Rhabdoviruses as vaccine platforms for infectious disease and cancer.

PubMed

Zemp, Franz; Rajwani, Jahanara; Mahoney, Douglas J

2018-05-21

The family Rhabdoviridae (RV) comprises a large, genetically diverse collection of single-stranded, negative sense RNA viruses from the order Mononegavirales. Several RV members are being developed as live-attenuated vaccine vectors for the prevention or treatment of infectious disease and cancer. These include the prototype recombinant Vesicular Stomatitis Virus (rVSV) and the more recently developed recombinant Maraba Virus, both species within the genus Vesiculoviridae. A relatively strong safety profile in humans, robust immunogenicity and genetic malleability are key features that make the RV family attractive vaccine platforms. Currently, the rVSV vector is in preclinical development for vaccination against numerous high-priority infectious diseases, with clinical evaluation underway for HIV/AIDS and Ebola virus disease. Indeed, the success of the rVSV-ZEBOV vaccine during the 2014-15 Ebola virus outbreak in West Africa highlights the therapeutic potential of rVSV as a vaccine vector for acute, life-threatening viral illnesses. The rVSV and rMaraba platforms are also being tested as 'oncolytic' cancer vaccines in a series of phase 1-2 clinical trials, after being proven effective at eliciting immune-mediated tumour regression in preclinical mouse models. In this review, we discuss the biological and genetic features that make RVs attractive vaccine platforms and the development and ongoing testing of rVSV and rMaraba strains as vaccine vectors for infectious disease and cancer.

Targeted Entry via Somatostatin Receptors Using a Novel Modified Retrovirus Glycoprotein That Delivers Genes at Levels Comparable to Those of Wild-Type Viral Glycoproteins

PubMed Central

Li, Fang; Ryu, Byoung Y.; Krueger, Robin L.; Heldt, Scott A.

2012-01-01

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 106 transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins. PMID:22013043

High degree of correlation between Ebola virus BSL-4 neutralization assays and pseudotyped VSV BSL-2 fluorescence reduction neutralization test.

PubMed

Konduru, Krishnamurthy; Shurtleff, Amy C; Bavari, Sina; Kaplan, Gerardo

2018-04-01

Ebola virus (EBOV), classified as a category A agent by the CDC and NIH, requires BSL-4 containment and induces high morbidity and mortality in humans. The 2013-2015 epidemic in West Africa underscored the urgent need to develop vaccines and therapeutics to prevent and treat EBOV disease. Neutralization assays are needed to evaluate the efficacy of EBOV vaccines and antibody therapies. Pseudotyped viruses based on nonpathogenic or attenuated vectors reduce the risks involved in the evaluation of neutralizing antibodies against highly pathogenic viruses. Selectable markers, fluorescent proteins, and luciferase have been introduced into pseudotyped viruses for detection and quantitation purposes. The current study describes the development of a BSL-2 fluorescence reduction neutralization test (FRNT) using a recombinant vesicular stomatitis virus (VSV) in which the VSV-G envelope gene was replaced with the EBOV glycoprotein (GP) and green fluorescent protein (GFP) genes (rVSV-EBOVgp-GFP). Cells infected with rVSV-EBOVgp-GFP express GFP. Anti-GP neutralizing monoclonal and polyclonal antibodies blocked rVSV-EBOVgp-GFP infection preventing or reducing GFP fluorescence. The high degree of correlation between the EBOV BSL-2 FRNT and the BSL-4 plaque reduction neutralization test (PRNT), the accepted standard of EBOV neutralization tests, supports the use of the EBOV BSL-2 FRNT to evaluate neutralizing antibodies in clinical trials. Published by Elsevier B.V.

Chikungunya, Influenza, Nipah, and Semliki Forest Chimeric Viruses with Vesicular Stomatitis Virus: Actions in the Brain.

PubMed

van den Pol, Anthony N; Mao, Guochao; Chattopadhyay, Anasuya; Rose, John K; Davis, John N

2017-03-15

Recombinant vesicular stomatitis virus (VSV)-based chimeric viruses that include genes from other viruses show promise as vaccines and oncolytic viruses. However, the critical safety concern is the neurotropic nature conveyed by the VSV glycoprotein. VSVs that include the VSV glycoprotein (G) gene, even in most recombinant attenuated strains, can still show substantial adverse or lethal actions in the brain. Here, we test 4 chimeric viruses in the brain, including those in which glycoprotein genes from Nipah, chikungunya (CHIKV), and influenza H5N1 viruses were substituted for the VSV glycoprotein gene. We also test a virus-like vesicle (VLV) in which the VSV glycoprotein gene is expressed from a replicon encoding the nonstructural proteins of Semliki Forest virus. VSVΔG-CHIKV, VSVΔG-H5N1, and VLV were all safe in the adult mouse brain, as were VSVΔG viruses expressing either the Nipah F or G glycoprotein. In contrast, a complementing pair of VSVΔG viruses expressing Nipah G and F glycoproteins were lethal within the brain within a surprisingly short time frame of 2 days. Intranasal inoculation in postnatal day 14 mice with VSVΔG-CHIKV or VLV evoked no adverse response, whereas VSVΔG-H5N1 by this route was lethal in most mice. A key immune mechanism underlying the safety of VSVΔG-CHIKV, VSVΔG-H5N1, and VLV in the adult brain was the type I interferon response; all three viruses were lethal in the brains of adult mice lacking the interferon receptor, suggesting that the viruses can infect and replicate and spread in brain cells if not blocked by interferon-stimulated genes within the brain. IMPORTANCE Vesicular stomatitis virus (VSV) shows considerable promise both as a vaccine vector and as an oncolytic virus. The greatest limitation of VSV is that it is highly neurotropic and can be lethal within the brain. The neurotropism can be mostly attributed to the VSV G glycoprotein. Here, we test 4 chimeric viruses of VSV with glycoprotein genes from Nipah, chikungunya, and influenza viruses and nonstructural genes from Semliki Forest virus. Two of the four, VSVΔG-CHIKV and VLV, show substantially attenuated neurotropism and were safe in the healthy adult mouse brain. VSVΔG-H5N1 was safe in the adult brain but lethal in the younger brain. VSVΔG Nipah F+G was even more neurotropic than wild-type VSV, evoking a rapid lethal response in the adult brain. These results suggest that while chimeric VSVs show promise, each must be tested with both intranasal and intracranial administration to ensure the absence of lethal neurotropism. Copyright © 2017 American Society for Microbiology.

Chikungunya, Influenza, Nipah, and Semliki Forest Chimeric Viruses with Vesicular Stomatitis Virus: Actions in the Brain

PubMed Central

Mao, Guochao; Chattopadhyay, Anasuya; Rose, John K.; Davis, John N.

2017-01-01

ABSTRACT Recombinant vesicular stomatitis virus (VSV)-based chimeric viruses that include genes from other viruses show promise as vaccines and oncolytic viruses. However, the critical safety concern is the neurotropic nature conveyed by the VSV glycoprotein. VSVs that include the VSV glycoprotein (G) gene, even in most recombinant attenuated strains, can still show substantial adverse or lethal actions in the brain. Here, we test 4 chimeric viruses in the brain, including those in which glycoprotein genes from Nipah, chikungunya (CHIKV), and influenza H5N1 viruses were substituted for the VSV glycoprotein gene. We also test a virus-like vesicle (VLV) in which the VSV glycoprotein gene is expressed from a replicon encoding the nonstructural proteins of Semliki Forest virus. VSVΔG-CHIKV, VSVΔG-H5N1, and VLV were all safe in the adult mouse brain, as were VSVΔG viruses expressing either the Nipah F or G glycoprotein. In contrast, a complementing pair of VSVΔG viruses expressing Nipah G and F glycoproteins were lethal within the brain within a surprisingly short time frame of 2 days. Intranasal inoculation in postnatal day 14 mice with VSVΔG-CHIKV or VLV evoked no adverse response, whereas VSVΔG-H5N1 by this route was lethal in most mice. A key immune mechanism underlying the safety of VSVΔG-CHIKV, VSVΔG-H5N1, and VLV in the adult brain was the type I interferon response; all three viruses were lethal in the brains of adult mice lacking the interferon receptor, suggesting that the viruses can infect and replicate and spread in brain cells if not blocked by interferon-stimulated genes within the brain. IMPORTANCE Vesicular stomatitis virus (VSV) shows considerable promise both as a vaccine vector and as an oncolytic virus. The greatest limitation of VSV is that it is highly neurotropic and can be lethal within the brain. The neurotropism can be mostly attributed to the VSV G glycoprotein. Here, we test 4 chimeric viruses of VSV with glycoprotein genes from Nipah, chikungunya, and influenza viruses and nonstructural genes from Semliki Forest virus. Two of the four, VSVΔG-CHIKV and VLV, show substantially attenuated neurotropism and were safe in the healthy adult mouse brain. VSVΔG-H5N1 was safe in the adult brain but lethal in the younger brain. VSVΔG Nipah F+G was even more neurotropic than wild-type VSV, evoking a rapid lethal response in the adult brain. These results suggest that while chimeric VSVs show promise, each must be tested with both intranasal and intracranial administration to ensure the absence of lethal neurotropism. PMID:28077641

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.

A recombinant virus vaccine that protects against both Chikungunya and Zika virus infections.

PubMed

Chattopadhyay, Anasuya; Aguilar, Patricia V; Bopp, Nathen E; Yarovinsky, Timur O; Weaver, Scott C; Rose, John K

2018-06-22

Chikungunya virus (CHIKV) and Zika virus (ZIKV) have recently expanded their range in the world and caused serious and widespread outbreaks of near pandemic proportions. There are no licensed vaccines that protect against these co-circulating viruses that are transmitted by invasive mosquito vectors. We report here on the development of a single-dose, bivalent experimental vaccine for CHIKV and ZIKV. This vaccine is based on a chimeric vesicular stomatitis virus (VSV) that expresses the CHIKV envelope polyprotein (E3-E2-6K-E1) in place of the VSV glycoprotein (G) and also expresses the membrane-envelope (ME) glycoproteins of ZIKV. This vaccine induced neutralizing antibody responses to both CHIKV and ZIKV in wild-type mice and in interferon receptor-deficient A129 mice, animal models for CHIKV and ZIKV infection. A single vaccination of A129 mice with the vector protected these mice against infection with both CHIKV and ZIKV. Our single-dose vaccine could provide durable, low-cost protection against both CHIKV and ZIKV for people traveling to or living in areas where both viruses are circulating, which include most tropical regions in the world. Copyright © 2018. Published by Elsevier Ltd.

The small non-coding RNA response to virus infection in the Leishmania vector Lutzomyia longipalpis.

PubMed

Ferreira, Flávia Viana; Aguiar, Eric Roberto Guimarães Rocha; Olmo, Roenick Proveti; de Oliveira, Karla Pollyanna Vieira; Silva, Emanuele Guimarães; Sant'Anna, Maurício Roberto Viana; Gontijo, Nelder de Figueiredo; Kroon, Erna Geessien; Imler, Jean Luc; Marques, João Trindade

2018-06-01

Sandflies are well known vectors for Leishmania but also transmit a number of arthropod-borne viruses (arboviruses). Few studies have addressed the interaction between sandflies and arboviruses. RNA interference (RNAi) mechanisms utilize small non-coding RNAs to regulate different aspects of host-pathogen interactions. The small interfering RNA (siRNA) pathway is a broad antiviral mechanism in insects. In addition, at least in mosquitoes, another RNAi mechanism mediated by PIWI interacting RNAs (piRNAs) is activated by viral infection. Finally, endogenous microRNAs (miRNA) may also regulate host immune responses. Here, we analyzed the small non-coding RNA response to Vesicular stomatitis virus (VSV) infection in the sandfly Lutzoymia longipalpis. We detected abundant production of virus-derived siRNAs after VSV infection in adult sandflies. However, there was no production of virus-derived piRNAs and only mild changes in the expression of vector miRNAs in response to infection. We also observed abundant production of virus-derived siRNAs against two other viruses in Lutzomyia Lulo cells. Together, our results suggest that the siRNA but not the piRNA pathway mediates an antiviral response in sandflies. In agreement with this hypothesis, pre-treatment of cells with dsRNA against VSV was able to inhibit viral replication while knock-down of the central siRNA component, Argonaute-2, led to increased virus levels. Our work begins to elucidate the role of RNAi mechanisms in the interaction between L. longipalpis and viruses and should also open the way for studies with other sandfly-borne pathogens.

Infection of Melanoplus sanguinipes Grasshoppers following Ingestion of Rangeland Plant Species Harboring Vesicular Stomatitis Virus▿

PubMed Central

Drolet, Barbara S.; Stuart, Melissa A.; Derner, Justin D.

2009-01-01

Knowledge of the many mechanisms of vesicular stomatitis virus (VSV) transmission is critical for understanding of the epidemiology of sporadic disease outbreaks in the western United States. Migratory grasshoppers [Melanoplus sanguinipes (Fabricius)] have been implicated as reservoirs and mechanical vectors of VSV. The grasshopper-cattle-grasshopper transmission cycle is based on the assumptions that (i) virus shed from clinically infected animals would contaminate pasture plants and remain infectious on plant surfaces and (ii) grasshoppers would become infected by eating the virus-contaminated plants. Our objectives were to determine the stability of VSV on common plant species of U.S. Northern Plains rangelands and to assess the potential of these plant species as a source of virus for grasshoppers. Fourteen plant species were exposed to VSV and assayed for infectious virus over time (0 to 24 h). The frequency of viable virus recovery at 24 h postexposure was as high as 73%. The two most common plant species in Northern Plains rangelands (western wheatgrass [Pascopyrum smithii] and needle and thread [Hesperostipa comata]) were fed to groups of grasshoppers. At 3 weeks postfeeding, the grasshopper infection rate was 44 to 50%. Exposure of VSV to a commonly used grasshopper pesticide resulted in complete viral inactivation. This is the first report demonstrating the stability of VSV on rangeland plant surfaces, and it suggests that a significant window of opportunity exists for grasshoppers to ingest VSV from contaminated plants. The use of grasshopper pesticides on pastures would decrease the incidence of a virus-amplifying mechanical vector and might also decontaminate pastures, thereby decreasing the inter- and intraherd spread of VSV. PMID:19286779

Leucine-rich repeat-containing G protein-coupled receptor 4 facilitates vesicular stomatitis virus infection by binding vesicular stomatitis virus glycoprotein.

PubMed

Zhang, Na; Huang, Hongjun; Tan, Binghe; Wei, Yinglei; Xiong, Qingqing; Yan, Yan; Hou, Lili; Wu, Nannan; Siwko, Stefan; Cimarelli, Andrea; Xu, Jianrong; Han, Honghui; Qian, Min; Liu, Mingyao; Du, Bing

2017-10-06

Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

PubMed Central

Felt, Sébastien A.; Droby, Gaith N.

2017-01-01

ABSTRACT Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma cell (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of resistance are still unclear. JAK1/2 inhibitors (such as ruxolitinib and JAK inhibitor I) strongly stimulate VSV replication and oncolysis in all resistant cell lines but only partially improve the susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication rather than on receptor specificity, with several ubiquitously expressed cell surface molecules playing a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDAC cells. Our data show a dramatically weaker attachment of VSV to HPAF-II cells, the most resistant human PDAC cell line. Although sequence analysis of low-density lipoprotein (LDL) receptor (LDLR) mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels but did not improve VSV attachment or LDL uptake in HPAF-II cells. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with Polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and Polybrene or DEAE-dextran successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC) cells. Although VSV is effective against most PDAC cells, some are highly resistant to VSV, and the mechanisms are still unclear. Here we examined if VSV attachment to cells was inhibited in resistant PDAC cells. Our data show very inefficient attachment of VSV to the most resistant human PDAC cell line, HPAF-II. However, VSV attachment to HPAF-II cells was dramatically improved by treating cells with polycations. Moreover, combining VSV with polycations and ruxolitinib (which inhibits antiviral signaling) successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. We envision that this novel triple-combination approach could be used in the future to treat PDAC tumors that are highly resistant to OV therapy. PMID:28566376

Vesicular stomatitis virus-based vaccines protect nonhuman primates against Bundibugyo ebolavirus.

PubMed

Mire, Chad E; Geisbert, Joan B; Marzi, Andrea; Agans, Krystle N; Feldmann, Heinz; Geisbert, Thomas W

2013-01-01

Ebola virus (EBOV) causes severe and often fatal hemorrhagic fever in humans and nonhuman primates (NHPs). Currently, there are no licensed vaccines or therapeutics for human use. Recombinant vesicular stomatitis virus (rVSV)-based vaccine vectors, which encode an EBOV glycoprotein in place of the VSV glycoprotein, have shown 100% efficacy against homologous Sudan ebolavirus (SEBOV) or Zaire ebolavirus (ZEBOV) challenge in NHPs. In addition, a single injection of a blend of three rVSV vectors completely protected NHPs against challenge with SEBOV, ZEBOV, the former Côte d'Ivoire ebolavirus, and Marburg virus. However, recent studies suggest that complete protection against the newly discovered Bundibugyo ebolavirus (BEBOV) using several different heterologous filovirus vaccines is more difficult and presents a new challenge. As BEBOV caused nearly 50% mortality in a recent outbreak any filovirus vaccine advanced for human use must be able to protect against this new species. Here, we evaluated several different strategies against BEBOV using rVSV-based vaccines. Groups of cynomolgus macaques were vaccinated with a single injection of a homologous BEBOV vaccine, a single injection of a blended heterologous vaccine (SEBOV/ZEBOV), or a prime-boost using heterologous SEBOV and ZEBOV vectors. Animals were challenged with BEBOV 29-36 days after initial vaccination. Macaques vaccinated with the homologous BEBOV vaccine or the prime-boost showed no overt signs of illness and survived challenge. In contrast, animals vaccinated with the heterologous blended vaccine and unvaccinated control animals developed severe clinical symptoms consistent with BEBOV infection with 2 of 3 animals in each group succumbing. These data show that complete protection against BEBOV will likely require incorporation of BEBOV glycoprotein into the vaccine or employment of a prime-boost regimen. Fortunately, our results demonstrate that heterologous rVSV-based filovirus vaccine vectors employed in the prime-boost approach can provide protection against BEBOV using an abbreviated regimen, which may have utility in outbreak settings.

Efficient transduction of equine adipose-derived mesenchymal stem cells by VSV-G pseudotyped lentiviral vectors.

PubMed

Petersen, Gayle F; Hilbert, Bryan; Trope, Gareth; Kalle, Wouter; Strappe, Padraig

2014-12-01

Equine adipose-derived mesenchymal stem cells (EADMSC) provide a unique cell-based approach for treatment of a variety of equine musculoskeletal injuries, via regeneration of diseased or damaged tissue, or the secretion of immunomodulatory molecules. These capabilities can be further enhanced by genetic modification using lentiviral vectors, which provide a safe and efficient method of gene delivery. We investigated the suitability of lentiviral vector technology for gene delivery into EADMSC, using GFP expressing lentiviral vectors pseudotyped with the G glycoprotein from the vesicular stomatitis virus (V-GFP) or, for the first time, the baculovirus gp64 envelope protein (G-GFP). In this study, we produced similarly high titre V-GFP and G-GFP lentiviral vectors. Flow cytometric analysis showed efficient transduction using V-GFP; however G-GFP exhibited a poor ability to transduce EADMSC. Transduction resulted in sustained GFP expression over four passages, with minimal effects on cell viability and doubling time, and an unaltered chondrogenic differentiation potential. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Development of viral vectors and the application for viral entry mechanisms].

PubMed

Tani, Hideki

2011-06-01

Virus is identified as one of the obligate intracellular parasites, which only amplify in cells of specific living things. Viral vectors, which are developed by utilizing these properties, are available in the various fields such as basic research of medical biology or application of gene therapy. Our research group has studied development of viral vectors using properties of baculovirus or vesicular stomatitis virus (VSV). Due to the development of new baculoviral vectors for mammalian cells, it is possible to be more efficient transduction of foreign gene in mammalian cells and animals. Furthermore, pseudotype or recombinant VSV possessing the envelope proteins of hepatitis C virus, Japanese encephalitis virus or baculovirus were constructed, and characteristics of the envelope proteins or entry mechanisms of these viruses were analyzed.

Cytopathogenesis of Vesicular Stomatitis virus is regulated by the PSAP motif of M protein in a species-dependent manner

USDA-ARS?s Scientific Manuscript database

Vesicular stomatitis virus (VSV) is an important vector-borne pathogen of bovine and equine species, causing a reportable vesicular disease. The matrix (M) protein of VSV is multifunctional and plays a key role in cytopathogenesis, apoptosis, host protein shut-off, and virion assembly/budding. Our ...

The p14 FAST Protein of Reptilian Reovirus Increases Vesicular Stomatitis Virus Neuropathogenesis▿

PubMed Central

Brown, Christopher W.; Stephenson, Kyle B.; Hanson, Stephen; Kucharczyk, Michael; Duncan, Roy; Bell, John C.; Lichty, Brian D.

2009-01-01

The fusogenic orthoreoviruses express nonstructural fusion-associated small transmembrane (FAST) proteins that induce cell-cell fusion and syncytium formation. It has been speculated that the FAST proteins may serve as virulence factors by promoting virus dissemination and increased or altered cytopathology. To directly test this hypothesis, the gene encoding the p14 FAST protein of reptilian reovirus was inserted into the genome of a heterologous virus that does not naturally form syncytia, vesicular stomatitis virus (VSV). Expression of the p14 FAST protein by the VSV/FAST recombinant gave the virus a highly fusogenic phenotype in cell culture. The growth of this recombinant fusogenic VSV strain was unaltered in vitro but was significantly enhanced in vivo. The VSV/FAST recombinant consistently generated higher titers of virus in the brains of BALB/c mice after intranasal or intravenous infection compared to the parental VSV/green fluorescent protein (GFP) strain that expresses GFP in place of p14. The VSV/FAST recombinant also resulted in an increased incidence of hind-limb paralysis, it infected a larger volume of brain tissue, and it induced more extensive neuropathology, thus leading to a lower maximum tolerable dose than that for the VSV/GFP parental virus. In contrast, an interferon-inducing mutant of VSV expressing p14 was still attenuated, indicating that this interferon-inducing phenotype is dominant to the fusogenic properties conveyed by the FAST protein. Based on this evidence, we conclude that the reovirus p14 FAST protein can function as a bona fide virulence factor. PMID:18971262

Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

PubMed Central

Kim, Yoon-Sang; Wielgosz, Matthew M; Hargrove, Phillip; Kepes, Steven; Gray, John; Persons, Derek A; Nienhuis, Arthur W

2010-01-01

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34+ peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34+ cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34+ cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45+ cells in total bone marrow were comparable to that of the control, mock-transduced group (37–45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the γ-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the γ-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector. PMID:20372106

Single-dose attenuated Vesiculovax vaccines protect primates against Ebola Makona virus.

PubMed

Mire, Chad E; Matassov, Demetrius; Geisbert, Joan B; Latham, Theresa E; Agans, Krystle N; Xu, Rong; Ota-Setlik, Ayuko; Egan, Michael A; Fenton, Karla A; Clarke, David K; Eldridge, John H; Geisbert, Thomas W

2015-04-30

The family Filoviridae contains three genera, Ebolavirus (EBOV), Marburg virus, and Cuevavirus. Some members of the EBOV genus, including Zaire ebolavirus (ZEBOV), can cause lethal haemorrhagic fever in humans. During 2014 an unprecedented ZEBOV outbreak occurred in West Africa and is still ongoing, resulting in over 10,000 deaths, and causing global concern of uncontrolled disease. To meet this challenge a rapid-acting vaccine is needed. Many vaccine approaches have shown promise in being able to protect nonhuman primates against ZEBOV. In response to the current ZEBOV outbreak several of these vaccines have been fast tracked for human use. However, it is not known whether any of these vaccines can provide protection against the new outbreak Makona strain of ZEBOV. One of these approaches is a first-generation recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing the ZEBOV glycoprotein (GP) (rVSV/ZEBOV). To address safety concerns associated with this vector, we developed two candidate, further-attenuated rVSV/ZEBOV vaccines. Both attenuated vaccines produced an approximately tenfold lower vaccine-associated viraemia compared to the first-generation vaccine and both provided complete, single-dose protection of macaques from lethal challenge with the Makona outbreak strain of ZEBOV.

Baculovirus GP64-mediated entry into mammalian cells.

PubMed

Kataoka, Chikako; Kaname, Yuuki; Taguwa, Shuhei; Abe, Takayuki; Fukuhara, Takasuke; Tani, Hideki; Moriishi, Kohji; Matsuura, Yoshiharu

2012-03-01

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.

Conserved Receptor-Binding Domains of Lake Victoria Marburgvirus and Zaire Ebolavirus Bind a Shared Receptor

DTIC Science & Technology

2006-04-14

virion, because of the functional importance of and limited variation in this region (44, 45). In some cases, such as murine and feline leukemia viruses ...murine leukemia virus ; PBS, phos- phate-buffered saline; RBD, receptor-binding domain; SARS, severe acute respiratory syndrome; VSV, vesicular stomatitis...entryofpseudotypedret- roviruses. A Moloney murine leukemia virus vector expressing GFP was pseudotyped with the GP1,2 of MARV-Mus (MARV/MLV), a mucin-like

Cytopathogenesis of vesicular stomatitis virus is regulated by the PSAP motif of M protein in a species-dependent manner.

PubMed

Irie, Takashi; Liu, Yuliang; Drolet, Barbara S; Carnero, Elena; García-Sastre, Adolfo; Harty, Ronald N

2012-09-01

Vesicular stomatitis virus (VSV) is an important vector-borne pathogen of bovine and equine species, causing a reportable vesicular disease. The matrix (M) protein of VSV is multifunctional and plays a key role in cytopathogenesis, apoptosis, host protein shut-off, and virion assembly/budding. Our previous findings indicated that mutations of residues flanking the (37)PSAP(40) motif within the M protein resulted in VSV recombinants having attenuated phenotypes in mice. In this report, we characterize the phenotype of VSV recombinant PS > A4 (which harbors four alanines (AAAA) in place of the PSAP motif without disruption of flanking residues) in both mice, and in Aedes albopictus C6/36 mosquito and Culicoides sonorensis KC cell lines. The PS > A4 recombinant displayed an attenuated phenotype in infected mice as judged by weight loss, mortality, and viral titers measured from lung and brain samples of infected animals. However, unexpectedly, the PS > A4 recombinant displayed a robust cytopathic phenotype in insect C6/36 cells compared to that observed with control viruses. Notably, titers of recombinant PS > A4 were approximately 10-fold greater than those of control viruses in infected C6/36 cells and in KC cells from Culicoides sonorensis, a known VSV vector species. In addition, recombinant PS > A4 induced a 25-fold increase in the level of C3 caspase activity in infected C6/36 cells. These findings indicate that the PSAP motif plays a direct role in regulating cytopathogenicity in a species-dependent manner, and suggest that the intact PSAP motif may be important for maintaining persistence of VSV in an insect host.

Heterogeneous Nuclear Ribonucleoprotein K Supports Vesicular Stomatitis Virus Replication by Regulating Cell Survival and Cellular Gene Expression

PubMed Central

Dinh, Phat X.; Das, Anshuman; Franco, Rodrigo

2013-01-01

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a member of the family of hnRNPs and was recently shown in a genome-wide small interfering RNA (siRNA) screen to support vesicular stomatitis virus (VSV) growth. To decipher the role of hnRNP K in VSV infection, we conducted studies which suggest that the protein is required for VSV spreading. Virus binding to cells, entry, and nucleocapsid uncoating steps were not adversely affected in the absence of hnRNP K, whereas viral genome transcription and replication were reduced slightly. These results indicate that hnRNP K is likely involved in virus assembly and/or release from infected cells. Further studies showed that hnRNP K suppresses apoptosis of virus-infected cells, resulting in increased cell survival during VSV infection. The increased survival of the infected cells was found to be due to the suppression of proapoptotic proteins such as Bcl-XS and Bik in a cell-type-dependent manner. Additionally, depletion of hnRNP K resulted in not only significantly increased levels of T-cell-restricted intracellular antigen 1 (TIA1) but also switching of the expression of the two isoforms of the protein (TIA1a and TIA1b), both of which inhibited VSV replication. hnRNP K was also found to support expression of several cellular proteins known to be required for VSV infection. Overall, our studies demonstrate hnRNP K to be a multifunctional protein that supports VSV infection via its role(s) in suppressing apoptosis of infected cells, inhibiting the expression of antiviral proteins, and maintaining the expression of proteins required for the virus. PMID:23843646

Carbohydrate-Based Ice Recrystallization Inhibitors Increase Infectivity and Thermostability of Viral Vectors

NASA Astrophysics Data System (ADS)

Ghobadloo, Shahrokh M.; Balcerzak, Anna K.; Gargaun, Ana; Muharemagic, Darija; Mironov, Gleb G.; Capicciotti, Chantelle J.; Briard, Jennie G.; Ben, Robert N.; Berezovski, Maxim V.

2014-07-01

The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL-1 during 40 days, and HSV-1, 2.7 Log10 PFU mL-1 during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL-1 after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors.

Carbohydrate-Based Ice Recrystallization Inhibitors Increase Infectivity and Thermostability of Viral Vectors

PubMed Central

Ghobadloo, Shahrokh M.; Balcerzak, Anna K.; Gargaun, Ana; Muharemagic, Darija; Mironov, Gleb G.; Capicciotti, Chantelle J.; Briard, Jennie G.; Ben, Robert N.; Berezovski, Maxim V.

2014-01-01

The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL−1 during 40 days, and HSV-1, 2.7 Log10 PFU mL−1 during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL−1 after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors. PMID:25078058

A novel, live-attenuated vesicular stomatitis virus vector displaying conformationally intact, functional HIV-1 envelope trimers that elicits potent cellular and humoral responses in mice.

PubMed

Rabinovich, Svetlana; Powell, Rebecca L R; Lindsay, Ross W B; Yuan, Maoli; Carpov, Alexei; Wilson, Aaron; Lopez, Mary; Coleman, John W; Wagner, Denise; Sharma, Palka; Kemelman, Marina; Wright, Kevin J; Seabrook, John P; Arendt, Heather; Martinez, Jennifer; DeStefano, Joanne; Chiuchiolo, Maria J; Parks, Christopher L

2014-01-01

Though vaccination with live-attenuated SIV provides the greatest protection from progressive disease caused by SIV challenge in rhesus macaques, attenuated HIV presents safety concerns as a vaccine; therefore, live viral vectors carrying HIV immunogens must be considered. We have designed a replication-competent vesicular stomatitis virus (VSV) displaying immunogenic HIV-1 Env trimers and attenuating quantities of the native surface glycoprotein (G). The clade B Env immunogen is an Env-VSV G hybrid (EnvG) in which the transmembrane and cytoplasmic tail regions are derived from G. Relocation of the G gene to the 5'terminus of the genome and insertion of EnvG into the natural G position induced a ∼1 log reduction in surface G, significant growth attenuation compared to wild-type, and incorporation of abundant EnvG. Western blot analysis indicated that ∼75% of incorporated EnvG was a mature proteolytically processed form. Flow cytometry showed that surface EnvG bound various conformationally- and trimer-specific antibodies (Abs), and in-vitro growth assays on CD4+CCR5+ cells demonstrated EnvG functionality. Neither intranasal (IN) or intramuscular (IM) administration in mice induced any observable pathology and all regimens tested generated potent Env-specific ELISA titers of 10(4)-10(5), with an IM VSV prime/IN VSV boost regimen eliciting the highest binding and neutralizing Ab titers. Significant quantities of Env-specific CD4+ T cells were also detected, which were augmented as much as 70-fold by priming with IM electroporated plasmids encoding EnvG and IL-12. These data suggest that our novel vector can achieve balanced safety and immunogenicity and should be considered as an HIV vaccine platform.

Baculovirus-mediated vascular endothelial growth factor-D(ΔNΔC) gene transfer induces angiogenesis in rabbit skeletal muscle.

PubMed

Heikura, Tommi; Nieminen, Tiina; Roschier, Miia M; Karvinen, Henna; Kaikkonen, Minna U; Mähönen, Anssi J; Lesch, Hanna P; Rissanen, Tuomas T; Laitinen, Olli H; Airenne, Kari J; Ylä-Herttuala, Seppo

2012-01-01

Occluded arteries and ischemic tissues cannot always be treated by angioplasty, stenting or by-pass-surgery. Under such circumstances, viral gene therapy may be useful in inducing increased blood supply to ischemic area. There is evidence of improved blood flow in ischemic skeletal muscle and myocardium in both animal and human studies using adenoviral vascular endothelial growth factor (VEGF) gene therapy. However, the expression is transient and repeated gene transfers with the same vector are inefficient due to immune responses. Different baculoviral vectors pseudotyped with or without vesicular stomatitis virus glycoprotein (VSV-G) and/or carrying woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) were tested both in vitro and in vivo. VEGF-D(ΔNΔC) was used as therapeutic transgene and lacZ as a control. In vivo efficacy was evaluated as capillary enlargement and transgene expression in New Zealand White (NZW) rabbit skeletal muscle. A statistically significant capillary enlargement was detected 6 days after gene transfer in transduced areas compared to the control gene transfers with baculovirus and adenovirus encoding β-galactosidase (lacZ). Substantially improved gene transfer efficiency was achieved with a modified baculovirus pseudotyped with VSV-G and carrying Wpre. Dose escalation experiments revealed that either too large volume or too many virus particles caused inflammation and necrosis in the target tissue, whereas 10(9) plaque forming units injected in multiple aliquots resulted in transgene expression with only mild immune reactions. We show the first evidence of biologically significant baculoviral gene transfer in skeletal muscle of NZW rabbits using VEGF-D(ΔNΔC) as a therapeutic transgene. Copyright © 2012 John Wiley & Sons, Ltd.

Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection.

PubMed

Liu, Xin; Li, Yun-Pan; Zhong, Zhen-Min; Tan, Hui-Qi; Lin, Hao-Peng; Chen, Shao-Jun; Fu, Yu-Cai; Xu, Wen-Can; Wei, Chi-Ju

2017-02-01

The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents. A luciferase expression construct (pGL3-control) and a DsRed expression cassette (pCMV-DsRed) were used to evaluate the delivery efficiency of DNA/EG/VSV-G complexes. VSV-G proteins could be incorporated into EG in static incubation under acidic conditions as evidenced by the Western blot analysis. Condensed plasmid DNA was bound mostly to the outer surface of EG, which could be detected by electromicroscopy and measured by electrophoresis. EG/VSV-G complexes stimulated the delivery of pGL3-control into Ad293 cells significantly with the luciferase activity increased about 4-fold as compared to that of the control. The delivery of pCMV-DsRed was also enhanced with the percentage of DsRed-positive Ad293 cells increased from 55 % to about 80 %. Moreover, the transfection efficiency in 3T3, HeLa, INS-1, and bone marrow stem cell (BMSC) cells increased about 2-3-fold. Finally, confocal microscopy analysis showed that incorporation of VSV-G significantly enhanced the endocytosis of EG into target cells. In the present study, a novel type of non-viral DNA delivery vehicle consisting of EG and fusogenic VSV-G proteins was formulated, which showed superior transfection efficiency even in cells resistant to classical transfection.

Contributions of hydrology to Vesicular Stomatitis Virus emergence in the Western United States

USDA-ARS?s Scientific Manuscript database

Relationships between environmental variables associated with the spread of vector-borne pathogens, such as RNA viruses transmitted to humans and animals, remain poorly understood. Vesicular stomatitis (VS) is caused by a vector-borne, zoonotic RNA virus (VSV), and is the most common vesicular dise...

Recombinant vesicular stomatitis virus-based vaccines against Ebola and Marburg virus infections.

PubMed

Geisbert, Thomas W; Feldmann, Heinz

2011-11-01

The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with a high mortality rate in humans and nonhuman primates. Among the most-promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Importantly, a single injection of blended rVSV-based filovirus vaccines was shown to completely protect nonhuman primates against Marburg virus and 3 different species of Ebola virus. These rVSV-based vaccines have also shown utility when administered as a postexposure treatment against filovirus infections, and a rVSV-based Ebola virus vaccine was recently used to treat a potential laboratory exposure. Here, we review the history of rVSV-based vaccines and pivotal animal studies showing their utility in combating Ebola and Marburg virus infections.

Recombinant Vesicular Stomatitis Virus–Based Vaccines Against Ebola and Marburg Virus Infections

PubMed Central

Feldmann, Heinz

2011-01-01

The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with a high mortality rate in humans and nonhuman primates. Among the most-promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Importantly, a single injection of blended rVSV-based filovirus vaccines was shown to completely protect nonhuman primates against Marburg virus and 3 different species of Ebola virus. These rVSV-based vaccines have also shown utility when administered as a postexposure treatment against filovirus infections, and a rVSV-based Ebola virus vaccine was recently used to treat a potential laboratory exposure. Here, we review the history of rVSV-based vaccines and pivotal animal studies showing their utility in combating Ebola and Marburg virus infections. PMID:21987744

Induction of stress granule-like structures in vesicular stomatitis virus-infected cells.

