMed Central

    Zheng, Fei-qun; Xu, Yin; Yang, Ren-jie; Wu, Bin; Tan, Xiao-hua; Qin, Yi-de; Zhang, Qun-wei

    2009-01-01

    Aim: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), can selectively propagate in tumor cells and cause cell lysis. The released viral progeny can infect neighboring cancer cells, initiating a cascade that can lead to the ultimate destruction of the tumor. Suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) offers a potential treatment strategy for cancer and is undergoing preclinical trials for a variety of tumors. We hypothesized that HSV-TK gene therapy combined with oncolytic adenoviral therapy would have an enhanced effect compared with the individual effects of the therapies and is a potential novel therapeutic strategy to treat liver cancer. Methods: To address our hypothesis, a novel CRAD was created, which consisted of a telomerase-dependent oncolytic adenovirus engineered to express E1A and HSV-TK genes (Ad-ETK). The combined effect of Ad-ETK and GCV was assessed both in vitro and in vivo in nude mice bearing HepG2 cell-derived tumors. Expression of the therapeutic genes by the transduced tumor cells was analyzed by RT-PCR and Western blotting. Results: We confirmed that Ad-ETK had antitumorigenic effects on human hepatocellular carcinoma (HCC) both in vitro and in vivo, and the TK/GCV system enhanced oncolytic adenoviral therapy. We confirmed that both E1A and HSV-TK genes were expressed in vivo. Conclusion: The Ad-ETK construct should provide a relatively safe and selective approach to killing cancer cells and should be investigated as an adjuvant therapy for hepatocellular carcinoma. PMID:19363518

  4. 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.

  5. The vaccinia virus I1 protein is essential for the assembly of mature virions.

    PubMed Central

    Klemperer, N; Ward, J; Evans, E; Traktman, P

    1997-01-01

    The product of the vaccinia virus I1 gene was characterized biochemically and genetically. This 35-kDa protein is conserved in diverse members of the poxvirus family but shows no homology to nonviral proteins. We show that recombinant I1 binds to both single-stranded and double-stranded DNA in a sequence-nonspecific manner in an electrophoretic mobility shift assay. The protein is expressed at late times during infection, and approximately 700 copies are encapsidated within the virion core. To determine the role of the I1 protein during the viral life cycle, a inducible viral recombinant in which the I1 gene was placed under the regulation of the Escherichia coli lac operator/repressor was constructed. In the absence of isopropyl-beta-D-thiogalactopyranoside, plaque formation was abolished and yields of infectious, intracellular virus were dramatically reduced. Although all phases of gene expression and DNA replication proceeded normally during nonpermissive infections, no mature virions were produced. Electron microscopic analysis confirmed the absence of mature virion assembly but revealed that apparently normal immature virions accumulated. Thus, I1 is an encapsidated DNA-binding protein required for the latest stages of vaccinia virion morphogenesis. PMID:9371587

  6. Directed cytokine expression in tumour cells in vivo using recombinant vaccinia virus.

    PubMed

    Acres, B; Dott, K; Stefani, L; Kieny, M P

    1994-01-01

    Athymic (Swiss nude) and euthymic (DBA) tumour-bearing mice were injected intravenously with various vaccinia virus (Copenhagen strain) recombinants. Several days after inoculation, tumour cells were found to be well infected with infective vaccinia particles, while organs such as liver, spleen, brain and bone marrow showed barely detectable levels or no signs at all of virus infection. Injection of tumour bearing mice with recombinant VV harbouring the cDNA for either huIL-2 or muIL-6 resulted in detectable lymphokine in the sera of injected animals. Injection of tumour-bearing nude mice with VV-IL-6, but not with VV-IL-2, resulted in significant reduction in growth rate of the tumour, and in some cases, complete rejection of the tumour. Tumour-bearing euthymic mice responded differently. Intravenous injection of VV-IL-2, but not VV-IL-6 resulted in reduced growth rate of 50% of tumours and complete rejection of 17% of tumours. PMID:7584475

  7. 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.

  8. Detection of Vaccinia virus in blood and faeces of experimentally infected cows.

    PubMed

    Guedes, M I M C; Rehfeld, I S; de Oliveira, T M L; Assis, F L; Matos, A C D; Abrahão, J S; Kroon, E G; Lobato, Z I P

    2013-12-01

    Bovine vaccinia (BV), a zoonosis caused by Vaccinia virus (VACV), affects dairy cattle and milkers, causing economic, veterinary and human health impacts. Despite such impacts, there are no experimental studies about the pathogenesis of BV in cows to assess whether there is a systemic spread of the virus and whether there are different ways of VACV shedding. Trying to answer some of these questions, a study was proposed using experimental inoculation of VACV in cows. All experimentally infected cows developed lesions compatible with VACV infection in cattle. Two of the six animals presented VACV DNA in blood and faecal samples, starting at the 2nd and the 3rd day post-infection (d.p.i.), respectively, and lasting until the 36th d.p.i., in an intermittent way. This study provides new evidence that VACV can be detected in blood and faeces of infected cows, suggesting that BV could be a systemic disease, and also bringing new information about the epidemiology and pathogenesis of BV.

  9. Molecular genetic analysis of a vaccinia virus gene with an essential role in DNA replication

    SciTech Connect

    Evans, E.V.A.

    1989-01-01

    The poxvirus, vaccinia, is large DNA virus which replicates in the cytoplasma of the host cell. The virus is believed to encode most or all of the functions required for the temporally regulated transcription and replication of its 186 kilobase genome. Physical and genetic autonomy from the host make vaccinia a useful eukaryotic organism in which to study replication genes and proteins, using a combination of biochemical and genetic techniques. Essential viral functions for replication are identified by conditional lethal mutants that fail to synthesize DNA at the non-permissive temperatures. One such group contains the non-complementing alleles ts17, ts24, ts69 (WR strain). Studies were undertaken to define the phenotype of ts mutants, and to identify and characterize the affected gene and protein. Mutant infection was essentially normal at 32{degree}C, but at 39{degree}C the mutants did not incorporate {sup 3}H-thymidine into nascent viral DNA or synthesize late viral proteins. If mutant cultures were shifted to non-permissive conditions at the height of replication, DNA synthesis was halted rapidly, implying that the mutants are defective in DNA elongation. The gene affected in the WR mutants and in ts6389, a DNA-minus mutant of the IHD strain, was mapped by marker rescue and corresponds to open reading frame 5 (orfD5) of the viral HindIII D fragment.

  10. 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

  11. 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.

  12. 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.

  13. 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. PMID:26812352

  14. Analysis of the L1 gene product of human papillomavirus type 16 by expression in a vaccinia virus recombinant.

    PubMed

    Browne, H M; Churcher, M J; Stanley, M A; Smith, G L; Minson, A C

    1988-06-01

    The L1 open reading frame of human papillomavirus type 16 (HPV16) has been expressed in vaccinia virus under the control of both the 7.5K early and late promoter, and the 4b major late promoter. Antibodies to a beta-galactosidase fusion protein containing a C-terminal portion of the HPV16 L1 gene product were used to compare the levels of L1 expression in the two recombinants, and showed that greater levels of expression were obtained when the gene was placed under the control of the 4b late promoter. Immunofluorescence studies revealed a nuclear location of the L1 gene product when expressed in vaccinia virus. Antibodies to the beta-galactosidase fusion protein detected a major polypeptide species of 57K and a minor species of 64K in Western blots of recombinant-infected cell lysates. The 64K species was not detected when cells were infected in the presence of tunicamycin, indicating that the primary translation product of the HPV16 L1 open reading frame is modified by N-linked glycosylation when expressed in vaccinia virus. Whereas antibodies to HPV16 L1 fusion proteins and to a peptide containing amino acids from the C terminus of HPV16 L1 reacted well in Western blots with the HPV16 L1 target expressed in vaccinia virus, no reactivity was observed with antibodies to bovine papillomavirus type 1 particles or to a HPV6b fusion protein.

  15. 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

  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. A36-dependent actin filament nucleation promotes release of vaccinia virus.

    PubMed

    Horsington, Jacquelyn; Lynn, Helena; Turnbull, Lynne; Cheng, Delfine; Braet, Filip; Diefenbach, Russell J; Whitchurch, Cynthia B; Karupiah, Guna; Newsome, Timothy P

    2013-03-01

    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 A36(YdF) virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36(YdF) 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 A36(YdF) 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 B5(P189S) 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

  18. Expression of the E3L Gene of Vaccinia Virus in Transgenic Mice Decreases Host Resistance to Vaccinia Virus and Leishmania major Infections▿

    PubMed Central

    Domingo-Gil, Elena; Pérez-Jiménez, Eva; Ventoso, Iván; Nájera, José L.; Esteban, Mariano

    2008-01-01

    The E3L gene of vaccinia virus (VACV) encodes the E3 protein that in cultured cells inhibits the activation of interferon (IFN)-induced proteins, double-stranded RNA-dependent protein kinase (PKR), 2′-5′-oligoadenylate synthetase/RNase L (2-5A system) and adenosine deaminase (ADAR-1), thus helping the virus to evade host responses. Here, we have characterized the in vivo E3 functions in a murine inducible cell culture system (E3L-TetOFF) and in transgenic mice (TgE3L). Inducible E3 expression in cultured cells conferred on cells resistance to the antiviral action of IFN against different viruses, while expression of the E3L gene in TgE3L mice triggered enhanced sensitivity of the animals to pathogens. Virus infection monitored in TgE3L mice by different inoculation routes (intraperitoneal and tail scarification) showed that transgenic mice became more susceptible to VACV infection than control mice. TgE3L mice were also more susceptible to Leishmania major infection, leading to an increase in parasitemia compared to control mice. The enhanced sensitivity of TgE3L mice to VACV and L. major infections occurred together with alterations in the host immune system, as revealed by decreased T-cell responses to viral antigens in the spleen and lymph nodes and by differences in the levels of specific innate cell populations. These results demonstrate that expression of the E3L gene in transgenic mice partly reverses the resistance of the host to viral and parasitic infections and that these effects are associated with immune alterations. PMID:17959665

  19. 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.

  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. 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

    2012-02-01

    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.

  3. Vaccinia virus gene H5R encodes a protein that is phosphorylated by the multisubstrate vaccinia virus B1R protein kinase.

    PubMed Central

    Beaud, G; Beaud, R; Leader, D P

    1995-01-01

    Vaccinia virus gene B1R encodes a protein kinase, the previously identified substrates of which include the proteins S2 and Sa of 40S ribosomal subunits. This work characterizes another substrate of the B1R kinase: a 36-kDa protein induced at the early stage of infection. Partially purified 36-kDa protein, eluted from a single-stranded DNA-cellulose column by 0.5 M NaCl, was separated by two-dimensional gel electrophoresis. Phosphorylation in vitro yielded multiple forms of the 36-kDa protein with approximate isoelectric points (pI) of 5.5, 5.7, 5.9, and 6.3, in addition to the apparently unphosphorylated form with a pI of approximately 6.8. The tryptic peptides derived from 36-kDa proteins with pI values of 5.7, 5.9, and 6.3 yielded almost identical high-pressure liquid chromatography profiles, strongly suggesting that the 36-kDa protein was modified by the phosphorylation of at least four sites, which were characterized as threonine residues. The amino acid sequence of several tryptic peptides derived from the 36-kDa protein showed that the 36-kDa protein was encoded by gene H5R of vaccinia virus. Consistent with this, the B1R kinase--either expressed in Escherichia coli or highly purified from HeLa cells--phosphorylated a recombinant trpE-H5R fusion protein in vitro. Fingerprints of the trpE-H5R and 36-kDa proteins phosphorylated by recombinant B1R kinase revealed common sites of phosphorylation, although some tryptic peptides were specific to either protein. Comparison was made of fingerprints of tryptic phosphopeptides derived from 36-kDa single-stranded DNA-binding protein labelled in vivo or in vitro. A common subset of peptides was observed, suggesting that some sites on H5R protein are phosphorylated by the B1R kinase in infected cells. These results suggest that some of the multiple threonine sites in the H5R protein are phosphorylated in vivo by the B1R protein kinase. PMID:7853522

  4. Vaccination of mice against canine distemper virus-induced encephalitis with vaccinia virus recombinants encoding measles or canine distemper virus antigens.

    PubMed

    Wild, T F; Bernard, A; Spehner, D; Villeval, D; Drillien, R

    1993-01-01

    Measles and canine distemper are caused by serologically related viruses. Although dogs immunized with measles virus (MV) do not elicit canine distemper virus (CDV) neutralizing antibodies, they are protected against the fatal disease. To investigate the potential role of the MV antigens in protection against CDV, we have immunized mice with vaccinia virus (VV) recombinants expressing the MV haemagglutinin (HA), fusion (F), nucleoprotein (NP) and matrix (M) antigens and challenged them with CDV. A partial protection was observed with the VV recombinants expressing the F, NP and M antigens, but not the HA. In contrast, immunization with a VV recombinant expressing the CDV F protein completely protected mice from CDV. PMID:8470428

  5. Long-lasting stability of Vaccinia virus strains in murine feces: implications for virus circulation and environmental maintenance.

    PubMed

    Abrahão, Jônatas S; Trindade, Giliane de Souza; Ferreira, Jaqueline M Siqueira; Campos, Rafael K; Bonjardim, Cláudio A; Ferreira, Paulo C Peregrino; Kroon, Erna Geessien

    2009-01-01

    Vaccinia virus (VACV) has been associated with several bovine vaccinia outbreaks in Brazil, causing exanthematic lesions in dairy cattle and humans. The way that VACV circulates in the environment is unknown, as is the way that this virus is transferred from wildlife to farms. Rodents are hypothetical VACV reservoirs, and murine feces has been identified as a potential source of viral shedding and transmission. In this work, we analyzed the stability of VACV infectious particles and DNA in feces from intranasally infected mice, exposed to environmental temperature and humidity, by titration assays and PCR, respectively. The results showed that VACV infectious particles were still detected at 20 days post-environmental-exposure (d.p.e.), while viral DNA was detected until 60 d.p.e. A gradual decrease in fecal viral load could be detected in all analyzed VACV strains. This work indicates long-lasting stability of VACV in murine feces and reinforces the idea that fecal matter may represent a potential source of circulating virus among rodents.

  6. 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.

  7. Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens

    PubMed Central

    Zajac, María Paula Del Médico; Taboga, Oscar Alberto; Calamante, Gabriela

    2012-01-01

    A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry. PMID:22705743

  8. 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

  9. 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.

  10. 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. PMID:17264852

  11. Comparative analysis of poxvirus orthologues of the vaccinia virus E3 protein: modulation of protein kinase R activity, cytokine responses, and virus pathogenicity.

    PubMed

    Myskiw, Chad; Arsenio, Janilyn; Hammett, Craig; van Bruggen, Rebekah; Deschambault, Yvon; Beausoleil, Nicole; Babiuk, Shawn; Cao, Jingxin

    2011-12-01

    Poxviruses are important human and animal pathogens that have evolved elaborate strategies for antagonizing host innate and adaptive immunity. The E3 protein of vaccinia virus, the prototypic member of the orthopoxviruses, functions as an inhibitor of innate immune signaling and is essential for vaccinia virus replication in vivo and in many human cell culture systems. However, the function of orthologues of E3 expressed by poxviruses of other genera with different host specificity remains largely unknown. In the present study, we characterized the E3 orthologues from sheeppox virus, yaba monkey tumor virus, swinepox virus, and myxoma virus for their ability to modulate protein kinase R (PKR) function, cytokine responses and virus pathogenicity. We found that the E3 orthologues of myxoma virus and swinepox virus could suppress PKR activation and interferon (IFN)-induced antiviral activities and restore the host range function of E3 in HeLa cells. In contrast, the E3 orthologues from sheeppox virus and yaba monkey tumor virus were unable to inhibit PKR activation. While the sheeppox orthologue was unable to restore the host range function of E3, the yaba monkey tumor virus orthologue partially restored E3-deficient vaccinia virus replication in HeLa cells, correlated with its ability to suppress IFN-induced antiviral activities. Moreover, poxvirus E3 orthologues show varying ability to inhibit the induction of antiviral and proinflammatory cytokines. Despite these in vitro results, none of the E3 orthologues tested was capable of restoring pathogenicity to E3-deficient vaccinia virus in vivo.

  12. 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

  13. 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

  14. ATN-224 enhances antitumor efficacy of oncolytic herpes virus against both local and metastatic head and neck squamous cell carcinoma

    PubMed Central

    Yoo, Ji Young; Yu, Jun-Ge; Kaka, Azeem; Pan, Quintin; Kumar, Pawan; Kumar, Bhavna; Zhang, Jianying; Mazar, Andrew; Teknos, Theodoros N; Kaur, Balveen; Old, Matthew O

    2015-01-01

    Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide, and the 5-year survival rates are among the worst of the major cancers. Oncolytic herpes simplex viruses (oHSV) have the potential to make a significant impact in the targeted treatment of these patients. Here, we tested antitumor efficacy of RAMBO, an oHSV armed with the antiangiogenic Vstat120, alone and in conjunction with ATN-224, a copper chelator against HNSCC in vitro and in vivo animal models. We found that all tested HNSCC cells responded well to virus treatment and were sensitive to RAMBO-mediated oncolytic destruction. In vivo, RAMBO had a significant antiangiogenic and antitumorigenic effect. Physiologic levels of copper inhibited viral replication and HNSCC cell killing. Chelation of copper using ATN-224 treatment significantly improved serum stability of RAMBO and permitted systemic delivery in HNSCC tumor xenografts models. Furthermore, our results show that the combination of ATN-224 and RAMBO strongly inhibits lung metastases in a mouse model of HNSCC. These findings suggest that combining ATN-224 with RAMBO has potential for clinical trials in both early and advanced HNSCC patients. PMID:27119105

  15. Armed oncolytic virus enhances immune functions of chimeric antigen receptor-modified T cells in solid tumors.

    PubMed

    Nishio, Nobuhiro; Diaconu, Iulia; Liu, Hao; Cerullo, Vincenzo; Caruana, Ignazio; Hoyos, Valentina; Bouchier-Hayes, Lisa; Savoldo, Barbara; Dotti, Gianpietro

    2014-09-15

    The clinical efficacy of chimeric antigen receptor (CAR)-redirected T cells remains marginal in solid tumors compared with leukemias. Failures have been attributed to insufficient T-cell migration and to the highly immunosuppressive milieu of solid tumors. To overcome these obstacles, we have combined CAR-T cells with an oncolytic virus armed with the chemokine RANTES and the cytokine IL15, reasoning that the modified oncolytic virus will both have a direct lytic effect on infected malignant cells and facilitate migration and survival of CAR-T cells. Using neuroblastoma as a tumor model, we found that the adenovirus Ad5Δ24 exerted a potent, dose-dependent, cytotoxic effect on tumor cells, whereas CAR-T cells specific for the tumor antigen GD2 (GD2.CAR-T cells) were not damaged. When used in combination, Ad5Δ24 directly accelerated the caspase pathways in tumor cells exposed to CAR-T cells, whereas the intratumoral release of both RANTES and IL15 attracted CAR-T cells and promoted their local survival, respectively, increasing the overall survival of tumor-bearing mice. These preclinical data support the use of this innovative biologic platform of immunotherapy for solid tumors. Cancer Res; 74(18); 5195-205. ©2014 AACR.

  16. 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.

  17. Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA

    SciTech Connect

    Jones, E.V.; Moss, B.

    1984-01-01

    The previous demonstration that a phosphonoacetate (PAA)-resistant (PAA/sup r/) vaccinia virus mutant synthesized an altered DNA polymerase provided the key to mapping this gene. Marker rescue was performed in cells infected with wild-type PAA-sensitive (PAA/sup s/) vaccinia by transfecting with calcium phosphate-precipitated DNA from a PAA/sup r/ mutant virus. Formation of PAA/sup r/ recombinants was measured by plaque assay in the presence of PAA. Of the 12 HindIII fragments cloned in plasmid or cosmid vectors, only fragment E conferred the PAA/sup r/ phenotype. Successive subcloning of the 15-kilobase HindIII fragment E localized the marker within a 7.5-kilobase BamHI-HindIII fragment and then within a 2.9-kilobase EcoRI fragment. The location of the DNA polymerase gene, about 57 kilobases from the left end of the genome, was confirmed by cell-free translation of mRNA selected by hybridization to plasmids containing regions of PAA/sup r/ vaccinia DNA active in marker rescue. A 100,000-dalton polypeptide that comigrated with authentic DNA polymerase was synthesized. Correspondence of the in vitro translation product with purified vaccinia DNA polymerase was established by peptide mapping.

  18. Vaccinia Virus A35R Inhibits MHC Class II Antigen Presentation

    PubMed Central

    Rehm, Kristina E.; Connor, Ramsey F.; Jones, Gwendolyn J.B.; Yimbu, Kenneth; Roper, Rachel L.

    2009-01-01

    The Vaccinia virus gene A35R (Copenhagen designation) is highly conserved in mammalian-tropic poxviruses and is an important virulence factor, but its function was unknown. We show herein that A35 does not affect viral infectivity, apoptosis induction, or replication; however, we found that A35 significantly inhibited MHC class II-restricted antigen presentation, immune priming of T lymphocytes, and subsequent chemokine and cytokine synthesis. A35 localized to endosomes and reduced the amount of a model antigenic peptide displayed in the cleft of class II MHC. In addition, A35 decreased VV specific T cell responses in vivo. Thus, this is the first report identifying a function for the A35 protein in virulence as well as the first report identifying a VV gene that inhibits peptide antigen presentation. PMID:19954808

  19. Environmental risk assessment of clinical trials involving modified vaccinia virus Ankara (MVA)-based vectors.

    PubMed

    Goossens, Martine; Pauwels, Katia; Willemarck, Nicolas; Breyer, Didier

    2013-12-01

    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.

