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Sample records for oncolytic vaccinia virus

  1. Anti-tumour activity of oncolytic Western Reserve vaccinia viruses in canine tumour cell lines, xenografts, and fresh tumour biopsies.

    PubMed

    Autio, K; Knuuttila, A; Kipar, A; Ahonen, M; Parviainen, S; Diaconu, I; Kanerva, A; Hakonen, T; Vähä-Koskela, M; Hemminki, A

    2014-10-10

    Cancer is one of the most common reasons for death in dogs. One promising approach is oncolytic virotherapy. We assessed the oncolytic effect of genetically modified vaccinia viruses in canine cancer cells, in freshly excised tumour biopsies, and in mice harbouring canine tumour xenografts. Tumour transduction efficacy was assessed using virus expressing luciferase or fluorescent marker genes and oncolysis was quantified by a colorimetric cell viability assay. Oncolytic efficacy in vivo was evaluated in a nude mouse xenograft model. Vaccinia virus was shown to infect most tested canine cancer cell lines and primary surgical tumour tissues. Virus infection significantly reduced tumour growth in the xenograft model. Oncolytic vaccinia virus has antitumour effects against canine cancer cells and experimental tumours and is able to replicate in freshly excised patient tumour tissue. Our results suggest that oncolytic vaccinia virus may offer an effective treatment option for otherwise incurable canine tumours. PMID:25302859

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

    PubMed

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

    2015-10-01

    Metastatic colorectal cancer (CRC) is complex clinical challenge for which there are limited treatment options. Chemotherapy with or without surgery provides moderate improvements in overall survival and quality of life; nevertheless the 5-year survival remains below 30%. Oncolytic vaccinia virus (VV) shows strong anti-tumour activity in models of CRC, however transient delays in disease progression are insufficient to lead to long-term survival. Here we examined the efficacy of VV with oxaliplatin or SN-38 (active metabolite of irinotecan) in CRC cell lines in vitro and VV with irinotecan in an orthotopic model of metastatic CRC. Synergistic improvements in in vitro cell killing were observed in multiple cell lines. Combination therapy was well tolerated in tumour-bearing mice and the median survival was significantly increased relative to monotherapy despite a drug-dependent decrease in the mean tumour titer. Increased apoptosis following in vitro and in vivo combination therapy was observed. In vitro cell cycle analysis showed increases in S-phase cells following infection occurred in both infected and uninfected cell populations. This corresponded to a 4-fold greater increase in apoptosis in the uninfected compared to infected cells following combination therapy. Combination treatment strategies are among the best options for patients with advanced cancers. VV is currently under clinical investigation in patients with CRC and the data presented here suggest that its combination with irinotecan may provide benefit to a subset of CRC patients. Further, investigation of this combination is necessary to determine the tumour characteristics responsible for mediating synergy. PMID:26004084

  3. Complement inhibition prevents oncolytic vaccinia virus neutralization in immune humans and cynomolgus macaques.

    PubMed

    Evgin, Laura; Acuna, Sergio A; Tanese de Souza, Christiano; Marguerie, Monique; Lemay, Chantal G; Ilkow, Carolina S; Findlay, C Scott; Falls, Theresa; Parato, Kelley A; Hanwell, David; Goldstein, Alyssa; Lopez, Roberto; Lafrance, Sandra; Breitbach, Caroline J; Kirn, David; Atkins, Harold; Auer, Rebecca C; Thurman, Joshua M; Stahl, Gregory L; Lambris, John D; Bell, John C; McCart, J Andrea

    2015-06-01

    Oncolytic viruses (OVs) have shown promising clinical activity when administered by direct intratumoral injection. However, natural barriers in the blood, including antibodies and complement, are likely to limit the ability to repeatedly administer OVs by the intravenous route. We demonstrate here that for a prototype of the clinical vaccinia virus based product Pexa-Vec, the neutralizing activity of antibodies elicited by smallpox vaccination, as well as the anamnestic response in hyperimmune virus treated cancer patients, is strictly dependent on the activation of complement. In immunized rats, complement depletion stabilized vaccinia virus in the blood and led to improved delivery to tumors. Complement depletion also enhanced tumor infection when virus was directly injected into tumors in immunized animals. The feasibility and safety of using a complement inhibitor, CP40, in combination with vaccinia virus was tested in cynomolgus macaques. CP40 pretreatment elicited an average 10-fold increase in infectious titer in the blood early after the infusion and prolonged the time during which infectious virus was detectable in the blood of animals with preexisting immunity. Capitalizing on the complement dependence of antivaccinia antibody with adjunct complement inhibitors may increase the infectious dose of oncolytic vaccinia virus delivered to tumors in virus in immune hosts. PMID:25807289

  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. PMID:26803055

  7. T-cell Engager-armed Oncolytic Vaccinia Virus Significantly Enhances Antitumor Therapy

    PubMed Central

    Yu, Feng; Wang, Xingbing; Guo, Z Sheng; Bartlett, David L; Gottschalk, Stephen M; Song, Xiao-Tong

    2014-01-01

    Oncolytic vaccinia virus (VV) therapy has shown promise in preclinical models and in clinical studies. However, complete responses have rarely been observed. This lack of efficacy is most likely due to suboptimal virus spread through the tumor resulting in limited tumor cell destruction. We reasoned that redirecting T cells to the tumor has the potential to improve the antitumor activity of oncolytic VVs. We, therefore, constructed a VV encoding a secretory bispecific T-cell engager consisting of two single- chain variable fragments specific for CD3 and the tumor cell surface antigen EphA2 (EphA2-T-cell engager-armed VV (EphA2-TEA-VV)). In vitro, EphA2-TEA-VV's ability to replicate and induce oncolysis was similar to that of unmodified virus. However, only tumor cells infected with EphA2-TEA-VV induced T-cell activation as judged by the secretion of interferon-γ and interleukin-2. In coculture assays, EphA2-TEA-VV not only killed infected tumor cells, but in the presence of T cells, it also induced bystander killing of noninfected tumor cells. In vivo, EphA2-TEA-VV plus T cells had potent antitumor activity in comparison with control VV plus T cells in a lung cancer xenograft model. Thus, arming oncolytic VVs with T-cell engagers may represent a promising approach to improve oncolytic virus therapy. PMID:24135899

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

    PubMed Central

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

    2016-01-01

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

  9. Inducible Gene Expression in Tumors Colonized by Modified Oncolytic Vaccinia Virus Strains

    PubMed Central

    Huppertz, Sascha; Zhang, Qian; Geissinger, Ulrike; Härtl, Barbara; Gentschev, Ivaylo

    2014-01-01

    ABSTRACT Exogenous gene induction of therapeutic, diagnostic, and safety mechanisms could be a considerable improvement in oncolytic virotherapy. Here, we introduced a doxycycline-inducible promoter system (comprised of a tetracycline repressor, several promoter constructs, and a tet operator sequence) into oncolytic recombinant vaccinia viruses (rVACV), which were further characterized in detail. Experiments in cell cultures as well as in tumor-bearing mice were analyzed to determine the role of the inducible-system components. To accomplish this, we took advantage of the optical reporter construct, which resulted in the production of click-beetle luciferase as well as a red fluorescent protein. The results indicated that each of the system components could be used to optimize the induction rates and had an influence on the background expression levels. Depending on the given gene to be induced in rVACV-colonized tumors of patients, we discuss the doxycycline-inducible promoter system adjustment and further optimization. IMPORTANCE Oncolytic virotherapy of cancer can greatly benefit from the expression of heterologous genes. It is reasonable that some of those heterologous gene products could have detrimental effects either on the cancer patient or on the oncolytic virus itself if they are expressed at the wrong time or if the expression levels are too high. Therefore, exogenous control of gene expression levels by administration of a nontoxic inducer will have positive effects on the safety as well as the therapeutic outcome of oncolytic virotherapy. In addition, it paves the way for the introduction of new therapeutic genes into the genome of oncolytic viruses that could not have been tested otherwise. PMID:25056902

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-04-01

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

  12. 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

  13. Oncolytic Immunotherapy Using Recombinant Vaccinia Virus GLV-1h68 Efficiently Kills Sorafenib-Resistant Hepatocellular Carcinoma

    PubMed Central

    Ady, Justin W.; Heffner, Jacqueline; Mojica, Kelly; Johnsen, Clark; Belin, Laurence J.; Love, Damon; Pugalenthi, Amudhan; Klein, Elizabeth; Chen, Nanhai G.; Yu, Yong A.; Szalay, Aladar A.; Fong, Yuman

    2014-01-01

    Background Sorafenib is the standard systemic therapy for un-resectable or recurrent hepatocellular carcinoma (HCC) with minimal increase in survival. Therefore, there is a great need to develop novel therapies for advanced or recurrent HCC. One emerging field of cancer treatment involves oncolytic viruses that specifically infect, replicate within, and kill cancer cells. In this study we look at the ability of GLV-1h68, a recombinant vaccinia virus derived from the vaccine strain that was used to eradicate smallpox, to kill sorafenib-resistant HCC. Methods Four sorafenib-resistant HCC cell lines were generated by repeated passage in the presence of sorafenib. Median inhibitory concentration was determined for all cell lines. The infectivity, viral replication and cytotoxicity of GLV-1h68 were assayed for both parental and sorafenib-resistant HCC cells. Results Infectivity increased in a time and concentration dependent manner in all cell lines. All cell lines supported efficient replication of virus. No significant difference between the rates of cell death between the parental and sorafenib-resistant cell lines was observed. Conclusions Our results demonstrate that oncolytic vaccinia virus GLV-1h68 efficiently kills both parental and sorafenib-resistant HCC cell lines. This study indicates that patients who have failed treatment with sorafenib remain viable candidates for oncolytic therapy. PMID:24957667

  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. 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

  16. A cancer-favoring oncolytic vaccinia virus shows enhanced suppression of stem-cell like colon cancer

    PubMed Central

    Yoo, So Young; Bang, Seo Young; Jeong, Su-Nam; Kang, Dae Hwan; Heo, Jeong

    2016-01-01

    Stem cell-like colon cancer cells (SCCs) pose a major challenge in colon cancer treatment because of their resistance to chemotherapy and radiotherapy. Oncolytic virus-based therapy has shown promising results in uncured cancer patients; however, its effects on SCCs are not well studied yet. Here, we engineered a cancer-favoring oncolytic vaccinia virus (CVV) as a potent biotherapeutic and investigated its therapeutic efficacy in terms of killing SCCs. CVV is an evolved Wyeth strain vaccinia virus (EVV) lacking the viral thymidine kinase. SCC models were established using human or mouse colon cancer spheres, which continuously expressed stemness markers. The cancer-favoring characteristics and different cytotoxic pathways for killing cancer cells successfully overrode general drug resistance, thereby killing colon cancer cells regardless of the presence of SCCs. Subcutaneously injected HT29 spheres showed lower growth in CVV-treated models than in 5-Fu-treated models. Intraperitoneally injected CT26 spheres induced tumor masses in the abdominal region. CVV-treated groups showed higher survival rates and smaller tumor mass formation, compared to 5-Fu-treated groups. Interestingly, the combined treatment of CVV with 5-Fu showed improved survival rates and complete suppression of tumor mass. The CVV developed in this study, thus, effectively suppresses SCCs, which can be synergistically enhanced by simultaneous treatment with the anticancer drug 5-Fu. Our novel CVV is highly advantageous as a next-generation therapeutic for treating colon cancer. PMID:26918725

  17. A cancer-favoring oncolytic vaccinia virus shows enhanced suppression of stem-cell like colon cancer.

    PubMed

    Yoo, So Young; Bang, Seo Young; Jeong, Su-Nam; Kang, Dae Hwan; Heo, Jeong

    2016-03-29

    Stem cell-like colon cancer cells (SCCs) pose a major challenge in colon cancer treatment because of their resistance to chemotherapy and radiotherapy. Oncolytic virus-based therapy has shown promising results in uncured cancer patients; however, its effects on SCCs are not well studied yet. Here, we engineered a cancer-favoring oncolytic vaccinia virus (CVV) as a potent biotherapeutic and investigated its therapeutic efficacy in terms of killing SCCs. CVV is an evolved Wyeth strain vaccinia virus (EVV) lacking the viral thymidine kinase. SCC models were established using human or mouse colon cancer spheres, which continuously expressed stemness markers. The cancer-favoring characteristics and different cytotoxic pathways for killing cancer cells successfully overrode general drug resistance, thereby killing colon cancer cells regardless of the presence of SCCs. Subcutaneously injected HT29 spheres showed lower growth in CVV-treated models than in 5-Fu-treated models. Intraperitoneally injected CT26 spheres induced tumor masses in the abdominal region. CVV-treated groups showed higher survival rates and smaller tumor mass formation, compared to 5-Fu-treated groups. Interestingly, the combined treatment of CVV with 5-Fu showed improved survival rates and complete suppression of tumor mass. The CVV developed in this study, thus, effectively suppresses SCCs, which can be synergistically enhanced by simultaneous treatment with the anticancer drug 5-Fu. Our novel CVV is highly advantageous as a next-generation therapeutic for treating colon cancer. PMID:26918725

  18. Recombinant vaccinia virus GLV-1h68 is a promising oncolytic vector in the treatment of cholangiocarcinoma.

    PubMed

    Pugalenthi, Amudhan; Mojica, Kelly; Ady, Justin W; Johnsen, Clark; Love, Damon; Chen, Nanhai G; Aguilar, Richard J; Szalay, Aladar A; Fong, Yuman

    2015-12-01

    Although early stage cholangiocarcinoma (CC) can be cured by surgical extirpation, the options for treatment of advanced stage CC are very few and suboptimal. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) is a promising new strategy to treat human cancers. The ability of oncolytic VACV GLV-1h68 to infect, replicate in, and lyse three human CC cell lines was assayed in vitro and in subcutaneous flank xenografts in athymic nude mice. In this study, we have demonstrated that GLV-1h68 effectively infects and lyses three CC cell lines (KMC-1, KMBC, and KMCH-1) in vitro. Expression of the viral marker gene ruc-gfp facilitated real-time monitoring of infection and replication. Furthermore in athymic nude mice, a single dose of GLV-1h68 significantly suppressed tumor growth. The treatment was well tolerated in all animals. Recombinant VACV GLV-1h68 has significant oncolytic ability against CC both in vitro and in vivo. GLV-1h68 has the potential to be used clinically as a therapeutic agent against CC. PMID:26584530

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

    PubMed Central

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

    2015-01-01

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

  20. Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene: an oncolytic virus superior to dl1520 (ONYX-015) for human head and neck cancer.

    PubMed

    Tysome, James R; Wang, Pengju; Alusi, Ghassan; Briat, Arnaud; Gangeswaran, Rathi; Wang, Jiwei; Bhakta, Vipul; Fodor, Istvan; Lemoine, Nick R; Wang, Yaohe

    2011-09-01

    Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC. PMID:21361787

  1. 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

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

    PubMed

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

    2015-02-15

    Isolated limb perfusion (ILP) is a treatment for advanced extremity sarcoma and in-transit melanoma. Advancing this procedure by investigating the addition of novel agents, such as cancer-selective oncolytic viruses, may improve both the therapeutic efficacy of ILP and the tumour-targeted delivery of oncolytic virotherapy. Standard in vitro assays were used to characterise single agent and combinatorial activities of melphalan, tumour necrosis factor-alpha (TNF-α) and Lister strain vaccinia virus (GLV-1h68) against BN175 rat sarcoma cells. An orthotopic model of advanced extremity sarcoma was used to evaluate survival of animals after ILP with combinations of TNF-α, melphalan and GLV-1h68. We investigated the efficiency of viral tumour delivery by ILP compared to intravenous therapy, the locoregional and systemic biodistribution of virus after ILP, and the effect of mode of administration on antibody response. The combination of melphalan and GLV-1h68 was synergistic in vitro. The addition of virus to standard ILP regimens was well tolerated and demonstrated superior tumour targeting compared to intravenous administration. Triple therapy (melphalan/TNF-α/GLV-1h68) resulted in increased tumour growth delay and enhanced survival compared to other treatment regimens. Live virus was recovered in large amounts from perfused regions, but in smaller amounts from systemic organs. The addition of oncolytic vaccinia virus to existing TNF-α/melphalan-based ILP strategies results in survival advantage in an immunocompetent rat model of advanced extremity sarcoma. Virus administered by ILP has superior tumour targeting compared to intravenous delivery. Further evaluation and clinical translation of this approach is warranted. PMID:24978211

  3. Gene Therapy Using Therapeutic and Diagnostic Recombinant Oncolytic Vaccinia Virus GLV-1h153 for Management of Colorectal Peritoneal Carcinomatosis

    PubMed Central

    Eveno, Clarisse; Mojica, Kelly; Ady, Justin W.; Thorek, Daniel L.J.; Longo, Valerie; Belin, Laurence J.; Gholami, Sepideh; Johnsen, Clark; Zanzonico, Pat; Chen, Nanhai; Yu, Tony; Szalay, Aladar A.; Fong, Yuman

    2015-01-01

    Background Peritoneal carcinomatosis (PC) is a terminal progression of colorectal cancer (CRC). Poor response to cytoreductive surgery and chemotherapy, coupled with the inability to reliably track disease progression using established diagnostic methods make this a deadly disease. This paper examines the effectiveness of the oncolytic vaccinia virus GLV-1h153 as a therapeutic and diagnostic vehicle. We believe that viral expression of the human sodium iodide transporter (hNIS) can provide both real-time monitoring of viral therapy and effective treatment of colorectal peritoneal carcinomatosis (CRPC). Methods Infectivity and cytotoxic effect of GLV-1h153 on CRC cell lines was assayed in-vitro. Viral replication was examined by standard viral plaque assays. Orthotopic CRPC xenografts were generated in athymic nude mice, and subsequently administered GLV-1h153 intraperitoneally. Reduction of tumor burden was assessed by mass. Orthotopic tumors were visualized by SPECT/CT after Iodine (131I) administration and by fluorescence optical imaging. Results GLV-1h153 infected and killed CRC cells in a time and concentration dependent manner. Viral replication demonstrated greater than a 2.35 log increase in titer over 4 days. Intraperitoneal treatment of orthotopic CRPC xenografts resulted in a significant reduction of tumor burden. Infection of orthotopic xenografts was both therapeutic and facilitated monitoring by 131I-SPECT/CT via expression of hNIS in infected tissue. Conclusions GLV-1h153 effectively kills CRC in-vitro and dramatically reduces tumor burden in-vivo. We demonstrate that GLV-1h153 can be used as an agent to provide accurate delineation of tumor burden in-vivo. These findings indicate that GLV-1h153 has significant potential for use as theragnostic agent in the treatment of CRPC. PMID:25616946

  4. 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

  5. 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

  6. 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

  7. 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

  8. A novel oncolytic viral therapy and imaging technique for gastric cancer using a genetically engineered vaccinia virus carrying the human sodium iodide symporter

    PubMed Central

    2014-01-01

    Background Gastric cancers have poor overall survival despite recent advancements in early detection methods, endoscopic resection techniques, and chemotherapy treatments. Vaccinia viral therapy has had promising therapeutic potential for various cancers and has a great safety profile. We investigated the therapeutic efficacy of a novel genetically-engineered vaccinia virus carrying the human sodium iodide symporter (hNIS) gene, GLV-1 h153, on gastric cancers and its potential utility for imaging with 99mTc pertechnetate scintigraphy and 124I positron emission tomography (PET). Methods GLV-1 h153 was tested against five human gastric cancer cell lines using cytotoxicity and standard viral plaque assays. In vivo, subcutaneous flank tumors were generated in nude mice with human gastric cancer cells, MKN-74. Tumors were subsequently injected with either GLV-1 h153 or PBS and followed for tumor growth. 99mTc pertechnetate scintigraphy and 124I microPET imaging were performed. Results GFP expression, a surrogate for viral infectivity, confirmed viral infection by 24 hours. At a multiplicity of infection (MOI) of 1, GLV-1 h153 achieved > 90% cytotoxicity in MNK-74, OCUM-2MD3, and AGS over 9 days, and >70% cytotoxicity in MNK- 45 and TMK-1. In vivo, GLV-1 h153 was effective in treating xenografts (p < 0.001) after 2 weeks of treatment. GLV-1 h153-infected tumors were readily imaged by 99mTc pertechnetate scintigraphy and 124I microPET imaging 2 days after treatment. Conclusions GLV-1 h153 is an effective oncolytic virus expressing the hNIS protein that can efficiently regress gastric tumors and allow deep-tissue imaging. These data encourages its continued investigation in clinical settings. PMID:24383569

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

    PubMed

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

    2015-07-01

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

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

    PubMed Central

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

    2015-01-01

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

  11. 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

  12. 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

  13. Vaccinia virus transcription.

    PubMed

    Broyles, Steven S

    2003-09-01

    Vaccinia virus replication takes place in the cytoplasm of the host cell. The nearly 200 kbp genome owes part of its complexity to encoding most of the proteins involved in genome and mRNA synthesis. The multisubunit vaccinia virus RNA polymerase requires a separate set of virus-encoded proteins for the transcription of the early, intermediate and late classes of genes. Cell fractionation studies have provided evidence for a role for host cell proteins in the initiation and termination of vaccinia virus intermediate and late gene transcription. Vaccinia virus resembles nuclear DNA viruses in the integration of viral and host proteins for viral mRNA synthesis, yet is markedly less reliant on host proteins than its nuclear counterparts. PMID:12917449

  14. Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells

    PubMed Central

    2012-01-01

    Background Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. Methods Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44+CD24+ESA+ cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. Results We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44+CD24+ESA+ cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44+CD24-ESA+ cells. Conclusions Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills

  15. 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

  16. Oncolytic viruses: finally delivering.

    PubMed

    Seymour, Leonard W; Fisher, Kerry D

    2016-02-16

    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

  17. Pexa-Vec double agent engineered vaccinia: oncolytic and active immunotherapeutic.

    PubMed

    Breitbach, Caroline J; Parato, Kelley; Burke, James; Hwang, Tae-Ho; Bell, John C; Kirn, David H

    2015-08-01

    Oncolytic immunotherapies (OI) selectively infect, amplify within and destroy cancer cells, thereby representing a novel class of anti-cancer therapy. In addition to this primary mechanism-of-action (MOA), OI based on vaccinia have been shown to selectively target tumor-associated vasculature, triggering an acute reduction in tumor perfusion. This review focuses on a third complementary MOA for this product class: the induction of active immunotherapy. While the active immunotherapy approach has been validated by recent product approvals, the field is still faced with significant challenges. Tumors have evolved diverse mechanisms to hide from immune-mediated destruction. Here we hypothesize that oncolytic immunotherapy replication within tumors may tip the immune balance to allow for the effective induction and execution of adaptive anti-tumor immunity, resulting in long-term tumor control following OI clearance. This immune activation against the cancer can be augmented through OI 'arming' for the expression of immunostimulatory transgene products from the virus genome. With the first vaccinia OI (Pexa-Vec, thymidine kinase-inactivated vaccinia expressing Granulocyte-colony stimulating factor [GM-CSF]) now in advanced-stage clinical trials, it has become more important than ever to understand the complimentary MOA that contributes to tumor destruction and control in patients. PMID:25900822

  18. Oncolytic virus therapies.

    PubMed

    Buonaguro, Franco Maria; Tornesello, Maria Lina; Izzo, Francesco; Buonaguro, Luigi

    2012-11-01

    Oncolytic virus (OV) therapy currently represents one of the most promising approaches to cancer treatment for their dual anticancer mechanisms: direct lysis of cancer cells (oncolytic feature) and activation of the immunosystem (cancer vaccine aspect). The latter demonstrates the advantage of a multi-target approach against multiple tumor-associated antigens. Since the 2005 SFDA (the Chinese FDA) approval for the clinical use of Oncorine™, the first human OV-based cancer treatment, more than 200 patents have been filed worldwide and several Phase I/II studies have been conducted. This patent review analyzes patents and clinical studies of the most promising OV products to highlight the pros and cons of this innovative anticancer approach, which is currently being tested in several cancers (i.e., hepatocellular carcinoma, melanoma and glioblastoma) by systemic as well as intratumoral injection. Clinical results, although effective only for a limited period of time, are encouraging. Combined treatments with radio or chemotherapeutic protocols are also in progress. PMID:24236929

  19. 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

  20. 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.

  1. 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

  2. Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer

    PubMed Central

    Heo, Jeong; Reid, Tony; Ruo, Leyo; Breitbach, Caroline J; Rose, Steven; Bloomston, Mark; Cho, Mong; Lim, Ho Yeong; Chung, Hyun Cheol; Kim, Chang Won; Burke, James; Lencioni, Riccardo; Hickman, Theresa; Moon, Anne; Lee, Yeon Sook; Kim, Mi Kyeong; Daneshmand, Manijeh; Dubois, Kara; Longpre, Lara; Ngo, Minhtran; Rooney, Cliona; Bell, John C; Rhee, Byung-Geon; Patt, Richard; Hwang, Tae-Ho; Kirn, David H

    2014-01-01

    Oncolytic viruses and active immunotherapeutics have complementary mechanisms of action (MOA) that are both self amplifying in tumors, yet the impact of dose on subject outcome is unclear. JX-594 (Pexa-Vec) is an oncolytic and immunotherapeutic vaccinia virus. To determine the optimal JX-594 dose in subjects with advanced hepatocellular carcinoma (HCC), we conducted a randomized phase 2 dose-finding trial (n = 30). Radiologists infused low-or high-dose JX-594 into liver tumors (days 1, 15 and 29); infusions resulted in acute detectable intravascular JX-594 genomes. Objective intrahepatic Modified Response Evaluation Criteria in Solid Tumors (mRECIST) (15%) and Choi (62%) response rates and intrahepatic disease control (50%) were equivalent in injected and distant noninjected tumors at both doses. JX-594 replication and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression preceded the induction of anticancer immunity. In contrast to tumor response rate and immune endpoints, subject survival duration was significantly related to dose (median survival of 14.1 months compared to 6.7 months on the high and low dose, respectively; hazard ratio 0.39; P = 0.020). JX-594 demonstrated oncolytic and immunotherapy MOA, tumor responses and dose-related survival in individuals with HCC. PMID:23396206

  3. Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer.

    PubMed

    Heo, Jeong; Reid, Tony; Ruo, Leyo; Breitbach, Caroline J; Rose, Steven; Bloomston, Mark; Cho, Mong; Lim, Ho Yeong; Chung, Hyun Cheol; Kim, Chang Won; Burke, James; Lencioni, Riccardo; Hickman, Theresa; Moon, Anne; Lee, Yeon Sook; Kim, Mi Kyeong; Daneshmand, Manijeh; Dubois, Kara; Longpre, Lara; Ngo, Minhtran; Rooney, Cliona; Bell, John C; Rhee, Byung-Geon; Patt, Richard; Hwang, Tae-Ho; Kirn, David H

    2013-03-01

    Oncolytic viruses and active immunotherapeutics have complementary mechanisms of action (MOA) that are both self amplifying in tumors, yet the impact of dose on subject outcome is unclear. JX-594 (Pexa-Vec) is an oncolytic and immunotherapeutic vaccinia virus. To determine the optimal JX-594 dose in subjects with advanced hepatocellular carcinoma (HCC), we conducted a randomized phase 2 dose-finding trial (n=30). Radiologists infused low- or high-dose JX-594 into liver tumors (days 1, 15 and 29); infusions resulted in acute detectable intravascular JX-594 genomes. Objective intrahepatic Modified Response Evaluation Criteria in Solid Tumors (mRECIST) (15%) and Choi (62%) response rates and intrahepatic disease control (50%) were equivalent in injected and distant noninjected tumors at both doses. JX-594 replication and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression preceded the induction of anticancer immunity. In contrast to tumor response rate and immune endpoints, subject survival duration was significantly related to dose (median survival of 14.1 months compared to 6.7 months on the high and low dose, respectively; hazard ratio 0.39; P=0.020). JX-594 demonstrated oncolytic and immunotherapy MOA, tumor responses and dose-related survival in individuals with HCC. PMID:23396206

  4. 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. PMID:25042001

  5. 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

  6. 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

  7. Targeting cancer stem cells with oncolytic virus

    PubMed Central

    Tong, Yin

    2014-01-01

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

  8. Oncolytic Poxviruses

    PubMed Central

    Chan, Winnie M.; McFadden, Grant

    2015-01-01

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

  9. Clinical development of oncolytic viruses in China.

    PubMed

    Liang, Min

    2012-07-01

    The oncolytic virus, being a promising new therapeutic strategy for cancer, has inspired a wave of recent clinical research and development in China. The first commercialized oncolytic virus, Oncorine, was approved by Chinese SFDA in November 2005 for nasopharyngeal carcinoma combined with chemotherapy. Since then, a number of oncolytic viruses have been moved into clinical trials. Among these are the armed oncolytic adenoviruses such as H103 (expressing the heat shock protein) currently has finished phase I trial, and KH901 (expressing GM-CSF) now launched in phase II trial In this review, we will discuss the current status of ongoing oncolytic virus projects being conducted at various clinical stages in China, including the preliminary market response for Oncorine after it was launched into the Chinese market in 2006. PMID:21740357

  10. MicroRNAs and oncolytic viruses.

    PubMed

    Ruiz, Autumn J; Russell, Stephen J

    2015-08-01

    MicroRNAs regulate gene expression in mammalian cells and often exhibit tissue-specific expression patterns. Incorporation of microRNA target sequences can be used to control exogenous gene expression and viral tropism in specific tissues to enhance the therapeutic indices of oncolytic viruses expressing therapeutic transgenes. Continued development of this targeting strategy has resulted in the generation of unattenuated oncolytic viruses with enhanced potency, broad species-tropisms and reduced off-target toxicities in multiple-tissues simultaneously. Furthermore, oncolytic viruses have been used to enhance the delivery, duration and therapeutic efficacy of microRNA-based therapeutics designed to either restore or inhibit the function of dysregulated microRNAs in cancer cells. Recent efforts focused on combining oncolytic virotherapy and microRNA regulation have generated increasingly potent and safe cancer therapeutics. PMID:25863717

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

    PubMed Central

    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

  12. 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

  13. Oncolytic Myxoma Virus: The path to clinic

    PubMed Central

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

    2013-01-01

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

  14. [Oncolytic viruses for therapy of malignant glioma].

    PubMed

    Sosnovtceva, A O; Grinenko, N F; Lipatova, A V; Chumakov, P M; Chekhonin, V P

    2016-05-01

    Effective treatment of malignant brain tumors is still an open problem. Location of tumor in vital areas of the brain significantly limits capasities of surgical treatment. The presence of tumor stem cells resistant to radiation and anticancer drugs in brain tumor complicates use of chemoradiotherapy and causes a high rate of disease recurrence. A technological improvement in bioselection and production of recombinant resulted in creation of viruses with potent oncolytic properties against glial tumors. Recent studies, including clinical trials, showed, that majority of oncolytic viruses are safe. Despite the impressive results of the viral therapy in some patients, the treatment of other patients is not effective; therefore, further improvement of the methods of oncolytic virotherapy is necessary. High genetic heterogeneity of glial tumor cells even within a single tumor determines differences in individual sensitivity of tumor cells to oncolytic viruses. This review analyses the most successful oncolytic virus strains, including those which had reached clinical trials, and discusses the prospects for new approaches to virotherapy of gliomas. PMID:27562991

  15. 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

  16. 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. PMID:21470473

  17. Cell carriers for oncolytic viruses: current challenges and future directions.

    PubMed

    Roy, Dominic G; Bell, John C

    2013-01-01

    The optimal route for clinical delivery of oncolytic viruses is thought to be systemic intravenous injection; however, the immune system is armed with several highly efficient mechanisms to remove pathogens from the circulatory system. To overcome the challenges faced in trying to delivery oncolytic viruses specifically to tumors via the bloodstream, carrier cells have been investigated to determine their suitability as delivery vehicles for systemic administration of oncolytic viruses. Cell carriers protect viruses from neutralization, one of the most limiting aspects of oncolytic virus interaction with the immune system. Cell carriers can also possess inherent tumor tropism, thus directing the delivery of the virus more specifically to a tumor. With preclinical studies already demonstrating the success and feasibility of this approach with multiple oncolytic viruses, clinical evaluation of cell-mediated delivery of viruses is on the horizon. Meanwhile, ongoing preclinical studies are aimed at identifying new cellular vehicles for oncolytic viruses and improving current promising cell carrier platforms. PMID:27512657

  18. 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

  19. 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

  20. 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

  1. Attacking Postoperative Metastases using Perioperative Oncolytic Viruses and Viral Vaccines

    PubMed Central

    Tai, Lee-Hwa; Auer, Rebecca

    2014-01-01

    Surgical resection of solid primary malignancies is a mainstay of therapy for cancer patients. Despite being the most effective treatment for these tumors, cancer surgery has been associated with impaired metastatic clearance due to immunosuppression. In preclinical surgery models and human cancer patients, we and others have demonstrated a profound suppression of both natural killer (NK) and T cell function in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Oncolytic viruses (OV) were originally designed to selectively infect and replicate in tumors, with the primary objective of directly lysing cancer cells. It is becoming increasingly clear, however, that OV infection results in a profound inflammatory reaction within the tumor, initiating innate and adaptive immune responses against it that is critical for its therapeutic benefit. This anti-tumor immunity appears to be mediated predominantly by NK and cytotoxic T cells. In preclinical models, we found that preoperative OV prevents postoperative NK cell dysfunction and attenuates tumor dissemination. Due to theoretical safety concerns of administering live virus prior to surgery in cancer patients, we characterized safe, attenuated versions of OV, and viral vaccines that could stimulate NK cells and reduce metastases when administered in the perioperative period. In cancer patients, we observed that in vivo infusion with oncolytic vaccinia virus and ex vivo stimulation with viral vaccines promote NK cell activation. These preclinical studies provide a novel and clinically relevant setting for OV therapy. Our challenge is to identify safe and promising OV therapies that will activate NK and T cells in the perioperative period preventing the establishment of micrometastatic disease in cancer patients. PMID:25161958

  2. 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

  3. Nucleotide Phosphohydrolase in Purified Vaccinia Virus

    PubMed Central

    Munyon, William; Paoletti, Enzo; Ospina, Julio; Grace, James T.

    1968-01-01

    Purified infectious vaccinia virus has been shown to contain an enzyme or enzymes that remove the terminal phosphate group from adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). The Km for ATP of this enzyme is 5.5 × 10−4m, and the relative rates of the reaction with ATP, GTP, UTP, and CTP are 1.00, 0.34, 0.15, and 0.29, respectively. The virus enzyme does not react with any of the diphosphates. The rate of the reaction is proportional to the amount of virus added and is linear for 130 min. The virus nucleotide phosphohydrolase activity is greatly stimulated by Mg++ and very slightly stimulated by Ca++. The small residual activity observed in the absence of divalent cations is completely inhibited by ethylenediaminetetraacetic acid. Neither Na+ nor K+ ions, nor any mixture of these, was found to stimulate the reaction significantly, and ouabain, at 10−4m, inhibited the reaction by only 27%. The response of the vaccinia enzyme to mono- and divalent cations and to ouabain indicates that the vaccinia enzyme has different properties from those associated with microsomes and mitochondria. PMID:4986904

  4. 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

  5. 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.

  6. Myxoma and Vaccinia Viruses Bind Differentially to Human Leukocytes

    PubMed Central

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

    2013-01-01

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

  7. 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. PMID:24274374

  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. 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

  10. Brazilian Vaccinia Viruses and Their Origins

    PubMed Central

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

    2007-01-01

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

  11. 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.

  12. Oncolytic Bluetongue Viruses: Promise, Progress, and Perspectives

    PubMed Central

    Li, Joseph K.-K.

    2011-01-01

    Humans are sero-negative toward bluetongue viruses (BTVs) since BTVs do not infect normal human cells. Infection and selective degradation of several human cancer cell lines but not normal ones by five US BTV serotypes have been investigated. We determined the susceptibilities of many normal and human cancer cells to BTV infections and made comparative kinetic analyses of their cytopathic effects, survival rates, ultra-structural changes, cellular apoptosis and necrosis, cell cycle arrest, cytokine profiles, viral genome, mRNAs, and progeny titers. The wild-type US BTVs, without any genetic modifications, could preferentially infect and degrade several types of human cancer cells but not normal cells. Their selective and preferential BTV-degradation of human cancer cells is viral dose–dependent, leading to effective viral replication, and induced apoptosis. Xenograft tumors in mice were substantially reduced by a single intratumoral BTV injection in initial in vivo experiments. Thus, wild-type BTVs, without genetic modifications, have oncolytic potentials. They represent an attractive, next generation of oncolytic viral approach for potential human cancer therapy combined with current anti-cancer agents and irradiation. PMID:21747785

  13. 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

  14. 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

  15. 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.