PubMed

Dinh, Phat X; Beura, Lalit K; Das, Phani B; Panda, Debasis; Das, Anshuman; Pattnaik, Asit K

2013-01-01

Previous studies from our laboratory revealed that cellular poly(C) binding protein 2 (PCBP2) downregulates vesicular stomatitis virus (VSV) gene expression. We show here that VSV infection induces the formation of granular structures in the cytoplasm containing cellular RNA-binding proteins, including PCBP2, T-cell-restricted intracellular antigen 1 (TIA1), and TIA1-related protein (TIAR). Depletion of TIA1 via small interfering RNAs (siRNAs), but not depletion of TIAR, results in enhanced VSV growth and gene expression. The VSV-induced granules appear to be similar to the stress granules (SGs) generated in cells triggered by heat shock or oxidative stress but do not contain some of the bona fide SG markers, such as eukaryotic initiation factor 3 (eIF3) or eIF4A, or the processing body (PB) markers, such as mRNA-decapping enzyme 1A (DCP1a), and thus may not represent canonical SGs or PBs. Our results revealed that the VSV-induced granules, called SG-like structures here, contain the viral replicative proteins and RNAs. The formation and maintenance of the SG-like structures required viral replication and ongoing protein synthesis, but an intact cytoskeletal network was not necessary. These results suggest that cells respond to VSV infection by aggregating the antiviral proteins, such as PCBP2 and TIA1, to form SG-like structures. The functional significance of these SG-like structures in VSV-infected cells is currently under investigation.

Ecological factors rather than temporal factors dominate the evolution of vesicular stomatitis virus.

PubMed

Rodríguez, L L; Fitch, W M; Nichol, S T

1996-11-12

Vesicular stomatitis New Jersey virus (VSV-NJ) is a rhabdovirus that causes economically important disease in cattle and other domestic animals in endemic areas from southeastern United States to northern South America. Its negatively stranded RNA genome is capable of undergoing rapid evolution, which allows phylogenetic analysis and molecular epidemiology studies to be performed. Previous epidemiological studies in Costa Rica showed the existence of at least two distinct ecological zones of high VSV-NJ activity, one located in the highlands (premontane tropical moist forest) and the other in the lowlands (tropical dry forest). We wanted to test the hypothesis that the viruses circulating in these ecological zones were genetically distinct. For this purpose, we sequenced the hypervariable region of the phosphoprotein gene for 50 VSV-NJ isolates from these areas. Phylogenetic analysis showed that viruses from each ecological zone had distinct genotypes. These genotypes were maintained in each area for periods of up to 8 years. This evolutionary pattern of VSV-NJ suggests an adaptation to ecological factors that could exert selective pressure on the virus. As previous data indicated an absence of virus adaptation to factors related to the bovine host (including immunological pressure), it appears that VSV genetic divergence represents positive selection to adapt to specific vectors and/or reservoirs at each ecological zone.

An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity.

PubMed

Morin, Benjamin; Liang, Bo; Gardner, Erica; Ross, Robin A; Whelan, Sean P J

2017-01-01

We report an in vitro RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression. The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L protein is lacking. This study reports the expression and purification of the full-length L protein of RABV and the characterization of its RdRP activity in vitro The study provides a new assay that has utility for screening inhibitors and understanding their mechanisms of action, as well as defining new interactions that are required for RdRP activity. Copyright © 2016 American Society for Microbiology.

Lentiviral transduction of rat Sertoli cells as a means to modify gene expression

PubMed Central

Nicholls, Peter K.; Stanton, Peter G.; Rainczuk, Katarzyna E.; Qian, Hongwei; Gregorevic, Paul; Harrison, Craig A.

2012-01-01

Primary cell culture is an established and widely used technique to study Sertoli cell function in vitro. However, the relative difficulty of stably overexpressing or knocking down genes in Sertoli cell culture has limited progress in the field. In this technical report, we present a method to transduce 20 dpp rat Sertoli cell cultures with VSV-G pseudotyped lentiviral based vectors at a high rate (~80%), with stable reporter gene expression. Although high transgene expression is desirable, it was noted that at transduction rates > 60% inter-Sertoli cell tight junction integrity and, hence, Sertoli cell function, were transiently compromised. We envisage that this optimized procedure has the potential to stimulate Sertoli cell research, and motivate the use of Sertoli cells in various cell therapy applications. PMID:23248769

Single-Injection Vaccine Protects Nonhuman Primates against Infection with Marburg Virus and Three Species of Ebola Virus▿

PubMed Central

Geisbert, Thomas W.; Geisbert, Joan B.; Leung, Anders; Daddario-DiCaprio, Kathleen M.; Hensley, Lisa E.; Grolla, Allen; Feldmann, Heinz

2009-01-01

The filoviruses Marburg virus and Ebola virus cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (VSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Here, we performed a proof-of-concept study in order to determine the potential of having one single-injection vaccine capable of protecting nonhuman primates against Sudan ebolavirus (SEBOV), Zaire ebolavirus (ZEBOV), Cote d'Ivoire ebolavirus (CIEBOV), and Marburgvirus (MARV). In this study, 11 cynomolgus monkeys were vaccinated with a blended vaccine consisting of equal parts of the vaccine vectors VSVΔG/SEBOVGP, VSVΔG/ZEBOVGP, and VSVΔG/MARVGP. Four weeks later, three of these animals were challenged with MARV, three with CIEBOV, three with ZEBOV, and two with SEBOV. Three control animals were vaccinated with VSV vectors encoding a nonfilovirus GP and challenged with SEBOV, ZEBOV, and MARV, respectively, and five unvaccinated control animals were challenged with CIEBOV. Importantly, none of the macaques vaccinated with the blended vaccine succumbed to a filovirus challenge. As expected, an experimental control animal vaccinated with VSVΔG/ZEBOVGP and challenged with SEBOV succumbed, as did the positive controls challenged with SEBOV, ZEBOV, and MARV, respectively. All five control animals challenged with CIEBOV became severely ill, and three of the animals succumbed on days 12, 12, and 14, respectively. The two animals that survived CIEBOV infection were protected from subsequent challenge with either SEBOV or ZEBOV, suggesting that immunity to CIEBOV may be protective against other species of Ebola virus. In conclusion, we developed an immunization scheme based on a single-injection vaccine that protects nonhuman primates against lethal challenge with representative strains of all human pathogenic filovirus species. PMID:19386702

Single-injection vaccine protects nonhuman primates against infection with marburg virus and three species of ebola virus.

PubMed

Geisbert, Thomas W; Geisbert, Joan B; Leung, Anders; Daddario-DiCaprio, Kathleen M; Hensley, Lisa E; Grolla, Allen; Feldmann, Heinz

2009-07-01

The filoviruses Marburg virus and Ebola virus cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (VSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Here, we performed a proof-of-concept study in order to determine the potential of having one single-injection vaccine capable of protecting nonhuman primates against Sudan ebolavirus (SEBOV), Zaire ebolavirus (ZEBOV), Cote d'Ivoire ebolavirus (CIEBOV), and Marburgvirus (MARV). In this study, 11 cynomolgus monkeys were vaccinated with a blended vaccine consisting of equal parts of the vaccine vectors VSVDeltaG/SEBOVGP, VSVDeltaG/ZEBOVGP, and VSVDeltaG/MARVGP. Four weeks later, three of these animals were challenged with MARV, three with CIEBOV, three with ZEBOV, and two with SEBOV. Three control animals were vaccinated with VSV vectors encoding a nonfilovirus GP and challenged with SEBOV, ZEBOV, and MARV, respectively, and five unvaccinated control animals were challenged with CIEBOV. Importantly, none of the macaques vaccinated with the blended vaccine succumbed to a filovirus challenge. As expected, an experimental control animal vaccinated with VSVDeltaG/ZEBOVGP and challenged with SEBOV succumbed, as did the positive controls challenged with SEBOV, ZEBOV, and MARV, respectively. All five control animals challenged with CIEBOV became severely ill, and three of the animals succumbed on days 12, 12, and 14, respectively. The two animals that survived CIEBOV infection were protected from subsequent challenge with either SEBOV or ZEBOV, suggesting that immunity to CIEBOV may be protective against other species of Ebola virus. In conclusion, we developed an immunization scheme based on a single-injection vaccine that protects nonhuman primates against lethal challenge with representative strains of all human pathogenic filovirus species.

Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells

PubMed Central

Shimizu, Akira; Nakatani, Yoko; Nakamura, Takako; Jinno-Oue, Atsushi; Ishikawa, Osamu; Boeke, Jef D.; Takeuchi, Yasuhiro; Hoshino, Hiroo

2014-01-01

The synthesis and subsequent genomic integration of DNA that is complementary to the genomes of non-retroviral RNA viruses are rarely observed. However, upon infection of various human cell lines and primary fibroblasts with the vesicular stomatitis virus (VSV), we detected DNA complementary to the VSV RNA. The VSV DNA was detected in the cytoplasm as single-stranded DNA fully complementary to the viral mRNA from the poly(A) region to the 7-methyl guanosine cap. The formation of this DNA was cell-dependent. Experimentally, we found that the transduction of cells that do not produce VSV DNA with the long interspersed nuclear element 1 and their infection with VSV could lead to the formation of VSV DNA. Viral DNA complementary to other RNA viruses was also detected in the respective infected human cells. Thus, the genetic information of the non-retroviral RNA virus genome can flow into the DNA of mammalian cells expressing LINE-1-like elements. PMID:24875540

Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice

PubMed Central

Konduru, Krishnamurthy; Bradfute, Steven B.; Jacques, Jerome; Manangeeswaran, Mohanraj; Nakamura, Siham; Morshed, Sufi; Wood, Steven C.; Bavari, Sina

2011-01-01

Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as "Category A bioterrorism agents", and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGP-Fc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use. PMID:21329775

A plasma membrane localization signal in the HIV-1 envelope cytoplasmic domain prevents localization at sites of vesicular stomatitis virus budding and incorporation into VSV virions.

PubMed

Johnson, J E; Rodgers, W; Rose, J K

1998-11-25

Previous studies showed that the HIV-1 envelope (Env) protein was not incorporated into vesicular stomatitis virus (VSV) virions unless its cytoplasmic tail was replaced with that of the VSV glycoprotein (G). To determine whether the G tail provided a positive incorporation signal for Env, or if sequences in the Env tail prevented incorporation, we generated mutants of Env with its 150-amino-acid tail shortened to 29, 10, or 3 amino acids (Envtr mutants). Cells infected with VSV recombinants expressing these proteins or an Env-G tail hybrid showed similar amounts of Env protein at the surface. The Env-G tail hybrid or the Envtr3 mutant were incorporated at the highest levels into budding VSV virions. In contrast, the Envtr29 or Envtr10 mutants were incorporated poorly. These results defined a signal preventing incorporation within the 10 membrane-proximal amino acids of the Env tail. Confocal microscopy revealed that this signal functioned by causing localization of human immunodeficiency virus type 1 Env to plasma membrane domains distinct from the VSV budding sites, where VSV proteins were concentrated. Copyright 1998 Academic Press.

Immunohistochemical Insights in Vector-Borne Disease Research

USDA-ARS?s Scientific Manuscript database

Culicoides sonorensis biting midges transmit several emerging and re-emerging arboviruses of domestic and wild ruminants, including bluetongue virus, epizootic hemorrhagic disease virus (EHDV), and vesicular stomatitis virus (VSV). We have utilized immunohistochemistry (IHC) to examine virus-vecto...

Clinical development of Ebola vaccines

PubMed Central

Sridhar, Saranya

2015-01-01

The ongoing outbreak of Ebola virus disease in West Africa highlighted the lack of a licensed drug or vaccine to combat the disease and has renewed the urgency to develop a pipeline of Ebola vaccines. A number of different vaccine platforms are being developed by assessing preclinical efficacy in animal models and expediting clinical development. Over 15 different vaccines are in preclinical development and 8 vaccines are now in different stages of clinical evaluation. These vaccines include DNA vaccines, virus-like particles and viral vectors such as live replicating vesicular stomatitis virus (rVSV), human and chimpanzee adenovirus, and vaccinia virus. Recently, in preliminary results reported from the first phase III trial of an Ebola vaccine, the rVSV-vectored vaccine showed promising efficacy. This review charts this rapidly advancing area of research focusing on vaccines in clinical development and discusses the future opportunities and challenges faced in the licensure and deployment of Ebola vaccines. PMID:26668751

Metabolic biotinylation of lentiviral pseudotypes for scalable paramagnetic microparticle-dependent manipulation.

PubMed

Nesbeth, Darren; Williams, Sharon L; Chan, Lucas; Brain, Tony; Slater, Nigel K H; Farzaneh, Farzin; Darling, David

2006-04-01

Nonviral, host-derived proteins on lentiviral vector surfaces can have a profound effect on the vector's biology as they can both promote infection and provide resistance to complement inactivation. We have exploited this to engineer a specific posttranslational modification of a "nonenvelope," virally associated protein. The bacterial biotin ligase (BirA) and a modified human DeltaLNGFR have been introduced into HEK293T cells and their protein products directed to the lumen of the endoplasmic reticulum. The BirA then couples biotin to an acceptor peptide that has been fused to the DeltaLNGFR. This results in the covalent linkage of biotin to the extracellular domain of the DeltaLNGFR expressed on the cell surface. Lentiviral vectors from these cells are metabolically labeled with biotin in the presence of free biotin. These biotinylated lentiviral vectors have a high affinity for streptavidin paramagnetic particles and, once captured, are easily manipulated in vitro. This is illustrated by the concentration of lentiviral vectors pseudotyped with either the VSV-G or an amphotropic envelope in excess of 4500-fold. This new cell line has the potential for widespread application to envelope pseudotypes compatible with lentiviral vector production.

Oncolytic vesicular stomatitis virus induces apoptosis in U87 glioblastoma cells by a type II death receptor mechanism and induces cell death and tumor clearance in vivo.

PubMed

Cary, Zachary D; Willingham, Mark C; Lyles, Douglas S

2011-06-01

Vesicular stomatitis virus (VSV) is a potential oncolytic virus for treating glioblastoma multiforme (GBM), an aggressive brain tumor. Matrix (M) protein mutants of VSV have shown greater selectivity for killing GBM cells versus normal brain cells than VSV with wild-type M protein. The goal of this research was to determine the contribution of death receptor and mitochondrial pathways to apoptosis induced by an M protein mutant (M51R) VSV in U87 human GBM tumor cells. Compared to controls, U87 cells expressing a dominant negative form of Fas (dnFas) or overexpressing Bcl-X(L) had reduced caspase-3 activation following infection with M51R VSV, indicating that both the death receptor pathway and mitochondrial pathways are important for M51R VSV-induced apoptosis. Death receptor signaling has been classified as type I or type II, depending on whether signaling is independent (type I) or dependent on the mitochondrial pathway (type II). Bcl-X(L) overexpression inhibited caspase activation in response to a Fas-inducing antibody, similar to the inhibition in response to M51R VSV infection, indicating that U87 cells behave as type II cells. Inhibition of apoptosis in vitro delayed, but did not prevent, virus-induced cell death. Murine xenografts of U87 cells that overexpress Bcl-X(L) regressed with a time course similar to that of control cells following treatment with M51R VSV, and tumors were not detectable at 21 days postinoculation. Immunohistochemical analysis demonstrated similar levels of viral antigen expression but reduced activation of caspase-3 following virus treatment of Bcl-X(L)-overexpressing tumors compared to controls. Further, the pathological changes in tumors following treatment with virus were quite different in the presence versus the absence of Bcl-X(L) overexpression. These results demonstrate that M51R VSV efficiently induces oncolysis in GBM tumor cells despite deregulation of apoptotic pathways, underscoring its potential use as a treatment for GBM.

Vesicular stomatitis virus infects resident cells of the central nervous system and induces replication-dependent inflammatory responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chauhan, Vinita S.; Furr, Samantha R.; Sterka, David G.

2010-05-10

Vesicular stomatitis virus (VSV) infection of mice via intranasal administration results in a severe encephalitis with rapid activation and proliferation of microglia and astrocytes. We have recently shown that these glial cells express RIG-I and MDA5, cytosolic pattern recognition receptors for viral RNA. However, it is unclear whether VSV can replicate in glial cells or if such replication is required for their inflammatory responses. Here we demonstrate that primary microglia and astrocytes are permissive for VSV infection and limited productive replication. Importantly, we show that viral replication is required for robust inflammatory mediator production by these cells. Finally, we havemore » confirmed that in vivo VSV administration can result in viral infection of glial cells in situ. These results suggest that viral replication within resident glial cells might play an important role in CNS inflammation following infection with VSV and possibly other neurotropic nonsegmented negative-strand RNA viruses.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kubo, Yoshinao; Yoshii, Hiroaki; Kamiyama, Haruka

Ezrin, radixin, and moesin (ERM) proteins supply functional linkage between integral membrane proteins and cytoskeleton in mammalian cells to regulate membrane protein dynamisms and cytoskeleton rearrangement. To assess potential role of the ERM proteins in HIV-1 lifecycle, we examined if suppression of ERM function in human cells expressing HIV-1 infection receptors influences HIV-1 envelope (Env)-mediated HIV-1-vector transduction and cell-cell fusion. Expression of an ezrin dominant negative mutant or knockdown of ezrin, radixin, or moesin with siRNA uniformly decreased transduction titers of HIV-1 vectors having X4-tropic Env. In contrast, transduction titers of R5-tropic Env HIV-1 vectors were decreased only by radixinmore » knockdown: ezrin knockdown had no detectable effects and moesin knockdown rather increased transduction titer. Each of the ERM suppressions had no detectable effects on cell surface expression of CD4, CCR5, and CXCR4 or VSV-Env-mediated HIV-1 vector transductions. Finally, the individual knockdown of ERM mRNAs uniformly decreased efficiency of cell-cell fusion mediated by X4- or R5-tropic Env and HIV-1 infection receptors. These results suggest that (i) the ERM proteins function as positive regulators of infection by X4-tropic HIV-1, (ii) moesin additionally functions as a negative regulator of R5-tropic HIV-1 virus infection at the early step(s) after the membrane fusion, and (iii) receptor protein dynamisms are regulated differently in R5- and X4-tropic HIV-1 infections.« less

RD2-MolPack-Chim3, a packaging cell line for stable production of lentiviral vectors for anti-HIV gene therapy.

PubMed

Stornaiuolo, Anna; Piovani, Bianca Maria; Bossi, Sergio; Zucchelli, Eleonora; Corna, Stefano; Salvatori, Francesca; Mavilio, Fulvio; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

2013-08-01

Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

Peripheral dendritic cells are essential for both the innate and adaptive antiviral immune responses in the central nervous system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steel, Christina D.; Hahto, Suzanne M.; Ciavarra, Richard P., E-mail: ciavarrp@evms.ed

2009-04-25

Intranasal application of vesicular stomatitis virus (VSV) causes acute infection of the central nervous system (CNS). However, VSV encephalitis is not invariably fatal, suggesting that the CNS may contain a professional antigen-presenting cell (APC) capable of inducing or propagating a protective antiviral immune response. To examine this possibility, we first characterized the cellular elements that infiltrate the brain as well as the activation status of resident microglia in the brains of normal and transgenic mice acutely ablated of peripheral dendritic cells (DCs) in vivo. VSV encephalitis was characterized by a pronounced infiltrate of myeloid cells (CD45{sup high}CD11b{sup +}) and CD8{supmore » +} T cells containing a subset that was specific for the immunodominant VSV nuclear protein epitope. This T cell response correlated temporally with a rapid and sustained upregulation of MHC class I expression on microglia, whereas class II expression was markedly delayed. Ablation of peripheral DCs profoundly inhibited the inflammatory response as well as infiltration of virus-specific CD8{sup +} T cells. Unexpectedly, the VSV-induced interferon-gamma (IFN-gamma) response in the CNS remained intact in DC-deficient mice. Thus, both the inflammatory and certain components of the adaptive primary antiviral immune response in the CNS are dependent on peripheral DCs in vivo.« less

Establishing a safe, rapid, convenient and low-cost antiviral assay of interferon bioactivity based on recombinant VSV expressing GFP.

PubMed

Chen, Weiye; Wen, Zhiyuan; Zhang, Jialin; Li, Cuicui; Huang, Kehe; Bu, Zhigao

2018-02-01

The methods of the quantitative assay of the antiviral activity of interferons (IFNs) (type I, II or III) are very important during carrying out of the research of them, since they were found. Here a recombinant vesicular stomatitis virus expressing green fluorescent protein (GFP) (VSV/GFP) and MDBK cells were used to develop an antiviral assay (AVA) for IFNs. This method was carried out on a 96-well cell culture plate, and the half reduction of virus replication was quantified by assaying GFP. To quantify GFP, cell lysis buffer was directly added to the wells infected with VSV/GFP to lyse cells, the VSV/GFP was then inactivated, and relative fluorescence unit (RFU) of GFP was measured and used to calculate the antiviral activity. This method needed only one step instead of three steps in the staining method with naphthol blue black, medium with phenol red can be used, and it had good reproducibility. The GFP-containing samples could be stored at 4°C in a wet box for at least 1 week without affecting the assay results. In addition, the results obtained with this method were similar to those obtained with the staining method. In conclusion, a safe, rapid, convenient and low-cost AVA of IFN based on recombinant VSV/GFP was established. Copyright © 2017. Published by Elsevier B.V.

Broad Neutralization of Ebolaviruses via a Fusion Loop Epitope Elicited by Immunization

DTIC Science & Technology

2017-03-31

overnight. After incubation with blocking buffer (BB, 2% non- fat milk , 5% FBS in PBS), the WT or mutant supernatant in five-fold serial dilution in BB was...replication competent rVSV pseudotyped with filovirus GP, which also expressed the reporter protein GFP (rVSV-GP-GFP) (Miller et al., 2012). CA45 potently...for proper protein folding and expression. The epitope mapping identified EBOV GP residues R64 within the N-terminus of GP1 in addition to Y517

Lutzomyia (Helcocyrtomyia) Apache Young and Perkins (Diptera: Psychodidae) feeds on reptiles

USDA-ARS?s Scientific Manuscript database

Phlebotomine sand flies are vectors of bacteria, parasites, and viruses. In the western USA a sand fly, Lutzomyia apache Young and Perkins, was initially associated with epizootics of vesicular stomatitis virus (VSV), because sand flies were trapped at sites of an outbreak. Additional studies indica...

Protein Transfer Into Human Cells by VSV-G-induced Nanovesicles

PubMed Central

Mangeot, Philippe-Emmanuel; Dollet, Sandra; Girard, Mathilde; Ciancia, Claire; Joly, Stéphane; Peschanski, Marc; Lotteau, Vincent

2011-01-01

Identification of new techniques to express proteins into mammal cells is of particular interest for both research and medical purposes. The present study describes the use of engineered vesicles to deliver exogenous proteins into human cells. We show that overexpression of the spike glycoprotein of the vesicular stomatitis virus (VSV-G) in human cells induces the release of fusogenic vesicles named gesicles. Biochemical and functional studies revealed that gesicles incorporated proteins from producer cells and could deliver them to recipient cells. This protein-transduction method allows the direct transport of cytoplasmic, nuclear or surface proteins in target cells. This was demonstrated by showing that the TetR transactivator and the receptor for the murine leukemia virus (MLV) envelope [murine cationic amino acid transporter-1 (mCAT-1)] were efficiently delivered by gesicles in various cell types. We further shows that gesicle-mediated transfer of mCAT-1 confers to human fibroblasts a robust permissiveness to ecotropic vectors, allowing the generation of human-induced pluripotent stem cells in level 2 biosafety facilities. This highlights the great potential of mCAT-1 gesicles to increase the safety of experiments using retro/lentivectors. Besides this, gesicles is a versatile tool highly valuable for the nongenetic delivery of functions such as transcription factors or genome engineering agents. PMID:21750535

Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms.

PubMed

Mundell, Nathan A; Beier, Kevin T; Pan, Y Albert; Lapan, Sylvain W; Göz Aytürk, Didem; Berezovskii, Vladimir K; Wark, Abigail R; Drokhlyansky, Eugene; Bielecki, Jan; Born, Richard T; Schier, Alexander F; Cepko, Constance L

2015-08-01

Current limitations in technology have prevented an extensive analysis of the connections among neurons, particularly within nonmammalian organisms. We developed a transsynaptic viral tracer originally for use in mice, and then tested its utility in a broader range of organisms. By engineering the vesicular stomatitis virus (VSV) to encode a fluorophore and either the rabies virus glycoprotein (RABV-G) or its own glycoprotein (VSV-G), we created viruses that can transsynaptically label neuronal circuits in either the retrograde or anterograde direction, respectively. The vectors were investigated for their utility as polysynaptic tracers of chicken and zebrafish visual pathways. They showed patterns of connectivity consistent with previously characterized visual system connections, and revealed several potentially novel connections. Further, these vectors were shown to infect neurons in several other vertebrates, including Old and New World monkeys, seahorses, axolotls, and Xenopus. They were also shown to infect two invertebrates, Drosophila melanogaster, and the box jellyfish, Tripedalia cystophora, a species previously intractable for gene transfer, although no clear evidence of transsynaptic spread was observed in these species. These vectors provide a starting point for transsynaptic tracing in most vertebrates, and are also excellent candidates for gene transfer in organisms that have been refractory to other methods. © 2015 Wiley Periodicals, Inc.

Independent of plasmacytoid dendritic cell (pDC) infection, pDC triggered by virus-infected cells mount enhanced type I IFN responses of different composition as opposed to pDC stimulated with free virus.

PubMed

Frenz, Theresa; Graalmann, Lukas; Detje, Claudia N; Döring, Marius; Grabski, Elena; Scheu, Stefanie; Kalinke, Ulrich

2014-09-01

Upon treatment with vesicular stomatitis virus (VSV) particles, plasmacytoid dendritic cells (pDC) are triggered to mount substantial type I IFN responses, whereas myeloid DC (mDC) are only minor producers. Interestingly, bone marrow-derived (BM-)mDC were more vulnerable to infection with enhanced GFP (eGFP)-expressing VSV (VSVeGFP) than BM-pDC. BM-pDC stimulated with wild-type VSV mounted TLR-dependent IFN responses that were independent of RIG-I-like helicase (RLH) signaling. In contrast, in BM-pDC the VSV variant M2 induced particularly high IFN responses triggered in a TLR- and RLH-dependent manner, whereas BM-mDC stimulation was solely RLH-dependent. Importantly, VSVeGFP treatment of BM-pDC derived from IFN-β yellow fluorescent protein (YFP) reporter mice (messenger of IFN-β) resulted in YFP(+) and eGFP(+) single-positive cells, whereas among messenger of IFN-β-BM-mDC most YFP(+) cells were also eGFP(+). This observation indicated that unlike mDC, direct virus infection was not required to trigger IFN responses of pDC. VSV-infected BM-mDC triggered BM-pDC to mount significantly higher IFN responses than free virus particles. Stimulation with infected cells enhanced the percentages of pDC subsets expressing either IFN-β(+) or IFN-α6(+) plus IFN-β(+). Irrespective of whether stimulated with free virus or infected cells, IFN induction was dependent on autophagy of pDC, whereas autophagy of the infected mDC was dispensable. Collectively, these results indicated that productive VSV infection was needed to trigger IFN responses of mDC, but not of pDC, and that IFN responses were primarily induced by virus-infected cells that stimulated pDC in a TLR-dependent manner. Copyright © 2014 by The American Association of Immunologists, Inc.

Multicomponent DNA carrier with a vesicular stomatitis virus G-peptide greatly enhances liver-targeted gene expression in mice.

PubMed

Schuster, M J; Wu, G Y; Walton, C M; Wu, C H

1999-01-01

Genes can be targeted to hepatocytes in vitro and in vivo by the use of asialoorosomucoid-polylysine conjugates. After systemic application, this nonviral vector is recognized by highly selective asialoglycoprotein (AsGP) receptors on the sinusoidal liver cell membrane and is taken up via receptor-mediated endocytosis. As most of the DNA is rapidly transferred to lysosomes where it is degraded, transfection efficiency is low and gene expression transient. To address this problem, we incorporated a pH-dependent synthetic hemolytic peptide derived of the G-protein of Vesicular Stomatitis Virus (VSV) into the gene transfer system, to increase endosomal escape of internalized DNA. The multicomponent carrier binds DNA in a nondamaging way, is still recognized by the AsGP receptor, and is targeted to the liver in vivo. Injection of DNA complexes containing a luciferase marker gene resulted in luciferase expression of 29 000 pg/g liver which corresponded to an increase of a factor of 10(3) overexpression after injection of DNA complexes without endosomolytic peptide. Furthermore, the amount of intact transgene within isolated liver cell nuclei was increased by a factor of 10(1)-10(2) by the use of the multicomponent carriers. These results demonstrate that incorporation of a hemolytic peptide into a nonviral vector can greatly increase gene expression while retaining cell type targetability in vivo.

Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial.

PubMed

Henao-Restrepo, Ana Maria; Longini, Ira M; Egger, Matthias; Dean, Natalie E; Edmunds, W John; Camacho, Anton; Carroll, Miles W; Doumbia, Moussa; Draguez, Bertrand; Duraffour, Sophie; Enwere, Godwin; Grais, Rebecca; Gunther, Stephan; Hossmann, Stefanie; Kondé, Mandy Kader; Kone, Souleymane; Kuisma, Eeva; Levine, Myron M; Mandal, Sema; Norheim, Gunnstein; Riveros, Ximena; Soumah, Aboubacar; Trelle, Sven; Vicari, Andrea S; Watson, Conall H; Kéïta, Sakoba; Kieny, Marie Paule; Røttingen, John-Arne

2015-08-29

A recombinant, replication-competent vesicular stomatitis virus-based vaccine expressing a surface glycoprotein of Zaire Ebolavirus (rVSV-ZEBOV) is a promising Ebola vaccine candidate. We report the results of an interim analysis of a trial of rVSV-ZEBOV in Guinea, west Africa. For this open-label, cluster-randomised ring vaccination trial, suspected cases of Ebola virus disease in Basse-Guinée (Guinea, west Africa) were independently ascertained by Ebola response teams as part of a national surveillance system. After laboratory confirmation of a new case, clusters of all contacts and contacts of contacts were defined and randomly allocated 1:1 to immediate vaccination or delayed (21 days later) vaccination with rVSV-ZEBOV (one dose of 2 × 10(7) plaque-forming units, administered intramuscularly in the deltoid muscle). Adults (age ≥18 years) who were not pregnant or breastfeeding were eligible for vaccination. Block randomisation was used, with randomly varying blocks, stratified by location (urban vs rural) and size of rings (≤20 vs >20 individuals). The study is open label and masking of participants and field teams to the time of vaccination is not possible, but Ebola response teams and laboratory workers were unaware of allocation to immediate or delayed vaccination. Taking into account the incubation period of the virus of about 10 days, the prespecified primary outcome was laboratory-confirmed Ebola virus disease with onset of symptoms at least 10 days after randomisation. The primary analysis was per protocol and compared the incidence of Ebola virus disease in eligible and vaccinated individuals in immediate vaccination clusters with the incidence in eligible individuals in delayed vaccination clusters. This trial is registered with the Pan African Clinical Trials Registry, number PACTR201503001057193. Between April 1, 2015, and July 20, 2015, 90 clusters, with a total population of 7651 people were included in the planned interim analysis. 48 of these clusters (4123 people) were randomly assigned to immediate vaccination with rVSV-ZEBOV, and 42 clusters (3528 people) were randomly assigned to delayed vaccination with rVSV-ZEBOV. In the immediate vaccination group, there were no cases of Ebola virus disease with symptom onset at least 10 days after randomisation, whereas in the delayed vaccination group there were 16 cases of Ebola virus disease from seven clusters, showing a vaccine efficacy of 100% (95% CI 74·7-100·0; p=0·0036). No new cases of Ebola virus disease were diagnosed in vaccinees from the immediate or delayed groups from 6 days post-vaccination. At the cluster level, with the inclusion of all eligible adults, vaccine effectiveness was 75·1% (95% CI -7·1 to 94·2; p=0·1791), and 76·3% (95% CI -15·5 to 95·1; p=0·3351) with the inclusion of everyone (eligible or not eligible for vaccination). 43 serious adverse events were reported; one serious adverse event was judged to be causally related to vaccination (a febrile episode in a vaccinated participant, which resolved without sequelae). Assessment of serious adverse events is ongoing. The results of this interim analysis indicate that rVSV-ZEBOV might be highly efficacious and safe in preventing Ebola virus disease, and is most likely effective at the population level when delivered during an Ebola virus disease outbreak via a ring vaccination strategy. WHO, with support from the Wellcome Trust (UK); Médecins Sans Frontières; the Norwegian Ministry of Foreign Affairs through the Research Council of Norway; and the Canadian Government through the Public Health Agency of Canada, Canadian Institutes of Health Research, International Development Research Centre, and Department of Foreign Affairs, Trade and Development. Copyright © 2015 World Health Organization. Published by Elsevier Ltd/Inc/BV. All rights reserved. Published by Elsevier Ltd.. All rights reserved.

The Respiratory Environment Diverts the Development of Antiviral Memory CD8 T Cells.

PubMed

Shane, Hillary L; Reagin, Katie L; Klonowski, Kimberly D

2018-06-01

Our understanding of memory CD8 + T cells has been largely derived from acute, systemic infection models. However, memory CD8 + T cells generated from mucosal infection exhibit unique properties and, following respiratory infection, are not maintained in the lung long term. To better understand how infection route modifies memory differentiation, we compared murine CD8 + T cell responses to a vesicular stomatitis virus (VSV) challenge generated intranasally (i.n.) or i.v. The i.n. infection resulted in greater peak expansion of VSV-specific CD8 + T cells. However, this numerical advantage was rapidly lost during the contraction phase of the immune response, resulting in memory CD8 + T cell numerical deficiencies when compared with i.v. infection. Interestingly, the antiviral CD8 + T cells generated in response to i.n. VSV exhibited a biased and sustained proportion of early effector cells (CD127 lo KLRG1 lo ) akin to the developmental program favored after i.n. influenza infection, suggesting that respiratory infection broadly favors an incomplete memory differentiation program. Correspondingly, i.n. VSV infection resulted in lower CD122 expression and eomesodermin levels by VSV-specific CD8 + T cells, further indicative of an inferior transition to bona fide memory. These results may be due to distinct (CD103 + CD11b + ) dendritic cell subsets in the i.n. versus i.v. T cell priming environments, which express molecules that regulate T cell signaling and the balance between tolerance and immunity. Therefore, we propose that distinct immunization routes modulate both the quality and quantity of antiviral effector and memory CD8 + T cells in response to an identical pathogen and should be considered in CD8 + T cell-based vaccine design. Copyright © 2018 by The American Association of Immunologists, Inc.

Durability of a vesicular stomatitis virus-based marburg virus vaccine in nonhuman primates.

PubMed

Mire, Chad E; Geisbert, Joan B; Agans, Krystle N; Satterfield, Benjamin A; Versteeg, Krista M; Fritz, Elizabeth A; Feldmann, Heinz; Hensley, Lisa E; Geisbert, Thomas W

2014-01-01

The filoviruses, Marburg virus (MARV) and Ebola virus, causes severe hemorrhagic fever with high mortality in humans and nonhuman primates. A promising filovirus vaccine under development is based on a recombinant vesicular stomatitis virus (rVSV) that expresses individual filovirus glycoproteins (GPs) in place of the VSV glycoprotein (G). These vaccines have shown 100% efficacy against filovirus infection in nonhuman primates when challenge occurs 28-35 days after a single injection immunization. Here, we examined the ability of a rVSV MARV-GP vaccine to provide protection when challenge occurs more than a year after vaccination. Cynomolgus macaques were immunized with rVSV-MARV-GP and challenged with MARV approximately 14 months after vaccination. Immunization resulted in the vaccine cohort of six animals having anti-MARV GP IgG throughout the pre-challenge period. Following MARV challenge none of the vaccinated animals showed any signs of clinical disease or viremia and all were completely protected from MARV infection. Two unvaccinated control animals exhibited signs consistent with MARV infection and both succumbed. Importantly, these data are the first to show 100% protective efficacy against any high dose filovirus challenge beyond 8 weeks after final vaccination. These findings demonstrate the durability of VSV-based filovirus vaccines.

Multigene Expression In Vivo: Supremacy of Large Versus Small Terminators for T7 RNA Polymerase

PubMed Central

Du, Liping; Villarreal, Seth; Forster, Anthony C.

2012-01-01

Designing and building multigene constructs is commonplace in synthetic biology. Yet functional successes at first attempts are rare because the genetic parts are not fully modular. In order to improve the modularity of transcription, we previously showed that transcription termination in vitro by bacteriophage T7 RNA polymerase could be made more efficient by substituting the standard, single, TΦ large (class I) terminator with adjacent copies of the Vesicular Stomatitis Virus (VSV) small (class II) terminator. However, in vitro termination at the downstream VSV terminator was less efficient than at the upstream VSV terminator, and multigene overexpression in vivo was complicated by unexpectedly inefficient VSV termination within E. coli cells. Here, we address hypotheses raised in that study by showing that VSV or preproparathyroid hormone (PTH) small terminators spaced further apart can work independently (i.e. more efficiently) in vitro, and that VSV and PTH terminations are severely inhibited in vivo. Surprisingly, the difference between class II terminator function in vivo versus in vitro is not due to differences in plasmid supercoiling, as supercoiling had a minimal effect on termination in vitro. We therefore turned to TΦ terminators for “BioBrick” synthesis of a pentameric gene construct suitable for overexpression in vivo. This indeed enabled coordinated overexpression and copurification of five His-tagged proteins using the first construct attempted, indicating that this strategy is more modular than other strategies. An application of this multigene overexpression and protein copurification method is demonstrated by supplying five of the six E. coli translation factors required for reconstitution of translation from a single cell line via copurification, greatly simplifying the reconstitution. PMID:22094962

Antibodies are necessary for rVSV/ZEBOV-GP-mediated protection against lethal Ebola virus challenge in nonhuman primates.