  20. Vaccinia virus binds to the scavenger receptor MARCO on the surface of keratinocytes.

    PubMed

    MacLeod, Daniel T; Nakatsuji, Teruaki; Wang, Zhenping; di Nardo, Anna; Gallo, Richard L

    2015-01-01

    Patients with altered skin immunity, such as individuals with atopic dermatitis (AD), can have a life-threatening disruption of the epidermis known as eczema vaccinatum after vaccinia virus (VV) infection of the skin. Here, we sought to better understand the mechanism(s) by which VV associates with keratinocytes. The class A scavenger receptor known as MARCO (macrophage receptor with collagenous structure) is expressed on human and mouse keratinocytes and found to be abundantly expressed in the skin of patients with AD. VV bound directly to MARCO, and overexpression of MARCO increased susceptibility to VV infection. Furthermore, ligands with affinity for MARCO, or excess soluble MARCO, competitively inhibited VV infection. These findings indicate that MARCO promotes VV infection and highlights potential new therapeutic strategies for prevention of VV infection in the skin.

  1. 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.

  2. Vaccinia virus A19 protein participates in the transformation of spherical immature particles to barrel-shaped infectious virions.

    PubMed

    Satheshkumar, P S; Weisberg, Andrea S; Moss, Bernard

    2013-10-01

    The A19L open reading frame of vaccinia virus encodes a 9-kDa protein that is conserved in all sequenced chordopoxviruses, yet until now it has not been specifically characterized in any species. We appended an epitope tag after the start codon of the A19L open reading frame without compromising infectivity. The protein was synthesized after viral DNA replication and was phosphorylated independently of the vaccinia virus F10 kinase. The A19 protein was present in purified virions and was largely resistant to nonionic detergent extraction, suggesting a location within the core. A conditional lethal mutant virus was constructed by placing the A19 open reading frame under the control of the Escherichia coli lac repressor system. A19 synthesis and infectious virus formation were dependent on inducer. In the absence of inducer, virion morphogenesis was interrupted, and spherical dense particles that had greatly reduced amounts of the D13 scaffold accumulated in place of barrel-shaped mature virions. The infectivity of purified A19-deficient particles was more than 2 log units less than that of A19-containing virions. Nevertheless, the A19-deficient particles contained DNA, and except for the absence of A19 and decreased core protein processing, they appeared to have a similar protein composition as A19-containing virions. Thus, the A19 protein participates in the maturation of immature vaccinia virus virions to infectious particles.

  3. A PCR-based method for manipulation of the vaccinia virus genome that eliminates the need for cloning.

    PubMed

    Turner, P C; Moyer, R W

    1992-11-01

    A general method is described for altering specific genes of vaccinia virus (VV). We demonstrate and evaluate the procedure by gene inactivation, using a dominant selectable marker in conjunction with recombinant polymerase chain reaction (PCR). Primers based on the sequence of the target gene enable amplification of flanking arms and their subsequent attachment to the gpt cassette that confers resistance to mycophenolic acid. Linear PCR constructs are transfected into cells infected with wild-type vaccinia virus. Mutant viruses with gpt inserted into the target gene by homologous recombination are then selected by growth in the presence of MPA. This technique was applied to the vaccinia virus thymidine kinase gene and compared to the traditional method of constructing gpt-containing plasmids by cloning. The PCR scheme was found to be highly efficient and could theoretically be used to insert any foreign DNA element into any nonessential target gene for which partial or complete sequence information is available. The procedure can potentially be used for a wide variety of genetic modifications, including the insertion of foreign genes, with poxviruses and other DNA viruses. Genomes of microorganisms, such as bacteria and yeast that can be transformed with linear DNA, are also candidates for manipulation by this methodology.

  4. Nucleotide sequence of XhoI O fragment of ectromelia virus DNA reveals significant differences from vaccinia virus.

    PubMed

    Senkevich, T G; Muravnik, G L; Pozdnyakov, S G; Chizhikov, V E; Ryazankina, O I; Shchelkunov, S N; Koonin, E V; Chernos, V I

    1993-10-01

    The nucleotide sequence of the 3913 base pair XhoI O fragment located in an evolutionary variable region adjacent to the right end of the genome of ectromelia virus (EMV) was determined. The sequence contains two long open reading frames coding for putative proteins of 559 amino acid residues (p65) and 344 amino acid residues (p39). Amino acid database searches showed that p39 is closely related to vaccinia virus (VV), strain WR, B22R gene product (C12L gene product of strain Copenhagen), which belongs to the family of serine protease inhibitors (serpins). Despite the overall high conservation, differences were observed in the sequences of p39, B22R, and C12L in the site known to interact with proteases in other serpins, suggesting that the serpins of EMV and two strains of VV may all inhibit proteases with different specificities. The gene coding for the ortholog of p65 is lacking in the Copenhagen strain of vaccinia virus; the WR strain contains a truncated variant of this gene (B21R) potentially coding for a small protein (p16) corresponding to the C-terminal region of p65. p65 is a new member of the family of poxvirus proteins including vaccinia virus proteins A55R, C2L and F3L, and a group of related proteins of leporipoxviruses, Shope fibroma and myxoma viruses (T6, T8, T9, M9). These proteins are homologous to the Drosophila protein Kelch involved in egg development. Both Kelch protein and the related poxvirus proteins contain two distinct domains. The N-terminal domain is related to the similarly located domains of transcription factors Ttk, Br-C (Drosophila), and KUP (human), and GCL protein involved in early development in Drosophila. The C-terminal domain consists of an array of four to five imperfect repeats and is related to human placental protein MIPP. Phylogenetic analysis of the family of poxvirus proteins showed that their genes have undergone a complex succession of duplications, and complete or partial deletions.

  5. Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus

    SciTech Connect

    Morgan, J.R.; Cohen, L.K.; Roberts, B.E.

    1984-10-01

    The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with (alpha-/sup 32/P)GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the /sup 32/P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the /sup 32/P-labeled large subunit and the (/sup 35/S)methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5 and 3 termini were precisely located by S1 nuclease analysis.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

    PubMed

    Burmeister, Wim P; Tarbouriech, Nicolas; Fender, Pascal; Contesto-Richefeu, Céline; Peyrefitte, Christophe N; Iseni, Frédéric

    2015-07-17

    Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action.

  11. Increased attenuation but decreased immunogenicity by deletion of multiple vaccinia virus immunomodulators.

    PubMed

    Sumner, Rebecca P; Ren, Hongwei; Ferguson, Brian J; Smith, Geoffrey L

    2016-09-14

    Vaccinia virus (VACV)-derived vectors are popular candidates for vaccination against diseases such as HIV-1, malaria and tuberculosis. However, their genomes encode a multitude of proteins with immunomodulatory functions, several of which reduce the immunogenicity of these vectors. Hitherto only limited studies have investigated whether the removal of these immunomodulatory genes in combination can increase vaccine efficacy further. To this end we constructed viruses based on VACV strain Western Reserve (WR) lacking up to three intracellular innate immunomodulators (N1, C6 and K7). These genes were selected because the encoded proteins had known functions in innate immunity and the deletion of each gene individually had caused a decrease in virus virulence in the murine intranasal and intradermal models of infection and an increase in immunogenicity. Data presented here demonstrate that deletion of two, or three of these genes in combination attenuated the virus further in an incremental manner. However, when vaccinated mice were challenged with VACV WR the double and triple gene deletion viruses provided weaker protection against challenge. This was accompanied by inferior memory CD8(+) T cell responses and lower neutralising antibody titres. This study indicates that, at least for the three genes studied in the context of VACV WR, the single gene deletion viruses are the best vaccine vectors, and that increased attenuation induced by deletion of additional genes decreased immunogenicity. These data highlight the fine balance and complex relationship between viral attenuation and immunogenicity. Given that the proteins encoded by the genes examined in this study are known to affect specific aspects of innate immunity, the set of viruses constructed here are interesting tools to probe the role of the innate immune response in influencing immune memory and vaccine efficacy. PMID:27544585

  12. Institutional Animal Care and Use Committee Considerations Regarding the Use of Virus-Induced Carcinogenesis and Oncolytic Viral Models.

    PubMed

    Lewis, Stephanie D; Hickman-Davis, Judy M; Bergdall, Valerie K

    2016-01-01

    The use of virus-induced carcinogenesis and oncologic experimental animal models is essential in understanding the mechanisms of cancer development to advance prevention, diagnosis, and treatment methods. The Institutional Animal Care and Use Committee (IACUC) is responsible for both the complex philosophical and practical considerations associated with animal models of cancer. Animal models of cancer carry their own unique issues that require special consideration from the IACUC. Many of the considerations to be discussed apply to cancer models in general; specific issues related to viral carcinogenesis or oncolytic viruses will be specifically discussed as they arise. Responsible animal use integrates good science, humane care, and regulatory compliance. To meet those standards, the IACUC, in conjunction with the research investigator and attending veterinarian, must address a wide range of issues, including animal model selection, cancer model selection, humane end point considerations, experimental considerations, postapproval monitoring, reporting requirements, and animal management and personnel safety considerations.

  13. Combination vascular delivery of herpes simplex oncolytic viruses and amplicon mediated cytokine gene transfer is effective therapy for experimental liver cancer.

    PubMed Central

    Zager, J. S.; Delman, K. A.; Malhotra, S.; Ebright, M. I.; Bennett, J. J.; Kates, T.; Halterman, M.; Federoff, H.; Fong, Y.

    2001-01-01

    BACKGROUND: Herpes simplex type I (HSV)-based vectors have been used experimentally for suicide gene therapy, immunomodulatory gene delivery, and direct oncolytic therapy. The current study utilizes the novel concept of regional delivery of an oncolytic virus in combination with or serving as the helper virus for packaging herpes-based amplicon vectors carrying a cytokine transgene, with the goal of identifying if this combination is more efficacious than either modality alone. MATERIALS AND METHODS: A replication competent oncolytic HSV (G207) and a replication incompetent HSV amplicon carrying the gene for the immunomodulatory cytokine IL-2 (HSV-IL2) were tested in murine syngeneic colorectal carcinoma and in rat hepatocellular carcinoma models. Liver tumors were treated with vascular delivery of (1) phosphate-buffered saline (PBS), (2) G207, (3) HSV-IL2, (4) G207 and HSV-IL2 mixed in combination (mG207/HSV- IL2), and (5) G207 as the helper virus for packaging the construct HSV-IL2 (pG207/HSV-IL2). RESULTS: Tumor burden was significantly reduced in all treatment groups in both rats and mice treated with high-dose G207, HSV-IL2, or both (p < 0.02). When a low dose of virus was used in mice, anti-tumor efficacy was improved by use of G207 and HSV-IL2 in combination or with HSV-IL2 packaged by G207 (p < 0.001). This improvement was abolished when CD4(+) and CD8(+) lymphocytes were depleted, implying that the enhanced anti-tumor response to low-dose combined therapy is immune mediated. CONCLUSIONS: Vascular regional delivery of oncolytic and amplicon HSV vectors can be used to induce improved anti-tumor efficacy by combining oncolytic and immunostimulatory strategies. PMID:11591892

  14. 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.

  15. 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.

  16. Protein B5 is required on extracellular enveloped vaccinia virus for repulsion of superinfecting virions.

    PubMed

    Doceul, Virginie; Hollinshead, Michael; Breiman, Adrien; Laval, Kathlyn; Smith, Geoffrey L

    2012-09-01

    Vaccinia virus (VACV) spreads across cell monolayers fourfold faster than predicted from its replication kinetics. Early after infection, infected cells repulse some superinfecting extracellular enveloped virus (EEV) particles by the formation of actin tails from the cell surface, thereby causing accelerated spread to uninfected cells. This strategy requires the expression of two viral proteins, A33 and A36, on the surface of infected cells and upon contact with EEV this complex induces actin polymerization. Here we have studied this phenomenon further and investigated whether A33 and A36 expression in cell lines causes an increase in VACV plaque size, whether these proteins are able to block superinfection by EEV, and which protein(s) on the EEV surface are required to initiate the formation of actin tails from infected cells. Data presented show that VACV plaque size was not increased by expression of A33 and A36, and these proteins did not block entry of the majority of EEV binding to these cells. In contrast, expression of proteins A56 and K2 inhibited entry of both EEV and intracellular mature virus. Lastly, VACV protein B5 was required on EEV to induce the formation of actin tails at the surface of cells expressing A33 and A36, and B5 short consensus repeat 4 is critical for this induction.

  17. Development and in vitro characterization of recombinant vaccinia viruses expressing bovine leukemia virus gp51 in combination with bovine IL4 or IL12.

    PubMed

    Von Beust, B R; Brown, W C; Estes, D M; Zarlenga, D S; McElwain, T F; Palmer, G H

    1999-01-28

    Type 1 and type 2 immune responses are modulated by IL12 or IL4, respectively, at the time of lymphocyte priming. Importantly, type 1 responses have been associated with resistance to retroviral infection in mice, humans, and ruminants. Specifically, vaccination of sheep with vaccinia virus expressing bovine leukemia virus (BLV) gp51 resulted in protective immunity with the characteristics of a type 1 response, whereas vaccination of cattle resulted in a non-protective type 2 response. In order to test the hypothesis that cattle inoculated with BLV gp51 and IL12 will respond with a type 1 response, a recombinant vaccinia virus expressing BLV gp51 together with bovine IL12 was developed and characterized in vitro. For induction of type 2 responses a recombinant vaccinia virus expressing gp51 with bovine IL4 was similarly constructed and characterized. In this study recombinant cassettes were developed containing either the BLVenv gene alone or in combination with bovine IL4 or the two genes, p35 and p40, encoding bovine IL12. Correct alignment with p7.5 or p11 vaccinia promoters and orientation was confirmed by complete sequencing. Recombinant vaccinia viruses were generated by homologous recombination, selected based on large plaque formation due to reconstitution of the vp37 gene, and structurally confirmed by Southern blotting. Transcription of recombinant BLVenv, bovine IL4, p35 and p40 was demonstrated by RT-PCR. Expression of BLVenv gp51 protein and bovine IL4 was shown by immunofluorescence and immunoblotting. Biologically active bovine IL4 expressed by vaccinia virus stimulated lymphoblast proliferation, B lymphocyte proliferation in the presence of CD40L, and inhibited IFN gamma secretion from PHA activated PBMC in a dose dependent fashion. Finally, bovine IL12 expression and biological function was confirmed by dose dependent induction of IFN gamma secretion by PHA activated PBMC and the moderate enhancement of lymphoblast proliferation. In conclusion

  18. 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

  19. 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.

  20. Nuclear localization of a double-stranded RNA-binding protein encoded by the vaccinia virus E3L gene.

    PubMed

    Yuwen, H; Cox, J H; Yewdell, J W; Bennink, J R; Moss, B

    1993-08-01

    We produced a B cell hybridoma (TW2.3) from vaccinia virus-infected mice that secreted a monoclonal antibody (MAb) reactive with a 25-kDA early viral protein that was localized by laser scanning confocal microscopy to the nucleus and cytoplasmic viral factory regions of infected cells. By cell-free translation of mRNA selected by hybridization to a complete library of vaccinia virus DNA fragments, the immunoreactive polypeptide was mapped to open reading frame E3L. The RNA start site of an early promoter was located 26 nucleotides upstream of the first methionine codon of E3L. Evidence was obtained that translation initiation occurs in vivo and in vitro at both the first and second methionine codons to produce major and minor polypeptides of 25 and 19 kDa, respectively. Both polypeptides bound double-stranded RNA, confirming the recent report of H.-W. Chang, J. C. Watson, and B. L. Jacobs (Proc. Natl. Acad. Sci. USA 89, 4825-4829, 1992). Other vaccinia virus proteins were not required for the nuclear localization of the E3L protein, since MAb TW2.3 bound to the nuclei of uninfected cells that were transfected with the E3L gene under the control of the SV40 early promoter. We also demonstrated that the E3L protein can bind to nuclei of aldehyde fixed and detergent permeabilized uninfected cells. This binding was abrogated by treatment of the cells with RNase but not DNase. The nuclear and cytoplasmic locations of the double-stranded RNA binding protein are consistent with multiple functions in the vaccinia virus infectious cycle.

  1. Imaging of Intratumoral Inflammation during Oncolytic Virotherapy of Tumors by 19F-Magnetic Resonance Imaging (MRI)

    PubMed Central

    Hess, Michael; Hofmann, Elisabeth; Seubert, Carolin; Langbein-Laugwitz, Johanna; Gentschev, Ivaylo; Sturm, Volker Jörg Friedrich; Ye, Yuxiang; Kampf, Thomas; Jakob, Peter Michael; Szalay, Aladar A.

    2013-01-01

    Background Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate 19F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy. Methodology/Principal Findings The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by 1H/19F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the 19F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the 19F signal hot spots and CD68+-macrophages. Thereby, the CD68+-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the 19F signal correlated with the extent of viral spreading within tumors. Conclusions/Significance These results suggest 19F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, 19F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response. PMID:23441176

  2. Extracts from rabbit skin inflamed by the vaccinia virus attenuate bupivacaine-induced spinal neurotoxicity in pregnant rats☆

    PubMed Central

    Cui, Rui; Xu, Shiyuan; Wang, Liang; Lei, Hongyi; Cai, Qingxiang; Zhang, Hongfei; Wang, Dongmei

    2013-01-01

    Extracts from rabbit skin inflamed by the vaccinia virus can relieve pain and promote repair of nerve injury. The present study intraperitoneally injected extracts from rabbit skin inflamed by the vaccinia virus for 3 and 4 days prior to and following intrathecal injection of bupivacaine into pregnant rats. The pain threshold test after bupivacaine injection showed that the maximum possible effect of tail-flick latency peaked 1 day after intrathecal injection of bupivacaine in the extract-pretreatment group, and gradually decreased, while the maximum possible effect in the bupivacaine group continued to increase after intrathecal injection of bupivacaine. Histological observation showed that after 4 days of intrathecal injection of bupivacaine, the number of shrunken, vacuolated, apoptotic and caspase-9-positive cells in the dorsal root ganglion in the extract-pretreatment group was significantly reduced compared with the bupivacaine group. These findings indicate that extracts from rabbit skin inflamed by the vaccinia virus can attenuate neurotoxicity induced by intrathecal injection of bupivacaine in pregnant rats, possibly by inhibiting caspase-9 protein expression and suppressing nerve cell apoptosis. PMID:25206391

  3. [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.

  4. Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence.

    PubMed

    Ferguson, Brian J; Benfield, Camilla T O; Ren, Hongwei; Lee, Vivian H; Frazer, Gordon L; Strnadova, Pavla; Sumner, Rebecca P; Smith, Geoffrey L

    2013-09-01

    Vaccinia virus (VACV) expresses many proteins that are non-essential for virus replication but promote virulence by inhibiting components of the host immune response to infection. These immunomodulators include a family of proteins that have, or are predicted to have, a structure related to the B-cell lymphoma (Bcl)-2 protein. Five members of the VACV Bcl-2 family (N1, B14, A52, F1 and K7) have had their crystal structure solved, others have been characterized and a function assigned (C6, A46), and others are predicted to be Bcl-2 proteins but are uncharacterized hitherto (N2, B22, C1). Data presented here show that N2 is a nuclear protein that is expressed early during infection and inhibits the activation of interferon regulatory factor (IRF)3. Consistent with its nuclear localization, N2 inhibits IRF3 downstream of the TANK-binding kinase (TBK)-1 and after IRF3 translocation into the nucleus. A mutant VACV strain Western Reserve lacking the N2L gene (vΔN2) showed normal replication and spread in cultured cells compared to wild-type parental (vN2) and revertant (vN2-rev) viruses, but was attenuated in two murine models of infection. After intranasal infection, the vΔN2 mutant induced lower weight loss and signs of illness, and virus was cleared more rapidly from the infected tissue. In the intradermal model of infection, vΔN2 induced smaller lesions that were resolved more rapidly. In summary, the N2 protein is an intracellular virulence factor that inhibits IRF3 activity in the nucleus.

  5. Science to Practice: Monitoring Oncolytic Virus Therapy with Chemical Exchange Saturation Transfer MR Imaging--Wishful Thinking?

    PubMed

    Choyke, Peter L

    2015-06-01

    Farrar et al demonstrate that modifying an oncolytic virus (OV) so that it produces excess protein when it infects a cancer cell is a process that can be detected both in vitro and in vivo in infected cancer cells by using chemical exchange saturation transfer (CEST) magnetic resonance (MR) imaging. The effect is at the limits of MR imaging detection (approximately 1%), but experience with functional MR imaging of the brain, with comparably small effects, should give pause to anyone who immediately writes this observation off as an exercise in wishful thinking. OVs are improving in their specificity, virulence, and ability to induce immune responses. Now, they have been modified to express proteins that are detectable with CEST MR imaging early after delivery into a tumor. This is clearly a surprising and hopeful development in the long road of OVs from the laboratory to the clinic.

  6. 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

  7. Treatment of medulloblastoma using an oncolytic measles virus encoding the thyroidal sodium iodide symporter shows enhanced efficacy with radioiodine

    PubMed Central

    2012-01-01

    Background Medulloblastoma is the most common malignant brain tumor of childhood. Although the clinical outcome for medulloblastoma patients has improved significantly, children afflicted with the disease frequently suffer from debilitating side effects related to the aggressive nature of currently available therapy. Alternative means for treating medulloblastoma are desperately needed. We have previously shown that oncolytic measles virus (MV) can selectively target and destroy medulloblastoma tumor cells in localized and disseminated models of the disease. MV-NIS, an oncolytic measles virus that encodes the human thyroidal sodium iodide symporter (NIS), has the potential to deliver targeted radiotherapy to the tumor site and promote a localized bystander effect above and beyond that achieved by MV alone. Methods We evaluated the efficacy of MV-NIS against medulloblastoma cells in vitro and examined their ability to incorporate radioiodine at various timepoints, finding peak uptake at 48 hours post infection. The effects of MV-NIS were also evaluated in mouse xenograft models of localized and disseminated medulloblastoma. Athymic nude mice were injected with D283med-Luc medulloblastoma cells in the caudate putamen (localized disease) or right lateral ventricle (disseminated disease) and subsequently treated with MV-NIS. Subsets of these mice were given a dose of 131I at 24, 48 or 72 hours later. Results MV-NIS treatment, both by itself and in combination with 131I, elicited tumor stabilization and regression in the treated mice and significantly extended their survival times. Mice given 131I were found to concentrate radioiodine at the site of their tumor implantations. In addition, mice with localized tumors that were given 131I either 24 or 48 hours after MV-NIS treatment exhibited a significant survival advantage over mice given MV-NIS alone. Conclusions These data suggest MV-NIS plus radioiodine may be a potentially useful therapy for the treatment of

  8. 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

  9. A vesicular stomatitis virus glycoprotein epitope-incorporated oncolytic adenovirus overcomes CAR-dependency and shows markedly enhanced cancer cell killing and suppression of tumor growth

    PubMed Central

    Yoon, A-Rum; Hong, Jinwoo; Yun, Chae-Ok

    2015-01-01

    Utility of traditional oncolytic adenovirus (Ad) has been limited due to low expression of coxsackie and adenovirus receptor (CAR) in cancer cells which results in poor infectivity of Ads. Here with an aim of improving the efficiency of Ad's entry to the cell, we generated a novel tropism-expanded oncolytic Ad which contains the epitope of vesicular stomatitis virus glycoprotein (VSVG) at the HI-loop of Ad fiber. We generated 9 variants of oncolytic Ads with varying linkers and partial deletion to the fiber. Only one VSVG epitope-incorporated variant, RdB-1L-VSVG, which contains 1 linker and no deletion to fiber, was produced efficiently. Production of 3-dimensionaly stable fiber in RdB-1L-VSVG was confirmed by immunoblot analysis. RdB-1L-VSVG shows a remarkable improvement in cytotoxicity and total viral yield in cancer cells. RdB-1L-VSVG demonstrates enhanced cytotoxicity in cancer cells with subdued CAR-expression as it can be internalized by an alternate pathway. Competition assays with a CAR-specific antibody (Ab) or VSVG receptor, phosphatidyl serine (PS), reveals that cell internalization of RdB-1L-VSVG is mediated by both CAR and PS. Furthermore, treatment with RdB-1L-VSVG significantly enhanced anti-tumor effect in vivo. These studies demonstrate that the strategy to expand oncolytic Ad tropism may significantly improve therapeutic profile for cancer treatment. PMID:26430798

  10. Chemical inactivation of recombinant vaccinia viruses and the effects on antigenicity and immunogenicity of recombinant simian immunodeficiency virus envelope glycoproteins.