  16. 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

  17. Oncolytic viruses-immunotherapeutics on the rise.

    PubMed

    Keller, Brian A; Bell, John C

    2016-09-01

    The oncolytic virus (OV) field has entered an exciting period in its evolution in which our basic understanding of viral biology and anti-cancer potential are being actively translated into viable therapeutic options for aggressive malignancies. OVs are naturally occurring or engineered viruses that are able to exploit cancer-specific changes in cellular signaling to specifically target cancers and their microenvironment. The direct cytolytic effect of OVs on cancer cells is known to release antigens, which can begin a cascade of events that results in the induction of anti-cancer adaptive immunity. This response is now regarded as the most critical mechanism of OV action and harnessing it can lead to the elimination of distant micrometastases as well as provide long-term anti-cancer immune surveillance. In this review, we highlight the development of the OV field, why OVs are gaining an increasingly elevated standing as members of the cancer immunotherapy armamentarium, and finally, ongoing clinical studies that are aimed at translating unique OV therapies into approved therapies for aggressive cancers. PMID:27492706

  18. 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

  19. Vaccinia Virus Infection Requires Maturation of Macropinosomes.

    PubMed

    Rizopoulos, Zaira; Balistreri, Giuseppe; Kilcher, Samuel; Martin, Caroline K; Syedbasha, Mohammedyaseen; Helenius, Ari; Mercer, Jason

    2015-08-01

    The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live-cell imaging, targeted RNAi screening and perturbants of endosome maturation, we analyzed the properties and maturation pathway of the macropinocytic vacuoles containing VACV MVs in HeLa cells. The vacuoles first acquired markers of early endosomes [Rab5, early endosome antigen 1 and phosphatidylinositol(3)P]. Prior to release of virus cores into the cytoplasm, they contained markers of late endosomes and lysosomes (Rab7a, lysosome-associated membrane protein 1 and sorting nexin 3). RNAi screening of endocytic cell factors emphasized the importance of late compartments for VACV infection. Follow-up perturbation analysis showed that infection required Rab7a and PIKfyve, confirming that VACV is a late-penetrating virus dependent on macropinosome maturation. VACV EV infection was inhibited by depletion of many of the same factors, indicating that both infectious particle forms share the need for late vacuolar conditions for penetration. PMID:25869659

  20. Targeting Cancer-initiating Cells With Oncolytic Viruses

    PubMed Central

    Cripe, Timothy P; Wang, Pin-Yi; Marcato, Paola; Mahller, Yonatan Y; Lee, Patrick WK

    2009-01-01

    Recent studies in a variety of leukemias and solid tumors indicate that there is significant heterogeneity with respect to tumor-forming ability within a given population of tumor cells, suggesting that only a subpopulation of cells is responsible for tumorigenesis. These cells have been commonly referred to as cancer stem cells (CSCs) or cancer-initiating cells (CICs). CICs have been shown to be relatively resistant to conventional anticancer therapies and are thus thought to be responsible for disease relapse. As such, they represent a potentially critical therapeutic target. Oncolytic viruses are in clinical trials for cancer and kill cells through mechanisms different from conventional therapeutics. Because these viruses are not susceptible to the same pathways of drug or radiation resistance, it is important to learn whether CICs are susceptible to oncolytic virus infection. Here we review the available data regarding the ability of several different oncolytic virus types to target CICs for destruction. PMID:19672244

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

    PubMed

    Tysome, J R; Briat, A; Alusi, G; Cao, F; Gao, D; Yu, J; Wang, P; Yang, S; Dong, Z; Wang, S; Deng, L; Francis, J; Timiryasova, T; Fodor, I; Lemoine, N R; Wang, Y

    2009-10-01

    Survival after pancreatic cancer remains poor despite incremental advances in surgical and adjuvant therapy, and new strategies for treatment are needed. Oncolytic virotherapy is an attractive approach for cancer treatment. In this study, we have evaluated the effectiveness of the Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapeutic approach for pancreatic cancer. The Lister vaccine strain of vaccinia virus was effective against all human pancreatic carcinoma cells tested in vitro, especially those insensitive to oncolytic adenovirus. The virus displayed inherently high selectivity for cancer cells, sparing normal cells both in vitro and in vivo, with effective infection of tumors after both intravenous (i.v.) and intratumoral (i.t.) administrations. The expression of the endostatin-angiostatin fusion protein was confirmed in a pancreatic cancer model both in vitro and in vivo, with evidence of inhibition of angiogenesis. This novel vaccinia virus showed significant antitumor potency in vivo against the Suit-2 model by i.t. administration. This study suggests that the novel Lister strain of vaccinia virus armed with the endostatin-angiostatin fusion gene is a potential therapeutic agent for pancreatic cancer. PMID:19587709

  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. PMID:22777090

  3. 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

  4. Clinical Trials with Oncolytic Viruses: Current and Future Prospects.

    PubMed

    Patil, Shankargouda; Rao, Roopa S; Majumdar, Barnali

    2015-08-01

    Reviewing the research in the field of oncolytic virus therapy (OVT) of the past two decades, it is inspiring to see the enormous amount of success accomplished by the scholars of this innovative therapeutic technique. Though the experimental trials have been ongoing from 1990s, however, it took a leap forward with approval of the clinical trials in China, 2005. The world's first oncolytic virus to be approved by their government was adenovirus (with E1B 55K gene deletion) for head and neck cancer therapy along with chemotherapy. PMID:26423510

  5. 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.

  6. Maraba Virus as a Potent Oncolytic Vaccine Vector

    PubMed Central

    Pol, Jonathan G; Zhang, Liang; Bridle, Byram W; Stephenson, Kyle B; Rességuier, Julien; Hanson, Stephen; Chen, Lan; Kazdhan, Natasha; Bramson, Jonathan L; Stojdl, David F; Wan, Yonghong; Lichty, Brian D

    2014-01-01

    The rhabdovirus Maraba has recently been characterized as a potent oncolytic virus. In the present study, we engineered an attenuated Maraba strain, defined as MG1, to express a melanoma-associated tumor antigen. Its ability to mount an antitumor immunity was evaluated in tumor-free and melanoma tumor-bearing mice. Alone, the MG1 vaccine appeared insufficient to prime detectable adaptive immunity against the tumor antigen. However, when used as a boosting vector in a heterologous prime-boost regimen, MG1 vaccine rapidly generated strong antigen-specific T-cell immune responses. Once applied for treating syngeneic murine melanoma tumors, our oncolytic prime-boost vaccination protocol involving Maraba MG1 dramatically extended median survival and allowed complete remission in more than 20% of the animals treated. This work describes Maraba virus MG1 as a potent vaccine vector for cancer immunotherapy displaying both oncolytic activity and a remarkable ability to boost adaptive antitumor immunity. PMID:24322333

  7. Vaccinia virus as a vaccine delivery system for marsupial wildlife.

    PubMed

    Cross, Martin L; Fleming, Stephen B; Cowan, Phil E; Scobie, Susie; Whelan, Ellena; Prada, Diana; Mercer, Andrew A; Duckworth, Janine A

    2011-06-20

    Vaccines based on recombinant poxviruses have proved successful in controlling diseases such as rabies and plague in wild eutherian mammals. They have also been trialled experimentally as delivery agents for fertility-control vaccines in rodents and foxes. In some countries, marsupial mammals represent a wildlife disease reservoir or a threat to conservation values but, as yet there has been no bespoke study of efficacy or immunogenicity of a poxvirus-based vaccine delivery system in a marsupial. Here, we report a study of the potential for vaccination using vaccinia virus in the Australian brushtail possum Trichosurus vulpecula, an introduced pest species in New Zealand. Parent-strain vaccinia virus (Lister) infected 8/8 possums following delivery of virus to the oral cavity and outer nares surfaces (oronasal immunisation), and persisted in the mucosal epithelium around the palatine tonsils for up to 2 weeks post-exposure. A recombinant vaccinia virus construct (VV399, which expresses the Eg95 antigen of the hydatid disease parasite Echinococcus granulosus) was shown to infect 10/15 possums after a single-dose oronasal delivery and to also persist. Both parent vaccinia virus and the VV399 construct virus induced peripheral blood lymphocyte reactivity against viral antigens in possums, first apparent at 4 weeks post-exposure and still detectable at 4 months post-exposure. Serum antibody reactivity to Eg95 was recorded in 7/8 possums which received a single dose of the VV399 construct and 7/7 animals which received triple-dose delivery, with titre end-points in the latter case exceeding 1/4000 dilution. This study demonstrates that vaccinia virus will readily infect possums via a delivery means used to deploy wildlife vaccines, and in doing is capable of generating immune reactivity against viral and heterologous antigens. This highlights the future potential of recombinant vaccinia virus as a vaccine delivery system in marsupial wildlife. PMID:21570435

  8. 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.

  9. Computational modeling approaches to the dynamics of oncolytic viruses.

    PubMed

    Wodarz, Dominik

    2016-05-01

    Replicating oncolytic viruses represent a promising treatment approach against cancer, specifically targeting the tumor cells. Significant progress has been made through experimental and clinical studies. Besides these approaches, however, mathematical models can be useful when analyzing the dynamics of virus spread through tumors, because the interactions between a growing tumor and a replicating virus are complex and nonlinear, making them difficult to understand by experimentation alone. Mathematical models have provided significant biological insight into the field of virus dynamics, and similar approaches can be adopted to study oncolytic viruses. The review discusses this approach and highlights some of the challenges that need to be overcome in order to build mathematical and computation models that are clinically predictive. WIREs Syst Biol Med 2016, 8:242-252. doi: 10.1002/wsbm.1332 For further resources related to this article, please visit the WIREs website. PMID:27001049

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

    PubMed

    Zainutdinov, Sergei S; Tikunov, Artem Y; Matveeva, Olga V; Netesov, Sergei V; Kochneva, Galina V

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. 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

  13. 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

  14. Herpes Simplex Virus Oncolytic Therapy for Pediatric Malignancies

    PubMed Central

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

    2009-01-01

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

  15. Protective efficacy of a recombinant vaccinia virus in vaccinia-immune mice.

    PubMed

    Andrew, M E

    1989-10-01

    Recombinant viral vectors offer a potential means of vaccinating against diseases for which there are no current safe vaccines. One of the criteria on which a viral vaccine vector would be selected is that it either circulates in the human or livestock population without producing overt disease (e.g. adenovirus) or has a history as a safe vaccine (e.g. vaccinia virus). However, this selection criterion also means that the target population is likely to have circulating antibodies that are specific to the vaccine vector. Since a percentage of the world's population has been vaccinated during the World Health Organization's Smallpox Eradication Campaign, such antibody titres, which are likely to lower vaccine efficacy, have been raised as an objection to the use of recombinant vaccinia viruses as vaccines. We have tested the effect of vaccinia-specific immunity on the protective efficacy of a recombinant virus, VV-PR8-HA6 (1) which expresses the haemagglutinin of the influenza virus A/PR/8/34. PMID:2613281

  16. 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.

  17. RESISTANCE OF PANCREATIC CANCER CELLS TO ONCOLYTIC VESICULAR STOMATITIS VIRUS: ROLE OF TYPE I INTERFERON SIGNALING

    PubMed Central

    Moerdyk-Schauwecker, Megan; Shah, Nirav R.; Murphy, Andrea M.; Hastie, Eric; Mukherjee, Pinku; Grdzelishvili, Valery Z.

    2012-01-01

    Oncolytic virus (OV) therapy takes advantage of common cancer characteristics, such as defective type I interferon (IFN) signaling, to preferentially infect and kill cancer cells with viruses. Our recent study (Murphy et al., 2012, J. Virol., 86: 3073-87) found human pancreatic ductal adenocarcinoma (PDA) cells were highly heterogeneous in their permissiveness to vesicular stomatitis virus (VSV) and suggested at least some resistant cell lines retained functional type I IFN responses. Here we examine cellular responses to infection by the oncolytic VSV recombinant VSV-ΔM51-GFP by analyzing a panel of 11 human PDA cell lines for expression of 33 genes associated with type I IFN pathways. Although all cell lines sensed infection by VSV-ΔM51-GFP and most activated IFN-α and β expression, only resistant cell lines displayed constitutive high-level expression of the IFN-stimulated antiviral genes MxA and OAS. Inhibition of JAK/STAT signaling decreased levels of MxA and OAS and increased VSV infection, replication and oncolysis, further implicating IFN responses in resistance. Unlike VSV, vaccinia and herpes simplex virus infectivity and killing of PDA cells was independent of the type I IFN signaling profile, possibly because these two viruses are better equipped to evade type I IFN responses. Our study demonstrates heterogeneity in the type I IFN signaling status of PDA cells and suggests MxA and OAS as potential biomarkers for PDA resistance to VSV and other OVs sensitive to type I IFN responses. PMID:23246628

  18. 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

  19. Immunostimulatory Gene Therapy Using Oncolytic Viruses as Vehicles

    PubMed Central

    Loskog, Angelica

    2015-01-01

    Immunostimulatory gene therapy has been developed during the past twenty years. The aim of immunostimulatory gene therapy is to tilt the suppressive tumor microenvironment to promote anti-tumor immunity. Hence, like a Trojan horse, the gene vehicle can carry warriors and weapons into enemy territory to combat the tumor from within. The most promising immune stimulators are those activating and sustaining Th1 responses, but even if potent effects were seen in preclinical models, many clinical trials failed to show objective responses in cancer patients. However, with new tools to control ongoing immunosuppression in cancer patients, immunostimulatory gene therapy is now emerging as an interesting option. In parallel, oncolytic viruses have been shown to be safe in patients. To prolong immune stimulation and to increase efficacy, these two fields are now merging and oncolytic viruses are armed with immunostimulatory transgenes. These novel agents are racing towards approval as established cancer immunotherapeutics. PMID:26561829

  20. Immunostimulatory Gene Therapy Using Oncolytic Viruses as Vehicles.

    PubMed

    Loskog, Angelica

    2015-11-01

    Immunostimulatory gene therapy has been developed during the past twenty years. The aim of immunostimulatory gene therapy is to tilt the suppressive tumor microenvironment to promote anti-tumor immunity. Hence, like a Trojan horse, the gene vehicle can carry warriors and weapons into enemy territory to combat the tumor from within. The most promising immune stimulators are those activating and sustaining Th1 responses, but even if potent effects were seen in preclinical models, many clinical trials failed to show objective responses in cancer patients. However, with new tools to control ongoing immunosuppression in cancer patients, immunostimulatory gene therapy is now emerging as an interesting option. In parallel, oncolytic viruses have been shown to be safe in patients. To prolong immune stimulation and to increase efficacy, these two fields are now merging and oncolytic viruses are armed with immunostimulatory transgenes. These novel agents are racing towards approval as established cancer immunotherapeutics. PMID:26561829

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

    PubMed Central

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

    2012-01-01

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

  2. Oncolytic viruses & their specific targeting to tumour cells

    PubMed Central

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

    2012-01-01

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

  3. 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

  4. 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.

  5. 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

  6. The therapeutic efficacy of the oncolytic virus Delta24-RGD in a murine glioma model depends primarily on antitumor immunity

    PubMed Central

    Kleijn, Anne; Kloezeman, Jenneke; Treffers-Westerlaken, Elike; Fulci, Giulia; Leenstra, Sieger; Dirven, Clemens; Debets, Reno; Lamfers, Martine

    2014-01-01

    Oncolytic viruses selectively lyse tumor cells, making these agents a promising treatment modality for glioma. Accumulating data suggest that the immune system plays an important role in the anti-glioma activity of oncolytic viruses. In an immune competent glioma model, the therapeutic effect of the oncolytic adenovirus Delta24-RGD was found to depend primarily on antitumor immune responses. PMID:25941622

  7. Progress in clinical oncolytic virus-based therapy for hepatocellular carcinoma.

    PubMed

    Jebar, Adel H; Errington-Mais, Fiona; Vile, Richard G; Selby, Peter J; Melcher, Alan A; Griffin, Stephen

    2015-07-01

    Hepatocellular carcinoma (HCC) carries a dismal prognosis, with advanced disease being resistant to both radiotherapy and conventional cytotoxic drugs, whilst anti-angiogenic drugs are marginally efficacious. Oncolytic viruses (OVs) offer the promise of selective cancer therapy through direct and immune-mediated mechanisms. The premise of OVs lies in their preferential genomic replication, protein expression and productive infection of malignant cells. Numerous OVs are being tested in preclinical models of HCC, with good evidence of direct and immune-mediated anti-tumour efficacy. Efforts to enhance the performance of these agents have concentrated on engineering OV cellular specificity, immune evasion, enhancing anti-tumour potency and improving delivery. The lead agent in HCC clinical trials, JX-594, a recombinant Wyeth strain vaccinia virus, has demonstrated evidence for significant benefit and earned orphan drug status. Thus, JX-594 appears to be transcending the barrier between novel laboratory science and credible clinical therapy. Relatively few other OVs have entered clinical testing, a hurdle that must be overcome if significant progress is to be made in this field. This review summarizes the preclinical and clinical experience of OV therapy in the difficult-to-treat area of HCC. PMID:25711964

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

    PubMed

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

    2016-03-01

    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

  9. 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

  10. Amalgamating oncolytic viruses to enhance their safety, consolidate their killing mechanisms, and accelerate their spread.

    PubMed

    Ayala-Breton, Camilo; Suksanpaisan, Lukkana; Mader, Emily K; Russell, Stephen J; Peng, Kah-Whye

    2013-10-01

    Oncolytic viruses are structurally and biologically diverse, spreading through tumors and killing them by various mechanisms and with different kinetics. Here, we created a hybrid vesicular stomatitis/measles virus (VSV/MV) that harnesses the safety of oncolytic MV, the speed of VSV, and the tumor killing mechanisms of both viruses. Oncolytic MV targets CD46 and kills by forcing infected cells to fuse with uninfected neighbors, but propagates slowly. VSV spreads rapidly, directly lysing tumor cells, but is neurotoxic and loses oncolytic potency when neuroattenuated by conventional approaches. The hybrid VSV/MV lacks neurotoxicity, replicates rapidly with VSV kinetics, and selectively targets CD46 on tumor cells. Its in vivo performance in a myeloma xenograft model was substantially superior to either MV or widely used recombinant oncolytic VSV-M51. PMID:23842448

  11. Oncolytic Immunotherapy: Where Are We Clinically?

    PubMed Central

    Hemminki, Akseli

    2014-01-01

    Following a century of preclinical and clinical work, oncolytic viruses are now proving themselves in randomized phase 3 trials. Interestingly, human data indicates that these agents have potent immunostimulatory activity, raising the possibility that the key consequence of oncolysis might be induction of antitumor immunity, especially in the context of viruses harboring immunostimulatory transgenes. While safety and efficacy of many types of oncolytic viruses, including adenovirus, herpes, reo, and vaccinia seem promising, few mechanisms of action studies have been performed with human substrates. Thus, the relative contribution of “pure” oncolysis, the immune response resulting from oncolysis, and the added benefit of adding a transgene remain poorly understood. Here, the available clinical data on oncolytic viruses is reviewed, with emphasis on immunological aspects. PMID:24551478

  12. Macrophage response to oncolytic paramyxoviruses potentiates virus-mediated tumor cell killing.

    PubMed

    Tan, Darren Qiancheng; Zhang, LiFeng; Ohba, Kenji; Ye, Min; Ichiyama, Koji; Yamamoto, Naoki

    2016-04-01

    Tumor-associated macrophages (TAMs) are known to regulate tumor response to many anti-cancer therapies, including oncolytic virotherapy. Oncolytic virotherapy employing oncolytic paramyxoviruses, such as attenuated measles (MeV) and mumps (MuV) viruses, has demonstrated therapeutic potential against various malignancies. However, the response of TAMs to oncolytic paramyxoviruses and the consequent effect on virotherapeutic efficacy remains to be characterized. Here, we demonstrate that the presence of human monocyte-derived macrophages (MDMs), irrespective of initial polarization state, enhances the virotherapeutic effect of MeV and MuV on breast cancer cells. Notably, our finding contrasts those of several studies involving other oncolytic viruses, which suggest that TAMs negatively impact virotherapeutic efficacy by impeding virus replication and dissemination. We found that the enhanced virotherapeutic effect in the presence of MDMs was due to slightly delayed proliferation and significantly elevated cell death that was not a result of increased virus replication. Instead, we found that the enhanced virotherapeutic effect involved several macrophage-associated anti-tumor mediators, and was associated with the modulation of MDMs towards an anti-tumor phenotype. Our findings present an alternative view on the role of TAMs in oncolytic virotherapy, and highlight the immunotherapeutic potential of oncolytic paramyxoviruses; possibly contributing towards the overall efficacy of oncolytic virotherapy. PMID:26763072

  13. 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

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

    PubMed

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

    2016-03-31

    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

  15. 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

  16. 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.

  17. 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

  18. 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.

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

    PubMed Central

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

    2013-01-01

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

  20. 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.

  1. 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

  2. [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

  3. 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.

  4. 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

  5. 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

  6. Vesicular Stomatitis Virus as an Oncolytic Agent against Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Murphy, Andrea M.; Besmer, Dahlia M.; Moerdyk-Schauwecker, Megan; Moestl, Natascha; Ornelles, David A.; Mukherjee, Pinku

    2012-01-01

    Vesicular stomatitis virus (VSV) is a promising oncolytic agent against a variety of cancers. However, it has never been tested in any pancreatic cancer model. Pancreatic ductal adenocarcinoma (PDA) is the most common and aggressive form of pancreatic cancer. In this study, the oncolytic potentials of several VSV variants were analyzed in a panel of 13 clinically relevant human PDA cell lines and compared to conditionally replicative adenoviruses (CRAds), Sendai virus and respiratory syncytial virus. VSV variants showed oncolytic abilities superior to those of other viruses, and some cell lines that exhibited resistance to other viruses were successfully killed by VSV. However, PDA cells were highly heterogeneous in their susceptibility to virus-induced oncolysis, and several cell lines were resistant to all tested viruses. Resistant cells showed low levels of very early VSV RNA synthesis, indicating possible defects at initial stages of infection. In addition, unlike permissive PDA cell lines, most of the resistant cell lines were able to both produce and respond to interferon, suggesting that intact type I interferon responses contributed to their resistance phenotype. Four cell lines that varied in their permissiveness to VSV-ΔM51 and CRAd dl1520 were tested in mice, and the in vivo results closely mimicked those in vitro. While our results demonstrate that VSV is a promising oncolytic agent against PDA, further studies are needed to better understand the molecular mechanisms of resistance of some PDAs to oncolytic virotherapy. PMID:22238308

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

    PubMed Central

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

    2012-01-01

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

  8. 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

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

    PubMed Central

    Buijs, Pascal RA; Verhagen, Judith HE; van Eijck, Casper HJ; 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

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

    PubMed

    Sebastian, Sarah; Gilbert, Sarah C

    2016-01-01

    A safe and effective malaria vaccine is a crucial part of the roadmap to malaria elimination/eradication by the year 2050. Viral-vectored vaccines based on adenoviruses and modified vaccinia virus Ankara (MVA) expressing malaria immunogens are currently being used in heterologous prime-boost regimes in clinical trials for induction of strong antigen-specific T-cell responses and high-titer antibodies. Recombinant MVA is a safe and well-tolerated attenuated vector that has consistently shown significant boosting potential. Advances have been made in large-scale MVA manufacture as high-yield producer cell lines and high-throughput purification processes have recently been developed. This review describes the use of MVA as malaria vaccine vector in both preclinical and clinical studies in the past 5 years. PMID:26511884

  11. Transcriptional complexity of vaccinia virus in vivo and in vitro.

    PubMed Central

    Paoletti, E; Grady, L J

    1977-01-01

    The transcriptional complexity of vaccinia virus both in vivo and in vitro has been measured by using DNA:RNA hybridization with RNA in excess. In vivo, "early" or prereplicative RNA was found to saturate at 25% or one-half of the viral genome. "Late" or postreplicative RNA from infected HeLa cells saturated at 52% or essentially the entire genome. This well-regulated transcriptional pattern of the virus in vivo was not maintained in vitro. In a number of experiments a range of saturation values from 40 to 50% was obtained for in vitro synthesized RNA. The complexity of polyadenylated and non-polyadenylated RNA, as well as total purified 8 to 12S RNA released from the virus, was indistinguishable from purified high-molecular-weight virion-associated RNA with a sedimentation value of greater than 20S and equivalent to total in vitro synthesized RNA. No additional hybrid formation was observed in experiments in which total in vitro RNA and late in vivo RNA from infected HeLa cells were combined, suggesting that the virus does not transcribe in vitro DNA sequences that are not also transcribed during productive infection. Approximately 15% complementary RNA was detected when radiolabeled total in vitro RNA was allowed to reanneal with late in vivo RNA, while as much as 8% of the in vitro synthesized RNA was found to be complementary. PMID:894791

  12. 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

  13. 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

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

    PubMed Central

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

    2015-01-01

    Epithelial ovarian cancer is unique among most carcinomas in that metastasis occurs by direct dissemination of malignant cells traversing throughout the intraperitoneal fluid. Accordingly, we test new therapeutic strategies using an in vitro three-dimensional spheroid suspension culture model that mimics key steps of this metastatic process. In the present study, we sought to uncover the differential oncolytic efficacy among three different viruses—Myxoma virus, double-deleted vaccinia virus, and Maraba virus—using three ovarian cancer cell lines in our metastasis model system. Herein, we demonstrate that Maraba virus effectively infects, replicates, and kills epithelial ovarian cancer (EOC) cells in proliferating adherent cells and with slightly slower kinetics in tumor spheroids. Myxoma virus and vaccinia viruses infect and kill adherent cells to a much lesser extent than Maraba virus, and their oncolytic potential is almost completely attenuated in spheroids. Myxoma virus and vaccinia are able to infect and spread throughout spheroids, but are blocked in the final stages of the lytic cycle, and oncolytic-mediated cell killing is reactivated upon spheroid reattachment. Alternatively, Maraba virus has a remarkably reduced ability to initially enter spheroid cells, yet rapidly infects and spreads throughout spheroids generating significant cell killing effects. We show that low-density lipoprotein receptor expression in ovarian cancer spheroids is reduced and this controls efficient Maraba virus binding and entry into infected cells. Taken together, these results are the first to implicate the potential impact of differential viral oncolytic properties at key steps of ovarian cancer metastasis. PMID:27119108

  15. 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.

  16. 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

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

    PubMed Central

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

    2007-01-01

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

  18. Oncolytic 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

  19. 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

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

    PubMed

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

    2016-01-01

    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

  1. 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

  2. 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

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

    PubMed Central

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

    2016-01-01

    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. PMID:26991092

  4. 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

  5. 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.

  6. 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

  7. 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. PMID:26290187

  8. Atomic Force Microscopy Investigation of Vaccinia Virus Structure▿

    PubMed Central

    Kuznetsov, Y.; Gershon, P. D.; McPherson, A.

    2008-01-01

    Vaccinia virus was treated in a controlled manner with various combinations of nonionic detergents, reducing agents, and proteolytic enzymes, and successive products of the reactions were visualized using atomic force microscopy (AFM). Following removal of the outer lipid/protein membrane, a layer 20 to 40 nm in thickness was encountered that was composed of fibrous elements which, under reducing conditions, rapidly decomposed into individual monomers on the substrate. Beneath this layer was the virus core and its prominent lateral bodies, which could be dissociated or degraded with proteases. The core, in addition to the lateral bodies, was composed of a thick, multilayered shell of proteins of diverse sizes and shapes. The shell, which was readily etched with proteases, was thoroughly permeated with pores, or channels. Prolonged exposure to proteases and reductants produced disgorgement of the viral DNA from the remainders of the cores and also left residual, flattened, protease-resistant sacs on the imaging substrate. The DNA was readily visualized by AFM, which revealed some regions to be “soldered” by proteins, others to be heavily complexed with protein, and yet other parts to apparently exist as bundled, naked DNA. Prolonged exposure to proteases deproteinized the DNA, leaving masses of extended, free DNA. Estimates of the interior core volume suggest moderate but not extreme compaction of the genome. PMID:18508898

  9. 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

  10. 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. PMID:24889230

  11. The Case of Oncolytic Viruses Versus the Immune System: Waiting on the Judgment of Solomon

    PubMed Central

    Prestwich, Robin J.; Errington, Fiona; Diaz, Rosa M.; Pandha, Hardev S.; Harrington, Kevin J.; Melcher, Alan A.

    2009-01-01

    Abstract The three-way interaction between oncolytic viruses, the tumor microenvironment, and the immune system is critical to the outcome of antitumor therapy. Classically, the immune system is thought to limit the efficacy of therapy, leading to viral clearance. However, preclinical and clinical data suggest that in some cases virotherapy may in fact act as cancer immunotherapy. In this review we discuss the ability of oncolytic viruses to alter the immunogenic milieu of the tumor microenvironment, and the role of innate and adaptive immunity in both restricting and augmenting therapy. Strategies to improve virotherapy by immunomodulation, including suppression or enhancement of the innate and adaptive responses, are discussed. PMID:19630549

  12. Oncolytic Newcastle Disease Virus for cancer therapy: old challenges and new directions

    PubMed Central

    Zamarin, Dmitriy; Palese, Peter

    2014-01-01

    Summary Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has been demonstrated to possess significant oncolytic activity against mammalian cancers. This review summarizes the research leading to the elucidation of the mechanisms of NDV-mediated oncolysis as well as the development of novel oncolytic agents through the use of genetic engineering. Clinical trials utilizing NDV strains and NDV-based autologous tumor cell vaccines will expand our knowledge of these novel anti-cancer strategies and will ultimately result in the successful use of the virus in the clinical setting. PMID:22393889

  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. 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.

  15. Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats.

    PubMed

    Altomonte, Jennifer; Muñoz-Álvarez, Kim A; Shinozaki, Katsunori; Baumgartner, Christine; Kaissis, Georgios; Braren, Rickmer; Ebert, Oliver

    2016-01-01

    Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC. PMID:27167921

  16. Chemovirotherapy of malignant melanoma with a targeted and armed oncolytic measles virus.

    PubMed

    Kaufmann, Johanna K; Bossow, Sascha; Grossardt, Christian; Sawall, Stefanie; Kupsch, Jörg; Erbs, Philippe; Hassel, Jessica C; von Kalle, Christof; Enk, Alexander H; Nettelbeck, Dirk M; Ungerechts, Guy

    2013-04-01

    Effective treatment modalities for advanced melanoma are desperately needed. An innovative approach is virotherapy, in which viruses are engineered to infect cancer cells, resulting in tumor cell lysis and an amplification effect by viral replication and spread. Ideally, tumor selectivity of these oncolytic viruses is already determined during viral cell binding and entry, which has not been reported for melanoma. We engineered an oncolytic measles virus entering melanoma cells through the high molecular weight melanoma-associated antigen (HMWMAA) and proved highly specific infection and spread in melanoma cells. We further enhanced this oncolytic virus by inserting the FCU1 gene encoding the yeast-derived prodrug convertases cytosine deaminase and uracil phosphoribosyltransferase. Combination treatment with armed and retargeted MV-FCU1-αHMWMAA and the prodrug 5-fluorocytosine (5-FC) led to effective prodrug conversion to 5-fluorouracil, extensive cytotoxicity to melanoma cells, and excessive bystander killing of noninfected cells. Importantly, HMWMAA-retargeted MV showed antitumor activity in a human xenograft mouse model, which was further increased by the FCU1/5-FC prodrug activation system. Finally, we demonstrated susceptibility of melanoma skin metastasis biopsies to HMWMAA-retargeted MV. The highly selective, entry-targeted and armed oncolytic virus MV-FCU1-αHMWMAA may become a potent building block of future melanoma therapies. PMID:23223133

  17. Animal movement and establishment of vaccinia virus Cantagalo strain in Amazon biome, Brazil.

    PubMed

    Quixabeira-Santos, Jociane Cristina; Medaglia, Maria Luiza G; Pescador, Caroline A; Damaso, Clarissa R

    2011-04-01

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

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

    PubMed

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

    2015-06-11

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

  19. 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

  20. Oncolytic Virotherapy: Molecular Targets in Tumor-Selective Replication and Carrier Cell-Mediated Delivery of Oncolytic Viruses

    PubMed Central

    Guo, Z. Sheng; Thorne, Stephen H.; Bartlett, David L.

    2010-01-01

    Tremendous advances have been made in developing oncolytic viruses (OVs) in the last few years. By taking advantage of current knowledge in cancer biology and virology, specific OVs have been genetically engineered to target specific molecules or signal transduction pathways in cancer cells in order to achieve efficient and selective replication. The viral infection and amplification eventually induces cancer cells into cell death pathways and elicits host anti-tumor immune responses to further help eliminate cancer cells. Specifically targeted molecules or signaling pathways (such as RB/E2F/p16, p53, IFN, PKR, EGFR, Ras, Wnt, anti-apoptosis or hypoxia) in cancer cells or tumor microenvironment have been studied and dissected with a variety of OVs such as adenovirus, herpes simplex virus, poxvirus, vesicular stomatitis virus, measles virus, Newcastle disease virus, influenza virus and reovirus, setting the molecular basis for further improvements in the near future. Another exciting new area of research has been the harnessing of naturally tumor-homing cells as carrier cells (or cellular vehicles) to deliver OVs to tumors. The trafficking of these tumor-homing cells (stem cells, immune cells and cancer cells), which support proliferation of the viruses, is mediated by specific chemokines and cell adhesion molecules and we are just beginning to understand the roles of these molecules. Finally, we will highlight some avenues deserving further study in order to achieve the ultimate goals of utilizing various OVs for effective cancer treatment. PMID:18328829

  1. 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

  2. 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

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

    PubMed

    Condit, Richard C; Moussatche, Nissin

    2015-08-01

    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

  4. Oncolytic vesicular stomatitis virus administered by isolated limb perfusion suppresses osteosarcoma growth.

    PubMed

    Kubo, Tadahiko; Shimose, Shoji; Matsuo, Toshihiro; Fujimori, Jun; Sakaguchi, Takemasa; Yamaki, Minoru; Shinozaki, Katsunori; Woo, Savio L C; Ochi, Mitsuo

    2011-05-01

    A significant limitation to oncolytic virotherapy in vivo is the lack of a clinically relevant means of delivering the virus. We evaluated the oncolytic activity of vesicular stomatitis virus (VSV) in human osteosarcoma cells and explored isolated limb perfusion (ILP) as a novel oncolytic virus delivery system to extremity sarcoma in immune-competent rats. Human and rat osteosarcoma cells transduced with rVSV-lacZ uniformly expressed β-gal. VSV was fully capable of replicating its RNA genome in all osteosarcoma cell lines, and efficiently killed them in time- and dose-dependent manners, whereas normal bone marrow stromal cells were refractory to the virus. VSV delivered by ILP inhibited growth of osteosarcoma xenografts more potently than that injected intravenously and intratumorally in the hind limb of immune-competent rats. Histopathological sections of tumor lesions treated by ILP-delivered VSV showed positive for VSV-G protein. There were no VSV-G expressions in perfused leg muscle, nonperfused leg muscle, brain, lung, and liver in VSV-treated rats. Our findings show efficient VSV gene expression and replication in osteosarcoma cells, suggesting that osteosarcoma may be a promising target for oncolytic virotherapy with VSV. Furthermore, we firstly showed that ILP of VSV against extremity sarcoma caused antitumor activity. PMID:21437961

  5. Selective oncolytic effect of an attenuated Newcastle disease virus (NDV-HUJ) in lung tumors.

    PubMed

    Yaacov, B; Eliahoo, E; Elihaoo, E; Lazar, I; Ben-Shlomo, M; Greenbaum, I; Panet, A; Zakay-Rones, Z

    2008-12-01

    Newcastle disease virus (NDV), an avian paramyxovirus, has a potential oncolytic effect that may be of significance in the treatment of a variety of cancer diseases. An attenuated lentogenic isolate of NDV (HUJ) demonstrated a selective cytopathic effect upon a panel of human and mouse lung tumor cells, as compared to human nontumorigenic lung cells. The virus-selective oncolytic effect is apoptosis dependent, and related to higher levels of viral transcription, translation and progeny virus formation. Furthermore, NDV-HUJ oncolytic activity is directed in-cis and not through induction of cytokines, that may act in-trans on neighboring cells. Development of primary lung tumors and of the consequent metastasis in mice inoculated with mouse lung tumor cells 3LL-D122 was decreased following treatment with NDV-HUJ. The preferential killing of the tumor cells is not due to a deficiency in the interferon (IFN) system, as expression of the IFN-beta gene, in the infected cells, is properly induced. Moreover, pretreatment with IFN effectively protected the tumor cells from the virus oncolytic effect. We conclude therefore, that NDV-HUJ should have a significant benefit in the treatment of lung cancer as well as other malignancies. PMID:18535620

  6. Vaccinia virus telomeres: interaction with the viral I1, I6, and K4 proteins.

    PubMed

    DeMasi, J; Du, S; Lennon, D; Traktman, P

    2001-11-01

    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

  7. The highly attenuated vaccinia virus strain modified virus Ankara induces apoptosis in melanoma cells and allows bystander dendritic cells to generate a potent anti-tumoral immunity

    PubMed Central

    Greiner, S; Humrich, J Y; Thuman, P; Sauter, B; Schuler, G; Jenne, L

    2006-01-01

    Vaccinia virus (VV) has been tested as oncolytic virus against malignant melanoma in clinical trials for more than 40 years. Until now, mainly strains comparable to viral strains used for smallpox vaccination have been probed for anti-tumoral therapy. We have shown recently that the wild-type strain Western Reserve (WR) can interfere with crucial functions of monocyte-derived dendritic cells (DCs). Our aim was to examine whether viral immune evasion mechanisms might be responsible for the ineffectiveness of WR-based vaccination strategies and whether the highly attenuated strain modified virus Ankara (MVA) differs from WR with respect to its possible immunostimulatory capacity after intratumoral injection. Using in vitro experiments, we compared the effect of both strains on melanoma cells and on local bystander DCs. We found that both VV-strains infected melanoma cells efficiently and caused disintegration of the actin cytoskeleton, as shown by fluorescence microscopy. In addition, both VV-strains caused apoptotic cell death in melanoma cells after infection. In contrast to MVA, WR underwent a complete viral replication cycle in melanoma cells. Bystander DCs were consecutively infected by newly generated WR virions and lost their capacity to induce allogeneic T cell proliferation. DCs in contact with MVA-infected melanoma cells retained their capacity to induce T cell proliferation. Immature DCs were capable of phagocytosing MVA-infected melanoma cells. Priming of autologous CD8+ T cells by DCs that had phagocytosed MVA-infected, MelanA positive melanoma cells resulted in the induction of T cell clones specifically reactive against the model antigen MelanA as shown by enzyme-linked immunospot (ELISPOT) analysis. We conclude that the clinical trials with oncolytic wild-type VV failed probably because of suppression of bystander DCs and consecutive suppression of T cell-mediated anti-melanoma immunity. The attenuated VV-strain MVA facilitates the generation of

  8. 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. PMID:25004098

  9. 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

  10. 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

  11. 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

  12. 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.