PubMed

Marzi, Andrea; Engelmann, Flora; Feldmann, Friederike; Haberthur, Kristen; Shupert, W Lesley; Brining, Douglas; Scott, Dana P; Geisbert, Thomas W; Kawaoka, Yoshihiro; Katze, Michael G; Feldmann, Heinz; Messaoudi, Ilhem

2013-01-29

Ebola viruses cause hemorrhagic disease in humans and nonhuman primates with high fatality rates. These viruses pose a significant health concern worldwide due to the lack of approved therapeutics and vaccines as well as their potential misuse as bioterrorism agents. Although not licensed for human use, recombinant vesicular stomatitis virus (rVSV) expressing the filovirus glycoprotein (GP) has been shown to protect macaques from Ebola virus and Marburg virus infections, both prophylactically and postexposure in a homologous challenge setting. However, the immune mechanisms of protection conferred by this vaccine platform remain poorly understood. In this study, we set out to investigate the role of humoral versus cellular immunity in rVSV vaccine-mediated protection against lethal Zaire ebolavirus (ZEBOV) challenge. Groups of cynomolgus macaques were depleted of CD4+ T, CD8+ T, or CD20+ B cells before and during vaccination with rVSV/ZEBOV-GP. Unfortunately, CD20-depleted animals generated a robust IgG response. Therefore, an additional group of vaccinated animals were depleted of CD4+ T cells during challenge. All animals were subsequently challenged with a lethal dose of ZEBOV. Animals depleted of CD8+ T cells survived, suggesting a minimal role for CD8+ T cells in vaccine-mediated protection. Depletion of CD4+ T cells during vaccination caused a complete loss of glycoprotein-specific antibodies and abrogated vaccine protection. In contrast, depletion of CD4+ T cells during challenge resulted in survival of the animals, indicating a minimal role for CD4+ T-cell immunity in rVSV-mediated protection. Our results suggest that antibodies play a critical role in rVSV-mediated protection against ZEBOV.

[Efficient packaging retrovirus and construction of transgenic chicken technical platform].

PubMed

Man, Chaolai; Zhang, Qing; Chen, Yan; Zhu, Dahai

2007-10-01

Transgenic chicken and oviduct bioreactor are growing to be one of the hotspot of scientific study in the field of biology. The most successful method of producing transgenic chicken is pseudotyped retrovirus vector system, but no one has reported the production of transgenic chicken by retrovirus system recently in our country. In order to accelerate our study in this field, we introduced the relevant technical methods such as packaging retrovirus and vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped retrovirus, optimizing the conditions of packaging retrovirus, concentrating VSV-G pseudotyped retrovirus, helper virus assays, and microinjection of retrovirus. Furthermore, we successfully conducted in vivo study for detecting the marker gene EGFP of chicken embryo as well as in vitro study for detecting that gene of chicken embryo myoblast (CFM), thus we have provided an applied technical platform for studies of transgenic chicken in the future.

Zinc finger protein designed to target 2-long terminal repeat junctions interferes with human immunodeficiency virus integration.

PubMed

Sakkhachornphop, Supachai; Barbas, Carlos F; Keawvichit, Rassamee; Wongworapat, Kanlaya; Tayapiwatana, Chatchai

2012-09-01

Integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host chromosome is a vital step in the HIV life cycle. The highly conserved cytosine-adenine (CA) dinucleotide sequence immediately upstream of the cleavage site is crucial for integrase (IN) activity. As this viral enzyme has an important role early in the HIV-1 replication cycle, interference with the IN substrate has become an attractive strategy for therapeutic intervention. We demonstrated that a designed zinc finger protein (ZFP) fused to green fluorescent protein (GFP) targets the 2-long terminal repeat (2-LTR) circle junctions of HIV-1 DNA with nanomolar affinity. We report now that 2LTRZFP-GFP stably transduced into 293T cells interfered with the expression of vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral red fluorescent protein (RFP), as shown by the suppression of RFP expression. We also used a third-generation lentiviral vector and pCEP4 expression vector to deliver the 2LTRZFP-GFP transgene into human T-lymphocytic cells, and a stable cell line for long-term expression studies was selected for HIV-1 challenge. HIV-1 integration and replication were inhibited as measured by Alu-gag real-time PCR and p24 antigen assay. In addition, the molecular activity of 2LTRZFP-GFP was evaluated in peripheral blood mononuclear cells. The results were confirmed by Alu-gag real-time PCR for integration interference. We suggest that the expression of 2LTRZFP-GFP limited viral integration on intracellular immunization, and that it has potential for use in HIV gene therapy in the future.

PEGylation of Vesicular Stomatitis Virus Extends Virus Persistence in Blood Circulation of Passively Immunized Mice

PubMed Central

Tesfay, Mulu Z.; Kirk, Amber C.; Hadac, Elizabeth M.; Griesmann, Guy E.; Federspiel, Mark J.; Barber, Glen N.; Henry, Stephen M.; Peng, Kah-Whye

2013-01-01

We are developing oncolytic vesicular stomatitis viruses (VSVs) for systemic treatment of multiple myeloma, an incurable malignancy of antibody-secreting plasma cells that are specifically localized in the bone marrow. One of the presumed advantages for using VSV as an oncolytic virus is that human infections are rare and preexisting anti-VSV immunity is typically lacking in cancer patients, which is very important for clinical success. However, our studies show that nonimmune human and mouse serum can neutralize clinical-grade VSV, reducing the titer by up to 4 log units in 60 min. In addition, we show that neutralizing anti-VSV antibodies negate the antitumor efficacy of VSV, a concern for repeat VSV administration. We have investigated the potential use of covalent modification of VSV with polyethylene glycol (PEG) or a function-spacer-lipid (FSL)–PEG construct to inhibit serum neutralization and to limit hepatosplenic sequestration of systemically delivered VSV. We report that in mice passively immunized with neutralizing anti-VSV antibodies, PEGylation of VSV improved the persistence of VSV in the blood circulation, maintaining a more than 1-log-unit increase in VSV genome copies for up to 1 h compared to the genome copy numbers for the non-PEGylated virus, which was mostly cleared within 10 min after intravenous injection. We are currently investigating if this increase in PEGylated VSV circulating half-life can translate to increased virus delivery and better efficacy in mouse models of multiple myeloma. PMID:23325695

Oncolytic recombinant vesicular stomatitis virus (VSV) is nonpathogenic and non-transmissible in pigs, a natural host of VSV

USDA-ARS?s Scientific Manuscript database

Vesicular stomatitis virus (VSV) is a negative stranded RNA virus that naturally causes disease in agricultural livestock including horses, cattle and pigs. The two main identified VSV strains are the New Jersey (VSNJV) and Indiana (VSIV) strains. VSV is a rapidly replicating, potently immunogenic v...

Oncotargeting by Vesicular Stomatitis Virus (VSV): Advances in Cancer Therapy.

PubMed

Bishnoi, Suman; Tiwari, Ritudhwaj; Gupta, Sharad; Byrareddy, Siddappa N; Nayak, Debasis

2018-02-23

Modern oncotherapy approaches are based on inducing controlled apoptosis in tumor cells. Although a number of apoptosis-induction approaches are available, site-specific delivery of therapeutic agents still remain the biggest hurdle in achieving the desired cancer treatment benefit. Additionally, systemic treatment-induced toxicity remains a major limiting factor in chemotherapy. To specifically address drug-accessibility and chemotherapy side effects, oncolytic virotherapy (OV) has emerged as a novel cancer treatment alternative. In OV, recombinant viruses with higher replication capacity and stronger lytic properties are being considered for tumor cell-targeting and subsequent cell lysing. Successful application of OVs lies in achieving strict tumor-specific tropism called oncotropism, which is contingent upon the biophysical interactions of tumor cell surface receptors with viral receptors and subsequent replication of oncolytic viruses in cancer cells. In this direction, few viral vector platforms have been developed and some of these have entered pre-clinical/clinical trials. Among these, the Vesicular stomatitis virus (VSV)-based platform shows high promise, as it is not pathogenic to humans. Further, modern molecular biology techniques such as reverse genetics tools have favorably advanced this field by creating efficient recombinant VSVs for OV; some have entered into clinical trials. In this review, we discuss the current status of VSV based oncotherapy, challenges, and future perspectives regarding its therapeutic applications in the cancer treatment.

Oncotargeting by Vesicular Stomatitis Virus (VSV): Advances in Cancer Therapy

PubMed Central

Bishnoi, Suman; Tiwari, Ritudhwaj; Gupta, Sharad; Byrareddy, Siddappa N.; Nayak, Debasis

2018-01-01

Modern oncotherapy approaches are based on inducing controlled apoptosis in tumor cells. Although a number of apoptosis-induction approaches are available, site-specific delivery of therapeutic agents still remain the biggest hurdle in achieving the desired cancer treatment benefit. Additionally, systemic treatment-induced toxicity remains a major limiting factor in chemotherapy. To specifically address drug-accessibility and chemotherapy side effects, oncolytic virotherapy (OV) has emerged as a novel cancer treatment alternative. In OV, recombinant viruses with higher replication capacity and stronger lytic properties are being considered for tumor cell-targeting and subsequent cell lysing. Successful application of OVs lies in achieving strict tumor-specific tropism called oncotropism, which is contingent upon the biophysical interactions of tumor cell surface receptors with viral receptors and subsequent replication of oncolytic viruses in cancer cells. In this direction, few viral vector platforms have been developed and some of these have entered pre-clinical/clinical trials. Among these, the Vesicular stomatitis virus (VSV)-based platform shows high promise, as it is not pathogenic to humans. Further, modern molecular biology techniques such as reverse genetics tools have favorably advanced this field by creating efficient recombinant VSVs for OV; some have entered into clinical trials. In this review, we discuss the current status of VSV based oncotherapy, challenges, and future perspectives regarding its therapeutic applications in the cancer treatment. PMID:29473868

Definitive Management of Oligometastatic Melanoma in a Murine Model Using Combined Ablative Radiation Therapy and Viral Immunotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blanchard, Miran; Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota; Shim, Kevin G.

Purpose: The oligometastatic state is an intermediate state between a malignancy that can be completely eradicated with conventional modalities and one in which a palliative approach is undertaken. Clinically, high rates of local tumor control are possible with stereotactic ablative radiation therapy (SABR), using precisely targeted, high-dose, low-fraction radiation therapy. However, in oligometastatic melanoma, virtually all patients develop progression systemically at sites not initially treated with ablative radiation therapy that cannot be managed with conventional chemotherapy and immunotherapy. We have demonstrated in mice that intravenous administration of vesicular stomatitis virus (VSV) expressing defined tumor-associated antigens (TAAs) generates systemic immune responsesmore » capable of clearing established tumors. Therefore, in the present preclinical study, we tested whether the combination of systemic VSV-mediated antigen delivery and SABR would be effective against oligometastatic disease. Methods and Materials: We generated a model of oligometastatic melanoma in C57BL/6 immunocompetent mice and then used a combination of SABR and systemically administered VSV-TAA viral immunotherapy to treat both local and systemic disease. Results: Our data showed that SABR generates excellent control or cure of local, clinically detectable, and accessible tumor through direct cell ablation. Also, the immunotherapeutic activity of systemically administered VSV-TAA generated T-cell responses that cleared subclinical metastatic tumors. We also showed that SABR induced weak T-cell-mediated tumor responses, which, particularly if boosted by VSV-TAA, might contribute to control of local and systemic disease. In addition, VSV-TAA therapy alone had significant effects on control of both local and metastatic tumors. Conclusions: We have shown in the present preliminary murine study using a single tumor model that this approach represents an effective, complementary combination therapy model that addresses the need for both systemic and local control in oligometastatic melanoma.« less

Zinc Finger Protein Designed to Target 2-Long Terminal Repeat Junctions Interferes with Human Immunodeficiency Virus Integration

PubMed Central

Sakkhachornphop, Supachai; Barbas, Carlos F.; Keawvichit, Rassamee; Wongworapat, Kanlaya

2012-01-01

Abstract Integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host chromosome is a vital step in the HIV life cycle. The highly conserved cytosine–adenine (CA) dinucleotide sequence immediately upstream of the cleavage site is crucial for integrase (IN) activity. As this viral enzyme has an important role early in the HIV-1 replication cycle, interference with the IN substrate has become an attractive strategy for therapeutic intervention. We demonstrated that a designed zinc finger protein (ZFP) fused to green fluorescent protein (GFP) targets the 2-long terminal repeat (2-LTR) circle junctions of HIV-1 DNA with nanomolar affinity. We report now that 2LTRZFP-GFP stably transduced into 293T cells interfered with the expression of vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral red fluorescent protein (RFP), as shown by the suppression of RFP expression. We also used a third-generation lentiviral vector and pCEP4 expression vector to deliver the 2LTRZFP-GFP transgene into human T-lymphocytic cells, and a stable cell line for long-term expression studies was selected for HIV-1 challenge. HIV-1 integration and replication were inhibited as measured by Alu-gag real-time PCR and p24 antigen assay. In addition, the molecular activity of 2LTRZFP-GFP was evaluated in peripheral blood mononuclear cells. The results were confirmed by Alu-gag real-time PCR for integration interference. We suggest that the expression of 2LTRZFP-GFP limited viral integration on intracellular immunization, and that it has potential for use in HIV gene therapy in the future. PMID:22429108

Transduction of ferret airway epithelia using a pre-treatment and lentiviral gene vector.

PubMed

Cmielewski, Patricia; Farrow, Nigel; Donnelley, Martin; McIntyre, Chantelle; Penny-Dimri, Jahan; Kuchel, Tim; Parsons, David

2014-11-21

The safety and efficiency of gene therapies for cystic fibrosis (CF) need to be assessed in pre-clinical models. Using the normal ferret, this study sought to determine whether ferret airway epithelia could be transduced with a lysophosphatidylcholine (LPC) pre-treatment followed by a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector, in preparation for future studies in CF ferrets. Six normal ferrets (7 -8 weeks old) were treated with a 150 μL LPC pre-treatment, followed one hour later by a 500 μL LV vector dose containing the LacZ transgene. LacZ gene expression in the conducting airways and lung was assessed by X-gal staining after 7 days. The presence of transduction in the lung, as well as off-target transduction in the liver, spleen and gonads, were assessed by qPCR. The levels of LV vector p24 protein bio-distribution in blood sera were assessed by ELISA at 0, 1, 3, 5 and 7 days. The dosing protocol was well tolerated. LacZ gene expression was observed en face in the trachea of all animals. Histology showed that ciliated and basal cells were transduced in the trachea, with rare LacZ transduced single cells noted in lung. p24 levels was not detectable in the sera of 5 of the 6 animals. The LacZ gene was not detected in the lung tissue and no off-target transduction was detected by qPCR. This study shows that ferret airway epithelia are transducible using our unique two-step pre-treatment and LV vector dosing protocol. We have identified a number of unusual anatomical factors that are likely to influence the level of transduction that can be achieved in ferret airways. The ability to transduce ferret airway epithelium is a promising step towards therapeutic LV-CFTR testing in a CF ferret model.

Group I but not group II NPV induces antiviral effects in mammalian cells.

PubMed

Liang, Changyong; Song, Jianhua; Hu, Zhihong; Chen, Xinwen

2006-10-01

Nucleopolyhedrovirus (NPV) is divided into Group I and Group II based on the phylogenetic analysis. It has been reported that Group I NPVs such as Autographa californica multiple NPV (AcMNPV) can transduce mammalian cells, while Group II NPVs such as Helicoverpa armigera single NPV (HaSNPV) cannot. Here we report that AcMNPV was capable of stimulating antiviral activity in human hepatoma cells (SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus (VSV) replication. In contrast, the HaSNPV and the Spodoptera exigua multiple NPV (SeMNPV) of group II had no inhibitory effect on VSV. Recombinant AcMNPV was shown to induce interferons alpha/beta even in the absence of transgene expression in human SMMC-7721 cells, while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.

Production of cytokines and stimulation of resistance to viral infection in human leukocytes by Scutellaria baicalensis flavones.

PubMed

Błach-Olszewska, Zofia; Jatczak, Bogna; Rak, Anna; Lorenc, Maria; Gulanowski, Bogdan; Drobna, Agnieszka; Lamer-Zarawska, Eliza

2008-09-01

Extracts of Scutellaria baicalensis display a wide spectrum of antiviral activity. It was of great interest to check the effect of baicalein and wogonin preparations on two important mechanisms of innate immunity: the secretion of cytokines and the natural resistance of human leukocytes to viral infection. To study the effect of S. baicalensis extracts on interferons (IFNs), tumor necrosis factor alpha (TNF-alpha), and interleukin (IL) production and virus replication, uninfected and vesicular stomatitis virus (VSV)-infected human peripheral blood leukocytes (PBLs) were used. Four pulverized preparations obtained from roots of Scutellaria and a Sigma-Aldrich preparation of purified baicalein were used in the study. RPMI extracts containing different amounts of baicalein and wogonin were used to study the effect on VSV replication in PBLs. PBLs express ex vivo individually differentiated cytokine-dependent resistance/innate immunity to viral infections. The degree of resistance was estimated on the basis of VSV replication in PBLs. The results obtained indicate that baicalein- and wogonin-containing extracts modulate cytokine production, that is inhibit IFN-alpha and IFN-gamma and stimulate TNF-alpha and IL (IL-12, IL-10) production. They also augment the resistance of PBLs to VSV. Extract from S. baicalensis containing baicalein and wogonin regulates the innate antiviral immunity by modulation of cytokine production and stimulation of human leukocyte resistance.

The establishment of polarized membrane traffic in Xenopus laevis embryos.

PubMed

Roberts, S J; Leaf, D S; Moore, H P; Gerhart, J C

1992-09-01

Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin was polarized. Oogenic prolactin was secreted only into the blastocoel (from the cleavage membrane), none could be detected in the external medium (from the original oocyte membrane). These results provide the first direct evidence that the oocyte synthesizes a cache of vesicles for specific recruitment to the embryonic cleavage membranes which are polarized beginning with the first cleavage division.

Downregulation of mouse CCR3 by lentiviral shRNA inhibits proliferation and induces apoptosis of mouse eosinophils.

PubMed

Zhu, Xin-Hua; Liao, Bing; Xu, Yi; Liu, Ke; Huang, Yun; Huang, Quan-Long; Liu, Yue-Hui

2017-02-01

RNA interference has been considered as an effective gene silencing method in basic and preclinical investigations. The aims of the present study were to construct a lentiviral vector expressing a short hairpin RNA (shRNA) targeting the murine CC chemokine receptor 3 (mCCR3), and to investigate its effects on the proliferation and apoptosis of mouse eosinophils. A recombinant lentiviral vector expressing four fragments of mouse CCR3 shRNA (pLVX‑mCCR3‑1+2+3+4‑shRNA) was constructed using subcloning techniques. This novel lentivirus was then packaged into 293T cells by co‑transduction with plasmids, including Baculo p35, pCMV R8.2 and VSV. The interference effects of the vector were verified using polymerase chain reaction (PCR) and western blot analyses. The effects of the interference on the proliferation and apoptosis of mouse eosinophils were investigated using 3‑(4,5‑dimethylthiazol‑2‑yl)‑5‑(3‑carboxymethoxyphenyl)‑2‑(4‑sulfophenyl)‑2H‑tetrazolium and terminal deoxynucleotidyl transferase dUTP nick end labeling methods, respectively. The results of the PCR and western blot analyses confirmed that the novel recombinant vector, pLVX‑mCCR3‑1+2+3+4‑shRNA, had high efficiency in inhibiting the mRNA and protein expression levels of mCCR3 in mouse eosinophils. The downregulation of mCCR3 significantly inhibited proliferation of the eosinophils. Furthermore, the present study found that the downregulation of mCCR3 significantly promoted apoptosis of the eosinophils. Therefore, the downregulation of mCCR3 led to the inhibition of proliferation and induction of apoptosis in mouse eosinophils. The predominant characteristics of allergic rhinitis are eosinophil infiltration and release of inflammatory mediators, which appear in a variety of clinical manifestations. The results of the present study indicate that mCCR3 silencing may serve as a putative approach for the treatment of allergic rhinitis.

Safety and immunogenicity of GamEvac-Combi, a heterologous VSV- and Ad5-vectored Ebola vaccine: An open phase I/II trial in healthy adults in Russia

PubMed Central

Dolzhikova, I. V.; Zubkova, O. V.; Tukhvatulin, A. I.; Dzharullaeva, A. S.; Tukhvatulina, N. M.; Shcheblyakov, D. V.; Shmarov, M. M.; Tokarskaya, E. A.; Simakova, Y. V.; Egorova, D. A.; Scherbinin, D. N.; Tutykhina, I. L.; Lysenko, A. A.; Kostarnoy, A. V.; Gancheva, P. G.; Ozharovskaya, T. A.; Belugin, B. V.; Kolobukhina, L. V.; Pantyukhov, V. B.; Syromyatnikova, S. I.; Shatokhina, I. V.; Sizikova, T. V.; Rumyantseva, I. G.; Andrus, A. F.; Boyarskaya, N. V.; Voytyuk, A. N.; Babira, V. F.; Volchikhina, S. V.; Kutaev, D. A.; Bel'skih, A. N.; Zhdanov, K. V.; Zakharenko, S. M.; Borisevich, S. V.; Logunov, D. Y.; Naroditsky, B. S.; Gintsburg, A. L.

2017-01-01

ABSTRACT Ebola hemorrhagic fever, also known as Ebola virus disease or EVD, is one of the most dangerous viral diseases in humans and animals. In this open-label, dose-escalation clinical trial, we assessed the safety, side effects, and immunogenicity of a novel, heterologous prime-boost vaccine against Ebola, which was administered in 2 doses to 84 healthy adults of both sexes between 18 and 55 years. The vaccine consists of live-attenuated recombinant vesicular stomatitis virus (VSV) and adenovirus serotype-5 (Ad5) expressing Ebola envelope glycoprotein. The most common adverse event was pain at the injection site, although no serious adverse events were reported. The vaccine did not significantly impact blood, urine, and immune indices. Seroconversion rate was 100 %. Antigen-specific IgG geometric mean titer at day 42 was 3,277 (95 % confidence interval 2,401–4,473) in volunteers immunized at full dose. Neutralizing antibodies were detected in 93.1 % of volunteers immunized at full dose, with geometric mean titer 20. Antigen-specific response in peripheral blood mononuclear cells was also detected in 100 % of participants, as well as in CD4+ and CD8+ T cells in 82.8 % and 58.6 % of participants vaccinated at full dose, respectively. The data indicate that the vaccine is safe and induces strong humoral and cellular immune response in up to 100 % of healthy adult volunteers, and provide a rationale for testing efficacy in Phase III trials. Indeed, the strong immune response to the vaccine may elicit long-term protection. This trial was registered with grls.rosminzdrav.ru (No. 495*), and with zakupki.gov.ru (No. 0373100043215000055). PMID:28152326

Safety and immunogenicity of GamEvac-Combi, a heterologous VSV- and Ad5-vectored Ebola vaccine: An open phase I/II trial in healthy adults in Russia.

PubMed

Dolzhikova, I V; Zubkova, O V; Tukhvatulin, A I; Dzharullaeva, A S; Tukhvatulina, N M; Shcheblyakov, D V; Shmarov, M M; Tokarskaya, E A; Simakova, Y V; Egorova, D A; Scherbinin, D N; Tutykhina, I L; Lysenko, A A; Kostarnoy, A V; Gancheva, P G; Ozharovskaya, T A; Belugin, B V; Kolobukhina, L V; Pantyukhov, V B; Syromyatnikova, S I; Shatokhina, I V; Sizikova, T V; Rumyantseva, I G; Andrus, A F; Boyarskaya, N V; Voytyuk, A N; Babira, V F; Volchikhina, S V; Kutaev, D A; Bel'skih, A N; Zhdanov, K V; Zakharenko, S M; Borisevich, S V; Logunov, D Y; Naroditsky, B S; Gintsburg, A L

2017-03-04

Ebola hemorrhagic fever, also known as Ebola virus disease or EVD, is one of the most dangerous viral diseases in humans and animals. In this open-label, dose-escalation clinical trial, we assessed the safety, side effects, and immunogenicity of a novel, heterologous prime-boost vaccine against Ebola, which was administered in 2 doses to 84 healthy adults of both sexes between 18 and 55 years. The vaccine consists of live-attenuated recombinant vesicular stomatitis virus (VSV) and adenovirus serotype-5 (Ad5) expressing Ebola envelope glycoprotein. The most common adverse event was pain at the injection site, although no serious adverse events were reported. The vaccine did not significantly impact blood, urine, and immune indices. Seroconversion rate was 100 %. Antigen-specific IgG geometric mean titer at day 42 was 3,277 (95 % confidence interval 2,401-4,473) in volunteers immunized at full dose. Neutralizing antibodies were detected in 93.1 % of volunteers immunized at full dose, with geometric mean titer 20. Antigen-specific response in peripheral blood mononuclear cells was also detected in 100 % of participants, as well as in CD4+ and CD8+ T cells in 82.8 % and 58.6 % of participants vaccinated at full dose, respectively. The data indicate that the vaccine is safe and induces strong humoral and cellular immune response in up to 100 % of healthy adult volunteers, and provide a rationale for testing efficacy in Phase III trials. Indeed, the strong immune response to the vaccine may elicit long-term protection. This trial was registered with grls.rosminzdrav.ru (No. 495*), and with zakupki.gov.ru (No. 0373100043215000055).

RNA Adenosine Deaminase ADAR1 Deficiency Leads to Increased Activation of Protein Kinase PKR and Reduced Vesicular Stomatitis Virus Growth Following Interferon Treatment

PubMed Central

Li, Zhiqun; Wolff, Karen C.; Samuel, Charles E.

2009-01-01

Two size forms of ADAR1 adenosine deaminase are known, one constitutively expressed (p110) and the other interferon (IFN)-induced (p150). To test the role of ADAR1 in viral infection, HeLa cells with ADAR1 stably knocked down and 293 cells over expressing ADAR1 were utilized. Over expression of p150 ADAR1 had no significant effect on the yield of vesicular stomatitis virus. Likewise, reduction of p110 and p150 ADAR1 proteins to less than ~10 to 15% of parental levels (ADAR1-deficient) had no significant effect on VSV growth in the absence of IFN treatment. However, inhibition of virus growth following IFN treatment was ~1 log10 further reduced compared to ADAR1-sufficient cells. The level of phosphorylated protein kinase PKR was increased in ADAR1-deficient cells compared to ADAR1-sufficient cells following IFN treatment, regardless of viral infection. These results suggest that ADAR1 suppresses activation of PKR and inhibition of VSV growth in response to IFN treatment. PMID:19913273

Lassa-Vesicular Stomatitis Chimeric Virus Safely Destroys Brain Tumors

PubMed Central

Wollmann, Guido; Drokhlyansky, Eugene; Davis, John N.; Cepko, Connie

2015-01-01

ABSTRACT High-grade tumors in the brain are among the deadliest of cancers. Here, we took a promising oncolytic virus, vesicular stomatitis virus (VSV), and tested the hypothesis that the neurotoxicity associated with the virus could be eliminated without blocking its oncolytic potential in the brain by replacing the neurotropic VSV glycoprotein with the glycoprotein from one of five different viruses, including Ebola virus, Marburg virus, lymphocytic choriomeningitis virus (LCMV), rabies virus, and Lassa virus. Based on in vitro infections of normal and tumor cells, we selected two viruses to test in vivo. Wild-type VSV was lethal when injected directly into the brain. In contrast, a novel chimeric virus (VSV-LASV-GPC) containing genes from both the Lassa virus glycoprotein precursor (GPC) and VSV showed no adverse actions within or outside the brain and targeted and completely destroyed brain cancer, including high-grade glioblastoma and melanoma, even in metastatic cancer models. When mice had two brain tumors, intratumoral VSV-LASV-GPC injection in one tumor (glioma or melanoma) led to complete tumor destruction; importantly, the virus moved contralaterally within the brain to selectively infect the second noninjected tumor. A chimeric virus combining VSV genes with the gene coding for the Ebola virus glycoprotein was safe in the brain and also selectively targeted brain tumors but was substantially less effective in destroying brain tumors and prolonging survival of tumor-bearing mice. A tropism for multiple cancer types combined with an exquisite tumor specificity opens a new door to widespread application of VSV-LASV-GPC as a safe and efficacious oncolytic chimeric virus within the brain. IMPORTANCE Many viruses have been tested for their ability to target and kill cancer cells. Vesicular stomatitis virus (VSV) has shown substantial promise, but a key problem is that if it enters the brain, it can generate adverse neurologic consequences, including death. We tested a series of chimeric viruses containing genes coding for VSV, together with a gene coding for the glycoprotein from other viruses, including Ebola virus, Lassa virus, LCMV, rabies virus, and Marburg virus, which was substituted for the VSV glycoprotein gene. Ebola and Lassa chimeric viruses were safe in the brain and targeted brain tumors. Lassa-VSV was particularly effective, showed no adverse side effects even when injected directly into the brain, and targeted and destroyed two different types of deadly brain cancer, including glioblastoma and melanoma. PMID:25878115

Phase 1 Trials of rVSV Ebola Vaccine in Africa and Europe.

PubMed

Agnandji, Selidji T; Huttner, Angela; Zinser, Madeleine E; Njuguna, Patricia; Dahlke, Christine; Fernandes, José F; Yerly, Sabine; Dayer, Julie-Anne; Kraehling, Verena; Kasonta, Rahel; Adegnika, Akim A; Altfeld, Marcus; Auderset, Floriane; Bache, Emmanuel B; Biedenkopf, Nadine; Borregaard, Saskia; Brosnahan, Jessica S; Burrow, Rebekah; Combescure, Christophe; Desmeules, Jules; Eickmann, Markus; Fehling, Sarah K; Finckh, Axel; Goncalves, Ana Rita; Grobusch, Martin P; Hooper, Jay; Jambrecina, Alen; Kabwende, Anita L; Kaya, Gürkan; Kimani, Domtila; Lell, Bertrand; Lemaître, Barbara; Lohse, Ansgar W; Massinga-Loembe, Marguerite; Matthey, Alain; Mordmüller, Benjamin; Nolting, Anne; Ogwang, Caroline; Ramharter, Michael; Schmidt-Chanasit, Jonas; Schmiedel, Stefan; Silvera, Peter; Stahl, Felix R; Staines, Henry M; Strecker, Thomas; Stubbe, Hans C; Tsofa, Benjamin; Zaki, Sherif; Fast, Patricia; Moorthy, Vasee; Kaiser, Laurent; Krishna, Sanjeev; Becker, Stephan; Kieny, Marie-Paule; Bejon, Philip; Kremsner, Peter G; Addo, Marylyn M; Siegrist, Claire-Anne

2016-04-28

The replication-competent recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing a Zaire ebolavirus (ZEBOV) glycoprotein was selected for rapid safety and immunogenicity testing before its use in West Africa. We performed three open-label, dose-escalation phase 1 trials and one randomized, double-blind, controlled phase 1 trial to assess the safety, side-effect profile, and immunogenicity of rVSV-ZEBOV at various doses in 158 healthy adults in Europe and Africa. All participants were injected with doses of vaccine ranging from 300,000 to 50 million plaque-forming units (PFU) or placebo. No serious vaccine-related adverse events were reported. Mild-to-moderate early-onset reactogenicity was frequent but transient (median, 1 day). Fever was observed in up to 30% of vaccinees. Vaccine viremia was detected within 3 days in 123 of the 130 participants (95%) receiving 3 million PFU or more; rVSV was not detected in saliva or urine. In the second week after injection, arthritis affecting one to four joints developed in 11 of 51 participants (22%) in Geneva, with pain lasting a median of 8 days (interquartile range, 4 to 87); 2 self-limited cases occurred in 60 participants (3%) in Hamburg, Germany, and Kilifi, Kenya. The virus was identified in one synovial-fluid aspirate and in skin vesicles of 2 other vaccinees, showing peripheral viral replication in the second week after immunization. ZEBOV-glycoprotein-specific antibody responses were detected in all the participants, with similar glycoprotein-binding antibody titers but significantly higher neutralizing antibody titers at higher doses. Glycoprotein-binding antibody titers were sustained through 180 days in all participants. In these studies, rVSV-ZEBOV was reactogenic but immunogenic after a single dose and warrants further evaluation for safety and efficacy. (Funded by the Wellcome Trust and others; ClinicalTrials.gov numbers, NCT02283099, NCT02287480, and NCT02296983; Pan African Clinical Trials Registry number, PACTR201411000919191.).

Lentiviral Protein Transfer Vectors Are an Efficient Vaccine Platform and Induce a Strong Antigen-Specific Cytotoxic T Cell Response

PubMed Central

Uhlig, Katharina M.; Schülke, Stefan; Scheuplein, Vivian A. M.; Malczyk, Anna H.; Reusch, Johannes; Kugelmann, Stefanie; Muth, Anke; Koch, Vivian; Hutzler, Stefan; Bodmer, Bianca S.; Schambach, Axel; Buchholz, Christian J.; Waibler, Zoe; Scheurer, Stephan

2015-01-01

ABSTRACT To induce and trigger innate and adaptive immune responses, antigen-presenting cells (APCs) take up and process antigens. Retroviral particles are capable of transferring not only genetic information but also foreign cargo proteins when they are genetically fused to viral structural proteins. Here, we demonstrate the capacity of lentiviral protein transfer vectors (PTVs) for targeted antigen transfer directly into APCs and thereby induction of cytotoxic T cell responses. Targeting of lentiviral PTVs to APCs can be achieved analogously to gene transfer vectors by pseudotyping the particles with truncated wild-type measles virus (MV) glycoproteins (GPs), which use human SLAM (signaling lymphocyte activation molecule) as a main entry receptor. SLAM is expressed on stimulated lymphocytes and APCs, including dendritic cells. SLAM-targeted PTVs transferred the reporter protein green fluorescent protein (GFP) or Cre recombinase with strict receptor specificity into SLAM-expressing CHO and B cell lines, in contrast to broadly transducing vesicular stomatitis virus G protein (VSV-G) pseudotyped PTVs. Primary myeloid dendritic cells (mDCs) incubated with targeted or nontargeted ovalbumin (Ova)-transferring PTVs stimulated Ova-specific T lymphocytes, especially CD8+ T cells. Administration of Ova-PTVs into SLAM-transgenic and control mice confirmed the observed predominant induction of antigen-specific CD8+ T cells and demonstrated the capacity of protein transfer vectors as suitable vaccines for the induction of antigen-specific immune responses. IMPORTANCE This study demonstrates the specificity and efficacy of antigen transfer by SLAM-targeted and nontargeted lentiviral protein transfer vectors into antigen-presenting cells to trigger antigen-specific immune responses in vitro and in vivo. The observed predominant activation of antigen-specific CD8+ T cells indicates the suitability of SLAM-targeted and also nontargeted PTVs as a vaccine for the induction of cytotoxic immune responses. Since cytotoxic CD8+ T lymphocytes are a mainstay of antitumoral immune responses, PTVs could be engineered for the transfer of specific tumor antigens provoking tailored antitumoral immunity. Therefore, PTVs can be used as safe and efficient alternatives to gene transfer vectors or live attenuated replicating vector platforms, avoiding genotoxicity or general toxicity in highly immunocompromised patients, respectively. Thereby, the potential for easy envelope exchange allows the circumventing of neutralizing antibodies, e.g., during repeated boost immunizations. PMID:26085166

Management of accidental exposure to Ebola virus in the biosafety level 4 laboratory, Hamburg, Germany.

PubMed

Günther, Stephan; Feldmann, Heinz; Geisbert, Thomas W; Hensley, Lisa E; Rollin, Pierre E; Nichol, Stuart T; Ströher, Ute; Artsob, Harvey; Peters, Clarence J; Ksiazek, Thomas G; Becker, Stephan; ter Meulen, Jan; Olschläger, Stephan; Schmidt-Chanasit, Jonas; Sudeck, Hinrich; Burchard, Gerd D; Schmiedel, Stefan

2011-11-01

A needlestick injury occurred during an animal experiment in the biosafety level 4 laboratory in Hamburg, Germany, in March 2009. The syringe contained Zaire ebolavirus (ZEBOV) mixed with Freund's adjuvant. Neither an approved treatment nor a postexposure prophylaxis (PEP) exists for Ebola hemorrhagic fever. Following a risk-benefit assessment, it was recommended the exposed person take an experimental vaccine that had shown PEP efficacy in ZEBOV-infected nonhuman primates (NHPs) [12]. The vaccine, which had not been used previously in humans, was a live-attenuated recombinant vesicular stomatitis virus (recVSV) expressing the glycoprotein of ZEBOV. A single dose of 5 × 10(7) plaque-forming units was injected 48 hours after the accident. The vaccinee developed fever 12 hours later and recVSV viremia was detectable by polymerase chain reaction (PCR) for 2 days. Otherwise, the person remained healthy, and ZEBOV RNA, except for the glycoprotein gene expressed in the vaccine, was never detected in serum and peripheral blood mononuclear cells during the 3-week observation period.