    PubMed

    Hulskotte, E G; Dings, M E; Norley, S G; Osterhaus, A D

    1997-12-01

    The efficiency of paraformaldehyde (PFA) and binary ethylenimine (BEI) in inactivating recombinant vaccinia virus (rVV), present in baby hamster kidney cells expressing simian immunodeficiency virus envelope glycoproteins (SIV-Env), was measured in a series of inactivation studies. Both compounds were shown to be effective in reducing rVV titres. The use of standard 3-day titration assays proved to be inadequate to measure PFA inactivation, since upon prolonged incubation, residual rVV infectivity was detected in cultures negative at 3 days. Different procedures using PFA or BEI were selected to assess their influence on the antigenicity and immunogenicity or rVV expressed SIV-Env. Antigenicity, as defined by the ability to react with a panel of monoclonal antibodies recognizing major antigenic sites, and immunogenicity, as defined by the ability to induce SIV envelope specific and virus neutralizing serum antibodies in rats, proved to be preserved after either inactivation procedure. These data show that both protocols using PFA or BEI can be used successfully as part of the procedures to remove residual rVV infectivity.

  11. Azathioprine inhibits vaccinia virus replication in both BSC-40 and RAG cell lines acting on different stages of virus cycle.

    PubMed

    Damaso, Clarissa R A; Oliveira, Marcus F; Massarani, Susana M; Moussatché, Nissin

    2002-08-15

    In the present study we demonstrate that azathioprine (AZA) inhibits vaccinia virus (VV) replication in both BSC-40 and RAG cell lines, acting on different stages of virus cycle. In BSC-40 cells, early protein synthesis was not significantly affected, but late gene expression was severely impaired. In RAG cells all stages of gene expression were completed during synchronous infection in the presence of the drug. The onset of DNA replication was not affected in RAG cells, but a severe inhibition was observed in BSC-40 cells. Electron microscopic analysis of VV-infected RAG cells treated with AZA revealed brick-shaped particles presenting abnormal definition of the internal structure. Purified virions from AZA-treated RAG cells presented several modifications of the protein content, a lesser amount of DNA, and a lower PFU:particle ratio. Our results suggest that in VV-infected RAG cells AZA interfered with virus morphogenesis, whereas in BSC-40 cells the replicative cycle was inhibited at the DNA replication stage.

  12. Increased antibody responses to human papillomavirus type 16 L1 protein expressed by recombinant vaccinia virus lacking serine protease inhibitor genes.

    PubMed

    Zhou, J; Crawford, L; McLean, L; Sun, X Y; Stanley, M; Almond, N; Smith, G L

    1990-09-01

    The L1 gene of human papillomavirus type 16 (HPV-16) driven by the vaccinia virus major late 4b gene promoter has been inserted into three different sites of the vaccinia virus genome. Insertion into the thymidine kinase (TK) gene was achieved by selection of TK- mutants in BUdR on TK- cells. Insertion into two vaccinia virus serine protease inhibitor (serpin) genes was achieved by co-insertion of the Escherichia coli xanthine guanine phosphoribosyltransferase gene linked to the vaccinia virus 7.5K promoter and selection of mycophenolic acid-resistant recombinant viruses. Each recombinant virus expressed a 57K L1 protein at similar levels and with similar kinetics. However, immunization of mice with these recombinant viruses induced different levels of antibody to the L1 protein. Viruses lacking serpin genes B13R and B24R induced significantly higher antibody levels than did viruses lacking the TK gene. The presence of functional B13R and B24R gene products is therefore somehow immunosuppressive at least for antibody responses to the L1 protein of HPV-16.

  13. Vaccinia virus inhibits NF-κB-dependent gene expression downstream of p65 translocation.

    PubMed

    Sumner, Rebecca P; Maluquer de Motes, Carlos; Veyer, David L; Smith, Geoffrey L

    2014-03-01

    The transcription factor nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) plays a critical role in host defense against viral infection by inducing the production of proinflammatory mediators and type I interferon. Consequently, viruses have evolved many mechanisms to block its activation. The poxvirus vaccinia virus (VACV) encodes numerous inhibitors of NF-κB activation that target multiple points in the signaling pathway. A derivative of VACV strain Copenhagen, called vv811, lacking 55 open reading frames in the left and right terminal regions of the genome was reported to still inhibit NF-κB activation downstream of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), suggesting the presence of one or more additional inhibitors. In this study, we constructed a recombinant vv811 lacking the recently described NF-κB inhibitor A49 (vv811ΔA49), yielding a virus that lacked all currently described inhibitors downstream of TNF-α and IL-1β. Unlike vv811, vv811ΔA49 no longer inhibited degradation of the phosphorylated inhibitor of κBα and p65 translocated into the nucleus. However, despite this translocation, vv811ΔA49 still inhibited TNF-α- and IL-1β-induced NF-κB-dependent reporter gene expression and the transcription and production of cytokines induced by these agonists. This inhibition did not require late viral gene expression. These findings indicate the presence of another inhibitor of NF-κB that is expressed early during infection and acts by a novel mechanism downstream of p65 translocation into the nucleus.

  14. A loss of function analysis of host factors influencing Vaccinia virus replication by RNA interference.

    PubMed

    Beard, Philippa M; Griffiths, Samantha J; 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.

  15. 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.

  16. Role of the vaccinia virus O3 protein in cell entry can be fulfilled by its Sequence flexible transmembrane domain

    SciTech Connect

    Satheshkumar, P.S.; Chavre, James; Moss, Bernard

    2013-09-15

    The vaccinia virus O3 protein, a component of the entry–fusion complex, is encoded by all chordopoxviruses. We constructed truncation mutants and demonstrated that the transmembrane domain, which comprises two-thirds of this 35 amino acid protein, is necessary and sufficient for interaction with the entry–fusion complex and function in cell entry. Nevertheless, neither single amino acid substitutions nor alanine scanning mutagenesis revealed essential amino acids within the transmembrane domain. Moreover, replication-competent mutant viruses were generated by randomization of 10 amino acids of the transmembrane domain. Of eight unique viruses, two contained only two amino acids in common with wild type and the remainder contained one or none within the randomized sequence. Although these mutant viruses formed normal size plaques, the entry–fusion complex did not co-purify with the mutant O3 proteins suggesting a less stable interaction. Thus, despite low specific sequence requirements, the transmembrane domain is sufficient for function in entry. - Highlights: • The 35 amino acid O3 protein is required for efficient vaccinia virus entry. • The transmembrane domain of O3 is necessary and sufficient for entry. • Mutagenesis demonstrated extreme sequence flexibility compatible with function.

  17. High cytokine production and effective antitumor activity of a recombinant vaccinia virus encoding murine interleukin 12.

    PubMed

    Meko, J B; Yim, J H; Tsung, K; Norton, J A

    1995-11-01

    We have constructed a recombinant vaccinia virus (recVV), vKT0334 mIL-12, containing the genes encoding the p35 and p40 subunits of murine interleukin-12 (mIL-12). In vitro experiments demonstrated that vKT0334 mIL-12 efficiently infected a variety of murine and human tumor cell lines and produced very high amounts (1.5 micrograms/10(6) cells/24 h) of biologically active mIL-12. Mice injected s.c. with 10(6) MCA 105 sarcoma cells, followed by injection at the same site with saline or a control recVV, vKT033, containing no mIL-12 genes, all developed progressively growing tumor, whereas 60% of animals injected with vKT0334 mIL-12 remained tumor free (P < 0.0005). Furthermore, tumor growth was significantly reduced in the remaining mice treated with vKT0334 mIL-12 that did develop tumor compared with mice treated with vKT033 (P < 0.03) or saline (P < 0.0001). We conclude that recVV expressing high levels of mIL-12 offers an effective in vivo method of cytokine gene delivery and expression in tumors with subsequent antitumor effect.

  18. Validation of an immunoperoxidase monolayer assay for total anti-Vaccinia virus antibody titration.

    PubMed

    Gerber, Priscilla F; Matos, Ana Carolina D; Guedes, Maria Isabel M C; Madureira, Marieta C; Silva, Marcos X; Lobato, Zélia I P

    2012-03-01

    Vaccinia virus (VACV) has been associated with zoonotic exanthemic outbreaks affecting bovids and human beings, with significant public health and economic impacts. Rapid and reliable diagnostic methods are needed to detect and epidemiologically monitor antibodies to VACV. The current study describes the development of an immunoperoxidase monolayer assay (IPMA) for detection of total VACV antibodies in bovine serum. The assay was validated by comparison with a plaque reduction neutralization test (PRNT). Kappa index of agreement, diagnostic sensitivity, specificity, and accuracy of the IPMA were -1.008, 100%, 96%, and 98%, respectively, when compared with PRNT on 148 field bovine sera. Repeatability tests on 32 field-positive serum samples revealed that intraclass coefficient correlation was 0.86. In experimentally infected cattle, VACV antibodies were detectable by IPMA 4 days postinfection, which was more than 2 weeks earlier than with the PRNT, indicating that IPMA could be a more sensitive test than the latter. In 4 naturally VACV-diseased cows monitored for 13 months, IPMA could detect VACV antibodies up to 13 months, a longer time than PRNT. The IPMA is simpler to produce and perform when compared with PRNT and is time saving and suitable for large-scale surveys of VACV infection in bovine.

  19. Modified vaccinia virus Ankara as a vector for suicide gene therapy.

    PubMed

    Erbs, P; Findeli, A; Kintz, J; Cordier, P; Hoffmann, C; Geist, M; Balloul, J-M

    2008-01-01

    Modified vaccinia virus Ankara (MVA) has been used successfully to express various antigens for the development of vaccines. Here we show that MVA can also be used as an efficient vector for the transfer of suicide genes to cancer cells. We have generated a new and highly potent suicide gene, FCU1, which encodes a fusion protein derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. We now describe the therapeutic benefit of using MVA to deliver and express the FCU1 gene in cancer cells. MVA-mediated transfer of the FCU1 gene to various human tumor cells results in the production of a bifunctional intracellular enzyme, such that exposure to the prodrug 5-FC suppresses the growth of the tumor cells both in vitro and in vivo. Moreover, we report a more potent tumor growth delay at lower doses of 5-FC using MVA-FCU1 in comparison to adenovirus encoding FCU1. Prolonged therapeutic levels of cytotoxic 5-FU were detected in tumors in mice treated with both MVA-FCU1 and 5-FC while no detectable 5-FU was found in the circulation. This original combination between MVA and FCU1 represents a potentially safe and attractive therapeutic option to test in man. PMID:17992203

  20. Delivery of Echinococcus granulosus antigen EG95 to mice and sheep using recombinant vaccinia virus.

    PubMed

    Dutton, S; Fleming, S B; Ueda, N; Heath, D D; Hibma, M H; Mercer, A A

    2012-06-01

    The tapeworm Echinococcus granulosus is the causative agent of hydatid disease and affects sheep, cattle, dogs and humans worldwide. It has a two-stage life cycle existing as worms in the gut of infected dogs (definitive host) and as cysts in herbivores and humans (intermediate host). The disease is debilitating and can be life threatening where the cysts interfere with organ function. Interruption of the hydatid life cycle in the intermediate host by vaccination may be a way to control the disease, and a protective oncosphere antigen EG95 has been shown to protect animals against challenge with E. granulosus eggs. We explored the use of recombinant vaccinia virus as a delivery vehicle for EG95. Mice and sheep were immunized with the recombinant vector, and the result monitored at the circulating antibody level. In addition, sera from immunized mice were assayed for the ability to kill E. granulosus oncospheres in vitro. Mice immunized once intranasally developed effective oncosphere-killing antibody by day 42 post-infection. Antibody responses and oncosphere killing were correlated and were significantly enhanced by boosting mice with either EG95 protein or recombinant vector. Sheep antibody responses to the recombinant vector or to EG95 protein mirrored those in mice.

  1. Study of Vaccinia and Cowpox viruses' replication in Rac1-N17 dominant-negative cells

    PubMed Central

    Salgado, Ana Paula Carneiro; Soares-Martins, Jamária Adriana Pinheiro; Andrade, Luciana Garcia; Albarnaz, Jonas Dutra; Ferreira, Paulo César Peregrino; Kroon, Erna Geessien; Bonjardim, Cláudio Antônio

    2013-01-01

    Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV) and Cowpox (CPXV) and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN) Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication. PMID:23903969

  2. Structural analysis of point mutations at the Vaccinia virus A20/D4 interface.

    PubMed

    Contesto-Richefeu, Céline; Tarbouriech, Nicolas; Brazzolotto, Xavier; Burmeister, Wim P; Peyrefitte, Christophe N; Iseni, Frédéric

    2016-09-01

    The Vaccinia virus polymerase holoenzyme is composed of three subunits: E9, the catalytic DNA polymerase subunit; D4, a uracil-DNA glycosylase; and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase cofactor, the function of which is essential for processive DNA synthesis. The recent crystal structure of D4 bound to the first 50 amino acids of A20 (D4/A201-50) revealed the importance of three residues, forming a cation-π interaction at the dimerization interface, for complex formation. These are Arg167 and Pro173 of D4 and Trp43 of A20. Here, the crystal structures of the three mutants D4-R167A/A201-50, D4-P173G/A201-50 and D4/A201-50-W43A are presented. The D4/A20 interface of the three structures has been analysed for atomic solvation parameters and cation-π interactions. This study confirms previous biochemical data and also points out the importance for stability of the restrained conformational space of Pro173. Moreover, these new structures will be useful for the design and rational improvement of known molecules targeting the D4/A20 interface. PMID:27599859

  3. Attenuation and immunogenicity of host-range extended modified vaccinia virus Ankara recombinants.

    PubMed

    Melamed, Sharon; Wyatt, Linda S; Kastenmayer, Robin J; Moss, Bernard

    2013-09-23

    Modified vaccinia virus Ankara (MVA) is being widely investigated as a safe smallpox vaccine and as an expression vector to produce vaccines against other infectious diseases and cancer. MVA was isolated following more than 500 passages in chick embryo fibroblasts and suffered several major deletions and numerous small mutations resulting in replication defects in human and most other mammalian cells as well as severe attenuation of pathogenicity. Due to the host range restriction, primary chick embryo fibroblasts are routinely used for production of MVA-based vaccines. While a replication defect undoubtedly contributes to safety of MVA, it is worth considering whether host range and attenuation are partially separable properties. Marker rescue transfection experiments resulted in the creation of recombinant MVAs with extended mammalian cell host range. Here, we characterize two host-range extended rMVAs and show that they (i) have acquired the ability to stably replicate in Vero cells, which are frequently used as a cell substrate for vaccine manufacture, (ii) are severely attenuated in immunocompetent and immunodeficient mouse strains following intranasal infection, (iii) are more pathogenic than MVA but less pathogenic than the ACAM2000 vaccine strain at high intracranial doses, (iv) do not form lesions upon tail scratch in mice in contrast to ACAM2000 and (v) induce protective humoral and cell-mediated immune responses similar to MVA. The extended host range of rMVAs may be useful for vaccine production.

  4. Products and substrate/template usage of vaccinia virus DNA primase

    SciTech Connect

    De Silva, Frank S.; Paran, Nir; Moss, Bernard

    2009-01-05

    Vaccinia virus encodes a 90-kDa protein conserved in all poxviruses, with DNA primase and nucleoside triphosphatase activities. DNA primase products, synthesized with a single stranded {phi}X174 DNA template, were resolved as dinucleotides and long RNAs on denaturing polyacrylamide and agarose gels. Following phosphatase treatment, the dinucleotides GpC and ApC in a 4:1 ratio were identified by nearest neighbor analysis in which {sup 32}P was transferred from [{alpha}-{sup 32}P]CTP to initiating purine nucleotides. Differences in the nucleotide binding sites for initiation and elongation were suggested by the absence of CpC and UpC dinucleotides as well as the inability of deoxynucleotides to mediate primer synthesis despite their incorporation into mixed RNA/DNA primers. Strong primase activity was detected with an oligo(dC) template. However, there was only weak activity with an oligo(dT) template and none with oligo(dA) or oligo(dG). The absence of stringent template specificity is consistent with a role for the enzyme in priming DNA synthesis at the replication fork.

  5. A tandemly-oriented late gene cluster within the vaccinia virus genome.

    PubMed

    Weinrich, S L; Hruby, D E

    1986-04-11

    The nucleotide sequence of a 5.1 kilobase-pair fragment from the central portion of the vaccinia virus genome has been determined. Within this region, five complete and two incomplete open reading frames (orfs) are tightly-clustered, tandemly-oriented, and read in the leftward direction. Late mRNA start sites for the five complete orfs and one incomplete orf were determined by S1 nuclease mapping. The two leftmost complete orfs correlated with late polypeptides of 65,000 and 32,000 molecular weight previously mapped to this region. When compared with each other and with sequences present in protein data banks, the five complete orfs showed no significant homology matches amongst themselves or any previously reported sequence. The six putative promoters were aligned with three previously sequenced late gene promoters. While all of the nine are A-T rich, the only apparent consensus sequence is TAA immediately preceeding the initiator ATG. Identification of this tandemly-oriented late gene cluster suggests local organization of the viral genome.

  6. Oncolytic virotherapy reaches adolescence.

    PubMed

    Hammill, Adrienne M; Conner, Joseph; Cripe, Timothy P

    2010-12-15

    Lytic viruses kill cells as a consequence of their normal replication life cycle. The idea of harnessing viruses to kill cancer cells arose over a century ago, before viruses were even discovered, from medical case reports of infections associated with cancer remissions. Since then, there has been no shortage of hype, hope, or fear regarding the prospect of oncolytic virotherapy for cancer. Early developments in the field included encouraging antitumor efficacy both in animal studies in the 1920s-1940s and in human clinical trials in the 1950s-1970s. Despite its long-standing history, oncolytic virotherapy was an idea ahead of its time. Without needed advances in molecular biology, virology, immunology, and clinical research ethics, early clinical trials resulted in infectious complications and were fraught with controversial research conduct, so that enthusiasm in the medical community waned. Oncolytic virotherapy is now experiencing a major growth spurt, having sustained numerous laboratory advances and undergone multiple encouraging adult clinical trials, and is now witnessing the emergence of pediatric trials. Here we review the history and salient biology of the field, including preclinical and clinical data, with a special emphasis on those agents now being tested in pediatric cancer patients. PMID:20734404

  7. Oncolytic Virotherapy of Canine and Feline Cancer

    PubMed Central

    Gentschev, Ivaylo; Patil, Sandeep S.; Petrov, Ivan; Cappello, Joseph; Adelfinger, Marion; Szalay, Aladar A.

    2014-01-01

    Cancer is the leading cause of disease-related death in companion animals such as dogs and cats. Despite recent progress in the diagnosis and treatment of advanced canine and feline cancer, overall patient treatment outcome has not been substantially improved. Virotherapy using oncolytic viruses is one promising new strategy for cancer therapy. Oncolytic viruses (OVs) preferentially infect and lyse cancer cells, without causing excessive damage to surrounding healthy tissue, and initiate tumor-specific immunity. The current review describes the use of different oncolytic viruses for cancer therapy and their application to canine and feline cancer. PMID:24841386

  8. Major neutralizing sites on vaccinia virus glycoprotein B5 are exposed differently on variola virus ortholog B6.

    PubMed

    Aldaz-Carroll, Lydia; Xiao, Yuhong; Whitbeck, J Charles; de Leon, Manuel Ponce; Lou, Huan; Kim, Mikyung; Yu, Jessica; Reinherz, Ellis L; Isaacs, Stuart N; Eisenberg, Roselyn J; Cohen, Gary H

    2007-08-01

    Immunization against smallpox (variola virus) with Dryvax, a live vaccinia virus (VV), was effective, but now safety is a major concern. To overcome this issue, subunit vaccines composed of VV envelope proteins from both forms of infectious virions, including the extracellular enveloped virion (EV) protein B5, are being developed. However, since B5 has 23 amino acid differences compared with its B6 variola virus homologue, B6 might be a better choice for such a strategy. Therefore, we compared the properties of both proteins using a panel of monoclonal antibodies (MAbs) to B5 that we had previously characterized and grouped according to structural and functional properties. The B6 gene was obtained from the Centers for Disease Control and Prevention, and the ectodomain was cloned and expressed in baculovirus as previously done with B5, allowing us to compare the antigenic properties of the proteins. Polyclonal antibodies to B5 or B6 cross-reacted with the heterologous protein, and 16 of 26 anti-B5 MAbs cross-reacted with B6. Importantly, 10 anti-B5 MAbs did not cross-react with B6. Of these, three have important anti-VV biologic properties, including their ability to neutralize EV infectivity and block comet formation. Here, we found that one of these three MAbs protected mice from a lethal VV challenge by passive immunization. Thus, epitopes that are present on B5 but not on B6 would generate an antibody response that would not recognize B6. Assuming that B6 contains similar variola virus-specific epitopes, our data suggest that a subunit vaccine using the variola virus homologues might exhibit improved protective efficacy against smallpox.