  13. Oral vaccination of the fox against rabies using a live recombinant vaccinia virus.

    PubMed

    Blancou, J; Kieny, M P; Lathe, R; Lecocq, J P; Pastoret, P P; Soulebot, J P; Desmettre, P

    Rabies, a viral disease affecting all warm-blooded animals, is prevalent in most parts of the world, where it propagates amongst wild animals, particularly the fox and dog. The public health and economic consequences of infection in man and livestock are well known. Attempts to control the disease by vaccinating wild carnivores with inactivated or attenuated rabies virus remain controversial, and we have instead evaluated here the potential of a recombinant vaccinia virus to protect foxes against the disease. We have found that the administration of vaccinia virus (VV) or a recombinant harbouring the rabies surface antigen gene (VVTGgRAB) is innocuous to foxes. The recombinant virus can elicit the production of titers of rabies-neutralizing antibodies equal or superior to those obtained with conventional vaccine, and 10(8) plaque-forming units (PFU) of VVTGgRAB administered subcutaneously, intradermally or orally confers complete protection to severe challenge infection with street rabies virus. PMID:3736663

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

    PubMed Central

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

    2007-01-01

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

  15. Vaccinia Virus Entry, Exit, and Interaction with Differentiated Human Airway Epithelia▿

    PubMed Central

    Vermeer, Paola D.; McHugh, Julia; Rokhlina, Tatiana; Vermeer, Daniel W.; Zabner, Joseph; Welsh, Michael J.

    2007-01-01

    Variola virus, the causative agent of smallpox, enters and exits the host via the respiratory route. To better understand the pathogenesis of poxvirus infection and its interaction with respiratory epithelia, we used vaccinia virus and examined its interaction with primary cultures of well-differentiated human airway epithelia. We found that vaccinia virus preferentially infected the epithelia through the basolateral membrane and released viral progeny across the apical membrane. Despite infection and virus production, epithelia retained tight junctions, transepithelial electrical conductance, and a steep transepithelial concentration gradient of virus, indicating integrity of the epithelial barrier. In fact, during the first four days of infection, epithelial height and cell number increased. These morphological changes and maintenance of epithelial integrity required vaccinia virus growth factor, which was released basolaterally, where it activated epidermal growth factor 1 receptors. These data suggest a complex interaction between the virus and differentiated airway epithelia; the virus preferentially enters the cells basolaterally, exits apically, and maintains epithelial integrity by stimulating growth factor receptors. PMID:17581984

  16. 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

  17. 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

  18. Targeting Human Medulloblastoma: Oncolytic Virotherapy with Myxoma Virus Is Enhanced by Rapamycin

    PubMed Central

    Lun, Xue Qing; Zhou, Hongyuan; Alain, Tommy; Sun, Beichen; Wang, Limei; Barrett, John W.; Stanford, Marianne M.; McFadden, Grant; Bell, John; Senger, Donna L.; Forsyth, Peter A.

    2015-01-01

    We have shown previously the oncolytic potential of myxoma virus in a murine xenograft model of human glioma. Here, we show that myxoma virus used alone or in combination with rapamycin is effective and safe when used in experimental models of medulloblastoma in vitro and in vivo. Nine of 10 medulloblastoma cell lines tested were susceptible to lethal myxoma virus infection, and pretreatment of cells with rapamycin increased the extent of in vitro oncolysis. Intratumoral injection of live myxoma virus when compared with control inactivated virus prolonged survival in D341 and Daoy orthotopic human medulloblastoma xenograft mouse models [D341 median survival: 21 versus 12.5 days; P = 0.0008; Daoy median survival: not reached (three of five mice apparently “cured” after 223 days) versus 75 days; P = 0.0021]. Rapamycin increased the extent of viral oncolysis, “curing” most Daoy tumor-bearing mice and reducing or eliminating spinal cord and ventricle metastases. Rapamycin enhanced tumor-specific myxoma virus replication in vivo and prolonged survival of D341 tumor-bearing mice (median survival of mice treated with live virus (LV) and rapamycin, versus LV alone, versus rapamycin alone, versus inactivated virus: 25 days versus 19, 13, and 11 days, respectively; P < 0.0001). Rapamycin increased the levels of constitutively activated Akt in Daoy and D341 cells, which may explain its ability to enhance myxoma virus oncolysis. These observations suggest that myxoma virus may be an effective oncolytic agent against medulloblastoma and that combination therapy with signaling inhibitors that modulate activity of the phosphatidylinositol 3-kinase/Akt pathway will further enhance the oncolytic potential of myxoma virus. PMID:17875723

  19. Engineering the vaccinia virus L1 protein for increased neutralizing antibody response after DNA immunization

    PubMed Central

    Shinoda, Kaori; Wyatt, Linda S; Irvine, Kari R; Moss, Bernard

    2009-01-01

    Background The licensed smallpox vaccine, comprised of infectious vaccinia virus, has associated adverse effects, particularly for immunocompromised individuals. Therefore, safer DNA and protein vaccines are being investigated. The L1 protein, a component of the mature virion membrane that is conserved in all sequenced poxviruses, is required for vaccinia virus entry into host cells and is a target for neutralizing antibody. When expressed by vaccinia virus, the unglycosylated, myristoylated L1 protein attaches to the viral membrane via a C-terminal transmembrane anchor without traversing the secretory pathway. The purpose of the present study was to investigate modifications of the gene expressing the L1 protein that would increase immunogenicity in mice when delivered by a gene gun. Results The L1 gene was codon modified for optimal expression in mammalian cells and potential N-glycosylation sites removed. Addition of a signal sequence to the N-terminus of L1 increased cell surface expression as shown by confocal microscopy and flow cytometry of transfected cells. Removal of the transmembrane domain led to secretion of L1 into the medium. Induction of binding and neutralizing antibodies in mice was enhanced by gene gun delivery of L1 containing the signal sequence with or without the transmembrane domain. Each L1 construct partially protected mice against weight loss caused by intranasal administration of vaccinia virus. Conclusion Modifications of the vaccinia virus L1 gene including codon optimization and addition of a signal sequence with or without deletion of the transmembrane domain can enhance the neutralizing antibody response of a DNA vaccine. PMID:19257896

  20. Analysis of vaccinia virus-host protein-protein interactions: validations of yeast two-hybrid screenings.

    PubMed

    Zhang, Leiliang; Villa, Nancy Y; Rahman, Masmudur M; Smallwood, Sherin; Shattuck, Donna; Neff, Chris; Dufford, Max; Lanchbury, Jerry S; Labaer, Joshua; McFadden, Grant

    2009-09-01

    Vaccinia virus, a large double-stranded DNA virus, is the prototype of the Orthopoxvirus genus, which includes several pathogenic poxviruses of humans, such as monkeypox virus and variola virus. Here, we report a comprehensive yeast two-hybrid (Y2H) screening for the protein-protein interactions between vaccinia and human proteins. A total of 109 novel vaccinia-human protein interactions were detected among 33 viral proteins. To validate subsets of those interactions, we constructed an ORFeome library of vaccinia virus strain WR using the Gateway plasmid cloning system. By co-expressing selected vaccinia and host proteins in a variety of expression systems, we found that at least 17 of the Y2H hits identified between vaccinia and human proteins can be verified by independent methods using GST pull-down assays, representing a 63% validation rate for the Y2H hits examined (17/27). Because the cloned ORFs are conveniently transferable from the entry vectors to various destination expression vectors, the vaccinia ORFeome library will be a useful resource for future high-throughput functional proteomic experiments. PMID:19637933

  1. 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

  2. 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

  3. 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

  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. PMID:24130722

  5. Oncolytic Semliki forest virus vector as a novel candidate against unresectable osteosarcoma.

    PubMed

    Ketola, Anna; Hinkkanen, Ari; Yongabi, Felicitas; Furu, Petra; Määttä, Ann-Marie; Liimatainen, Timo; Pirinen, Risto; Björn, Marko; Hakkarainen, Tanja; Mäkinen, Kimmo; Wahlfors, Jarmo; Pellinen, Riikka

    2008-10-15

    Oncolytic viruses are a promising tool for treatment of cancer. We studied an oncolytic Semliki Forest virus (SFV) vector, VA7, carrying the enhanced green fluorescent protein gene (EGFP), as a novel virotherapy candidate against unresectable osteosarcoma. The efficiency and characteristics of the VA7-EGFP treatment were compared with a widely studied oncolytic adenovirus, Ad5Delta24, both in vitro and in vivo. VA7-EGFP resulted in more rapid oncolysis and was more efficient at low multiplicities of infection (MOI) when compared with Ad5Delta24 in vitro. Yet, in MG-63 cells, a subpopulation resistant to the VA7-EGFP vector emerged. In subcutaneous human osteosarcoma xenografts in nude mice treatment with either vector reduced tumor size, whereas tumors in control mice expanded quickly. The VA7-EGFP-treated tumors were either completely abolished or regressed to pinpoint size. The efficacy of VA7-EGFP vector was studied also in an orthotopic osteosarcoma nude mouse model characterized by highly aggressive tumor growth. Treatment with oncolytic SFV extended survival of the animals significantly (P < 0.01), yet none of the animals were finally cured. Sera from SFV-treated mice contained neutralizing antibodies, and as nude mice are not able to establish IgG response, the result points out the role of IgM class antibodies in clearance of virus from peripheral tumors. Furthermore, biodistribution analysis at the survival end point verified the presence of virus in some of the brain samples, which is in line with previous studies demonstrating that IgG is required for clearance of SFV from central nervous system. PMID:18922906

  6. Mantle cell lymphoma salvage regimen: synergy between a reprogrammed oncolytic virus and two chemotherapeutics

    PubMed Central

    Ungerechts, Guy; Frenzke, Marie E; Yaiw, Koon-Chu; Miest, Tanner; Johnston, Patrick B; Cattaneo, Roberto

    2010-01-01

    MV-PNP HblindantiCD20 is a CD20-targeted and prodrug convertase-armed measles virus (MV) that temporarily controls growth of lymphoma xenografts in SCID mice in combination with fludarabine phosphate. Herein, we examine the replication of this targeted virus and of a vaccine-lineage MV in disease bulks and circulating cells from mantle cell lymphoma (MCL) patients, and show that only the targeted virus is specific for CD20-expressing cells. We then assessed the efficacy of different regimens of administration of this virus in combination with fludarabine and cyclophosphamide (CPA) in a MCL xenograft model. We show that CPA administration before virus enhances oncolytic efficacy, likely through temporary immunosuppression. An interval of one-week between intravenous virus administration and fludarabine treatment further enhanced oncolysis, by synchronizing maximum prodrug convertase expression with fludarabine availability. Finally, three 23-day courses of triple sequential treatment with CPA, virus and fludarabine treatment resulted in complete regression of the xenografts. Secondary disease symptoms interfered with survival, but average survival times increased from 22 to 77 days. These studies document a reprogrammed oncolytic virus consolidating the effects of two chemotherapeutics, a concept well-suited for a phase I clinical trial for MCL patients for whom conventional therapies have failed. PMID:20686506

  7. 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

  8. Oncolytic measles virus prolongs survival in a murine model of cerebral spinal fluid–disseminated medulloblastoma

    PubMed Central

    Studebaker, Adam W.; Hutzen, Brian; Pierson, Christopher R.; Russell, Stephen J.; Galanis, Evanthia; Raffel, Corey

    2012-01-01

    Medulloblastoma is the most common malignant brain tumor of childhood. Although the survival rate of afflicted children has improved considerably over the past several years, a subset of these patients will present with disseminated disease and face a much bleaker prognosis. In addition, patients may present with disseminated disease at recurrence. We previously demonstrated the efficacy of a recombinant oncolytic measles virus (MV) to treat localized medulloblastoma in a mouse xenograft model. In the present study, we sought to extend our findings to the treatment of disseminated disease. To this end, we developed and characterized a mouse xenograft model of disseminated medulloblastoma using serial bioluminescent imaging techniques in combination with histopathological examination. Mice injected with medulloblastoma cells into their right lateral ventricle showed tumor growth in their ventricles and in both intracranial and spinal subarachnoid spaces, closely recapitulating the human disease. Subsequent intraventricular administration of MV resulted in stabilization and shrinkage of the tumor, significantly prolonging the survival of the treated animals, compared with those treated with an inactivated virus. These data demonstrate that oncolytic MV may be of use in treating disseminated medulloblastoma. In addition, our protocol of intraventricular tumor cell injection, followed by bioluminescent imaging coupled with histopathological examination, provides a model for use in evaluating future recombinant oncolytic viruses and other preclinical therapeutic approaches for disseminated medulloblastoma. PMID:22307474

  9. Entry of Oncolytic Herpes Simplex Virus into Human Squamous Cell Carcinoma Cells by Ultrasound.

    PubMed

    Okunaga, Shusuke; Takasu, Ayako; Meshii, Noritoshi; Imai, Tomoaki; Hamada, Masakagu; Iwai, Soichi; Yura, Yoshiaki

    2015-10-01

    Low-intensity ultrasound is a useful method to introduce materials into cells due to the transient formation of micropores, called sonoporations, on the cell membrane. Whether oncolytic herpes simplex virus type 1 (HSV-1) can be introduced into oral squamous cell carcinoma (SCC) cells through membrane pores remains undetermined. Human SCC cell line SAS and oncolytic HSV-1 RH2, which was deficient in the 134.5 gene and fusogenic, were used. Cells were exposed to ultrasound in the presence or absence of microbubbles. The increase of virus entry was estimated by plaque numbers. Viral infection was hardly established without the adsorption step, but plaque number was increased by the exposure of HSV-1-inoculated cells to ultrasound. Plaque number was also increased even if SAS cells were exposed to ultrasound and inoculated with RH2 without the adsorption step. This effect was abolished when the interval from ultrasound exposure to virus inoculation was prolonged. Scanning electron microscopy revealed depressed spots on the cell surface after exposure to ultrasound. These results suggest that oncolytic HSV-1 RH2 can be introduced into SAS cells through ultrasound-mediated pores of the cell membrane that are resealed after an interval. PMID:26516901

  10. Entry of Oncolytic Herpes Simplex Virus into Human Squamous Cell Carcinoma Cells by Ultrasound

    PubMed Central

    Okunaga, Shusuke; Takasu, Ayako; Meshii, Noritoshi; Imai, Tomoaki; Hamada, Masakagu; Iwai, Soichi; Yura, Yoshiaki

    2015-01-01

    Low-intensity ultrasound is a useful method to introduce materials into cells due to the transient formation of micropores, called sonoporations, on the cell membrane. Whether oncolytic herpes simplex virus type 1 (HSV-1) can be introduced into oral squamous cell carcinoma (SCC) cells through membrane pores remains undetermined. Human SCC cell line SAS and oncolytic HSV-1 RH2, which was deficient in the γ134.5 gene and fusogenic, were used. Cells were exposed to ultrasound in the presence or absence of microbubbles. The increase of virus entry was estimated by plaque numbers. Viral infection was hardly established without the adsorption step, but plaque number was increased by the exposure of HSV-1-inoculated cells to ultrasound. Plaque number was also increased even if SAS cells were exposed to ultrasound and inoculated with RH2 without the adsorption step. This effect was abolished when the interval from ultrasound exposure to virus inoculation was prolonged. Scanning electron microscopy revealed depressed spots on the cell surface after exposure to ultrasound. These results suggest that oncolytic HSV-1 RH2 can be introduced into SAS cells through ultrasound-mediated pores of the cell membrane that are resealed after an interval. PMID:26516901

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

    PubMed Central

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

    1995-01-01

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

  12. Glycosylated and Nonglycosylated Complement Control Protein of the Lister Strain of Vaccinia Virus

    PubMed Central

    Kuhn, Jordan; Atukorale, Vajini; Campbell, Joseph; Weir, Jerry P.

    2014-01-01

    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. PMID:25030055

  13. 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

  14. 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 Central

    2013-01-01

    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. PMID:23842430

  15. 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

  16. 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

  17. A Novel Oncolytic Herpes Simplex Virus Type 2 Has Potent Anti-Tumor Activity

    PubMed Central

    Zhuang, Xiufen; Lu, Haizhen; Liang, Jing; Li, Jie; Zhang, Yu; Dong, Ying; Zhang, Youhui; Zhang, Shuren; Liu, Shangmei; Liu, Binlei

    2014-01-01

    Oncolytic viruses are promising treatments for many kinds of solid tumors. In this study, we constructed a novel oncolytic herpes simplex virus type 2: oHSV2. We investigated the cytopathic effects of oHSV2 in vitro and tested its antitumor efficacy in a 4T1 breast cancer model. We compared its effect on the cell cycle and its immunologic impact with the traditional chemotherapeutic agent doxorubicin. In vitro data showed that oHSV2 infected most of the human and murine tumor cell lines and was highly oncolytic. oHSV2 infected and killed 4T1 tumor cells independent of their cell cycle phase, whereas doxorubicin mainly blocked cells that were in S and G2/M phase. In vivo study showed that both oHSV2 and doxorubicin had an antitumor effect, though the former was less toxic. oHSV2 treatment alone not only slowed down the growth of tumors without causing weight loss but also induced an elevation of NK cells and mild decrease of Tregs in spleen. In addition, combination therapy of doxorubicin followed by oHSV2 increased survival with weight loss than oHSV2 alone. The data showed that the oncolytic activity of oHSV2 was similar to oHSV1 in cell lines examined and in vivo. Therefore, we concluded that our virus is a safe and effective therapeutic agent for 4T1 breast cancer and that the sequential use of doxorubicin followed by oHSV2 could improve antitumor activity without enhancing doxorubicin’s toxicity. PMID:24671154

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

    PubMed Central

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

    2014-01-01

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

  19. 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.

  20. 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

  1. Identification of a Bohle iridovirus thymidine kinase gene and demonstration of activity using vaccinia virus.

    PubMed

    Coupar, B E H; Goldie, S G; Hyatt, A D; Pallister, J A

    2005-09-01

    In recent years interest in the family Iridoviridae has been renewed by the identification of a number of viruses, particularly from the genus Ranavirus, associated with disease in a range of poikilotherms. Ranaviruses have been isolated from amphibian, piscine and reptilian species. Here we describe an open reading frame (ORF) identified in the genome of Bohle iridovirus (BIV) which contains a nucleotide binding motif conserved within the thymidine kinase (TK) genes of iridoviruses from other genera (lymphocystis disease virus, LCDV, type species of the genus Lymphocystivirus; Chilo iridescent virus, CIV, type species of the genus Iridovirus). The ability of this putative gene to express a functional TK was confirmed by rescue of a TK negative mutant vaccinia virus in the presence of selective media, when expression was controlled by a vaccinia virus promoter. The sequence of the BIV TK was compared with the homologous sequences from epizootic haematopoietic necrosis virus (EHNV), a virus associated with disease in fish, from Wamena iridovirus (WIV) associated with systemic disease in green pythons, and from frog virus 3 (FV3) the ranavirus type species. Comparisons between these sequences and those available from other ranaviruses, other iridoviruses, other DNA viruses and cellular TKs are presented. PMID:15883656

  2. Human cytomegalovirus UL97 kinase confers ganciclovir susceptibility to recombinant vaccinia virus.

    PubMed Central

    Metzger, C; Michel, D; Schneider, K; Lüske, A; Schlicht, H J; Mertens, T

    1994-01-01

    We analyzed whether the phosphotransferase encoded by the UL97 open reading frame of human cytomegalovirus (HCMV) alone is sufficient to confer ganciclovir (GCV) susceptibility to a foreign virus. Two vaccinia virus recombinants (T1 and A5) containing the UL97 open reading frames from a GCV-sensitive HCMV and from a GCV-resistant strain were constructed. T1 exhibited a GCV-sensitive phenotype in plaque reduction assays, whereas A5 did not. Moreover, T1-infected cell cultures showed a strongly increased incorporation of [14C]GCV triphosphate into macromolecular DNA, compared with recombinant A5 or vaccinia virus controls, which could be inhibited by the addition of guanosine. This shows that UL97 kinase is the only additional gene product required to make vaccinia virus susceptible to GCV, and guanosine seems to be one natural substrate for the enzyme. The system described here should be very helpful for fast and detailed functional analyses of UL97 mutations found in GCV-resistant HCMV isolates. Images PMID:7966639

  3. Promising oncolytic agents for metastatic breast cancer treatment

    PubMed Central

    Cody, James J; Hurst, Douglas R

    2015-01-01

    New therapies for metastatic breast cancer patients are urgently needed. The long-term survival rates remain unacceptably low for patients with recurrent disease or disseminated metastases. In addition, existing therapies often cause a variety of debilitating side effects that severely impact quality of life. Oncolytic viruses constitute a developing therapeutic modality in which interest continues to build due to their ability to spare normal tissue while selectively destroying tumor cells. A number of different viruses have been used to develop oncolytic agents for breast cancer, including herpes simplex virus, adenovirus, vaccinia virus, measles virus, reovirus, and others. In general, clinical trials for several cancers have demonstrated excellent safety records and evidence of efficacy. However, the impressive tumor responses often observed in preclinical studies have yet to be realized in the clinic. In order for the promise of oncolytic virotherapy to be fully realized for breast cancer patients, effectiveness must be demonstrated in metastatic disease. This review provides a summary of oncolytic virotherapy strategies being developed to target metastatic breast cancer.

  4. 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

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

    PubMed

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

    2016-01-01

    Short-term nutritional restriction (fasting) has been shown to enhance the efficacy of chemotherapy by sensitizing cancer cells and protecting normal cells in a variety of cancer models, including glioblastoma (GBM). Cancer cells, unlike normal cells, respond to fasting by promoting oncogenic signaling and protein synthesis. We hypothesized that fasting would increase the replication of oncolytic herpes simplex virus (oHSV) in GBM. Patient-derived GBM cell lines were fasted by growth in glucose and fetal calf serum restricted culture medium. "Transient fasting", 24-hour fasting followed by 24-hour recovery in complete medium, increased late virus gene expression and G47Δ yields about 2-fold in GBM cells, but not in human astrocytes, and enhanced G47Δ killing of GBM cells. Mechanistically, "transient fasting" suppressed phosphorylation of the subunit of eukaryotic initiation factor 2α (eIF2α) and c-Jun N-terminal kinases (JNK) in GBM cells, but not in astrocytes. Pharmacological inhibition of JNK also increased G47Δ yield. In vivo, transient fasting (48-hour food restriction and 24-hour recovery) doubled luciferase activity after intratumoral G47Δ-US11fluc injection into orthotopic GBM xenografts. Thus, "transient fasting" increases G47Δ replication and oncolytic activity in human GBM cells. These results suggest that "transient fasting" may be effectively combined to enhance oncolytic HSV therapy of GBM. PMID:27186404

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

    PubMed Central

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

    2016-01-01

    Short-term nutritional restriction (fasting) has been shown to enhance the efficacy of chemotherapy by sensitizing cancer cells and protecting normal cells in a variety of cancer models, including glioblastoma (GBM). Cancer cells, unlike normal cells, respond to fasting by promoting oncogenic signaling and protein synthesis. We hypothesized that fasting would increase the replication of oncolytic herpes simplex virus (oHSV) in GBM. Patient-derived GBM cell lines were fasted by growth in glucose and fetal calf serum restricted culture medium. “Transient fasting”, 24-hour fasting followed by 24-hour recovery in complete medium, increased late virus gene expression and G47Δ yields about 2-fold in GBM cells, but not in human astrocytes, and enhanced G47Δ killing of GBM cells. Mechanistically, “transient fasting” suppressed phosphorylation of the subunit of eukaryotic initiation factor 2α (eIF2α) and c-Jun N-terminal kinases (JNK) in GBM cells, but not in astrocytes. Pharmacological inhibition of JNK also increased G47Δ yield. In vivo, transient fasting (48-hour food restriction and 24-hour recovery) doubled luciferase activity after intratumoral G47Δ-US11fluc injection into orthotopic GBM xenografts. Thus, “transient fasting” increases G47Δ replication and oncolytic activity in human GBM cells. These results suggest that “transient fasting” may be effectively combined to enhance oncolytic HSV therapy of GBM. PMID:27186404

  7. 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

  8. 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

  9. Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase.

    PubMed Central

    Broyles, S S; Moss, B

    1987-01-01

    Vaccinia virus encapsidates a DNA-dependent ATPase known as nucleoside triphosphate phosphohydrolase I (NPH I). A bacteriophage lambda gt11 expression library of poxvirus DNA was screened with antibodies specific for NPH I. Positive clones were used to probe restriction fragments of vaccinia virus genomic DNA to locate the NPH I gene. The identity of the open reading frame (ORF) was confirmed by placing it downstream of a bacteriophage T7 promoter, transcribing the ORF in vitro, and translating the RNA in a reticulocyte lysate. A polypeptide of the correct molecular weight, which was recognized by anti-NPH I antibody, was synthesized. Inspection of the deduced amino acid sequence of the NPH I ORF revealed consensus ATP-binding sites. Images PMID:2437324

  10. 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

  11. 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

  12. Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

    PubMed Central

    Vassef, A; Mars, M; Dru, A; Plucienniczak, A; Streeck, R E; Beaud, G

    1985-01-01

    Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs. Images PMID:2989553

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

    PubMed Central

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

    2014-01-01

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

  14. 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

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

    PubMed Central

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

    2016-01-01

    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

  16. 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

  17. 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

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

    PubMed Central

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

    1995-01-01

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

  19. Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

    PubMed Central

    Bolden, A; Aucker, J; Weissbach, A

    1975-01-01

    Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha. PMID:172658

  20. Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65.

    PubMed Central

    Sodeik, B; Griffiths, G; Ericsson, M; Moss, B; Doms, R W

    1994-01-01

    The cytoplasmic assembly of vaccinia virus is reversibly blocked by the antibiotic rifampin, leading to the accumulation of partially membrane-delineated rifampin bodies in infected cells. Rifampin-resistant vaccinia virus mutants have point mutations in the D13L gene, which is controlled by a late promoter and expresses a 65-kDa protein, designated p65. To further characterize the mechanism of rifampin inhibition and the function of p65 in virus assembly, we raised antibodies to this protein. Immunoreactive p65 was expressed at late times of infection, and neither its expression nor its turnover was affected by rifampin. Virus-associated p65 could be extracted only with denaturing detergents from purified virions, suggesting that it is an integral viral component. Immunofluorescence studies showed that p65 is localized to the sites of virus assembly. Also, immunoelectron microscopy showed p65 to be associated with viral crescents as well as spherical, immature virions, in both cases predominantly on the inner or concave surface. In the presence of rifampin, p65 was found in large, cytoplasmic inclusion bodies that were distinct from rifampin bodies. The rifampin bodies themselves were labeled with p65 antibodies only after reversal of the rifampin block, predominantly on the viral crescents which rapidly formed following removal of the drug. We propose that p65 functions as an internal scaffold in the formation of viral crescents and immature virions, analogously to the matrix proteins of other viruses. Images PMID:8289340

  1. Velogenic newcastle disease virus as an oncolytic virotherapeutics: in vitro characterization.

    PubMed

    Kumar, Rajiv; Tiwari, Ashok K; Chaturvedi, Uttara; Kumar, G Ravi; Sahoo, Aditya P; Rajmani, R S; Saxena, Lovleen; Saxena, Shikha; Tiwari, Sangeeta; Kumar, Sudesh

    2012-08-01

    Cancer is one of the killer diseases in humans and needs alternate curative measures despite recent improvement in modern treatment modalities. Oncolytic virotherapy seems to be a promising nonconventional way to treat cancers. Newcastle disease virus (NDV), a poultry virus, is nonpathogenic to human and domestic animals and has a long history of being used in oncotherapy research in several preclinical studies. The ability of NDV to successfully infect and destroy cancer cells is dependent on the strain and the pathotype of the virus. Adaptation of viruses to heterologous hosts without losing its replicative and oncolytic potential is prerequisite for use as cancer virotherapeutics. In the present study, velogenic NDV was adapted for replication in HeLa cells, and its cytotoxic potential was evaluated by observing morphological, biochemical, and nuclear landmarks of apoptosis. Our results indicated that the NDV-induced apoptosis in HeLa cells was dependent on upregulation of TNF-related apoptosis-inducing ligand (TRAIL) and caspases activation. Different determinants of apoptosis evaluated in the present study indicated that this strain could be a promising candidate for cancer therapy in future. PMID:22644640

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

    PubMed Central

    Msaouel, Pavlos; Dispenzieri, Angela; Galanis, Evanthia

    2009-01-01

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

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

    PubMed Central

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

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8+ T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine. PMID:26018172

  4. Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to vaccinia virus killing.

    PubMed Central

    Bair, C H; Chung, C S; Vasilevskaya, I A; Chang, W

    1996-01-01

    The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and

  5. 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.

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

    PubMed Central

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

    2014-01-01

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

  7. 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.

  8. Suitability of vaccinia virus and bovine viral diarrhea virus (BVDV) for determining activities of three commonly-used alcohol-based hand rubs against enveloped viruses

    PubMed Central

    Kampf, Günter; Steinmann, Jochen; Rabenau, Holger

    2007-01-01

    Background A procedure for including activity against enveloped viruses in the post-contamination treatment of hands has been recommended, but so far no European standard is available to implement it. In 2004, the German Robert Koch-Institute (RKI) and the German Association for the Control of Virus Disease (DVV) suggested that vaccinia virus and bovine viral diarrhea virus (BVDV) should be used as test viruses in a quantitative suspension test to determine the activity of a disinfectant against all enveloped viruses. Methods We have studied the activities of three commonly-used alcohol-based hand rubs (hand rub A, based on 45% propan-2-ol, 30% propan-1-ol and 0.2% mecetronium etilsulfate; hand rub B, based on 80% ethanol; hand rub C, based on 95% ethanol) against vaccinia virus and BVDV, and in addition against four other clinically relevant enveloped viruses: herpes simplex virus (HSV) types 1 and 2, and human and avian influenza A virus. The hand rubs were challenged with different organic loads at exposure time of 15, 30 and 60 s. According to the guidelines of both BGA/RKI and DVV, and EN 14476:2005, the reduction of infectivity of each test virus was measured on appropriate cell lines using a quantitative suspension test. Results All three alcohol-based hand rubs reduced the infectivity of vaccinia virus and BVDV by ≥ 4 log10-steps within 15 s, irrespective of the type of organic load. Similar reductions of infectivity were seen against the other four enveloped viruses within 15 s in the presence of different types of organic load. Conclusion Commonly used alcohol-based hand rubs with a total alcohol concentration ≥ 75% can be assumed to be active against clinically relevant enveloped viruses if they effectively reduce the infectivities of vaccinia virus and BVDV in a quantitative suspension test. PMID:17291338

  9. Structural Insight into BH3 Domain Binding of Vaccinia Virus Antiapoptotic F1L

    PubMed Central

    Campbell, Stephanie; Thibault, John; Mehta, Ninad; Colman, Peter M.