Whole-body voxel-based personalized dosimetry: Multiple voxel S-value approach for heterogeneous media with non-uniform activity distributions.

PubMed

Lee, Min Sun; Kim, Joong Hyun; Paeng, Jin Chul; Kang, Keon Wook; Jeong, Jae Min; Lee, Dong Soo; Lee, Jae Sung

2017-12-14

Personalized dosimetry with high accuracy is becoming more important because of the growing interests in personalized medicine and targeted radionuclide therapy. Voxel-based dosimetry using dose point kernel or voxel S-value (VSV) convolution is available. However, these approaches do not consider medium heterogeneity. Here, we propose a new method for whole-body voxel-based personalized dosimetry for heterogeneous media with non-uniform activity distributions, which is referred to as the multiple VSV approach. Methods: The multiple numbers (N) of VSVs for media with different densities covering the whole-body density ranges were used instead of using only a single VSV for water. The VSVs were pre-calculated using GATE Monte Carlo simulation; those were convoluted with the time-integrated activity to generate density-specific dose maps. Computed tomography-based segmentation was conducted to generate binary maps for each density region. The final dose map was acquired by the summation of N segmented density-specific dose maps. We tested several sets of VSVs with different densities: N = 1 (single water VSV), 4, 6, 8, 10, and 20. To validate the proposed method, phantom and patient studies were conducted and compared with direct Monte Carlo, which was considered the ground truth. Finally, patient dosimetry (10 subjects) was conducted using the multiple VSV approach and compared with the single VSV and organ-based dosimetry approaches. Errors at the voxel- and organ-levels were reported for eight organs. Results: In the phantom and patient studies, the multiple VSV approach showed significant improvements regarding voxel-level errors, especially for the lung and bone regions. As N increased, voxel-level errors decreased, although some overestimations were observed at lung boundaries. In the case of multiple VSVs ( N = 8), we achieved voxel-level errors of 2.06%. In the dosimetry study, our proposed method showed much improved results compared to the single VSV and organ-based dosimetry. Errors at the organ-level were -6.71%, 2.17%, and 227.46% for the single VSV, multiple VSV, and organ-based dosimetry, respectively. Conclusion: The multiple VSV approach for heterogeneous media with non-uniform activity distributions offers fast personalized dosimetry at whole-body level, yielding results comparable to those of the direct Monte Carlo approach. Copyright © 2017 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Interferon-Induced Ifit2/ISG54 Protects Mice from Lethal VSV Neuropathogenesis

PubMed Central

Fensterl, Volker; Wetzel, Jaime L.; Ramachandran, Srividya; Ogino, Tomoaki; Stohlman, Stephen A.; Bergmann, Cornelia C.; Diamond, Michael S.; Virgin, Herbert W.; Sen, Ganes C.

2012-01-01

Interferon protects mice from vesicular stomatitis virus (VSV) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (ISG) mediate the antiviral effect. A prominent family of ISGs is the interferon-induced with tetratricopeptide repeats (Ifit) genes comprising three members in mice, Ifit1/ISG56, Ifit2/ISG54 and Ifit3/ISG49. Intranasal infection with a low dose of VSV is not lethal to wild-type mice and all three Ifit genes are induced in the central nervous system of the infected mice. We tested their potential contributions to the observed protection of wild-type mice from VSV pathogenesis, by taking advantage of the newly generated knockout mice lacking either Ifit2 or Ifit1. We observed that in Ifit2 knockout (Ifit2 −/−) mice, intranasal VSV infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. In contrast, wild-type and Ifit1 −/− mice were highly protected and survived without developing such disease. However, when VSV was injected intracranially, virus replication and survival were not significantly different between wild-type and Ifit2−/− mice. When administered intranasally, VSV entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and Ifit2 −/− mice and induced interferon-β. However, as the infection spread to other regions of the brain, VSV titers rose several hundred folds higher in Ifit2 −/− mice as compared to wild-type mice. This was not caused by a broadened cell tropism in the brains of Ifit2 −/− mice, where VSV still replicated selectively in neurons. Surprisingly, this advantage for VSV replication in the brains of Ifit2−/− mice was not observed in other organs, such as lung and liver. Pathogenesis by another neurotropic RNA virus, encephalomyocarditis virus, was not enhanced in the brains of Ifit2 −/− mice. Our study provides a clear demonstration of tissue-, virus- and ISG-specific antiviral action of interferon. PMID:22615570

Comparative oncology evaluation of intravenous recombinant oncolytic Vesicular Stomatitis Virus therapy in spontaneous canine cancer

PubMed Central

Naik, Shruthi; Galyon, Gina D.; Jenks, Nathan J.; Steele, Michael B.; Miller, Amber C.; Allstadt, Sara D.; Suksanpaisan, Lukkana; Peng, Kah Whye; Federspiel, Mark J.; Russell, Stephen J.; LeBlanc, Amy K.

2017-01-01

Clinical translation of intravenous therapies to treat disseminated or metastatic cancer is imperative. Comparative oncology, the evaluation of novel cancer therapies in animals with spontaneous cancer, can be utilized to inform and accelerate clinical translation. Preclinical murine studies demonstrate that single shot systemic therapy with a VSV-IFNβ-NIS, a novel recombinant oncolytic Vesicular stomatitis virus (VSV), can induce curative remission in tumor bearing mice. Clinical translation of VSV-IFNβ-NIS therapy is dependent on comprehensive assessment of clinical toxicities, virus shedding, pharmacokinetics, and efficacy in clinically relevant models. Dogs spontaneously develop cancer with comparable etiology, clinical progression and response to therapy as human malignancies. A comparative oncology study was carried out to investigate feasibility and tolerability of intravenous oncolytic VSV-IFNβ-NIS therapy in pet dogs with spontaneous cancer. Nine dogs with various malignancies were treated with a single intravenous dose of VSV-IFNβ-NIS. Two dogs with high-grade peripheral T-cell lymphoma had rapid but transient remission of disseminated disease and transient hepatotoxicity that resolved spontaneously. There was no shedding of infectious virus. Correlative pharmacokinetic studies revealed elevated levels of VSV RNA in blood in dogs with measurable disease remission. This is the first evaluation of intravenous oncolytic virus therapy for spontaneous canine cancer, demonstrating that VSV-IFNβ-NIS is well-tolerated and safe in dogs with advanced or metastatic disease. This approach has informed clinical translation, including dose and target indication selection, leading to a clinical investigation of intravenous VSV-IFNβ-NIS therapy, and provided preliminary evidence of clinical efficacy, and potential biomarkers that correlate with therapeutic response. PMID:29158470

Pseudotyped Lentiviral Vectors for Retrograde Gene Delivery into Target Brain Regions

PubMed Central

Kobayashi, Kenta; Inoue, Ken-ichi; Tanabe, Soshi; Kato, Shigeki; Takada, Masahiko; Kobayashi, Kazuto

2017-01-01

Gene transfer through retrograde axonal transport of viral vectors offers a substantial advantage for analyzing roles of specific neuronal pathways or cell types forming complex neural networks. This genetic approach may also be useful in gene therapy trials by enabling delivery of transgenes into a target brain region distant from the injection site of the vectors. Pseudotyping of a lentiviral vector based on human immunodeficiency virus type 1 (HIV-1) with various fusion envelope glycoproteins composed of different combinations of rabies virus glycoprotein (RV-G) and vesicular stomatitis virus glycoprotein (VSV-G) enhances the efficiency of retrograde gene transfer in both rodent and nonhuman primate brains. The most recently developed lentiviral vector is a pseudotype with fusion glycoprotein type E (FuG-E), which demonstrates highly efficient retrograde gene transfer in the brain. The FuG-E–pseudotyped vector permits powerful experimental strategies for more precisely investigating the mechanisms underlying various brain functions. It also contributes to the development of new gene therapy approaches for neurodegenerative disorders, such as Parkinson’s disease, by delivering genes required for survival and protection into specific neuronal populations. In this review article, we report the properties of the FuG-E–pseudotyped vector, and we describe the application of the vector to neural circuit analysis and the potential use of the FuG-E vector in gene therapy for Parkinson’s disease. PMID:28824385

Reversal of papilloma growth in rabbits therapeutically vaccinated against E6 with naked DNA and/or vesicular stomatitis virus vectors

PubMed Central

Brandsma, Janet L.; Shlyankevich, Mark; Su, Yuhua; Zelterman, Daniel; Rose, John K.; Buonocore, Linda

2009-01-01

Persistent infection with high-risk human papillomaviruses (HPVs) is the greatest risk factor for the development of HPV-associated cancers. In this study rabbits bearing persistent and potentially malignant papillomas were used to test the efficacy of vaccination with a recombinant DNA and/or vesicular stomatitis virus (VSV) targeting the cottontail rabbit papillomavirus (CRPV) E6 protein. Immune responses were primed with either vector and boosted twice with the homologous or heterologous E6 vector. Over the course of 18 weeks, E6 vaccination reduced papilloma volumes to one third the volume in the controls, and the rabbits boosted with an heterologous vector tended to mount stronger responses. Small and medium-sized papillomas responded significantly but only slightly better than large papillomas. Finally the initial papilloma burden per rabbit, ranging from <100 mm3 to >1000 mm3, was not prognostic of antitumor efficacy. In summary both E6 vaccines elicited significant therapeutic immunity, and their sequential use tended to be advantageous. PMID:19615481

Therapeutic Effect of Duck Interferon-Alpha Against H5N1 Highly Pathogenic Avian Influenza Virus Infection in Peking Ducks.

PubMed

Gao, Pei; Xiang, Bin; Li, Yulian; Li, Yaling; Sun, Minhua; Kang, Yinfeng; Xie, Peng; Chen, Libin; Lin, Qiuyan; Liao, Ming; Ren, Tao

2018-04-01

The antiviral cytokine interferon-alpha (IFN-α) plays a critical role in the innate immune system. Previous studies have shown that recombinant chicken IFN-α inhibits avian influenza virus (AIV) replication in vivo; however, the antiviral effect of recombinant duck IFN-α (rDuIFN-α) on highly pathogenic AIV remains unknown. In this study, the duck IFN-α gene was cloned, expressed, and purified. The antiviral effects of the resulting rDuIFN-α were further evaluated in vitro and in vivo. Our results showed that rDuIFN-α inhibited the replication of vesicular stomatitis virus (VSV) and AIV in duck embryo fibroblasts in vitro, with antiviral activities against VSV and AIV of 2.1 × 10 5 and 4.1 × 10 5 U/mg, respectively. We next investigated the anti-H5N1 AIV effect of intramuscular injection of rDuIFN-α in vivo. rDuIFN-α reduced viral titers in the brains, lungs, and spleens of 2-day-old (2D) ducks compared with that in the virus-challenged control group, and pretreatment with rDuIFN-α reduced mortality from 60% to 10% in 2D ducks. Moreover, rDuIFN-α increased the expression of IFN-stimulated genes in the brains and spleens of 2D ducks. Our results demonstrate that rDuIFN-α blocks VSV and H5N1 influenza virus infection in vitro and exhibits antiviral effects against H5N1 influenza virus infection in 2D ducks.

Characterization of a 3rd Generation Lentiviral Vector Pseudotyped With Nipah Virus Envelope Proteins For Endothelial Cell Transduction

PubMed Central

Witting, Scott R.; Vallanda, Priya; Gamble, Aisha L.

2013-01-01

Lentiviruses are becoming progressively more popular as gene therapy vectors due to their ability to integrate into quiescent cells and recent clinical trial successes. Directing these vectors to specific cell types and limiting off-target transduction in vivo remains a challenge. Replacing the viral envelope proteins responsible for cellular binding, or pseudotyping, remains a common method to improve lentiviral targeting. Here, we describe the development of a high titer, 3rd generation lentiviral vector pseudotyped with Nipah virus fusion protein (NiV-F) and attachment protein (NiV-G). Critical to high titers was truncation of the cytoplasmic domains of both NiV-F and NiV-G. As known targets of wild-type Nipah virus, primary endothelial cells are shown to be effectively transduced by the Nipah pseudotype. In contrast, human CD34+ hematopoietic progenitors were not significantly transduced. Additionally, the Nipah pseudotype has increased stability in human serum compared to VSV pseudotyped lentivirus. These findings suggest that the use of Nipah virus envelope proteins in 3rd generation lentiviral vectors would be a valuable tool for gene delivery targeted to endothelial cells. PMID:23698741

Transgenic chickens expressing human urokinase-type plasminogen activator.

PubMed

Lee, Sung Ho; Gupta, Mukesh Kumar; Ho, Young Tae; Kim, Teoan; Lee, Hoon Taek

2013-09-01

Urokinase-type plasminogen activator is a serine protease that is clinically used in humans for the treatment of thrombolytic disorders and vascular diseases such as acute ischemic stroke and acute peripheral arterial occlusion. This study explored the feasibility of using chickens as a bioreactor for producing human urokinase-type plasminogen activator (huPA). Recombinant huPA gene, under the control of a ubiquitous Rous sarcoma virus promoter, was injected into the subgerminal cavity of freshly laid chicken eggs at stage X using the replication-defective Moloney murine leukemia virus (MoMLV)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein. A total of 38 chicks, out of 573 virus-injected eggs, hatched and contained the huPA gene in their various body parts. The mRNA transcript of the huPA gene was present in various organs, including blood and egg, and was germ-line transmitted to the next generation. The level of active huPA protein was 16-fold higher in the blood of the transgenic chicken than in the nontransgenic chicken (P < 0.05). The expression of huPA protein in eggs increased from 7.82 IU/egg in the G0 generation to 17.02 IU/egg in the G1 generation. However, huPA-expressing embryos had reduced survival and hatchability at d 18 and 21 of incubation, respectively, and the blood clotting time was significantly higher in transgenic chickens than their nontransgenic counterparts (P < 0.05). Furthermore, adult transgenic rooster showed reduced (P < 0.05) fertility, as revealed by reduced volume of semen ejaculate, sperm concentration, and sperm viability. Taken together, our data suggest that huPA transgenic chickens could be successfully produced by the retroviral vector system. Transgenic chickens, expressing the huPA under the control of a ubiquitous promoter, may not only be used as a bioreactor for pharming of the huPA drug but also be useful for studying huPA-induced bleeding and other disorders.

A Recombinant Vesicular Stomatitis Virus Ebola Vaccine.

PubMed

Regules, Jason A; Beigel, John H; Paolino, Kristopher M; Voell, Jocelyn; Castellano, Amy R; Hu, Zonghui; Muñoz, Paula; Moon, James E; Ruck, Richard C; Bennett, Jason W; Twomey, Patrick S; Gutiérrez, Ramiro L; Remich, Shon A; Hack, Holly R; Wisniewski, Meagan L; Josleyn, Matthew D; Kwilas, Steven A; Van Deusen, Nicole; Mbaya, Olivier Tshiani; Zhou, Yan; Stanley, Daphne A; Jing, Wang; Smith, Kirsten S; Shi, Meng; Ledgerwood, Julie E; Graham, Barney S; Sullivan, Nancy J; Jagodzinski, Linda L; Peel, Sheila A; Alimonti, Judie B; Hooper, Jay W; Silvera, Peter M; Martin, Brian K; Monath, Thomas P; Ramsey, W Jay; Link, Charles J; Lane, H Clifford; Michael, Nelson L; Davey, Richard T; Thomas, Stephen J

2017-01-26

The worst Ebola virus disease (EVD) outbreak in history has resulted in more than 28,000 cases and 11,000 deaths. We present the final results of two phase 1 trials of an attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV)-based vaccine candidate designed to prevent EVD. We conducted two phase 1, placebo-controlled, double-blind, dose-escalation trials of an rVSV-based vaccine candidate expressing the glycoprotein of a Zaire strain of Ebola virus (ZEBOV). A total of 39 adults at each site (78 participants in all) were consecutively enrolled into groups of 13. At each site, volunteers received one of three doses of the rVSV-ZEBOV vaccine (3 million plaque-forming units [PFU], 20 million PFU, or 100 million PFU) or placebo. Volunteers at one of the sites received a second dose at day 28. Safety and immunogenicity were assessed. The most common adverse events were injection-site pain, fatigue, myalgia, and headache. Transient rVSV viremia was noted in all the vaccine recipients after dose 1. The rates of adverse events and viremia were lower after the second dose than after the first dose. By day 28, all the vaccine recipients had seroconversion as assessed by an enzyme-linked immunosorbent assay (ELISA) against the glycoprotein of the ZEBOV-Kikwit strain. At day 28, geometric mean titers of antibodies against ZEBOV glycoprotein were higher in the groups that received 20 million PFU or 100 million PFU than in the group that received 3 million PFU, as assessed by ELISA and by pseudovirion neutralization assay. A second dose at 28 days after dose 1 significantly increased antibody titers at day 56, but the effect was diminished at 6 months. This Ebola vaccine candidate elicited anti-Ebola antibody responses. After vaccination, rVSV viremia occurred frequently but was transient. These results support further evaluation of the vaccine dose of 20 million PFU for preexposure prophylaxis and suggest that a second dose may boost antibody responses. (Funded by the National Institutes of Health and others; rVSV∆G-ZEBOV-GP ClinicalTrials.gov numbers, NCT02269423 and NCT02280408 .).

Rhabdovirus evasion of the interferon system.

PubMed

Rieder, Martina; Conzelmann, Karl-Klaus

2009-09-01

The family Rhabdoviridae contains important pathogens of humans, livestock, and crops, including the insect-transmitted vesicular stomatitis virus (VSV) and the neurotropic rabies virus (RV), which is directly transmitted between mammals. In spite of a highly similar organization of RNA genomes, proteins, and virus particles, cell biology of VSV and RV is divergent in several aspects, particularly with respect to their interplay with the cellular host defense. While infection with both rhabdoviruses is recognized via viral triphosphate RNAs by the cytoplasmic RNA helicase/translocase RIG-I, the viral counteractions to limit the response are contrasting. VSV infection is characterized by a rapid general shutdown of host gene expression and severe cytopathic effects, due to multiple activities of the matrix (M) protein affecting host polymerase functions and mRNA nuclear export, and by rapid and high-level virus replication. In contrast, RV spread and transmission relies on preserving the integrity of host cells, particularly of neurons. While a general cell shutdown by RV M is not observed, RV phosphoprotein (P) has developed independent functions to interfere with activation of IRFs and with STAT signaling. The molecular mechanisms employed are different from those of the paramyxovirus P gene products serving similar functions, and illustrate evolution of IFN antagonists to specifically support virus survival in the natural niches.

Efficacy of Vesicular Stomatitis Virus-Ebola Virus Postexposure Treatment in Rhesus Macaques Infected With Ebola Virus Makona.

PubMed

Marzi, Andrea; Hanley, Patrick W; Haddock, Elaine; Martellaro, Cynthia; Kobinger, Gary; Feldmann, Heinz

2016-10-15

The Ebola virus (EBOV) epidemic in West Africa increased the focus on vaccine development against this hemorrhagic fever-causing pathogen, and as a consequence human clinical trials for a few selected platforms were accelerated. One of these vaccines is vesicular stomatitis virus (VSV)-EBOV, also known as rVSV-ZEBOV, a fast-acting vaccine against EBOV and so far the only vaccine with reported efficacy against EBOV infections in humans in phase III clinical trials. In this study, we analyzed the potential of VSV-EBOV for postexposure treatment of rhesus macaques infected with EBOV-Makona. We treated groups of animals with 1 dose of VSV-EBOV either in a single injection at 1 or 24 hours after EBOV exposure or with 2 injections, half the dose at each time point; 1 control group received the same dose of the VSV-based Marburg virus vaccine at both time points; another group remained untreated. Although all untreated animals succumbed to EBOV infection, 33%-67% of the animals in each treatment group survived the infection, including the group treated with the VSV-based Marburg virus vaccine. This result suggests that protection from postexposure vaccination may be antigen unspecific and due rather to an early activation of the innate immune system. In conclusion, VSV-EBOV remains a potent and fast-acting prophylactic vaccine but demonstrates only limited efficacy in postexposure treatment. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Filone, Claire Marie; Heise, Mark; Doms, Robert W.

2006-12-20

Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expressionmore » of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by {beta}-galactosidase {alpha}-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.« less

Sensitivity of Breast Tumors to Oncolytic Viruses

DTIC Science & Technology

2006-08-01

therapies for breast cancer based on the oncolytic virus, vesicular stomatitis virus (VSV). Studies have shown that matrix (M) protein mutants of VSV, such...more resistant to VSV-induced cytopathic effect than breast cancer cells. However, in syngeneic breast cancer system in vivo, rM51R-M virus is only...interleukin 12, breast cancer , interferon 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE

Safety studies on intravenous administration of oncolytic recombinant vesicular stomatitis virus in purpose-bred beagle dogs.

PubMed

LeBlanc, Amy K; Naik, Shruthi; Galyon, Gina D; Jenks, Nathan; Steele, Mike; Peng, Kah-Whye; Federspiel, Mark J; Donnell, Robert; Russell, Stephen J

2013-12-01

VSV-IFNβ-NIS is a novel recombinant oncolytic vesicular stomatitis virus (VSV) with documented efficacy and safety in preclinical murine models of cancer. To facilitate clinical translation of this promising oncolytic therapy in patients with disseminated cancer, we are utilizing a comparative oncology approach to gather data describing the safety and efficacy of systemic VSV-IFNβ-NIS administration in dogs with naturally occurring cancer. In support of this, we executed a dose-escalation study in purpose-bred dogs to determine the maximum tolerated dose (MTD) of systemic VSV-hIFNβ-NIS, characterize the adverse event profile, and describe routes and duration of viral shedding in healthy, immune-competent dogs. The data indicate that an intravenous dose of 10(10) TCID50 is well tolerated in dogs. Expected adverse events were mild to moderate fever, self-limiting nausea and vomiting, lymphopenia, and oral mucosal lesions. Unexpected adverse events included prolongation of partial thromboplastin time, development of bacterial urinary tract infection, and scrotal dermatitis, and in one dog receiving 10(11) TCID50 (10 × the MTD), the development of severe hepatotoxicity and symptoms of shock leading to euthanasia. Viral shedding data indicate that detectable viral genome in blood diminishes rapidly with anti-VSV neutralizing antibodies detectable in blood as early as day 5 postintravenous virus administration. While low levels of viral genome copies were detectable in plasma, urine, and buccal swabs of dogs treated at the MTD, no infectious virus was detectable in plasma, urine, or buccal swabs at any of the doses tested. These studies confirm that VSV can be safely administered systemically in dogs, justifying the use of oncolytic VSV as a novel therapy for the treatment of canine cancer.

Fluvastatin delays propagation of viral infection in isolated rat FDB myofibers but does not affect exocytic membrane trafficking.

PubMed

Nevalainen, Mika; Metsikkö, Kalervo

2015-11-01

We have utilized the enveloped viral model to study the effect of fluvastatin on membrane trafficking in isolated rat myofibers. Our immunofluorescence studies constantly showed that infections in myofibers, which were treated with fluvastatin prior and during the infection with either vesicular stomatitis virus (VSV) or influenza A virus, propagated more slowly than in control myofibers without drug treatment. Experiments with a virus expressing Dad1 tagged with green fluorescent protein (GFP-Dad1) showed that fluvastatin did not affect its distribution within the ER/SR network and immunofluorescence staining for GM130 did not show any marked effect on the structure of the Golgi components. Furthermore, fluvastatin did not inhibit trafficking of the chimeric transport marker VSV temperature sensitive G protein (tsG-GFP) from the ER to the Golgi. We next subjected VSV infected myofibers for pulse-chase labeling experiments and found that fluvastatin did not slow down the ER-to-Golgi trafficking or Golgi to plasma membrane trafficking of the viral glycoprotein. These studies show that fluvastatin inhibited the propagation of viral infection in skeletal myofibers but no adverse effect on the exocytic trafficking could be demonstrated. These results suggest that other effects of statins rather than inhibition of ER-to-Golgi trafficking might be behind the myotoxic effects of the statins. © 2015 International Federation for Cell Biology.

Immunogenicity and efficacy of immunodeficiency virus-like particles pseudotyped with the G protein of vesicular stomatitis virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuate, Seraphin; Stahl-Hennig, Christiane; Stoiber, Heribert

2006-07-20

Vaccination with exogenous antigens such as recombinant viral proteins, immunodeficiency virus-derived whole inactivated virus particles, or virus-like particles (VLP) has generally failed to provide sufficient protection in animal models for AIDS. Pseudotyping VLPs with the vesicular stomatitis virus G protein (VSV-G), which is known to mediate entry into dendritic cells, might allow more efficient stimulation of immune responses. Therefore, we pseudotyped noninfectious immunodeficiency virus-like particles with VSV-G and carried out a preliminary screen of their immunogenicity and vaccination efficacy. Incorporation of VSV-G into HIV-1 VLPs led to hundred-fold higher antibody titers to HIV-1 Gag and enhancement of T cell responsesmore » in mice. Repeated vaccination of rhesus monkeys for 65 weeks with VSV-G pseudotyped simian immunodeficiency virus (SIV)-like particles (VLP[G]) provided initial evidence for efficient suppression of viral load after mucosal challenge with the SIVmac239 virus. Challenge of monkeys after a 28 week vaccination regimen with VLP[G] led to a reduction in peak viremia, but persistent suppression of viral load was not achieved. Due to limitations in the number of animals available for this study, improved efficacy of VSV-G pseudotyped VLPs in nonhuman primates could not be demonstrated. However, mouse experiments revealed that pseudotyping of VLPs with fusion-competent VSV-G clearly improves their immunogenicity. Additional strategies, particularly adjuvants, should be considered to provide greater protection against a challenge with pathogenic immunodeficiency virus.« less

Mutated BRAF Emerges as a Major Effector of Recurrence in a Murine Melanoma Model After Treatment With Immunomodulatory Agents.

PubMed

Zaidi, Shane; Blanchard, Miran; Shim, Kevin; Ilett, Elizabeth; Rajani, Karishma; Parrish, Christopher; Boisgerault, Nicolas; Kottke, Tim; Thompson, Jill; Celis, Esteban; Pulido, Jose; Selby, Peter; Pandha, Hardev; Melcher, Alan; Harrington, Kevin; Vile, Richard

2015-05-01

We used a VSV-cDNA library to treat recurrent melanoma, identifying immunogenic antigens, allowing us to target recurrences with immunotherapy or chemotherapy. Primary B16 melanoma tumors were induced to regress by frontline therapy. Mice with recurrent tumors were treated with VSV-cDNA immunotherapy. A Th17 recall response was used to screen the VSV-cDNA library for individual viruses encoding rejection antigens, subsequently targeted using immunotherapy or chemotherapy. Recurrent tumors were effectively treated with a VSV-cDNA library using cDNA from recurrent B16 tumors. Recurrence-associated rejection antigens identified included Topoisomerase-IIα, YB-1, cdc7 kinase, and BRAF. Fourteen out of 16 recurrent tumors carried BRAF mutations (595-605 region) following frontline therapy, even though the parental B16 tumors were BRAF wild type. The emergence of mutated BRAF-containing recurrences served as an excellent target for BRAF-specific immune-(VSV-BRAF), or chemo-(PLX-4720) therapies. Successful PLX-4720 therapy of recurrent tumors was associated with the development of a broad spectrum of T-cell responses. VSV-cDNA technology can be used to identify recurrence specific antigens. Emergence of mutated BRAF may be a major effector of melanoma recurrence which could serve as a target for chemo or immune therapy. This study suggests a rationale for offering patients with initially wild-type BRAF melanomas an additional biopsy to screen for mutant BRAF upon recurrence.

Mutated BRAF Emerges as a Major Effector of Recurrence in a Murine Melanoma Model After Treatment With Immunomodulatory Agents

PubMed Central

Zaidi, Shane; Blanchard, Miran; Shim, Kevin; Ilett, Elizabeth; Rajani, Karishma; Parrish, Christopher; Boisgerault, Nicolas; Kottke, Tim; Thompson, Jill; Celis, Esteban; Pulido, Jose; Selby, Peter; Pandha, Hardev; Melcher, Alan; Harrington, Kevin; Vile, Richard

2015-01-01

We used a VSV-cDNA library to treat recurrent melanoma, identifying immunogenic antigens, allowing us to target recurrences with immunotherapy or chemotherapy. Primary B16 melanoma tumors were induced to regress by frontline therapy. Mice with recurrent tumors were treated with VSV-cDNA immunotherapy. A Th17 recall response was used to screen the VSV-cDNA library for individual viruses encoding rejection antigens, subsequently targeted using immunotherapy or chemotherapy. Recurrent tumors were effectively treated with a VSV-cDNA library using cDNA from recurrent B16 tumors. Recurrence-associated rejection antigens identified included Topoisomerase-IIα, YB-1, cdc7 kinase, and BRAF. Fourteen out of 16 recurrent tumors carried BRAF mutations (595–605 region) following frontline therapy, even though the parental B16 tumors were BRAF wild type. The emergence of mutated BRAF-containing recurrences served as an excellent target for BRAF-specific immune-(VSV-BRAF), or chemo-(PLX-4720) therapies. Successful PLX-4720 therapy of recurrent tumors was associated with the development of a broad spectrum of T-cell responses. VSV-cDNA technology can be used to identify recurrence specific antigens. Emergence of mutated BRAF may be a major effector of melanoma recurrence which could serve as a target for chemo or immune therapy. This study suggests a rationale for offering patients with initially wild-type BRAF melanomas an additional biopsy to screen for mutant BRAF upon recurrence. PMID:25544599

Biarsenical labeling of vesicular stomatitis virus encoding tetracysteine-tagged m protein allows dynamic imaging of m protein and virus uncoating in infected cells.

PubMed

Das, Subash C; Panda, Debasis; Nayak, Debasis; Pattnaik, Asit K

2009-03-01

A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-DeltaM-Mtc and VSV-DeltaM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-DeltaM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-DeltaM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

A recombinant vesicular stomatitis virus-based Lassa fever vaccine protects guinea pigs and macaques against challenge with geographically and genetically distinct Lassa viruses.

PubMed

Safronetz, David; Mire, Chad; Rosenke, Kyle; Feldmann, Friederike; Haddock, Elaine; Geisbert, Thomas; Feldmann, Heinz

2015-04-01

Lassa virus (LASV) is endemic in several West African countries and is the etiological agent of Lassa fever. Despite the high annual incidence and significant morbidity and mortality rates, currently there are no approved vaccines to prevent infection or disease in humans. Genetically, LASV demonstrates a high degree of diversity that correlates with geographic distribution. The genetic heterogeneity observed between geographically distinct viruses raises concerns over the potential efficacy of a "universal" LASV vaccine. To date, several experimental LASV vaccines have been developed; however, few have been evaluated against challenge with various genetically unique Lassa virus isolates in relevant animal models. Here we demonstrate that a single, prophylactic immunization with a recombinant vesicular stomatitis virus (VSV) expressing the glycoproteins of LASV strain Josiah from Sierra Leone protects strain 13 guinea pigs from infection / disease following challenge with LASV isolates originating from Liberia, Mali and Nigeria. Similarly, the VSV-based LASV vaccine yields complete protection against a lethal challenge with the Liberian LASV isolate in the gold-standard macaque model of Lassa fever. Our results demonstrate the VSV-based LASV vaccine is capable of preventing morbidity and mortality associated with non-homologous LASV challenge in two animal models of Lassa fever. Additionally, this work highlights the need for the further development of disease models for geographical distinct LASV strains, particularly those from Nigeria, in order to comprehensively evaluate potential vaccines and therapies against this prominent agent of viral hemorrhagic fever.

Resistance to Virus Infection Conferred by the Interferon-Induced Promyelocytic Leukemia Protein

PubMed Central

Chelbi-Alix, Mounira K.; Quignon, Frédérique; Pelicano, Luis; Koken, Marcel H. M.; de Thé, Hugues

1998-01-01

The interferon (IFN)-induced promyelocytic leukemia (PML) protein is specifically associated with nuclear bodies (NBs) whose functions are yet unknown. Two of the NB-associated proteins, PML and Sp100, are induced by IFN. Here we show that overexpression of PML and not Sp100 induces resistance to infections by vesicular stomatitis virus (VSV) (a rhabdovirus) and influenza A virus (an orthomyxovirus) but not by encephalomyocarditis virus (a picornavirus). Inhibition of viral multiplication was dependent on both the level of PML expression and the multiplicity of infection and reached 100-fold. PML was shown to interfere with VSV mRNA and protein synthesis. Compared to the IFN mediator MxA protein, PML had less powerful antiviral activity. While nuclear body localization of PML did not seem to be required for the antiviral effect, deletion of the PML coiled-coil domain completely abolished it. Taken together, these results suggest that PML can contribute to the antiviral state induced in IFN-treated cells. PMID:9444998

Elicitation of anti-vesicular stomatitis virus cytotoxic T lymphocytes by using purified viral and cellular antigens incorporated into phospholipid vesicles.

PubMed

Ruebush, M J; Hale, A H; Harris, D T

1981-05-01

We evaluated the minimal molecular and cellular requirements for elicitation of anti-vesicular stomatitis virus (VSV) cytotoxic T lymphocytes (CTL). The results indicated that lipid vesicles containing the purified major surface glyco-protein of VSV (G protein) and purified H-2K(k) glycoproteins elicited specific H-2K(k)-restricted anti-VSV CTL. These antiviral CTL were shown to be Ly 1(-),2(+). However, both Ly 1(+),2(-) and Ly1(-),2(+) T-cell subpopulations were shown to be required for elicitation of these CTL.

From bench to almost bedside: the long road to a licensed Ebola virus vaccine.

PubMed

Wong, Gary; Mendoza, Emelissa J; Plummer, Francis A; Gao, George F; Kobinger, Gary P; Qiu, Xiangguo

2018-02-01

The Ebola virus (EBOV) disease epidemic during 2014-16 in West Africa has accelerated the clinical development of several vaccine candidates that have demonstrated efficacy in the gold standard nonhuman primate (NHP) model, namely cynomolgus macaques. Areas covered: This review discusses the pre-clinical research and if available, clinical evaluation of the currently available EBOV vaccine candidates, while emphasizing the translatability of pre-clinical data generated in the NHP model to clinical data in humans. Expert opinion: Despite the existence of many successful EBOV vaccine candidates in the pre-clinical stages, only two platforms became the focus of Phase 2/3 efficacy trials in Liberia, Sierra Leone, and Guinea near the peak of the epidemic: the Vesicular stomatitis virus (VSV)-vectored vaccine and the chimpanzee adenovirus type 3 (ChAd3)-vectored vaccine. The results of three distinct clinical trials involving these candidates may soon pave the way for a licensed, safe and efficacious EBOV vaccine to help combat future epidemics.

From bench to almost bedside: The long road to a licensed Ebola virus vaccine

PubMed Central

Wong, Gary; Mendoza, Emelissa J.; Plummer, Francis A.; Gao, George F.; Kobinger, Gary P.; Qiu, Xiangguo

2018-01-01

Introduction The Ebola virus (EBOV) disease epidemic during 2014-16 in West Africa has accelerated the clinical development of several vaccine candidates that have demonstrated efficacy in the gold standard nonhuman primate (NHP) model, namely cynomolgus macaques. Areas covered This review discusses the pre-clinical research and if available, clinical evaluation of the currently available EBOV vaccine candidates, while emphasizing the translatability of pre-clinical data generated in the NHP model to clinical data in humans. Expert opinion Despite the existence of many successful EBOV vaccine candidates in the pre-clinical stages, only two platforms became the focus of Phase 2/3 efficacy trials in Liberia, Sierra Leone, and Guinea near the peak of the epidemic: the Vesicular stomatitis virus (VSV)-vectored vaccine and the chimpanzee adenovirus type 3 (ChAd3)-vectored vaccine. The results of three distinct clinical trials involving these candidates may soon pave the way for a licensed, safe and efficacious EBOV vaccine to help combat future epidemics. PMID:29148858

Viral fusion efficacy of specific H3N2 influenza virus reassortant combinations at single-particle level

NASA Astrophysics Data System (ADS)

Hsu, Hung-Lun; Millet, Jean K.; Costello, Deirdre A.; Whittaker, Gary R.; Daniel, Susan

2016-10-01

Virus pseudotyping is a useful and safe technique for studying entry of emerging strains of influenza virus. However, few studies have compared different reassortant combinations in pseudoparticle systems, or compared entry kinetics of native viruses and their pseudotyped analogs. Here, vesicular stomatitis virus (VSV)-based pseudovirions displaying distinct influenza virus envelope proteins were tested for fusion activity. We produced VSV pseudotypes containing the prototypical X-31 (H3) HA, either alone or with strain-matched or mismatched N2 NAs. We performed single-particle fusion assays using total internal reflection fluorescence microscopy to compare hemifusion kinetics among these pairings. Results illustrate that matching pseudoparticles behaved very similarly to native virus. Pseudoparticles harboring mismatched HA-NA pairings fuse at significantly slower rates than native virus, and NA-lacking pseudoparticles exhibiting the slowest fusion rates. Relative viral membrane HA density of matching pseudoparticles was higher than in mismatching or NA-lacking pseudoparticles. An equivalent trend of HA expression level on cell membranes of HA/NA co-transfected cells was observed and intracellular trafficking of HA was affected by NA co-expression. Overall, we show that specific influenza HA-NA combinations can profoundly affect the critical role played by HA during entry, which may factor into viral fitness and the emergence of new pandemic influenza viruses.

Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector.

PubMed

Chakkaramakkil Verghese, Santhosh; Goloviznina, Natalya A; Kurre, Peter

2016-11-19

Fanconi anemia (FA) is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC)-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV) particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.

The brain parenchyma has a type I interferon response that can limit virus spread.

PubMed

Drokhlyansky, Eugene; Göz Aytürk, Didem; Soh, Timothy K; Chrenek, Ryan; O'Loughlin, Elaine; Madore, Charlotte; Butovsky, Oleg; Cepko, Constance L

2017-01-03

The brain has a tightly regulated environment that protects neurons and limits inflammation, designated "immune privilege." However, there is not an absolute lack of an immune response. We tested the ability of the brain to initiate an innate immune response to a virus, which was directly injected into the brain parenchyma, and to determine whether this response could limit viral spread. We injected vesicular stomatitis virus (VSV), a transsynaptic tracer, or naturally occurring VSV-derived defective interfering particles (DIPs), into the caudate-putamen (CP) and scored for an innate immune response and inhibition of virus spread. We found that the brain parenchyma has a functional type I interferon (IFN) response that can limit VSV spread at both the inoculation site and among synaptically connected neurons. Furthermore, we characterized the response of microglia to VSV infection and found that infected microglia produced type I IFN and uninfected microglia induced an innate immune response following virus injection.

A Recombinant Vesicular Stomatitis Virus-Based Lassa Fever Vaccine Protects Guinea Pigs and Macaques against Challenge with Geographically and Genetically Distinct Lassa Viruses

PubMed Central

Safronetz, David; Mire, Chad; Rosenke, Kyle; Feldmann, Friederike; Haddock, Elaine; Geisbert, Thomas; Feldmann, Heinz

2015-01-01

Background Lassa virus (LASV) is endemic in several West African countries and is the etiological agent of Lassa fever. Despite the high annual incidence and significant morbidity and mortality rates, currently there are no approved vaccines to prevent infection or disease in humans. Genetically, LASV demonstrates a high degree of diversity that correlates with geographic distribution. The genetic heterogeneity observed between geographically distinct viruses raises concerns over the potential efficacy of a “universal” LASV vaccine. To date, several experimental LASV vaccines have been developed; however, few have been evaluated against challenge with various genetically unique Lassa virus isolates in relevant animal models. Methodologies/principle findings Here we demonstrate that a single, prophylactic immunization with a recombinant vesicular stomatitis virus (VSV) expressing the glycoproteins of LASV strain Josiah from Sierra Leone protects strain 13 guinea pigs from infection / disease following challenge with LASV isolates originating from Liberia, Mali and Nigeria. Similarly, the VSV-based LASV vaccine yields complete protection against a lethal challenge with the Liberian LASV isolate in the gold-standard macaque model of Lassa fever. Conclusions/significance Our results demonstrate the VSV-based LASV vaccine is capable of preventing morbidity and mortality associated with non-homologous LASV challenge in two animal models of Lassa fever. Additionally, this work highlights the need for the further development of disease models for geographical distinct LASV strains, particularly those from Nigeria, in order to comprehensively evaluate potential vaccines and therapies against this prominent agent of viral hemorrhagic fever. PMID:25884628

Analysis of the Contribution of Hemocytes and Autophagy to Drosophila Antiviral Immunity.

PubMed

Lamiable, Olivier; Arnold, Johan; de Faria, Isaque Joao da Silva; Olmo, Roenick Proveti; Bergami, Francesco; Meignin, Carine; Hoffmann, Jules A; Marques, Joao Trindade; Imler, Jean-Luc

2016-06-01

Antiviral immunity in the model organism Drosophila melanogaster involves the broadly active intrinsic mechanism of RNA interference (RNAi) and virus-specific inducible responses. Here, using a panel of six viruses, we investigated the role of hemocytes and autophagy in the control of viral infections. Injection of latex beads to saturate phagocytosis, or genetic depletion of hemocytes, resulted in decreased survival and increased viral titers following infection with Cricket paralysis virus (CrPV), Flock House virus (FHV), and vesicular stomatitis virus (VSV) but had no impact on Drosophila C virus (DCV), Sindbis virus (SINV), and Invertebrate iridescent virus 6 (IIV6) infection. In the cases of CrPV and FHV, apoptosis was induced in infected cells, which were phagocytosed by hemocytes. In contrast, VSV did not trigger any significant apoptosis but we confirmed that the autophagy gene Atg7 was required for full virus resistance, suggesting that hemocytes use autophagy to recognize the virus. However, this recognition does not depend on the Toll-7 receptor. Autophagy had no impact on DCV, CrPV, SINV, or IIV6 infection and was required for replication of the sixth virus, FHV. Even in the case of VSV, the increases in titers were modest in Atg7 mutant flies, suggesting that autophagy does not play a major role in antiviral immunity in Drosophila Altogether, our results indicate that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in insects. Phagocytosis and autophagy are two cellular processes that involve lysosomal degradation and participate in Drosophila immunity. Using a panel of RNA and DNA viruses, we have addressed the contribution of phagocytosis and autophagy in the control of viral infections in this model organism. We show that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in Drosophila This work brings to the front a novel facet of antiviral host defense in insects, which may have relevance in the control of virus transmission by vector insects or in the resistance of beneficial insects to viral pathogens. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Analysis of the Contribution of Hemocytes and Autophagy to Drosophila Antiviral Immunity

PubMed Central

Lamiable, Olivier; Arnold, Johan; de Faria, Isaque Joao da Silva; Olmo, Roenick Proveti; Bergami, Francesco; Meignin, Carine; Hoffmann, Jules A.; Marques, Joao Trindade

2016-01-01

ABSTRACT Antiviral immunity in the model organism Drosophila melanogaster involves the broadly active intrinsic mechanism of RNA interference (RNAi) and virus-specific inducible responses. Here, using a panel of six viruses, we investigated the role of hemocytes and autophagy in the control of viral infections. Injection of latex beads to saturate phagocytosis, or genetic depletion of hemocytes, resulted in decreased survival and increased viral titers following infection with Cricket paralysis virus (CrPV), Flock House virus (FHV), and vesicular stomatitis virus (VSV) but had no impact on Drosophila C virus (DCV), Sindbis virus (SINV), and Invertebrate iridescent virus 6 (IIV6) infection. In the cases of CrPV and FHV, apoptosis was induced in infected cells, which were phagocytosed by hemocytes. In contrast, VSV did not trigger any significant apoptosis but we confirmed that the autophagy gene Atg7 was required for full virus resistance, suggesting that hemocytes use autophagy to recognize the virus. However, this recognition does not depend on the Toll-7 receptor. Autophagy had no impact on DCV, CrPV, SINV, or IIV6 infection and was required for replication of the sixth virus, FHV. Even in the case of VSV, the increases in titers were modest in Atg7 mutant flies, suggesting that autophagy does not play a major role in antiviral immunity in Drosophila. Altogether, our results indicate that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in insects. IMPORTANCE Phagocytosis and autophagy are two cellular processes that involve lysosomal degradation and participate in Drosophila immunity. Using a panel of RNA and DNA viruses, we have addressed the contribution of phagocytosis and autophagy in the control of viral infections in this model organism. We show that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in Drosophila. This work brings to the front a novel facet of antiviral host defense in insects, which may have relevance in the control of virus transmission by vector insects or in the resistance of beneficial insects to viral pathogens. PMID:27009948

Prospects for immunisation against Marburg and Ebola viruses.

PubMed

Geisbert, Thomas W; Bausch, Daniel G; Feldmann, Heinz

2010-11-01

For more than 30 years the filoviruses, Marburg virus and Ebola virus, have been associated with periodic outbreaks of hemorrhagic fever that produce severe and often fatal disease. The filoviruses are endemic primarily in resource-poor regions in Central Africa and are also potential agents of bioterrorism. Although no vaccines or antiviral drugs for Marburg or Ebola are currently available, remarkable progress has been made over the last decade in developing candidate preventive vaccines against filoviruses in nonhuman primate models. Due to the generally remote locations of filovirus outbreaks, a single-injection vaccine is desirable. Among the prospective vaccines that have shown efficacy in nonhuman primate models of filoviral hemorrhagic fever, two candidates, one based on a replication-defective adenovirus serotype 5 and the other on a recombinant VSV (rVSV), were shown to provide complete protection to nonhuman primates when administered as a single injection. The rVSV-based vaccine has also shown utility when administered for postexposure prophylaxis against filovirus infections. A VSV-based Ebola vaccine was recently used to manage a potential laboratory exposure. 2010 John Wiley & Sons, Ltd.

Preimplantation bovine embryos: Pathobiology of Haemophilus somnus exposure and resistance mechanisms to vesicular stomatitis virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thomson, M.S.

1988-01-01

Preimplantation bovine embryos were exposed in vitro to H. somnus to determine if the bacteria would adhere to zona pellucida-intact (ZP-I) embryos or adhere to or infect ZP-free embryos. The effect of H. somnus on embryonic development in vitro was also investigated. Electrophoretic comparisons of outer membrane proteins of H. somnus revealed 2 major protein bands common to 10 H. somnus isolates. A monoclonal antibody produced against the outer membrane proteins reacted to one of the major protein bands. The sensitivity of a nucleic acid probe for detection of vesicular stomatitis virus (VSV) was validated in cells in culture andmore » used to determine if the synthetic double-stranded complex of polyriboinosinic and polyribocytidylic acids (poly I:C) would induce viral resistance in cultured bovine embryos. Two {sup 32}P-nick translated probes of high specific activity prepared from plasmids containing nucleic acid sequences of VSV virus were employed for viral mRNA detection in the tissue culture cells using a DNA-hybridization dot-blot technique. Using one of the probes, the technique was applied to detect differences in viral replication between four groups of bovine embryos (nonexposed, exposed to VSV virus, poly I:C-treated, and poly I:C-treated and exposed to VSV). The nucleic acid probe was sufficiently sensitive to detect differences in quantities of VSV mRNA among embryo treatment groups, resulting in the demonstration that resistance to viral infection was induced in day 9 bovine embryos.« less

The effect of dose on the safety and immunogenicity of the VSV Ebola candidate vaccine: a randomised double-blind, placebo-controlled phase 1/2 trial.

PubMed

Huttner, Angela; Dayer, Julie-Anne; Yerly, Sabine; Combescure, Christophe; Auderset, Floriane; Desmeules, Jules; Eickmann, Markus; Finckh, Axel; Goncalves, Ana Rita; Hooper, Jay W; Kaya, Gürkan; Krähling, Verena; Kwilas, Steve; Lemaître, Barbara; Matthey, Alain; Silvera, Peter; Becker, Stephan; Fast, Patricia E; Moorthy, Vasee; Kieny, Marie Paule; Kaiser, Laurent; Siegrist, Claire-Anne

2015-10-01

Safe and effective vaccines against Ebola could prevent or control outbreaks. The safe use of replication-competent vaccines requires a careful dose-selection process. We report the first safety and immunogenicity results in volunteers receiving 3 × 10(5) plaque-forming units (pfu) of the recombinant vesicular stomatitis virus-based candidate vaccine expressing the Zaire Ebola virus glycoprotein (rVSV-ZEBOV; low-dose vaccinees) compared with 59 volunteers who had received 1 ×10(7) pfu (n=35) or 5 × 10(7) pfu (n=16) of rVSV-ZEBOV (high-dose vaccinees) or placebo (n=8) before a safety-driven study hold. The Geneva rVSV-ZEBOV study, an investigator-initiated phase 1/2, dose-finding, placebo-controlled, double-blind trial conducted at the University Hospitals of Geneva, Switzerland, enrolled non-pregnant, immunocompetent, and otherwise healthy adults aged 18-65 years. Participants from the low-dose group with no plans to deploy to Ebola-aff5cted regions (non-deployable) were randomised 9:1 in a double-blind fashion using randomly permuted blocks of varying sizes to a single injection of 3 × 10(5) pfu or placebo, whereas deployable participants received single-injection 3 × 10(5) pfu open-label. Primary safety and immunogenicity outcomes were the incidence of adverse events within 14 days of vaccination and day-28 antibody titres, respectively, analysed by intention to treat. After viral oligoarthritis was observed in 11 of the first 51 vaccinees (22%) receiving 10(7) or 5 × 10(7) pfu, 56 participants were given a lower dose (3 × 10(5) pfu, n=51) or placebo (n=5) to assess the effect of dose reduction on safety and immunogenicity. This trial is ongoing with a follow-up period of 12 months; all reported results are from interim databases. This study is registered with ClinicalTrials.gov, number NCT02287480. Between Jan 5 and Jan 26, 2015, 43 non-deployable participants received low-dose rVSV-ZEBOV (3 × 10(5) pfu) or placebo in a double-blind fashion, whereas 13 deployable participants received 3 × 10(5) pfu open-label. Altogether, in the low-dose group, 51 participants received rVSV-ZEBOV and five received placebo. No serious adverse events occurred. At 3 × 10(5) pfu, early-onset reactogenicity remained frequent (45 [88%] of 51 compared with 50 [98%] of 51 high dose and two [15%] of 13 placebo recipients), but mild. Objective fever was present in one (2%) of 51 low-dose versus 13 (25%) of 51 high-dose vaccinees receiving at least 1 ×10(7) pfu (p<0·0001). Subjective fever (p<0·0001), myalgia (p=0·036), and chills (p=0·026) were significantly reduced and their time of onset delayed, reflecting significantly lower viraemia (p<0·0001) and blood monocyte-activation patterns (p=0·0233). Although seropositivity rates remained similarly high (48 [94%] of 51), day-28 EBOV-glycoprotein-binding and neutralising antibody titres were lower in low-dose versus high-dose vaccinees (geometric mean titres 344·5 [95% CI 229·7-516·4] vs 1064·2 [757·6-1495·1]; p<0·0001; and 35·1 [24·7-50·7] vs 127·0 [86·0-187·6]; p<0·0001, respectively). Furthermore, oligoarthritis again occurred on day 10 (median; IQR 9-14) in 13 (25%) of 51 low-dose vaccinees, with maculopapular, vesicular dermatitis, or both in seven (54%) of 13; arthritis was associated with increasing age in low-dose but not high-dose vaccinees. Two vaccinees presented with purpura of the lower legs; histological findings indicated cutaneous vasculitis. The presence of rVSV in synovial fluid and skin lesions confirmed causality. Reducing the dose of rVSV-ZEBOV improved its early tolerability but lowered antibody responses and did not prevent vaccine-induced arthritis, dermatitis, or vasculitis. Like its efficacy, the safety of rVSV-ZEBOV requires further definition in the target populations of Africa. Wellcome Trust through WHO. Copyright © 2015 Elsevier Ltd. All rights reserved.

Viral fusion efficacy of specific H3N2 influenza virus reassortant combinations at single-particle level

PubMed Central

Hsu, Hung-Lun; Millet, Jean K.; Costello, Deirdre A.; Whittaker, Gary R.; Daniel, Susan

2016-01-01

Virus pseudotyping is a useful and safe technique for studying entry of emerging strains of influenza virus. However, few studies have compared different reassortant combinations in pseudoparticle systems, or compared entry kinetics of native viruses and their pseudotyped analogs. Here, vesicular stomatitis virus (VSV)-based pseudovirions displaying distinct influenza virus envelope proteins were tested for fusion activity. We produced VSV pseudotypes containing the prototypical X-31 (H3) HA, either alone or with strain-matched or mismatched N2 NAs. We performed single-particle fusion assays using total internal reflection fluorescence microscopy to compare hemifusion kinetics among these pairings. Results illustrate that matching pseudoparticles behaved very similarly to native virus. Pseudoparticles harboring mismatched HA-NA pairings fuse at significantly slower rates than native virus, and NA-lacking pseudoparticles exhibiting the slowest fusion rates. Relative viral membrane HA density of matching pseudoparticles was higher than in mismatching or NA-lacking pseudoparticles. An equivalent trend of HA expression level on cell membranes of HA/NA co-transfected cells was observed and intracellular trafficking of HA was affected by NA co-expression. Overall, we show that specific influenza HA-NA combinations can profoundly affect the critical role played by HA during entry, which may factor into viral fitness and the emergence of new pandemic influenza viruses. PMID:27752100

Sleep and behavior during vesicular stomatitis virus induced encephalitis in BALB/cJ and C57BL/6J mice

PubMed Central

Machida, Mayumi; Ambrozewicz, Marta A.; Breving, Kimberly; Wellman, Laurie L.; Yang, Linghui; Ciavarra, Richard P.; Sanford, Larry D.

2013-01-01

Intranasal application of vesicular stomatitis virus (VSV) produces a well-characterized model of viral encephalitis in mice. Within one day post-infection (PI), VSV travels to the olfactory bulb and, over the course of 7 days, it infects regions and tracts extending into the brainstem followed by clearance and recovery in most mice by PI day 14 (PI 14). Infectious diseases are commonly accompanied by excessive sleepiness; thus, sleep is considered a component of the acute phase response to infection. In this project, we studied the relationship between sleep and VSV infection using C57BL/6 (B6) and BALB/c mice. Mice were implanted with transmitters for recording EEG, activity and temperature by telemetry. After uninterrupted baseline recordings were collected for 2 days, each animal was infected intranasally with a single low dose of VSV (5 × 104 PFU). Sleep was recorded for 15 consecutive days and analyzed on PI 0, 1, 3, 5, 7, 10, and 14. Compared to baseline, amounts of non-rapid eye movement sleep (NREM) were increased in B6 mice during the dark period of PI 1–5, whereas rapid eye movement sleep (REM) was significantly reduced during the light periods of PI 0–14. In contrast, BALB/c mice showed significantly fewer changes in NREM and REM. These data demonstrate sleep architecture is differentially altered in these mouse strains and suggests that, in B6 mice, VSV can alter sleep before virus progresses into brain regions that control sleep. PMID:24055862

Membrane fusion activity of vesicular stomatitis virus glycoprotein G is induced by low pH but not by heat or denaturant.

PubMed

Yao, Yi; Ghosh, Kakoli; Epand, Raquel F; Epand, Richard M; Ghosh, Hara P

2003-06-05

The fusogenic envelope glycoprotein G of the rhabdovirus vesicular stomatitis virus (VSV) induces membrane fusion at acidic pH. At acidic pH the G protein undergoes a major structural reorganization leading to the fusogenic conformation. However, unlike other viral fusion proteins, the low-pH-induced conformational change of VSV G is completely reversible. As well, the presence of an alpha-helical coiled-coil motif required for fusion by a number of viral and cellular fusion proteins was not predicted in VSV G protein by using a number of algorithms. Results of pH dependence of the thermal stability of G protein as determined by intrinsic Trp fluorescence and circular dichroism (CD) spectroscopy show that the G protein is equally stable at neutral or acidic pH. Destabilization of G structure at neutral pH with either heat or urea did not induce membrane fusion or conformational change(s) leading to membrane fusion. Taken together, these data suggest that the mechanism of VSV G-induced fusion is distinct from the fusion mechanism of fusion proteins that involve a coiled-coil motif.

Molecular Interactions of the Hantaan Virus Nucleocapsid Protein

DTIC Science & Technology

1991-09-12

stomatitis virus (VSV; a rhabdovirus ), where the phosphoprotein NS (analogous to Sendai P protein) contains a single carboxyl-terminal domain...structures reminiscent of rhabdovirus capsomeres were clearly visible in the electron microscope (Blumberg et ai-, 1983). This innate ability of viral...passaged virus (Weiss et. al.., 1989) . In addition, a soluble form of VSV ( rhabdovirus ) N protein was found, in the presence of various RNAs, to

The Bioelectromagnetics Society Annual Scientific Session (5th) Abstracts,

DTIC Science & Technology

1983-01-01

cm ) for one hour. Extended survival of hamsters from VSV injection could be due to activated macropha- ges destroying Injected virus and perhaps...to increased anti-VSV antibody production. These results are consistent with our previous work with vaccinia virus (Bloelectromagnetics, In press...guinea pigs and cats reflect intracranial rever- berations of a pressure pulse induced within the brain. Results from single-unit and psychophysical

Shear wave velocity and radial anisotropy beneath the Wyoming craton: craton destruction and lithospheric layering

NASA Astrophysics Data System (ADS)

Dave, R.; Li, A.

2016-12-01

The Wyoming craton has evolved under an intriguing geological history with suture zones, accreted margins, flat-slab subduction, orogeny and an encroaching hotspot. Whether and how the cratonic root has been widely destroyed by the series of tectonic events remain controversial. Aiming to address these questions using a craton-wide model, we have analyzed Rayleigh and Love wave data from 75 earthquakes recorded by 103 USArray TA stations in the Wyoming craton. 2-D phase velocity maps are constructed for 18 periods from 20 s to 166 s using the two-plane-wave tomography. The Yellowstone hotspot and the Cheyenne belt are characterized by low velocity anomalies at all periods in both Rayleigh and Love wave models. The northern craton in Montana is broadly fast at periods < 70 s and is relatively slow at longer periods, suggesting a shallower lithosphere. The fast anomaly in Wyoming has a NE-SW trend and extends to more than 200 km in the VSV model. However, such a fast anomaly is largely absent in the Love wave images at long periods. The association of VSV>VSH with this deep fast anomaly indicates mantle downwelling beneath south-central Wyoming. Mantle upwelling likely happens in slow regions at the hotspot, the Cheyenne belt, and the northeastern craton. The overall pattern of velocity anomaly and radial anisotropy suggests that small-scale mantle convection is vigorously acting beneath the Wyoming craton and continuously destructing the cratonic lithosphere. In addition, the average VSV and VSH models show a strong positive radial anisotropy of 5% (VSH>VSV) above 100 km and a weak negative anisotropy (VSV>VSH) below 120 km. Such a significant change in radial anisotropy could contribute to the observed mid-lithosphere discontinuity (MLD) from receiver functions. Both VSV and VSH reveal a fast lid above 100 km and a large velocity reduction at the depths of 115-190 km, corresponding with a lithosphere-asthenosphere boundary (LAB) at 150 km. These observations suggest different origins of the MLD and the LAB.

Rapid Titration of Measles and Other Viruses: Optimization with Determination of Replication Cycle Length

PubMed Central

Grigorov, Boyan; Rabilloud, Jessica; Lawrence, Philip; Gerlier, Denis

2011-01-01

Background Measles virus (MV) is a member of the Paramyxoviridae family and an important human pathogen causing strong immunosuppression in affected individuals and a considerable number of deaths worldwide. Currently, measles is a re-emerging disease in developed countries. MV is usually quantified in infectious units as determined by limiting dilution and counting of plaque forming unit either directly (PFU method) or indirectly from random distribution in microwells (TCID50 method). Both methods are time-consuming (up to several days), cumbersome and, in the case of the PFU assay, possibly operator dependent. Methods/Findings A rapid, optimized, accurate, and reliable technique for titration of measles virus was developed based on the detection of virus infected cells by flow cytometry, single round of infection and titer calculation according to the Poisson's law. The kinetics follow up of the number of infected cells after infection with serial dilutions of a virus allowed estimation of the duration of the replication cycle, and consequently, the optimal infection time. The assay was set up to quantify measles virus, vesicular stomatitis virus (VSV), and human immunodeficiency virus type 1 (HIV-1) using antibody labeling of viral glycoprotein, virus encoded fluorescent reporter protein and an inducible fluorescent-reporter cell line, respectively. Conclusion Overall, performing the assay takes only 24–30 hours for MV strains, 12 hours for VSV, and 52 hours for HIV-1. The step-by-step procedure we have set up can be, in principle, applicable to accurately quantify any virus including lentiviral vectors, provided that a virus encoded gene product can be detected by flow cytometry. PMID:21915289

Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms

PubMed Central

Mundell, Nathan A.; Beier, Kevin T.; Pan, Y. Albert; Lapan, Sylvain W.; Göz Aytürk, Didem; Berezovskii, Vladimir K.; Wark, Abigail R.; Drokhlyansky, Eugene; Bielecki, Jan; Born, Richard T.; Schier, Alexander F.

2015-01-01

Current limitations in technology have prevented an extensive analysis of the connections among neurons, particularly within nonmammalian organisms. We developed a transsynaptic viral tracer originally for use in mice, and then tested its utility in a broader range of organisms. By engineering the vesicular stomatitis virus (VSV) to encode a fluorophore and either the rabies virus glycoprotein (RABV‐G) or its own glycoprotein (VSV‐G), we created viruses that can transsynaptically label neuronal circuits in either the retrograde or anterograde direction, respectively. The vectors were investigated for their utility as polysynaptic tracers of chicken and zebrafish visual pathways. They showed patterns of connectivity consistent with previously characterized visual system connections, and revealed several potentially novel connections. Further, these vectors were shown to infect neurons in several other vertebrates, including Old and New World monkeys, seahorses, axolotls, and Xenopus. They were also shown to infect two invertebrates, Drosophila melanogaster, and the box jellyfish, Tripedalia cystophora, a species previously intractable for gene transfer, although no clear evidence of transsynaptic spread was observed in these species. These vectors provide a starting point for transsynaptic tracing in most vertebrates, and are also excellent candidates for gene transfer in organisms that have been refractory to other methods. J. Comp. Neurol. 523:1639–1663, 2015. © 2015 Wiley Periodicals, Inc. PMID:25688551

Function of duck RIG-I in induction of antiviral response against IBDV and avian influenza virus on chicken cells.

PubMed

Shao, Qiang; Xu, Wenpin; Yan, Li; Liu, Jinhua; Rui, Lei; Xiao, Xiao; Yu, Xiaoxue; Lu, Yanan; Li, Zandong

2014-10-13

The avian influenza (AI) H9N2 virus and IBDV are two major problems in the poultry industry. They have been prevalent among domestic poultry in Asia for many years and have caused considerable economic losses. Retinoic-acid-induced gene I (RIG-I) is a cytoplasmic sensor of dsRNA and ssRNA. It can detect Encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) in human cells, influenza virus in duck leads to production of IFN-β and IFN-stimulated antiviral genes and reductions in the replication of RNA virus. Chickens, which lack RIG-I, are more sensitive to influenza virus than ducks. However, little is known about the roles of duck RIG-I (dRIG-I) in the detection of IBDV and AI H9N2 in chicken cells DF-1. The purpose of this study was to examine the function of dRIG-I in the recognition of IBDV Ts strain and H9N2 A/Chicken/Shandong/ZB/2007(ZB07) and in the induction of antiviral gene expression to gain an understanding of antiviral ability of dRIG-I in chicken cells against dsRNA virus IBDV and ssRNA virus ZB07. After challenge with the IBDV Ts strain and ZB07 the expression levels of Type I IFN (IFN-β and IFN-α) and IFN-induced antiviral genes (Mx and PKR) were significantly up-regulated in dRIG-I-transfected DF-1cells compared with the empty-vector-transfected control. dRIG-I knockdown experiments further proved that dRIG-I is essential to sensing IBDV and ZB07 in duck embryo fibroblasts (DEF). Growth curves showed that dRIG-I repressed the replication of IBDV and almost blunted the growth of ZB07 in DF-1. Apoptosis analysis revealed that dRIG-I increase the number of the survival cells after IBDV Ts strain or ZB07 infection relative to the empty-vector-transfected control. These results indicate that dRIG-I can up-regulates type I IFN and reduce viral gene expression and viral replication and protect chicken cells from virus-induced apoptosis during ZB07 and IBDV infection. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetic and Molecular Studies of the Phlebotomus Fever Group of Viruses.

DTIC Science & Technology

1980-08-01

unrelated rhabdovirus , VSV. Two sources of LAC viral antigens were used, those from virus grown in BHK-21 cells and those obtained from LAC virus grown in...KAR, SFS and CHG, as well as those of the bunyaviruses LAC, ORI and BUN, and the viral antigens of the unrelated rhabdovirus , VSV. Again two sources...and Shope, R.E., 1977b, Oligonucleotide fingerprints of RNA obtained from rhabdoviruses belonging to the vesicular stornatitis virus subgroup. J

Digital sensing and sizing of vesicular stomatitis virus pseudotypes in complex media: a model for Ebola and Marburg detection.

PubMed

Daaboul, George G; Lopez, Carlos A; Chinnala, Jyothsna; Goldberg, Bennett B; Connor, John H; Ünlü, M Selim

2014-06-24

Rapid, sensitive, and direct label-free capture and characterization of nanoparticles from complex media such as blood or serum will broadly impact medicine and the life sciences. We demonstrate identification of virus particles in complex samples for replication-competent wild-type vesicular stomatitis virus (VSV), defective VSV, and Ebola- and Marburg-pseudotyped VSV with high sensitivity and specificity. Size discrimination of the imaged nanoparticles (virions) allows differentiation between modified viruses having different genome lengths and facilitates a reduction in the counting of nonspecifically bound particles to achieve a limit-of-detection (LOD) of 5 × 10(3) pfu/mL for the Ebola and Marburg VSV pseudotypes. We demonstrate the simultaneous detection of multiple viruses in a single sample (composed of serum or whole blood) for screening applications and uncompromised detection capabilities in samples contaminated with high levels of bacteria. By employing affinity-based capture, size discrimination, and a "digital" detection scheme to count single virus particles, we show that a robust and sensitive virus/nanoparticle sensing assay can be established for targets in complex samples. The nanoparticle microscopy system is termed the Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and is capable of high-throughput and rapid sizing of large numbers of biological nanoparticles on an antibody microarray for research and diagnostic applications.

Use of a flow-cell system to investigate virucidal dimethylmethylene blue phototreatment in two RBC additive solutions.

PubMed

Wagner, Stephen; Skripchenko, Andrey; Thompson-Montgomery, Dedeene

2002-09-01

Limited photoinactivation kinetics, use of low-volume 30 percent Hct RBCs, and hemolysis have restricted the practicality of the use of dimethylmethylene blue (DMMB) and light for RBC decontamination. A flow-cell system was developed to rapidly treat larger volumes of oxygenated 45 percent Hct RBCs with high-intensity red light. CPD-whole blood was WBC reduced, RBCs were diluted in additive solutions (either Adsol or Erythrosol), and suspensions were subsequently oxygenated by gas overlay. Intracellular or extracellular VSV and DMMB were sequentially added. VSV-infected RBC suspensions (45% Hct) were passed through 1-mm-thick flow cells and illuminated. Samples were titered for VSV, stored for up to 42 days, and assayed for Hb, supernatant potassium, ATP, and MCV. The use of oxygenated RBCs resulted in rapid and reproducible photoinactivaton of > or = 6.6 log extracellular and approximately 4.0 log intracellular VSV independent of additive solution. Phototreated Adsol RBCs exhibited more than 10 times greater hemolysis and 30 percent greater MCV during storage than identically treated Erythrosol RBCs. Phototreatment caused RBC potassium leakage from RBCs in both additive solutions. ATP levels were better preserved in Erythrosol than Adsol RBCs. A rapid, reproducible, and robust method for photoinactivating model virus in RBC suspensions was developed. Despite improved hemolysis and ATP levels in Erythrosol-phototreated RBCs, storage properties were not maintained for 42 days.

Assessment of the ability of V920 recombinant vesicular stomatitis-Zaire ebolavirus vaccine to replicate in relevant arthropod cell cultures and vector species

PubMed Central

2018-01-01

ABSTRACT V920, rVSVΔG-ZEBOV-GP, is a recombinant vesicular stomatitis-Zaire ebolavirus vaccine which has shown an acceptable safety profile and provides a protective immune response against Ebola virus disease (EVD) induced by Zaire ebolavirus in humans. The purpose of this study was to determine whether the V920 vaccine is capable of replicating in arthropod cell cultures of relevant vector species and of replicating in live mosquitoes. While the V920 vaccine replicated well in Vero cells, no replication was observed in Anopheles or Aedes mosquito, Culicoides biting midge, or Lutzomyia sand fly cells, nor in live Culex or Aedes mosquitoes following exposure through intrathoracic inoculation or feeding on a high-titer infectious blood meal. The insect taxa selected for use in this study represent actual and potential epidemic vectors of VSV. V920 vaccine inoculated into Cx. quinquefasciatus and Ae. aegypti mosquitoes demonstrated persistence of replication-competent virus following inoculation, consistent with the recognized biological stability of the vaccine, but no evidence for active virus replication in live mosquitoes was observed. Following administration of an infectious blood meal to Ae. aegypti and Cx. quinquefasciatus mosquitoes at a titer several log10 PFU more concentrated than would be observed in vaccinated individuals, no infection or dissemination of V920 was observed in either mosquito species. In vitro and in vivo data gathered during this study support minimal risk of the vector-borne potential of the V920 vaccine. PMID:29206076

Assessment of the ability of V920 recombinant vesicular stomatitis-Zaire ebolavirus vaccine to replicate in relevant arthropod cell cultures and vector species.

PubMed

Bergren, Nicholas A; Miller, Megan R; Monath, Thomas P; Kading, Rebekah C

2018-04-03

V920, rVSVΔG-ZEBOV-GP, is a recombinant vesicular stomatitis-Zaire ebolavirus vaccine which has shown an acceptable safety profile and provides a protective immune response against Ebola virus disease (EVD) induced by Zaire ebolavirus in humans. The purpose of this study was to determine whether the V920 vaccine is capable of replicating in arthropod cell cultures of relevant vector species and of replicating in live mosquitoes. While the V920 vaccine replicated well in Vero cells, no replication was observed in Anopheles or Aedes mosquito, Culicoides biting midge, or Lutzomyia sand fly cells, nor in live Culex or Aedes mosquitoes following exposure through intrathoracic inoculation or feeding on a high-titer infectious blood meal. The insect taxa selected for use in this study represent actual and potential epidemic vectors of VSV. V920 vaccine inoculated into Cx. quinquefasciatus and Ae. aegypti mosquitoes demonstrated persistence of replication-competent virus following inoculation, consistent with the recognized biological stability of the vaccine, but no evidence for active virus replication in live mosquitoes was observed. Following administration of an infectious blood meal to Ae. aegypti and Cx. quinquefasciatus mosquitoes at a titer several log 10 PFU more concentrated than would be observed in vaccinated individuals, no infection or dissemination of V920 was observed in either mosquito species. In vitro and in vivo data gathered during this study support minimal risk of the vector-borne potential of the V920 vaccine.

Enhancing the Oncolytic Activity of CD133-Targeted Measles Virus: Receptor Extension or Chimerism with Vesicular Stomatitis Virus Are Most Effective

PubMed Central

Kleinlützum, Dina; Hanauer, Julia D. S.; Muik, Alexander; Hanschmann, Kay-Martin; Kays, Sarah-Katharina; Ayala-Breton, Camilo; Peng, Kah-Whye; Mühlebach, Michael D.; Abel, Tobias; Buchholz, Christian J.

2017-01-01

Therapy resistance and tumor recurrence are often linked to a small refractory and highly tumorigenic subpopulation of neoplastic cells, known as cancer stem cells (CSCs). A putative marker of CSCs is CD133 (prominin-1). We have previously described a CD133-targeted oncolytic measles virus (MV-CD133) as a promising approach to specifically eliminate CD133-positive tumor cells. Selectivity was introduced at the level of cell entry by an engineered MV hemagglutinin (H). The H protein was blinded for its native receptors and displayed a CD133-specific single-chain antibody fragment (scFv) as targeting domain. Interestingly, MV-CD133 was more active in killing CD133-positive tumors than the unmodified MV-NSe despite being highly selective for its target cells. To further enhance the antitumoral activity of MV-CD133, we here pursued arming technologies, receptor extension, and chimeras between MV-CD133 and vesicular stomatitis virus (VSV). All newly generated viruses including VSV-CD133 were highly selective in eliminating CD133-positive cells. MV-CD46/CD133 killed in addition CD133-negative cells being positive for the MV receptors. In an orthotopic glioma model, MV-CD46/CD133 and MVSCD-CD133, which encodes the super cytosine deaminase, were most effective. Notably, VSV-CD133 caused fatal neurotoxicity in this tumor model. Use of CD133 as receptor could be excluded as being causative. In a subcutaneous tumor model of hepatocellular cancer, VSV-CD133 revealed the most potent oncolytic activity and also significantly prolonged survival of the mice when injected intravenously. Compared to MV-CD133, VSV-CD133 infected a more than 104-fold larger area of the tumor within the same time period. Our data not only suggest new concepts and approaches toward enhancing the oncolytic activity of CD133-targeted oncolytic viruses but also raise awareness about careful toxicity testing of novel virus types. PMID:28695108

Upper Mantle Anisotropy Under Fast Spreading Mid-ocean Ridges: 2-D Whole Mantle Convection Model With Subduction

NASA Astrophysics Data System (ADS)

Lee, C.; Zhou, Y.; King, S. D.