  9. Vaccinia Virus Morphogenesis: A13 Phosphoprotein Is Required for Assembly of Mature Virions

    PubMed Central

    Unger, Bethany; Traktman, Paula

    2004-01-01

    The 70-amino-acid A13L protein is a component of the vaccinia virus membrane. We demonstrate here that the protein is expressed at late times of infection, undergoes phosphorylation at a serine residue(s), and becomes encapsidated in a monomeric form. Phosphorylation is dependent on Ser40, which lies within the proline-rich motif SPPP. Because phosphorylation of the A13 protein is only minimally affected by disruption of the viral F10 kinase or H1 phosphatase, a cellular kinase is likely to be involved. We generated an inducible recombinant in which A13 protein expression is dependent upon the inclusion of tetracycline in the culture medium. Repression of the A13L protein spares the biochemical progression of the viral life cycle but arrests virion morphogenesis. Virion assembly progresses through the formation of immature virions (IVs); however, these virions do not acquire nucleoids, and DNA crystalloids accumulate in the cytoplasm. Further development into intracellular mature virions is blocked, causing a 1,000-fold decrease in the infectious virus yield relative to that obtained in the presence of the inducer. We also determined that the temperature-sensitive phenotype of the viral mutant Cts40 is due to a nucleotide transition within the A13L gene that causes a Thr48→Ile substitution. This substitution disrupts the function of the A13 protein but does not cause thermolability of the protein; at the nonpermissive temperature, virion morphogenesis arrests at the stage of IV formation. The A13L protein, therefore, is part of a newly recognized group of membrane proteins that are dispensable for the early biogenesis of the virion membrane but are essential for virion maturation. PMID:15280497

  10. Vaccinia virus DNA replication occurs in endoplasmic reticulum-enclosed cytoplasmic mini-nuclei.

    PubMed

    Tolonen, N; Doglio, L; Schleich, S; Krijnse Locker, J

    2001-07-01

    Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ~45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [(3)H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.

  11. Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens.

    PubMed

    Volz, Asisa; Lim, Stephanie; Kaserer, Martina; Lülf, Anna; Marr, Lisa; Jany, Sylvia; Deeg, Cornelia A; Pijlman, Gorben P; Koraka, Penelope; Osterhaus, Albert D M E; Martina, Byron E; Sutter, Gerd

    2016-04-01

    West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA-WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA-WNV vaccines in other preclinical models and use them as candidate vaccine in humans. PMID:26939903

  12. Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens.

    PubMed

    Volz, Asisa; Lim, Stephanie; Kaserer, Martina; Lülf, Anna; Marr, Lisa; Jany, Sylvia; Deeg, Cornelia A; Pijlman, Gorben P; Koraka, Penelope; Osterhaus, Albert D M E; Martina, Byron E; Sutter, Gerd

    2016-04-01

    West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA-WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA-WNV vaccines in other preclinical models and use them as candidate vaccine in humans.

  13. Secondary and tertiary transfer of vaccinia virus among U.S. military personnel--United States and worldwide, 2002-2004.

    PubMed

    2004-02-13

    In December 2002, the Department of Defense (DoD) began vaccinating military personnel as part of the pre-event vaccination program. Because vaccinia virus is present on the skin at the site of vaccination, it can spread to other parts of the body (i.e., autoinoculation) or to contacts of vaccinees (i.e., contact transfer). To prevent autoinoculation and contact transfer, DoD gave vaccinees printed information that focused on hand washing, covering the vaccination site, and limiting contact with infants (1,2). This report describes cases of contact transfer of vaccinia virus among vaccinated military personnel since December 2002; findings indicate that contact transfer of vaccinia virus is rare. Continued efforts are needed to educate vaccinees about the importance of proper vaccination-site care in preventing contact transmission, especially in household settings.

  14. Bioluminescent imaging of vaccinia virus infection in immunocompetent and immunodeficient rats as a model for human smallpox

    PubMed Central

    Liu, Qiang; Fan, Changfa; Zhou, Shuya; Guo, Yanan; Zuo, Qin; Ma, Jian; Liu, Susu; Wu, Xi; Peng, Zexu; Fan, Tao; Guo, Chaoshe; Shen, Yuelei; Huang, Weijin; Li, Baowen; He, Zhengming; Wang, Youchun

    2015-01-01

    Due to the increasing concern of using smallpox virus as biological weapons for terrorist attack, there is renewed interest in studying the pathogenesis of human smallpox and development of new therapies. Animal models are highly demanded for efficacy and safety examination of new vaccines and therapeutic drugs. Here, we demonstrated that both wild type and immunodeficient rats infected with an engineered vaccinia virus carrying Firefly luciferase reporter gene (rTV-Fluc) could recapitulate infectious and clinical features of human smallpox. Vaccinia viral infection in wild type Sprague-Dawley (SD) rats displayed a diffusible pattern in various organs, including liver, head and limbs. The intensity of bioluminescence generated from rTV-Fluc correlated well with viral loads in tissues. Moreover, neutralizing antibodies had a protective effect against virus reinfection. The recombination activating gene 2 (Rag2) knockout rats generated by transcription activator-like effector nucleases (TALENs) technology were further used to examine the infectivity of the rTV-Fluc in immunodeficient populations. Here we demonstrated that Rag2-/- rats were more susceptible to rTV-Fluc than SD rats with a slower virus clearance rate. Therefore, the rTV-Fluc/SD rats and rTV-Fluc/Rag2-/- rats are suitable visualization models, which recapitulate wild type or immunodeficient populations respectively, for testing human smallpox vaccine and antiviral drugs. PMID:26235050

  15. Bioluminescent imaging of vaccinia virus infection in immunocompetent and immunodeficient rats as a model for human smallpox.

    PubMed

    Liu, Qiang; Fan, Changfa; Zhou, Shuya; Guo, Yanan; Zuo, Qin; Ma, Jian; Liu, Susu; Wu, Xi; Peng, Zexu; Fan, Tao; Guo, Chaoshe; Shen, Yuelei; Huang, Weijin; Li, Baowen; He, Zhengming; Wang, Youchun

    2015-01-01

    Due to the increasing concern of using smallpox virus as biological weapons for terrorist attack, there is renewed interest in studying the pathogenesis of human smallpox and development of new therapies. Animal models are highly demanded for efficacy and safety examination of new vaccines and therapeutic drugs. Here, we demonstrated that both wild type and immunodeficient rats infected with an engineered vaccinia virus carrying Firefly luciferase reporter gene (rTV-Fluc) could recapitulate infectious and clinical features of human smallpox. Vaccinia viral infection in wild type Sprague-Dawley (SD) rats displayed a diffusible pattern in various organs, including liver, head and limbs. The intensity of bioluminescence generated from rTV-Fluc correlated well with viral loads in tissues. Moreover, neutralizing antibodies had a protective effect against virus reinfection. The recombination activating gene 2 (Rag2) knockout rats generated by transcription activator-like effector nucleases (TALENs) technology were further used to examine the infectivity of the rTV-Fluc in immunodeficient populations. Here we demonstrated that Rag2-/- rats were more susceptible to rTV-Fluc than SD rats with a slower virus clearance rate. Therefore, the rTV-Fluc/SD rats and rTV-Fluc/Rag2-/- rats are suitable visualization models, which recapitulate wild type or immunodeficient populations respectively, for testing human smallpox vaccine and antiviral drugs. PMID:26235050

  16. Protective Effect of Surfactant Protein D in Pulmonary Vaccinia Virus Infection: Implication of A27 Viral Protein

    PubMed Central

    Julien, Perino; Thielens, Nicole M.; Crouch, Erika; Spehner, Danièle; Crance, Jean-Marc; Favier, Anne-Laure

    2013-01-01

    Vaccinia virus (VACV) was used as a surrogate of variola virus (VARV) (genus Orthopoxvirus), the causative agent of smallpox, to study Orthopoxvirus infection. VARV is principally transmitted between humans by aerosol droplets. Once inhaled, VARV first infects the respiratory tract where it could encounter surfactant components, such as soluble pattern recognition receptors. Surfactant protein D (SP-D), constitutively present in the lining fluids of the respiratory tract, plays important roles in innate host defense against virus infection. We investigated the role of SP-D in VACV infection and studied the A27 viral protein involvement in the interaction with SP-D. Interaction between SP-D and VACV caused viral inhibition in a lung cell model. Interaction of SP-D with VACV was mediated by the A27 viral protein. Binding required Ca2+ and interactions were blocked in the presence of excess of SP-D saccharide ligands. A27, which lacks glycosylation, directly interacted with SP-D. The interaction between SP-D and the viral particle was also observed using electron microscopy. Infection of mice lacking SP-D (SP-D-/-) resulted in increased mortality compared to SP-D+/+ mice. Altogether, our data show that SP-D participates in host defense against the vaccinia virus infection and that the interaction occurs with the viral surface protein A27. PMID:23518578

  17. Protective effect of surfactant protein d in pulmonary vaccinia virus infection: implication of A27 viral protein.

    PubMed

    Perino, Julien; Thielens, Nicole M; Crouch, Erika; Spehner, Danièle; Crance, Jean-Marc; Favier, Anne-Laure

    2013-03-21

    Vaccinia virus (VACV) was used as a surrogate of variola virus (VARV) (genus Orthopoxvirus), the causative agent of smallpox, to study Orthopoxvirus infection. VARV is principally transmitted between humans by aerosol droplets. Once inhaled, VARV first infects the respiratory tract where it could encounter surfactant components, such as soluble pattern recognition receptors. Surfactant protein D (SP-D), constitutively present in the lining fluids of the respiratory tract, plays important roles in innate host defense against virus infection. We investigated the role of SP-D in VACV infection and studied the A27 viral protein involvement in the interaction with SP-D. Interaction between SP-D and VACV caused viral inhibition in a lung cell model. Interaction of SP-D with VACV was mediated by the A27 viral protein. Binding required Ca2+ and interactions were blocked in the presence of excess of SP-D saccharide ligands. A27, which lacks glycosylation, directly interacted with SP-D. The interaction between SP-D and the viral particle was also observed using electron microscopy. Infection of mice lacking SP-D (SP-D-/-) resulted in increased mortality compared to SP-D+/+ mice. Altogether, our data show that SP-D participates in host defense against the vaccinia virus infection and that the interaction occurs with the viral surface protein A27.

  18. Bioluminescent imaging of vaccinia virus infection in immunocompetent and immunodeficient rats as a model for human smallpox.

    PubMed

    Liu, Qiang; Fan, Changfa; Zhou, Shuya; Guo, Yanan; Zuo, Qin; Ma, Jian; Liu, Susu; Wu, Xi; Peng, Zexu; Fan, Tao; Guo, Chaoshe; Shen, Yuelei; Huang, Weijin; Li, Baowen; He, Zhengming; Wang, Youchun

    2015-08-03

    Due to the increasing concern of using smallpox virus as biological weapons for terrorist attack, there is renewed interest in studying the pathogenesis of human smallpox and development of new therapies. Animal models are highly demanded for efficacy and safety examination of new vaccines and therapeutic drugs. Here, we demonstrated that both wild type and immunodeficient rats infected with an engineered vaccinia virus carrying Firefly luciferase reporter gene (rTV-Fluc) could recapitulate infectious and clinical features of human smallpox. Vaccinia viral infection in wild type Sprague-Dawley (SD) rats displayed a diffusible pattern in various organs, including liver, head and limbs. The intensity of bioluminescence generated from rTV-Fluc correlated well with viral loads in tissues. Moreover, neutralizing antibodies had a protective effect against virus reinfection. The recombination activating gene 2 (Rag2) knockout rats generated by transcription activator-like effector nucleases (TALENs) technology were further used to examine the infectivity of the rTV-Fluc in immunodeficient populations. Here we demonstrated that Rag2-/- rats were more susceptible to rTV-Fluc than SD rats with a slower virus clearance rate. Therefore, the rTV-Fluc/SD rats and rTV-Fluc/Rag2-/- rats are suitable visualization models, which recapitulate wild type or immunodeficient populations respectively, for testing human smallpox vaccine and antiviral drugs.

  19. Broad Protection against Avian Influenza Virus by Using a Modified Vaccinia Ankara Virus Expressing a Mosaic Hemagglutinin Gene

    PubMed Central

    Kamlangdee, Attapon; Kingstad-Bakke, Brock; Anderson, Tavis K.; Goldberg, Tony L.

    2014-01-01

    ABSTRACT A critical failure in our preparedness for an influenza pandemic is the lack of a universal vaccine. Influenza virus strains diverge by 1 to 2% per year, and commercially available vaccines often do not elicit protection from one year to the next, necessitating frequent formulation changes. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. We have constructed a recombinant modified vaccinia virus Ankara (MVA) that expresses an H5N1 mosaic hemagglutinin (H5M) (MVA-H5M). This mosaic was generated in silico using 2,145 field-sourced H5N1 isolates. A single dose of MVA-H5M provided 100% protection in mice against clade 0, 1, and 2 avian influenza viruses and also protected against seasonal H1N1 virus (A/Puerto Rico/8/34). It also provided short-term (10 days) and long-term (6 months) protection postvaccination. Both neutralizing antibodies and antigen-specific CD4+ and CD8+ T cells were still detected at 5 months postvaccination, suggesting that MVA-H5M provides long-lasting immunity. IMPORTANCE Influenza viruses infect a billion people and cause up to 500,000 deaths every year. A major problem in combating influenza is the lack of broadly effective vaccines. One solution from the field of human immunodeficiency virus vaccinology involves a novel in silico mosaic approach that has been shown to provide broad and robust protection against highly variable viruses. Unlike a consensus algorithm which picks the most frequent residue at each position, the mosaic method chooses the most frequent T-cell epitopes and combines them to form a synthetic antigen. These studies demonstrated that a mosaic influenza virus H5 hemagglutinin expressed by a viral vector can elicit full protection against diverse H5N1 challenges as well as induce broader immunity than a wild-type hemagglutinin. PMID:25210173

  20. Immunization with vaccinia virus recombinants that express the surface glycoproteins of human parainfluenza virus type 3 (PIV3) protects patas monkeys against PIV3 infection.

    PubMed Central

    Spriggs, M K; Collins, P L; Tierney, E; London, W T; Murphy, B R

    1988-01-01

    Patas monkeys (Eryphrocebus patas) were immunized intradermally with two vaccinia virus recombinants that individually express the hemagglutinin-neuraminidase glycoprotein or the fusion glycoprotein of human parainfluenza virus type 3 (PIV3). These immunizations induced a high titer of PIV3 serum-neutralizing antibodies. At 1 month after immunization, monkeys were challenged intratracheally with PIV3. Subsequent virus replication was reduced in these monkeys by 3.2 log10 and 1.9 log10 (mean peak virus titers) in the upper and lower respiratory tracts, respectively, compared with control animals. The average duration of virus shedding was also reduced from 9.0 to 3.4 days in the upper respiratory tract and from 5.3 to 1.2 days in the lower respiratory tract. These findings demonstrate that a single intradermal dose of live recombinant vaccinia viruses can significantly restrict the replication of a virus which primarily infects the epithelial cells of the respiratory tract. PMID:2831389

  1. Live-vaccinia virus encapsulation in pH-sensitive polymer increases safety of a reservoir-targeted Lyme disease vaccine by targeting gastrointestinal release.

    PubMed

    Kern, Aurelie; Zhou, Chensheng W; Jia, Feng; Xu, Qiaobing; Hu, Linden T

    2016-08-31

    The incidence of Lyme disease has continued to rise despite attempts to control its spread. Vaccination of zoonotic reservoirs of human pathogens has been successfully used to decrease the incidence of rabies in raccoons and foxes. We have previously reported on the efficacy of a vaccinia virus vectored vaccine to reduce carriage of Borrelia burgdorferi in reservoir mice and ticks. One potential drawback to vaccinia virus vectored vaccines is the risk of accidental infection of humans. To reduce this risk, we developed a process to encapsulate vaccinia virus with a pH-sensitive polymer that inactivates the virus until it is ingested and dissolved by stomach acids. We demonstrate that the vaccine is inactive both in vitro and in vivo until it is released from the polymer. Once released from the polymer by contact with an acidic pH solution, the virus regains infectivity. Vaccination with coated vaccinia virus confers protection against B. burgdorferi infection and reduction in acquisition of the pathogen by naïve feeding ticks. PMID:27502570

  2. [Hypoparathyroidismus following L-asparaginase and vaccinia virus infection. Effect of hypocalcemia on phagocytosis and the function of lymphocytes].

    PubMed

    Ricken, K H

    1975-11-21

    Rabbits, treated with injections of 4000 IU of L-Asparaginase, develop the clinical and chemical signs of hypoparathyroidism. A simultaneous vaccination with vaccinia virus (strain "Elstree") markedly increase the tetanic symptoms ("conditioned deficiency"). L-Asparaginase may influence the cellular immunity by hypocalcemia. Two mechanisms are discussed: 1. the suppression of the phagocytosis, recognizable by the absence of signs for vaccinal allergy by deficiency of macrophages in the intradermal test with inactivated small-pox vaccine. 2. the inhibition of the PHA-induced lymphocyte transformation caused by deficiency of calcium ions.

  3. Identification of viral membrane proteins required for cell fusion and viral dissemination that are modified during vaccinia virus persistence.

    PubMed

    Ortiz, M A; Paez, E

    1994-01-01

    Wild-type vaccinia virus WR strain forms non-fusogenic (F-) large plaques and is hemagglutinin positive (HA+) under normal conditions of virus infection. We have analyzed a collection of spontaneous, highly attenuated mutants of vaccinia virus isolated from persistently infected Friend erythroleukemia cells (E. Paez, S. Dallo, and M. Esteban, J. Virol. 61, 2642-2647, 1987) for the ability to express HA during virus infection. After 14 cell passages, all the mutants isolated were hemadsorption negative (HAD-) and did not synthesize a HA that could be recognized by anti-HA monoclonal antibodies. All these HA- mutants induced extensive cell-cell fusion (F+), with the exception of two mutants (65-16 and 101-14) isolated from late cell passages. Nucleotide sequence analysis of the HA gene in these two mutants confirmed the HA- phenotype. A frameshift mutation very close to the initiation codon resulted in premature translational termination. The truncated gene now only encodes the first 25 amino acids. Analysis of progeny from "wild-type," like early serial passage virus (5-3) X mutant back crosses, shows that for one late passage non-fusogenic small-plaque mutant (101-14) among large plaque progeny there is good correspondence between the ability to fuse and the absence of a viral HA and that each large plaque mutant contains a normal 14 kDa membrane protein. However, with a second serial passage mutant 65-16, which, like 101-14, is a nonfusogenic small-plaque variant, there is again an excellent correlation between the inability to synthesize HA and the ability to fuse, but there is no correlation of plaque size with a normal 14 kDa viral membrane protein, as most large plaque mutants encode a larger, i.e., 17 kDa protein. Rescue experiments of 65-16 with bona fide cloned 14 kDa protein gene confirm that the ability to regulate plaque size and cell fusion in this mutant is due to a protein other than the 14 kDa protein. Marker rescue experiments indicated that the map

  4. Spread of vaccinia virus through shaving during military training, Joint Base San Antonio-Lackland, TX, June 2014.

    PubMed

    Webber, Bryant J; Montgomery, Jay R; Markelz, Ana E; Allen, Kahtonna C; Hunninghake, John C; Ritchie, Simon A; Pawlak, Mary T; Johnston, Lindsay A; Oliver, Tiffany A; Winterton, Brad S

    2014-08-01

    Although naturally occurring smallpox virus was officially declared eradicated in 1980, concern for biological warfare prompted the U.S. Government in 2002 to recommend smallpox vaccination for select individuals. Vaccinia, the smallpox vaccine virus, is administered into the skin, typically on the upper arm, where the virus remains viable and infectious until the scab falls off and the epidermis is fully intact - typically 2-4 weeks. Adverse events following smallpox vaccination may occur in the vaccinee, in individuals who have contact with the vaccinee (i.e., secondary transmission), or in individuals who have contact with the vaccinee's contact (i.e., tertiary transmission). In June 2014 at Joint Base San Antonio-Lackland, TX, two cases of inadvertent inoculation of vaccinia and one case of a non-viral reaction following vaccination occurred in the security forces training squadron. This includes the first reported case of shaving as the likely source of autoinoculation after contact transmission. This paper describes the diagnosis and treatment of these cases, the outbreak investigation, and steps taken to prevent future transmission.