    2014-01-01

    ABSTRACT Apoptosis is a tightly regulated process that plays a crucial role in the removal of virus-infected cells, a process controlled by both pro- and antiapoptotic members of the Bcl-2 family. The proapoptotic proteins Bak and Bax are regulated by antiapoptotic Bcl-2 proteins and are also activated by a subset of proteins known as BH3-only proteins that perform dual functions by directly activating Bak and Bax or by sequestering and neutralizing antiapoptotic family members. Numerous viruses express proteins that prevent premature host cell apoptosis. Vaccinia virus encodes F1L, an antiapoptotic protein essential for survival of infected cells that bears no discernible sequence homology to mammalian cell death inhibitors. Despite the limited sequence similarities, F1L has been shown to adopt a novel dimeric Bcl-2-like fold that enables hetero-oligomeric binding to both Bak and the proapoptotic BH3-only protein Bim that ultimately prevents Bak and Bax homo-oligomerization. However, no structural data on the mode of engagement of F1L and its Bcl-2 counterparts are available. Here we solved the crystal structures of F1L in complex with two ligands, Bim and Bak. Our structures indicate that F1L can engage two BH3 ligands simultaneously via the canonical Bcl-2 ligand binding grooves. Furthermore, by structure-guided mutagenesis, we generated point mutations within the binding pocket of F1L in order to elucidate the residues responsible for both Bim and Bak binding and prevention of apoptosis. We propose that the sequestration of Bim by F1L is primarily responsible for preventing apoptosis during vaccinia virus infection. IMPORTANCE Numerous viruses have adapted strategies to counteract apoptosis by encoding proteins responsible for sequestering proapoptotic components. Vaccinia virus, the prototypical member of the family Orthopoxviridae, encodes a protein known as F1L that functions to prevent apoptosis by interacting with Bak and the BH3-only protein Bim. Despite

  10. 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

  11. 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

  12. Oncolytic Viruses in Head and Neck Cancer: A New Ray of Hope in the Management Protocol

    PubMed Central

    Shilpa, PS; Kaul, R; Bhat, S; Sultana, N; Pandeshwar, P

    2014-01-01

    This paper intends to highlight the different types of oncolytic viruses (OVs), mechanism of tumor specificity, its safety, and various obstacles in the design of treatment and combination therapy utilizing oncotherapy. Search was conducted using the internet-based search engines and scholarly bibliographic databases with key words such as OVs, head and neck cancer, viruses, oral squamous cell carcinoma, and gene therapy. Revolutionary technologies in the field of cancer treatment have gone through a series changes leading to the development of innovative therapeutic strategies. Oncolytic virotherapy is one such therapeutic approach that has awaited phase III clinical trial validation. OVs are self-replicating, tumor selective and lyse cancer cells following viral infection. By modifying the viral genome, it is possible to direct their toxicity toward cancer cells. Viruses that are used for treatment of head and neck cancer are either naturally occurring or genetically modified. OVs are tumor selective and potential anticancer agents. Virotherapy may become the standard of care and part of combination therapy in the management of head and neck cancer in the future. PMID:25364586

  13. Analysis of a mathematical model for tumor therapy with a fusogenic oncolytic virus.

    PubMed

    Jacobsen, Karly; Pilyugin, Sergei S

    2015-12-01

    Oncolytic virotherapy is a tumor treatment which uses viruses to selectively target and destroy cancer cells. Fusogenic viruses, capable of causing cell-to-cell fusion upon infection of a tumor cell, have shown promise in experimental studies. Fusion causes the formation of large, multinucleated syncytia which eventually leads to cell death. We formulate a partial differential equations model with a moving boundary to describe the treatment of a spherical tumor with a fusogenic oncolytic virus. Fusion, lysis, and budding are incorporated as mechanisms of viral spread, resulting in nonlocal integral terms. A proof is presented for existence and uniqueness of global solutions to the nonlinear hyperbolic-parabolic system. Numerical simulations demonstrate convergence to spatially homogeneous solutions and exponential growth or decay of the tumor radius depending on viral burst size and rate of fusion. Long-term tumor radius is shown to decrease with increasing values of viral burst size while the effect of the rate of fusion on tumor growth is demonstrated to be nonmonotonic. PMID:25744606

  14. 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

  15. Genome Sequence of WAU86/88-1, a New Variant of Vaccinia Virus Lister Strain from Poland.

    PubMed

    Mavian, Carla; López-Bueno, Alberto; Alcamí, Antonio

    2014-01-01

    The poxviruses Warsaw Agricultural University 86 (WAU86) and 88-1 (WAU88-1) were isolated in 1986 to 1988 from separate outbreaks in laboratory mice in Poland and described as ectromelia virus isolates. The genome sequences of these poxviruses reveal that they are almost identical and represent a novel variant of the vaccinia virus Lister strain. PMID:24407630

  16. 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

  17. 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. PMID:27155322

  18. 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

  19. Treatment of colon cancer with oncolytic herpes simplex virus in preclinical models.

    PubMed

    Yang, H; Peng, T; Li, J; Wang, Y; Zhang, W; Zhang, P; Peng, S; Du, T; Li, Y; Yan, Q; Liu, B

    2016-05-01

    Cancer stem cells (CSCs), which are a rare population in any type of cancer, including colon cancer, are tumorigenic and responsible for cancer recurrence and metastasis. CSCs have been isolated from a number of different solid tumors recently, although the isolation of CSCs in colon cancer is still challenging. We cultured colon cancer cells in stem cell medium to obtain colonosphere cells. These cells possessed the characteristics of CSCs, with a high capacity of tumorigenicity, migration and invasion in vitro and in vivo. The isolation and identification of CSCs have provided new targets for the therapeutics. Oncolytic herpes simplex viruses (oHSV) are an effective strategy for killing colon cancer cells in preclinical models. Here, we examined the efficacy of an oncolytic herpes simplex virus type 2 (oHSV2) in killing colon cancer cells and colon cancer stem-like cells (CSLCs). oHSV2 was found to be highly cytotoxic to the adherent and sphere cells in vitro, and oHSV2 treatment in vivo significantly inhibited tumor growth. This study demonstrates that oHSV2 is effective against colon cancer cells and colon CSLCs and could be a promising strategy for treating colon cancer patients. PMID:26871935

  20. Cell-Type-Specific Innate Immune Response to Oncolytic Newcastle Disease Virus

    PubMed Central

    Biswas, Moanaro; Kumar, Sandeep R.P.; Allen, Adria; Yong, Wang; Nimmanapalli, Ramadevi; Samal, Siba K.

    2012-01-01

    Abstract Virotherapy of cancer exploits the potential of naturally occurring and engineered oncolytic viruses to selectively replicate in and cause cytotoxicity to tumor cells without affecting healthy normal cells. The tumor selectivity of Newcastle disease virus (NDV), a member of the family Paramyxoviridae, depends on the differential type I interferon (IFN) response. Further understanding of the key mechanisms and immune effector molecules involved will aid in augmenting the oncolytic properties of NDV. Here we report on the infection kinetics and innate immune responses to a recombinant LaSota strain of NDV (rLaSota eGFP) in human tumor and normal cells. We observed varying replicative fit and cytotoxicity of rLaSota eGFP depending on the tumor cell type, with severely restricted replication in normal cells. The absence of retinoic acid-inducible gene I (RIG-I), a cytosolic RNA sensor, determined sensitivity to NDV. Productive NDV infection with a moderate IFN-α induction in human multiple myeloma cells suggested a role for IFN-independent mechanisms or lack of type I IFN reinforcement by RIG-I. Proinflammatory cytokines and chemokines were altered differentially in infected normal and tumor cells. Our results suggest that tumor selectivity is dependent on variations in the cellular antiviral response to infection with NDV and RIG-I expression. PMID:22808996

  1. Fine structure marker rescue of temperature-sensitive mutations of vaccinia virus within a central conserved region of the genome.

    PubMed Central

    Ensinger, M J; Weir, J P; Moss, B

    1985-01-01

    Fine structure marker rescue involving the use of subfragments of vaccinia virus HindIII DNA fragments L, J, and H has been used to map the mutations in eight temperature-sensitive mutants of vaccinia virus representing four complementation groups. Comparison of their map locations with the positions of the open reading frames and RNA transcripts that have been mapped within this region has allowed the identification of one or two polypeptides as the temperature-sensitive gene product of each mutant. PMID:4068140

  2. Nucleotide sequence of a cluster of early and late genes in a conserved segment of the vaccinia virus genome.

    PubMed Central

    Plucienniczak, A; Schroeder, E; Zettlmeissl, G; Streeck, R E

    1985-01-01

    The nucleotide sequence of a 7.6 kb vaccinia DNA segment from a genomic region conserved among different orthopox virus has been determined. This segment contains a tight cluster of 12 partly overlapping open reading frames most of which can be correlated with previously identified early and late proteins and mRNAs. Regulatory signals used by vaccinia virus have been studied. Presumptive promoter regions are rich in A, T and carry the consensus sequences TATA and AATAA spaced at 20-24 base pairs. Tandem repeats of a CTATTC consensus sequence are proposed to be involved in the termination of early transcription. PMID:2987815

  3. 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

  4. Protective Immunity to Vaccinia Virus Induced by Vaccination with Multiple Recombinant Outer Membrane Proteins of Intracellular and Extracellular Virions

    PubMed Central

    Fogg, Christiana; Lustig, Shlomo; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.; Moss, Bernard

    2004-01-01

    Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped virus. The recombinant proteins, in 10-μg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine. PMID:15367588

  5. Efficiently Editing the Vaccinia Virus Genome by Using the CRISPR-Cas9 System

    PubMed Central

    Yuan, Ming; Zhang, Wensheng; Wang, Jun; Al Yaghchi, Chadwan; Ahmed, Jahangir; Chard, Louisa

    2015-01-01

    Vaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions. PMID:25741005

  6. Efficiently editing the vaccinia virus genome by using the CRISPR-Cas9 system.

    PubMed

    Yuan, Ming; Zhang, Wensheng; Wang, Jun; Al Yaghchi, Chadwan; Ahmed, Jahangir; Chard, Louisa; Lemoine, Nick R; Wang, Yaohe

    2015-05-01

    Vaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions. PMID:25741005

  7. Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome.

    PubMed Central

    Wittek, R; Hänggi, M; Hiller, G

    1984-01-01

    Cell-free translation of total RNA isolated from vaccinia virus-infected cells late in infection results in a complex mixture of polypeptides. A monospecific antibody directed against one of the major structural proteins of the virus particle immunoprecipitated a single polypeptide with a molecular weight of 11,000 (11K) from this mixture. Immunoprecipitation was therefore used to identify the structural polypeptide among the in vitro translation products of RNA purified by hybridization selection to restriction fragments of the vaccinia virus genome. This allowed us to map the mRNA coding for the 11K polypeptide to the extreme left-hand end of the HindIII E fragment. Detailed transcriptional mapping of this region of the genome by nuclease S1 analysis revealed the presence of a late RNA transcribed from the rightward-reading strand. Its 5' end mapped at ca. 130 base pairs to the left of the HindIII site at the junction between the HindIII F and E fragments. The map position of this RNA coincided precisely with the map position of the late message coding for the 11K polypeptide. Images PMID:6319738

  8. Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus

    PubMed Central

    Cyrklaff, Marek; Linaroudis, Alexandros; Boicu, Marius; Chlanda, Petr; Baumeister, Wolfgang; Griffiths, Gareth; Krijnse-Locker, Jacomine

    2007-01-01

    At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, which is followed by its cytoplasmic replication. Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction. PMID:17487274

  9. Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses.

    PubMed

    Ralston, R; Thudium, K; Berger, K; Kuo, C; Gervase, B; Hall, J; Selby, M; Kuo, G; Houghton, M; Choo, Q L

    1993-11-01

    We constructed recombinant vaccinia virus vectors for expression of the structural region of hepatitis C virus (HCV). Infection of mammalian cells with a vector (vv/HCV1-906) encoding C-E1-E2-NS2 generated major protein species of 22 kDa (C), 33 to 35 kDa (E1), and 70 to 72 kDa (E2), as observed previously with other mammalian expression systems. The bulk of the E1 and E2 expressed by vv/HCV1-906 was found integrated into endoplasmic reticulum membranes as core-glycosylated species, suggesting that these E1 and E2 species represent intracellular forms of the HCV envelope proteins. HCV E1 and E2 formed E1-E2 complexes which were precipitated by either anti-E1 or anti-E2 serum and which sedimented at approximately 15 S on glycerol density gradients. No evidence of intermolecular disulfide bonding between E1 and E2 was detected. E1 and E2 were copurified to approximately 90% purity by mild detergent extraction followed by chromatography on Galanthus nivalus lectin-agarose and DEAE-Fractogel. Immunization of chimpanzees with purified E1-E2 generated high titers of anti-E1 and anti-E2 antibodies. Further studies, to be reported separately, demonstrated that purified E1-E2 complexes were recognized at high frequency by HCV+ human sera (D. Y. Chien, Q.-L. Choo, R. Ralston, R. Spaete, M. Tong, M. Houghton, and G. Kuo, Lancet, in press) and generated protective immunity in chimpanzees (Q.-L. Choo, G. Kuo, R. Ralston, A. Weiner, D. Chien, G. Van Nest, J. Han, K. Berger, K. Thudium, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Mass, L. B. Cummins, E. Muchmore, and M. Houghton, submitted for publication), suggesting that these purified HCV envelope proteins display native HCV epitopes. PMID:8411378

  10. 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.

  11. Combination therapy of oncolytic herpes simplex virus HF10 and bevacizumab against experimental model of human breast carcinoma xenograft.

    PubMed

    Tan, Gewen; Kasuya, Hideki; Sahin, Tevfik Tolga; Yamamura, Kazuo; Wu, Zhiwen; Koide, Yusuke; Hotta, Yoshihiro; Shikano, Toshio; Yamada, Suguru; Kanzaki, Akiyuki; Fujii, Tsutomu; Sugimoto, Hiroyuki; Nomoto, Shuji; Nishikawa, Yoko; Tanaka, Maki; Tsurumaru, Naoko; Kuwahara, Toshie; Fukuda, Saori; Ichinose, Toru; Kikumori, Toyone; Takeda, Shin; Nakao, Akimasa; Kodera, Yasuhiro

    2015-04-01

    Breast cancer is one of the most common and feared cancers faced by women. The prognosis of patients with advanced or recurrent breast cancer remains poor despite refinements in multimodality therapies involving chemotherapeutic and hormonal agents. Multimodal therapy with more specific and effective strategy is urgently needed. The oncolytic herpes simplex virus (HSV) has potential to become a new effective treatment option because of its broad host range and tumor selective viral distribution. Bevacizumab is a monoclonal antibody against VEGFA, which inhibits angiogenesis and therefore tumor growth. Our approach to enhance the antitumor effect of the oncolytic HSV is to combine oncolytic HSV HF10 and bevacizumab in the treatment of breast cancer. Our results showed that bevacizumab enhanced viral distribution as well as tumor hypoxia and expanded the population of apoptotic cells and therefore induced a synergistic antitumor effect. HF10 is expected to be a promising agent in combination with bevacizumab in the anticancer treatment. PMID:25156870

  12. A Novel High-Throughput Vaccinia Virus Neutralization Assay and Preexisting Immunity in Populations from Different Geographic Regions in China

    PubMed Central

    Liu, Qiang; Huang, Weijin; Nie, Jianhui; Zhu, Rong; Gao, Dongying; Song, Aijing; Meng, Shufang; Xu, Xuemei; Wang, Youchun

    2012-01-01

    Background Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China. Methodology/Principal Findings A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30–55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin. Conclusion A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui

  13. Oncolytic Measles Virus Encoding Thyroidal Sodium Iodide Symporter for Squamous Cell Cancer of the Head and Neck Radiovirotherapy

    PubMed Central

    Li, Hongtao; Peng, Kah-Whye

    2012-01-01

    Abstract Oncolytic measles virus (MV) encoding the human thyroidal sodium iodide symporter (MV-NIS) has proved to be safe after intraperitoneal or intravenous administration in patients with ovarian cancer or multiple myeloma, respectively, but it has not yet been administered through intratumoral injection in humans. Squamous cell carcinoma (SCC) of the head and neck (SCCHN) usually is locally invasive and spreads to the cervical lymph nodes, which are suitable for the intratumoral administration of oncolytic viruses. To test whether oncolytic MV is an effective treatment for SCCHN, we used oncolytic MV-NIS to infect SCCHN in vitro and in vivo. The data show that SCCHN cells were infected and killed by MV-NIS in vitro. Permissiveness of the tumor cells to MV infection was not affected by irradiation after viral addition. Monitored noninvasively through radioiodine-based single-photon emission computed tomography/computed tomography, intratumorally virus-delivered NIS has concentrated the radioiodine in the MV-NIS–treated tumors in the FaDu mouse xenograft model of human SCCHN, and the antitumor effect could be boosted significantly (p<0.05) either with concomitant cyclophosphamide therapy or with appropriately timed administration of radioiodine 131I. MV-NIS could be a promising new anticancer agent that may substantially enhance the outcomes of standard therapy after intratumoral administration in patients with locally advanced SCCHN. PMID:22235810

  14. 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.

  15. 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.

  16. Evaluation of vesicular stomatitis virus mutant as an oncolytic agent against prostate cancer

    PubMed Central

    Zhao, Xin; Huang, Shengsong; Luo, Huarong; Wan, Xiaodong; Gui, Yaping; Li, Junliang; Wu, Denglong

    2014-01-01

    Background: To date, limited options are available to treat malignant prostate cancer, and novel strategies need to be developed. Oncolytic viruses (OV) that have preferential replication capabilities in cancer cells rather than normal cells represent one promising alternative for treating malignant tumors. Vesicular stomatitis virus (VSV) is a non-segmented, negative-strand RNA virus with the inherent capability to selectively kill tumor cells. The aim of this study was to evaluate the potential of VSV-ΔM51-GFP as an effective therapeutic agent for treating prostate tumors. Methods: For in vitro experiments, DU145 and PC3 cell lines were treated with VSV-ΔM51-GFP. Viral titers were quantified using plaque assays. Cytotoxicity was performed by MTT analysis. IFN-β production was measured using a Human IFN-β detection ELISA Kit. The detection of apoptosis was performed via Annexin-V-FITC staining method and analyzed with flow cytometry. The in vivo antitumor efficacy of VSV-ΔM51-GFP in a xenograft mice prostate tumor model. Results: It was observed that VSV-ΔM51-GFP can efficiently replicate and lyse human prostate cancer cells and that this virus has reduced toxicity against normal human prostate epithelial cells in vitro. VSV-ΔM51-GFP in the induction of apoptosis in DU145 cells and PC3 cells. Furthermore, in a xenograft tumor animal model, nude mice bearing replication-competent VSV-ΔM51-GFP were able to eradicate malignant cells while leaving normal tissue relatively unaffected. The survival of the tumor-burdened animals treated with VSV-ΔM51-GFP may also be significantly prolonged compared to mock-infected animals. Conclusions: VSV-ΔM51-GFP showed promising oncolytic activity for treating prostate cancer. PMID:24995075

  17. Oral Vaccination With Vaccinia Virus Expressing the Tick Antigen Subolesin Inhibits Tick Feeding and Transmission of Borrelia burgdorferi Vaccination

    PubMed Central

    Bensaci, Mekki; Bhattacharya, Debaditya; Clark, Roger; Hu, Linden T.

    2014-01-01

    Immunization with the Ixodes scapularis protein, subolesin, has previously been shown to protect hosts against tick infestation and to decrease acquisition of Anaplsma marginale and Babesia bigemina. Here we report the efficacy of subolesin expressed from Vaccinia virus for use as an orally delivered reservoir–targeted vaccine for prevention of tick infestation and acquisition/transmission of Borrelia burgdorferi to its tick and mouse hosts. We cloned subolesin into Vaccinia virus and showed that it is expressed from mammalian cells infected with the recombinant virus in vitro. We then vaccinated mice by oral gavage. A single dose of the vaccine was sufficient for mice to generate antibody response to subolesin. Vaccination with the subolesin expressing Vaccinia virus inhibited tick infestation by 52% compared to control vaccination with Vaccinia virus and reduced uptake of B. burgdorferi among the surviving ticks that fed to repletion by 34%. There was a reduction in transmission of B. burgdorferi to uninfected vaccinated mice of 40% compared to controls. These results suggest that subolesin has potential as a component of a reservoir targeted vaccine to decrease B. burgdorferi, Babesia and Anaplasma species infections in their natural hosts. PMID:22864146

  18. 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

  19. 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

  20. 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.

  1. Modified vaccinia virus Ankara as a vaccine against feline coronavirus: immunogenicity and efficacy.

    PubMed

    Hebben, Matthias; Duquesne, Véronique; Cronier, Joëlle; Rossi, Bernard; Aubert, André

    2004-04-01

    Feline infectious peritonitis virus (FIPV) is a coronavirus that induces a fatal systemic disease mediated by an inappropriate immune response. Most previous vaccination attempts against FIPV were unsuccessful because IgG antibodies against the surface protein enhance the infection. However, two studies have shown that poxvirus vectors (vaccinia WR and canarypox) expressing only the FIPV membrane (M) protein can elicit a partially protective immunity which is supposed to be cell-mediated (Virology 181 (1991) 327; International patent WO 97/20054 (1997)). In our study, we report the construction of another poxvirus, the modified vaccinia virus Ankara (MVA), as an expression vector for the FIPV M protein. In this vector, the M gene has been inserted downstream a strong early/late promoter, whereas the two previously described poxviruses expressed the M protein during their early stage only. The immunogenicity of the recombinant MVA-M was evaluated in the murine model which revealed an effect of the vector on the Th1/Th2 balance. The vaccine was then tested in cats to evaluate its efficacy in an FIPV 79-1146 challenge. Vaccinated kittens developed FIPV-specific antibodies after immunization, however, none of them was protected against FIPV. Our results suggest a crucial role for the type of poxviral promoter that must be used to induce an effective immune response against FIPV. PMID:15123156

  2. 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. PMID:22909142

  3. 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

  4. Nonreplicating Vaccinia Virus Vectors Expressing the H5 Influenza Virus Hemagglutinin Produced in Modified Vero Cells Induce Robust Protection▿

    PubMed Central

    Mayrhofer, Josef; Coulibaly, Sogue; Hessel, Annett; Holzer, Georg W.; Schwendinger, Michael; Brühl, Peter; Gerencer, Marijan; Crowe, Brian A.; Shuo, Shen; Hong, Wanjing; Tan, Yee Joo; Dietrich, Barbara; Sabarth, Nicolas; Savidis-Dacho, Helga; Kistner, Otfried; Barrett, P. Noel; Falkner, Falko G.

    2009-01-01

    The timely development of safe and effective vaccines against avian influenza virus of the H5N1 subtype will be of the utmost importance in the event of a pandemic. Our aim was first to develop a safe live vaccine which induces both humoral and cell-mediated immune responses against human H5N1 influenza viruses and second, since the supply of embryonated eggs for traditional influenza vaccine production may be endangered in a pandemic, an egg-independent production procedure based on a permanent cell line. In the present article, the generation of a complementing Vero cell line suitable for the production of safe poxviral vaccines is described. This cell line was used to produce a replication-deficient vaccinia virus vector H5N1 live vaccine, dVV-HA5, expressing the hemagglutinin of a virulent clade 1 H5N1 strain. This experimental vaccine was compared with a formalin-inactivated whole-virus vaccine based on the same clade and with different replicating poxvirus-vectored vaccines. Mice were immunized to assess protective immunity after high-dose challenge with the highly virulent A/Vietnam/1203/2004(H5N1) strain. A single dose of the defective live vaccine induced complete protection from lethal homologous virus challenge and also full cross-protection against clade 0 and 2 challenge viruses. Neutralizing antibody levels were comparable to those induced by the inactivated vaccine. Unlike the whole-virus vaccine, the dVV-HA5 vaccine induced substantial amounts of gamma interferon-secreting CD8 T cells. Thus, the nonreplicating recombinant vaccinia virus vectors are promising vaccine candidates that induce a broad immune response and can be produced in an egg-independent and adjuvant-independent manner in a proven vector system. PMID:19279103

  5. 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

  6. 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

  7. 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

  8. Vesicular stomatitis virus as a flexible platform for oncolytic virotherapy against cancer

    PubMed Central

    Hastie, Eric

    2012-01-01

    Oncolytic virus (OV) therapy is an emerging anti-cancer approach that utilizes viruses to preferentially infect and kill cancer cells, while not harming healthy cells. Vesicular stomatitis virus (VSV) is a prototypic non-segmented, negative-strand RNA virus with inherent OV qualities. Antiviral responses induced by type I interferon pathways are believed to be impaired in most cancer cells, making them more susceptible to VSV than normal cells. Several other factors make VSV a promising OV candidate for clinical use, including its well-studied biology, a small, easily manipulated genome, relative independence of a receptor or cell cycle, cytoplasmic replication without risk of host-cell transformation, and lack of pre-existing immunity in humans. Moreover, various VSV-based recombinant viruses have been engineered via reverse genetics to improve oncoselectivity, safety, oncotoxicity and stimulation of tumour-specific immunity. Alternative delivery methods are also being studied to minimize premature immune clearance of VSV. OV treatment as a monotherapy is being explored, although many studies have employed VSV in combination with radiotherapy, chemotherapy or other OVs. Preclinical studies with various cancers have demonstrated that VSV is a promising OV; as a result, a human clinical trial using VSV is currently in progress. PMID:23052398

  9. 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

  10. 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.

  11. 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.

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

    PubMed Central

    Evans, E; Traktman, P

    1987-01-01

    We have identified a gene encoded by vaccinia virus which is essential for DNA replication. The gene, located in the HindIII D fragment of the viral genome, is transcribed early after infection into two transcripts of 3.0 and 3.7 kilobases which share a 3' terminus. The lesions of three temperature-sensitive DNA replication mutants with defects in this gene have been localized by marker rescue with progressively smaller DNA fragments. We have determined by hybrid selection that the gene encodes an 82-kilodalton protein. An antibody has been prepared against this polypeptide and used to quantitate expression of the protein after infection with wild-type virus or with a viral mutant whose lesion maps within this gene. The temporal pattern of expression in the mutant is unaffected, but the product encoded by the mutant is significantly more thermolabile than the wild-type protein. Images PMID:3041037

  13. Enhanced lysis by bispecific oncolytic measles viruses simultaneously using HER2/neu or EpCAM as target receptors

    PubMed Central

    Hanauer, Jan RH; Gottschlich, Lisa; Riehl, Dennis; Rusch, Tillmann; Koch, Vivian; Friedrich, Katrin; Hutzler, Stefan; Prüfer, Steffen; Friedel, Thorsten; Hanschmann, Kay-Martin; Münch, Robert C; Jost, Christian; Plückthun, Andreas; Cichutek, Klaus; Buchholz, Christian J; Mühlebach, Michael D

    2016-01-01

    To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass. PMID:27119117

  14. Sorting Out Pandora's Box: Discerning the Dynamic Roles of Liver Microenvironment in Oncolytic Virus Therapy for Hepatocellular Carcinoma.

    PubMed

    Altomonte, Jennifer; Ebert, Oliver

    2014-01-01

    Oncolytic viral therapies have recently found their way into clinical application for hepatocellular carcinoma (HCC), a disease with limited treatment options and poor prognosis. Adding to the many intrinsic challenges of in vivo oncolytic viral therapy, is the complex microenvironment of the liver, which imposes unique limitations to the successful delivery and propagation of the virus. The normal liver milieu is characterized by an intricate network of hepatocytes and non-parenchymal cells including Kupffer cells, stellate cells, and sinusoidal endothelial cells, which can secrete anti-viral cytokines, provide a platform for non-specific uptake, and form a barrier to efficient viral spread. In addition, natural killer cells are greatly enriched in the liver, contributing to the innate defense against viruses. The situation is further complicated when HCC arises in the setting of underlying hepatitis virus infection and/or hepatic cirrhosis, which occurs in more than 90% of clinical cases. These conditions pose further inhibitory effects on oncolytic virus (OV) therapy due to the presence of chronic inflammation, constitutive cytokine expression, altered hepatic blood flow, and extracellular matrix deposition. In addition, OVs can modulate the hepatic microenvironment, resulting in a complex interplay between virus and host. The immune system undoubtedly plays a substantial role in the outcome of OV therapy, both as an inhibitor of viral replication, and as a potent mechanism of virus-mediated tumor cell killing. This review will discuss the particular challenges of oncolytic viral therapy for HCC, as well as some potential strategies for modulating the immune system and synergizing with the hepatic microenvironment to improve therapeutic outcome. PMID:24795862

  15. Sorting Out Pandora’s Box: Discerning the Dynamic Roles of Liver Microenvironment in Oncolytic Virus Therapy for Hepatocellular Carcinoma

    PubMed Central

    Altomonte, Jennifer; Ebert, Oliver

    2014-01-01

    Oncolytic viral therapies have recently found their way into clinical application for hepatocellular carcinoma (HCC), a disease with limited treatment options and poor prognosis. Adding to the many intrinsic challenges of in vivo oncolytic viral therapy, is the complex microenvironment of the liver, which imposes unique limitations to the successful delivery and propagation of the virus. The normal liver milieu is characterized by an intricate network of hepatocytes and non-parenchymal cells including Kupffer cells, stellate cells, and sinusoidal endothelial cells, which can secrete anti-viral cytokines, provide a platform for non-specific uptake, and form a barrier to efficient viral spread. In addition, natural killer cells are greatly enriched in the liver, contributing to the innate defense against viruses. The situation is further complicated when HCC arises in the setting of underlying hepatitis virus infection and/or hepatic cirrhosis, which occurs in more than 90% of clinical cases. These conditions pose further inhibitory effects on oncolytic virus (OV) therapy due to the presence of chronic inflammation, constitutive cytokine expression, altered hepatic blood flow, and extracellular matrix deposition. In addition, OVs can modulate the hepatic microenvironment, resulting in a complex interplay between virus and host. The immune system undoubtedly plays a substantial role in the outcome of OV therapy, both as an inhibitor of viral replication, and as a potent mechanism of virus-mediated tumor cell killing. This review will discuss the particular challenges of oncolytic viral therapy for HCC, as well as some potential strategies for modulating the immune system and synergizing with the hepatic microenvironment to improve therapeutic outcome. PMID:24795862

  16. 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

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

    PubMed

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

    2016-03-26

    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

  18. Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.

    PubMed Central

    Giavedoni, L D; Jones, L; Gardner, M B; Gibson, H L; Ng, C T; Barr, P J; Yilma, T

    1992-01-01

    We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41. All fusion proteins retained the antigenic characteristics of both IFN-gamma and HIV as shown by immunoblot analysis. However, the antiviral activity of IFN-gamma could be demonstrated only for the IFN-gamma-gag fusion protein. In contrast, the attenuating activity of IFN-gamma for nude mice was retained by all of the recombinants, albeit at various rates. Unlike the antiviral activity, the attenuating activity of IFN-gamma was not species specific. Implications for the development of attenuated live recombinant vaccines for AIDS are discussed. Images PMID:1565633

  19. 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

  20. Reciprocal cellular cross-talk within the tumor microenvironment promotes oncolytic virus activity.

    PubMed

    Ilkow, Carolina S; Marguerie, Monique; Batenchuk, Cory; Mayer, Justin; Ben Neriah, Daniela; Cousineau, Sophie; Falls, Theresa; Jennings, Victoria A; Boileau, Meaghan; Bellamy, David; Bastin, Donald; de Souza, Christiano Tanese; Alkayyal, Almohanad; Zhang, Jiqing; Le Boeuf, Fabrice; Arulanandam, Rozanne; Stubbert, Lawton; Sampath, Padma; Thorne, Steve H; Paramanthan, Piriya; Chatterjee, Avijit; Strieter, Robert M; Burdick, Marie; Addison, Christina L; Stojdl, David F; Atkins, Harold L; Auer, Rebecca C; Diallo, Jean-Simon; Lichty, Brian D; Bell, John C

    2015-05-01

    Tumors are complex ecosystems composed of networks of interacting 'normal' and malignant cells. It is well recognized that cytokine-mediated cross-talk between normal stromal cells, including cancer-associated fibroblasts (CAFs), vascular endothelial cells, immune cells, and cancer cells, influences all aspects of tumor biology. Here we demonstrate that the cross-talk between CAFs and cancer cells leads to enhanced growth of oncolytic virus (OV)-based therapeutics. Transforming growth factor-β (TGF-β) produced by tumor cells reprogrammed CAFs, dampened their steady-state level of antiviral transcripts and rendered them sensitive to virus infection. In turn, CAFs produced high levels of fibroblast growth factor 2 (FGF2), initiating a signaling cascade in cancer cells that reduced retinoic acid-inducible gene I (RIG-I) expression and impeded the ability of malignant cells to detect and respond to virus. In xenografts derived from individuals with pancreatic cancer, the expression of FGF2 correlated with the susceptibility of the cancer cells to OV infection, and local application of FGF2 to resistant tumor samples sensitized them to virotherapy both in vitro and in vivo. An OV engineered to express FGF2 was safe in tumor-bearing mice, showed improved therapeutic efficacy compared to parental virus and merits consideration for clinical testing. PMID:25894825

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

    PubMed

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

    2012-06-01

    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

  2. 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

  3. Capturing the Natural Diversity of the Human Antibody Response against Vaccinia Virus

    PubMed Central

    Lantto, Johan; Haahr Hansen, Margit; Rasmussen, Søren Kofoed; Steinaa, Lucilla; Poulsen, Tine R.; Duggan, Jackie; Dennis, Mike; Naylor, Irene; Easterbrook, Linda; Bregenholt, Søren; Haurum, John; Jensen, Allan

    2011-01-01

    The eradication of smallpox (variola) and the subsequent cessation of routine vaccination have left modern society vulnerable to bioterrorism employing this devastating contagious disease. The existing, licensed vaccines based on live vaccinia virus (VACV) are contraindicated for a substantial number of people, and prophylactic vaccination of large populations is not reasonable when there is little risk of exposure. Consequently, there is an emerging need to develop efficient and safe therapeutics to be used shortly before or after exposure, either alone or in combination with vaccination. We have characterized the human antibody response to smallpox vaccine (VACV Lister) in immunized volunteers and isolated a large number of VACV-specific antibodies that recognize a variety of different VACV antigens. Using this broad antibody panel, we have generated a fully human, recombinant analogue to plasma-derived vaccinia immunoglobulin (VIG), which mirrors the diversity and specificity of the human antibody immune response and offers the advantage of unlimited supply and reproducible specificity and activity. The recombinant VIG was found to display a high specific binding activity toward VACV antigens, potent in vitro VACV neutralizing activity, and a highly protective efficacy against VACV challenge in the mouse tail lesion model when given either prophylactically or therapeutically. Altogether, the results suggest that this compound has the potential to be used as an effective postexposure prophylaxis or treatment of disease caused by orthopoxviruses. PMID:21147924

  4. Vaccinia Virus Virulence Factor N1L is a Novel Promising Target for Antiviral Therapeutic Intervention

    PubMed Central

    Cheltsov, Anton V.; Aoyagi, Mika; Aleshin, Alexander; Chi-Wang, Yu Eric; Gilliland, Taylor; Zhai, Dayong; Bobkov, Andrey A.; Reed, John C.; Liddington, Robert C.; Abagyan, Ruben

    2010-01-01

    The 14 kDa homodimeric N1L protein is a potent vaccinia and variola (smallpox) virulence factor. It is not essential for viral replication, but it causes a strong attenuation of viral production in culture when deleted. The N1L protein is predicted to contain the BH3-like binding domain characteristic of Bcl-2 family proteins, and it is able to bind the BH3 peptides. Its overexpression has been reported to prevent infected cells from committing apoptosis. Therefore, interfering with the N1L apoptotic blockade may be a legitimate therapeutic strategy affecting the viral growth. By using in silico ligand docking and an array of in vitro assays, we have identified sub-micromolar (600 nM) N1L antagonists, belonging to the family of polyphenols. Their affinity is comparable to that of the BH3 peptides (70 nM ÷ 1000 nM). We have also identified the natural polyphenol resveratrol as a moderate N1L inhibitor. Finally, we show that our ligands efficiently inhibit growth of vaccinia virus. PMID:20441222

  5. 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

  6. Vaccinia Virus Tropism for Primary Hematolymphoid Cells Is Determined by Restricted Expression of a Unique Virus Receptor

    PubMed Central

    Chahroudi, Ann; Chavan, Rahul; Koyzr, Natalia; Waller, Edmund K.; Silvestri, Guido; Feinberg, Mark B.

    2005-01-01

    The presumed broad tropism of poxviruses has stymied attempts to identify both the cellular receptor(s) and the viral determinant(s) for binding. Detailed studies of poxvirus binding to and infection of primary human cells have not been conducted. In particular, the determinants of target cell infection and the consequences of infection for cells involved in the generation of antiviral immune responses are incompletely understood. In this report, we show that vaccinia virus (VV) exhibits a more restricted tropism for primary hematolymphoid human cells than has been previously recognized. We demonstrate that vaccinia virus preferentially infects antigen-presenting cells (dendritic cells, monocytes/macrophages, and B cells) and activated T cells, but not resting T cells. The infection of activated T cells is permissive, with active viral replication and production of infectious progeny. Susceptibility to infection is determined by restricted expression of a cellular receptor that is induced de novo upon T-cell activation and can be removed from the cell surface by either trypsin or pronase treatment. The VV receptor expressed on activated T cells displays unique characteristics that distinguish it from the receptor used to infect cell lines in culture. The observed restricted tropism of VV may have significant consequences for the understanding of natural poxvirus infection and immunity and for poxvirus-based vaccine development. PMID:16051832

  7. Regulation of Vaccinia Virus E3 Protein by Small Ubiquitin-Like Modifier Proteins ▿ †

    PubMed Central

    González-Santamaría, José; Campagna, Michela; García, María Angel; Marcos-Villar, Laura; González, Dolores; Gallego, Pedro; Lopitz-Otsoa, Fernando; Guerra, Susana; Rodríguez, Manuel S.; Esteban, Mariano; Rivas, Carmen

    2011-01-01

    The vaccinia virus (VACV) E3 protein is essential for virulence and has antiapoptotic activity and the ability to impair the host innate immune response. Here we demonstrate that E3 interacts with SUMO1 through a small ubiquitin-like modifier (SUMO)-interacting motif (SIM). SIM integrity is required for maintaining the stability of the viral protein and for the covalent conjugation of E3 to SUMO1 or SUMO2, a modification that has a negative effect on the E3 transcriptional transactivation of the p53-upregulated modulator of apoptosis (PUMA) and APAF-1 genes. We also demonstrate that E3 is ubiquitinated, a modification that does not destabilize the wild-type protein but triggers the degradation of an E3-ΔSIM mutant. This report constitutes the first demonstration of the important roles that both SUMO and ubiquitin play in the regulation of the VACV protein E3. PMID:21957283

  8. 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

  9. Effect of suspending media on freeze-drying and preservation of vaccinia virus

    PubMed Central

    Suzuki, Masatoshi

    1970-01-01

    Unpurified and purified smallpox vaccines were prepared from calf dermal pulp, or chorioallantoic membrane (CAM) of hen eggs infected with vaccinia virus, and freeze-dried. The protective effect of various suspending media was investigated both in the course of the freeze-drying and in the period of subsequent storage of the dried product at different temperatures, including 100° C. Single media consisting of either sodium glutamate or peptone were effective in the preservation of both unpurified and purified vaccines prepared from calf dermal pulp or CAM. It was shown that there was an optimal concentration of sodium glutamate for the preservation of the vaccine preparations, especially of the purified vaccine. Combined media, consisting of soluble starch, polyvinylpyrrolidone or sodium carboxymethyl cellulose with sodium glutamate, were effective with the purified vaccine when the concentration of sodium glutamate exceeded the optimum necessary for preservation. PMID:5266585

  10. 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.