2008-12-01

Analyses of seismic anisotropy caused by spatial alignments of anisotropic minerals (e.g., olivine) have been widely used to infer mantle flow directions in the upper mantle. Deep seismic anisotropy beneath fast spreading mid-ocean ridges (e.g., East Pacific Rise) has been recently observed at depths of 200-300 km and even down to the transition zone, with polarization changes in radial anisotropy from VSH < VSV (shallow) to VSH < VSV (deep). We investigate the origin of the observed deep seismic anisotropy and polarization changes beneath the EPR in 2-D Cartesian numerical models using both kinematically (prescribed velocity) and dynamically (negative buoyancy) driven ridge spreading. Because subduction is thought to be an important controlling factor in the style of ridge spreading and mantle convection, we consider a subduction zone developing at the prescribed weak zone. A whole mantle domain expressed by a one by four box (2890 by 11560 km) is used to minimize the boundary effects on the subducting slab. For the upper mantle rheology, we consider composite viscosity of diffusion and dislocation creep for dry olivine to evaluate the effects of lateral variation of mantle viscosity and the rheological changes from dislocation to diffusion creep under the mid-ocean ridge. For the lower mantle rheology, we use diffusion creep for dry olivine by increasing grain size to match relevant lower mantle viscosity. We also consider the 660 km phase transition with density and viscosity jump as well as Clapeyron slope. Anisotropy is evaluated using finite-strain ellipses based on the assumption that a-axes of olivine crystals are parallel to the major axes of the finite-strain ellipses. Our preliminary results show 1) in general, the development of VSH < VSV anisotropy is confined only in a narrow region under the ridge axis at depths of 200- 300 km; 2) strong VSH > VSV anisotropy can be found in the 'asthenosphere' beneath the entire spreading oceanic lithosphere; and 3) the dominate creep mechanism changes from dislocation creep to diffusion creep at depths of 300-400 km; indicating a more isotropic lower upper mantle. We conclude that our geodynamical modeling in a passive ridge spreading system does not produce the deep seismic anisotropy recently observed beneath the EPR. However, we do not consider partial melting, dynamic recrystallization and anisotropic viscosity which would change seismic interpretation and mantle flow, and thus further study is required.

In vitro evolution of high-titer, virus-like vesicles containing a single structural protein

PubMed Central

Rose, Nina F.; Buonocore, Linda; Schell, John B.; Chattopadhyay, Anasuya; Bahl, Kapil; Liu, Xinran; Rose, John K.

2014-01-01

Self-propagating, infectious, virus-like vesicles (VLVs) are generated when an alphavirus RNA replicon expresses the vesicular stomatitis virus glycoprotein (VSV G) as the only structural protein. The mechanism that generates these VLVs lacking a capsid protein has remained a mystery for over 20 years. We present evidence that VLVs arise from membrane-enveloped RNA replication factories (spherules) containing VSV G protein that are largely trapped on the cell surface. After extensive passaging, VLVs evolve to grow to high titers through acquisition of multiple point mutations in their nonstructural replicase proteins. We reconstituted these mutations into a plasmid-based system from which high-titer VLVs can be recovered. One of these mutations generates a late domain motif (PTAP) that is critical for high-titer VLV production. We propose a model in which the VLVs have evolved in vitro to exploit a cellular budding pathway that is hijacked by many enveloped viruses, allowing them to bud efficiently from the cell surface. Our results suggest a basic mechanism of propagation that may have been used by primitive RNA viruses lacking capsid proteins. Capsids may have evolved later to allow more efficient packaging of RNA, greater virus stability, and evasion of innate immunity. PMID:25385608

Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

PubMed

Hunt, L A

1980-08-01

To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein.

Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

PubMed Central

Hunt, L A

1980-01-01

To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein. Images PMID:6255177

Analysis of the entry mechanism of Crimean-Congo hemorrhagic fever virus, using a vesicular stomatitis virus pseudotyping system.

PubMed

Suda, Yuto; Fukushi, Shuetsu; Tani, Hideki; Murakami, Shin; Saijo, Masayuki; Horimoto, Taisuke; Shimojima, Masayuki

2016-06-01

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease causing severe hemorrhagic symptoms with a nearly 30 % case-fatality rate in humans. The experimental use of CCHF virus (CCHFV), which causes CCHF, requires high-biosafety-level (BSL) containment. In contrast, pseudotyping of various viral glycoproteins (GPs) onto vesicular stomatitis virus (VSV) can be used in facilities with lower BSL containment, and this has facilitated studies on the viral entry mechanism and the measurement of neutralizing activity, especially for highly pathogenic viruses. In the present study, we generated high titers of pseudotyped VSV bearing the CCHFV envelope GP and analyzed the mechanisms involved in CCHFV infection. A partial deletion of the CCHFV GP cytoplasmic domain increased the titer of the pseudotyped VSV, the entry mechanism of which was dependent on the CCHFV envelope GP. Using the pseudotype virus, DC-SIGN (a calcium-dependent [C-type] lectin cell-surface molecule) was revealed to enhance viral infection and act as an entry factor for CCHFV.

Virus recognition by Toll-7 activates antiviral autophagy in Drosophila.

PubMed

Nakamoto, Margaret; Moy, Ryan H; Xu, Jie; Bambina, Shelly; Yasunaga, Ari; Shelly, Spencer S; Gold, Beth; Cherry, Sara

2012-04-20

Innate immunity is highly conserved and relies on pattern recognition receptors (PRRs) such as Toll-like receptors (identified through their homology to Drosophila Toll) for pathogen recognition. Although Drosophila Toll is vital for immune recognition and defense, roles for the other eight Drosophila Tolls in immunity have remained elusive. Here we have shown that Toll-7 is a PRR both in vitro and in adult flies; loss of Toll-7 led to increased vesicular stomatitis virus (VSV) replication and mortality. Toll-7, along with additional uncharacterized Drosophila Tolls, was transcriptionally induced by VSV infection. Furthermore, Toll-7 interacted with VSV at the plasma membrane and induced antiviral autophagy independently of the canonical Toll signaling pathway. These data uncover an evolutionarily conserved role for a second Drosophila Toll receptor that links viral recognition to autophagy and defense and suggest that other Drosophila Tolls may restrict specific as yet untested pathogens, perhaps via noncanonical signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

Determinants of antibody persistence across doses and continents after single-dose rVSV-ZEBOV vaccination for Ebola virus disease: an observational cohort study.

PubMed

Huttner, Angela; Agnandji, Selidji Todagbe; Combescure, Christophe; Fernandes, José F; Bache, Emmanuel Bache; Kabwende, Lumeka; Ndungu, Francis Maina; Brosnahan, Jessica; Monath, Thomas P; Lemaître, Barbara; Grillet, Stéphane; Botto, Miriam; Engler, Olivier; Portmann, Jasmine; Siegrist, Denise; Bejon, Philip; Silvera, Peter; Kremsner, Peter; Siegrist, Claire-Anne

2018-04-04

The recombinant vesicular stomatitis virus (rVSV) vaccine expressing the Zaire Ebola virus (ZEBOV) glycoprotein is efficacious in the weeks following single-dose injection, but duration of immunity is unknown. We aimed to assess antibody persistence at 1 and 2 years in volunteers who received single-dose rVSV-ZEBOV in three previous trials. In this observational cohort study, we prospectively followed-up participants from the African and European phase 1 rVSV-ZEBOV trials, who were vaccinated once in 2014-15 with 300 000 (low dose) or 10-50 million (high dose) plaque-forming units (pfu) of rVSV-ZEBOV vaccine to assess ZEBOV glycoprotein (IgG) antibody persistence. The primary outcome was ZEBOV glycoprotein-specific IgG geometric mean concentrations (GMCs) measured yearly by ELISA compared with 1 month (ie, 28 days) after immunisation. We report GMCs up to 2 years (Geneva, Switzerland, including neutralising antibodies up to 6 months) and 1 year (Lambaréné, Gabon; Kilifi, Kenya) after vaccination and factors associated with higher antibody persistence beyond 6 months, according to multivariable analyses. Trials and the observational study were registered at ClinicalTrials.gov (Geneva: NCT02287480 and NCT02933931; Kilifi: NCT02296983) and the Pan-African Clinical Trials Registry (Lambaréné PACTR201411000919191). Of 217 vaccinees from the original studies (102 from the Geneva study, 75 from the Lambaréné study, and 40 from the Kilifi study), 197 returned and provided samples at 1 year (95 from the Geneva study, 63 from the Lambaréné, and 39 from the Kilifi study) and 90 at 2 years (all from the Geneva study). In the Geneva group, 44 (100%) of 44 participants who had been given a high dose (ie, 10-50 million pfu) of vaccine and who were seropositive at day 28 remained seropositive at 2 years, whereas 33 (89%) of 37 who had been given the low dose (ie, 300 000 pfu) remained seropositive for 2 years (p=0·042). In participants who had received a high dose, ZEBOV glycoprotein IgG GMCs decreased significantly between their peak (at 1-3 months) and month 6 after vaccination in Geneva (p<0·0001) and Lambaréné (p=0·0298) but not in Kilifi (p=0·5833) and subsequently remained stable at all sites apart from Geneva, where GMC in those given a high dose of vaccine increased significantly between 6 months and 1 year (p=0·0264). Antibody persistence was similar at 1 year and at 6 months in those who had received a low dose of vaccine, with lower titres among participants from the Geneva study at 2 years than at 1 year after vaccination (GMC ratio 0·61, 95% CI 0·49-0·77; p<0·0001). In multivariable analyses, predictors of increased IgG GMCs beyond 6 months included high-dose versus low-dose vaccination (Geneva p=0·0133; Lambaréné p=0·008) and vaccine-related arthritis (p=0·0176), but not sex, age, or baseline seropositivity (all p>0·05). Neutralising antibodies seem to be less durable, with seropositivity dropping from 64-71% at 28 days to 27-31% at 6 months in participants from the Geneva study. Antibody responses to single-dose rVSV-ZEBOV vaccination are sustained across dose ranges and settings, a key criterion in countries where booster vaccinations would be impractical. The Wellcome Trust and Innovative Medicines Initiative 2 Joint Undertaking. Copyright © 2018 Elsevier Ltd. All rights reserved.

W17_geowave “3D full waveform geophysical models”

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larmat, Carene; Maceira, Monica; Roy, Corinna

2018-02-12

Performance of the MCMC inversion according to the number of cores for the computation. A) 64 cores. B) 480 cores. C) 816 cores. The true model is represented by the black line. Vsv is the wave speed of S waves polarized in the vertical plane, ξ is an anisotropy parameter. The Earth is highly anisotropics; the wavespeed of seismic waves depends on the polarization of the wave. Seismic inversion of the elastic structure is usually limited to isotropic information such as Vsv. Our research looked at the inversion of Earth anisotropy.

CD133-targeted gene transfer into long-term repopulating hematopoietic stem cells.

PubMed

Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwäble, Joachim; Kaufmann, Kerstin B; Müller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J; Grez, Manuel

2015-01-01

Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.

Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

PubMed Central

Farr, Glen A.; Hull, Michael; Mellman, Ira

2009-01-01

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells. PMID:19620635

Antiviral activity of some South American medicinal plants.

PubMed

Abad, M J; Bermejo, P; Sanchez Palomino, S; Chiriboga, X; Carrasco, L

1999-03-01

Folk medicinal plants are potential sources of useful therapeutic compounds including some with antiviral activities. Extracts prepared from 10 South American medicinal plants (Baccharis trinervis, Baccharis teindalensis, Eupatorium articulatum, Eupatorium glutinosum, Tagetes pusilla, Neurolaena lobata, Conyza floribunda, Phytolacca bogotensis, Phytolacca rivinoides and Heisteria acuminata) were screened for in vitro antiviral activity against herpes simplex type I (HSV-1), vesicular stomatitis virus (VSV) and poliovirus type 1. The most potent inhibition was observed with an aqueous extract of B. trinervis, which inhibited HSV-1 replication by 100% at 50-200 micrograms/mL, without showing cytotoxic effects. Good activities were also found with the ethanol extract of H. acuminata and the aqueous extract of E. articulatum, which exhibited antiviral effects against both DNA and RNA viruses (HSV-1 and VSV, respectively) at 125-250 micrograms/mL. The aqueous extracts of T. pusilla (100-250 micrograms/mL), B. teindalensis (50-125 micrograms/mL) and E. glutinosum (50-125 micrograms/mL) also inhibited the replication of VSV, but none of the extracts tested had any effect on poliovirus replication.

MicroRNA-466l inhibits antiviral innate immune response by targeting interferon-alpha

PubMed Central

Li, Yingke; Fan, Xiaohua; He, Xingying; Sun, Haijing; Zou, Zui; Yuan, Hongbin; Xu, Haitao; Wang, Chengcai; Shi, Xueyin

2012-01-01

Effective recognition of viral infections and subsequent triggering of antiviral innate immune responses are essential for the host antiviral defense, which is tightly regulated by multiple regulators, including microRNAs (miRNAs). A previous study showed that miR-466l upregulates IL-10 expression in macrophages by antagonizing RNA-binding protein tristetraprolin-mediated IL-10 mRNA degradation. However, the ability of miR-466l to regulate antiviral immune responses remains unknown. Here, we found that interferon-alpha (IFN-α) expression was repressed in Sendai virus (SeV)- and vesicular stomatitis virus (VSV)-infected macrophages and in dendritic cells transfected with miR-466l expression. Moreover, multiple IFN-α species can be directly targeted by miR-466l through their 3′ untranslated region (3′UTR). This study has demonstrated that miR-466l could directly target IFN-α expression to inhibit host antiviral innate immune response. PMID:23042536

Quantitative proteomic profiling for clarification of the crucial roles of lysosomes in microbial infections.

PubMed

Xu, Benhong; Gao, Yanpan; Zhan, Shaohua; Ge, Wei

2017-07-01

Lysosomes play vital roles in both innate and adaptive immunity. It is widely accepted that lysosomes do not function exclusively as a digestive organelle. It is also involved in the process of immune cells against pathogens. However, the changes in the lysosomal proteome caused by infection with various microbes are still largely unknown, and our understanding of the proteome of the purified lysosome is another obstacle that needs to be resolved. Here, we performed a proteomic study on lysosomes enriched from THP1 cells after infection with Listeria monocytogenes (L.m), Herpes Simplex Virus 1 (HSV-1) and Vesicular Stomatitis Virus (VSV). In combination with the gene ontology (GO) analysis, we identified 284 lysosomal-related proteins from a total of 4560 proteins. We also constructed the protein-protein interaction networks for the differentially expressed proteins and revealed the core lysosomal proteins, including SRC in the L. m treated group, SRC, GLB1, HEXA and HEXB in the HSV-1 treated group and GLB1, CTSA, CTSB, HEXA and HEXB in the VSV treated group, which are involved in responding to diverse microbial infections. This study not only reveals variable lysosome responses depending on the bacterial or virus infection, but also provides the evidence based on which we propose a novel approach to proteome research for investigation of the function of the enriched organelles. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of the Bas-Congo virus glycoprotein and its function in pseudotyped viruses.

PubMed

Steffen, Imke; Liss, Nathan M; Schneider, Bradley S; Fair, Joseph N; Chiu, Charles Y; Simmons, Graham

2013-09-01

Bas-Congo virus (BASV) is a novel rhabdovirus recently identified from a patient with acute hemorrhagic fever in the Bas-Congo province of the Democratic Republic of Congo (DRC). Here we show that the BASV glycoprotein (BASV-G) can be successfully used to pseudotype glycoprotein-deficient vesicular stomatitis virus (VSV), allowing studies of BASV-G-driven membrane fusion and viral entry into target cells without replication-competent virus. BASV-G displayed broad tissue and species tropism in vitro, and BASV-G-mediated membrane fusion was pH dependent. The conformational changes induced in BASV-G by acidification were fully reversible and did not lead to inactivation of the viral fusion protein. Our data combined with comparative sequence similarity analyses suggest that BASV-G shares structural and functional features with other rhabdovirus glycoproteins and falls into the group of class III viral fusion proteins. However, activation of BASV-G-driven fusion required a lower pH and higher temperatures than did VSV-G-mediated fusion. Moreover, in contrast to VSV-G, mature BASV-G in VSV pseudotypes consists of a mixture of high-mannose and complex glycans that enables it to bind to certain C-type lectins, thereby enhancing its attachment to target cells. Taken together, the results presented in this study will facilitate future investigations of BASV-G-mediated cell entry and its inhibition in the absence of an infectious cell culture assay for BASV and at lower biosafety levels. Moreover, serology testing based on BASV-G pseudotype neutralization can be used to uncover the prevalence and importance of BASV as a potential novel human pathogen in the DRC and throughout Central Africa.

1-Cinnamoyl-3,11-dihydroxymeliacarpin is a natural bioactive compound with antiviral and nuclear factor-{kappa}B modulating properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barquero, Andrea A.; Michelini, Flavia M.; Alche, Laura E.

2006-06-09

We have reported the isolation of the tetranortriterpenoid 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM) from partially purified leaf extracts of Melia azedarach L. (MA) that reduced both, vesicular stomatitis virus (VSV) and Herpes simplex virus type 1 (HSV-1) multiplication. CDM blocks VSV entry and the intracellular transport of VSV-G protein, confining it to the Golgi apparatus, by pre- or post-treatment, respectively. Here, we report that HSV-1 glycoproteins were also confined to the Golgi apparatus independently of the nature of the host cell. Considering that MA could be acting as an immunomodulator preventing the development of herpetic stromal keratitis in mice, we also examined anmore » eventual effect of CDM on NF-{kappa}B signaling pathway. CDM is able to impede NF-{kappa}B activation in HSV-1-infected conjunctival cells and leads to the accumulation of p65 NF-{kappa}B subunit in the cytoplasm of uninfected treated Vero cells. In conclusion, CDM is a pleiotropic agent that not only inhibits the multiplication of DNA and RNA viruses by the same mechanism of action but also modulates the NF-{kappa}B signaling pathway.« less

5'-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping.

PubMed

Ogino, Minako; Ogino, Tomoaki

2017-03-15

The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5'-phospho-RNA (pRNA) from 5'-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5'-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m 7 G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m 7 GpppA (cap 0), respectively. Furthermore, either the 2'- or 3'-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5'-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as essential for the substrate activity. The findings presented here are useful not only for understanding the mechanism of the substrate recognition with PRNTase but also for designing antiviral agents targeting this enzyme. Copyright © 2017 American Society for Microbiology.

5′-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping

PubMed Central

Ogino, Minako

2017-01-01

ABSTRACT The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5′-phospho-RNA (pRNA) from 5′-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5′-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2′- or 3′-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5′-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as essential for the substrate activity. The findings presented here are useful not only for understanding the mechanism of the substrate recognition with PRNTase but also for designing antiviral agents targeting this enzyme. PMID:28053102

Development of novel types of plastid transformation vectors and evaluation of factors controlling expression.

PubMed

Herz, Stefan; Füssl, Monika; Steiger, Sandra; Koop, Hans-Ulrich

2005-12-01

Two new vector types for plastid transformation were developed and uidA reporter gene expression was compared to standard transformation vectors. The first vector type does not contain any plastid promoter, instead it relies on extension of existing plastid operons and was therefore named "operon-extension" vector. When a strongly expressed plastid operon like psbA was extended by the reporter gene with this vector type, the expression level was superior to that of a standard vector under control of the 16S rRNA promoter. Different insertion sites, promoters and 5'-UTRs were analysed for their effect on reporter gene expression with standard and operon-extension vectors. The 5'-UTR of phage 7 gene 10 in combination with a modified N-terminus was found to yield the highest expression levels. Expression levels were also strongly dependent on external factors like plant or leaf age or light intensity. In the second vector type, named "split" plastid transformation vector, modules of the expression cassette were distributed on two separate vectors. Upon co-transformation of plastids with these vectors, the complete expression cassette became inserted into the plastome. This result can be explained by successive co-integration of the split vectors and final loop-out recombination of the duplicated sequences. The split vector concept was validated with different vector pairs.

Differential Sensitivity of Bat Cells to Infection by Enveloped RNA Viruses: Coronaviruses, Paramyxoviruses, Filoviruses, and Influenza Viruses

PubMed Central

Hoffmann, Markus; Müller, Marcel Alexander; Drexler, Jan Felix; Glende, Jörg; Erdt, Meike; Gützkow, Tim; Losemann, Christoph; Binger, Tabea; Deng, Hongkui; Schwegmann-Weßels, Christel; Esser, Karl-Heinz; Drosten, Christian; Herrler, Georg

2013-01-01

Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed. PMID:24023659

Antiviral screening of forty-two Egyptian medicinal plants.

PubMed

Soltan, Maha Mohamed; Zaki, Adel Kamal

2009-10-29

Egyptian medicinal plants are well known by their diverse uses in traditional folk medicine to cure various ailments including infectious diseases. Forty-two Egyptian medicinal plant species were selected from local market and were subjected to antiviral screening bioassay to investigate and to evaluate their biological activities. Hydro-alcoholic extracts of each species were separately prepared and tested against three viruses: herpes simplex-1 virus (HSV), poliomyelitis-1 virus (POLIO) and vesicular stomatitis virus (VSV). The antiviral activity were determined by means of the end point titration technique (EPTT) that depends on the ability of plant extract dilutions to inhibit the produced cytopathogenic effect (CPE) and expressed as reduction factor (Rf) of the viral titer. Achillea fragrantissima, Jasonia montana and Globularia arabica are found to have antiviral activity against POLIO in a concentration dependent manner at complete non-toxic concentration range 10-100 microg/ml (Rf 10(6)), 10-100 microg/ml (Rf 10(5)) and 50-100 microg/ml (Rf 10(4)), respectively while Tanacetum sinaicum are found to have moderate antiviral activity against POLIO at concentration of 50-100 microg/ml (Rf 10(2)). Ephedra alata and Moringa peregrina are found to have antiviral activity against HSV (Rf 10(4)). Also, the results revealed that Capparis sinaica, Tamarix nilotica and Cyperus rotundus are found to have virucidal effect against HSV. All the forty-two plant species are found to have no reliable antiviral activity against VSV. The specific indications claimed by the traditional healers are confirmed by antiviral test.

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

PubMed Central

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors. Images PMID:2404285

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

PubMed

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.

Crust and Mantle Deformation Revealed from High-Resolution Radially Anisotropic Velocity Models

NASA Astrophysics Data System (ADS)

Li, A.; Dave, R.; Yao, Y.

2017-12-01

Love wave tomography, which can achieve a similar model resolution as Rayleigh wave, so far has limited applications to the USArray data. Recently, we have developed high-resolution Love wave phase velocity maps in the Wyoming craton and Texas using data at the Transportable Array stations. 3-D, radially anisotropic velocity models are obtained by jointly inverting Love and Rayleigh wave phase velocities. A high-velocity anomaly extending to about 200 km depth beneath central Wyoming correlates with negative radial anisotropy (Vsv>Vsh), suggesting that mantle downwelling develops under the cratonic lithosphere. Surprisingly, the significantly low velocity beneath the Yellowstone hotspot, which has been interpreted as partial melting and asthenospheric upwelling, is associated with the largest radial anisotropy (Vsh>Vsv) in the area. This observation does not support mantle upwelling. Instead, it indicates that the upper mantle beneath the hotspot has experienced strong shear deformation probably by the plate motion and large-scale mantle flow. In Texas, positive radial anisotropy in the lower crust extends from the coast to the Ouachita belt, which is characterized by high velocity and negative radial anisotropy. In the upper mantle, large variations of velocity and anisotropy exit under the coastal plain. A common feature in these anisotropic models is that high-velocity anomalies in the upper mantle often correlate with negative anisotropy (Vsv>Vsh) while low-velocity anomalies are associated with positive anisotropy (Vsh>Vsv). The manifestation of mantle downweling as negative radial anisotropy is largely due to the relatively high viscosity of the high-velocity mantle block, which is less affected by the surrounding large-scale horizontal flow. However, mantle upwelling, which is often associated with low-velocity anomalies, presumably low-viscosity mantle blocks, is invisible in radial anisotropy models. Such upwelling may happen too quickly to make last effects or too slow to alter the dominant shear deformation in the asthenosphere.

Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

PubMed

Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

2014-11-01

Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates heterologous expression of large gene clusters for drug discovery. Copyright © 2014 Elsevier Inc. All rights reserved.

Stability of Lentiviral Vector-Mediated Transgene Expression in the Brain in the Presence of Systemic Antivector Immune Responses

PubMed Central

ABORDO-ADESIDA, EVELYN; FOLLENZI, ANTONIA; BARCIA, CARLOS; SCIASCIA, SANDRA; CASTRO, MARIA G.; NALDINI, LUIGI; LOWENSTEIN, PEDRO R.

2009-01-01

Lentiviral vectors are promising tools for gene therapy in the CNS. It is therefore important to characterize their interactions with the immune system in the CNS. This work characterizes transgene expression and brain inflammation in the presence or absence of immune responses generated after systemic immunization with lentiviral vectors. We characterized transduction with SIN-LV vectors in the CNS. A dose—response curve using SIN-LV-GFP demonstrated detectable transgene expression in the striatum at a dose of 102, and maximum expression at 106, transducing units of lentiviral vector, with minimal increase in inflammatory markers between the lowest and highest dose of vector injected. Our studies demonstrate that injection of a lentiviral vector into the CNS did not cause a measurable inflammatory response. Systemic immunization after CNS injection, with the lentiviral vector expressing the same transgene as a vector injected into the CNS, caused a decrease in transgene expression in the CNS, concomitantly with an infiltration of inflammatory cells into the CNS parenchyma at the injection site. However, peripheral immunization with a lentiviral vector carrying a different transgene did not diminish transgene expression, or cause CNS inflammation. Systemic immunization preceding injection of lentiviral vectors into the CNS determined that preexisting antilentiviral immunity, regardless of the transgene, did not affect transgene expression. Furthermore, we showed that the transgene, but not the virion or vector components, is responsible for providing antigenic epitopes to the activated immune system, on systemic immunization with lentivirus. Low immunogenicity and prolonged transgene expression in the presence of preexisting lentiviral immunity are encouraging data for the future use of lentiviral vectors in CNS gene therapy. In summary, the lentiviral vectors tested induced undetectable activation of innate immune responses, and stimulation of adaptive immune responses against lentiviral vectors was effective in causing a decrease in transgene expression only if the immune response was directed against the transgene. A systemic immune response against vector components alone did not cause brain inflammation, possibly because vector-derived epitopes were not being presented in the CNS. PMID:15960605

Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons

PubMed Central

Morral, Núria; O’Neal, Wanda; Rice, Karen; Leland, Michele; Kaplan, Johanne; Piedra, Pedro A.; Zhou, Heshan; Parks, Robin J.; Velji, Rizwan; Aguilar-Córdova, Estuardo; Wadsworth, Samuel; Graham, Frank L.; Kochanek, Stefan; Carey, K. Dee; Beaudet, Arthur L.

1999-01-01

The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human α1-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3–5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helper-dependent vectors with sequential delivery of vectors of different serotypes. PMID:10536005

Chimeric Filoviruses for Identification and Characterization of Monoclonal Antibodies.

PubMed

Ilinykh, Philipp A; Shen, Xiaoli; Flyak, Andrew I; Kuzmina, Natalia; Ksiazek, Thomas G; Crowe, James E; Bukreyev, Alexander

2016-04-01

Recent experiments suggest that some glycoprotein (GP)-specific monoclonal antibodies (MAbs) can protect experimental animals against the filovirus Ebola virus (EBOV). There is a need for isolation of MAbs capable of neutralizing multiple filoviruses. Antibody neutralization assays for filoviruses frequently use surrogate systems such as the rhabdovirus vesicular stomatitis Indiana virus (VSV), lentiviruses or gammaretroviruses with their envelope proteins replaced with EBOV GP or pseudotyped with EBOV GP. It is optimal for both screening and in-depth characterization of newly identified neutralizing MAbs to generate recombinant filoviruses that express a reporter fluorescent protein in order to more easily monitor and quantify the infection. Our study showed that unlike neutralization-sensitive chimeric VSV, authentic filoviruses are highly resistant to neutralization by MAbs. We used reverse genetics techniques to replace EBOV GP with its counterpart from the heterologous filoviruses Bundibugyo virus (BDBV), Sudan virus, and even Marburg virus and Lloviu virus, which belong to the heterologous genera in the filovirus family. This work resulted in generation of multiple chimeric filoviruses, demonstrating the ability of filoviruses to tolerate swapping of the envelope protein. The sensitivity of chimeric filoviruses to neutralizing MAbs was similar to that of authentic biologically derived filoviruses with the same GP. Moreover, disabling the expression of the secreted GP (sGP) resulted in an increased susceptibility of an engineered virus to the BDBV52 MAb isolated from a BDBV survivor, suggesting a role for sGP in evasion of antibody neutralization in the context of a human filovirus infection. The study demonstrated that chimeric rhabdoviruses in which G protein is replaced with filovirus GP, widely used as surrogate targets for characterization of filovirus neutralizing antibodies, do not accurately predict the ability of antibodies to neutralize authentic filoviruses, which appeared to be resistant to neutralization. However, a recombinant EBOV expressing a fluorescent protein tolerated swapping of GP with counterparts from heterologous filoviruses, allowing high-throughput screening of B cell lines to isolate MAbs of any filovirus specificity. Human MAb BDBV52, which was isolated from a survivor of BDBV infection, was capable of partially neutralizing a chimeric EBOV carrying BDBV GP in which expression of sGP was disabled. In contrast, the parental virus expressing sGP was resistant to the MAb. Thus, the ability of filoviruses to tolerate swapping of GP can be used for identification of neutralizing MAbs specific to any filovirus and for the characterization of MAb specificity and mechanism of action. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Chimeric Filoviruses for Identification and Characterization of Monoclonal Antibodies

PubMed Central

Ilinykh, Philipp A.; Shen, Xiaoli; Flyak, Andrew I.; Kuzmina, Natalia; Ksiazek, Thomas G.; Crowe, James E.

2016-01-01

ABSTRACT Recent experiments suggest that some glycoprotein (GP)-specific monoclonal antibodies (MAbs) can protect experimental animals against the filovirus Ebola virus (EBOV). There is a need for isolation of MAbs capable of neutralizing multiple filoviruses. Antibody neutralization assays for filoviruses frequently use surrogate systems such as the rhabdovirus vesicular stomatitis Indiana virus (VSV), lentiviruses or gammaretroviruses with their envelope proteins replaced with EBOV GP or pseudotyped with EBOV GP. It is optimal for both screening and in-depth characterization of newly identified neutralizing MAbs to generate recombinant filoviruses that express a reporter fluorescent protein in order to more easily monitor and quantify the infection. Our study showed that unlike neutralization-sensitive chimeric VSV, authentic filoviruses are highly resistant to neutralization by MAbs. We used reverse genetics techniques to replace EBOV GP with its counterpart from the heterologous filoviruses Bundibugyo virus (BDBV), Sudan virus, and even Marburg virus and Lloviu virus, which belong to the heterologous genera in the filovirus family. This work resulted in generation of multiple chimeric filoviruses, demonstrating the ability of filoviruses to tolerate swapping of the envelope protein. The sensitivity of chimeric filoviruses to neutralizing MAbs was similar to that of authentic biologically derived filoviruses with the same GP. Moreover, disabling the expression of the secreted GP (sGP) resulted in an increased susceptibility of an engineered virus to the BDBV52 MAb isolated from a BDBV survivor, suggesting a role for sGP in evasion of antibody neutralization in the context of a human filovirus infection. IMPORTANCE The study demonstrated that chimeric rhabdoviruses in which G protein is replaced with filovirus GP, widely used as surrogate targets for characterization of filovirus neutralizing antibodies, do not accurately predict the ability of antibodies to neutralize authentic filoviruses, which appeared to be resistant to neutralization. However, a recombinant EBOV expressing a fluorescent protein tolerated swapping of GP with counterparts from heterologous filoviruses, allowing high-throughput screening of B cell lines to isolate MAbs of any filovirus specificity. Human MAb BDBV52, which was isolated from a survivor of BDBV infection, was capable of partially neutralizing a chimeric EBOV carrying BDBV GP in which expression of sGP was disabled. In contrast, the parental virus expressing sGP was resistant to the MAb. Thus, the ability of filoviruses to tolerate swapping of GP can be used for identification of neutralizing MAbs specific to any filovirus and for the characterization of MAb specificity and mechanism of action. PMID:26819310

Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, cluster-randomised trial (Ebola Ça Suffit!).

PubMed

Henao-Restrepo, Ana Maria; Camacho, Anton; Longini, Ira M; Watson, Conall H; Edmunds, W John; Egger, Matthias; Carroll, Miles W; Dean, Natalie E; Diatta, Ibrahima; Doumbia, Moussa; Draguez, Bertrand; Duraffour, Sophie; Enwere, Godwin; Grais, Rebecca; Gunther, Stephan; Gsell, Pierre-Stéphane; Hossmann, Stefanie; Watle, Sara Viksmoen; Kondé, Mandy Kader; Kéïta, Sakoba; Kone, Souleymane; Kuisma, Eewa; Levine, Myron M; Mandal, Sema; Mauget, Thomas; Norheim, Gunnstein; Riveros, Ximena; Soumah, Aboubacar; Trelle, Sven; Vicari, Andrea S; Røttingen, John-Arne; Kieny, Marie-Paule

2017-02-04

rVSV-ZEBOV is a recombinant, replication competent vesicular stomatitis virus-based candidate vaccine expressing a surface glycoprotein of Zaire Ebolavirus. We tested the effect of rVSV-ZEBOV in preventing Ebola virus disease in contacts and contacts of contacts of recently confirmed cases in Guinea, west Africa. We did an open-label, cluster-randomised ring vaccination trial (Ebola ça Suffit!) in the communities of Conakry and eight surrounding prefectures in the Basse-Guinée region of Guinea, and in Tomkolili and Bombali in Sierra Leone. We assessed the efficacy of a single intramuscular dose of rVSV-ZEBOV (2×10 7 plaque-forming units administered in the deltoid muscle) in the prevention of laboratory confirmed Ebola virus disease. After confirmation of a case of Ebola virus disease, we definitively enumerated on a list a ring (cluster) of all their contacts and contacts of contacts including named contacts and contacts of contacts who were absent at the time of the trial team visit. The list was archived, then we randomly assigned clusters (1:1) to either immediate vaccination or delayed vaccination (21 days later) of all eligible individuals (eg, those aged ≥18 years and not pregnant, breastfeeding, or severely ill). An independent statistician generated the assignment sequence using block randomisation with randomly varying blocks, stratified by location (urban vs rural) and size of rings (≤20 individuals vs >20 individuals). Ebola response teams and laboratory workers were unaware of assignments. After a recommendation by an independent data and safety monitoring board, randomisation was stopped and immediate vaccination was also offered to children aged 6-17 years and all identified rings. The prespecified primary outcome was a laboratory confirmed case of Ebola virus disease with onset 10 days or more from randomisation. The primary analysis compared the incidence of Ebola virus disease in eligible and vaccinated individuals assigned to immediate vaccination versus eligible contacts and contacts of contacts assigned to delayed vaccination. This trial is registered with the Pan African Clinical Trials Registry, number PACTR201503001057193. In the randomised part of the trial we identified 4539 contacts and contacts of contacts in 51 clusters randomly assigned to immediate vaccination (of whom 3232 were eligible, 2151 consented, and 2119 were immediately vaccinated) and 4557 contacts and contacts of contacts in 47 clusters randomly assigned to delayed vaccination (of whom 3096 were eligible, 2539 consented, and 2041 were vaccinated 21 days after randomisation). No cases of Ebola virus disease occurred 10 days or more after randomisation among randomly assigned contacts and contacts of contacts vaccinated in immediate clusters versus 16 cases (7 clusters affected) among all eligible individuals in delayed clusters. Vaccine efficacy was 100% (95% CI 68·9-100·0, p=0·0045), and the calculated intraclass correlation coefficient was 0·035. Additionally, we defined 19 non-randomised clusters in which we enumerated 2745 contacts and contacts of contacts, 2006 of whom were eligible and 1677 were immediately vaccinated, including 194 children. The evidence from all 117 clusters showed that no cases of Ebola virus disease occurred 10 days or more after randomisation among all immediately vaccinated contacts and contacts of contacts versus 23 cases (11 clusters affected) among all eligible contacts and contacts of contacts in delayed plus all eligible contacts and contacts of contacts never vaccinated in immediate clusters. The estimated vaccine efficacy here was 100% (95% CI 79·3-100·0, p=0·0033). 52% of contacts and contacts of contacts assigned to immediate vaccination and in non-randomised clusters received the vaccine immediately; vaccination protected both vaccinated and unvaccinated people in those clusters. 5837 individuals in total received the vaccine (5643 adults and 194 children), and all vaccinees were followed up for 84 days. 3149 (53·9%) of 5837 individuals reported at least one adverse event in the 14 days after vaccination; these were typically mild (87·5% of all 7211 adverse events). Headache (1832 [25·4%]), fatigue (1361 [18·9%]), and muscle pain (942 [13·1%]) were the most commonly reported adverse events in this period across all age groups. 80 serious adverse events were identified, of which two were judged to be related to vaccination (one febrile reaction and one anaphylaxis) and one possibly related (influenza-like illness); all three recovered without sequelae. The results add weight to the interim assessment that rVSV-ZEBOV offers substantial protection against Ebola virus disease, with no cases among vaccinated individuals from day 10 after vaccination in both randomised and non-randomised clusters. WHO, UK Wellcome Trust, the UK Government through the Department of International Development, Médecins Sans Frontières, Norwegian Ministry of Foreign Affairs (through the Research Council of Norway's GLOBVAC programme), and the Canadian Government (through the Public Health Agency of Canada, Canadian Institutes of Health Research, International Development Research Centre and Department of Foreign Affairs, Trade and Development). Copyright © 2017 World Health Organization. Published by Elsevier Ltd/Inc/BV. All rights reserved. Published by Elsevier Ltd.. All rights reserved.