  5. Modified Vaccinia Virus Ankara (MVA) as Production Platform for Vaccines against Influenza and Other Viral Respiratory Diseases

    PubMed Central

    Altenburg, Arwen F.; Kreijtz, Joost H. C. M.; de Vries, Rory D.; Song, Fei; Fux, Robert; Rimmelzwaan, Guus F.; Sutter, Gerd; Volz, Asisa

    2014-01-01

    Respiratory viruses infections caused by influenza viruses, human parainfluenza virus (hPIV), respiratory syncytial virus (RSV) and coronaviruses are an eminent threat for public health. Currently, there are no licensed vaccines available for hPIV, RSV and coronaviruses, and the available seasonal influenza vaccines have considerable limitations. With regard to pandemic preparedness, it is important that procedures are in place to respond rapidly and produce tailor made vaccines against these respiratory viruses on short notice. Moreover, especially for influenza there is great need for the development of a universal vaccine that induces broad protective immunity against influenza viruses of various subtypes. Modified Vaccinia Virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform. MVA can encode one or more foreign antigens and thus functions as a multivalent vaccine. The vector can be used at biosafety level 1, has intrinsic adjuvant capacities and induces humoral and cellular immune responses. However, there are some practical and regulatory issues that need to be addressed in order to develop MVA-based vaccines on short notice at the verge of a pandemic. In this review, we discuss promising novel influenza virus vaccine targets and the use of MVA for vaccine development against various respiratory viruses. PMID:25036462

  6. Seven major genomic deletions of vaccinia virus Tiantan strain are sufficient to decrease pathogenicity.

    PubMed

    Li, Yiquan; Sheng, Yuan; Chu, Yunjie; Ji, Huifan; Jiang, Shuang; Lan, Tian; Li, Min; Chen, Shuang; Fan, Yuanyuan; Li, Wenjie; Li, Xiao; Sun, Lili; Jin, Ningyi

    2016-05-01

    Attenuated strain TTVAC7, as a multi-gene-deleted vaccinia virus Tiantan strain (VTT), was constructed by knocking out parts of non-essential genes related to virulence, host range and immunomodulation of VTT, and by combining double marker screening with exogenous selectable marker knockout techniques. In this study, shuttle vector plasmids pTC-EGFP, pTA35-EGFP, pTA66-EGFP, pTE-EGFP, pTB-EGFP, pTI-EGFP and pTJ-EGFP were constructed, which contained seven pairs of recombinant arms linked to the early and late strong promoter pE/L, as well as to enhanced green fluorescent protein (EGFP) as an exogenous selectable marker. BHK cells were co-transfected/infected successively with the above plasmids and VTT or gene-deleted VTT, and homologous recombination and fluorescence plaque screening methods were used to knock out the gene fragments (TC: TC7L ∼ TK2L; TA35: TA35L; TA66: TA66R; TE: TE3L ∼ TE4L; TB: TB13R; TI: TI4L; TJ: TJ2R). The Cre/LoxP system was then applied to knock out the exogenous selectable marker, and ultimately the gene-deleted attenuated strain TTVAC7 was obtained. A series of in vivo and in vitro experiments demonstrated that not only the host range of TTVAC7 could be narrowed and its toxicity weakened significantly, but its high immunogenicity was maintained at the same time. These results support the potential of TTVAC7 to be developed as a safe viral vector or vaccine. PMID:26821204

  7. Apoptosis and necrosis in vaccinia virus-infected HeLa G and BSC-40 cells.

    PubMed

    Liskova, Jana; Knitlova, Jarmila; Honner, Richard; Melkova, Zora

    2011-09-01

    In most cells, vaccinia virus (VACV) infection is considered to cause a lytic cell death, an equivalent of necrosis. However, upon infection of the epithelial cell lines HeLa G and BSC-40 with VACV strain Western Reserve (WR), we have previously observed an increased activation of and activity attributable to caspases, a typical sign of apoptosis. In this paper, we have further analyzed the type of cell death in VACV-infected cells HeLa G and BSC-40. In a cell-based flow cytometric assay, we showed a specific activation of caspase-2 and 4 in HeLa G and BSC-40 cells infected with VACV, strain WR, while we did not find any effects of inhibitors of calpain and cathepsin D and E. The actual activity of the two caspases, but also of caspase-3, was then confirmed in lysates of infected HeLa G, but not in BSC-40 cells. Accordingly, poly(ADP)-ribose polymerase (PARP) cleavage was found increased only in infected HeLa G cells. Consequently, we have determined morphological features of apoptosis and/or activity of the executioner caspase-3 in infected HeLa G cells in situ, while only a background apoptosis was observed in infected BSC-40 cells. Finally, vaccination strains Dryvax and Praha were found to induce apoptosis in both HeLa G and BSC-40 cells, as characterized morphologically and by PARP cleavage. These findings may be important for understanding the differences in VACV-host interactions and post-vaccination complications in different individuals.

  8. Administration of vaccinia virus complement control protein shows significant cognitive improvement in a mild injury model.

    PubMed

    Pillay, Nirvana S; Kellaway, Laurie A; Kotwal, Girish J

    2005-11-01

    Previous studies have shown that traumatic mild brain injury in a rat model is accompanied by breakdown of the blood brain barrier and the accumulation of inflammatory cells. A therapeutic agent, vaccinia virus complement control protein (VCP), inhibits both the classic and the alternative pathways of the complement system and, in so doing, prevents cell death and inflammation. With the use of a rat mild injury model, the effects of VCP on spatial learning and memory were tested. Training in a Morris water maze consisted of a total of 16 trials over a 2-day period before rats were anesthetized and subjected to mild (1.0-1.1 atm) lateral fluid percussion injury (FPI) 3.0 mm lateral to the sagittal suture and 4.5 mm posterior to bregma. Ten microl of VCP (1.7 mg/ml) was injected into the injury site immediately after FPI. Two weeks post-FPI the rats were assessed in the Morris water maze for spatial learning and memory. Neurologic motor function tests were carried out after FPI for 14 consecutive days and again after 28 days. The Morris water maze data show that FPI plus saline-injected rats spent a significantly (P <0.05) larger amount of time in one of the incorrect quadrants than did the FPI plus VCP-injected group. Neurologic evaluations 24 hours postinjury revealed differences in sensorimotor function between groups. The results suggest that in a mild injury model, VCP influences neurologic outcome and offers some enhancement in spatial memory and learning.

  9. Seven major genomic deletions of vaccinia virus Tiantan strain are sufficient to decrease pathogenicity.

    PubMed

    Li, Yiquan; Sheng, Yuan; Chu, Yunjie; Ji, Huifan; Jiang, Shuang; Lan, Tian; Li, Min; Chen, Shuang; Fan, Yuanyuan; Li, Wenjie; Li, Xiao; Sun, Lili; Jin, Ningyi

    2016-05-01

    Attenuated strain TTVAC7, as a multi-gene-deleted vaccinia virus Tiantan strain (VTT), was constructed by knocking out parts of non-essential genes related to virulence, host range and immunomodulation of VTT, and by combining double marker screening with exogenous selectable marker knockout techniques. In this study, shuttle vector plasmids pTC-EGFP, pTA35-EGFP, pTA66-EGFP, pTE-EGFP, pTB-EGFP, pTI-EGFP and pTJ-EGFP were constructed, which contained seven pairs of recombinant arms linked to the early and late strong promoter pE/L, as well as to enhanced green fluorescent protein (EGFP) as an exogenous selectable marker. BHK cells were co-transfected/infected successively with the above plasmids and VTT or gene-deleted VTT, and homologous recombination and fluorescence plaque screening methods were used to knock out the gene fragments (TC: TC7L ∼ TK2L; TA35: TA35L; TA66: TA66R; TE: TE3L ∼ TE4L; TB: TB13R; TI: TI4L; TJ: TJ2R). The Cre/LoxP system was then applied to knock out the exogenous selectable marker, and ultimately the gene-deleted attenuated strain TTVAC7 was obtained. A series of in vivo and in vitro experiments demonstrated that not only the host range of TTVAC7 could be narrowed and its toxicity weakened significantly, but its high immunogenicity was maintained at the same time. These results support the potential of TTVAC7 to be developed as a safe viral vector or vaccine.

  10. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    SciTech Connect

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This also represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.

  11. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    DOE PAGES

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

  12. Targeting the vaccinia virus L1 protein to the cell surface enhances production of neutralizing antibodies.

    PubMed

    Golden, Joseph W; Josleyn, Matthew D; Hooper, Jay W

    2008-06-25

    The current live-orthopoxvirus vaccine is associated with minor to serious adverse affects, and is contraindicated for use in a significant portion of the population. As an alternative vaccine, we have previously shown that a DNA subunit vaccine (4pox) based on four orthopoxvirus immunogens (L1R, B5R, A27L and A33R) can produce protective immunity against lethal orthopoxvirus challenges in mice and nonhuman primates. Because antibodies are critical for protection against secondary orthopoxvirus infections, we are now interested in strategies that will enhance the humoral immune response against vaccine targets. Here, we tested the immunogenicity of an L1R construct to which a tissue plasminogen activator signal sequence was placed in frame with the full-length L1R gene. The tPA-L1R construct produced a more robust neutralizing antibody response in vaccinated mice when the DNA vaccine was administered by gene-gun as a prime/single boost. When the tPA-L1R construct was substituted for the unmodified L1R gene in the 4pox vaccine, given as a prime and single boost, animals were better protected from lethal challenge with vaccinia virus (VACV). These findings indicate that adding a tPA-leader sequence can enhance the immunogenicity of the L1R gene when given as a DNA vaccine. Furthermore, our results demonstrate that a DNA-based vaccine is capable of establishing protection from lethal orthopoxvirus challenges when administered as a prime and single boost without requiring adjuvant. PMID:18485547

  13. Cleavage of Dicer protein by I7 protease during vaccinia virus infection.

    PubMed

    Chen, Jhih-Si; Li, Hui-Chun; Lin, Shu-I; Yang, Chee-Hing; Chien, Wan-Yu; Syu, Ciao-Ling; Lo, Shih-Yen

    2015-01-01

    Dicer is the key component in the miRNA pathway. Degradation of Dicer protein is facilitated during vaccinia virus (VV) infection. A C-terminal cleaved product of Dicer protein was detected in the presence of MG132 during VV infection. Thus, it is possible that Dicer protein is cleaved by a viral protease followed by proteasome degradation of the cleaved product. There is a potential I7 protease cleavage site in the C-terminus of Dicer protein. Indeed, reduction of Dicer protein was detected when Dicer was co-expressed with I7 protease but not with an I7 protease mutant protein lack of the protease activity. Mutation of the potential I7 cleavage site in the C-terminus of Dicer protein resisted its degradation during VV infection. Furthermore, Dicer protein was reduced dramatically by recombinant VV vI7Li after the induction of I7 protease. If VV could facilitate the degradation of Dicer protein, the process of miRNA should be affected by VV infection. Indeed, accumulation of precursor miR122 was detected after VV infection or I7 protease expression. Reduction of miR122 would result in the suppression of HCV sub-genomic RNA replication, and, in turn, the amount of viral proteins. As expected, significant reduction of HCVNS5A protein was detected after VV infection and I7 protease expression. Therefore, our results suggest that VV could cleave Dicer protein through I7 protease to facilitate Dicer degradation, and in turn, suppress the processing of miRNAs. Effect of Dicer protein on VV replication was also studied. Exogenous expression of Dicer protein suppresses VV replication slightly while knockdown of Dicer protein does not affect VV replication significantly.

  14. Complete nucleotide sequences of two adjacent early vaccinia virus genes located within the inverted terminal repetition.

    PubMed

    Venkatesan, S; Gershowitz, A; Moss, B

    1982-11-01

    The proximal part of the 10,000-base pair (bp) inverted terminal repetition of vaccinia virus DNA encodes at least three early mRNAs. A 2,236-bp segment of the repetition was sequenced to characterize two of the genes. This task was facilitated by constructing a series of recombinants containing overlapping deletions; oligonucleotide linkers with synthetic restriction sites provided points for radioactive labeling before sequencing by the chemical degradation method of Maxam and Gilbert (Methods Enzymol. 65:499-560, 1980). The ends of the transcripts were mapped by hybridizing labeled DNA fragments to early viral RNA and resolving nuclease S1-protected fragments in sequencing gels, by sequencing cDNA clones, and from the lengths of the RNAs. The nucleotide sequences for at least 60 bp upstream of both transcriptional initiation sites are more than 80% adenine . thymine rich and contain long runs of adenines and thymines with some homology to procaryotic and eucaryotic consensus sequences. The gene transcribed in the rightward direction encodes an RNA of approximately 530 nucleotides with a single open reading frame of 420 nucleotides. Preceding the first AUG, there is a heptanucleotide that can hybridize to the 3' end of 18S rRNA with only one mismatch. The derived amino acid sequence of the protein indicated a molecular weight of 15,500. The gene transcribed in the leftward direction encodes an RNA 1,000 to 1,100 nucleotides long with an open reading frame of 996 nucleotides and a leader sequence of only 5 to 6 nucleotides. The derived amino acid sequence of this protein indicated a molecular weight of 38,500. The 3' ends of the two transcripts were located within 100 bp of each other. Although there are adenine . thymine-rich clusters near the putative transcriptional termination sites, specific AATAAA polyadenylic acid signal sequences are absent.

  15. IL-18 Expression Results in a Recombinant Vaccinia Virus That Is Highly Attenuated and Immunogenic

    PubMed Central

    Verardi, Paulo H.; Legrand, Fatema A.; Chan, Kenneth S.; Peng, Yue; Jones, Leslie A.

    2014-01-01

    Interferon-γ (IFN-γ) is an attenuating factor for vaccinia virus (VACV), decreasing its virulence in vivo by more than a million fold. It is also a highly effective adjuvant when administered at the time of immunization with protein antigens. However, recombinant VACV (rVACV) vaccines expressing IFN-γ do not induce enhanced immune responses. It is possible that the IFN-γ expressed by rVACVs induces both an antiviral state and increased immunological clearance, thus resulting in decreased levels of antigen expression due to reduced viral replication and spread. We conjectured that delaying expression of IFN-γ would result in enhanced production of antigens by rVACVs thus resulting in increased immune responses to foreign antigens. Interleukin (IL)-18, also known as IFN-γ inducing factor, is a cytokine that induces T and NK cells to produce IFN-γ. In this study, we demonstrated that an rVACV expressing bioactive murine IL-18 replicated to low but detectable levels in vivo, unlike an rVACV expressing IFN-γ. Moreover, the rVACV expressing IL-18 was significantly attenuated in both immunocompromised and immunocompetent mice. This attenuation was dependent on IFN-γ, as IL-18 expression failed to attenuate VACV in IFN-γ knock-out mice. Cytotoxic T-cell (CTL) and anamnestic antibody responses were slightly increased in animals vaccinated with the rVACV expressing IL-18. Thus, induction of IFN-γ because of IL-18 expression resulted in an rVACV that replicated to low but detectable levels in vivo, yet elicited slightly better CTL and anamnestic humoral immune responses. PMID:24168450

  16. Sickle Cells Abolish Melanoma Tumorigenesis in Hemoglobin SS Knockin Mice and Augment the Tumoricidal Effect of Oncolytic Virus In Vivo.

    PubMed

    Sun, Chiang Wang; Willmon, Candice; Wu, Li-Chen; Knopick, Peter; Thoerner, Jutta; Vile, Richard; Townes, Tim M; Terman, David S

    2016-01-01

    Insights from the study of cancer resistance in animals have led to the discovery of novel anticancer pathways and opened new venues for cancer prevention and treatment. Sickle cells (SSRBCs) from subjects with homozygous sickle cell anemia (SCA) have been shown to target hypoxic tumor niches, induce diffuse vaso-occlusion, and potentiate a tumoricidal response in a heme- and oxidant-dependent manner. These findings spawned the hypothesis that SSRBCs and the vasculopathic microenvironment of subjects with SCA might be inimical to tumor outgrowth and thereby constitute a natural antitumor defense. We therefore implanted the B16F10 melanoma into humanized hemoglobin SS knockin mice which exhibit the hematologic and vasculopathic sequelae of human SCA. Over the 31-day observation period, hemoglobin SS mice showed no significant melanoma outgrowth. By contrast, 68-100% of melanomas implanted in background and hemoglobin AA knockin control mice reached the tumor growth end point (p < 0.0001). SS knockin mice also exhibited established markers of underlying vasculopathy, e.g., chronic hemolysis (anemia, reticulocytosis) and vascular inflammation (leukocytosis) that differed significantly from all control groups. Genetic differences or normal AA gene knockin do not explain the impaired tumor outgrowth in SS knockin mice. These data point instead to the chronic pro-oxidative vasculopathic network in these mice as the predominant cause. In related studies, we demonstrate the ability of the sickle cell component of this system to function as a therapeutic vehicle in potentiating the oncolytic/vasculopathic effect of RNA reovirus. Sickle cells were shown to efficiently adsorb and transfer the virus to melanoma cells where it induced apoptosis even in the presence of anti-reovirus neutralizing antibodies. In vivo, SSRBCs along with their viral cargo rapidly targeted the tumor and initiated a tumoricidal response exceeding that of free virus and similarly loaded normal RBCs

  17. Sickle Cells Abolish Melanoma Tumorigenesis in Hemoglobin SS Knockin Mice and Augment the Tumoricidal Effect of Oncolytic Virus In Vivo.

    PubMed

    Sun, Chiang Wang; Willmon, Candice; Wu, Li-Chen; Knopick, Peter; Thoerner, Jutta; Vile, Richard; Townes, Tim M; Terman, David S

    2016-01-01

    Insights from the study of cancer resistance in animals have led to the discovery of novel anticancer pathways and opened new venues for cancer prevention and treatment. Sickle cells (SSRBCs) from subjects with homozygous sickle cell anemia (SCA) have been shown to target hypoxic tumor niches, induce diffuse vaso-occlusion, and potentiate a tumoricidal response in a heme- and oxidant-dependent manner. These findings spawned the hypothesis that SSRBCs and the vasculopathic microenvironment of subjects with SCA might be inimical to tumor outgrowth and thereby constitute a natural antitumor defense. We therefore implanted the B16F10 melanoma into humanized hemoglobin SS knockin mice which exhibit the hematologic and vasculopathic sequelae of human SCA. Over the 31-day observation period, hemoglobin SS mice showed no significant melanoma outgrowth. By contrast, 68-100% of melanomas implanted in background and hemoglobin AA knockin control mice reached the tumor growth end point (p < 0.0001). SS knockin mice also exhibited established markers of underlying vasculopathy, e.g., chronic hemolysis (anemia, reticulocytosis) and vascular inflammation (leukocytosis) that differed significantly from all control groups. Genetic differences or normal AA gene knockin do not explain the impaired tumor outgrowth in SS knockin mice. These data point instead to the chronic pro-oxidative vasculopathic network in these mice as the predominant cause. In related studies, we demonstrate the ability of the sickle cell component of this system to function as a therapeutic vehicle in potentiating the oncolytic/vasculopathic effect of RNA reovirus. Sickle cells were shown to efficiently adsorb and transfer the virus to melanoma cells where it induced apoptosis even in the presence of anti-reovirus neutralizing antibodies. In vivo, SSRBCs along with their viral cargo rapidly targeted the tumor and initiated a tumoricidal response exceeding that of free virus and similarly loaded normal RBCs

  18. Sickle Cells Abolish Melanoma Tumorigenesis in Hemoglobin SS Knockin Mice and Augment the Tumoricidal Effect of Oncolytic Virus In Vivo

    PubMed Central

    Sun, Chiang Wang; Willmon, Candice; Wu, Li-Chen; Knopick, Peter; Thoerner, Jutta; Vile, Richard; Townes, Tim M.; Terman, David S.

    2016-01-01

    Insights from the study of cancer resistance in animals have led to the discovery of novel anticancer pathways and opened new venues for cancer prevention and treatment. Sickle cells (SSRBCs) from subjects with homozygous sickle cell anemia (SCA) have been shown to target hypoxic tumor niches, induce diffuse vaso-occlusion, and potentiate a tumoricidal response in a heme- and oxidant-dependent manner. These findings spawned the hypothesis that SSRBCs and the vasculopathic microenvironment of subjects with SCA might be inimical to tumor outgrowth and thereby constitute a natural antitumor defense. We therefore implanted the B16F10 melanoma into humanized hemoglobin SS knockin mice which exhibit the hematologic and vasculopathic sequelae of human SCA. Over the 31-day observation period, hemoglobin SS mice showed no significant melanoma outgrowth. By contrast, 68–100% of melanomas implanted in background and hemoglobin AA knockin control mice reached the tumor growth end point (p < 0.0001). SS knockin mice also exhibited established markers of underlying vasculopathy, e.g., chronic hemolysis (anemia, reticulocytosis) and vascular inflammation (leukocytosis) that differed significantly from all control groups. Genetic differences or normal AA gene knockin do not explain the impaired tumor outgrowth in SS knockin mice. These data point instead to the chronic pro-oxidative vasculopathic network in these mice as the predominant cause. In related studies, we demonstrate the ability of the sickle cell component of this system to function as a therapeutic vehicle in potentiating the oncolytic/vasculopathic effect of RNA reovirus. Sickle cells were shown to efficiently adsorb and transfer the virus to melanoma cells where it induced apoptosis even in the presence of anti-reovirus neutralizing antibodies. In vivo, SSRBCs along with their viral cargo rapidly targeted the tumor and initiated a tumoricidal response exceeding that of free virus and similarly loaded normal

  19. Safety and Immunogenicity of Modified Vaccinia Ankara-Bavarian Nordic Smallpox Vaccine in Vaccinia-Naive and Experienced Human Immunodeficiency Virus-Infected Individuals: An Open-Label, Controlled Clinical Phase II Trial

    PubMed Central

    Overton, Edgar Turner; Stapleton, Jack; Frank, Ian; Hassler, Shawn; Goepfert, Paul A.; Barker, David; Wagner, Eva; von Krempelhuber, Alfred; Virgin, Garth; Meyer, Thomas Peter; Müller, Jutta; Bädeker, Nicole; Grünert, Robert; Young, Philip; Rösch, Siegfried; Maclennan, Jane; Arndtz-Wiedemann, Nathaly; Chaplin, Paul

    2015-01-01

    Background. First- and second-generation smallpox vaccines are contraindicated in individuals infected with human immunodeficiency virus (HIV). A new smallpox vaccine is needed to protect this population in the context of biodefense preparedness. The focus of this study was to compare the safety and immunogenicity of a replication-deficient, highly attenuated smallpox vaccine modified vaccinia Ankara (MVA) in HIV-infected and healthy subjects. Methods. An open-label, controlled Phase II trial was conducted at 36 centers in the United States and Puerto Rico for HIV-infected and healthy subjects. Subjects received 2 doses of MVA administered 4 weeks apart. Safety was evaluated by assessment of adverse events, focused physical exams, electrocardiogram recordings, and safety laboratories. Immune responses were assessed using enzyme-linked immunosorbent assay (ELISA) and a plaque reduction neutralization test (PRNT). Results. Five hundred seventy-nine subjects were vaccinated at least once and had data available for analysis. Rates of ELISA seropositivity were comparably high in vaccinia-naive healthy and HIV-infected subjects, whereas PRNT seropositivity rates were higher in healthy compared with HIV-infected subjects. Modified vaccinia Ankara was safe and well tolerated with no adverse impact on viral load or CD4 counts. There were no cases of myo-/pericarditis reported. Conclusions. Modified vaccinia Ankara was safe and immunogenic in subjects infected with HIV and represents a promising smallpox vaccine candidate for use in immunocompromised populations. PMID:26380340

  20. The generation of CD8+ T-cell population specific for vaccinia virus epitope involved in the antiviral protection against ectromelia virus challenge.