  11. 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. PMID:26458834

  12. Vesicular stomatitis virus expressing tumor suppressor p53 is a highly attenuated, potent oncolytic agent.

    PubMed

    Heiber, Joshua F; Barber, Glen N

    2011-10-01

    Vesicular stomatitis virus (VSV), a negative-strand RNA rhabdovirus, preferentially replicates in and eradicates transformed versus nontransformed cells and is thus being considered for use as a potential anticancer treatment. The genetic malleability of VSV also affords an opportunity to develop more potent agents that exhibit increased therapeutic activity. The tumor suppressor p53 has been shown to exert potent antitumor properties, which may in part involve stimulating host innate immune responses to malignancies. To evaluate whether VSV expressing p53 exhibited enhanced oncolytic action, the murine p53 (mp53) gene was incorporated into recombinant VSVs with or without a functional viral M gene-encoded protein that could either block (VSV-mp53) or enable [VSV-M(mut)-mp53] host mRNA export following infection of susceptible cells. Our results indicated that VSV-mp53 and VSV-M(mut)-mp53 expressed high levels of functional p53 and retained the ability to lyse transformed versus normal cells. In addition, we observed that VSV-ΔM-mp53 was extremely attenuated in vivo due to p53 activating innate immune genes, such as type I interferon (IFN). Significantly, immunocompetent animals with metastatic mammary adenocarcinoma exhibited increased survival following treatment with a single inoculation of VSV-ΔM-mp53, the mechanisms of which involved enhanced CD49b+ NK and tumor-specific CD8+ T cell responses. Our data indicate that VSV incorporating p53 could provide a safe, effective strategy for the design of VSV oncolytic therapeutics and VSV-based vaccines. PMID:21813611

  13. 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

  14. 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. PMID:26045555

  15. From lesions to viral clones: biological and molecular diversity amongst autochthonous Brazilian vaccinia virus.

    PubMed

    Oliveira, Graziele; Assis, Felipe; Almeida, Gabriel; Albarnaz, Jonas; Lima, Maurício; Andrade, Ana Cláudia; Calixto, Rafael; Oliveira, Cairo; Diomedes Neto, José; Trindade, Giliane; Ferreira, Paulo César; Kroon, Erna Geessien; Abrahão, Jônatas

    2015-03-01

    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

  16. 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

  17. 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.

  18. Genomic Analysis of the Vaccinia Virus Strain Variants Found in Dryvax Vaccine▿†

    PubMed Central

    Qin, Li; Upton, Chris; Hazes, Bart; Evans, David H.

    2011-01-01

    Smallpox was eradicated using variant forms of vaccinia virus-based vaccines. One of these was Dryvax, a calf lymph vaccine derived from the New York City Board of Health strain. We used genome-sequencing technology to examine the genetic diversity of the population of viruses present in a sample of Dryvax. These studies show that the conserved cores of these viruses exhibit a lower level of sequence variation than do the telomeres. However, even though the ends of orthopoxviruses are more genetically plastic than the cores, there are still many telomeric genes that are conserved as intact open reading frames in the 11 genomes that we, and 4 genomes that others, have sequenced. Most of these genes likely modulate inflammation. Our sequencing also detected an evolving pattern of mutation, with some genes being highly fragmented by randomly assorting mutations (e.g., M1L), while other genes are intact in most viruses but have been disrupted in individual strains (e.g., I4L in strain DPP17). Over 85% of insertion and deletion mutations are associated with repeats, and a rare new isolate bearing a large deletion in the right telomere was identified. All of these strains cluster in dendrograms consistent with their origin but which also surprisingly incorporate horsepox virus. However, these viruses also exhibit a “patchy” pattern of polymorphic sites characteristic of recombinants. There is more genetic diversity detected within a vial of Dryvax than between variola virus major and minor strains, and our study highlights how propagation methods affect the genetics of orthopoxvirus populations. PMID:21976639

  19. Genomic analysis of the vaccinia virus strain variants found in Dryvax vaccine.

    PubMed

    Qin, Li; Upton, Chris; Hazes, Bart; Evans, David H

    2011-12-01

    Smallpox was eradicated using variant forms of vaccinia virus-based vaccines. One of these was Dryvax, a calf lymph vaccine derived from the New York City Board of Health strain. We used genome-sequencing technology to examine the genetic diversity of the population of viruses present in a sample of Dryvax. These studies show that the conserved cores of these viruses exhibit a lower level of sequence variation than do the telomeres. However, even though the ends of orthopoxviruses are more genetically plastic than the cores, there are still many telomeric genes that are conserved as intact open reading frames in the 11 genomes that we, and 4 genomes that others, have sequenced. Most of these genes likely modulate inflammation. Our sequencing also detected an evolving pattern of mutation, with some genes being highly fragmented by randomly assorting mutations (e.g., M1L), while other genes are intact in most viruses but have been disrupted in individual strains (e.g., I4L in strain DPP17). Over 85% of insertion and deletion mutations are associated with repeats, and a rare new isolate bearing a large deletion in the right telomere was identified. All of these strains cluster in dendrograms consistent with their origin but which also surprisingly incorporate horsepox virus. However, these viruses also exhibit a "patchy" pattern of polymorphic sites characteristic of recombinants. There is more genetic diversity detected within a vial of Dryvax than between variola virus major and minor strains, and our study highlights how propagation methods affect the genetics of orthopoxvirus populations. PMID:21976639

  20. Oncolytic reovirus induces intracellular redistribution of Ras to promote apoptosis and progeny virus release.

    PubMed

    Garant, K A; Shmulevitz, M; Pan, L; Daigle, R M; Ahn, D-G; Gujar, S A; Lee, P W K

    2016-02-11

    Reovirus is a naturally oncolytic virus that preferentially replicates in Ras-transformed cells and is currently undergoing clinical trials as a cancer therapeutic. Ras transformation promotes reovirus oncolysis by enhancing virion disassembly during entry, viral progeny production, and virus release through apoptosis; however, the mechanism behind the latter is not well understood. Here, we show that reovirus alters the intracellular location of oncogenic Ras to induce apoptosis of H-RasV12-transformed fibroblasts. Reovirus infection decreases Ras palmitoylation levels and causes accumulation of Ras in the Golgi through Golgi fragmentation. With the Golgi being the site of Ras palmitoylation, treatment of target cells with the palmitoylation inhibitor, 2-bromopalmitate (2BP), prompts a greater accumulation of H-RasV12 in the Golgi, and a dose-dependent increase in progeny virus release and subsequent spread. Conversely, tethering H-RasV12 to the plasma membrane (thereby preventing its movement to the Golgi) allows for efficient virus production, but results in basal levels of reovirus-induced cell death. Analysis of Ras downstream signaling reveals that cells expressing cycling H-RasV12 have elevated levels of phosphorylated JNK (c-Jun N-terminal kinase), and that Ras retained at the Golgi body by 2BP increases activation of the MEKK1/MKK4/JNK signaling pathway to promote cell death. Collectively, our data suggest that reovirus induces Golgi fragmentation of target cells, and the subsequent accumulation of oncogenic Ras in the Golgi body initiates apoptotic signaling events required for virus release and spread. PMID:25961930

  1. Comparison of the Cowpox Virus and Vaccinia Virus Mature Virion Proteome: Analysis of the Species- and Strain-Specific Proteome.

    PubMed

    Doellinger, Joerg; Schaade, Lars; Nitsche, Andreas

    2015-01-01

    Cowpox virus (CPXV) causes most zoonotic orthopoxvirus (OPV) infections in Europe and Northern as well as Central Asia. The virus has the broadest host range of OPV and is transmitted to humans from rodents and other wild or domestic animals. Increasing numbers of human CPXV infections in a population with declining immunity have raised concerns about the virus' zoonotic potential. While there have been reports on the proteome of other human-pathogenic OPV, namely vaccinia virus (VACV) and monkeypox virus (MPXV), the protein composition of the CPXV mature virion (MV) is unknown. This study focused on the comparative analysis of the VACV and CPXV MV proteome by label-free single-run proteomics using nano liquid chromatography and high-resolution tandem mass spectrometry (nLC-MS/MS). The presented data reveal that the common VACV and CPXV MV proteome contains most of the known conserved and essential OPV proteins and is associated with cellular proteins known to be essential for viral replication. While the species-specific proteome could be linked mainly to less genetically-conserved gene products, the strain-specific protein abundance was found to be of high variance in proteins associated with entry, host-virus interaction and protein processing. PMID:26556597

  2. Oncolytic Virus-Mediated Targeting of PGE2 in the Tumor Alters the Immune Status and Sensitizes Established and Resistant Tumors to Immunotherapy.

    PubMed

    Hou, Weizhou; Sampath, Padma; Rojas, Juan J; Thorne, Steve H

    2016-07-11

    Immunotherapies are highly promising cancer treatments, but understanding the factors mediating their resistance remains critical. Successes in randomized clinical testing have supported the growing appreciation that oncolytic virotherapies primarily act as immunotherapies. Here we identified prostaglandin E2 (PGE2) in the tumor as a key mediator of resistance to immunotherapies, including oncolytic vaccinia virotherapy. Elevated levels of PGE2 coupled to suppressive chemokine profiles and high levels of granulocytic myeloid-derived suppressor cells resulted in loss of immunotherapeutic potential. Viral vectors engineered to target PGE2 were capable of overcoming localized immunosuppression leading to profound changes in the tumor's immune status. This allowed the viral vectors to raise robust anti-tumor adaptive immune responses and sensitized established and previously resistant tumors to immunotherapies. PMID:27374223

  3. 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.

  4. Bacterial-Mediated Knockdown of Tumor Resistance to an Oncolytic Virus Enhances Therapy

    PubMed Central

    Cronin, Michelle; Le Boeuf, Fabrice; Murphy, Carola; Roy, Dominic G; Falls, Theresa; Bell, John C; Tangney, Mark

    2014-01-01

    Oncolytic viruses (OVs) and bacteria share the property of tumor-selective replication following systemic administration. In the case of nonpathogenic bacteria, tumor selectivity relates to their ability to grow extracellularly within tumor stroma and is therefore ideally suited to restricting the production of bacterially produced therapeutic agents to tumors. We have previously shown the ability of the type 1 interferon antagonist B18R to enhance the replication and spread of vesicular stomatitis virus (VSV) by overcoming related cellular innate immunity. In this study, we utilized nonpathogenic bacteria (E. coli) expressing B18R to facilitate tumor-specific production of B18R, resulting in a microenvironment depleted of bioactive antiviral cytokine, thus “preconditioning” the tumor to enhance subsequent tumor destruction by the OV. Both in vitro and in vivo infection by VSVΔ51 was greatly enhanced by B18R produced from E. coli. Moreover, a significant increase in therapeutic efficacy resulted from intravenous (IV) injection of bacteria to tumor-bearing mice 5 days prior to IV VSVΔ51 administration, as evidenced by a significant reduction in tumor growth and increased survival in mice. Our strategy is the first example where two such diverse microorganisms are rationally combined and demonstrates the feasibility of combining complementary microorganisms to improve therapeutic outcome. PMID:24569832

  5. The oncolytic virus ΔPK has multimodal anti-tumor activity.

    PubMed

    Aurelian, Laure; Bollino, Dominique; Colunga, Aric

    2016-07-01

    Oncolytic viruses (OVs) are an emerging cancer therapeutic, with a near complete absence of serious adverse effects. However, clinical efficacy is relatively modest, related to poor tumor penetration, failure to lyse cancer stem cells (CSCs) and blockade of immunogenic cell death by the immunosuppressive tumor microenvironment. To overcome such limitations, we developed an OV (known as ΔPK) with multimodal anti-tumor activity. ΔPK has potent anti-tumor activity both in melanoma cell lines and xenograft animal models, associated with virus replication and the induction of multiple independent programmed cell death pathways. It lyses CSCs through autophagy modulation and it reverses the immunosuppressive tumor microenvironment by altering the balance of cytokines secreted by the tumor cells. This includes decreased tumor cell secretion of the immunosuppressive and procancerous cytokines IL-10 and IL-18 and concomitant increased secretion of the proinflammatory cytokines TNF-α, GM-CSF, IL-6 and IL-1β. ΔPK also upregulates the NKG2D ligand, MICA expressed by cytotoxic NK and T cells, and downregulates the negative immune checkpoint regulator cytotoxic T-lymphocyte antigen-4 (CTLA-4). ΔPK is well tolerated in human patients in whom it also alters the Th1/Th2 balance. Further studies are designed to elucidate the role of these contributions in different tumor types. PMID:27242376

  6. [Oncolytic vesicular stomatitis viruses as intravesical agents against non-muscle-invasive bladder cancer].

    PubMed

    Hadaschik, B A; Zhang, K; So, A I; Bell, J C; Thüroff, J W; Rennie, P S; Gleave, M E

    2008-09-01

    Patients with high-risk bladder cancer who do not respond to bacillus Calmette-Guerin (BCG) immunotherapy represent a significant therapeutic challenge. The addition of interferon to BCG has recently evolved as a second-line treatment option; however, many high-grade tumors are nonresponsive to interferon. Thus, replication-competent oncolytic vesicular stomatitis viruses (VSV) that selectively target interferon-refractory tumors are promising intravesical agents. In vitro, wild-type VSV as well as a mutant variant (AV3) that has an impaired ability to shut down innate immunity preferentially killed undifferentiated, interferon-nonresponsive bladder cancer cells. Testing of these viruses in an orthotopic murine model of high-grade bladder cancer, which we have recently validated, revealed that both AV3 and wild-type VSV significantly inhibited orthotopic tumor growth. Despite the use of immunocompromised nude mice, there was no evidence of toxicity. In conclusion, VSV instillation therapy demonstrated strong antitumor activity and safety in an orthotopic model of high-risk disease. These findings provide preclinical proof-of-principle for the intravesical use of VSV, especially in interferon-refractory patients. PMID:18670747

  7. 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

  8. Dendritic Cells in Oncolytic Virus-Based Anti-Cancer Therapy.

    PubMed

    Kim, Youra; Clements, Derek R; Sterea, Andra M; Jang, Hyun Woo; Gujar, Shashi A; Lee, Patrick W K

    2015-12-01

    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

  9. 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

  10. Activation of Cyclic Adenosine Monophosphate Pathway Increases the Sensitivity of Cancer Cells to the Oncolytic Virus M1.

    PubMed

    Li, Kai; Zhang, Haipeng; Qiu, Jianguang; Lin, Yuan; Liang, Jiankai; Xiao, Xiao; Fu, Liwu; Wang, Fang; Cai, Jing; Tan, Yaqian; Zhu, Wenbo; Yin, Wei; Lu, Bingzheng; Xing, Fan; Tang, Lipeng; Yan, Min; Mai, Jialuo; Li, Yuan; Chen, Wenli; Qiu, Pengxin; Su, Xingwen; Gao, Guangping; Tai, Phillip W L; Hu, Jun; Yan, Guangmei

    2016-02-01

    Oncolytic virotherapy is a novel and emerging treatment modality that uses replication-competent viruses to destroy cancer cells. Although diverse cancer cell types are sensitive to oncolytic viruses, one of the major challenges of oncolytic virotherapy is that the sensitivity to oncolysis ranges among different cancer cell types. Furthermore, the underlying mechanism of action is not fully understood. Here, we report that activation of cyclic adenosine monophosphate (cAMP) signaling significantly sensitizes refractory cancer cells to alphavirus M1 in vitro, in vivo, and ex vivo. We find that activation of the cAMP signaling pathway inhibits M1-induced expression of antiviral factors in refractory cancer cells, leading to prolonged and severe endoplasmic reticulum (ER) stress, and cell apoptosis. We also demonstrate that M1-mediated oncolysis, which is enhanced by cAMP signaling, involves the factor, exchange protein directly activated by cAMP 1 (Epac1), but not the classical cAMP-dependent protein kinase A (PKA). Taken together, cAMP/Epac1 signaling pathway activation inhibits antiviral factors and improves responsiveness of refractory cancer cells to M1-mediated virotherapy. PMID:26373347

  11. A Novel Armed Oncolytic Measles Vaccine Virus for the Treatment of Cholangiocarcinoma

    PubMed Central

    Lange, Sebastian; Lampe, Johanna; Bossow, Sascha; Zimmermann, Martina; Neubert, Wolfgang; Bitzer, Michael

    2013-01-01

    Abstract Cholangiocarcinoma (CC) is curable only in early stages by complete surgical resection. Thus, in advanced disease stages in which a complete removal of the tumor mass is no longer possible and palliative chemotherapy achieves only modest success, therapeutics employing new methods of action are desperately needed. Oncolytic viruses employed in clinical studies have been shown to spread preferentially in cancer cells. Beyond that, virotherapeutic cell killing can be enhanced by virus-based expression of suicide genes. We engineered a measles vaccine virus (MeV) vector expressing super cytosine deaminase (SCD), a fusion protein of yeast cytosine deaminase and uracil phosphoribosyltransferase, which converts the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) and subsequently to 5-fluorouridine-monophosphate. This novel vector was evaluated using three different human-derived CC cell lines. In vitro, all CC cell lines were found to be permissive to MeV infection. Partial blocking of MeV-mediated oncolysis could be overcome by employment of the SCD transgene together with administration of 5-FC. In vivo, intratumoral application of SCD-armed MeV together with a systemic 5-FC treatment showed a significant reduction in tumor size in a TFK-1 xenograft mouse model when compared with virus-only treatment. In a second animal experiment employing a HuCCT1 xenograft tumor model, an enhanced SCD-armed MeV vector, in which the SCD transgene was expressed from a different genomic position, led not only to reduced tumor volumes, but also to a significant survival benefit. On the basis of these encouraging preclinical data on employment of SCD-armed MeV for the virotherapeutic treatment of chemotherapy-resistant CC, a clinical virotherapy trial is set up currently. PMID:23550539

  12. A novel armed oncolytic measles vaccine virus for the treatment of cholangiocarcinoma.

    PubMed

    Lange, Sebastian; Lampe, Johanna; Bossow, Sascha; Zimmermann, Martina; Neubert, Wolfgang; Bitzer, Michael; Lauer, Ulrich M

    2013-05-01

    Cholangiocarcinoma (CC) is curable only in early stages by complete surgical resection. Thus, in advanced disease stages in which a complete removal of the tumor mass is no longer possible and palliative chemotherapy achieves only modest success, therapeutics employing new methods of action are desperately needed. Oncolytic viruses employed in clinical studies have been shown to spread preferentially in cancer cells. Beyond that, virotherapeutic cell killing can be enhanced by virus-based expression of suicide genes. We engineered a measles vaccine virus (MeV) vector expressing super cytosine deaminase (SCD), a fusion protein of yeast cytosine deaminase and uracil phosphoribosyltransferase, which converts the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) and subsequently to 5-fluorouridine-monophosphate. This novel vector was evaluated using three different human-derived CC cell lines. In vitro, all CC cell lines were found to be permissive to MeV infection. Partial blocking of MeV-mediated oncolysis could be overcome by employment of the SCD transgene together with administration of 5-FC. In vivo, intratumoral application of SCD-armed MeV together with a systemic 5-FC treatment showed a significant reduction in tumor size in a TFK-1 xenograft mouse model when compared with virus-only treatment. In a second animal experiment employing a HuCCT1 xenograft tumor model, an enhanced SCD-armed MeV vector, in which the SCD transgene was expressed from a different genomic position, led not only to reduced tumor volumes, but also to a significant survival benefit. On the basis of these encouraging preclinical data on employment of SCD-armed MeV for the virotherapeutic treatment of chemotherapy-resistant CC, a clinical virotherapy trial is set up currently. PMID:23550539

  13. Comparison of the Cowpox Virus and Vaccinia Virus Mature Virion Proteome: Analysis of the Species- and Strain-Specific Proteome

    PubMed Central

    Doellinger, Joerg; Schaade, Lars; Nitsche, Andreas

    2015-01-01

    Cowpox virus (CPXV) causes most zoonotic orthopoxvirus (OPV) infections in Europe and Northern as well as Central Asia. The virus has the broadest host range of OPV and is transmitted to humans from rodents and other wild or domestic animals. Increasing numbers of human CPXV infections in a population with declining immunity have raised concerns about the virus’ zoonotic potential. While there have been reports on the proteome of other human-pathogenic OPV, namely vaccinia virus (VACV) and monkeypox virus (MPXV), the protein composition of the CPXV mature virion (MV) is unknown. This study focused on the comparative analysis of the VACV and CPXV MV proteome by label-free single-run proteomics using nano liquid chromatography and high-resolution tandem mass spectrometry (nLC-MS/MS). The presented data reveal that the common VACV and CPXV MV proteome contains most of the known conserved and essential OPV proteins and is associated with cellular proteins known to be essential for viral replication. While the species-specific proteome could be linked mainly to less genetically-conserved gene products, the strain-specific protein abundance was found to be of high variance in proteins associated with entry, host-virus interaction and protein processing. PMID:26556597

  14. 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.

  15. Combined Cytolytic Effects of a Vaccinia Virus Encoding a Single Chain Trimer of MHC-I with a Tax-Epitope and Tax-Specific CTLs on HTLV-I-Infected Cells in a Rat Model

    PubMed Central

    Nakamura, Takafumi; Kidokoro, Minoru; Zhang, Xianfeng; Shida, Hisatoshi

    2014-01-01

    Adult T cell leukemia (ATL) is a malignant lymphoproliferative disease caused by human T cell leukemia virus type I (HTLV-I). To develop an effective therapy against the disease, we have examined the oncolytic ability of an attenuated vaccinia virus (VV), LC16m8Δ (m8Δ), and an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL) line, 4O1/C8, against an HTLV-I-infected rat T cell line, FPM1. Our results demonstrated that m8Δ was able to replicate in and lyse tumorigenic FPM1 cells but was incompetent to injure 4O1/C8 cells, suggesting the preferential cytolytic activity toward tumor cells. To further enhance the cytolysis of HTLV-I-infected cells, we modified m8Δ and obtained m8Δ/RT1AlSCTax180L, which can express a single chain trimer (SCT) of rat major histocompatibility complex class I with a Tax-epitope. Combined treatment with m8Δ/RT1AlSCTax180L and 4O1/C8 increased the cytolysis of FPM1V.EFGFP/8R cells, a CTL-resistant subclone of FPM1, compared with that using 4O1/C8 and m8Δ presenting an unrelated peptide, suggesting that the activation of 4O1/C8 by m8Δ/RT1AlSCTax180L further enhanced the killing of the tumorigenic HTLV-I-infected cells. Our results indicate that combined therapy of oncolytic VVs with SCTs and HTLV-I-specific CTLs may be effective for eradication of HTLV-I-infected cells, which evade from CTL lysis and potentially develop ATL. PMID:24791004

  16. [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. PMID:26697713

  17. Analysis of canine herpesvirus gB, gC and gD expressed by a recombinant vaccinia virus.

    PubMed

    Xuan, X; Kojima, A; Murata, T; Mikami, T; Otsuka, H

    1997-01-01

    The genes encoding the canine herpesvirus (CHV) glycoprotein B (gB), gC and gD homologues have been reported already. However, products of these genes have not been identified yet. Previously, we have identified three CHV glycoproteins, gp 145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gB, gC or gD, the putative genes of gB, gC, and gD of CHV were inserted into the thymidine kinase gene of vaccinia virus LC16mO strain under the control of the early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. We demonstrated here that gp145/112, gp80 and gp47 were the translation products of the CHV gB, gC and gD genes, respectively. The antigenic authenticity of recombinant gB, gC and gD were confirmed by a panel of MAbs specific for each glycoprotein produced in CHV-infected cells. Immunization of mice with these recombinants produced high titers of neutralizing antibodies against CHV. These results suggest that recombinant vaccinia viruses expressing CHV gB, gC and gD may be useful to develop a vaccine to control CHV infection. PMID:9191864

  18. Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication.

    PubMed Central

    Rempel, R E; Anderson, M K; Evans, E; Traktman, P

    1990-01-01

    Vaccinia virus mutants ts2 and ts25, members of the same complementation group, exhibit a temperature-dependent arrest at the stage of viral DNA replication. The lesions responsible for the mutant phenotypes have been localized to the far left region of the HindIII B genomic fragment by marker rescue studies. Hybrid selection analyses established that the DNA fragments positive for rescue represented the first open reading frame of the HindIII B fragment and encoded a 30-kilodalton protein. The gene is expressed early after infection as a rightwardly transcribed 1-kilobase-pair mRNA whose coordinates were determined by S1 nuclease mapping. To further the phenotypic analysis of the mutants, the accumulation of viral DNA sequences during permissive and nonpermissive infections was quantitated. The extent of the DNA- phenotype was shown to vary in different cell types. In mouse L cells at either high or low multiplicity of infection, nonpermissive DNA synthesis was less than 5% of that seen in permissive infections. This severe defect was mirrored by correspondingly low viral yields. In infections of BSC40 monkey cells, however, the deficiencies in both DNA synthesis and virus production were far less severe. For one mutant (ts2), the temperature sensitivity in BSC40 cells varied inversely with the multiplicity of infection. Images PMID:2296077

  19. High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus.

    PubMed Central

    Stunnenberg, H G; Lange, H; Philipson, L; van Miltenburg, R T; van der Vliet, P C

    1988-01-01

    Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system. Images PMID:3362670

  20. Dissociation between Epitope Hierarchy and Immunoprevalence in CD8 Responses to Vaccinia Virus Western Reserve1

    PubMed Central

    Oseroff, Carla; Peters, Bjoern; Pasquetto, Valerie; Moutaftsi, Magdalini; Sidney, John; Panchanathan, Vijay; Tscharke, David C.; Maillere, Bernard; Grey, Howard; Sette, Alessandro

    2008-01-01

    Understanding immunity to vaccinia virus (VACV) is important for the development of safer vaccines for smallpox- and poxvirus-vectored recombinant vaccines. VACV is also emerging as an outstanding model for studying CD8+ T cell immunodominance because of the large number of CD8+ T cell epitopes known for this virus in both mice and humans. In this study, we characterize the CD8+ T cell response in vaccinated BALB/c mice by a genome-wide mapping approach. Responses to each of 54 newly identified H-2d-restricted T cell epitopes could be detected after i.p. and dermal vaccination routes. Analysis of these new epitopes in the context of those already known for VACV in mice and humans revealed two important findings. First, CD8+ T cell epitopes are not randomly distributed across the VACV proteome, with some proteins being poorly or nonimmunogenic, while others are immunoprevalent, being frequently recognized across diverse MHC haplotypes. Second, some proteins constituted the major targets of the immune response by a specific haplotype as they recruited the majority of the specific CD8+ T cells but these proteins did not correspond to the immunoprevalent Ags. Thus, we found a dissociation between immunoprevalence and immunodominance, implying that different sets of rules govern these two phenomena. Together, these findings have clear implications for the design of CD8+ T cell subunit vaccines and in particular raise the exciting prospect of being able to choose subunits without reference to MHC restriction. PMID:18490718

  1. Vaccinia viruses isolated from cutaneous disease in horses are highly virulent for rabbits.

    PubMed

    Felipetto Cargnelutti, Juliana; Schmidt, Candice; Masuda, Eduardo Kenji; Braum, Lisiane Danusa; Weiblen, Rudi; Furtado Flores, Eduardo

    2012-03-01

    Two genotypically distinct Vaccinia viruses (VACV), named P1V and P2V, were isolated from an outbreak of cutaneous disease in horses in Southern Brazil. We herein investigated the susceptibility of rabbits, a proposed animal model, to P1V and P2V infection. Groups of weanling rabbits were inoculated intranasally (IN) with P1V or P2V at low (10(2.5) TCID50), medium (10(4.5)TCID50), or high titer (10(6.5)TCID50). Rabbits inoculated with medium and high titers shed virus in nasal secretions and developed serous to hemorrhagic nasal discharge and severe respiratory distress, followed by progressive apathy and high lethality. Clinical signs appeared around days 3-6 post-inoculation (pi) and lasted up to the day of death or euthanasia (around days 5-10). Virus shedding and clinical signs were less frequent in rabbits inoculated with low virus titers. Viremia was detected in all groups, with different frequencies. Viral DNA was detected in the feces of a few animals inoculated with P1V and P2V, low titer, and with P2V at high titer. Gross necropsy findings and histological examination showed diffuse interstitial fibrousing pneumonia with necrosuppurative bronchopneumonia and intestinal liquid content. Neutralizing antibodies were detected in all inoculated animals surviving beyond day 9 pi. These results show that rabbits are highly susceptible to VACV isolated from horses, and develop severe respiratory and systemic disease upon IN inoculation. Thus, rabbits may be used to study selected aspects of VACV infection and disease. PMID:22226666

  2. Involvement of the Cellular Phosphatase DUSP1 in Vaccinia Virus Infection

    PubMed Central

    Cáceres, Ana; Perdiguero, Beatriz; Gómez, Carmen E.; Cepeda, Maria Victoria; Caelles, Carme; Sorzano, Carlos Oscar; Esteban, Mariano

    2013-01-01

    Poxviruses encode a large variety of proteins that mimic, block or enhance host cell signaling pathways on their own benefit. It has been reported that mitogen-activated protein kinases (MAPKs) are specifically upregulated during vaccinia virus (VACV) infection. Here, we have evaluated the role of the MAPK negative regulator dual specificity phosphatase 1 (DUSP1) in the infection of VACV. We demonstrated that DUSP1 expression is enhanced upon infection with the replicative WR virus and with the attenuated VACV viruses MVA and NYVAC. This upregulation is dependent on early viral gene expression. In the absence of DUSP1 in cultured cells, there is an increased activation of its molecular targets JNK and ERK and an enhanced WR replication. Moreover, DUSP1 knock-out (KO) mice are more susceptible to WR infection as a result of enhanced virus replication in the lungs. Significantly, MVA, which is known to produce non-permissive infections in most mammalian cell lines, is able to grow in DUSP1 KO immortalized murine embryo fibroblasts (MEFs). By confocal and electron microscopy assays, we showed that in the absence of DUSP1 MVA morphogenesis is similar as in permissive cell lines and demonstrated that DUSP1 is involved at the stage of transition between IVN and MV in VACV morphogenesis. In addition, we have observed that the secretion of pro-inflammatory cytokines at early times post-infection in KO mice infected with MVA and NYVAC is increased and that the adaptive immune response is enhanced in comparison with WT-infected mice. Altogether, these findings reveal that DUSP1 is involved in the replication and host range of VACV and in the regulation of host immune responses through the modulation of MAPKs. Thus, in this study we demonstrate that DUSP1 is actively involved in the antiviral host defense mechanism against a poxvirus infection. PMID:24244156

  3. Vaccinia Virus E3 Protein Prevents the Antiviral Action of ISG15

    PubMed Central

    Guerra, Susana; Cáceres, Ana; Knobeloch, Klaus-Peter; Horak, Ivan; Esteban, Mariano

    2008-01-01

    The ubiquitin-like modifier ISG15 is one of the most predominant proteins induced by type I interferons (IFN). In this study, murine embryo fibroblast (MEFs) and mice lacking the gene were used to demonstrate a novel role of ISG15 as a host defense molecule against vaccinia virus (VACV) infection. In MEFs, the growth of replication competent Western Reserve (WR) VACV strain was affected by the absence of ISG15, but in addition, virus lacking E3 protein (VVΔE3L) that is unable to grow in ISG15+/+ cells replicated in ISG15-deficient cells. Inhibiting ISG15 with siRNA or promoting its expression in ISG15−/− cells with a lentivirus vector showed that VACV replication was controlled by ISG15. Immunoprecipitation analysis revealed that E3 binds ISG15 through its C-terminal domain. The VACV antiviral action of ISG15 and its interaction with E3 are events independent of PKR (double-stranded RNA-dependent protein kinase). In mice lacking ISG15, infection with VVΔE3L caused significant disease and mortality, an effect not observed in VVΔE3L-infected ISG15+/+ mice. Pathogenesis in ISG15-deficient mice infected with VVΔE3L or with an E3L deletion mutant virus lacking the C-terminal domain triggered an enhanced inflammatory response in the lungs compared with ISG15+/+-infected mice. These findings showed an anti-VACV function of ISG15, with the virus E3 protein suppressing the action of the ISG15 antiviral factor. PMID:18604270

  4. A rationally designed A34R mutant oncolytic poxvirus: improved efficacy in peritoneal carcinomatosis.

    PubMed

    Thirunavukarasu, Pragatheeshwar; Sathaiah, Magesh; Gorry, Michael C; O'Malley, Mark E; Ravindranathan, Roshni; Austin, Frances; Thorne, Steven H; Guo, Zong Sheng; Bartlett, David L

    2013-05-01

    Oncolytic poxviruses have demonstrated initial promising results in patients with cancer in clinical trials, yet further improvements are needed. It has been shown that a single point mutation in the A34R gene resulted in the production of more total progeny virus and more extracellular enveloped virus (EEV), a form that can be immune-evasive and with enhanced spread. We have genetically engineered a new oncolytic poxvirus (designated vA34R) by incorporating this mutated A34R gene into a viral backbone (vvDD) which was designed for tumor-selective replication. This rationally designed virus can evade neutralization from antipoxvirus antibodies and is highly cytotoxic to cancer cells. It demonstrates improved spread and increased replication within the peritoneal cavity resulting in improved antitumor effects in a peritoneal carcinomatosis (PC) model of MC38 colon cancer. Impressively, after carrier cell-mediated delivery in the preimmunized host, vA34R displayed high replication in tumor nodules yet low accumulation in normal tissues thus enhancing the therapeutic index leading to 70% long-term cures. These results demonstrate that vA34R gains an enhanced therapeutic index for PC via immune evasion, increased spread, and production of more progeny virus. Thus, vA34R may be a potent oncolytic virus (OV) for patients with PC, even after prior exposure to vaccinia virus (VV). PMID:23439499

  5. Oncolytic newcastle disease virus triggers cell death of lung cancer spheroids and is enhanced by pharmacological inhibition of autophagy

    PubMed Central

    Hu, Lulu; Sun, Sulan; Wang, Tianpeng; Li, Yingchun; Jiang, Ke; Lin, Guibin; Ma, Yan; Barr, Martin P; Song, Fei; Zhang, Guirong; Meng, Songshu

    2015-01-01

    Lung cancer stem cells (CSCs) have recently been isolated from lung cancer patient samples and have been reported to be responsible for tumor initiation, treatment resistance and tumor recurrence. We have previously shown that oncolytic Newcastle disease virus (NDV), strain FMW (NDV/FMW) induces apoptosis in drug-resistant lung cancer cells. However, how NDV exerts its oncolytic effect on lung CSCs remains to be investigated. Here we show that NDV/FMW replicates in, and lyses CSC-enriched lung cancer spheroids and inhibits the 3D growth potential of lung cancer spheroid and agar colonies. We demonstrate that NDV/FMW triggers caspase-dependent apoptosis in lung cancer spheroids as shown by increased caspase-3 processing and Poly (ADP-ribose) polymerase (PARP) cleavage. Notably, NDV/FMW infection results in the degradation of microtubule-associated protein 1 light chain 3 (LC3) II and P62, two hallmarks of autophagy maturation, indicating that NDV/FMW promotes autophagy flux in lung cancer cell spheroids. This was further confirmed by the appearance of an increased number of double-membrane vesicles as detected by transmission electron microscopy. We also show that NDV/FMW promotes autophagy degradation in lung cancer spheroids via inhibition of the AKT/mTOR pathway. In addition, treatment of spheroids with the autophagy inhibitor, chloroquine increases NDV/FMW-induced cytotoxicity. Collectively, our data show that oncolytic NDV/FMW may be a potential strategy in targeting lung CSCs. PMID:26885450

  6. 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

  7. Oncolytic Activity of a Recombinant Measles Virus, Blind to Signaling Lymphocyte Activation Molecule, Against Colorectal Cancer Cells

    PubMed Central

    Amagai, Yosuke; Fujiyuki, Tomoko; Yoneda, Misako; Shoji, Koichiro; Furukawa, Yoichi; Sato, Hiroki; Kai, Chieko

    2016-01-01

    Oncolytic virotherapy is a distinctive antitumor therapy based on the cancer-cell-specific infectivity and killing activity of viruses, which exert a considerable antitumor effect with only a few treatments. Because colorectal cancer cells often acquire resistance to the molecular-targeted therapies and alternative treatments are called for, in this study, we evaluated the oncolytic activity against colorectal cancer cells of a recombinant measles virus (rMV-SLAMblind), which is blind to signaling lymphocytic activation molecule (SLAM) and infects target cells via nectin-4/poliovirus receptor-related 4 protein. We examined 10 cell lines including 8 cell lines that were resistant to epidermal-growth-factor-receptor (EGFR) targeted therapy. rMV-SLAMblind infected and lysed the nectin-4-positive cell lines dependently on nectin-4 expression, in spite of mutation in EGFR cascade. Tumour progression in xenograft models was also abrogated by the virus, and the infection of cancer cells in vivo by the virus was demonstrated with both flow cytometry and a histological analysis. Therefore, rMV-SLAMblind is considered a novel therapeutic agent for colorectal cancers, including those resistant to molecular-targeted therapies. PMID:27090874

  8. Oncolytic Activity of a Recombinant Measles Virus, Blind to Signaling Lymphocyte Activation Molecule, Against Colorectal Cancer Cells.