Long-term preservation of retinal function in the RCS rat model of retinitis pigmentosa following lentivirus-mediated gene therapy.

PubMed

Tschernutter, M; Schlichtenbrede, F C; Howe, S; Balaggan, K S; Munro, P M; Bainbridge, J W B; Thrasher, A J; Smith, A J; Ali, R R

2005-04-01

The Royal College of Surgeons (RCS) rat is a well-characterized model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding , a receptor tyrosine kinase found in RPE cells, that is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. In order to evaluate the efficacy of lentiviral-mediated gene replacement therapy in the RCS rat, we produced recombinant VSV-G pseudotyped HIV-1-based lentiviruses containing a murine Mertk cDNA driven by a spleen focus forming virus (SFFV) promoter. The vector was subretinally injected into the right eye of 10-day-old RCS rats; the left eye was left untreated as an internal control. Here, we present a detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits. We examined animals at various time points over a period of 7 months by light and electron microscopy, and electroretinography. We observed correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function for up to 7 months. This study demonstrates the potential of gene therapy approaches for the treatment of retinal degenerations caused by defects specific to the RPE and supports the use of lentiviral vectors for the treatment of such disorders.

Design, synthesis, and biological evaluation of novel iso-D-2',3'-dideoxy-3'-fluorothianucleoside derivatives.

PubMed

Kim, Kyung Ran; Moon, Hyung Ryong; Park, Ah-Young; Chun, Moon Woo; Jeong, Lak Shin

2007-01-01

Novel iso-d-2',3'-dideoxythianucleoside derivatives 1-4 were designed and asymmetrically synthesized as a bioisostere of lamivudine to search for new anti-HIV agents. The information about using sulfur participation occurred on DAST fluorination and Mitsunobu reaction will be of great help in synthesizing sulfur-containing compounds. Final compounds 1-4 were evaluated against HIV-1 and 2, HSV-1 and 2, EMCV, Cox. B3, VSV, FluA (Taiwan), FluA (Johan.), FCV, and FIP. Only cytosine analogue 3 showed a potent anti-VSV activity (EC(50)=9.43microg/mL). This result implies that iso-2',3'-dideoxy sugar templates might play a role of a sugar surrogate of nucleosides for the development of anti-RNA virus agent.

Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression.

PubMed

Jiang, Xiaoou; Yu, Han; Teo, Cui Rong; Tan, Genim Siu Xian; Goh, Sok Chin; Patel, Parasvi; Chua, Yiqiang Kevin; Hameed, Nasirah Banu Sahul; Bertoletti, Antonio; Patzel, Volker

2016-09-01

Dumbbell-shaped DNA minimal vectors lacking nontherapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, nonviral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of noncoding or coding RNA. We minimized small hairpin RNA (shRNA) or microRNA (miRNA) expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimized shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimized miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA.

Geminivirus vectors for high-level expression of foreign proteins in plant cells.

PubMed

Mor, Tsafrir S; Moon, Yong-Sun; Palmer, Kenneth E; Mason, Hugh S

2003-02-20

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430-437, 2003.

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields

PubMed Central

Carnes, Aaron E.; Luke, Jeremy M.; Vincent, Justin M.; Anderson, Sheryl; Schukar, Angela; Hodgson, Clague P.; Williams, James A.

2010-01-01

Background For safety considerations, regulatory agencies recommend elimination of antibiotic resistance markers and nonessential sequences from plasmid DNA-based gene medicines. In the present study we analyzed antibiotic-free (AF) vector design criteria impacting bacterial production and mammalian transgene expression. Methods Both CMV-HTLV-I R RNA Pol II promoter (protein transgene) and murine U6 RNA Pol III promoter (RNA transgene) vector designs were studied. Plasmid production yield was assessed through inducible fed-batch fermentation. RNA Pol II-directed EGFP and RNA Pol III-directed RNA expression were quantified by fluorometry and quantitative real-time polymerase chain reaction (RT-PCR), respectively, after transfection of human HEK293 cells. Results Sucrose-selectable minimalized protein and therapeutic RNA expression vector designs that combined an RNA-based AF selection with highly productive fermentation manufacturing (>1,000 mg/L plasmid DNA) and high level in vivo expression of encoded products were identified. The AF selectable marker was also successfully applied to convert existing kanamycin-resistant DNA vaccine plasmids gWIZ and pVAX1 into AF vectors, demonstrating a general utility for retrofitting existing vectors. A minimum vector size for high yield plasmid fermentation was identified. A strategy for stable fermentation of plasmid dimers with improved vector potency and fermentation yields up to 1,740 mg/L was developed. Conclusions We report the development of potent high yield AF gene medicine expression vectors for protein or RNA (e.g. short hairpin RNA or microRNA) products. These AF expression vectors were optimized to exceed a newly identified size threshold for high copy plasmid replication and direct higher transgene expression levels than alternative vectors. PMID:20806425

Modular protein expression by RNA trans-splicing enables flexible expression of antibody formats in mammalian cells from a dual-host phage display vector.

PubMed

Shang, Yonglei; Tesar, Devin; Hötzel, Isidro

2015-10-01

A recently described dual-host phage display vector that allows expression of immunoglobulin G (IgG) in mammalian cells bypasses the need for subcloning of phage display clone inserts to mammalian vectors for IgG expression in large antibody discovery and optimization campaigns. However, antibody discovery and optimization campaigns usually need different antibody formats for screening, requiring reformatting of the clones in the dual-host phage display vector to an alternative vector. We developed a modular protein expression system mediated by RNA trans-splicing to enable the expression of different antibody formats from the same phage display vector. The heavy-chain region encoded by the phage display vector is directly and precisely fused to different downstream heavy-chain sequences encoded by complementing plasmids simply by joining exons in different pre-mRNAs by trans-splicing. The modular expression system can be used to efficiently express structurally correct IgG and Fab fragments or other antibody formats from the same phage display clone in mammalian cells without clone reformatting. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Disclosing the Parameters Leading to High Productivity of Retroviral Producer Cells Lines: Evaluating Random Versus Targeted Integration.

PubMed

Bandeira, Vanessa S; Tomás, Hélio A; Alici, Evren; Carrondo, Manuel J T; Coroadinha, Ana S

2017-04-01

Gammaretrovirus and lentivirus are the preferred viral vectors to genetically modify T and natural killer cells to be used in immune cell therapies. The transduction efficiency of hematopoietic and T cells is more efficient using gibbon ape leukemia virus (GaLV) pseudotyping. In this context gammaretroviral vector producer cells offer competitive higher titers than transient lentiviral vectors productions. The main aim of this work was to identify the key parameters governing GaLV-pseudotyped gammaretroviral vector productivity in stable producer cells, using a retroviral vector expression cassette enabling positive (facilitating cell enrichment) and negative cell selection (allowing cell elimination). The retroviral vector contains a thymidine kinase suicide gene fused with a ouabain-resistant Na + ,K + -ATPase gene, a potential safer and faster marker. The establishment of retroviral vector producer cells is traditionally performed by randomly integrating the retroviral vector expression cassette codifying the transgene. More recently, recombinase-mediated cassette exchange methodologies have been introduced to achieve targeted integration. Herein we compared random and targeted integration of the retroviral vector transgene construct. Two retroviral producer cell lines, 293 OuaS and 293 FlexOuaS, were generated by random and targeted integration, respectively, producing high titers (on the order of 10 7 infectious particles·ml -1 ). Results showed that the retroviral vector transgene cassette is the key retroviral vector component determining the viral titers notwithstanding, single-copy integration is sufficient to provide high titers. The expression levels of the three retroviral constructs (gag-pol, GaLV env, and retroviral vector transgene) were analyzed. Although gag-pol and GaLV env gene expression levels should surpass a minimal threshold, we found that relatively modest expression levels of these two expression cassettes are required. Their levels of expression should not be maximized. We concluded, to establish a high producer retroviral vector cell line only the expression level of the genomic retroviral RNA, that is, the retroviral vector transgene cassette, should be maximized, both through (1) the optimization of its design (i.e., genetic elements composition) and (2) the selection of high expressing chromosomal locus for its integration. The use of methodologies identifying and promoting integration into high-expression loci, as targeted integration or high-throughput screening are in this perspective highly valuable.

Inhalation of Nebulized Perfluorochemical Enhances Recombinant Adenovirus and Adeno-Associated Virus-Mediated Gene Expression in Lung Epithelium

PubMed Central

Beckett, Travis; Bonneau, Laura; Howard, Alan; Blanchard, James; Borda, Juan; Weiner, Daniel J.; Wang, Lili; Gao, Guang Ping; Kolls, Jay K.; Bohm, Rudolf; Liggitt, Denny

2012-01-01

Abstract Use of perfluorochemical liquids during intratracheal vector administration enhances recombinant adenovirus and adeno-associated virus (AAV)-mediated lung epithelial gene expression. We hypothesized that inhalation of nebulized perfluorochemical vapor would also enhance epithelial gene expression after subsequent intratracheal vector administration. Freely breathing adult C57BL/6 mice were exposed for selected times to nebulized perflubron or sterile saline in a sealed Plexiglas chamber. Recombinant adenoviral vector was administered by transtracheal puncture at selected times afterward and mice were killed 3 days after vector administration to assess transgene expression. Mice tolerated the nebulized perflubron without obvious ill effects. Vector administration 6 hr after nebulized perflubron exposure resulted in an average 540% increase in gene expression in airway and alveolar epithelium, compared with that with vector alone or saline plus vector control (p<0.05). However, vector administration 1 hr, 1 day, or 3 days after perflubron exposure was not different from either nebulized saline with vector or vector alone and a 60-min exposure to nebulized perflubron is required. In parallel pilot studies in macaques, inhalation of nebulized perflubron enhanced recombinant AAV2/5 vector expression throughout the lung. Serial chest radiographs, bronchoalveolar lavages, and results of complete blood counts and serum biochemistries demonstrated no obvious adverse effects of nebulized perflubron. Further, one macaque receiving nebulized perflubron only was monitored for 1 year with no obvious adverse effects of exposure. These results demonstrate that inhalation of nebulized perflubron, a simple, clinically more feasible technique than intratracheal administration of liquid perflubron, safely enhances lung gene expression. PMID:22568624

Definition of Contravariant Velocity Components

NASA Technical Reports Server (NTRS)

Hung, Ching-moa; Kwak, Dochan (Technical Monitor)

2002-01-01

In this paper we have reviewed the basics of tensor analysis in an attempt to clarify some misconceptions regarding contravariant and covariant vector components as used in fluid dynamics. We have indicated that contravariant components are components of a given vector expressed as a unique combination of the covariant base vector system and, vice versa, that the covariant components are components of a vector expressed with the contravariant base vector system. Mathematically, expressing a vector with a combination of base vector is a decomposition process for a specific base vector system. Hence, the contravariant velocity components are decomposed components of velocity vector along the directions of coordinate lines, with respect to the covariant base vector system. However, the contravariant (and covariant) components are not physical quantities. Their magnitudes and dimensions are controlled by their corresponding covariant (and contravariant) base vectors.

Development of siRNA expression vector utilizing rock bream beta-actin promoter: a potential therapeutic tool against viral infection in fish.

PubMed

Zenke, Kosuke; Nam, Yoon Kwon; Kim, Ki Hong

2010-01-01

In the present study, we have developed short interfering RNA (siRNA) expression vector utilizing rock bream beta-actin promoter and examined the possible use for the inhibition of highly pathogenic fish virus, rock bream iridovirus (RBIV), replication in vitro. Initially, in order to express siRNA effectively, we added several modifications to wild-type rock bream beta-actin promoter. Next, we succeeded in knocking down the expression of enhanced green fluorescent protein reporter gene expression in fish cells using newly developed vector more effectively than the fugu U6 promoter-driven vector we described previously. Finally, we could observe that cells transfected with modified rock bream beta-actin promoter-driven siRNA expression vector targeting major capsid protein (MCP) gene of RBIV exhibited more resistance to RBIV challenge than other control cells. Our results indicate that this novel siRNA expression vector can be used as a new tool for therapeutics in virus infection in fish species.

Novel Nonreplicating Vaccinia Virus Vector Enhances Expression of Heterologous Genes and Suppresses Synthesis of Endogenous Viral Proteins.

PubMed

Wyatt, Linda S; Xiao, Wei; Americo, Jeffrey L; Earl, Patricia L; Moss, Bernard

2017-06-06

Viruses are used as expression vectors for protein synthesis, immunology research, vaccines, and therapeutics. Advantages of poxvirus vectors include the accommodation of large amounts of heterologous DNA, the presence of a cytoplasmic site of transcription, and high expression levels. On the other hand, competition of approximately 200 viral genes with the target gene for expression and immune recognition may be disadvantageous. We describe a vaccinia virus (VACV) vector that uses an early promoter to express the bacteriophage T7 RNA polymerase; has the A23R intermediate transcription factor gene deleted, thereby restricting virus replication to complementing cells; and has a heterologous gene regulated by a T7 promoter. In noncomplementing cells, viral early gene expression and DNA replication occurred normally but synthesis of intermediate and late proteins was prevented. Nevertheless, the progeny viral DNA provided templates for abundant expression of heterologous genes regulated by a T7 promoter. Selective expression of the Escherichia coli lac repressor gene from an intermediate promoter reduced transcription of the heterologous gene specifically in complementing cells, where large amounts might adversely impact VACV replication. Expression of heterologous proteins mediated by the A23R deletion vector equaled that of a replicating VACV, was higher than that of a nonreplicating modified vaccinia virus Ankara (MVA) vector used for candidate vaccines in vitro and in vivo , and was similarly immunogenic in mice. Unlike the MVA vector, the A23R deletion vector still expresses numerous early genes that can restrict immunogenicity as demonstrated here by the failure of the prototype vector to induce interferon alpha. By deleting immunomodulatory genes, we anticipate further improvements in the system. IMPORTANCE Vaccines provide an efficient and effective way of preventing infectious diseases. Nevertheless, new and better vaccines are needed. Vaccinia virus, which was used successfully as a live vaccine to eradicate smallpox, has been further attenuated and adapted as a recombinant vector for immunization against other pathogens. However, since the initial description of this vector system, only incremental improvements largely related to safety have been implemented. Here we described novel modifications of the platform that increased expression of the heterologous target gene and decreased expression of endogenous vaccinia virus genes while providing safety by preventing replication of the candidate vaccine except in complementing cells used for vector propagation. Copyright © 2017 Wyatt et al.

Use of vacuum-steam-vacuum and ionizing radiation to eliminate Listeria innocua from ham.

PubMed

Sommers, Christopher; Kozempel, Michael; Fan, Xuetong; Radewonuk, E Richard

2002-12-01

Listeria spp. are a frequent postprocess contaminant of ready-to-eat (RTE) meat products, including ham. Vacuum-steam-vacuum (VSV) technology has been used successfully to eliminate Listeria innocua from hot dogs. Ionizing radiation can eliminate Listeria spp. from RTE meats. However, the excessive application of either technology can cause changes in product quality, including structural changes, changes in cure color (redness), and lipid oxidation. In this study, two cycles of VSV were combined with 2.0 kGy of ionizing radiation to obtain 4.40- and 4.85-log10 reductions of L. innocua on ham meat and skin, respectively. The use of both treatments resulted in an additive, as opposed to synergistic, reduction of L. innocua on ham. The combination treatment did not cause statistically significant changes in product structure, color (redness), or lipid oxidation.

Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters.

PubMed

Ferreira, Joshua P; Peacock, Ryan W S; Lawhorn, Ingrid E B; Wang, Clifford L

2011-12-01

The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines. The online version of this article (doi:10.1007/s11693-011-9089-0) contains supplementary material, which is available to authorized users.

TMV-Gate vectors: Gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins

PubMed Central

Kagale, Sateesh; Uzuhashi, Shihomi; Wigness, Merek; Bender, Tricia; Yang, Wen; Borhan, M. Hossein; Rozwadowski, Kevin

2012-01-01

Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts. PMID:23166857

EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors.

PubMed

Martella, Andrea; Matjusaitis, Mantas; Auxillos, Jamie; Pollard, Steven M; Cai, Yizhi

2017-07-21

Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology.

Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline-inducible vectors.

PubMed

Nakashima, N; Tamura, T

2013-06-01

Here, we report on the construction of doxycycline (tetracycline analogue)-inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl-β-D-galactopyranoside-inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline-inducible promoter with the T7 promoter-T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline-inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications. A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose. © 2013 The Society for Applied Microbiology.

Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

PubMed

Hong, Y K; Kim, D H; Beletskii, A; Lee, C; Memili, E; Strauss, W M

2001-04-01

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells. Copyright 2001 Academic Press.

TimesVector: a vectorized clustering approach to the analysis of time series transcriptome data from multiple phenotypes.

PubMed

Jung, Inuk; Jo, Kyuri; Kang, Hyejin; Ahn, Hongryul; Yu, Youngjae; Kim, Sun

2017-12-01

Identifying biologically meaningful gene expression patterns from time series gene expression data is important to understand the underlying biological mechanisms. To identify significantly perturbed gene sets between different phenotypes, analysis of time series transcriptome data requires consideration of time and sample dimensions. Thus, the analysis of such time series data seeks to search gene sets that exhibit similar or different expression patterns between two or more sample conditions, constituting the three-dimensional data, i.e. gene-time-condition. Computational complexity for analyzing such data is very high, compared to the already difficult NP-hard two dimensional biclustering algorithms. Because of this challenge, traditional time series clustering algorithms are designed to capture co-expressed genes with similar expression pattern in two sample conditions. We present a triclustering algorithm, TimesVector, specifically designed for clustering three-dimensional time series data to capture distinctively similar or different gene expression patterns between two or more sample conditions. TimesVector identifies clusters with distinctive expression patterns in three steps: (i) dimension reduction and clustering of time-condition concatenated vectors, (ii) post-processing clusters for detecting similar and distinct expression patterns and (iii) rescuing genes from unclassified clusters. Using four sets of time series gene expression data, generated by both microarray and high throughput sequencing platforms, we demonstrated that TimesVector successfully detected biologically meaningful clusters of high quality. TimesVector improved the clustering quality compared to existing triclustering tools and only TimesVector detected clusters with differential expression patterns across conditions successfully. The TimesVector software is available at http://biohealth.snu.ac.kr/software/TimesVector/. sunkim.bioinfo@snu.ac.kr. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

PubMed

Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

2009-12-30

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

A tetracycline inducible expression vector for Corynebacterium glutamicum allowing tightly regulable gene expression.

PubMed

Lausberg, Frank; Chattopadhyay, Ava Rebecca; Heyer, Antonia; Eggeling, Lothar; Freudl, Roland

2012-09-01

Here we report on the construction of a tetracycline inducible expression vector that allows a tightly regulable gene expression in Corynebacterium glutamicum which is used in industry for production of small molecules such as amino acids. Using the green fluorescent protein (GFP) as a reporter protein we show that this vector, named pCLTON1, is characterized by tight repression under non-induced conditions as compared to a conventional IPTG inducible expression vector, and that it allows gradual GFP synthesis upon gradual increase of anhydrotetracycline addition. Copyright © 2012 Elsevier Inc. All rights reserved.

A Novel Role for the Receptor of the Complement Cleavage Fragment C5a, C5aR1, in CCR5-Mediated Entry of HIV into Macrophages.

PubMed

Moreno-Fernandez, Maria E; Aliberti, Julio; Groeneweg, Sander; Köhl, Jörg; Chougnet, Claire A

2016-04-01

The complement system is an ancient pattern recognition system that becomes activated during all stages of HIV infection. Previous studies have shown that C5a can enhance the infection of monocyte-derived macrophages and T cells indirectly through the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and the attraction of dendritic cells. C5a exerts its multiple biologic functions mainly through activation of C5a receptor 1 (C5aR1). Here, we assessed the role of C5aR1 as an enhancer of CCR5-mediated HIV infection. We determined CCR5 and C5aR1 heterodimer formation in myeloid cells and the impact of C5aR1 blockade on HIV entry and genomic integration. C5aR1/CCR5 heterodimer formation was identified by immunoprecipitation and western blotting. THP-1 cells and monocyte-derived macrophages (MDM) were infected by R5 laboratory strains or HIV pseudotyped for the vesicular stomatitis virus (VSV) envelope. Levels of integrated HIV were measured by quantitative PCR after targeting of C5aR1 by a C5aR antagonist, neutralizing C5aR1 monoclonal antibody (mAb) or hC5a. C5aR1 was also silenced by specific siRNA prior to viral entry. We found that C5aR1 forms heterodimers with the HIV coreceptor CCR5 in myeloid cells. Targeting C5aR1 significantly decreased integration by R5 viruses but not by VSV-pseudotyped viruses, suggesting that C5aR1 is critical for viral entry. The level of inhibition achieved with C5aR1-blocking reagents was comparable to that of CCR5 antagonists. Mechanistically, C5aR1 targeting decreased CCR5 expression. MDM from CCR5Δ32 homozygous subjects expressed levels of C5aR1 similar to CCR5 WT individuals, suggesting that mere C5aR1 expression is not sufficient for HIV infection. HIV appeared to preferentially enter THP-1 cells expressing high levels of both C5aR1 and CCR5. Targeted reduction of C5aR1 expression in such cells reduced HIV infection by ~50%. Our data thus suggest that C5aR1 acts as an enhancer of CCR5-mediated HIV entry into macrophages, the targeting of which may prove useful to reduce HIV infection by R5 strains.

[Use of a novel baculovirus vector to express nucleoprotein gene of Crimean-Congo hemorrhagic fever virus in both insect and mammalian cells].

PubMed

Ma, Benjiang; Hang, Changshou; Zhao, Yun; Wang, Shiwen; Xie, Yanxiang

2002-09-01

To construct a novel baculovirus vector which is capable of promoting the high-yield expression of foreign gene in mammalian cells and to express by this vector the nucleoprotein (NP) gene of Crimean-Congo hemorrhagic fever virus (CCHFV) Chinese isolate (Xinjiang hemorrhagic fever virus, XHFV) BA88166 in insect and Vero cells. Human cytomegalovirus (CMV) immediate early (IE) promoter was ligated to the baculovirus vector pFastBac1 downstream of the polyhedrin promoter to give rise to the novel vector pCB1. XHFV NP gene was cloned to this vector and was well expressed in COS-7 cells and Vero cells by means of recombinant plasmid transfection and baculovirus infection. The XHFV NP gene in vector pCB1 could be well expressed in mammalian cells. Vero cells infected with recombinant baculovirus harboring NP gene could be employed as antigens to detect XHF serum specimens whose results were in good correlation with those of ELISA and in parallel with clinical diagnoses. This novel baculovirus vector is able to express the foreign gene efficiently in both insect and mammalian cells, which provides not only the convenient diagnostic antigens but also the potential for developing recombinant virus vaccines and gene therapies.

Development of the gateway recycling cloning system for multiple linking of expression cassettes in a defined order, and direction on gateway compatible binary vectors.

PubMed

Kimura, Tetsuya; Nakao, Akihide; Murata, Sachiko; Kobayashi, Yasuyuki; Tanaka, Yuji; Shibahara, Kenta; Kawazu, Tetsu; Nakagawa, Tsuyoshi

2013-01-01

We developed the Gateway recycling cloning system, which allows multiple linking of expression cassettes by multiple rounds of the Gateway LR reaction. Employing this system, the recycling donor vector pRED419 was subjected to the first LR reaction with an attR1-attR2 type destination vector. Then conversion vector pCON was subjected to an LR reaction to restore the attR1-attR2 site on the destination vector for the next cloning cycle. By repetition of these two simple steps, we linked four expression cassettes of a reporter gene in Gateway binary vector pGWB1, introduced the constructs into tobacco BY-2 cells, and observed the expression of transgenes.

Human T Cell Leukemia Virus Type 1 Tax Inhibits Innate Antiviral Signaling via NF-κB-Dependent Induction of SOCS1▿

PubMed Central

Charoenthongtrakul, Soratree; Zhou, Qinjie; Shembade, Noula; Harhaj, Nicole S.; Harhaj, Edward W.

2011-01-01

Human T cell leukemia virus type 1 (HTLV-1) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-1 Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-1-transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-I-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-1-transformed cell line MT-2 resulted in increased IFN-β expression accompanied by reduced HTLV-1 replication and p19Gag levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein. PMID:21593151

Identification of a broad-spectrum inhibitor of virus RNA synthesis: validation of a prototype virus-based approach

PubMed Central

Filone, Claire Marie; Hodges, Erin N.; Honeyman, Brian; Bushkin, G. Guy; Boyd, Karla; Platt, Andrew; Ni, Feng; Strom, Kyle; Hensley, Lisa; Snyder, John K.; Connor, John H.

2013-01-01

There are no approved therapeutics for the most deadly nonsegmented negative-strand (NNS) RNA viruses, including Ebola (EBOV). To identify new chemical scaffolds for development of broad-spectrum antivirals, we undertook a prototype-based lead identification screen. Using the prototype NNS virus, vesicular stomatitis virus (VSV), multiple inhibitory compounds were identified. Three compounds were investigated for broad-spectrum activity, and inhibited EBOV infection. The most potent, CMLDBU3402, was selected for further study. CMLDBU3402 did not show significant activity against segmented negative-strand RNA viruses suggesting proscribed broad-spectrum activity. Mechanistic analysis indicated that CMLDBU3402 blocked VSV viral RNA synthesis and inhibited EBOV RNA transcription, demonstrating a consistent mechanism of action against genetically distinct viruses. The identification of this chemical backbone as a broad-spectrum inhibitor of viral RNA synthesis offers significant potential for the development of new therapies for highly pathogenic viruses. PMID:23521799

AAVPG: A vigilant vector where transgene expression is induced by p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bajgelman, Marcio C.; Medrano, Ruan F.V.; Carvalho, Anna Carolina P.V.

2013-12-15

Using p53 to drive transgene expression from viral vectors may provide on demand expression in response to physiologic stress, such as hypoxia or DNA damage. Here we introduce AAVPG, an adeno-associated viral (AAV) vector where a p53-responsive promoter, termed PG, is used to control transgene expression. In vitro assays show that expression from the AAVPG-luc vector was induced specifically in the presence of functional p53 (1038±202 fold increase, p<0.001). The AAVPG-luc vector was an effective biosensor of p53 activation in response to hypoxia (4.48±0.6 fold increase in the presence of 250 µM CoCl{sub 2}, p<0.001) and biomechanical stress (2.53±0.4 foldmore » increase with stretching, p<0.05). In vivo, the vigilant nature of the AAVPG-luc vector was revealed after treatment of tumor-bearing mice with doxorubicin (pre-treatment, 3.4×10{sup 5}±0.43×10{sup 5} photons/s; post-treatment, 6.6×10{sup 5}±2.1×10{sup 5} photons/s, p<0.05). These results indicate that the AAVPG vector is an interesting option for detecting p53 activity both in vitro and in vivo. - Highlights: • AAV vector where transgene expression is controlled by the tumor suppressor p53. • The new vector, AAVPG, shown to function as a biosensor of p53 activity, in vitro and in vivo. • The p53 activity monitored by the AAVPG vector is relevant to cancer and other diseases. • AAVPG reporter gene expression was activated upon DNA damage, hypoxia and mechanical stress.« less

Construction of two vectors for gene expression in Trichoderma reesei.

PubMed

Lv, Dandan; Wang, Wei; Wei, Dongzhi

2012-01-01

We report the construction of two filamentous fungi Trichoderma reesei expression vectors, pWEF31 and pWEF32. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase 1 gene (cbh1) from T. reesei. The two newly constructed vectors can be efficiently transformed into T. reesei with Agrobacterium-mediated transformation. The difference between pWEF31 and pWEF32 is that pWEF32 has two longer homologous arms. As a result, pWEF32 easily undergoes homologous recombination. On the other hand, pWEF31 undergoes random recombination. The applicability of both vectors was tested by first generating the expression vectors pWEF31-red and pWEF32-red and then detecting the expression of the DsRed2 gene in T. reesei Rut C30. Additionally, we measured the exo-1,4-β-glucanase activity of the recombinant cells. Our work provides an effective transformation system for homologous and heterologous gene expression and gene knockout in T. reesei. It also provides a method for recombination at a specific chromosomal location. Finally, both vectors will be useful for the large-scale gene expression industry. Copyright © 2011 Elsevier Inc. All rights reserved.

Molecular cloning and characterization of a novel bovine IFN-ε.

PubMed

Guo, Yongli; Gao, Mingchun; Bao, Jun; Luo, Xiuxin; Liu, Ying; An, Dong; Zhang, Haili; Ma, Bo; Wang, Junwei

2015-03-01

A bovine IFN-ε (BoIFN-ε) gene was amplified from bovine liver genomic DNA consisting of a 463bp partial 5'UTR, 582bp complete ORF and 171bp partial 3'UTR, which encodes a protein of 193 amino acids with a 21-amino acid signal peptide and shares 61 to 87% identity with other species IFN-ε. Then BoIFN-ε gene was characterized, and it can be transcribed in EBK cells at a high level after being infected by VSV. Recombinant proteins were expressed in Escherichia coli and the antiviral activity was determined in vitro, which revealed that bovine IFN-ε has less antiviral activity than bovine IFN-α. In addition, an immunofluorescence assay indicated that BoIFN-ε expressed in MDBK cells could be detected by polyclonal antibody against BoIFN-ε. Furthermore, the BoIFN-ε gene can be constitutively expressed in the liver, thymus, kidney, small intestine and testis, but not in the heart. This study revealed that BoIFN-ε has the typical characteristics of type I interferon and can be expressed constitutively in certain tissue, which not only can be a likely candidate for a novel, effective therapeutic agent, but also facilitate further research on the role of bovine IFN system. Copyright © 2014 Elsevier B.V. All rights reserved.

Configurations of a two-tiered amplified gene expression system in adenoviral vectors designed to improve the specificity of in vivo prostate cancer imaging

PubMed Central

Sato, M; Figueiredo, ML; Burton, JB; Johnson, M; Chen, M; Powell, R; Gambhir, SS; Carey, M; Wu, L

2009-01-01

Effective treatment for recurrent, disseminated prostate cancer is notably limited. We have developed adenoviral vectors with a prostate-specific two-step transcriptional amplification (TSTA) system that would express therapeutic genes at a robust level to target metastatic disease. The TSTA system employs the prostate-specific antigen (PSA) promoter/enhancer to drive a potent synthetic activator, which in turn activates the expression of the therapeutic gene. In this study, we explored different configurations of this bipartite system and discovered that physical separation of the two TSTA components into E1 and E3 regions of adenovirus was able to enhance androgen regulation and cell-discriminatory expression. The TSTA vectors that express imaging reporter genes were assessed by noninvasive imaging technologies in animal models. The improved selectivity of the E1E3 configured vector was reflected in silenced ectopic expression in the lung. Significantly, the enhanced specificity of the E1E3 vector enabled the detection of lung metastasis of prostate cancer. An E1E3 TSTA vector that expresses the herpes simplex virus thymidine kinase gene can effectively direct positron emission tomography (PET) imaging of the tumor. The prostate-targeted gene delivery vectors with robust and cell-specific expression capability will advance the development of safe and effective imaging guided therapy for recurrent metastatic stages of prostate cancer. PMID:18305574

[Construction, identification and expression of three kinds of shuttle plasmids of adenovirus expression vector of hepatitis C virus structure gene].

PubMed

Cao, Yi-zhan; Hao, Chun-qiu; Feng, Zhi-hua; Zhou, Yong-xing; Li, Jin-ge; Jia, Zhan-sheng; Wang, Ping-zhong

2003-02-01

To construct three recombinant shuttle plasmids of adenovirus expression vector which can express hepatitis C virus(HCV) different structure genes(C, C+E1, C+E1+E2) in order to pack adenovirus expression vectors which can express HCV different structure gene effectively. The different HCV structure genes derived from the plasmid pBRTM/HCV1-3011 by using polymerase chain reaction (PCR) were inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of shuttle plasmid(pAd.CMV-Link.1) of adenovirus expression vector respectively, then the three recombinant plasmids (pAd.HCV-C, pAd.HCV-CE1, pAd.HCV-S) were obtained. The recombinant plasmids were identified by endonuclease, PCR and sequencing. HCV structure genes were expressed transiently with Lipofectamine 2000 coated in HepG2 cells which were confirmed by immunofluorescence and Western-Blot. Insert DNAs of the three recombinant plasmids' were confirmed to be HCV different structure genes by endonuclease, PCR and sequencing. The three recombinant plasmids can express HCV structure gene (C, C+E1, C+E1+E2) transiently in HepG2 cells which were confirmed by immunofluorescence and Western-Blot. The three recombinant shuttle plasmids of adenovirus expression vector can express HCV structure gene(C, C+E1, C+E1+E2) transiently. This should be useful to pack adenovirus expression vector which can express HCV structure genes.

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design.

PubMed

Ahmadi, Samira; Davami, Fatemeh; Davoudi, Noushin; Nematpour, Fatemeh; Ahmadi, Maryam; Ebadat, Saeedeh; Azadmanesh, Kayhan; Barkhordari, Farzaneh; Mahboudi, Fereidoun

2017-01-01

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios.

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design

PubMed Central

Ahmadi, Samira; Davami, Fatemeh; Davoudi, Noushin; Nematpour, Fatemeh; Ahmadi, Maryam; Ebadat, Saeedeh; Azadmanesh, Kayhan; Barkhordari, Farzaneh

2017-01-01

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios. PMID:28662065

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

PubMed Central

2009-01-01

Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. Conclusion These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency. PMID:20042112

Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

PubMed Central

Blanc, Andrea Maria; Berois, Mabel Beatriz; Tomé, Lorena Magalí; Epstein, Alberto L.

2012-01-01

Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease. PMID:22437537

Synthetically derived bat influenza A-like viruses reveal a cell type- but not species-specific tropism.

PubMed

Moreira, Étori Aguiar; Locher, Samira; Kolesnikova, Larissa; Bolte, Hardin; Aydillo, Teresa; García-Sastre, Adolfo; Schwemmle, Martin; Zimmer, Gert

2016-10-24

Two novel influenza A-like viral genome sequences have recently been identified in Central and South American fruit bats and provisionally designated "HL17NL10" and "HL18NL11." All efforts to isolate infectious virus from bats or to generate these viruses by reverse genetics have failed to date. Recombinant vesicular stomatitis virus (VSV) encoding the hemagglutinin-like envelope glycoproteins HL17 or HL18 in place of the VSV glycoprotein were generated to identify cell lines that are susceptible to bat influenza A-like virus entry. More than 30 cell lines derived from various species were screened but only a few cell lines were found to be susceptible, including Madin-Darby canine kidney type II (MDCK II) cells. The identification of cell lines susceptible to VSV chimeras allowed us to recover recombinant HL17NL10 and HL18NL11 viruses from synthetic DNA. Both influenza A-like viruses established a productive infection in MDCK II cells; however, HL18NL11 replicated more efficiently than HL17NL10 in this cell line. Unlike conventional influenza A viruses, bat influenza A-like viruses started the infection preferentially at the basolateral membrane of polarized MDCK II cells; however, similar to conventional influenza A viruses, bat influenza A-like viruses were released primarily from the apical site. The ability of HL18NL11 or HL17NL10 viruses to infect canine and human cells might reflect a zoonotic potential of these recently identified bat viruses.

Synthetically derived bat influenza A-like viruses reveal a cell type- but not species-specific tropism

PubMed Central

Moreira, Étori Aguiar; Locher, Samira; Kolesnikova, Larissa; Bolte, Hardin; Aydillo, Teresa; García-Sastre, Adolfo; Schwemmle, Martin; Zimmer, Gert

2016-01-01

Two novel influenza A-like viral genome sequences have recently been identified in Central and South American fruit bats and provisionally designated “HL17NL10” and “HL18NL11.” All efforts to isolate infectious virus from bats or to generate these viruses by reverse genetics have failed to date. Recombinant vesicular stomatitis virus (VSV) encoding the hemagglutinin-like envelope glycoproteins HL17 or HL18 in place of the VSV glycoprotein were generated to identify cell lines that are susceptible to bat influenza A-like virus entry. More than 30 cell lines derived from various species were screened but only a few cell lines were found to be susceptible, including Madin–Darby canine kidney type II (MDCK II) cells. The identification of cell lines susceptible to VSV chimeras allowed us to recover recombinant HL17NL10 and HL18NL11 viruses from synthetic DNA. Both influenza A-like viruses established a productive infection in MDCK II cells; however, HL18NL11 replicated more efficiently than HL17NL10 in this cell line. Unlike conventional influenza A viruses, bat influenza A-like viruses started the infection preferentially at the basolateral membrane of polarized MDCK II cells; however, similar to conventional influenza A viruses, bat influenza A-like viruses were released primarily from the apical site. The ability of HL18NL11 or HL17NL10 viruses to infect canine and human cells might reflect a zoonotic potential of these recently identified bat viruses. PMID:27791106

Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

PubMed

Stanton, Richard J; McSharry, Brian P; Armstrong, Melanie; Tomasec, Peter; Wilkinson, Gavin W G

2008-12-01

With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

Lentivirus Vectors Incorporating the Immunoglobulin Heavy Chain Enhancer and Matrix Attachment Regions Provide Position-Independent Expression in B Lymphocytes

PubMed Central

Lutzko, Carolyn; Senadheera, Dinithi; Skelton, Dianne; Petersen, Denise; Kohn, Donald B.