    PubMed

    Gierynska, Malgorzata; Szulc-Dabrowska, Lidia; Dzieciatkowski, Tomasz; Golke, Anna; Schollenberger, Ada

    2015-12-01

    Eradication of smallpox has led to cessation of vaccination programs. This has rendered the human population increasingly susceptible not only to variola virus infection but also to infections with other representatives of Poxviridae family that cause zoonotic variola-like diseases. Thus, new approaches for designing improved vaccine against smallpox are required. Discovering that orthopoxviruses, e.g. variola virus, vaccinia virus, ectromelia virus, share common immunodominant antigen, may result in the development of such a vaccine. In our study, the generation of antigen-specific CD8(+) T cells in mice during the acute and memory phase of the immune response was induced using the vaccinia virus immunodominant TSYKFESV epitope and CpG oligodeoxynucleotides as adjuvants. The role of the generated TSYKFESV-specific CD8(+) T cells was evaluated in mice during ectromelia virus infection using systemic and mucosal model. Moreover, the involvement of dendritic cells subsets in the adaptive immune response stimulation was assessed. Our results indicate that the TSYKFESV epitope/TLR9 agonist approach, delivered systemically or mucosally, generated strong CD8(+) T-cell response when measured 10 days after immunization. Furthermore, the TSYKFESV-specific cell population remained functionally active 2 months post-immunization, and gave cross-protection in virally challenged mice, even though the numbers of detectable antigen-specific T cells decreased.

  1. The generation of CD8+ T-cell population specific for vaccinia virus epitope involved in the antiviral protection against ectromelia virus challenge.

    PubMed

    Gierynska, Malgorzata; Szulc-Dabrowska, Lidia; Dzieciatkowski, Tomasz; Golke, Anna; Schollenberger, Ada

    2015-12-01

    Eradication of smallpox has led to cessation of vaccination programs. This has rendered the human population increasingly susceptible not only to variola virus infection but also to infections with other representatives of Poxviridae family that cause zoonotic variola-like diseases. Thus, new approaches for designing improved vaccine against smallpox are required. Discovering that orthopoxviruses, e.g. variola virus, vaccinia virus, ectromelia virus, share common immunodominant antigen, may result in the development of such a vaccine. In our study, the generation of antigen-specific CD8(+) T cells in mice during the acute and memory phase of the immune response was induced using the vaccinia virus immunodominant TSYKFESV epitope and CpG oligodeoxynucleotides as adjuvants. The role of the generated TSYKFESV-specific CD8(+) T cells was evaluated in mice during ectromelia virus infection using systemic and mucosal model. Moreover, the involvement of dendritic cells subsets in the adaptive immune response stimulation was assessed. Our results indicate that the TSYKFESV epitope/TLR9 agonist approach, delivered systemically or mucosally, generated strong CD8(+) T-cell response when measured 10 days after immunization. Furthermore, the TSYKFESV-specific cell population remained functionally active 2 months post-immunization, and gave cross-protection in virally challenged mice, even though the numbers of detectable antigen-specific T cells decreased. PMID:26474845

  2. Multiple Viral Ligands Naturally Presented by Different Class I Molecules in Transporter Antigen Processing-Deficient Vaccinia Virus-Infected Cells

    PubMed Central

    Lorente, Elena; Infantes, Susana; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Vilches, Carlos; Lemonnier, François A.; Admon, Arie

    2012-01-01

    The transporter associated with antigen processing (TAP) delivers the viral proteolytic products generated by the proteasome in the cytosol to the endoplasmic reticulum lumen that are subsequently recognized by cytotoxic T lymphocytes (CTLs). However, several viral epitopes have been identified in TAP-deficient models. Using mass spectrometry to analyze complex human leukocyte antigen (HLA)-bound peptide pools isolated from large numbers of TAP-deficient vaccinia virus-infected cells, we identified 11 ligands naturally presented by four different HLA-A, HLA-B, and HLA-C class I molecules. Two of these ligands were presented by two different HLA class I alleles, and, as a result, 13 different HLA-peptide complexes were formed simultaneously in the same vaccinia virus-infected cells. In addition to the high-affinity ligands, one low-affinity peptide restricted by each of the HLA-A, HLA-B, and HLA-C class I molecules was identified. Both high- and low-affinity ligands generated long-term memory CTL responses to vaccinia virus in an HLA-A2-transgenic mouse model. The processing and presentation of two vaccinia virus-encoded HLA-A2-restricted antigens took place via proteasomal and nonproteasomal pathways, which were blocked in infected cells with chemical inhibitors specific for different subsets of metalloproteinases. These data have implications for the study of the effectiveness of early empirical vaccination with cowpox virus against smallpox disease. PMID:22031944

  3. Multiple viral ligands naturally presented by different class I molecules in transporter antigen processing-deficient vaccinia virus-infected cells.

    PubMed

    Lorente, Elena; Infantes, Susana; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Vilches, Carlos; Lemonnier, François A; Admon, Arie; López, Daniel

    2012-01-01

    The transporter associated with antigen processing (TAP) delivers the viral proteolytic products generated by the proteasome in the cytosol to the endoplasmic reticulum lumen that are subsequently recognized by cytotoxic T lymphocytes (CTLs). However, several viral epitopes have been identified in TAP-deficient models. Using mass spectrometry to analyze complex human leukocyte antigen (HLA)-bound peptide pools isolated from large numbers of TAP-deficient vaccinia virus-infected cells, we identified 11 ligands naturally presented by four different HLA-A, HLA-B, and HLA-C class I molecules. Two of these ligands were presented by two different HLA class I alleles, and, as a result, 13 different HLA-peptide complexes were formed simultaneously in the same vaccinia virus-infected cells. In addition to the high-affinity ligands, one low-affinity peptide restricted by each of the HLA-A, HLA-B, and HLA-C class I molecules was identified. Both high- and low-affinity ligands generated long-term memory CTL responses to vaccinia virus in an HLA-A2-transgenic mouse model. The processing and presentation of two vaccinia virus-encoded HLA-A2-restricted antigens took place via proteasomal and nonproteasomal pathways, which were blocked in infected cells with chemical inhibitors specific for different subsets of metalloproteinases. These data have implications for the study of the effectiveness of early empirical vaccination with cowpox virus against smallpox disease.

  4. Protein of macaques against infection with simian type D retrovirus (SRV-1) by immunization with recombinant vaccinia virus expressing the envelope glycoproteins of either SRV-1 or Mason-Pfizer monkey virus (SRV-3).

    PubMed Central

    Brody, B A; Hunter, E; Kluge, J D; Lasarow, R; Gardner, M; Marx, P A

    1992-01-01

    Rhesus macaques were immunized with live vaccinia virus recombinants expressing the envelope glycoproteins (gp70 and gp22) of simian type D retrovirus (SRV), serotype 1 or 3. All of the animals immunized with either the SRV-1 env or the SRV-3 env vaccinia virus recombinant developed neutralizing antibodies against the homologous SRV. In addition, both groups developed cross-reactive antibodies and were protected against an intravenous live-virus challenge with SRV-1. The four control animals immunized with a vaccinia virus recombinant expressing the G protein of respiratory syncytial virus were not protected against the same SRV-1 challenge. Although SRV-1 and SRV-3 immune sera showed cross-neutralization, they failed to neutralize a separate, more distantly related serotype, SRV-2, in an in vitro assay. These findings are consistent with the known degree of serologic and genetic relatedness of these three SRV strains. Images PMID:1316495

  5. Vaccinia virus A17L gene product is essential for an early step in virion morphogenesis.

    PubMed

    Rodríguez, D; Esteban, M; Rodríguez, J R

    1995-08-01

    Vaccinia virus (VV) A17L gene encodes a 23-kDa protein that is proteolytically cleaved to generate a 21-kDa product that is incorporated into the viral particles. We have previously shown that the 21-kDa protein forms a stable complex with the VV 14-kDa envelope protein and suggested that the 21-kDa protein may serve to anchor the 14-kDa protein to the envelope of the virion (D. Rodríguez, J. R. Rodríguez, and M. Esteban, J. Virol. 67:3435-3440, 1993). To study the role of the 21-kDa protein in virion assembly, in this investigation we generated a VV recombinant, VVindA17L, that contains an inducible A17L gene regulated by the E. coli repressor/operator system. In the absence of the inducer, shutoff of the A17L gene was complete, and this shutoff correlated with a reduction in virus yields of about 3 log units. Although early and late viral polypeptides are normally synthesized in the absence of the A17L gene product, proteolytic processing of the major p4a and p4b core proteins was clearly impaired under these conditions. Electron microscopy examination of cells infected in the absence of isopropylthiogalactopyranoside (IPTG) revealed that virion morphogenesis was completely arrested at a very early stage, even prior to the formation of crescent-shaped membranes, which are the first distinguishable viral structures. Only electron-dense structures similar to rifampin bodies, but devoid of membranes, could be observed in the cytoplasm of cells infected with VVindA17L under nonpermissive conditions. Considering the most recent assembly model presented by Sodeik et al. (B. Sodeik, R. W. Doms, M. Ericsson, G. Hiller, C. E. Machamer, W. van't Hof, G. van Meer, B. Moss, and G. Griffiths, J. Cell Biol. 121:521-541, 1993), we propose that this protein is targeted to the intermediate compartment and is involved in the recruitment of these membranes to the viral factories, where it forms the characteristic crescent structures that subsequently result in the formation of

  6. Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

    PubMed Central

    Alkhatib, G; Shen, S H; Briedis, D; Richardson, C; Massie, B; Weinberg, R; Smith, D; Taylor, J; Paoletti, E; Roder, J

    1994-01-01

    The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes. Images PMID:8107215

  7. Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies

    SciTech Connect

    Nelson, Gretchen E.; Sisler, Jerry R.; Chandran, Dev; Moss, Bernard

    2008-10-25

    The vaccinia virus entry/fusion complex (EFC) is comprised of at least eight transmembrane proteins that are conserved in all poxviruses. However, neither the physical structure of the EFC nor the immunogenicity of the individual components has been determined. We prepared soluble forms of two EFC components, A28 and H2, by replacing the transmembrane domain with a signal peptide and adding a polyhistidine tail. The proteins were expressed by baculoviruses, secreted from insect cells, purified by affinity chromatography and used to raise antibodies in rabbits. The antibodies recognized the viral proteins but only the antibody to recombinant A28 bound intact virions and neutralized infectivity. Analyses with a set of overlapping peptides revealed a neutralizing epitope between residues 73 and 92 of A28. Passive immunization of mice with IgG purified from the anti-A28 serum provided partial protection against a vaccinia virus intranasal challenge, whereas IgG from the anti-H2 serum did not.

  8. The NYCBH vaccinia virus deleted for the innate immune evasion gene, E3L, protects rabbits against lethal challenge by rabbitpox virus.

    PubMed

    Denzler, Karen L; Rice, Amanda D; MacNeill, Amy L; Fukushima, Nobuko; Lindsey, Scott F; Wallace, Greg; Burrage, Andrew M; Smith, Andrew J; Manning, Brandi R; Swetnam, Daniele M; Gray, Stacey A; Moyer, R W; Jacobs, Bertram L

    2011-10-13

    Vaccinia virus deleted for the innate immune evasion gene, E3L, has been shown to be highly attenuated and yet induces a protective immune response against challenge by homologous virus in a mouse model. In this manuscript the NYCBH vaccinia virus vaccine strain was compared to NYCBH vaccinia virus deleted for E3L (NYCBHΔE3L) in a rabbitpox virus (RPV) challenge model. Upon scarification, both vaccines produced a desired skin lesion, although the lesion produced by NYCBHΔE3L was smaller. Both vaccines fully protected rabbits against lethal challenge by escalating doses of RPV, from 10LD(50) to 1000LD(50). A single dose of NYCBHΔE3L protected rabbits from weight loss, fever, and clinical symptoms following the lowest dose challenge of 10LD(50), however it allowed a moderate level of RPV replication at the challenge site, some spread to external skin and mucosal surfaces, and increased numbers of secondary lesions as compared to vaccination with NYCBH. Alternately, two doses of NYCBHΔE3L fully protected rabbits from weight loss, fever, and clinical symptoms, following challenge with 100-1000LD(50) RPV, and it prevented development of secondary lesions similar to protection seen with NYCBH. Finally, vaccination with either one or two doses of NYCBHΔE3L resulted in similar neutralizing antibody titers following RPV challenge as compared to titers obtained by vaccination with NYCBH. These results support the efficacy of the attenuated NYCBHΔE3L in protection against an orthologous poxvirus challenge.

  9. Extent of Systemic Spread Determines CD8+ T Cell Immunodominance for Laboratory Strains, Smallpox Vaccines, and Zoonotic Isolates of Vaccinia Virus.

    PubMed

    Flesch, Inge E A; Hollett, Natasha A; Wong, Yik Chun; Quinan, Bárbara Resende; Howard, Debbie; da Fonseca, Flávio G; Tscharke, David C

    2015-09-01

    CD8(+) T cells that recognize virus-derived peptides presented on MHC class I are vital antiviral effectors. Such peptides presented by any given virus vary greatly in immunogenicity, allowing them to be ranked in an immunodominance hierarchy. However, the full range of parameters that determine immunodominance and the underlying mechanisms remain unknown. In this study, we show across a range of vaccinia virus strains, including the current clonal smallpox vaccine, that the ability of a strain to spread systemically correlated with reduced immunodominance. Reduction in immunodominance was observed both in the lymphoid system and at the primary site of infection. Mechanistically, reduced immunodominance was associated with more robust priming and especially priming in the spleen. Finally, we show this is not just a property of vaccine and laboratory strains of virus, because an association between virulence and immunodominance was also observed in isolates from an outbreak of zoonotic vaccinia virus that occurred in Brazil.

  10. Genetic Analysis of the Vaccinia Virus I6 Telomere-Binding Protein Uncovers a Key Role in Genome Encapsidation

    PubMed Central

    Grubisha, Olivera; Traktman, Paula

    2003-01-01

    The linear, double-stranded DNA genome of vaccinia virus contains covalently closed hairpin termini. These hairpin termini comprise a terminal loop and an A+T-rich duplex stem that has 12 extrahelical bases. DeMasi et al. have shown previously that proteins present in infected cells and in virions form distinct complexes with the telomeric hairpins and that these interactions require the extrahelical bases. The vaccinia virus I6 protein was identified as the protein showing the greatest specificity and affinity for interaction with the viral hairpins (J. DeMasi, S. Du, D. Lennon, and P. Traktman, J. Virol. 75:10090-10105, 2001). To gain insight into the role of I6 in vivo, we generated eight recombinant viruses bearing altered alleles of I6 in which clusters of charged amino acids were changed to alanine residues. One allele (temperature-sensitive I6-12 [tsI6-12]) conferred a tight ts phenotype and was used to examine the stage(s) of the viral life cycle that was affected at the nonpermissive temperature. Gene expression, DNA replication, and genome resolution proceeded normally in this mutant. However, proteolytic processing of structural proteins, which accompanies virus maturation, was incomplete. Electron microscopic studies confirmed a severe block in morphogenesis in which immature, but no mature, virions were observed. Instead, aberrant spherical virions and large crystalloids were seen. When purified, these aberrant virions were found to have normal protein content but to be devoid of viral DNA. We propose that the binding of I6 to viral telomeres directs genome encapsidation into the virus particle. PMID:14512543

  11. Some Attenuated Variants of Vesicular Stomatitis Virus Show Enhanced Oncolytic Activity against Human Glioblastoma Cells relative to Normal Brain Cells▿

    PubMed Central

    Wollmann, Guido; Rogulin, Vitaliy; Simon, Ian; Rose, John K.; van den Pol, Anthony N.

    2010-01-01

    Vesicular stomatitis virus (VSV) has been shown in laboratory studies to be effective against a variety of tumors, including malignant brain tumors. However, attenuation of VSV may be necessary to balance the potential toxicity toward normal cells, particularly when targeting brain tumors. Here we compared 10 recombinant VSV variants resulting from different attenuation strategies. Attenuations included gene shifting (VSV-p1-GFP/RFP), M protein mutation (VSV-M51), G protein cytoplasmic tail truncations (VSV-CT1/CT9), G protein deletions (VSV-dG-GFP/RFP), and combinations thereof (VSV-CT9-M51). Using in vitro viability and replication assays, the VSV variants were grouped into three categories, based on their antitumor activity and non-tumor-cell attenuation. In the first group, wild-type-based VSV-G/GFP, tumor-adapted VSV-rp30, and VSV-CT9 showed a strong antitumor profile but also retained some toxicity toward noncancer control cells. The second group, VSV-CT1, VSV-dG-GFP, and VSV-dG-RFP, had significantly diminished toxicity toward normal cells but showed little oncolytic action. The third group displayed a desired combination of diminished general toxicity and effective antitumor action; this group included VSV-M51, VSV-CT9-M51, VSV-p1-GFP, and VSV-p1-RFP. A member of the last group, VSV-p1-GFP, was then compared in vivo against wild-type-based VSV-G/GFP. Intranasal inoculation of young, postnatal day 16 mice with VSV-p1-GFP showed no adverse neurological effects, whereas VSV-G/GFP was associated with high lethality (80%). Using an intracranial tumor xenograft model, we further demonstrated that attenuated VSV-p1-GFP targets and kills human U87 glioblastoma cells after systemic application. We concluded that some, but not all, attenuated VSV mutants display a favorable oncolytic profile and merit further investigation. PMID:19906910

  12. Biophysical analysis of bacterial and viral systems. A shock tube study of bio-aerosols and a correlated AFM/nanosims investigation of vaccinia virus

    SciTech Connect

    Gates, Sean Damien

    2013-05-01

    The work presented herein is concerned with the development of biophysical methodology designed to address pertinent questions regarding the behavior and structure of select pathogenic agents. Two distinct studies are documented: a shock tube analysis of endospore-laden bio-aerosols and a correlated AFM/NanoSIMS study of the structure of vaccinia virus.

  13. Prime-boost vaccination with plasmid DNA followed by recombinant vaccinia virus expressing BgGARP induced a partial protective immunity to inhibit Babesia gibsoni proliferation in dogs.

    PubMed

    Cao, Shinuo; Mousa, Ahmed Abdelmoniem; Aboge, Gabriel Oluga; Kamyingkird, Ketsarin; Zhou, Mo; Moumouni, Paul Franck Adjou; Terkawi, Mohamad Alaa; Masatani, Tatsunori; Nishikawa, Yoshifumi; Suzuki, Hiroshi; Fukumoto, Shinya; Xuan, Xuenan

    2013-12-01

    A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rvv) vectors expressing relevant antigens has been shown to induce effective immune responses against several infectious pathogens. In this study, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a recombinant plasmid followed by vaccinia virus, both of which expressed the glutamic acid-rich protein (BgGARP) of Babesia gibsoni. The dogs immunized with the prime-boost regime developed a significantly high level of specific antibodies against BgGARP when compared with the control groups. The antibody level was strongly increased after a booster immunization with a recombinant vaccinia virus. Two weeks after the booster immunization with a recombinant vaccinia virus expressing BgGARP, the dogs were challenged with B. gibsoni parasite. The dogs immunized with the prime-boost regime showed partial protection, manifested as a significantly low level of parasitemia. These results indicated that this type of DNA/rvv prime-boost immunization approach may have use against B. gibsoni infection in dogs. PMID:24338330

  14. NPF motifs in the vaccinia virus protein A36 recruit intersectin-1 to promote Cdc42:N-WASP-mediated viral release from infected cells.

    PubMed

    Snetkov, Xenia; Weisswange, Ina; Pfanzelter, Julia; Humphries, Ashley C; Way, Michael

    2016-01-01

    During its egress, vaccinia virus transiently recruits AP-2 and clathrin after fusion with the plasma membrane. This recruitment polarizes the viral protein A36 beneath the virus, enhancing actin polymerization and the spread of infection. We now demonstrate that three NPF motifs in the C-terminus of A36 recruit AP-2 and clathrin by interacting directly with the Epsin15 homology domains of Eps15 and intersectin-1. A36 is the first identified viral NPF motif containing protein shown to interact with endocytic machinery. Vaccinia still induces actin tails in the absence of the A36 NPF motifs. Their loss, however, reduces the cell-to-cell spread of vaccinia. This is due to a significant reduction in virus release from infected cells, as the lack of intersectin-1 recruitment leads to a loss of Cdc42 activation, impairing N-WASP-driven Arp2/3-mediated actin polymerization. Our results suggest that initial A36-mediated virus release plays a more important role than A36-driven super-repulsion in promoting the cell-to-cell spread of vaccinia. PMID:27670116

  15. Use of Vaccinia Virus Smallpox Vaccine in Laboratory and Health Care Personnel at Risk for Occupational Exposure to Orthopoxviruses - Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2015.

    PubMed

    Petersen, Brett W; Harms, Tiara J; Reynolds, Mary G; Harrison, Lee H

    2016-03-18

    On June 25, 2015, the Advisory Committee on Immunization Practices (ACIP) recommended routine vaccination with live smallpox (vaccinia) vaccine (ACAM2000) for laboratory personnel who directly handle 1) cultures or 2) animals contaminated or infected with replication-competent vaccinia virus, recombinant vaccinia viruses derived from replication-competent vaccinia strains (i.e., those that are capable of causing clinical infection and producing infectious virus in humans), or other orthopoxviruses that infect humans (e.g., monkeypox, cowpox, and variola) (recommendation category: A, evidence type 2 [Box]). Health care personnel (e.g., physicians and nurses) who currently treat or anticipate treating patients with vaccinia virus infections and whose contact with replication-competent vaccinia viruses is limited to contaminated materials (e.g., dressings) and persons administering ACAM2000 smallpox vaccine who adhere to appropriate infection prevention measures can be offered vaccination with ACAM2000 (recommendation category: B, evidence type 2 [Box]). These revised recommendations update the previous ACIP recommendations for nonemergency use of vaccinia virus smallpox vaccine for laboratory and health care personnel at risk for occupational exposure to orthopoxviruses (1). Since 2001, when the previous ACIP recommendations were developed, ACAM2000 has replaced Dryvax as the only smallpox vaccine licensed by the U.S. Food and Drug Administration (FDA) and available for use in the United States (2). These recommendations contain information on ACAM2000 and its use in laboratory and health care personnel at risk for occupational exposure to orthopoxviruses.