    PubMed

    Amagai, Yosuke; Fujiyuki, Tomoko; Yoneda, Misako; Shoji, Koichiro; Furukawa, Yoichi; Sato, Hiroki; Kai, Chieko

    2016-01-01

    Oncolytic virotherapy is a distinctive antitumor therapy based on the cancer-cell-specific infectivity and killing activity of viruses, which exert a considerable antitumor effect with only a few treatments. Because colorectal cancer cells often acquire resistance to the molecular-targeted therapies and alternative treatments are called for, in this study, we evaluated the oncolytic activity against colorectal cancer cells of a recombinant measles virus (rMV-SLAMblind), which is blind to signaling lymphocytic activation molecule (SLAM) and infects target cells via nectin-4/poliovirus receptor-related 4 protein. We examined 10 cell lines including 8 cell lines that were resistant to epidermal-growth-factor-receptor (EGFR) targeted therapy. rMV-SLAMblind infected and lysed the nectin-4-positive cell lines dependently on nectin-4 expression, in spite of mutation in EGFR cascade. Tumour progression in xenograft models was also abrogated by the virus, and the infection of cancer cells in vivo by the virus was demonstrated with both flow cytometry and a histological analysis. Therefore, rMV-SLAMblind is considered a novel therapeutic agent for colorectal cancers, including those resistant to molecular-targeted therapies. PMID:27090874

  9. Direct comparison of antigen production and induction of apoptosis by canarypox virus- and modified vaccinia virus ankara-human immunodeficiency virus vaccine vectors.

    PubMed

    Zhang, Xiugen; Cassis-Ghavami, Farah; Eller, Mike; Currier, Jeff; Slike, Bonnie M; Chen, Xuemin; Tartaglia, James; Marovich, Mary; Spearman, Paul

    2007-07-01

    Recombinant poxvirus vectors are undergoing intensive evaluation as vaccine candidates for a variety of infectious pathogens. Avipoxviruses, such as canarypox virus, are replication deficient in mammalian cells by virtue of a poorly understood species-specific restriction. Highly attenuated vaccinia virus strains such as modified vaccinia virus Ankara (MVA) are similarly unable to complete replication in most mammalian cells but have an abortive-late phenotype, in that the block to replication occurs post-virus-specific DNA replication. In this study, an identical expression cassette for human immunodeficiency virus gag, pro, and env coding sequences was placed in canarypox virus and MVA vector backbones in order to directly compare vector-borne expression and to analyze differences in vector-host cell interactions. Antigen production by recombinant MVA was shown to be greater than that from recombinant canarypox virus in the mammalian cell lines and in the primary human cells tested. This observation was primarily due to a longer duration of antigen production in recombinant MVA-infected cells. Apoptosis induction was found to be more profound with the empty canarypox virus vector than with MVA. Remarkably, however, the inclusion of a gag/pro/env expression cassette altered the kinetics of apoptosis induction in recombinant MVA-infected cells to levels equal to those found in canarypox virus-infected cells. Antigen production by MVA was noted to be greater in human dendritic cells and resulted in enhanced T-cell stimulation in an in vitro antigen presentation assay. These results reveal differences in poxvirus vector-host cell interactions that should be relevant to their use as immunization vehicles. PMID:17409140

  10. Direct Comparison of Antigen Production and Induction of Apoptosis by Canarypox Virus- and Modified Vaccinia Virus Ankara-Human Immunodeficiency Virus Vaccine Vectors▿

    PubMed Central

    Zhang, Xiugen; Cassis-Ghavami, Farah; Eller, Mike; Currier, Jeff; Slike, Bonnie M.; Chen, Xuemin; Tartaglia, James; Marovich, Mary; Spearman, Paul

    2007-01-01

    Recombinant poxvirus vectors are undergoing intensive evaluation as vaccine candidates for a variety of infectious pathogens. Avipoxviruses, such as canarypox virus, are replication deficient in mammalian cells by virtue of a poorly understood species-specific restriction. Highly attenuated vaccinia virus strains such as modified vaccinia virus Ankara (MVA) are similarly unable to complete replication in most mammalian cells but have an abortive-late phenotype, in that the block to replication occurs post-virus-specific DNA replication. In this study, an identical expression cassette for human immunodeficiency virus gag, pro, and env coding sequences was placed in canarypox virus and MVA vector backbones in order to directly compare vector-borne expression and to analyze differences in vector-host cell interactions. Antigen production by recombinant MVA was shown to be greater than that from recombinant canarypox virus in the mammalian cell lines and in the primary human cells tested. This observation was primarily due to a longer duration of antigen production in recombinant MVA-infected cells. Apoptosis induction was found to be more profound with the empty canarypox virus vector than with MVA. Remarkably, however, the inclusion of a gag/pro/env expression cassette altered the kinetics of apoptosis induction in recombinant MVA-infected cells to levels equal to those found in canarypox virus-infected cells. Antigen production by MVA was noted to be greater in human dendritic cells and resulted in enhanced T-cell stimulation in an in vitro antigen presentation assay. These results reveal differences in poxvirus vector-host cell interactions that should be relevant to their use as immunization vehicles. PMID:17409140

  11. Potentiation of immunomodulatory antibody therapy with oncolytic viruses for treatment of cancer

    PubMed Central

    Zamarin, Dmitriy; Wolchok, Jedd D

    2014-01-01

    Identification of the immune suppressive mechanisms active within the tumor microenvironment led to development of immunotherapeutic strategies aiming to reverse the immunosuppression and to enhance the function of tumor-infiltrating lymphocytes. Of those, cancer therapy with antibodies targeting the immune costimulatory and coinhibitory receptors has demonstrated significant promise in the recent years, with multiple antibodies entering clinical testing. The responses to these agents, however, have not been universal and have not been observed in all cancer types, calling for identification of appropriate predictive biomarkers and development of combinatorial strategies. Pre-existing immune infiltration in tumors has been demonstrated to have a strong association with response to immunotherapies, with the type I interferon (IFN) pathway emerging as a key player in tumor innate immune recognition and activation of adaptive immunity. These findings provide a rationale for evaluation of strategies targeting the type I IFN pathway as a means to enhance tumor immune recognition and infiltration, which could potentially make them susceptible to therapeutics targeting the cosignaling receptors. To this end in particular, oncolytic viruses (OVs) have been demonstrated to enhance tumor recognition by the immune system through multiple mechanisms, which include upregulation of major histocompatibility complex and costimulatory molecules on cancer cells, immunogenic cell death and antigen release, and activation of the type I IFN pathway. Evidence is now emerging that combination therapies using OVs and agents targeting immune cosignaling receptors such as 4-1BB, PD-1, and CTLA-4 may work in concert to enhance antitumor immunity and therapeutic efficacy. Our evolving understanding of the interplay between OVs and the immune system demonstrates that the virus-induced antitumor immune responses can be harnessed to drive the efficacy of the agents targeting cosignaling

  12. 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. PMID:23593246

  13. Ectopic Expression of Vaccinia Virus E3 and K3 Cannot Rescue Ectromelia Virus Replication in Rabbit RK13 Cells

    PubMed Central

    Peng, Chen; Rothenburg, Stefan; Hersperger, Adam R.

    2015-01-01

    As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV. PMID:25734776

  14. Ectopic expression of vaccinia virus E3 and K3 cannot rescue ectromelia virus replication in rabbit RK13 cells.

    PubMed

    Hand, Erin S; Haller, Sherry L; Peng, Chen; Rothenburg, Stefan; Hersperger, Adam R

    2015-01-01

    As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV. PMID:25734776

  15. The Oncolytic Poxvirus JX-594 Selectively Replicates in and Destroys Cancer Cells Driven by Genetic Pathways Commonly Activated in Cancers

    PubMed Central

    Parato, Kelley A; Breitbach, Caroline J; Le Boeuf, Fabrice; Wang, Jiahu; Storbeck, Chris; Ilkow, Carolina; Diallo, Jean-Simon; Falls, Theresa; Burns, Joseph; Garcia, Vanessa; Kanji, Femina; Evgin, Laura; Hu, Kang; Paradis, Francois; Knowles, Shane; Hwang, Tae-Ho; Vanderhyden, Barbara C; Auer, Rebecca; Kirn, David H; Bell, John C

    2012-01-01

    Oncolytic viruses are generally designed to be cancer selective on the basis of a single genetic mutation. JX-594 is a thymidine kinase (TK) gene-inactivated oncolytic vaccinia virus expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and lac-Z transgenes that is designed to destroy cancer cells through replication-dependent cell lysis and stimulation of antitumoral immunity. JX-594 has demonstrated a favorable safety profile and reproducible tumor necrosis in a variety of solid cancer types in clinical trials. However, the mechanism(s) responsible for its cancer-selectivity have not yet been well described. We analyzed the replication of JX-594 in three model systems: primary normal and cancer cells, surgical explants, and murine tumor models. JX-594 replication, transgene expression, and cytopathic effects were highly cancer-selective, and broad spectrum activity was demonstrated. JX-594 cancer-selectivity was multi-mechanistic; replication was activated by epidermal growth factor receptor (EGFR)/Ras pathway signaling, cellular TK levels, and cancer cell resistance to type-I interferons (IFNs). These findings confirm a large therapeutic index for JX-594 that is driven by common genetic abnormalities in human solid tumors. This appears to be the first description of multiple selectivity mechanisms, both inherent and engineered, for an oncolytic virus. These findings have implications for oncolytic viruses in general, and suggest that their cancer targeting is a complex and multifactorial process. PMID:22186794

  16. Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity.

    PubMed Central

    Langford, C J; Edwards, S J; Smith, G L; Mitchell, G F; Moss, B; Kemp, D J; Anders, R F

    1986-01-01

    We show that the subcellular location of foreign antigens expressed in recombinant vaccinia viruses influences their effectiveness as immunogens. Live recombinant viruses induced very poor antibody responses to a secreted repetitive plasmodial antigen (the S-antigen) in rabbits and mice. The poor response accords with epidemiological data suggesting that S-antigens are poorly immunogenic. Appending the transmembrane domain of a membrane immunoglobulin (immunoglobulin G1) to its carboxy terminus produced a hybrid S-antigen that was no longer secreted but was located on the surface of virus-infected cells. This recombinant virus elicited high antibody titers to the S-antigen. This approach will facilitate the use of live virus delivery systems to immunize against a wide range of foreign nonsurface antigens. Images PMID:3537732

  17. Combinations of Polyclonal or Monoclonal Antibodies to Proteins of the Outer Membranes of the Two Infectious Forms of Vaccinia Virus Protect Mice against a Lethal Respiratory Challenge

    PubMed Central

    Lustig, Shlomo; Fogg, Christiana; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.; Moss, Bernard

    2005-01-01

    Previous studies demonstrated that antibodies to live vaccinia virus infection are needed for optimal protection against orthopoxvirus infection. The present report is the first to compare the protective abilities of individual and combinations of specific polyclonal and monoclonal antibodies that target proteins of the intracellular (IMV) and extracellular (EV) forms of vaccinia virus. The antibodies were directed to one IMV membrane protein, L1, and to two outer EV membrane proteins, A33 and B5. In vitro studies showed that the antibodies to L1 neutralized IMV and that the antibodies to A33 and B5 prevented the spread of EV in liquid medium. Prophylactic administration of individual antibodies to BALB/c mice partially protected them against disease following intranasal challenge with lethal doses of vaccinia virus. Combinations of antibodies, particularly anti-L1 and -A33 or -L1 and -B5, provided enhanced protection when administered 1 day before or 2 days after challenge. Furthermore, the protection was superior to that achieved with pooled immune gamma globulin from human volunteers inoculated with live vaccinia virus. In addition, single injections of anti-L1 plus anti-A33 antibodies greatly delayed the deaths of severe combined immunodeficiency mice challenged with vaccinia virus. These studies suggest that antibodies to two or three viral membrane proteins optimally derived from the outer membranes of IMV and EV, may be beneficial for prophylaxis or therapy of orthopoxvirus infections. PMID:16227266

  18. Protection of rhesus monkeys from fatal Lassa fever by vaccination with a recombinant vaccinia virus containing the Lassa virus glycoprotein gene.

    PubMed Central

    Fisher-Hoch, S P; McCormick, J B; Auperin, D; Brown, B G; Castor, M; Perez, G; Ruo, S; Conaty, A; Brammer, L; Bauer, S

    1989-01-01

    Lassa fever is an acute febrile disease of West Africa, where there are as many as 300,000 infections a year and an estimated 3000 deaths. As control of the rodent host is impracticable at present, the best immediate prospect is vaccination. We tested as potential vaccines in rhesus monkeys a closely related virus, Mopeia virus (two monkeys), and a recombinant vaccinia virus containing the Lassa virus glycoprotein gene, V-LSGPC (four monkeys). Two monkeys vaccinated with the New York Board of Health strain of vaccinia virus as controls died after challenge with Lassa virus. The two monkeys vaccinated with Mopeia virus developed antibodies measurable by radioimmunoprecipitation prior to challenge, and they survived challenge by Lassa virus with minimal physical or physiologic disturbances. However, both showed a transient, low-titer Lassa viremia. Two of the four animals vaccinated with V-LSGPC had antibodies to both Lassa glycoproteins, as determined by radioimmunoprecipitation. All four animals survived a challenge of Lassa virus but experienced a transient febrile illness and moderate physiologic changes following challenge. Virus was recoverable from each of these animals, but at low titer and only during a brief period, as observed for the Mopeia-protected animals. We conclude that V-LSGPC can protect rhesus monkeys against death from Lassa fever. PMID:2911575

  19. Introduction of the six major genomic deletions of modified vaccinia virus Ankara (MVA) into the parental vaccinia virus is not sufficient to reproduce an MVA-like phenotype in cell culture and in mice.

    PubMed

    Meisinger-Henschel, Christine; Späth, Michaela; Lukassen, Susanne; Wolferstätter, Michael; Kachelriess, Heike; Baur, Karen; Dirmeier, Ulrike; Wagner, Markus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

    2010-10-01

    Modified vaccinia virus Ankara (MVA) has a highly restricted host range in cell culture and is apathogenic in vivo. MVA was derived from the parental chorioallantois vaccinia virus Ankara (CVA) by more than 570 passages in chicken embryo fibroblast (CEF) cells. During CEF cell passaging, six major deletions comprising 24,668 nucleotides occurred in the CVA genome. We have cloned both the MVA and the parental CVA genome as bacterial artificial chromosomes (BACs) and have sequentially introduced the six major MVA deletions into the cloned CVA genome. Reconstituted mutant CVA viruses containing up to six major MVA deletions showed no detectable replication restriction in 12 of 14 mammalian cell lines tested; the exceptions were rabbit cell lines RK13 and SIRC. In mice, CVA mutants with up to three deletions showed slightly enhanced virulence, suggesting that gene deletion in replicating vaccinia virus (VACV) can result in gain of fitness in vivo. CVA mutants containing five or all six deletions were still pathogenic, with a moderate degree of attenuation. Deletion V was mainly responsible for the attenuated phenotype of these mutants. In conclusion, loss or truncation of all 31 open reading frames in the six major deletions is not sufficient to reproduce the specific MVA phenotype of strong attenuation and highly restricted host range. Mutations in viral genes outside or in association with the six major deletions appear to contribute significantly to this phenotype. Host range restriction and avirulence of MVA are most likely a cooperative effect of gene deletions and mutations involving the major deletions. PMID:20668072

  20. Introduction of the Six Major Genomic Deletions of Modified Vaccinia Virus Ankara (MVA) into the Parental Vaccinia Virus Is Not Sufficient To Reproduce an MVA-Like Phenotype in Cell Culture and in Mice▿

    PubMed Central

    Meisinger-Henschel, Christine; Späth, Michaela; Lukassen, Susanne; Wolferstätter, Michael; Kachelriess, Heike; Baur, Karen; Dirmeier, Ulrike; Wagner, Markus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

    2010-01-01

    Modified vaccinia virus Ankara (MVA) has a highly restricted host range in cell culture and is apathogenic in vivo. MVA was derived from the parental chorioallantois vaccinia virus Ankara (CVA) by more than 570 passages in chicken embryo fibroblast (CEF) cells. During CEF cell passaging, six major deletions comprising 24,668 nucleotides occurred in the CVA genome. We have cloned both the MVA and the parental CVA genome as bacterial artificial chromosomes (BACs) and have sequentially introduced the six major MVA deletions into the cloned CVA genome. Reconstituted mutant CVA viruses containing up to six major MVA deletions showed no detectable replication restriction in 12 of 14 mammalian cell lines tested; the exceptions were rabbit cell lines RK13 and SIRC. In mice, CVA mutants with up to three deletions showed slightly enhanced virulence, suggesting that gene deletion in replicating vaccinia virus (VACV) can result in gain of fitness in vivo. CVA mutants containing five or all six deletions were still pathogenic, with a moderate degree of attenuation. Deletion V was mainly responsible for the attenuated phenotype of these mutants. In conclusion, loss or truncation of all 31 open reading frames in the six major deletions is not sufficient to reproduce the specific MVA phenotype of strong attenuation and highly restricted host range. Mutations in viral genes outside or in association with the six major deletions appear to contribute significantly to this phenotype. Host range restriction and avirulence of MVA are most likely a cooperative effect of gene deletions and mutations involving the major deletions. PMID:20668072

  1. 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

  2. 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.

  3. Vaccinia virus infection induces dendritic cell maturation but inhibits antigen presentation by MHC class II

    PubMed Central

    Yao, Yongxue; Li, Ping; Singh, Pratibha; Thiele, Allison T.; Wilkes, David S.; Renukaradhya, Gourapura J.; Brutkiewicz, Randy R.; Travers, Jeffrey B.; Luker, Gary D.; Hong, Soon-Cheol; Blum, Janice S.; Chang, Cheong-Hee

    2007-01-01

    Vaccinia virus (VV) infection is known to inhibit dendritic cells (DC) functions in vitro. Paradoxically, VV is also highly immunogenic and thus has been used as a vaccine. In the present study, we investigated the effects of an in vivo VV infection on DC function by focusing on early innate immunity. Our data indicated that DC are activated upon in vivo VV infection of mice. Splenic DC from VV-infected mice expressed elevated levels of MHC class I and co-stimulatory molecules on their cell surface and exhibited the enhanced potential to produce cytokines upon LPS stimulation. DC from VV-infected mice also expressed a high level of interferon-β. However, a VV infection resulted in the down-regulation of MHC class II expression and the impairment of antigen presentation to CD4 T cells by DC. Thus, during the early stage of a VV infection, although DC are impaired in some of the critical antigen presentation functions, they can promote innate immune defenses against viral infection. PMID:17678637

  4. Poly(A) Polymerase from Vaccinia Virus-Infected Cells I. Partial Purification and Characterization

    PubMed Central

    Brakel, Christine; Kates, Joseph R.

    1974-01-01

    Poly(A) polymerase activity is induced during vaccinia virus infection of HeLa cells. The enzyme is maximally induced at 3.5 h postinfection. Partial purification frees the preparation of RNase activity and RNA polymerase activity. ATP is the substrate for poly(A) synthesis. A small amount of poly(A) is produced from added adenosine diphosphate due to the production of ATP by an adenylate kinase present in the preparation. The incorporation of ATP into poly(A) is dependent on divalent cations (Mg2+ or Mn2+) and is not inhibited by UTP, CTP, or GTP. Poly(U) stimulates ATP incorporation; poly(A) and poly(C) have little effect on ATP incorporation, and poly(dT) is extremely inhibitory. RNA prepared from HeLa cells and from the partially purified poly(A) polymerase (the enzyme preparation contains endogenous RNA [Brakel and Kates]) stimulates ATP incorporation by poly(A) polymerase which was subjected to DEAE-cellulose chromatography. RNase's, pancreatic and T1, inhibit the production of poly(A). DNase has little effect. Poly(U) is able to stimulate poly(A) production in the presence of T1 RNase. PMID:4417406

  5. 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.

  6. The effects of targeting the vaccinia virus B5R protein to the endoplasmic reticulum on virus morphogenesis and dissemination.

    PubMed

    Mathew, E C; Sanderson, C M; Hollinshead, R; Hollinshead, M; Grimley, R; Smith, G L

    1999-12-01

    The consequence of redirecting the vaccinia virus (VV) B5R protein to the endoplasmic reticulum (ER) has been investigated by the addition of an ER retrieval signal KKSL (K(2)X(2)) to the B5R C-terminus. This mutant B5R gene and a version of the gene with the inactive ER retrieval sequence KKSLAL (K(2)X(4)) were inserted into the thymidine kinase locus of a VV mutant lacking the B5R gene, vDeltaB5R. Similar levels of B5R protein were made by each virus, but the B5R-K(2)X(2) protein remained sensitive to endoglycosidase H and colocalised with protein disulphide isomerase in the ER. In contrast, the B5R-K(2)X(4) protein colocalised with 1, 4-galactosyltransferase in the trans-Golgi network. Electron microscopy revealed that even when the B5R protein was redirected to the ER, intracellular mature virus particles were wrapped by cellular membranes to form intracellular enveloped virus particles, although more incompletely wrapped particles were evident compared with wild type. These intracellular enveloped virus particles were, however, unable to efficiently induce the polymerisation of actin and the plaque size formed by vB5R-K(2)X(2) was small. Nevertheless, the amount and specific infectivity of EEV produced by vB5R-K(2)X(2) were similar to those of wild type, despite the dramatic reduction in the amount of B5R protein present in vB5R-K(2)X(2) EEV. PMID:10603324

  7. Sensitivity of human pleural mesothelioma to oncolytic measles virus depends on defects of the type I interferon response.

    PubMed

    Achard, Carole; Boisgerault, Nicolas; Delaunay, Tiphaine; Roulois, David; Nedellec, Steven; Royer, Pierre-Joseph; Pain, Mallory; Combredet, Chantal; Mesel-Lemoine, Mariana; Cellerin, Laurent; Magnan, Antoine; Tangy, Frédéric; Grégoire, Marc; Fonteneau, Jean-François

    2015-12-29

    Attenuated measles virus (MV) is currently being evaluated as an oncolytic virus in clinical trials and could represent a new therapeutic approach for malignant pleural mesothelioma (MPM). Herein, we screened the sensitivity to MV infection and replication of twenty-two human MPM cell lines and some healthy primary cells. We show that MV replicates in fifteen of the twenty-two MPM cell lines. Despite overexpression of CD46 by a majority of MPM cell lines compared to healthy cells, we found that the sensitivity to MV replication did not correlate with this overexpression. We then evaluated the antiviral type I interferon (IFN) responses of MPM cell lines and healthy cells. We found that healthy cells and the seven insensitive MPM cell lines developed a type I IFN response in presence of the virus, thereby inhibiting replication. In contrast, eleven of the fifteen sensitive MPM cell lines were unable to develop a complete type I IFN response in presence of MV. Finally, we show that addition of type I IFN onto MV sensitive tumor cell lines inhibits replication. These results demonstrate that defects in type I IFN response are frequent in MPM and that MV takes advantage of these defects to exert oncolytic activity. PMID:26539644

  8. Sensitivity of human pleural mesothelioma to oncolytic measles virus depends on defects of the type I interferon response

    PubMed Central

    Achard, Carole; Boisgerault, Nicolas; Delaunay, Tiphaine; Roulois, David; Nedellec, Steven; Royer, Pierre-Joseph; Pain, Mallory; Combredet, Chantal; Mesel-Lemoine, Mariana; Cellerin, Laurent; Magnan, Antoine; Tangy, Frédéric; Grégoire, Marc; Fonteneau, Jean-François

    2015-01-01

    Attenuated measles virus (MV) is currently being evaluated as an oncolytic virus in clinical trials and could represent a new therapeutic approach for malignant pleural mesothelioma (MPM). Herein, we screened the sensitivity to MV infection and replication of twenty-two human MPM cell lines and some healthy primary cells. We show that MV replicates in fifteen of the twenty-two MPM cell lines. Despite overexpression of CD46 by a majority of MPM cell lines compared to healthy cells, we found that the sensitivity to MV replication did not correlate with this overexpression. We then evaluated the antiviral type I interferon (IFN) responses of MPM cell lines and healthy cells. We found that healthy cells and the seven insensitive MPM cell lines developed a type I IFN response in presence of the virus, thereby inhibiting replication. In contrast, eleven of the fifteen sensitive MPM cell lines were unable to develop a complete type I IFN response in presence of MV. Finally, we show that addition of type I IFN onto MV sensitive tumor cell lines inhibits replication. These results demonstrate that defects in type I IFN response are frequent in MPM and that MV takes advantage of these defects to exert oncolytic activity. PMID:26539644

  9. Primary Human Leukocyte Subsets Differentially Express Vaccinia Virus Receptors Enriched in Lipid Rafts

    PubMed Central

    Byrd, Daniel; Amet, Tohti; Hu, Ningjie; Lan, Jie; Hu, Sishun

    2013-01-01

    Poxviruses, including vaccinia virus (VV) and canarypox virus (ALVAC), do not indiscriminately infect all cell types of the primary human leukocytes (PHLs) that they encounter but instead demonstrate an extremely strong bias toward infection of monocytes and monocyte lineage cells. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias of cell surface protein-dependent binding to monocytes, B cells, and activated T cells to a similar degree and to neutrophils to a much lesser extent. Resting T cells and resting NK cells exhibited only trace amounts of VV binding. Activated T cells, however, became permissive to VV binding, infection, and replication, while activated NK cells still resisted VV binding. VV binding strongly colocalized with lipid rafts on the surfaces of all VV binding-susceptible PHL subsets, even when lipid rafts were relocated to cell uropods upon cell polarization. Immunosera raised against detergent-resistant membranes (DRMs) from monocytes or activated T cells, but not resting T cells, effectively cross-blocked VV binding to and infection of PHL subsets. CD29 and CD98, two lipid raft-associated membrane proteins that had been found to be important for VV entry into HeLa cells, had no effect on VV binding to and infection of primary activated T cells. Our data indicate that PHL subsets express VV protein receptors enriched in lipid rafts and that receptors are cross-presented on all susceptible PHLs. PMID:23785200

  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. 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. 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

  13. 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

  14. Protective Properties of Vaccinia Virus-Based Vaccines: Skin Scarification Promotes a Nonspecific Immune Response That Protects against Orthopoxvirus Disease

    PubMed Central

    Rice, Amanda D.; Adams, Mathew M.; Lindsey, Scott F.; Swetnam, Daniele M.; Manning, Brandi R.; Smith, Andrew J.; Burrage, Andrew M.; Wallace, Greg; MacNeill, Amy L.

    2014-01-01

    ABSTRACT The process of vaccination introduced by Jenner generated immunity against smallpox and ultimately led to the eradication of the disease. Procedurally, in modern times, the virus is introduced into patients via a process called scarification, performed with a bifurcated needle containing a small amount of virus. What was unappreciated was the role that scarification itself plays in generating protective immunity. In rabbits, protection from lethal disease is induced by intradermal injection of vaccinia virus, whereas a protective response occurs within the first 2 min after scarification with or without virus, suggesting that the scarification process itself is a major contributor to immunoprotection. IMPORTANCE These results show the importance of local nonspecific immunity in controlling poxvirus infections and indicate that the process of scarification should be critically considered during the development of vaccination protocols for other infectious agents. PMID:24760885

  15. 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

  16. 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. PMID:25060519

  17. [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. PMID:1214695

  18. Oncolytic vaccine virus harbouring the IL-24 gene suppresses the growth of lung cancer by inducing apoptosis.

    PubMed

    Lv, Chunwei; Su, Qunshu; Liang, Yupei; Hu, Jinqing; Yuan, Sujing

    2016-07-15

    Lung cancer has an especially high incidence rate worldwide, and its resistance to cell death and chemotherapeutic drugs increases its intractability. The vaccinia virus has been shown to destroy neoplasm within a short time and disseminate rapidly and extensively as an enveloped virion throughout the circulatory system, and this virus has also demonstrated a strong ability to overexpress exogenous genes. Interleukin-24 (IL-24/mda-7) is an important cytokine that belongs to the activating caspase family and facilitates the inhibition of STAT3 when a cell enters the apoptosis pathway. In this study, we constructed a cancer-targeted vaccinia virus carrying the IL-24 gene knocked in the region of the viral thymidine kinase (TK) gene (VV-IL-24). Our results showed that VV-IL-24 efficiently infected and destroyed lung cancer cells via caspase-dependent apoptosis and decreased the expression of STAT3. In vivo, VV-IL-24 expressed IL-24 at a high level in the transplanted tumour, reduced STAT3 activity, and eventually led to apoptosis. In conclusion, we demonstrated that vv-IL-24 has the potential for use as a new human lung cancer treatment. PMID:27208781

  19. Structure and Function of A41, a Vaccinia Virus Chemokine Binding Protein

    PubMed Central

    Abrescia, Nicola G. A; Pease, James E; Wise, Emma L; Stuart, David I; Smith, Geoffrey L; Grimes, Jonathan M

    2008-01-01

    The vaccinia virus (VACV) A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI), and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 Å resolution. The protein has a globular β sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI–chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (μM–nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine–chemokine receptor interactions. PMID:18208323

  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. Marker rescue of temperature-sensitive mutations of vaccinia virus WR: correlation of genetic and physical maps.

    PubMed Central

    Ensinger, M J; Rovinsky, M

    1983-01-01

    The physical map locations of 62 temperature-sensitive mutations of vaccinia virus WR have been determined by marker rescue experiments, using cloned HindIII fragments of wild-type DNA. Since vaccinia virus DNA is not infectious, marker rescue was performed by infecting monolayers of cells at the nonpermissive temperature with a low multiplicity of the mutant to be rescued and transfecting with calcium phosphate-precipitated recombinant DNA. Wild-type recombinants were measured by using either a direct plaque assay technique or a two-step procedure in which the final yield of virus from the transfected cells was assayed at the permissive and nonpermissive temperatures. Mutants that had been previously assigned to the same complementation-recombination group were rescued by the same HindIII fragment, with the exception of three mutants in one group that were rescued by either one of two adjacent fragments. A comparison between the genetic linkage map of the temperature-sensitive mutations in 30 mutants with their physical locations demonstrated that not only was the order of the genetic map correct but also recombination frequencies generally reflected actual physical distances. PMID:6312100

  3. The vaccinia virus E8R gene product is required for formation of transcriptionally active virions.

    PubMed

    Kato, Sayuri E M; Condit, Richard C; Moussatché, Nissin

    2007-10-25

    Two vaccinia virus temperature-sensitive mutants were mapped to the E8R gene and subjected to phenotypic characterization. Dts23 contains a missense mutation in the coding region of E8R (L81F), and in Cts19 the initiating methionine codon is changed from ATG to ATA (M1I). The two ts mutants display normal patterns of gene expression and DNA replication during infection. The E8 protein is synthesized exclusively late during infection and packaged into virion cores Western blot analysis revealed that E8 synthesis is reduced in Dts23 infected cells at permissive (31 degrees C) and non-permissive temperature (39.7 degrees C) and absent in Cts19 infection under both conditions. Dts23 virions produced at 39.7 degrees C were indistinguishable in appearance from wt virions. Cts19 fails to produce identifiable viral structures when incubated at 39.7 degrees C. Purified Dts23 virions produced at 39.7 degrees C contain reduced amounts of E8 and have a high particle to infectivity ratio; purified Cts19 virions grown at 31 degrees C also show reduced infectivity and do not contain detectable E8. Dts23 grown at 39.7 degrees C could enter cells but failed to synthesize early mRNA or produce CPE. Soluble extracts from mutant virions were active in a promoter dependent in vitro transcription assay, however intact mutant cores were defective in transcription. We suggest that E8 plays a subtle role in virion core structure that impacts directly or indirectly on core transcription. PMID:17619043

  4. 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.

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

    DOE PAGESBeta

    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

  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. 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

  8. Vaccinia Virus Extracellular Enveloped Virion Neutralization In Vitro and Protection In Vivo Depend on Complement▿

    PubMed Central

    Benhnia, Mohammed Rafii-El-Idrissi; McCausland, Megan M.; Moyron, Juan; Laudenslager, John; Granger, Steven; Rickert, Sandra; Koriazova, Lilia; Kubo, Ralph; Kato, Shinichiro; Crotty, Shane

    2009-01-01

    Antibody neutralization is an important component of protective immunity against vaccinia virus (VACV). Two distinct virion forms, mature virion and enveloped virion (MV and EV, respectively), possess separate functions and nonoverlapping immunological properties. In this study we examined the mechanics of EV neutralization, focusing on EV protein B5 (also called B5R). We show that neutralization of EV is predominantly complement dependent. From a panel of high-affinity anti-B5 monoclonal antibodies (MAbs), the only potent neutralizer in vitro (90% at 535 ng/ml) was an immunoglobulin G2a (IgG2a), and neutralization was complement mediated. This MAb was the most protective in vivo against lethal intranasal VACV challenge. Further studies demonstrated that in vivo depletion of complement caused a >50% loss of anti-B5 IgG2a protection, directly establishing the importance of complement for protection against the EV form. However, the mechanism of protection is not sterilizing immunity via elimination of the inoculum as the viral inoculum consisted of a purified MV form. The prevention of illness in vivo indicated rapid control of infection. We further demonstrate that antibody-mediated killing of VACV-infected cells expressing surface B5 is a second protective mechanism provided by complement-fixing anti-B5 IgG. Cell killing was very efficient, and this effector function was highly isotype specific. These results indicate that anti-B5 antibody-directed cell lysis via complement is a powerful mechanism for clearance of infected cells, keeping poxvirus-infected cells from being invisible to humoral immune responses. These findings highlight the importance of multiple mechanisms of antibody-mediated protection against VACV and point to key immunobiological differences between MVs and EVs that impact the outcome of infection. PMID:19019965

  9. 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

  10. Modified vaccinia virus ankara (MVA) as production platform for vaccines against influenza and other viral respiratory diseases.

    PubMed

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

    2014-07-01

    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

  11. Systemically administered DNA and fowlpox recombinants expressing four vaccinia virus genes although immunogenic do not protect mice against the highly pathogenic IHD-J vaccinia strain.

    PubMed

    Bissa, Massimiliano; Pacchioni, Sole Maria; Zanotto, Carlo; De Giuli Morghen, Carlo; Illiano, Elena; Granucci, Francesca; Zanoni, Ivan; Broggi, Achille; Radaelli, Antonia

    2013-12-26

    The first-generation smallpox vaccine was based on live vaccinia virus (VV) and it successfully eradicated the disease worldwide. Therefore, it was not administered any more after 1980, as smallpox no longer existed as a natural infection. However, emerging threats by terrorist organisations has prompted new programmes for second-generation vaccine development based on attenuated VV strains, which have been shown to cause rare but serious adverse events in immunocompromised patients. Considering the closely related animal poxviruses that might also be used as bioweapons, and the increasing number of unvaccinated young people and AIDS-affected immunocompromised subjects, a safer and more effective smallpox vaccine is still required. New avipoxvirus-based vectors should improve the safety of conventional vaccines, and protect from newly emerging zoonotic orthopoxvirus diseases and from the threat of deliberate release of variola or monkeypox virus in a bioterrorist attack. In this study, DNA and fowlpox recombinants expressing the L1R, A27L, A33R and B5R genes were constructed and evaluated in a pre-clinical trial in mouse, following six prime/boost immunisation regimens, to compare their immunogenicity and protective efficacy against a challenge with the lethal VV IHD-J strain. Although higher numbers of VV-specific IFNγ-producing T lymphocytes were observed in the protected mice, the cytotoxic T-lymphocyte response and the presence of neutralising antibodies did not always correlate with protection. In spite of previous successful results in mice, rabbits and monkeys, where SIV/HIV transgenes were expressed by the fowlpox vector, the immune response elicited by these recombinants was low, and most of the mice were not protected. PMID:24050999

  12. Substrate specificity of three viral thymidine kinases (TK): vaccinia virus TK, feline herpesvirus TK, and canine herpesvirus TK.

    PubMed

    Solaroli, N; Johansson, M; Balzarini, J; Karlsson, A

    2006-01-01

    In search of novel suicide gene candidates we have cloned and characterized thymidine kinases from three viruses; vaccinia virus TK (VVTK), feline herpesvirus TK (FHV-TK), and canine herpesvirus TK (CHV-TK). Our studies showed that VVTK primarily is a thymidine kinase, with a substrate specificity mainly restricted to dThd and only minor affinity for dCyd. VVTK also is related closely to mammalian thymidine kinase 1 (TK1), with 66% identity and 75% general homology. Although CHV-TK and FHV-TK are sequence related to herpes simplex virus types 1 thymidine kinase (HSV1-TK), with 31% and 35% identity and a general similarity of 54%, the substrate specificity of these enzymes was restricted to dThd and thymidine analogs. PMID:17065088

  13. 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

  14. 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

    2016-03-31

    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. PMID:27034398

  15. Analysis of glioblastoma tumor coverage by oncolytic virus-loaded neural stem cells using MRI-based tracking and histological reconstruction.

    PubMed

    Morshed, R A; Gutova, M; Juliano, J; Barish, M E; Hawkins-Daarud, A; Oganesyan, D; Vazgen, K; Yang, T; Annala, A; Ahmed, A U; Aboody, K S; Swanson, K R; Moats, R A; Lesniak, M S

    2015-01-01

    In preclinical studies, neural stem cell (NSC)-based delivery of oncolytic virus has shown great promise in the treatment of malignant glioma. Ensuring the success of this therapy will require critical evaluation of the spatial distribution of virus after NSC transplantation. In this study, the patient-derived GBM43 human glioma line was established in the brain of athymic nude mice, followed by the administration of NSCs loaded with conditionally replicating oncolytic adenovirus (NSC-CRAd-S-pk7). We determined the tumor coverage potential of oncolytic adenovirus by examining NSC distribution using magnetic resonance (MR) imaging and by three-dimensional reconstruction from ex vivo tissue specimens. We demonstrate that unmodified NSCs and NSC-CRAd-S-pk7 exhibit a similar distribution pattern with most prominent localization occurring at the tumor margins. We were further able to visualize the accumulation of these cells at tumor sites via T2-weighted MR imaging as well as the spread of viral particles using immunofluorescence. Our analyses reveal that a single administration of oncolytic virus-loaded NSCs allows for up to 31% coverage of intracranial tumors. Such results provide valuable insights into the therapeutic potential of this novel viral delivery platform. PMID:25525033

  16. Production of HIV-1 gp120 in packed-bed bioreactor using the vaccinia virus/T7 expression system.

    PubMed

    Hu, Y C; Kaufman, J; Cho, M W; Golding, H; Shiloach, J

    2000-01-01

    The HeLa cell-vaccinia virus system is an attractive method for producing recombinant mammalian proteins with proper post-translation modifications. This approach is especially important for the production of HIV-1 envelope glycoprotein, gp120, since more than half of its total mass is due to carbohydrates. A recombinant vaccinia virus/T7 RNA polymerase expression system was developed to express and produce large amounts of gp120 tagged with six histidine residues. In this system, the expressed T7 RNA polymerase from one virus drives the transcription of the gp120 encoded in the second virus. During the process development phase, the following parameters were studied: infection time, infection duration, multiplicity of infection, ratio of the two viruses, medium composition, and medium replacement strategy during the infection phase. The chosen production method was based on using the packed-bed bioreactor. The HeLa cells were immobilized on fibrous disks (Fibra-Cel) packed in an internal basket positioned in a vertically mixed bioreactor (Celligen Plus), and 25 g of carriers were packed in a 1.6-L (working volume) reactor. The process included a growth stage followed by a production stage. In the growth stage, the bed was perfused with a serum-containing medium, allowing the cells to grow to saturation, and in the production stage, done using serum-free medium, the cells were infected with the two recombinant viruses. The expressed protein was secreted, collected from the culture fluid, and purified. The specific production was found to be between 2 and 3 microg of protein/10(6) cells, and the volumetric production was around 10 mg/50 g carriers. PMID:11027165

  17. Choindroitinase ABC I-Mediated Enhancement of Oncolytic Virus Spread and Anti Tumor Efficacy: A Mathematical Model

    PubMed Central

    Kim, Yangjin; Lee, Hyun Geun; Dmitrieva, Nina; Kim, Junseok; Kaur, Balveen; Friedman, Avner

    2014-01-01

    Oncolytic viruses are genetically engineered viruses that are designed to kill cancer cells while doing minimal damage to normal healthy tissue. After being injected into a tumor, they infect cancer cells, multiply inside them, and when a cancer cell is killed they move on to spread and infect other cancer cells. Chondroitinase ABC (Chase-ABC) is a bacterial enzyme that can remove a major glioma ECM component, chondroitin sulfate glycosoamino glycans from proteoglycans without any deleterious effects in vivo. It has been shown that Chase-ABC treatment is able to promote the spread of the viruses, increasing the efficacy of the viral treatment. In this paper we develop a mathematical model to investigate the effect of the Chase-ABC on the treatment of glioma by oncolytic viruses (OV). We show that the model's predictions agree with experimental results for a spherical glioma. We then use the model to test various treatment options in the heterogeneous microenvironment of the brain. The model predicts that separate injections of OV, one into the center of the tumor and another outside the tumor will result in better outcome than if the total injection is outside the tumor. In particular, the injection of the ECM-degrading enzyme (Chase-ABC) on the periphery of the main tumor core need to be administered in an optimal strategy in order to infect and eradicate the infiltrating glioma cells outside the tumor core in addition to proliferative cells in the bulk of tumor core. The model also predicts that the size of tumor satellites and distance between the primary tumor and multifocal/satellite lesions may be an important factor for the efficacy of the viral therapy with Chase treatment. PMID:25047810

  18. Choindroitinase ABC I-mediated enhancement of oncolytic virus spread and anti tumor efficacy: a mathematical model.

    PubMed

    Kim, Yangjin; Lee, Hyun Geun; Dmitrieva, Nina; Kim, Junseok; Kaur, Balveen; Friedman, Avner

    2014-01-01

    Oncolytic viruses are genetically engineered viruses that are designed to kill cancer cells while doing minimal damage to normal healthy tissue. After being injected into a tumor, they infect cancer cells, multiply inside them, and when a cancer cell is killed they move on to spread and infect other cancer cells. Chondroitinase ABC (Chase-ABC) is a bacterial enzyme that can remove a major glioma ECM component, chondroitin sulfate glycosoamino glycans from proteoglycans without any deleterious effects in vivo. It has been shown that Chase-ABC treatment is able to promote the spread of the viruses, increasing the efficacy of the viral treatment. In this paper we develop a mathematical model to investigate the effect of the Chase-ABC on the treatment of glioma by oncolytic viruses (OV). We show that the model's predictions agree with experimental results for a spherical glioma. We then use the model to test various treatment options in the heterogeneous microenvironment of the brain. The model predicts that separate injections of OV, one into the center of the tumor and another outside the tumor will result in better outcome than if the total injection is outside the tumor. In particular, the injection of the ECM-degrading enzyme (Chase-ABC) on the periphery of the main tumor core need to be administered in an optimal strategy in order to infect and eradicate the infiltrating glioma cells outside the tumor core in addition to proliferative cells in the bulk of tumor core. The model also predicts that the size of tumor satellites and distance between the primary tumor and multifocal/satellite lesions may be an important factor for the efficacy of the viral therapy with Chase treatment. PMID:25047810

  19. De novo Fatty Acid Biosynthesis Contributes Significantly to Establishment of a Bioenergetically Favorable Environment for Vaccinia Virus Infection

    PubMed Central

    Greseth, Matthew D.; Traktman, Paula

    2014-01-01

    The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis). Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis) that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia infection, and in

  20. De novo fatty acid biosynthesis contributes significantly to establishment of a bioenergetically favorable environment for vaccinia virus infection.

    PubMed

    Greseth, Matthew D; Traktman, Paula

    2014-03-01

    The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis). Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis) that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia infection, and in

  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. 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

  4. 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

  5. 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.

  6. Expression of anti-VEGF antibody together with anti-EGFR or anti-FAP enhances tumor regression as a result of vaccinia virotherapy

    PubMed Central

    Huang, Ting; Wang, Huiqiang; Chen, Nanhai G; Frentzen, Alexa; Minev, Boris; Szalay, Aladar A

    2015-01-01

    The tumor microenvironment plays an important role in tumor growth and progression. Here we demonstrate that vaccinia virus-mediated, constitutively expressed intratumoral antibodies against vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), and fibroblast activation protein (FAP) significantly improved tumor regression and oncolytic virotherapy through suppression of angiogenesis, cell proliferation, and stromagenesis in virus-colonized tumors. In contrast to the tumor growth inhibition by the three tumor growth-inhibiting antibodies individually, when two of the three antibodies were expressed simultaneously by single vaccinia virus strains tumor regression was further enhanced. These findings strongly indicate that interference with the two tumor growth-stimulating mechanisms did in fact result in enhanced therapeutic efficacy in tumor xenograft models and may lead to an effective therapy in patients with cancer. PMID:27119102

  7. 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. PMID:26195812

  8. MicroRNA-145 regulates oncolytic herpes simplex virus-1 for selective killing of human non-small cell lung cancer cells

    PubMed Central

    2013-01-01

    Background Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide, and novel treatment modalities to improve the prognosis of patients with advanced disease are highly desirable. Oncolytic virotherapy is a promising approach for the treatment of advanced NSCLC. MicroRNAs (miRNAs) may be a factor in the regulation of tumor-specific viral replication. The purpose of this study was to investigate whether miRNA-145 regulated oncolytic herpes simplex virus-1 (HSV-1) can selectively kill NSCLC cells with reduced collateral damage to normal cells. Methods We incorporated 4 copies of miRNA-145 target sequences into the 3′-untranslated region of an HSV-1 essential viral gene, ICP27, to create AP27i145 amplicon viruses and tested their target specificity and toxicity on normal cells and lung cancer cells in vitro. Results miRNA-145 expression in normal cells was higher than that in NSCLC cells. AP27i145 replication was inversely correlated with the expression of miRNA-145 in infected cells. This oncolytic HSV-1 selectively reduced cell proliferation and prevented the colony formation of NSCLC cells. The combination of radiotherapy and AP27i145 infection was significantly more potent in killing cancer cells than each therapy alone. Conclusions miRNA-145-regulated oncolytic HSV-1 is a promising agent for the treatment of NSCLC. PMID:23876001

  9. 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.

  10. 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

  11. 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. PMID:22244689

  12. 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.

  13. 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

  14. Characterization and Structure of the Vaccinia Virus NF-κB Antagonist A46 *

    PubMed Central

    Fedosyuk, Sofiya; Grishkovskaya, Irina; de Almeida Ribeiro, Euripedes; Skern, Tim

    2014-01-01

    Successful vaccinia virus (VACV) replication in the host requires expression of viral proteins that interfere with host immunity, such as antagonists of the activation of the proinflammatory transcription factor NF-κB. Two such VACV proteins are A46 and A52. A46 interacts with the Toll-like receptor/interleukin-1R (TIR) domain of Toll-like receptors and intracellular adaptors such as MAL (MyD88 adapter-like), TRAM (TIR domain-containing adapter-inducing interferon-β (TRIF)-related adaptor molecule), TRIF, and MyD88, whereas A52 binds to the downstream signaling components TRAF6 and IRAK2. Here, we characterize A46 biochemically, determine by microscale thermophoresis binding constants for the interaction of A46 with the TIR domains of MyD88 and MAL, and present the 2.0 Å resolution crystal structure of A46 residues 87–229. Full-length A46 behaves as a tetramer; variants lacking the N-terminal 80 residues are dimeric. Nevertheless, both bind to the Toll-like receptor domains of MAL and MyD88 with KD values in the low μm range. Like A52, A46 also shows a Bcl-2-like fold but with biologically relevant differences from that of A52. Thus, A46 uses helices α4 and α6 to dimerize, compared with the α1-α6 face used by A52 and other Bcl-2 like VACV proteins. Furthermore, the loop between A46 helices α4-α5 is flexible and shorter than in A52; there is also evidence for an intramolecular disulfide bridge between consecutive cysteine residues. We used molecular docking to propose how A46 interacts with the BB loop of the TRAM TIR domain. Comparisons of A46 and A52 exemplify how subtle changes in viral proteins with the same fold lead to crucial differences in biological activity. PMID:24356965

  15. 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

  16. Duplication of the A17L Locus of Vaccinia Virus Provides an Alternate Route to Rifampin Resistance

    PubMed Central

    Erlandson, Karl J.; Cotter, Catherine A.; Charity, James C.; Martens, Craig; Fischer, Elizabeth R.; Ricklefs, Stacy M.; Porcella, Stephen F.

    2014-01-01

    ABSTRACT Specific gene duplications can enable double-stranded DNA viruses to adapt rapidly to environmental pressures despite the low mutation rate of their high-fidelity DNA polymerases. We report on the rapid positive selection of a novel vaccinia virus genomic duplication mutant in the presence of the assembly inhibitor rifampin. Until now, all known rifampin-resistant vaccinia virus isolates have contained missense mutations in the D13L gene, which encodes a capsid-like scaffold protein required for stabilizing membrane curvature during the early stage of virion assembly. Here we describe a second pathway to rifampin resistance involving A17, a membrane protein that binds and anchors D13 to the immature virion. After one round of selection, a rifampin-resistant virus that contained a genomic duplication in the A17L-A21L region was recovered. The mutant had both C-terminally truncated and full-length A17L open reading frames. Expression of the truncated A17 protein was retained when the virus was passaged in the presence of rifampin but was lost in the absence of the drug, suggesting that the duplication decreased general fitness. Both forms of A17 were bound to the virion membrane and associated with D13. Moreover, insertion of an additional truncated or inducible full-length A17L open reading frame into the genome of the wild-type virus was sufficient to confer rifampin resistance. In summary, this report contains the first evidence of an alternate mechanism for resistance of poxviruses to rifampin, indicates a direct relationship between A17 levels and the resistance phenotype, and provides further evidence of the ability of double-stranded DNA viruses to acquire drug resistance through gene duplication. IMPORTANCE The present study provides the first evidence of a new mechanism of resistance of a poxvirus to the antiviral drug rifampin. In addition, it affirms the importance of the interaction between the D13 scaffold protein and the A17 membrane protein

  17. Natural Killer Cells and Innate Interferon Gamma Participate in the Host Defense against Respiratory Vaccinia Virus Infection

    PubMed Central

    Abboud, Georges; Tahiliani, Vikas; Desai, Pritesh; Varkoly, Kyle; Driver, John; Hutchinson, Tarun E.

    2015-01-01

    ABSTRACT In establishing a respiratory infection, vaccinia virus (VACV) initially replicates in airway epithelial cells before spreading to secondary sites of infection, mainly the draining lymph nodes, spleen, gastrointestinal tract, and reproductive organs. We recently reported that interferon gamma (IFN-γ) produced by CD8 T cells ultimately controls this disseminated infection, but the relative contribution of IFN-γ early in infection is unknown. Investigating the role of innate immune cells, we found that the frequency of natural killer (NK) cells in the lung increased dramatically between days 1 and 4 postinfection with VACV. Lung NK cells displayed an activated cell surface phenotype and were the primary source of IFN-γ prior to the arrival of CD8 T cells. In the presence of an intact CD8 T cell compartment, depletion of NK cells resulted in increased lung viral load at the time of peak disease severity but had no effect on eventual viral clearance, disease symptoms, or survival. In sharp contrast, RAG−/− mice devoid of T cells failed to control VACV and succumbed to infection despite a marked increase in NK cells in the lung. Supporting an innate immune role for NK cell-derived IFN-γ, we found that NK cell-depleted or IFN-γ-depleted RAG−/− mice displayed increased lung VACV titers and dissemination to ovaries and a significantly shorter mean time to death compared to untreated NK cell-competent RAG−/− controls. Together, these findings demonstrate a role for IFN-γ in aspects of both the innate and adaptive immune response to VACV and highlight the importance of NK cells in T cell-independent control of VACV in the respiratory tract. IMPORTANCE Herein, we provide the first systematic evaluation of natural killer (NK) cell function in the lung after infection with vaccinia virus, a member of the Poxviridae family. The respiratory tract is an important mucosal site for entry of many human pathogens, including poxviruses, but precisely how our

  18. 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. PMID:25796990

  19. Isolated limb perfusion with biochemotherapy and oncolytic virotherapy combines with radiotherapy and surgery to overcome treatment resistance in an animal model of extremity soft tissue sarcoma.

    PubMed

    Wilkinson, Michelle J; Smith, Henry G; Pencavel, Timothy D; Mansfield, David C; Kyula-Currie, Joan; Khan, Aadil A; McEntee, Gráinne; Roulstone, Victoria; Hayes, Andrew J; Harrington, Kevin J

    2016-09-15

    The management of locally advanced or recurrent extremity sarcoma often necessitates multimodal therapy to preserve a limb, of which isolated limb perfusion (ILP) is a key component. However, with standard chemotherapeutic agents used in ILP, the duration of response is limited. Novel agents or treatment combinations are urgently needed to improve outcomes. Previous work in an animal model has demonstrated the efficacy of oncolytic virotherapy when delivered by ILP and, in this study, we report further improvements from combining ILP-delivered oncolytic virotherapy with radiation and surgical resection. In vitro, the combination of radiation with an oncolytic vaccinia virus (GLV-1h68) and melphalan demonstrated increased cytotoxicity in a panel of sarcoma cell lines. The effects were mediated through activation of the intrinsic apoptotic pathway. In vivo, combinations of radiation, oncolytic virotherapy and standard ILP resulted in delayed tumour growth and prolonged survival when compared with standard ILP alone. However, local disease control could only be secured when such treatment was combined with surgical resection, the timing of which was crucial in determining outcome. Combinations of oncolytic virotherapy with surgical resection and radiation have direct clinical relevance in extremity sarcoma and represent an exciting prospect for improving outcomes in this pathology. PMID:27116656

  20. Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis.

    PubMed Central

    Falgout, B; Bray, M; Schlesinger, J J; Lai, C J

    1990-01-01

    The protective immunity conferred by a set of recombinant vaccinia viruses containing the entire coding sequence of dengue virus type 4 nonstructural glycoprotein NS1 plus various flanking sequences was evaluated by using a mouse encephalitis model. Mice immunized with recombinant vNS1-NS2a, which expresses authentic NS1, were solidly protected against intracerebral dengue virus challenge. However, mice immunized with recombinants vNS1-15%NS2a and vRSVG/NS1-15%NS2a, which express aberrant forms of NS1, were only partially protected (63 to 67% survival rate). Serologic analysis showed that mice immunized with vNS1-NS2a developed high titers of antibodies to NS1 as measured by radioimmunoprecipitation, enzyme-linked immunosorbent assay, and complement-mediated cytolytic assays. In addition, a pool of sera from these animals was protective in a passive transfer experiment. Lower titers of NS1-specific antibodies were detected in sera of animals immunized with vNS1-15%NS2a or vRSVG/NS1-15%NS2a by all three assays. These data support the view that protection against dengue virus infection in mice may be mediated at least in part by NS1-specific antibodies through a mechanism of complement-mediated lysis of infected cells. Additionally, immunization with two recombinant viruses expressing authentic NS1 of dengue virus type 2 conferred partial protection (30-50%) against dengue virus type 2 challenge. Images PMID:2143542

  1. Characterization of a temperature-sensitive mutant of vaccinia virus reveals a novel function that prevents virus-induced breakdown of RNA.

    PubMed Central

    Pacha, R F; Condit, R C

    1985-01-01

    We have attempted to characterize the molecular defect in a temperature-sensitive mutant of vaccinia virus, ts22, which has an abortive late phenotype. At the nonpermissive temperature, ts22 displays normal viral protein synthesis until 8 h postinfection. Between 8 and 10 h after infection all viral protein synthesis ceases abruptly. Characterization of ts22 revealed that (i) primary transcription of late viral genes was not grossly impaired, (ii) late viral mRNA was biologically inactive since it could not stimulate in vitro protein synthesis, and (iii) extensive cleavage of rRNA and late viral mRNA occurred at the time that viral protein synthesis aborted in vivo. These data suggest that ts22 is defective in a function which prevents host rRNA and viral mRNA from being degraded. Inhibitor studies with cytosine arabinoside and cycloheximide showed that induction of and protection from rRNA breakdown occurred at approximately the same time during infection and required late viral gene expression. The viral protein synthesis pattern observed in vaccinia virus-infected cells treated with the drug isatin-beta-thiosemicarbazone was strikingly similar to that observed in ts22-infected cells at the nonpermissive temperature (J. Cooper, B. Moss, and E. Katz, Virology 96:381-392, 1979). Analysis of rRNA integrity in isatin-beta-thiosemicarbazone-treated, vaccinia virus-infected cells revealed extensive cleavage of rRNA, suggesting that the ts22 and drug inhibitor may function in the same pathway. Images PMID:4057355

  2. 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

  3. Oncolytic HSV virotherapy in murine sarcomas differentially triggers an antitumor T-cell response in the absence of virus permissivity

    PubMed Central

    Leddon, Jennifer L; Chen, Chun-Yu; Currier, Mark A; Wang, Pin-Yi; Jung, Francesca A; Denton, Nicholas L; Cripe, Kevin M; Haworth, Kellie B; Arnold, Michael A; Gross, Amy C; Eubank, Timothy D; Goins, William F; Glorioso, Joseph C; Cohen, Justus B; Grandi, Paola; Hildeman, David A; Cripe, Timothy P

    2015-01-01

    Multiple studies have indicated that in addition to direct oncolysis, virotherapy promotes an antitumor cytotoxic T cell response important for efficacy. To study this phenomenon further, we tested three syngeneic murine sarcoma models that displayed varied degrees of permissiveness to oncolytic herpes simplex virus replication and cytotoxicity in vitro, with the most permissive being comparable to some human sarcoma tumor lines. The in vivo antitumor effect ranged from no or modest response to complete tumor regression and protection from tumor rechallenge. The in vitro permissiveness to viral oncolysis was not predictive of the in vivo antitumor effect, as all three tumors showed intact interferon signaling and minimal permissiveness to virus in vivo. Tumor shrinkage was T-cell mediated with a tumor-specific antigen response required for maximal antitumor activity. Further analysis of the innate and adaptive immune microenvironment revealed potential correlates of susceptibility and resistance, including favorable and unfavorable cytokine profiles, differential composition of intratumoral myeloid cells, and baseline differences in tumor cell immunogenicity and tumor-infiltrating T-cell subsets. It is likely that a more complete understanding of the interplay between the immunologic immune microenvironment and virus infection will be necessary to fully leverage the antitumor effects of this therapeutic platform. PMID:27119100

  4. 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.

  5. 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.

  6. 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

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

    1994-06-01

    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

  7. Disparity between Levels of In Vitro Neutralization of Vaccinia Virus by Antibody to the A27 Protein and Protection of Mice against Intranasal Challenge▿

    PubMed Central

    Fogg, Christiana N.; Americo, Jeffrey L.; Earl, Patricia L.; Resch, Wolfgang; Aldaz-Carroll, Lydia; Eisenberg, Roselyn J.; Cohen, Gary H.; Moss, Bernard

    2008-01-01

    Immunization with recombinant proteins may provide a safer alternative to live vaccinia virus for prophylaxis of poxvirus infections. Although antibody protects against vaccinia virus infection, the mechanism is not understood and the selection of immunogens is daunting as there are dozens of surface proteins and two infectious forms known as the mature virion (MV) and the enveloped virion (EV). Our previous studies showed that mice immunized with soluble forms of EV membrane proteins A33 and B5 and MV membrane protein L1 or passively immunized with antibodies to these proteins survived an intranasal challenge with vaccinia virus. The present study compared MV protein A27, which has a role in virus attachment to glycosaminoglycans on the cell surface, to L1 with respect to immunogenicity and protection. Although mice developed similar levels of neutralizing antibody after immunizations with A27 or L1, A27-immunized mice exhibited more severe disease upon an intranasal challenge with vaccinia virus. In addition, mice immunized with A27 and A33 were not as well protected as mice receiving L1 and A33. Polyclonal rabbit anti-A27 and anti-L1 IgG had equivalent MV-neutralizing activities when measured by the prevention of infection of human or mouse cells or cells deficient in glycosaminoglycans or by adding antibody prior to or after virus adsorption. Nevertheless, the passive administration of antibody to A27 was poorly protective compared to the antibody to L1. These studies raise questions regarding the basis for antibody protection against poxvirus disease and highlight the importance of animal models for the early evaluation of vaccine candidates. PMID:18524827

  8. Vaccinia virus protein K7 is a virulence factor that alters the acute immune response to infection

    PubMed Central

    Benfield, Camilla T. O.; Ren, Hongwei; Lucas, Stuart J.; Bahsoun, Basma

    2013-01-01

    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. PMID:23580427

  9. Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid

    PubMed Central

    Moussatche, Nissin; Condit, Richard C.

    2014-01-01

    ABSTRACT Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core, and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the L4R gene. Using a cryo-fixation method for electron microscopy (high-pressure freezing followed by freeze-substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structures are produced under nonpermissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed. IMPORTANCE The vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like high-pressure freezing/freeze-substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. Enhancement of CD8+ T-cell memory by removal of a vaccinia virus nuclear factor-κB inhibitor

    PubMed Central

    Ren, Hongwei; Ferguson, Brian J; de Motes, Carlos Maluquer; Sumner, Rebecca P; Harman, Laura E R; Smith, Geoffrey L

    2015-01-01

    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. PMID:25382035

  15. Structure Function Studies of Vaccinia Virus Host Range Protein K1 Reveal a Novel Functional Surface for Ankyrin Repeat Proteins▿

    PubMed Central

    Li, Yongchao; Meng, Xiangzhi; Xiang, Yan; Deng, Junpeng

    2010-01-01

    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 Å, 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. PMID:20089642

  16. A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9

    PubMed Central

    Yuan, Ming; Gao, Xuefei; Chard, Louisa S; Ali, Zarah; Ahmed, Jahangir; Li, Yunqing; Liu, Pentao; Lemoine, Nick R; Wang, Yaohe

    2015-01-01

    The current method for creation of vaccinia virus (VACV) vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK) region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (~90%) in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP) could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP) that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application. PMID:26417609

  17. Development of an oncolytic HSV vector fully retargeted specifically to cellular EpCAM for virus entry and cell-to-cell spread.

    PubMed

    Shibata, T; Uchida, H; Shiroyama, T; Okubo, Y; Suzuki, T; Ikeda, H; Yamaguchi, M; Miyagawa, Y; Fukuhara, T; Cohen, J B; Glorioso, J C; Watabe, T; Hamada, H; Tahara, H

    2016-06-01

    Oncolytic herpes simplex virus (HSV) vectors have attracted increasing attention as novel anti-cancer agents. HSV entry is triggered by the binding of glycoprotein D (gD) to its receptors, such as herpesvirus entry mediator or nectin-1. We have recently reported the construction of a fully retargeted HSV platform that incorporates single-chain antibodies (scFv) into gD to mediate entry exclusively via tumor-associated antigens. In this study, we created an scFv directed against epithelial cell adhesion molecule (EpCAM), a recognized carcinoma-associated antigen, and inserted it into the retargeted HSV platform that is ablated for gD recognition of its canonical receptors and contains the entry-enhancing mutations in gB we previously identified. We observed that both initial entry and subsequent cell-to-cell spread of the retargeted virus were stringently dependent on cellular EpCAM expression. Interestingly, the retargeted virus developed larger plaques on some of the human tumor lines tested than the control virus bearing wild-type gD. Intratumoral injection of the retargeted virus revealed antitumor activity in a mouse xenograft model. These observations illustrate the versatility of our retargeted HSV platform as it allows expansion of the oncolytic virus toolbox for the treatment of diverse cancers. PMID:26905369

  18. 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

  19. The heterogeneity of human antibody responses to vaccinia virus revealed through use of focused protein arrays.

    PubMed

    Duke-Cohan, Jonathan S; Wollenick, Kristin; Witten, Elizabeth A; Seaman, Michael S; Baden, Lindsey R; Dolin, Raphael; Reinherz, Ellis L

    2009-02-18

    The renewed interest in strategies to combat infectious agents with epidemic potential has led to a re-examination of vaccination protocols against smallpox. To help define which antigens elicit a human antibody response, we have targeted proteins known or predicted to be presented on the surface of the intracellular mature virion (IMV) or the extracellular enveloped virion (EEV). The predicted ectodomains were expressed in a mammalian in vitro coupled transcription/translation reaction using tRNA(lys) precharged with lysine-epsilon-biotin followed by solid phase immobilization on 384-well neutravidin-coated plates. The generated array is highly specific and sensitive in a micro-ELISA format. By comparison of binding of vaccinia-immune sera to the reticulocyte lysate-produced proteins and to secreted post-translationally modified proteins, we demonstrate that for several proteins including the EEV proteins B5 and A33, proper recognition is dependent upon appropriate folding, with little dependence upon glycosylation per se. We further demonstrate that the humoral immune response to vaccinia among different individuals is not uniform in specificity or strength, as different IMV and EEV targets predominate within the group of immunogenic proteins. This heterogeneity likely results from the diversity of HLA Class II alleles and CD4 T helper cell epitopes stimulating B cell antibody production. Our findings have important implications both for design of new recombinant subunit vaccines as well as for methods of assaying the human antibody response utilizing recombinant proteins produced in vitro. PMID:19146908

  20. 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

  1. 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

  2. Patterns of viral replication correlate with outcome in simian immunodeficiency virus (SIV)-infected macaques: effect of prior immunization with a trivalent SIV vaccine in modified vaccinia virus Ankara.

    PubMed Central

    Hirsch, V M; Fuerst, T R; Sutter, G; Carroll, M W; Yang, L C; Goldstein, S; Piatak, M; Elkins, W R; Alvord, W G; Montefiori, D C; Moss, B; Lifson, J D

    1996-01-01

    The dynamics of plasma viremia were explored in a group of 12 simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) that had received prior immunization with either nonrecombinant or trivalent (gag-pol, env) SIV-recombinant vaccinia viruses. Three distinct patterns of viral replication observed during and following primary viremia accounted for significant differences in survival times. High-level primary plasma viremia with subsequently increasing viremia was associated with rapid progression to AIDS (n = 2). A high-level primary plasma virus load with a transient decline and subsequent progressive increase in viremia in the post-acute phase of infection was associated with progression to AIDS within a year (n = 6). Low levels of primary plasma viremia followed by sustained restriction of virus replication were associated with maintenance of normal lymphocyte subsets and intact lymphoid architecture (n = 4), reminiscent of the profile observed in human immunodeficiency virus type 1-infected long-term nonprogressors. Three of four macaques that showed this pattern had been immunized with an SIV recombinant derived from the attenuated vaccinia virus, modified vaccinia virus Ankara. These data link the dynamics and extent of virus replication to disease course and suggest that sustained suppression of virus promotes long-term, asymptomatic survival of SIV-infected macaques. These findings also suggest that vaccine modulation of host immunity may have profound beneficial effects on the subsequent disease course, even if sterilizing immunity is not achieved. PMID:8648709

  3. Inhibition of Translation Initiation by Protein 169: A Vaccinia Virus Strategy to Suppress Innate and Adaptive Immunity and Alter Virus Virulence

    PubMed Central

    Strnadova, Pavla; Ren, Hongwei; Valentine, Robert; Mazzon, Michela; Sweeney, Trevor R.; Brierley, Ian; Smith, Geoffrey L.

    2015-01-01

    Vaccinia virus (VACV) is the prototypic orthopoxvirus and the vaccine used to eradicate smallpox. Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation. Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways. A virus lacking 169 (vΔ169) replicates and spreads normally in cell culture but is more virulent than parental and revertant control viruses in intranasal and intradermal murine models of infection. Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight. These alterations in innate immunity resulted in a stronger CD8+ T-cell memory response and better protection against virus challenge. This work illustrates how inhibition of host protein synthesis can be a strategy for virus suppression of innate and adaptive immunity. PMID:26334635

  4. Inhibition of Translation Initiation by Protein 169: A Vaccinia Virus Strategy to Suppress Innate and Adaptive Immunity and Alter Virus Virulence.

    PubMed

    Strnadova, Pavla; Ren, Hongwei; Valentine, Robert; Mazzon, Michela; Sweeney, Trevor R; Brierley, Ian; Smith, Geoffrey L

    2015-09-01

    Vaccinia virus (VACV) is the prototypic orthopoxvirus and the vaccine used to eradicate smallpox. Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation. Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways. A virus lacking 169 (vΔ169) replicates and spreads normally in cell culture but is more virulent than parental and revertant control viruses in intranasal and intradermal murine models of infection. Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight. These alterations in innate immunity resulted in a stronger CD8+ T-cell memory response and better protection against virus challenge. This work illustrates how inhibition of host protein synthesis can be a strategy for virus suppression of innate and adaptive immunity. PMID:26334635

  5. Strong, but Age-Dependent, Protection Elicited by a Deoxyribonucleic Acid/Modified Vaccinia Ankara Simian Immunodeficiency Virus Vaccine

    PubMed Central

    Chamcha, Venkateswarlu; Kannanganat, Sunil; Gangadhara, Sailaja; Nabi, Rafiq; Kozlowski, Pamela A.; Montefiori, David C.; LaBranche, Celia C.; Wrammert, Jens; Keele, Brandon F.; Balachandran, Harikrishnan; Sahu, Sujata; Lifton, Michelle; Santra, Sampa; Basu, Rahul; Moss, Bernard; Robinson, Harriet L.; Amara, Rama Rao

    2016-01-01

    Background. In this study, we analyzed the protective efficacy of a simian immunodeficiency virus (SIV) macaque 239 (SIVmac239) analogue of the clinically tested GOVX-B11 deoxyribonucleic acid (DNA)/modified vaccinia Ankara (MVA) human immunodeficiency virus vaccine. Methods. The tested vaccine used a DNA immunogen mutated to mimic the human vaccine and a regimen with DNA deliveries at weeks 0 and 8 and MVA deliveries at weeks 16 and 32. Twelve weekly rectal challenges with 0.3 animal infectious doses of SIV sootey mangabey E660 (SIVsmE660) were administered starting at 6 months after the last immunization. Results. Over the first 6 rectal exposures to SIVsmE660, <10-year-old tripartite motif-containing protein 5 (TRIM5)α-permissive rhesus macaques showed an 80% reduction in per-exposure risk of infection as opposed to a 46% reduction in animals over 10 years old; and, over the 12 challenges, they showed a 72% as opposed to a 10% reduction. Analyses of elicited immune responses suggested that higher antibody responses in the younger animals had played a role in protection. Conclusions. The simian analogue of the GOVX-B11 HIV provided strong protection against repeated rectal challenges in young adult macaques. PMID:27006959

  6. Strong, but Age-Dependent, Protection Elicited by a Deoxyribonucleic Acid/Modified Vaccinia Ankara Simian Immunodeficiency Virus Vaccine.