  16. Use of Vaccinia Virus Smallpox Vaccine in Laboratory and Health Care Personnel at Risk for Occupational Exposure to Orthopoxviruses - Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2015.

    PubMed

    Petersen, Brett W; Harms, Tiara J; Reynolds, Mary G; Harrison, Lee H

    2016-03-18

    On June 25, 2015, the Advisory Committee on Immunization Practices (ACIP) recommended routine vaccination with live smallpox (vaccinia) vaccine (ACAM2000) for laboratory personnel who directly handle 1) cultures or 2) animals contaminated or infected with replication-competent vaccinia virus, recombinant vaccinia viruses derived from replication-competent vaccinia strains (i.e., those that are capable of causing clinical infection and producing infectious virus in humans), or other orthopoxviruses that infect humans (e.g., monkeypox, cowpox, and variola) (recommendation category: A, evidence type 2 [Box]). Health care personnel (e.g., physicians and nurses) who currently treat or anticipate treating patients with vaccinia virus infections and whose contact with replication-competent vaccinia viruses is limited to contaminated materials (e.g., dressings) and persons administering ACAM2000 smallpox vaccine who adhere to appropriate infection prevention measures can be offered vaccination with ACAM2000 (recommendation category: B, evidence type 2 [Box]). These revised recommendations update the previous ACIP recommendations for nonemergency use of vaccinia virus smallpox vaccine for laboratory and health care personnel at risk for occupational exposure to orthopoxviruses (1). Since 2001, when the previous ACIP recommendations were developed, ACAM2000 has replaced Dryvax as the only smallpox vaccine licensed by the U.S. Food and Drug Administration (FDA) and available for use in the United States (2). These recommendations contain information on ACAM2000 and its use in laboratory and health care personnel at risk for occupational exposure to orthopoxviruses. PMID:26985679

  17. Vaccinia viruses isolated from skin infection in horses produced cutaneous and systemic disease in experimentally infected rabbits.

    PubMed

    Cargnelutti, Juliana Felipetto; Schmidt, Candice; Masuda, Eduardo Kenji; Nogueira, Paula Rochelle Kurrle; Weiblen, Rudi; Flores, Eduardo Furtado

    2012-10-01

    The susceptibility of rabbits to two isolates of Vaccinia virus (VACV) recovered from cutaneous disease in horses in Southern Brazil was investigated. Rabbits were inoculated in the ear skin with both VACV isolates, either in single or mixed infection. All inoculated animals presented local skin lesions characterized by hyperaemia, papules, vesicles, pustules and ulcers. Infectious virus was detected in the lungs and intestine of rabbits that died during acute disease. Histological examination of the skin revealed changes characteristic of those associated with members of the genus Orthopoxvirus. These results demonstrate that rabbits develop skin disease accompanied by systemic signs upon intradermal inoculation of these two equine VACV isolates, either alone or in combination, opening the way for using rabbits to study selected aspects of the biology and pathogenesis of VACV infection.

  18. Evaluation of radiation effects against C6 glioma in combination with vaccinia virus-p53 gene therapy

    NASA Technical Reports Server (NTRS)

    Gridley, D. S.; Andres, M. L.; Li, J.; Timiryasova, T.; Chen, B.; Fodor, I.; Nelson, G. A. (Principal Investigator)

    1998-01-01

    The primary objective of this study was to evaluate the antitumor effects of recombinant vaccinia virus-p53 (rVV-p53) in combination with radiation therapy against the C6 rat glioma, a p53 deficient tumor that is relatively radioresistant. VV-LIVP, the parental virus (Lister strain), was used as a control. Localized treatment of subcutaneous C6 tumors in athymic mice with either rVV-p53 or VV-LIVP together with tumor irradiation resulted in low tumor incidence and significantly slower tumor progression compared to the agents given as single modalities. Assays of blood and spleen indicated that immune system activation may account, at least partly, for the enhance tumor inhibition seen with combined treatment. No overt signs of treatment-related toxicity were noted.

  19. The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

    PubMed Central

    Lai, C F; Gong, S C; Esteban, M

    1991-01-01

    The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor. Images PMID:1985213

  20. Immunogenicity and virulence of attenuated vaccinia virus Tian Tan encoding HIV-1 muti-epitope genes, p24 and cholera toxin B subunit in mice.

    PubMed

    Du, Shouwen; Wang, Yuhang; Liu, Cunxia; Wang, Maopeng; Zhu, Yilong; Tan, Peng; Ren, Dayong; Li, Xiao; Tian, Mingyao; Yin, Ronglan; Li, Chang; Jin, Ningyi

    2015-07-01

    No effective prophylactic or therapeutic vaccine against HIV-1 in humans is currently available. This study analyzes the immunogenicity and safety of a recombinant attenuated vaccinia virus. A chimeric gene of HIV-1 multi-epitope genes containing CpG ODN and cholera toxin B subunit (CTB) was inserted into Chinese vaccinia virus Tian Tan strain (VTT) mutant strain. The recombinant virus rddVTT(-CCMp24) was assessed for immunogenicity and safety in mice. Results showed that the protein CCMp24 was expressed stably in BHK-21 infected with rddVTT(-CCMp24). And the recombinant virus induced the production of HIV-1 p24 specific immunoglobulin G (IgG), IL-2 and IL-4. The recombinant vaccine induced γ-interferon secretion against HIV peptides, and elicited a certain levels of immunological memory. Results indicated that the recombinant virus had certain immunogenicity to HIV-1. Additionally, the virulence of the recombinant virus was been attenuated in vivo of mice compared with wild type VTT (wtVTT), and the introduction of CTB and HIV Mp24 did not alter the infectivity and virulence of defective vaccinia virus.

  1. Primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and immunogenicity of recombinant vaccinia viruses in kittens.

    PubMed

    Vennema, H; de Groot, R J; Harbour, D A; Horzinek, M C; Spaan, W J

    1991-03-01

    Feline infectious peritonitis virus (FIPV) causes a mostly fatal, immunologically mediated disease in cats. Previously, we demonstrated that immunization with a recombinant vaccinia virus expressing the FIPV spike protein (S) induced early death after challenge with FIPV (Vennema et al., 1990, J. Virol. 64, 1407-1409). In this paper we describe similar immunizations with the FIPV membrane (M) and nucleocapsid (N) proteins. The genes encoding these proteins were cloned and sequenced. Comparison of the amino acid sequences with the corresponding sequences of porcine transmissible gastroenteritis virus revealed 84.7 and 77% identity for M and N, respectively. Vaccinia virus recombinants expressing the cloned genes induced antibodies in immunized kittens. Immunization with neither recombinant induced early death after challenge with FIPV, strongly suggesting that antibody-dependent enhancement is mediated by antibodies against S only. Immunization with the N protein recombinant had no apparent effect on the outcome of challenge. However, three of eight kittens immunized with the M protein recombinant survived the challenge, as compared to one of eight kittens of the control group.

  2. Vaccinia virus F5 is required for normal plaque morphology in multiple cell lines but not replication in culture or virulence in mice.

    PubMed

    Dobson, Bianca M; Procter, Dean J; Hollett, Natasha A; Flesch, Inge E A; Newsome, Timothy P; Tscharke, David C

    2014-05-01

    Vaccinia virus (VACV) gene F5L was recently identified as a determinant of plaque morphology that is truncated in Modified Vaccinia virus Ankara (MVA). Here we show that F5L also affects plaque morphology of the virulent VACV strain Western Reserve (WR) in some, but not all cell lines, and not via previously described mechanisms. Further, despite a reduction in plaque size for VACV WR lacking F5L there was no evidence of reduced virus replication or spread in vitro or in vivo. In vivo we examined two mouse models, each with more than one dose and measured signs of disease and virus burden. These data provide an initial characterization of VACV F5L in a virulent strain of VACV. Further they show the necessity of testing plaque phenotypes in more than one cell type and provide an example of a VACV gene required for normal plaque morphology but not replication and spread.

  3. Type I interferon mimetics bypass vaccinia virus decoy receptor virulence factor for protection of mice against lethal infection.

    PubMed

    Ahmed, Chulbul M; Johnson, Howard M

    2014-08-01

    The canonical model of interferon (IFN) signaling focuses solely on the activation of STAT transcription factors which, according to the model, are initiated by the singular event of cross-linkage of the receptor extracellular domain by the IFN. The IFN has no further function beyond this. The model thus provides no approach to circumventing poxviruses decoy receptors that compete with the IFN receptors for IFNs. This simple event has allowed smallpox virus to decimate human populations throughout the ages. We have developed a noncanonical model of IFN signaling that has resulted in the development of small peptide mimetics to both types I and II IFNs. In this report, we focus on a type I IFN mimetic at positions 152 to 189, IFN-α1(152-189), which corresponds to the C terminus of human IFN-α1. This mimetic functions intracellularly and is thus not recognized by the B18R vaccinia virus decoy receptor. Mimetic synthesized with an attached palmitate (lipo-) for cell penetration protects mice from a lethal dose of vaccinia virus, while the parent IFN-α1 is ineffective. Unlike IFN-α1, the mimetic does not bind to the B18R decoy receptor. It further differs from the parent IFN in that it lacks the toxicity of weight loss and bone marrow suppression in mice while at the same time possessing a strong adjuvant effect on the immune system. The mimetic is thus an innate and adaptive immune regulator that is evidence of the dynamic nature of the noncanonical model of IFN signaling, in stark contrast to the canonical or classical model of signaling.

  4. Antibiotic-dependent expression of early transcription factor subunits leads to stringent control of vaccinia virus replication.

    PubMed

    Hagen, Caitlin J; Titong, Allison; Sarnoski, Ethan A; Verardi, Paulo H

    2014-03-01

    The use of vaccinia virus (VACV) as the vaccine against variola virus resulted in the eradication of smallpox. VACV has since been used in the development of recombinant vaccine and therapeutic vectors, but complications associated with uncontrolled viral replication have constrained its use as a live viral vector. We propose to improve the safety of VACV as a live-replicating vector by using elements of the tet operon to control the transcription of genes that are essential for viral growth. Poxviruses encode all enzymes and factors necessary for their replication within the host cell cytoplasm. One essential VACV factor is the vaccinia early transcription factor (VETF) packaged into the viral core. This heterodimeric protein is required for expression of early VACV genes. VETF is composed of a large subunit encoded by the A7L gene and a small subunit encoded by the D6R gene. Two recombinant VACVs were generated in which either the A7L or D6R gene was placed under the control of tet operon elements to allow their transcription, and therefore viral replication, to be dependent on tetracycline antibiotics such as doxycycline. In the absence of inducers, no plaques were produced but abortively infected cells could be identified by expression of a reporter gene. In the presence of doxycycline, both recombinant viruses replicated indistinguishably from the wild-type strain. This stringent control of VACV replication can be used for the development of safer, next-generation VACV vaccines and therapeutic vectors. Such replication-inducible VACVs would only replicate when administered with tetracycline antibiotics, and if adverse events were to occur, treatment would be as simple as antibiotic cessation.

  5. The 3'-to-5' exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination.

    PubMed

    Gammon, Don B; Evans, David H

    2009-05-01

    Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3'-to-5' proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3' ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDV(r)) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3'-to-5' exonuclease domain of the viral polymerase. Viruses encoding a CDV(r) mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3' ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3'-to-5' exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

  6. A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases

    PubMed Central

    Zhang, Jianying; Chen, Charlie; Chen, Luxi; Wang, Youwei; Wang, Hongwei; Yi, Long; Elder, J. Bradley; Wang, Qi-En; He, Xiaoming; Kaur, Balveen; Chiocca, E. Antonio; Yu, Jianhua

    2016-01-01

    Breast cancer brain metastases (BCBMs) are common in patients with metastatic breast cancer and indicate a poor prognosis. These tumors are especially resistant to currently available treatments due to multiple factors. However, the combination of chimeric antigen receptor (CAR)-modified immune cells and oncolytic herpes simplex virus (oHSV) has not yet been explored in this context. In this study, NK-92 cells and primary NK cells were engineered to express the second generation of EGFR-CAR. The efficacies of anti-BCBMs of EGFR-CAR NK cells, oHSV-1, and their combination were tested in vitro and in a breast cancer intracranial mouse model. In vitro, compared with mock-transduced NK-92 cells or primary NK cells, EGFR-CAR-engineered NK-92 cells and primary NK cells displayed enhanced cytotoxicity and IFN-γ production when co-cultured with breast cancer cell lines MDA-MB-231, MDA-MB-468, and MCF-7. oHSV-1 alone was also capable of lysing and destroying these cells. However, a higher cytolytic effect of EGFR-CAR NK-92 cells was observed when combined with oHSV-1 compared to the monotherapies. In the mice intracranially pre-inoculated with EGFR-expressing MDA-MB-231 cells, intratumoral administration of either EGFR-CAR-transduced NK-92 cells or oHSV-1 mitigated tumor growth. Notably, the combination of EGFR-CAR NK-92 cells with oHSV-1 resulted in more efficient killing of MDA-MB-231 tumor cells and significantly longer survival of tumor-bearing mice when compared to monotherapies. These results demonstrate that regional administration of EGFR-CAR NK-92 cells combined with oHSV-1 therapy is a potentially promising strategy to treat BCBMs. PMID:27050072

  7. A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases.

    PubMed

    Chen, Xilin; Han, Jianfeng; Chu, Jianhong; Zhang, Lingling; Zhang, Jianying; Chen, Charlie; Chen, Luxi; Wang, Youwei; Wang, Hongwei; Yi, Long; Elder, J Bradley; Wang, Qi-En; He, Xiaoming; Kaur, Balveen; Chiocca, E Antonio; Yu, Jianhua

    2016-05-10

    Breast cancer brain metastases (BCBMs) are common in patients with metastatic breast cancer and indicate a poor prognosis. These tumors are especially resistant to currently available treatments due to multiple factors. However, the combination of chimeric antigen receptor (CAR)-modified immune cells and oncolytic herpes simplex virus (oHSV) has not yet been explored in this context. In this study, NK-92 cells and primary NK cells were engineered to express the second generation of EGFR-CAR. The efficacies of anti-BCBMs of EGFR-CAR NK cells, oHSV-1, and their combination were tested in vitro and in a breast cancer intracranial mouse model. In vitro, compared with mock-transduced NK-92 cells or primary NK cells, EGFR-CAR-engineered NK-92 cells and primary NK cells displayed enhanced cytotoxicity and IFN-γ production when co-cultured with breast cancer cell lines MDA-MB-231, MDA-MB-468, and MCF-7. oHSV-1 alone was also capable of lysing and destroying these cells. However, a higher cytolytic effect of EGFR-CAR NK-92 cells was observed when combined with oHSV-1 compared to the monotherapies. In the mice intracranially pre-inoculated with EGFR-expressing MDA-MB-231 cells, intratumoral administration of either EGFR-CAR-transduced NK-92 cells or oHSV-1 mitigated tumor growth. Notably, the combination of EGFR-CAR NK-92 cells with oHSV-1 resulted in more efficient killing of MDA-MB-231 tumor cells and significantly longer survival of tumor-bearing mice when compared to monotherapies. These results demonstrate that regional administration of EGFR-CAR NK-92 cells combined with oHSV-1 therapy is a potentially promising strategy to treat BCBMs.

  8. A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases.

    PubMed

    Chen, Xilin; Han, Jianfeng; Chu, Jianhong; Zhang, Lingling; Zhang, Jianying; Chen, Charlie; Chen, Luxi; Wang, Youwei; Wang, Hongwei; Yi, Long; Elder, J Bradley; Wang, Qi-En; He, Xiaoming; Kaur, Balveen; Chiocca, E Antonio; Yu, Jianhua

    2016-05-10

    Breast cancer brain metastases (BCBMs) are common in patients with metastatic breast cancer and indicate a poor prognosis. These tumors are especially resistant to currently available treatments due to multiple factors. However, the combination of chimeric antigen receptor (CAR)-modified immune cells and oncolytic herpes simplex virus (oHSV) has not yet been explored in this context. In this study, NK-92 cells and primary NK cells were engineered to express the second generation of EGFR-CAR. The efficacies of anti-BCBMs of EGFR-CAR NK cells, oHSV-1, and their combination were tested in vitro and in a breast cancer intracranial mouse model. In vitro, compared with mock-transduced NK-92 cells or primary NK cells, EGFR-CAR-engineered NK-92 cells and primary NK cells displayed enhanced cytotoxicity and IFN-γ production when co-cultured with breast cancer cell lines MDA-MB-231, MDA-MB-468, and MCF-7. oHSV-1 alone was also capable of lysing and destroying these cells. However, a higher cytolytic effect of EGFR-CAR NK-92 cells was observed when combined with oHSV-1 compared to the monotherapies. In the mice intracranially pre-inoculated with EGFR-expressing MDA-MB-231 cells, intratumoral administration of either EGFR-CAR-transduced NK-92 cells or oHSV-1 mitigated tumor growth. Notably, the combination of EGFR-CAR NK-92 cells with oHSV-1 resulted in more efficient killing of MDA-MB-231 tumor cells and significantly longer survival of tumor-bearing mice when compared to monotherapies. These results demonstrate that regional administration of EGFR-CAR NK-92 cells combined with oHSV-1 therapy is a potentially promising strategy to treat BCBMs. PMID:27050072

  9. Evaluation of pseudorabies virus glycoprotein gp50 as a vaccine for Aujeszky's disease in mice and swine: expression by vaccinia virus and Chinese hamster ovary cells.

    PubMed Central

    Marchioli, C C; Yancey, R J; Petrovskis, E A; Timmins, J G; Post, L E

    1987-01-01

    Pseudorabies virus (PRV) is an alphaherpesvirus which causes an economically important disease of swine. One of the PRV glycoproteins, gp50, was previously identified as the sequence homolog of herpes simplex virus glycoprotein gD (E.A. Petrovskis, J.G. Timmins, M.A. Armentrout, C.C. Marchioli, R.J. Yancey, Jr., and L.E. Post, J. Virol. 59:216-223, 1986). gp50 was evaluated as a PRV subunit vaccine candidate. gp50 protected mice from PRV-induced mortality either when delivered via infection with a recombinant vaccinia virus or when administered as a subunit vaccine produced in a eucaryotic cell line, Chinese hamster ovary (CHO) cells. In addition, gp50 synthesized in CHO cells protected pigs from lethal infection with PRV. This result demonstrates that a single viral glycoprotein could induce a protective immune response in the natural host of a herpesvirus infection. Images PMID:2824827

  10. Protection of Mice from Fatal Measles Encephalitis by Vaccination with Vaccinia Virus Recombinants Encoding Either the Hemagglutinin or the Fusion Protein

    NASA Astrophysics Data System (ADS)

    Drillien, Robert; Spehner, Daniele; Kirn, Andre; Giraudon, Pascale; Buckland, Robin; Wild, Fabian; Lecocq, Jean-Pierre

    1988-02-01

    Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.

  11. Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge.

    PubMed Central

    Lawson, C M; Bennink, J R; Restifo, N P; Yewdell, J W; Murphy, B R

    1994-01-01

    The nucleoprotein (NP) of influenza A virus is the dominant antigen recognized by influenza virus-specific cytotoxic T lymphocytes (CTLs), and adoptive transfer of NP-specific CTLs protects mice from influenza A virus infection. BALB/c mouse cells (H-2d) recognize a single Kd-restricted CTL epitope of NP consisting of amino acids 147 to 155. In the present study, mice were immunized with various vaccinia virus recombinant viruses to examine the effect of the induction of primary pulmonary CTLs on resistance to challenge with influenza A/Puerto Rico/8/34 virus. The minigene ESNP(147-155)-VAC construct, composed of a signal sequence from the adenovirus E3/19K glycoprotein (designated ES) and expressing the 9-amino-acid NP natural determinant (amino acids 147 to 155) preceded by an alanine residue, a similar minigene NP(Met 147-155)-VAC lacking ES, and a full-length NP-VAC recombinant of influenza virus were analyzed. The two minigene NP-VAC recombinants induced a greater primary pulmonary CTL response than the full-length NP-VAC recombinant. However, NP-specific CTLs induced by immunization with ESNP(147-155)-VAC did not decrease peak virus titer or accelerate clearance of virus in the lungs of mice challenged intranasally with A/PR/8/34. Furthermore, NP-specific CTLs induced by immunization did not protect mice challenged intranasally with a lethal dose of A/PR/8/34. Sequence analysis of the NP CTL epitope of A/PR/8/34 challenge virus obtained from lungs after 8 days of replication in ESNP(147-155)-VAC-immunized mice showed identity with that of the input virus, demonstrating that an escape mutant had not emerged during replication in vivo. Thus, in contrast to adoptively transferred CTLs, pulmonary NP-specific CTLs induced by recombinant vaccinia virus immunization do not have protective in vivo antiviral activity against influenza virus infection. PMID:7514677

  12. Kinetics and intracellular location of intramolecular disulfide bond formation mediated by the cytoplasmic redox system encoded by vaccinia virus

    SciTech Connect

    Bisht, Himani; Brown, Erica; Moss, Bernard

    2010-03-15

    Poxviruses encode a redox system for intramolecular disulfide bond formation in cytoplasmic domains of viral proteins. Our objectives were to determine the kinetics and intracellular location of disulfide bond formation. The vaccinia virus L1 myristoylated membrane protein, used as an example, has three intramolecular disulfide bonds. Reduced and disulfide-bonded forms of L1 were distinguished by electrophoretic mobility and reactivity with monoclonal and polyclonal antibodies. Because disulfide bonds formed during 5 min pulse labeling with radioactive amino acids, a protocol was devised in which dithiothreitol was present at this step. Disulfide bond formation was detected by 2 min after removal of reducing agent and was nearly complete in 10 min. When the penultimate glycine residue was mutated to prevent myristoylation, L1 was mistargeted to the endoplasmic reticulum and disulfide bond formation failed to occur. These data suggested that viral membrane association was required for oxidation of L1, providing specificity for the process.

  13. Structural analysis of the extracellular domain of vaccinia virus envelope protein, A27L, by NMR and CD spectroscopy.

    PubMed

    Lin, Ta-Hsien; Chia, Chih-Ming; Hsiao, Jye-Chian; Chang, Wen; Ku, Chiao-Chu; Hung, Shang-Cheng; Tzou, Der-Lii M

    2002-06-01

    This study presents the molecular structure of the extracellular domain of vaccinia virus envelope protein, A27L, determined by NMR and CD spectroscopy. A recombinant protein, eA27L-aa, containing this domain in which cysteines 71 and 72 were replaced with alanine, was constructed to prevent self-assembly due to intermolecular disulfide bonds between these two cysteines. The soluble eA27L-aa protein forms an oligomer resembling that of A27L on vaccinia virions. Heteronuclear correlation NMR spectroscopy was carried out on eA27L-aa in the presence or absence of urea to determine backbone resonance assignments. Chemical shift index (CSI) propensity analysis showed that eA27L-aa has two distinct structural domains, a relatively flexible 22-amino acid random coil in the N-terminal region and a fairly rigid alpha-helix structure in the remainder of the structure. Binding interaction studies using isothermal titration calorimetry suggest that a 12-amino acid lysine/arginine-rich segment in the N-terminal region is responsible for glycosaminoglycan binding. The rigid alpha-helix portion of eA27L-aa is probably involved in the intrinsic self-assembly, and CSI propensity analysis suggests that region N37-E49, with a residual alpha-helix tendency, is probably the self-assembly core. Self-assembly was ascribed to three hydrophobic leucine residues (Leu(41), Leu(45), and Leu(48)) in this segment. The folding mechanism of eA27L-aa was analyzed by CD spectroscopy, which revealed a two-step transition with a Gibbs free energy of 2.5 kcal/mol in the absence of urea. Based on these NMR and CD studies, a residue-specific molecular model of the extracellular domain of A27L is proposed. These studies on the molecular structure of eA27L-aa will help in understanding how vaccinia virus enters cells.

  14. Tyrosine Phosphorylation of A17 during Vaccinia Virus Infection: Involvement of the H1 Phosphatase and the F10 Kinase

    PubMed Central

    Derrien, M.; Punjabi, A.; Khanna, M.; Grubisha, O.; Traktman, P.

    1999-01-01

    Vaccinia virus encodes two protein kinases (B1 and F10) and a dual-specificity phosphatase (VH1), suggesting that phosphorylation and dephosphorylation of substrates on serine/threonine and tyrosine residues are important in regulating diverse aspects of the viral life cycle. Using a recombinant in which expression of the H1 phosphatase can be regulated experimentally (vindH1), we have previously demonstrated that repression of H1 leads to the maturation of noninfectious virions that contain several hyperphosphorylated substrates (K. Liu et al., J. Virol. 69:7823–7834). In this report, we demonstrate that among these is a 25-kDa protein that is phosphorylated on tyrosine residues in H1-deficient virions and can be dephosphorylated by recombinant H1. We demonstrate that the 25-kDa phosphoprotein represents the product of the A17 gene and that A17 is phosphorylated on serine, threonine, and tyrosine residues during infection. Detection of phosphotyrosine within A17 is abrogated when Tyr203 (but not Tyr3, Tyr6, or Tyr7) is mutated to phenylalanine, suggesting strongly that this amino acid is the site of tyrosine phosphorylation. Phosphorylation of A17 fails to occur during nonpermissive infections performed with temperature-sensitive mutants defective in the F10 kinase. Our data suggest that this enzyme, which was initially characterized as a serine/threonine kinase, might in fact have dual specificity. This hypothesis is strengthened by the observation that Escherichia coli induced to express F10 contain multiple proteins which are recognized by antiphosphotyrosine antiserum. This study presents the first evidence for phosphotyrosine signaling during vaccinia virus infection and implicates the F10 kinase and the H1 phosphatase as the dual-specificity enzymes that direct this cycle of reversible phosphorylation. PMID:10438817

  15. Characterization of the vaccinia virus D10 decapping enzyme provides evidence for a two-metal-ion mechanism.

    PubMed

    Soulière, Marie F; Perreault, Jean-Pierre; Bisaillon, Martin

    2009-05-15

    Decapping enzymes are required for the removal of the 5'-end cap of mRNAs. These enzymes exhibit a specific hydrolase activity, resulting in cleavage between the alpha- and beta-phosphates of the m7GpppN cap to generate both m7GDP and monophosphorylated RNA products. Decapping enzymes have been found in humans, plants and yeasts, and have been discovered more recently in vaccinia virus (D10 protein). Although experimental evidences are lacking, three-metal- and two-metal-ion mechanisms have been proposed so far for the decapping enzymes. In the present study, we performed a biochemical characterization of the interaction of bivalent cations with the vaccinia virus D10 protein. Synergistic activation of the enzyme was observed in the presence of Mg2+ and Mn2+ ions, suggesting the existence of two metal-ion-binding sites on the D10 protein. Moreover, dual-ligand titration experiments using fluorescence spectroscopy demonstrated the presence of two metal-ion-binding sites on the enzyme. A three-dimensional structural model of the active site of the enzyme was generated which highlighted the importance of three glutamate residues involved in the co-ordination of two metal ions and a water molecule. Mutational analyses confirmed the role of two glutamate residues for the binding of metal ions. We demonstrate that one metal ion is co-ordinated by Glu132, while the second metal ion is co-ordinated by Glu145. Taken together, these results support the proposed two-metal-ion mechanistic model for the D10 decapping enzyme.

  16. Vaccinia virus B1 kinase: phenotypic analysis of temperature-sensitive mutants and enzymatic characterization of recombinant proteins.

    PubMed Central

    Rempel, R E; Traktman, P

    1992-01-01

    The vaccinia virus B1 gene encodes a 34-kDa protein with homology to protein kinases. In L cells infected nonpermissively with mutants containing lesions in the B1 gene (ts2 and ts25), the infectious cycle arrests prior to DNA replication. In this report, we demonstrate that DNA synthesis ceases when cultures infected with these mutants at 32 degrees C are shifted to the nonpermissive temperature (39.5 degrees C) in the midst of DNA replication. We also show that B1 protein is synthesized transiently during the early phase of infection, even when the progression to later stages of gene expression is prevented. Although wild-type (wt) B1 is stable, the ts B1 proteins are markedly labile in both L and BSC40 cells at both permissive and nonpermissive temperatures. These results suggest that the ts phenotype of the mutants is complex and may in part reflect a temperature-dependent requirement for kinase activity, an induction of temperature sensitivity in B1 substrates under nonpermissive conditions, and/or ts complementation by host factors. To facilitate biochemical analyses, recombinant wt B1, ts2 B1, and ts25 B1 were produced in Escherichia coli. The wt protein was able to phosphorylate serine and threonine residues on several exogenous substrates in vitro. The activity of ts25 B1 was 3% that of the wt enzyme, and no detectable kinase activity was associated with ts2 B1. In light of the inactivity of the ts2 B1 protein in vitro and its extreme lability in vivo, we attempted to isolate a vaccinia virus B1 null mutant by targeted interruption of the B1 gene at 32 degrees C. No null mutants were isolated. These results indicate that the B1 protein kinase provides a vital function which cannot be supplied by the host or circumvented by incubation at 32 degrees C. Images PMID:1602551

  17. Detection of Vaccinia Virus in Milk: Evidence of a Systemic and Persistent Infection in Experimentally Infected Cows.

    PubMed

    de Oliveira, Tércia Moreira Ludolfo; Guedes, Maria Isabel Maldonado Coelho; Rehfeld, Izabelle Silva; Matos, Ana Carolina Diniz; Rivetti, Anselmo Vasconcelos; Alves, Pedro Augusto; Galinari, Grazielle Cossenzo Florentino; Cerqueira, Mônica Maria Oliveira Pinho; Abrahão, Jônatas Santos; Lobato, Zélia Inês Portela

    2015-11-01

    Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects lactating cows and milkers. VACV DNA and infectious particles have been detected in milk of naturally infected cows. However, the period and pattern of VACV shedding in milk is unknown, as is whether the presence of VACV in milk is due to a localized or a systemic infection. To address those questions, eight lactating cows were inoculated with VACV in previously scarified teats. The experiment was divided in two phases. In Phase 1, milk samples were collected daily for 33 days, and in Phase 2, four animals from the first phase were immunosuppressed. In both phases, milk was collected with a sterile catheter on even days and by hand milking on odd days. All animals showed typical BV lesions in the inoculated teats. All milk samples were subjected to nested polymerase chain reaction (PCR) and real-time quantitative PCR to detect VACV DNA. PCR-positive samples were subjected to virus isolation. VACV DNA was intermittently detected in milk in both phases and infectious viral particles could be detected only in phase 2, on the 69th, 73rd, 74th, 77th, 79th, and 81st days postinfection. Despite the possibility of propagation of VACV through milk, it is known that milk continues to be drawn and marketed normally during outbreaks of the disease. The detection of both VACV DNA and infectious particles in milk samples draws attention to the potential public health risk associated with the consumption of milk from BV outbreaks. Detection of VACV in the milk from noninfected teats demonstrated that VACV shedding in milk might be related to a systemic infection. Moreover, it was shown that VACV DNA and viral infectious particles could be detected in milk even after healing of the lesions, demonstrating that VACV may cause a persistent infection in cattle.

  18. The amino terminus of the vaccinia virus E3 protein is necessary to inhibit the interferon response.

    PubMed

    White, Stacy D; Jacobs, Bertram L

    2012-05-01

    Vaccinia virus (VACV) encodes a multifunctional protein, E3L, that is necessary for interferon (IFN) resistance in cells in culture. Interferon resistance has been mapped to the well-characterized carboxy terminus of E3L, which contains a conserved double-stranded RNA binding domain. The amino terminus of E3L has a Z-form nucleic acid binding domain, which has been shown to be dispensable for replication and IFN resistance in HeLa and RK13 cells; however, a virus expressing E3L deleted of the amino terminus has reduced pathogenicity in an animal model. In this study, we demonstrate that the pathogenicity of a virus expressing E3L deleted of the amino terminus was fully rescued in type I IFN receptor knockout (IFN-α/βR(-/-)) mice. Furthermore, this virus was IFN sensitive in primary mouse embryo fibroblasts (MEFs). This virus induced the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) in MEFs in an IFN-dependent manner. The depletion of double-stranded RNA-dependent protein kinase (PKR) from these MEFs restored the IFN resistance of this virus. Furthermore, the virus expressing E3L deleted of the amino terminus was also IFN resistant in PKR(-/-) MEFs. Thus, our data demonstrate that the amino terminus of E3L is necessary to inhibit the type I IFN response both in mice and in MEFs and that in MEFs, the amino terminus of E3L functions to inhibit the PKR pathway.

  19. Vaccinia virus protein K7 is a virulence factor that alters the acute immune response to infection.

    PubMed

    Benfield, Camilla T O; Ren, Hongwei; Lucas, Stuart J; Bahsoun, Basma; Smith, Geoffrey L

    2013-07-01

    Vaccinia virus (VACV) encodes many proteins that antagonize the innate immune system including a family of intracellular proteins with a B-cell lymphoma (Bcl)-2-like structure. One of these Bcl-2 proteins called K7 binds Toll-like receptor-adaptor proteins and the DEAD-box RNA helicase DDX3 and thereby inhibits the activation of NF-κB and interferon regulatory factor 3. However, the contribution of K7 to virus virulence is not known. Here a VACV lacking the K7R gene (vΔK7) was constructed and compared with control viruses that included a plaque purified wt (vK7), a revertant with the K7R gene reinserted (vK7-rev) and a frame-shifted virus in which the translational initiation codon was mutated to prevent K7 protein expression (vK7-fs). Data presented show that loss of K7 does not affect virus replication in cell culture or in vivo; however, viruses lacking the K7 protein were less virulent than controls in murine intradermal (i.d.) and intranasal (i.n.) infection models and there was an altered acute immune response to infection. In the i.d. model, vΔK7 induced smaller lesions than controls, and after i.n. infection vΔK7 induced a reduced weight loss and signs of illness, and more rapid clearance of virus from infected tissue. Concomitantly, the intrapulmonary innate immune response to infection with vΔK7 showed increased infiltration of NK cells and CD8⁺ T-cells, enhanced MHC class II expression by macrophages, and enhanced cytolysis of target cells by NK cells and VACV-specific CD8⁺ T-cells. Thus protein K7 is a virulence factor that affects the acute immune response to infection.

  20. Nucleotide sequence analysis of the bovine respiratory syncytial virus fusion protein mRNA and expression from a recombinant vaccinia virus.

    PubMed

    Lerch, R A; Anderson, K; Amann, V L; Wertz, G W

    1991-03-01

    inserted into the thymidine kinase gene of vaccinia virus and following homologous recombination, a recombinant virus containing the BRS virus F gene was isolated. The BRS virus F protein was expressed in recombinant virus infected cells as demonstrated by immunoprecipitation and was transported to and expressed on the surface of infected cells as shown by indirect immunofluorescence.

  1. Complete genome sequence analysis of Seneca Valley virus-001, a novel oncolytic picornavirus.

    PubMed

    Hales, Laura M; Knowles, Nick J; Reddy, P Seshidar; Xu, Ling; Hay, Carl; Hallenbeck, Paul L

    2008-05-01

    The complete genome sequence of Seneca Valley virus-001 (SVV-001), a small RNA virus, was determined and was shown to have typical picornavirus features. The 7280 nt long genome was predicted to contain a 5' untranslated region (UTR) of 666 nt, followed by a single long open reading frame consisting of 6543 nt, which encodes a 2181 aa polyprotein. This polyprotein could potentially be cleaved into 12 polypeptides in the standard picornavirus L-4-3-4 layout. A 3' UTR of 71 nt was followed by a poly(A) tail of unknown length. Comparisons with other picornaviruses showed that the P1, 2C, 3C and 3D polypeptides of SVV-001 were related most closely to those of the cardioviruses, although they were not related as closely to those of encephalomyocarditis virus and Theiler's murine encephalomyelitis virus as the latter were to each other. Most other regions of the polyprotein differed considerably from those of all other known picornaviruses. SVV-001 contains elements of an internal ribosome entry site reminiscent of that found in hepatitis C virus and a number of genetically diverse picornaviruses. SVV-001 is a novel picornavirus and it is proposed that it be classified as the prototype species in a novel genus named 'Senecavirus'.

  2. Long-Term Sterilizing Immunity to Rinderpest in Cattle Vaccinated with a Recombinant Vaccinia Virus Expressing High Levels of the Fusion and Hemagglutinin Glycoproteins

    PubMed Central

    Verardi, Paulo H.; Aziz, Fatema H.; Ahmad, Shabbir; Jones, Leslie A.; Beyene, Berhanu; Ngotho, Rosemary N.; Wamwayi, Henry M.; Yesus, Mebratu G.; Egziabher, Berhe G.; Yilma, Tilahun D.

    2002-01-01

    Rinderpest is an acute and highly contagious viral disease of ruminants, often resulting in greater than 90% mortality. We have constructed a recombinant vaccinia virus vaccine (v2RVFH) that expresses both the fusion (F) and hemagglutinin (H) genes of rinderpest virus (RPV) under strong synthetic vaccinia virus promoters. v2RVFH-infected cells express high levels of the F and H glycoproteins and show extensive syncytium formation. Cattle vaccinated intramuscularly with as little as 103 PFU of v2RVFH and challenged 1 month later with a lethal dose of RPV were completely protected from clinical disease; the 50% protective dose was determined to be 102 PFU. Animals vaccinated with v2RVFH did not develop pock lesions and did not transmit the recombinant vaccinia virus to contact animals. Intramuscular vaccination of cattle with 108 PFU of v2RVFH provided long-term sterilizing immunity against rinderpest. In addition to being highly safe and efficacious, v2RVFH is a heat-stable, inexpensive, and easily administered vaccine that allows the serological differentiation between vaccinated and naturally infected animals. Consequently, mass vaccination of cattle with v2RVFH could eradicate rinderpest. PMID:11752138

  3. Structure Function Studies of Vaccinia Virus Host Range Protein K1 Reveal a Novel Functional Surface for Ankyrin Repeat Proteins

    SciTech Connect

    Li, Yongchao; Meng, Xiangzhi; Xiang, Yan; Deng, Junpeng

    2010-06-15

    Poxvirus host tropism at the cellular level is regulated by virus-encoded host range proteins acting downstream of virus entry. The functioning mechanisms of most host range proteins are unclear, but many contain multiple ankyrin (ANK) repeats, a motif that is known for ligand interaction through a concave surface. We report here the crystal structure of one of the ANK repeat-containing host range proteins, the vaccinia virus K1 protein. The structure, at a resolution of 2.3 {angstrom}, showed that K1 consists entirely of ANK repeats, including seven complete ones and two incomplete ones, one each at the N and C terminus. Interestingly, Phe82 and Ser83, which were previously shown to be critical for K1's function, are solvent exposed and located on a convex surface, opposite the consensus ANK interaction surface. The importance of this convex surface was further supported by our additional mutagenesis studies. We found that K1's host range function was negatively affected by substitution of either Asn51 or Cys47 and completely abolished by substitution of both residues. Cys47 and Asn51 are also exposed on the convex surface, spatially adjacent to Phe82 and Ser83. Altogether, our data showed that K1 residues on a continuous convex ANK repeat surface are critical for the host range function, suggesting that K1 functions through ligand interaction and does so with a novel ANK interaction surface.

  4. Enhancement of CD8(+) T-cell memory by removal of a vaccinia virus nuclear factor-κB inhibitor.

    PubMed

    Ren, Hongwei; Ferguson, Brian J; Maluquer de Motes, Carlos; Sumner, Rebecca P; Harman, Laura E R; Smith, Geoffrey L

    2015-05-01

    Factors influencing T-cell responses are important for vaccine development but are incompletely understood. Here, vaccinia virus (VACV) protein N1 is shown to impair the development of both effector and memory CD8(+) T cells and this correlates with its inhibition of nuclear factor-κB (NF-κB) activation. Infection with VACVs that either have the N1L gene deleted (vΔN1) or contain a I6E mutation (vN1.I6E) that abrogates its inhibition of NF-κB resulted in increased central and memory CD8(+) T-cell populations, increased CD8(+) T-cell cytotoxicity and lower virus titres after challenge. Furthermore, CD8(+) memory T-cell function was increased following infection with vN1.I6E, with more interferon-γ production and greater protection against VACV infection following passive transfer to naive mice, compared with CD8(+) T cells from mice infected with wild-type virus (vN1.WT). This demonstrates the importance of NF-κB activation within infected cells for long-term CD8(+) T-cell memory and vaccine efficacy. Further, it provides a rationale for deleting N1 from VACV vectors to enhance CD8(+) T-cell immunogenicity, while simultaneously reducing virulence to improve vaccine safety.

  5. Vaccinia Virus N1l Protein Resembles a B Cell Lymphoma-2 (Bcl-2) Family Protein

    SciTech Connect

    Aoyagi, M.; Zhai, D.; Jin, C.; Aleshin, A.E.; Stec, B.; Reed, J.C.; Liddington, R.C.; /Burnham Inst.

    2007-07-03

    Poxviruses encode immuno-modulatory proteins capable of subverting host defenses. The poxvirus vaccinia expresses a small 14-kDa protein, N1L, that is critical for virulence. We report the crystal structure of N1L, which reveals an unexpected but striking resemblance to host apoptotic regulators of the B cell lymphoma-2 (Bcl-2) family. Although N1L lacks detectable Bcl-2 homology (BH) motifs at the sequence level, we show that N1L binds with high affinity to the BH3 peptides of pro-apoptotic Bcl-2 family proteins in vitro, consistent with a role for N1L in modulating host antiviral defenses.

  6. Ultraviolet-irradiated vaccinia virus recombinants, exposing HIV-envelope on their outer membrane, induce antibodies against this antigen in rabbits.

    PubMed

    Loewinger, M; Katz, E

    2002-01-01

    The construction and isolation of recombinants of vaccinia virus (IHD-J strain), bearing on their outer membrane a chimeric protein consisting of the cytoplasmic and transmembrane domains of vaccinia B5R protein and the external domain of HIV envelope, has been previously described by us. The present study aimed to investigate the potential use of such recombinants as a vaccine, following inactivation of their infectivity by ultraviolet (UV) irradiation. The minimal dose of UV irradiation, required for the complete inactivation of the infectivity of these recombinants, was determined. Injections of rabbits with the irradiated noninfectious recombinant viruses successfully induced specific antibodies against the HIV envelope antigen, in addition to those against the poxvirus. PMID:12479396

  7. Multiple 3' ends of mRNA encoding vaccinia virus growth factor occur within a series of repeated sequences downstream of T clusters.

    PubMed

    Yuen, L; Moss, B

    1986-10-01

    Analysis of the 5' ends of six apparently full-length cloned cDNA copies of the vaccinia virus growth factor gene suggested precise transcriptional initiation at the first purine following a run of five pyrimidines in the noncoding strand. By contrast, the 3' ends exhibited heterogeneity and were distributed over a 46-base-pair region. In each of the six cDNAs, the nucleotide immediately preceding the retained copy of the poly(A) tail corresponded to the first or second T of a TATGT repeat. Clusters of Ts occurred upstream of the 3' ends, but the AATAAA polyadenylation consensus sequence of higher eucaryotes was absent. Determination of the complete sequence of one cDNA revealed exact correspondence with the vaccinia virus growth factor gene, indicating the absence of internal RNA processing.

  8. The combined effects of oncolytic reovirus plus Newcastle disease virus and reovirus plus parvovirus on U87 and U373 cells in vitro and in vivo.

    PubMed

    Alkassar, Muhannad; Gärtner, Barbara; Roemer, Klaus; Graesser, Friedrich; Rommelaere, Jean; Kaestner, Lars; Haeckel, Isabelle; Graf, Norbert

    2011-09-01

    Previous results had documented oncolytic capacity of reovirus, parvovirus and Newcastle disease virus (NDV) on several tumor cell types. To test whether combinations of these viruses may increase this capacity, human U87- and U373-glioblastoma cells, in vitro or xenografted into immuno-compromised mice, were subjected to simultaneous double infections and analyzed. Our results show that reovirus (serotype-3) plus NDV (Hitcher-B1) and reovirus plus parvovirus-H1 lead to a significant increase in tumor cell killing in vitro in both cell lines (Kruskal-Wallis test, P < 0.01) and in vivo. Immunofluorescence and flow cytometry analyses demonstrated the simul