    PubMed

    Chamcha, Venkateswarlu; Kannanganat, Sunil; Gangadhara, Sailaja; Nabi, Rafiq; Kozlowski, Pamela A; Montefiori, David C; LaBranche, Celia C; Wrammert, Jens; Keele, Brandon F; Balachandran, Harikrishnan; Sahu, Sujata; Lifton, Michelle; Santra, Sampa; Basu, Rahul; Moss, Bernard; Robinson, Harriet L; Amara, Rama Rao

    2016-01-01

    Background.  In this study, we analyzed the protective efficacy of a simian immunodeficiency virus (SIV) macaque 239 (SIVmac239) analogue of the clinically tested GOVX-B11 deoxyribonucleic acid (DNA)/modified vaccinia Ankara (MVA) human immunodeficiency virus vaccine. Methods.  The tested vaccine used a DNA immunogen mutated to mimic the human vaccine and a regimen with DNA deliveries at weeks 0 and 8 and MVA deliveries at weeks 16 and 32. Twelve weekly rectal challenges with 0.3 animal infectious doses of SIV sootey mangabey E660 (SIVsmE660) were administered starting at 6 months after the last immunization. Results.  Over the first 6 rectal exposures to SIVsmE660, <10-year-old tripartite motif-containing protein 5 (TRIM5)α-permissive rhesus macaques showed an 80% reduction in per-exposure risk of infection as opposed to a 46% reduction in animals over 10 years old; and, over the 12 challenges, they showed a 72% as opposed to a 10% reduction. Analyses of elicited immune responses suggested that higher antibody responses in the younger animals had played a role in protection. Conclusions.  The simian analogue of the GOVX-B11 HIV provided strong protection against repeated rectal challenges in young adult macaques. PMID:27006959

  7. Myristoylation increases the CD8+T-cell response to a GFP prototype antigen delivered by modified vaccinia virus Ankara.

    PubMed

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

    2016-04-01

    Activation of CD8(+)T-cells is an essential part of immune responses elicited by recombinant modified vaccinia virus Ankara (MVA). Strategies to enhance T-cell responses to antigens may be particularly necessary for broadly protective immunization against influenza A virus infections or for candidate vaccines targeting chronic infections and cancer. Here, we tested recombinant MVAs that targeted a model antigen, GFP, to different localizations in infected cells. In vitro characterization demonstrated that GFP accumulated in the nucleus (MVA-nls-GFP), associated with cellular membranes (MVA-myr-GFP) or was equally distributed throughout the cell (MVA-GFP). On vaccination, we found significantly higher levels of GFP-specific CD8(+)T-cells in MVA-myr-GFP-vaccinated BALB/c mice than in those immunized with MVA-GFP or MVA-nls-GFP. Thus, myristoyl modification may be a useful strategy to enhance CD8(+)T-cell responses to MVA-delivered target antigens. PMID:26864442

  8. Redirected cellular cytotoxicity by infection of effector cells with a recombinant vaccinia virus encoding a tumor-specific monoclonal antibody.

    PubMed

    Paul, S; Bizouarne, N; Dott, K; Ruet, L; Dufour, P; Acres, R B; Kieny, M P

    2000-04-01

    Cytotoxicity is an important function of the immune system that results in the destruction of cellular targets by humoral and/or cellular mechanisms. We wanted to assess the possibility of targeting the lytic function of immune cells toward cancer cells, which express the gene coding for a known tumor antigen (Ag) (GA733-2/epithelial cell adhesion molecule), using a viral vector encoding a monoclonal antibody (mAb) specific for said tumor Ag (CO17-1A). To this end, we have constructed recombinant vaccinia viruses expressing the sequences corresponding to mAb CO17-1A, which recognizes a specific Ag (GA733-2) that is present on the surface of most gastrointestinal carcinomas. The recombinant vectors encoding either a secreted or membrane-anchored form of CO17-1A mAb were used to infect effector cells, which were subsequently assessed for their cytotoxic activity. The recombinant viruses were able to infect both granulocyte-macrophage colony-stimulating factor-activated human macrophages and Ag-stimulated murine cytotoxic T lymphocytes. Infected granulocyte-macrophage colony-stimulating factor-activated macrophages were found to be able to kill GA733-2-expressing tumor cells. Likewise, infected cytotoxic T lymphocytes, although conserving their original alloreactivity, gained the capability of killing GA733-2-expressing cancer cells. PMID:10811480

  9. Oncolytic virotherapy for urological cancers.

    PubMed

    Delwar, Zahid; Zhang, Kaixin; Rennie, Paul S; Jia, William

    2016-06-01

    Oncolytic virotherapy is a cancer treatment in which replication-competent viruses are used that specifically infect, replicate in and lyse malignant tumour cells, while minimizing harm to normal cells. Anecdotal evidence of the effectiveness of this strategy has existed since the late nineteenth century, but advances and innovations in biotechnological methods in the 1980s and 1990s led to a renewed interest in this type of therapy. Multiple clinical trials investigating the use of agents constructed from a wide range of viruses have since been performed, and several of these enrolled patients with urological malignancies. Data from these clinical trials and from preclinical studies revealed a number of challenges to the effectiveness of oncolytic virotherapy that have prompted the development of further sophisticated strategies. Urological cancers have a range of distinctive features, such as specific genetic mutations and cell surface markers, which enable improving both effectiveness and safety of oncolytic virus treatments. The strategies employed in creating advanced oncolytic agents include alteration of the virus tropism, regulating transcription and translation of viral genes, combination with chemotherapy, radiotherapy or gene therapy, arming viruses with factors that stimulate the immune response against tumour cells and delivery technologies to ensure that the viral agent reaches its target tissue. PMID:27215429

  10. Vaccination of horses with a recombinant modified vaccinia Ankara virus (MVA) expressing African horse sickness (AHS) virus major capsid protein VP2 provides complete clinical protection against challenge

    PubMed Central

    Alberca, Berta; Bachanek-Bankowska, Katarzyna; Cabana, Marta; Calvo-Pinilla, Eva; Viaplana, Elisenda; Frost, Lorraine; Gubbins, Simon; Urniza, Alicia; Mertens, Peter; Castillo-Olivares, Javier

    2014-01-01

    African horse sickness virus (AHSV) is an arthropod-borne pathogen that infects all species of equidae and causes high mortality in horses. Previously, a recombinant modified vaccinia Ankara (MVA) virus expressing the protein VP2 of AHSV serotype 4 was shown to induce virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (IFNAR −/−) against virulent AHSV challenge. This study builds on the previous work, examining the protective efficacy of MVA-VP2 vaccination in the natural host of AHSV infection. A study group of 4 horses was vaccinated twice with a recombinant MVA virus expressing the major capsid protein (VP2) of AHSV serotype 9. Vaccinated animals and a control group of unvaccinated horses were then challenged with a virulent strain of AHSV-9. The vaccinated animals were completely protected against clinical disease and also against viraemia as measured by standard end-point dilution assays. In contrast, all control horses presented viraemia after challenge and succumbed to the infection. These results demonstrate the potential of recombinant MVA viruses expressing the outer capsid VP2 of AHSV as a protective vaccine against AHSV infection in the field. PMID:24837765

  11. Armed Oncolytic Virus Enhances Immune Functions of Chimeric Antigen Receptor-Modified T Cells in Solid Tumors

    PubMed Central

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

    2014-01-01

    The clinical efficacy of chimeric antigen receptor (CAR)-redirected T cells remains marginal in solid tumors compared to 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 (OV) armed with the chemokine RANTES and the cytokine IL-15, reasoning that the modified OV will have both a direct lytic effect on infected malignant cells and facilitate migration and survival of CAR-T cells. Using neuroblastoma (NB) as a tumor model we found that the adenovirus Ad5Δ24 exerted a potent, dose-dependent, cytotoxic effect on tumor cells, while 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, while the intratumoral release of both RANTES and IL-15 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 biological platform of immunotherapy for solid tumors. PMID:25060519

  12. Ultrasound-induced cavitation enhances the delivery and therapeutic efficacy of an oncolytic virus in an in vitro model.

    PubMed

    Bazan-Peregrino, Miriam; Arvanitis, Costas D; Rifai, Bassel; Seymour, Leonard W; Coussios, Constantin-C

    2012-01-30

    We investigated whether ultrasound-induced cavitation at 0.5 MHz could improve the extravasation and distribution of a potent breast cancer-selective oncolytic adenovirus, AdEHE2F-Luc, to tumour regions that are remote from blood vessels. We developed a novel tumour-mimicking model consisting of a gel matrix containing human breast cancer cells traversed by a fluid channel simulating a tumour blood vessel, through which the virus and microbubbles could be made to flow. Ultrasonic pressures were chosen to maximize either broadband emissions, associated with inertial cavitation, or ultraharmonic emissions, associated with stable cavitation, while varying duty cycle to keep the total acoustic energy delivered constant for comparison across exposures. None of the exposure conditions tested affected cell viability in the absence of the adenovirus. When AdEHE2F-Luc was delivered via the vessel, inertial cavitation increased transgene expression in tumour cells by up to 200 times. This increase was not observed in the absence of Coxsackie and Adenovirus Receptor cell expression, discounting sonoporation as the mechanism of action. In the presence of inertial cavitation, AdEHE2F-Luc distribution was greatly improved in the matrix surrounding the vessel, particularly in the direction of the ultrasound beam; this enabled AdEHE2F-Luc to kill up to 80% of cancer cells within the ultrasound focal volume in the gel 24 hours after delivery, compared to 0% in the absence of cavitation. PMID:21982902

  13. A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

    PubMed Central

    Vijayan, Aneesh; García-Arriaza, Juan; C. Raman, Suresh; Conesa, José Javier; Chichón, Francisco Javier; Santiago, César; Sorzano, Carlos Óscar S.; Carrascosa, José L.; Esteban, Mariano

    2015-01-01

    In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive

  14. Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors.

    PubMed

    Mulryan, Kate; Ryan, Matthew G; Myers, Kevin A; Shaw, David; Wang, Who; Kingsman, Susan M; Stern, Peter L; Carroll, Miles W

    2002-10-01

    The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression. Such properties make 5T4 an excellent putative target for cancer immunotherapy. The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in "self-antigen" in vivo tumor models. We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ). Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively. C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4. MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS. In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumor and to a lesser extent B16.h5T4 s.c. tumors. Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals. Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity. These data support the use of MVA-5T4 for tumor immunotherapy. PMID:12481437

  15. CXCL11-Armed oncolytic poxvirus elicits potent antitumor immunity and shows enhanced therapeutic efficacy

    PubMed Central

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

    2016-01-01

    ABSTRACT We have armed a tumor-selective oncolytic vaccinia virus (vvDD) with the chemokine (CK) CXCL11, in order to enhance its ability to attract CXCR3+ antitumor CTLs and possibly NK cells to the tumor microenvironment (TME) and improve its therapeutic efficacy. As expected, vvDD-CXCL11 attracted high numbers of tumor-specific T cells to the TME in a murine AB12 mesothelioma model. Intratumoral virus-directed CXCL11 expression enhanced local numbers of CD8+ CTLs and levels of granzyme B, while reducing expression of several suppressive molecules, TGF-β, COX2, and CCL22 in the TME. Unexpectedly, we observed that vvDD-CXCL11, but not parental vvDD, induced a systemic increase in tumor-specific IFNγ-producing CD8+ T cells in the spleen and other lymph organs, indicating the induction of systemic antitumor immunity. This effect was associated with enhanced therapeutic efficacy and a survival benefit in tumor-bearing mice treated with vvDD-CXCL11, mediated by CD8+ T cells and IFNγ, but not CD4+ T cells. These results demonstrate that intratumoral expression of CXCL11, in addition to promoting local trafficking of T cells and to a lesser extent NK cells, has a novel function as a factor eliciting systemic immunity to cancer-associated antigens. Our data provide a rationale for expressing CXCL11 to enhance the therapeutic efficacy of oncolytic viruses (OVs) and cancer vaccines. PMID:27141352

  16. The Membrane Fusion Step of Vaccinia Virus Entry Is Cooperatively Mediated by Multiple Viral Proteins and Host Cell Components

    PubMed Central

    Laliberte, Jason P.; Weisberg, Andrea S.; Moss, Bernard

    2011-01-01

    For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry. PMID:22194690

  17. Protective Efficacy of the Conserved NP, PB1, and M1 Proteins as Immunogens in DNA- and Vaccinia Virus-Based Universal Influenza A Virus Vaccines in Mice.

    PubMed

    Wang, Wenling; Li, Renqing; Deng, Yao; Lu, Ning; Chen, Hong; Meng, Xin; Wang, Wen; Wang, Xiuping; Yan, Kexia; Qi, Xiangrong; Zhang, Xiangmin; Xin, Wei; Lu, Zhenhua; Li, Xueren; Bian, Tao; Gao, Yingying; Tan, Wenjie; Ruan, Li

    2015-06-01

    The conventional hemagglutinin (HA)- and neuraminidase (NA)-based influenza vaccines need to be updated most years and are ineffective if the glycoprotein HA of the vaccine strains is a mismatch with that of the epidemic strain. Universal vaccines targeting conserved viral components might provide cross-protection and thus complement and improve conventional vaccines. In this study, we generated DNA plasmids and recombinant vaccinia viruses expressing the conserved proteins nucleoprotein (NP), polymerase basic 1 (PB1), and matrix 1 (M1) from influenza virus strain A/Beijing/30/95 (H3N2). BALB/c mice were immunized intramuscularly with a single vaccine based on NP, PB1, or M1 alone or a combination vaccine based on all three antigens and were then challenged with lethal doses of the heterologous influenza virus strain A/PR/8/34 (H1N1). Vaccines based on NP, PB1, and M1 provided complete or partial protection against challenge with 1.7 50% lethal dose (LD50) of PR8 in mice. Of the three antigens, NP-based vaccines induced protection against 5 LD50 and 10 LD50 and thus exhibited the greatest protective effect. Universal influenza vaccines based on the combination of NP, PB1, and M1 induced a strong immune response and thus might be an alternative approach to addressing future influenza virus pandemics. PMID:25834017

  18. NK Cell-extrinsic IL-18 Signaling Is Required for Efficient NK Cell Activation to Vaccinia Virus

    PubMed Central

    Brandstadter, Joshua D.; Huang, Xiaopei; Yang, Yiping

    2014-01-01

    Summary NK cells are important for the control of vaccinia virus (VV) in vivo. Recent studies have shown that multiple pathways are required for effective activation of NK cells. These include both TLR-dependent and -independent pathways, as well as the NKG2D activating receptor that recognizes host stress-induced NKG2D ligands. However, it remains largely unknown what controls the upregulation of NKG2D ligands in response to VV infection. In this study, we first showed that IL-18 is critical for NK cell activation and viral clearance. We then demonstrated that IL-18 signaling on both NK cells and DCs is required for efficient NK cell activation upon VV infection in vitro. We further showed in vivo that efficient NK cell activation to VV is dependent on DCs and IL-18 signaling in non-NK cells, suggesting an essential role for NK cell-extrinsic IL-18 signaling in NK cell activation. Mechanistically, IL-18 signaling in DCs promotes expression of Rae-1, an NKG2D ligand. Collectively, our data reveal a previously unrecognized role for NK cell-extrinsic IL-18 signaling in NK cell activation through upregulation of NKG2D ligands. These observations may provide insights into the design of effective NK cell-based therapies for viral infections and cancer. PMID:24846540

  19. Interaction between the G3 and L5 proteins of the vaccinia virus entry-fusion complex

    SciTech Connect

    Wolfe, Cindy L.; Moss, Bernard

    2011-04-10

    The vaccinia virus entry-fusion complex (EFC) consists of 10 to 12 proteins that are embedded in the viral membrane and individually required for fusion with the cell and entry of the core into the cytoplasm. The architecture of the EFC is unknown except for information regarding two pair-wise interactions: A28 with H2 and A16 with G9. Here we used a technique to destabilize the EFC by repressing the expression of individual components and identified a third pair-wise interaction: G3 with L5. These two proteins remained associated under several different EFC destabilization conditions and in each case were immunopurified together as demonstrated by Western blotting. Further evidence for the specific interaction of G3 and L5 was obtained by mass spectrometry. This interaction also occurred when G3 and L5 were expressed in uninfected cells, indicating that no other viral proteins were required. Thus, the present study extends our knowledge of the protein interactions important for EFC assembly and stability.

  20. Genome-wide lentiviral shRNA screen identifies serine/arginine-rich splicing factor 2 as a determinant of oncolytic virus activity in breast cancer cells.

    PubMed

    Workenhe, S T; Ketela, T; Moffat, J; Cuddington, B P; Mossman, K L

    2016-05-12

    Oncolytic human herpes simplex virus type 1 (HSV-1) shows promising treatment efficacy in late-stage clinical trials. The anticancer activity of oncolytic viruses relies on deregulated pathways in cancer cells, which make them permissive to oncolysis. To identify pathways that restrict HSV-1 KM100-mediated oncolysis, this study used a pooled genome-wide short hairpin RNA library and found that depletion of the splicing factor arginine-rich splicing factor 2 (SRSF2) leads to enhanced cytotoxicity of breast cancer cells by KM100. Serine/arginine-rich (SR) proteins are a family of RNA-binding phosphoproteins that control both constitutive and alternative pre-mRNA splicing. Further characterization showed that KM100 infection of HS578T cells under conditions of low SRSF2 leads to pronounced apoptosis without a corresponding increase in virus replication. As DNA topoisomerase I inhibitors can limit the phosphorylation of SRSF2, we combined a topoisomerase I inhibitor chemotherapeutic with KM100 and observed synergistic anticancer effect in vitro and prolonged survival of tumor-bearing mice in vivo. PMID:26257065

  1. Induction of Potent Humoral and Cell-Mediated Immune Responses by Attenuated Vaccinia Virus Vectors with Deleted Serpin Genes

    PubMed Central

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

    2004-01-01

    Vaccinia virus (VV) has been effectively utilized as a live vaccine against smallpox as well as a vector for vaccine development and immunotherapy. Increasingly there is a need for a new generation of highly attenuated and efficacious VV vaccines, especially in light of the AIDS pandemic and the threat of global bioterrorism. We therefore developed recombinant VV (rVV) vaccines that are significantly attenuated and yet elicit potent humoral and cell-mediated immune responses. B13R (SPI-2) and B22R (SPI-1) are two VV immunomodulating genes with sequence homology to serine protease inhibitors (serpins) that possess antiapoptotic and anti-inflammatory properties. We constructed and characterized rVVs that have the B13R or B22R gene insertionally inactivated (vΔB13R and vΔB22R) and coexpress the vesicular stomatitis virus glycoprotein (v50ΔB13R and v50ΔB22R). Virulence studies with immunocompromised BALB/cBy nude mice indicated that B13R or B22R gene deletion decreases viral replication and significantly extends time of survival. Viral pathogenesis studies in immunocompetent CB6F1 mice further demonstrated that B13R or B22R gene inactivation diminishes VV virulence, as measured by decreased levels of weight loss and limited viral spread. Finally, rVVs with B13R and B22R deleted elicited potent humoral, T-helper, and cytotoxic T-cell immune responses, revealing that the observed attenuation did not reduce immunogenicity. Therefore, inactivation of immunomodulating genes such as B13R or B22R represents a general method for enhancing the safety of rVV vaccines while maintaining a high level of immunogenicity. Such rVVs could serve as effective vectors for vaccine development and immunotherapy. PMID:14990697

  2. Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence.

    PubMed

    Stack, Julianne; Haga, Ismar R; Schröder, Martina; Bartlett, Nathan W; Maloney, Geraldine; Reading, Patrick C; Fitzgerald, Katherine A; Smith, Geoffrey L; Bowie, Andrew G

    2005-03-21

    Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like-interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain-containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain-containing adaptor inducing IFN-beta (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor kappaB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-beta by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence. PMID:15767367

  3. Immunological Characterization of a Modified Vaccinia Virus Ankara Vector Expressing the Human Papillomavirus 16 E1 Protein

    PubMed Central

    Remy-Ziller, Christelle; Germain, Claire; Spindler, Anita; Hoffmann, Chantal; Silvestre, Nathalie; Rooke, Ronald; Bonnefoy, Jean-Yves

    2014-01-01

    Women showing normal cytology but diagnosed with a persistent high-risk human papillomavirus (HR-HPV) infection have a higher risk of developing high-grade cervical intraepithelial neoplasia and cervical cancer than noninfected women. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in the HPV life cycle, represent very interesting candidates. Both proteins are necessary for maintaining coordinated viral replication and gene synthesis during the differentiation process of the epithelium and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we evaluated a new active targeted immunotherapeutic, a modified vaccinia virus Ankara (MVA) vector containing the E1 sequence of HPV16, aimed at inducing cellular immune responses with the potential to help and clear persistent HPV16-related infection. We carried out an extensive comparative time course analysis of the cellular immune responses induced by different schedules of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16 E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16 E1-specific CD8+ T cells, but they led to the differentiation of multifunctional effector T cells with high cytotoxic capacity. This study provides proof of concept that an MVA expressing HPV16 E1 can induce robust and long-lasting E1-specific responses and warrants further development of this candidate. PMID:24307238

  4. Immunological characterization of a modified vaccinia virus Ankara vector expressing the human papillomavirus 16 E1 protein.

    PubMed

    Remy-Ziller, Christelle; Germain, Claire; Spindler, Anita; Hoffmann, Chantal; Silvestre, Nathalie; Rooke, Ronald; Bonnefoy, Jean-Yves; Préville, Xavier

    2014-02-01

    Women showing normal cytology but diagnosed with a persistent high-risk human papillomavirus (HR-HPV) infection have a higher risk of developing high-grade cervical intraepithelial neoplasia and cervical cancer than noninfected women. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in the HPV life cycle, represent very interesting candidates. Both proteins are necessary for maintaining coordinated viral replication and gene synthesis during the differentiation process of the epithelium and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we evaluated a new active targeted immunotherapeutic, a modified vaccinia virus Ankara (MVA) vector containing the E1 sequence of HPV16, aimed at inducing cellular immune responses with the potential to help and clear persistent HPV16-related infection. We carried out an extensive comparative time course analysis of the cellular immune responses induced by different schedules of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16 E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16 E1-specific CD8⁺ T cells, but they led to the differentiation of multifunctional effector T cells with high cytotoxic capacity. This study provides proof of concept that an MVA expressing HPV16 E1 can induce robust and long-lasting E1-specific responses and warrants further development of this candidate. PMID:24307238

  5. Rabies vaccination: comparison of neutralizing antibody responses after priming and boosting with different combinations of DNA, inactivated virus, or recombinant vaccinia virus vaccines.

    PubMed

    Lodmell, D L; Ewalt, L C

    2000-05-01

    Long-term levels of neutralizing antibody were evaluated in mice after a single immunization with experimental DNA or recombinant vaccinia virus (RVV) vaccines encoding the rabies virus glycoprotein (G), or the commercially available inactivated virus human diploid cell vaccine (HDCV). Anamnestic antibody titers were also evaluated after two booster immunizations with vaccines that were identical to or different from the priming vaccine. Five hundred and forty days (1.5 year) after a single immunization with any of the three vaccines, neutralizing antibody titers remained greater than the minimal acceptable human level of antibody titer (0.5 International Units (IU)/ml). In addition, either an HDCV or DNA booster elicited early and elevated anamnestic antibody responses in mice that had been primed with any of the three vaccines. In contrast, RVV boosters failed to elevate titers in mice that had been previously primed with RVV, and elicited slowly rising titers in mice that had been primed with either DNA or HDCV. Thus, a single vaccination with any of the three different vaccines elicited long-term levels of neutralizing antibody that exceeded 0.5 IU/ml. In contrast, different prime-booster vaccine combinations elicited anamnestic neutralizing antibody responses that increased quickly, increased slowly or failed to increase. PMID:10738096

  6. Envelope formation is blocked by mutation of a sequence related to the HKD phospholipid metabolism motif in the vaccinia virus F13L protein.

    PubMed

    Roper, R L; Moss, B

    1999-02-01

    The outer envelope of the extracellular form of vaccinia virus is derived from Golgi membranes that have been modified by the insertion of specific viral proteins, of which the major component is the 37-kDa, palmitylated, nonglycosylated product of the F13L gene. The F13L protein contains a variant of the HKD (His-Lys-Asp) motif, which is conserved in numerous enzymes of phospholipid metabolism. Vaccinia virus mutants with a conservative substitution of either the K (K314R) or the D (D319E) residue of the F13L protein formed only tiny plaques similar to those produced by an F13L deletion mutant, were unable to produce extracellular enveloped virions, and failed to mediate low-pH-induced fusion of infected cells. Membrane-wrapped forms of intracellular virus were rarely detected in electron microscopic images of cells infected with either of the mutants. Western blotting and pulse-chase experiments demonstrated that the D319E protein was less stable than either the K314R or wild-type F13L protein. Most striking, however, was the failure of either of the two mutated proteins to concentrate in the Golgi compartment. Palmitylation, oleation, and partitioning of the F13L protein in Triton X-114 detergent were unaffected by the K314R substitution. These results indicated that the F13L protein must retain the K314 and D319 for it to localize in the Golgi compartment and function in membrane envelopment of vaccinia virus. PMID:9882312

  7. Potent and Broadly Reactive HIV-2 Neutralizing Antibodies Elicited by a Vaccinia Virus Vector Prime-C2V3C3 Polypeptide Boost Immunization Strategy▿ †

    PubMed Central

    Marcelino, José Maria; Borrego, Pedro; Rocha, Cheila; Barroso, Helena; Quintas, Alexandre; Novo, Carlos; Taveira, Nuno

    2010-01-01

    Human immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism. PMID:20844029

  8. Potent and broadly reactive HIV-2 neutralizing antibodies elicited by a vaccinia virus vector prime-C2V3C3 polypeptide boost immunization strategy.

    PubMed

    Marcelino, José Maria; Borrego, Pedro; Rocha, Cheila; Barroso, Helena; Quintas, Alexandre; Novo, Carlos; Taveira, Nuno

    2010-12-01

    Human immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism. PMID:20844029

  9. The Vaccinia Virus O1 Protein Is Required for Sustained Activation of Extracellular Signal-Regulated Kinase 1/2 and Promotes Viral Virulence

    PubMed Central

    Lukassen, Susanne; Späth, Michaela; Wolferstätter, Michael; Babel, Eveline; Brinkmann, Kay; Wielert, Ursula; Chaplin, Paul; Suter, Mark

    2012-01-01

    Sustained activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway in infected cells has been shown to be crucial for full replication efficiency of orthopoxviruses in cell culture. In infected cells, this pathway is mainly activated by the vaccinia virus growth factor (VGF), an epidermal growth factor (EGF)-like protein. We show here that chorioallantois vaccinia virus Ankara (CVA), but not modified vaccinia virus Ankara (MVA), induced sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in infected human 293 cells, although both viruses direct secretion of functional VGF. A CVA mutant lacking the O1L gene (CVA-ΔO1L) demonstrated that the O1 protein was required for sustained upregulation of the ERK1/2 pathway in 293 cells as well as in other mammalian cell lines. The highly conserved orthopoxvirus O1L gene encodes a predicted 78-kDa protein with a hitherto-unknown function. CVA-ΔO1L showed reduced plaque size and an attenuated cytopathic effect (CPE) in infected cell cultures and reduced virulence and spread from lungs to ovaries in intranasally infected BALB/c mice. Reinsertion of an intact O1L gene into MVA, which in its original form harbors a fragmented O1L open reading frame (ORF), restored ERK1/2 activation in 293 cells but did not increase replication and spread of MVA in human or other mammalian cell lines. Thus, the O1 protein was crucial for sustained ERK1/2 activation in CVA- and MVA-infected human cells, complementing the autocrine function of VGF, and enhanced virulence in vivo. PMID:22171261

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

    PubMed

    Guo, Z S; Parimi, V; O'Malley, M E; Thirunavukarasu, P; Sathaiah, M; Austin, F; Bartlett, D L

    2010-12-01

    Pre-existing antipoxvirus immunity in cancer patients presents a severe barrier to poxvirus-mediated oncolytic virotherapy. We have explored strategies of immunosuppression (IS) and/or immune evasion for efficient delivery of an oncolytic double-deleted vaccinia virus (vvDD) to tumors in the pre-immunized mice. Transient IS using immunosuppressive drugs, including tacrolimus, mycophenolate mofetil and methylprednisolone sodium succinate, have been used successfully in organ transplantation. This drug cocktail alone did not enhance viral recovery from subcutaneous tumor after systemic viral delivery. Using B-cell knockout mice, we confirmed that the neutralizing antibodies had a significant role in preventing poxvirus infection. Using a MC38 peritoneal carcinomatosis model, we found that the combination of IS and tumor cells as carriers led to the most effective viral delivery, viral replication and viral spread inside the tumor mass. We found that our immunosuppressive drug cocktail facilitated recruitment of tumor-associated macrophages and conversion into an immunosuppressive M2 phenotype (interleukin (IL)-10(hi)/IL-12(low)) in the tumor microenvironment. A combination of IS and carrier cells led to significantly prolonged survival in the tumor model. These results showed the feasibility of treating pre-vaccinated patients with peritoneal carcinomatosis using an oncolytic poxvirus and a combined immune intervention strategy. PMID:20703311

  11. Preclinical Evaluation of Oncolytic Δγ134.5 Herpes Simplex Virus Expressing Interleukin-12 for Therapy of Breast Cancer Brain Metastases

    PubMed Central

    Cody, James J.; Scaturro, Pietro; Cantor, Alan B.; Yancey Gillespie, G.; Parker, Jacqueline N.; Markert, James M.

    2012-01-01

    The metastasis of breast cancer to the brain and central nervous system (CNS) is a problem of increasing importance. As improving treatments continue to extend patient survival, the incidence of CNS metastases from breast cancer is on the rise. New treatments are needed, as current treatments are limited by deleterious side effects and are generally palliative. We have previously described an oncolytic herpes simplex virus (HSV), designated M002, which lacks both copies of the γ134.5 neurovirulence gene and carries a murine interleukin 12 (IL-12) expression cassette, and have validated its antitumor efficacy in a variety of preclinical models of primary brain tumors. However, M002 has not been yet evaluated for use against metastatic brain tumors. Here, we demonstrate the following: both human breast cancer and murine mammary carcinoma cells support viral replication and IL-12 expression from M002; M002 replicates in and destroys breast cancer cells from a variety of histological subtypes, including “triple-negative” and HER2 overexpressing; M002 improves survival in an immunocompetent model more effectively than does a non-cytokine control virus. Thus, we conclude from this proof-of-principle study that a γ134.5-deleted IL-12 expressing oncolytic HSV may be a potential new therapy for breast cancer brain metastases. PMID:23346408

  12. The detection of Vaccinia virus confirms the high circulation of Orthopoxvirus in buffaloes living in geographical isolation, Marajó Island, Brazilian Amazon.

    PubMed

    Franco-Luiz, Ana Paula Moreira; Fagundes Pereira, Alexandre; de Oliveira, Cairo Henrique Sousa; Barbosa, José Diomedes; Oliveira, Danilo Bretas; Bonjardim, Cláudio Antônio; Ferreira, Paulo César Peregrino; de Souza Trindade, Giliane; Abrahão, Jônatas Santos; Kroon, Erna Geessien

    2016-06-01

    In Brazil, serologic evidence of Orthopoxvirus (OPV) circulation showed positivity around 20% in cattle, humans, monkeys and rodents. Although OPV seropositivity has been described in buffalo herds in southeastern Brazil, no Vaccinia virus (VACV) (member of genus OPV) outbreaks in buffalo herds have been described in this country. This study aimed to investigate the detection of anti-OPV antibodies and to study the OPV genome in Brazilian buffalo herds. Our results demonstrated a high OPV seropositivity in buffalo herds on Marajó Island and molecular data confirmed the circulation of VACV. The geographical isolation conditionmight be a sine qua non condition to explain our results. PMID:27260805

  13. The immune response to vaccinia virus is significantly reduced after scarification with TK- recombinants as compared to wild-type virus.

    PubMed

    Phillpotts, R J; Lescott, T; Gates, A J; Jones, L

    2000-01-01

    Although it is unlikely that large-scale vaccination against smallpox will ever be required again, it is conceivable that the need may arise to vaccinate against a human orthopoxvirus infection. A possible example could be the emergence of monkey poxvirus (MPV) as a significant human disease in Africa. Vaccinia virus (VV) recombinants, genetically modified to carry the immunogenic proteins of other pathogenic organisms, have potential use as vaccines against other diseases present in this region. The immune response to parental wild-type (wt) or recombinant VV was examined by binding and functional assays, relevant to protection: total IgG, IgG subclass profile, B5R gene product (gp42)-specific IgG, neutralizing antibodies and class 1-mediated cytotoxic lymphocyte activity. There was a substantial reduction in the immune response to VV after scarification with about 10(8) PFU of recombinant as compared to wt virus. These data suggest that to achieve the levels of immunity associated with protection against human orthopoxvirus infection, and to control a possible future outbreak of orthopoxvirus disease, the use of wt VV would be necessary. PMID:11155357

  14. Oncolytic virotherapy for ovarian cancer

    PubMed Central

    Li, Shoudong; Tong, Jessica; Rahman, Masmudur M; Shepherd, Trevor G; McFadden, Grant

    2012-01-01

    In the past two decades, more than 20 viruses with selective tropism for tumor cells have been developed as oncolytic viruses (OVs) for treatments of a variety of malignancies. Of these viruses, eleven have been tested in human ovarian cancer models in preclinical studies. So far, nine phase I or II clinical trials have been conducted or initiated using four different types of OVs in patients with recurrent ovarian cancers. In this article, we summarize the different OVs that are being assessed as therapeutics for ovarian cancer. We also present an overview of recent advances in identification of key genetic or immune-response pathways involved in tumorigenesis of ovarian cancer, which provides a better understanding of the tumor specificities and oncolytic properties of OVs. In addition, we discuss how next-generation OVs could be genetically modified or integrated into multimodality regimens to improve clinical outcomes based on recent advances in ovarian cancer biology. PMID:25977900

  15. Expression of a biotinylated human thyrotropin receptor in HeLa cells using recombinant vaccinia virus and its application for the detection of Graves' autoantibodies.

    PubMed

    Minich, W B; Weymayer, J D; Loos, U

    1998-01-01

    We have prepared a biotinylated thyrotropin receptor (TSHR-BIO), and characterized its activity in cells and when bound to solid phase (streptavidin agarose). TSHR-BIO consists of the N-terminal 725 amino acids of the human thyrotropin (TSH) receptor linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotinylation of the protein. TSHR-BIO was expressed using a vaccinia virus expression system. HeLa cells infected with recombinant virus produced large amounts of TSH receptor of approximately 120,000 molecules per cell. Vaccinia virus produced TSHR-BIO was fully functional interacting with TSH (Kd of 2.3+/-0.1 x 10(-10) M) and coupling to cyclic adenosine monophosphate (cAMP) second messenger system. The expressed protein was biotinylated with high efficiency; more than 90% of TSHR-BIO was bound to streptavidin. We have shown the application of streptavidin agarose immobilized TSHR-BIO for the detection of thyroid-binding inhibiting immunoglobulines in unfractionated sera. There was a good positive correlation between the results obtained in this assay and the commercially available TRAK assay performed with solubilized porcine TSH receptor (r = 0.71; p < 0.001, in 45 sera of patients with Graves' disease and 17 normal sera). PMID:9492146

  16. A novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses.

    PubMed

    Wennier, Sonia T; Brinkmann, Kay; Steinhäußer, Charlotte; Mayländer, Nicole; Mnich, Claudia; Wielert, Ursula; Dirmeier, Ulrike; Hausmann, Jürgen; Chaplin, Paul; Steigerwald, Robin

    2013-01-01

    Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines. PMID:23951355

  17. A Novel Naturally Occurring Tandem Promoter in Modified Vaccinia Virus Ankara Drives Very Early Gene Expression and Potent Immune Responses

    PubMed Central

    Wennier, Sonia T.; Brinkmann, Kay; Steinhäußer, Charlotte; Mayländer, Nicole; Mnich, Claudia; Wielert, Ursula; Dirmeier, Ulrike; Hausmann, Jürgen; Chaplin, Paul; Steigerwald, Robin

    2013-01-01

    Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines. PMID:23951355

  18. Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B sub 1

    SciTech Connect

    Aoyama, Toshifumi; Yamano, Shigeru; Gelboin, H.V.; Gonzalez, F.J. ); Guzelian, P.S. )

    1990-06-01

    Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B{sub 1} to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B{sub 1} to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B{sub 1} to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cells expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, and IIIA5, and IVB1, did not significantly activate aflatoxin B{sub 1} as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B{sub 1} activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B{sub 1} in human liver involves the contribution of multiple forms of P450.

  19. Structural and Biochemical Characterization of the Vaccinia Virus Envelope Protein D8 and Its Recognition by the Antibody LA5