2003-01-01

In the present studies we developed lentivirus vectors with regulated, consistent transgene expression in B lymphocytes by incorporating the immunoglobulin heavy chain enhancer (Eμ) with and without associated matrix attachment regions (EμMAR) into lentivirus vectors. Incorporation of these fragments upstream of phosphoglycerate kinase (PGK) or cytomegalovirus promoters resulted in a two- to threefold increase in enhanced green fluorescent protein (EGFP) mean fluorescence intensity (MFI) in B-lymphoid but not T-lymphoid, myeloid, fibroblast, or carcinoma cell lines. A 1-log increase in EGFP expression was observed in B-lymphoid cells (but not myeloid cells) differentiated from human CD34+ progenitors in vitro transduced with Eμ- and EμMAR-containing lentivectors. Lastly, we evaluated the expression from the EμMAR element in mice 2 to 24 weeks posttransplant with transduced hematopoietic stem cells. In mice receiving vectors with the Eμ and EμMAR elements upstream of the PGK promoter, there was a 2- to 10-fold increase in EGFP expression in B cells (but not other cell types). Evaluation of the coefficient of variation of expression among different cell types demonstrated that consistent, position-independent transgene expression was observed exclusively in B cells transduced with the EμMAR-containing vector and not other cells types or vectors. Proviral genomes with the EμMAR element had increased chromatin accessibility, which likely contributed to the position independence of expression in B lymphocytes. In summary, incorporation of the EμMAR element in lentivirus vectors resulted in enhanced, position-independent expression in primary B lymphocytes. These vectors provide a useful tool for the study of B-lymphocyte biology and the development of gene therapy for disorders affecting B lymphocytes, such as immune deficiencies. PMID:12805432

Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites.

PubMed

Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

2006-03-06

Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics.

Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites

PubMed Central

Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

2006-01-01

Background Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. Results To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. Conclusion The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics. PMID:16519801

Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

PubMed

Tagliavia, Marcello; Cuttitta, Angela

2016-01-01

High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

Expression of Rous sarcoma virus-derived retroviral vectors in the avian blastoderm: Potential as stable genetic markers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reddy, S.T.; Stoker, A.W.; Bissell, M.J.

1991-12-01

Retroviruses are valuable tools in studies of embryonic development, both as gene expression vectors and as cell lineage markers. In this study early chicken blastoderm cells are shown to be permissive for infection by Rous sarcoma virus and derivative replication-defective by Rous sarcoma virus and derivative replication-defective vectors, and, in contrast to previously published data, these cells will readily express viral genes. In cultured blastoderm cells, Rous sarcoma virus stably integrates and is transcribed efficiently, producing infectious virus particles. Using replication-defective vectors encoding the bacterial lacZ gene, the authors further show that blastoderms can be infected in culture and inmore » ovo. In ovo, lacZ expression is seen within 24 hours of virus inoculation, and by 96 hours stably expressing clones of cells are observed in diverse tissues throughout the embryo, including epidermis, somites, and heart, as well as in extraembryonic membranes. Given the rapid onset of vector expression and the broad range of permissive cell types, it should be feasible to use Rous sarcoma virus-derived retroviruses as early lineage markers and expression vectors beginning at the blastoderm stage of avian embryogenesis.« less

Inhibition of highly productive HIV-1 infection in T cells, primary human macrophages, microglia, and astrocytes by Sargassum fusiforme

PubMed Central

Paskaleva, Elena E; Lin, Xudong; Li, Wen; Cotter, Robin; Klein, Michael T; Roberge, Emily; Yu, Er K; Clark, Bruce; Veille, Jean-Claude; Liu, Yanze; Lee, David Y-W; Canki, Mario

2006-01-01

Background The high rate of HIV-1 mutation and increasing resistance to currently available antiretroviral (ART) therapies highlight the need for new antiviral agents. Products derived from natural sources have been shown to inhibit HIV-1 replication during various stages of the virus life cycle, and therefore represent a potential source of novel therapeutic agents. To expand our arsenal of therapeutics against HIV-1 infection, we investigated aqueous extract from Sargassum fusiforme (S. fusiforme) for ability to inhibit HIV-1 infection in the periphery, in T cells and human macrophages, and for ability to inhibit in the central nervous system (CNS), in microglia and astrocytes. Results S. fusiforme extract blocked HIV-1 infection and replication by over 90% in T cells, human macrophages and microglia, and it also inhibited pseudotyped HIV-1 (VSV/NL4-3) infection in human astrocytes by over 70%. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5)-tropic HIV-1, was dose dependant and long lasting, did not inhibit cell growth or viability, was not toxic to cells, and was comparable to inhibition by the nucleoside analogue 2', 3'-didoxycytidine (ddC). S. fusiforme treatment blocked direct cell-to-cell infection spread. To investigate at which point of the virus life cycle this inhibition occurs, we infected T cells and CD4-negative primary human astrocytes with HIV-1 pseudotyped with envelope glycoprotein of vesicular stomatitis virus (VSV), which bypasses the HIV receptor requirements. Infection by pseudotyped HIV-1 (VSV/NL4-3) was also inhibited in a dose dependant manner, although up to 57% less, as compared to inhibition of native NL4-3, indicating post-entry interferences. Conclusion This is the first report demonstrating S. fusiforme to be a potent inhibitor of highly productive HIV-1 infection and replication in T cells, in primary human macrophages, microglia, and astrocytes. Results with VSV/NL4-3 infection, suggest inhibition of both entry and post-entry events of the virus life cycle. Absence of cytotoxicity and high viability of treated cells also suggest that S. fusiforme is a potential source of novel naturally occurring antiretroviral compounds that inhibit HIV-1 infection and replication at more than one site of the virus life cycle. PMID:16725040

Adenoviral vector tethering to metal surfaces via hydrolysable cross-linkers for the modulation of vector release and transduction

PubMed Central

Fishbein, Ilia; Forbes, Scott P.; Chorny, Michael; Connolly, Jeanne M.; Adamo, Richard F.; Corrales, Ricardo; Alferiev, Ivan S.; Levy, Robert J.

2013-01-01

The use of arterial stents and other medical implants as a delivery platform for surface immobilized gene vectors allows for safe and efficient localized expression of therapeutic transgenes. In this study we investigate the use of hydrolysable cross-linkers with distinct kinetics of hydrolysis for delivery of gene vectors from polyallylamine bisphosphonate-modified metal surfaces. Three cross-linkers with the estimated t1/2 of ester bonds hydrolysis of 5, 12 and 50 days demonstrated a cumulative 20%, 39% and 45% vector release, respectively, after 30 days exposure to physiological buffer at 37°C. Transgene expression in endothelial and smooth muscles cells transduced with substrate immobilized adenovirus resulted in significantly different expression profiles for each individual cross-linker. Furthermore, immobilization of adenoviral vectors effectively extended their transduction effectiveness beyond the initial phase of release. Transgene expression driven by adenovirus-tethered stents in rat carotid arteries demonstrated that a faster rate of cross-linker hydrolysis resulted in higher expression levels at day 1, which declined by day 8 after stent implantation, while inversely, slower hydrolysis was associated with increased arterial expression at day 8 in comparison with day 1. In conclusion, adjustable release of transduction-competent adenoviral vectors from metallic surfaces can be achieved, both in vitro and in vivo, through surface immobilization of adenoviral vectors using hydrolysable cross-linkers with structure-specific release kinetics. PMID:23777912

Kinematic sensitivity of robot manipulators

NASA Technical Reports Server (NTRS)

Vuskovic, Marko I.

1989-01-01

Kinematic sensitivity vectors and matrices for open-loop, n degrees-of-freedom manipulators are derived. First-order sensitivity vectors are defined as partial derivatives of the manipulator's position and orientation with respect to its geometrical parameters. The four-parameter kinematic model is considered, as well as the five-parameter model in case of nominally parallel joint axes. Sensitivity vectors are expressed in terms of coordinate axes of manipulator frames. Second-order sensitivity vectors, the partial derivatives of first-order sensitivity vectors, are also considered. It is shown that second-order sensitivity vectors can be expressed as vector products of the first-order sensitivity vectors.

Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits.

PubMed

Arazi, T; Slutsky, S G; Shiboleth, Y M; Wang, Y; Rubinstein, M; Barak, S; Yang, J; Gal-On, A

2001-04-27

Plant virus vectors provide an attractive biotechnological tool for the transient expression of foreign genes in whole plants. As yet there has been no use of recombinant viruses for the improvement of commercial crops. This is mainly because the viruses used to create vectors usually cause significant yield loss and can be transmitted in the field. A novel attenuated zucchini yellow mosaic potyvirus (AG) was used for the development of an environmentally safe non-pathogenic virus vector. The suitability of AG as an expression vector in plants was tested by analysis of two infectious viral constructs, each containing a distinct gene insertion site. Introduction of a foreign viral coat protein gene into AG genome between the P1 and HC-Pro genes, resulted in no expression in planta. In contrast, the same gene was stably expressed when inserted between NIb and CP genes, suggesting that this site is more suitable for a gene vector. Virus-mediated expression of reporter genes was observed in squash and cucumber leaves, stems, roots and edible fruit. Furthermore, AG stably expressed human interferon-alpha 2, an important human anti-viral drug, without affecting plant development and yield. Interferon biological activity was measured in cucumber and squash fruit. Together, these data corroborate a biotechnological utility of AG as a non-pathogenic vector for the expression of a foreign gene, as a benefit trait, in cucurbits and their edible fruit.

Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector.

PubMed

Rhee, Sun-Ju; Jang, Yoon Jeong; Lee, Gung Pyo

2016-06-01

Heterologous gene expression using plant virus vectors enables research on host-virus interactions and the production of useful proteins, but the host range of plant viruses limits the practical applications of such vectors. Here, we aimed to develop a viral vector based on cucumber fruit mottle mosaic virus (CFMMV), a member of the genus Tobamovirus, whose members infect cucurbits. The subgenomic promoter (SGP) in the coat protein (CP) gene, which was used to drive heterologous expression, was mapped by analyzing deletion mutants from a CaMV 35S promoter-driven infectious CFMMV clone. The region from nucleotides (nt) -55 to +160 relative to the start codon of the open reading frame (ORF) of CP was found to be a fully active promoter, and the region from nt -55 to +100 was identified as the active core promoter. Based on these SGPs, we constructed a cloning site in the CFMMV vector and successfully expressed enhanced green fluorescent protein (EGFP) in Nicotiana benthamiana and watermelon (Citrullus lanatus). Co-inoculation with the P19 suppressor increased EGFP expression and viral replication by blocking degradation of the viral genome. Our CFMMV vector will be useful as an expression vector in cucurbits.

Cone-Specific Promoters for Gene Therapy of Achromatopsia and Other Retinal Diseases

PubMed Central

Ye, Guo-Jie; Budzynski, Ewa; Sonnentag, Peter; Nork, T. Michael; Sheibani, Nader; Gurel, Zafer; Boye, Sanford L.; Peterson, James J.; Boye, Shannon E.; Hauswirth, William W.; Chulay, Jeffrey D.

2016-01-01

Adeno-associated viral (AAV) vectors containing cone-specific promoters have rescued cone photoreceptor function in mouse and dog models of achromatopsia, but cone-specific promoters have not been optimized for use in primates. Using AAV vectors administered by subretinal injection, we evaluated a series of promoters based on the human L-opsin promoter, or a chimeric human cone transducin promoter, for their ability to drive gene expression of green fluorescent protein (GFP) in mice and nonhuman primates. Each of these promoters directed high-level GFP expression in mouse photoreceptors. In primates, subretinal injection of an AAV-GFP vector containing a 1.7-kb L-opsin promoter (PR1.7) achieved strong and specific GFP expression in all cone photoreceptors and was more efficient than a vector containing the 2.1-kb L-opsin promoter that was used in AAV vectors that rescued cone function in mouse and dog models of achromatopsia. A chimeric cone transducin promoter that directed strong GFP expression in mouse and dog cone photoreceptors was unable to drive GFP expression in primate cones. An AAV vector expressing a human CNGB3 gene driven by the PR1.7 promoter rescued cone function in the mouse model of achromatopsia. These results have informed the design of an AAV vector for treatment of patients with achromatopsia. PMID:26603570

Cone-Specific Promoters for Gene Therapy of Achromatopsia and Other Retinal Diseases.

PubMed

Ye, Guo-Jie; Budzynski, Ewa; Sonnentag, Peter; Nork, T Michael; Sheibani, Nader; Gurel, Zafer; Boye, Sanford L; Peterson, James J; Boye, Shannon E; Hauswirth, William W; Chulay, Jeffrey D

2016-01-01

Adeno-associated viral (AAV) vectors containing cone-specific promoters have rescued cone photoreceptor function in mouse and dog models of achromatopsia, but cone-specific promoters have not been optimized for use in primates. Using AAV vectors administered by subretinal injection, we evaluated a series of promoters based on the human L-opsin promoter, or a chimeric human cone transducin promoter, for their ability to drive gene expression of green fluorescent protein (GFP) in mice and nonhuman primates. Each of these promoters directed high-level GFP expression in mouse photoreceptors. In primates, subretinal injection of an AAV-GFP vector containing a 1.7-kb L-opsin promoter (PR1.7) achieved strong and specific GFP expression in all cone photoreceptors and was more efficient than a vector containing the 2.1-kb L-opsin promoter that was used in AAV vectors that rescued cone function in mouse and dog models of achromatopsia. A chimeric cone transducin promoter that directed strong GFP expression in mouse and dog cone photoreceptors was unable to drive GFP expression in primate cones. An AAV vector expressing a human CNGB3 gene driven by the PR1.7 promoter rescued cone function in the mouse model of achromatopsia. These results have informed the design of an AAV vector for treatment of patients with achromatopsia.

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

PubMed

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Lagoumintzis, George; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

PubMed Central

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors. PMID:29091919

Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein.

PubMed

Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo

2013-07-19

RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways. Copyright © 2013 Elsevier Inc. All rights reserved.

A Partial E3 Deletion in Replication-Defective Adenoviral Vectors Allows for Stable Expression of Potentially Toxic Transgene Products.

PubMed

Haut, Larissa H; Gill, Amanda L; Kurupati, Raj K; Bian, Ang; Li, Yan; Giles-Davis, Wynetta; Xiang, Zhiquan; Zhou, Xiang Yang; Ertl, Hildegund C J

2016-10-01

Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert envelope proteins (Env) of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1- and E3-deleted AdC vector of serotype C6 expressing Env of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis. The same partial E3 deletion also allows for the generation of stable glycoprotein 140 (gp140)- and gp160-expressing Ad vectors based on AdC7, a distinct AdC serotype. Env-expressing AdC vectors containing the partial E3 deletion are genetically stable upon serial cell culture passaging, produce yields comparable to those of other AdC vectors, and induce transgene product-specific antibody responses in mice. A partial E3 deletion thereby allows expansion of the repertoire of transgenes that can be expressed by Ad vectors.

Alpharetroviral Self-inactivating Vectors: Long-term Transgene Expression in Murine Hematopoietic Cells and Low Genotoxicity

PubMed Central

Suerth, Julia D; Maetzig, Tobias; Brugman, Martijn H; Heinz, Niels; Appelt, Jens-Uwe; Kaufmann, Kerstin B; Schmidt, Manfred; Grez, Manuel; Modlich, Ute; Baum, Christopher; Schambach, Axel

2012-01-01

Comparative integrome analyses have highlighted alpharetroviral vectors with a relatively neutral, and thus favorable, integration spectrum. However, previous studies used alpharetroviral vectors harboring viral coding sequences and intact long-terminal repeats (LTRs). We recently developed self-inactivating (SIN) alpharetroviral vectors with an advanced split-packaging design. In a murine bone marrow (BM) transplantation model we now compared alpharetroviral, gammaretroviral, and lentiviral SIN vectors and showed that all vectors transduced hematopoietic stem cells (HSCs), leading to comparable, sustained multilineage transgene expression in primary and secondary transplanted mice. Alpharetroviral integrations were decreased near transcription start sites, CpG islands, and potential cancer genes compared with gammaretroviral, and decreased in genes compared with lentiviral integrations. Analyzing the transcriptome and intragenic integrations in engrafting cells, we observed stronger correlations between in-gene integration targeting and transcriptional activity for gammaretroviral and lentiviral vectors than for alpharetroviral vectors. Importantly, the relatively “extragenic” alpharetroviral integration pattern still supported long-term transgene expression upon serial transplantation. Furthermore, sensitive genotoxicity studies revealed a decreased immortalization incidence compared with gammaretroviral and lentiviral SIN vectors. We conclude that alpharetroviral SIN vectors have a favorable integration pattern which lowers the risk of insertional mutagenesis while supporting long-term transgene expression in the progeny of transplanted HSCs. PMID:22334016

“Stealth” Adenoviruses Blunt Cell-Mediated and Humoral Immune Responses against the Virus and Allow for Significant Gene Expression upon Readministration in the Lung

PubMed Central

Croyle, Maria A.; Chirmule, Narendra; Zhang, Yi; Wilson, James M.

2001-01-01

Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy. PMID:11312351

Transgene Expression and Host Cell Responses to Replication-Defective, Single-Cycle, and Replication-Competent Adenovirus Vectors.

PubMed

Crosby, Catherine M; Barry, Michael A

2017-02-18

Most adenovirus (Ad) vectors are E1 gene deleted replication defective (RD-Ad) vectors that deliver one transgene to the cell and all expression is based on that one gene. In contrast, E1-intact replication-competent Ad (RC-Ad) vectors replicate their DNA and their transgenes up to 10,000-fold, amplifying transgene expression markedly higher than RD-Ad vectors. While RC-Ad are more potent, they run the real risk of causing adenovirus infections in vector recipients and those that administer them. To gain the benefits of transgene amplification, but avoid the risk of Ad infections, we developed "single cycle" Ad (SC-Ad) vectors. SC-Ads amplify transgene expression and generated markedly stronger and more persistent immune responses than RD-Ad as expected. However, they also unexpectedly generated stronger immune responses than RC-Ad vectors. To explore the basis of this potency here, we compared gene expression and the cellular responses to infection to these vectors in vitro and in vivo. In vitro, in primary human lung epithelial cells, SC- and RC-Ad amplified their genomes more than 400-fold relative to RD-Ad with higher replication by SC-Ad. This replication translated into higher green fluorescent protein (GFP) expression for 48 h by SC- and RC-Ad than by RD-Ad. In vitro, in the absence of an immune system, RD-Ad expression became higher by 72 h coincident with cell death mediated by SC- and RC-Ad and release of transgene product from the dying cells. When the vectors were compared in human THP-1 Lucia- interferon-stimulated gene (ISG) cells, which are a human monocyte cell line that have been modified to quantify ISG activity, RC-Ad6 provoked significantly stronger ISG responses than RD- or SC-Ad. In mice, intravenous or intranasal injection produced up to 100-fold genome replication. Under these in vivo conditions in the presence of the immune system, luciferase expression by RC and SC-Ad was markedly higher than that by RD-Ad. In immunodeficient mice, SC-Ad drove stronger luciferase expression than RC- or RD-Ad. These data demonstrate better transgene expression by SC- and RC-Ad in vitro and in vivo than RD-Ad. This higher expression by the replicating vectors results in a peak of expression within 1 to 2 days followed by cell death of infected cells and release of transgene products. While SC- and RC-Ad expression were similar in mice and in Syrian hamsters, RC-Ad provoked much stronger ISG induction which may explain in part SC-Ad's ability to generate stronger and more persistent immune responses than RC-Ad in Ad permissive hamsters.

A novel system for the production of high levels of functional human therapeutic proteins in stable cells with a Semliki Forest virus noncytopathic vector.

PubMed

Casales, Erkuden; Aranda, Alejandro; Quetglas, Jose I; Ruiz-Guillen, Marta; Rodriguez-Madoz, Juan R; Prieto, Jesus; Smerdou, Cristian

2010-05-31

Semliki Forest virus (SFV) vectors lead to high protein expression in mammalian cells, but expression is transient due to vector cytopathic effects, inhibition of host cell proteins and RNA-based expression. We have used a noncytopathic SFV mutant (ncSFV) RNA vector to generate stable cell lines expressing two human therapeutic proteins: insulin-like growth factor I (IGF-I) and cardiotrophin-1 (CT-1). Therapeutic genes were fused at the carboxy-terminal end of Puromycin N-acetyl-transferase gene by using as a linker the sequence coding for foot-and-mouth disease virus (FMDV) 2A autoprotease. These cassettes were cloned into the ncSFV vector. Recombinant ncSFV vectors allowed rapid and efficient selection of stable BHK cell lines with puromycin. These cells expressed IGF-I and CT-1 in supernatants at levels reaching 1.4 and 8.6 microg/10(6)cells/24 hours, respectively. Two cell lines generated with each vector were passaged ten times during 30 days, showing constant levels of protein expression. Recombinant proteins expressed at different passages were functional by in vitro signaling assays. Stability at RNA level was unexpectedly high, showing a very low mutation rate in the CT-1 sequence, which did not increase at high passages. CT-1 was efficiently purified from supernatants of ncSFV cell lines, obtaining a yield of approximately 2mg/L/24 hours. These results indicate that the ncSFV vector has a great potential for the production of recombinant proteins in mammalian cells. 2010 Elsevier B.V. All rights reserved.

Optimized Lentiviral Vector Design Improves Titer and Transgene Expression of Vectors Containing the Chicken β-Globin Locus HS4 Insulator Element

PubMed Central

Hanawa, Hideki; Yamamoto, Motoko; Zhao, Huifen; Shimada, Takashi; Persons, Derek A

2009-01-01

Hematopoietic cell gene therapy using retroviral vectors has achieved success in clinical trials. However, safety issues regarding vector insertional mutagenesis have emerged. In two different trials, vector insertion resulted in the transcriptional activation of proto-oncogenes. One strategy for potentially diminishing vector insertional mutagenesis is through the use of self-inactivating lentiviral vectors containing the 1.2-kb insulator element derived from the chicken β-globin locus. However, use of this element can dramatically decrease both vector titer and transgene expression, thereby compromising its practical use. Here, we studied lentiviral vectors containing either the full-length 1.2-kb insulator or the smaller 0.25-kb core element in both orientations in the partially deleted long-terminal repeat. We show that use of the 0.25-kb core insulator rescued vector titer by alleviating a postentry block to reverse transcription associated with the 1.2-kb element. In addition, in an orientation-dependent manner, the 0.25-kb core element significantly increased transgene expression from an internal promoter due to improved transcriptional termination. This element also demonstrated barrier activity, reducing variability of expression due to position effects. As it is known that the 0.25-kb core insulator has enhancer-blocking activity, this particular insulated lentiviral vector design may be useful for clinical application. PMID:19223867

Design of a muscle cell-specific expression vector utilising human vascular smooth muscle alpha-actin regulatory elements.

PubMed

Keogh, M C; Chen, D; Schmitt, J F; Dennehy, U; Kakkar, V V; Lemoine, N R

1999-04-01

The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.

Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming.

PubMed

Schmitt, Christopher E; Morales, Blanca M; Schmitz, Ellen M H; Hawkins, John S; Lizama, Carlos O; Zape, Joan P; Hsiao, Edward C; Zovein, Ann C

2017-06-05

Non-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the "Yamanaka reprogramming factors" (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluripotent state. However, the reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors. We modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for reprogramming efficiency and for the ability to enrich for stoichiometric ratios of factors. The reengineered vectors allow for cell sorting based on reprogramming factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the reprogramming factor dosage. Together, these modified vectors are a useful tool for understanding the reprogramming process and improving induced pluripotent stem cell isolation efficiency.

Design and construction of functional AAV vectors.

PubMed

Gray, John T; Zolotukhin, Serge

2011-01-01

Using the basic principles of molecular biology and laboratory techniques presented in this chapter, researchers should be able to create a wide variety of AAV vectors for both clinical and basic research applications. Basic vector design concepts are covered for both protein coding gene expression and small non-coding RNA gene expression cassettes. AAV plasmid vector backbones (available via AddGene) are described, along with critical sequence details for a variety of modular expression components that can be inserted as needed for specific applications. Protocols are provided for assembling the various DNA components into AAV vector plasmids in Escherichia coli, as well as for transferring these vector sequences into baculovirus genomes for large-scale production of AAV in the insect cell production system.

In situ reprogramming to transdifferentiate fibroblasts into cardiomyocytes using adenoviral vectors: Implications for clinical myocardial regeneration.

PubMed

Mathison, Megumi; Singh, Vivek P; Chiuchiolo, Maria J; Sanagasetti, Deepthi; Mao, Yun; Patel, Vivekkumar B; Yang, Jianchang; Kaminsky, Stephen M; Crystal, Ronald G; Rosengart, Todd K

2017-02-01

The reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells improves ventricular function in myocardial infarction models. Only integrating persistent expression vectors have thus far been used to induce reprogramming, potentially limiting its clinical applicability. We therefore tested the reprogramming potential of nonintegrating, acute expression adenoviral (Ad) vectors. Ad or lentivirus vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) were validated in vitro. Sprague-Dawley rats then underwent coronary ligation and Ad-mediated administration of vascular endothelial growth factor to generate infarct prevascularization. Three weeks later, animals received Ad or lentivirus encoding G, M, or T (AdGMT or LentiGMT) or an equivalent dose of a null vector (n = 11, 10, and 10, respectively). Outcomes were analyzed by echocardiography, magnetic resonance imaging, and histology. Ad and lentivirus vectors provided equivalent G, M, and T expression in vitro. AdGMT and LentiGMT both likewise induced expression of the cardiomyocyte marker cardiac troponin T in approximately 6% of cardiac fibroblasts versus <1% cardiac troponin T expression in AdNull (adenoviral vector that does not encode a transgene)-treated cells. Infarcted myocardium that had been treated with AdGMT likewise demonstrated greater density of cells expressing the cardiomyocyte marker beta myosin heavy chain 7 compared with AdNull-treated animals. Echocardiography demonstrated that AdGMT and LentiGMT both increased ejection fraction compared with AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: -0.4% ± 2%; P < .05). Ad vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into induced cardiomyocyte-like cells and improving cardiac function in postinfarct rat hearts. Short-term expression Ad vectors may represent an important means to induce cardiac cellular reprogramming in humans. Copyright © 2016 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Construction of siRNA/miRNA expression vectors based on a one-step PCR process

PubMed Central

Xu, Jun; Zeng, Jie Qiong; Wan, Gang; Hu, Gui Bin; Yan, Hong; Ma, Li Xin

2009-01-01

Background RNA interference (RNAi) has become a powerful means for silencing target gene expression in mammalian cells and is envisioned to be useful in therapeutic approaches to human disease. In recent years, high-throughput, genome-wide screening of siRNA/miRNA libraries has emerged as a desirable approach. Current methods for constructing siRNA/miRNA expression vectors require the synthesis of long oligonucleotides, which is costly and suffers from mutation problems. Results Here we report an ingenious method to solve traditional problems associated with construction of siRNA/miRNA expression vectors. We synthesized shorter primers (< 50 nucleotides) to generate a linear expression structure by PCR. The PCR products were directly transformed into chemically competent E. coli and converted to functional vectors in vivo via homologous recombination. The positive clones could be easily screened under UV light. Using this method we successfully constructed over 500 functional siRNA/miRNA expression vectors. Sequencing of the vectors confirmed a high accuracy rate. Conclusion This novel, convenient, low-cost and highly efficient approach may be useful for high-throughput assays of RNAi libraries. PMID:19490634

A Modular Lentiviral and Retroviral Construction System to Rapidly Generate Vectors for Gene Expression and Gene Knockdown In Vitro and In Vivo

PubMed Central

Geiling, Benjamin; Vandal, Guillaume; Posner, Ada R.; de Bruyns, Angeline; Dutchak, Kendall L.; Garnett, Samantha; Dankort, David

2013-01-01

The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems. PMID:24146852

[Prokaryotic expression and immunological activity of human neutrophil gelatinase associated lipocalin].

PubMed

Wu, Jianwei; Cai, Lei; Qian, Wei; Jiao, Liyuan; Li, Jiangfeng; Song, Xiaoli; Wang, Jihua

2015-07-01

To construct a prokaryotic expression vector of human neutrophil gelatinase associated lipocalin (NGAL) and identify the bioactivity of the fusion protein. The cDNA of human NGAL obtained from GenBank was linked to a cloning vector to construct the prokaryotic expression vector pCold-NGAL. Then the vector was transformed into E.coli BL21(DE3) plysS. Under the optimal induction condition, the recombinant NGAL (rNGAL) was expressed and purified by Ni Sepharose 6 Fast Flow affinity chromatography. The purity and activity of the rNGAL were respectively identified by SDS-PAGE and Western blotting combined with NGAL reagent (Latex enhanced immunoturbidimetry). Restriction enzyme digestion and nucleotide sequencing proved that the expression vector pCold-NGAL was successfully constructed. Under the optimal induction condition that we determined, the rNGAL was expressed in soluble form in E.coli BL21(DE3) plysS. The relative molecular mass of the rNGAL was 25 000, and its purity was more than 98.0%. Furthermore, Western blotting and immunoturbidimetry indicated that the rNGAL reacted with NGAL mAb specifically. Human rNGAL of high purity and bioactivity was successfully constructed in E.coli BL21(DE3) plysS using the expression vector pCold-NGAL.

[Construction and expression of the targeting super-antigen EGF-SEA fusion gene].

PubMed

Xie, Yang; Peng, Shaoping; Liao, Zhiying; Liu, Jiafeng; Liu, Xuemei; Chen, Weifeng

2014-05-01

To construct expression vector for the SEA-EGF fusion gene. Clone the SEA gene and the EGF gene segment with PCR and RT-PCR independently, and connect this two genes by the bridge PCR. Insert the fusion gene EGF-SEA into the expression vector PET-44. Induced the secretion of the fusion protein SEA-EGF by the antileptic. The gene fragment encoding EGF and SEA mature peptide was successfully cloned. The fusion gene EGF-SEA was successfully constructed and was inserted into expression vector. The new recombinant expression vector for fusion gene EGF-SEA is specific for head and neck cancer, laid the foundation for the further study of fusion protein SEA-EGF targeting immune therapy in head and neck tumors.

Retrovirus-based vectors for transient and permanent cell modification.

PubMed

Schott, Juliane W; Hoffmann, Dirk; Schambach, Axel

2015-10-01

Retroviral vectors are commonly employed for long-term transgene expression via integrating vector technology. However, three alternative retrovirus-based platforms are currently available that allow transient cell modification. Gene expression can be mediated from either episomal DNA or RNA templates, or selected proteins can be directly transferred through retroviral nanoparticles. The different technologies are functionally graded with respect to safety, expression magnitude and expression duration. Improvement of the initial technologies, including modification of vector designs, targeted increase in expression strength and duration as well as improved safety characteristics, has allowed maturation of retroviral systems into efficient and promising tools that meet the technological demands of a wide variety of potential application areas. Copyright © 2015 Elsevier Ltd. All rights reserved.

Development of nonhuman adenoviruses as vaccine vectors

PubMed Central

Bangari, Dinesh S.; Mittal, Suresh K.

2006-01-01

Human adenoviral (HAd) vectors have demonstrated great potential as vaccine vectors. Preclinical and clinical studies have demonstrated the feasibility of vector design, robust antigen expression and protective immunity using this system. However, clinical use of adenoviral vectors for vaccine purposes is anticipated to be limited by vector immunity that is either preexisting or develops rapidly following the first inoculation with adenoviral vectors. Vector immunity inactivates the vector particles and rapidly removes the transduced cells, thereby limiting the duration of transgene expression. Due to strong vector immunity, subsequent use of the same vector is usually less efficient. In order to circumvent this limitation, nonhuman adenoviral vectors have been proposed as alternative vectors. In addition to eluding HAd immunity, these vectors possess most of the attractive features of HAd vectors. Several replication-competent or replication-defective nonhuman adenoviral vectors have been developed and investigated for their potential as vaccine delivery vectors. Here, we review recent advances in the design and characterization of various nonhuman adenoviral vectors, and discuss their potential applications for human and animal vaccination. PMID:16297508

A dual host vector for Fab phage display and expression of native IgG in mammalian cells.

PubMed

Tesar, Devin; Hötzel, Isidro

2013-10-01

A significant bottleneck in antibody discovery by phage display is the transfer of immunoglobulin variable regions from phage clones to vectors that express immunoglobulin G (IgG) in mammalian cells for screening. Here, we describe a novel phagemid vector for Fab phage display that allows expression of native IgG in mammalian cells without sub-cloning. The vector uses an optimized mammalian signal sequence that drives robust expression of Fab fragments fused to an M13 phage coat protein in Escherichia coli and IgG expression in mammalian cells. To allow the expression of Fab fragments fused to a phage coat protein in E.coli and full-length IgG in mammalian cells from the same vector without sub-cloning, the sequence encoding the phage coat protein was embedded in an optimized synthetic intron within the immunoglobulin heavy chain gene. This intron is removed from transcripts in mammalian cells by RNA splicing. Using this vector, we constructed a synthetic Fab phage display library with diversity in the heavy chain only and selected for clones binding different antigens. Co-transfection of mammalian cells with DNA from individual phage clones and a plasmid expressing the invariant light chain resulted in the expression of native IgG that was used to assay affinity, ligand blocking activity and specificity.

Nephron segment-specific gene expression using AAV vectors.

PubMed

Asico, Laureano D; Cuevas, Santiago; Ma, Xiaobo; Jose, Pedro A; Armando, Ines; Konkalmatt, Prasad R

2018-02-26

AAV9 vector provides efficient gene transfer in all segments of the renal nephron, with minimum expression in non-renal cells, when administered retrogradely via the ureter. It is important to restrict the transgene expression to the desired cell type within the kidney, so that the physiological endpoints represent the function of the transgene expressed in that specific cell type within kidney. We hypothesized that segment-specific gene expression within the kidney can be accomplished using the highly efficient AAV9 vectors carrying the promoters of genes that are expressed exclusively in the desired segment of the nephron in combination with administration by retrograde infusion into the kidney via the ureter. We constructed AAV vectors carrying eGFP under the control of: kidney-specific cadherin (KSPC) gene promoter for expression in the entire nephron; Na + /glucose co-transporter (SGLT2) gene promoter for expression in the S1 and S2 segments of the proximal tubule; sodium, potassium, 2 chloride co-transporter (NKCC2) gene promoter for expression in the thick ascending limb of Henle's loop (TALH); E-cadherin (ECAD) gene promoter for expression in the collecting duct (CD); and cytomegalovirus (CMV) early promoter that provides expression in most of the mammalian cells, as control. We tested the specificity of the promoter constructs in vitro for cell type-specific expression in mouse kidney cells in primary culture, followed by retrograde infusion of the AAV vectors via the ureter in the mouse. Our data show that AAV9 vector, in combination with the segment-specific promoters administered by retrograde infusion via the ureter, provides renal nephron segment-specific gene expression. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Interleukin 10 mediated by herpes simplex virus vectors suppresses neuropathic pain induced by human immunodeficiency virus gp120 in rats.

PubMed

Zheng, Wenwen; Huang, Wan; Liu, Shue; Levitt, Roy C; Candiotti, Keith A; Lubarsky, David A; Hao, Shuanglin

2014-09-01

Human immunodeficiency virus (HIV)-associated sensory neuropathy is a common neurological complication of HIV infection affecting up to 30% of HIV-positive individuals. However, the exact neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments for HIV-related neuropathic pain (NP). In this study, we tested the hypothesis that inhibition of proinflammatory factors with overexpression of interleukin (IL)-10 reduces HIV-related NP in a rat model. NP was induced by the application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve. The hindpaws of rats were inoculated with nonreplicating herpes simplex virus (HSV) vectors expressing anti-inflammatory cytokine IL-10 or control vector. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The mechanical threshold response was assessed over time using the area under curves. The expression of phosphorylated p38 mitogen-activated kinase, tumor necrosis factor-α, stromal cell-derived factor-1α, and C-X-C chemokine receptor type 4 in both the lumbar spinal cord and the L4/5 dorsal root ganglia (DRG), was examined at 14 and 28 days after vector inoculation using Western blots. We found that in the gp120-induced NP model, IL-10 overexpression mediated by the HSV vector resulted in a significant elevation of the mechanical threshold that was apparent on day 3 after vector inoculation compared with the control vector (P < 0.001). The antiallodynic effect of the single HSV vector inoculation expressing IL-10 lasted >28 days. The area under curve in the HSV vector expressing IL-10 was increased compared with that in the control vector (P < 0.0001). HSV vectors expressing IL-10 reversed the upregulation of phosphorylated p38 mitogen-activated kinase, tumor necrosis factor-α, stromal cell-derived factor-1α, and C-X-C chemokine receptor type 4 expression at 14 and/or 28 days in the DRG and/or the spinal dorsal horn. Our studies demonstrate that blocking the signaling of these proinflammatory molecules in the DRG and/or the spinal cord using the HSV vector expressing IL-10 is able to reduce HIV-related NP. These results provide new insights on the potential mechanisms of HIV-associated NP and a proof of concept for treating painful HIV sensory neuropathy with this type of gene therapy.

Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors

PubMed Central

Giritch, Anatoli; Marillonnet, Sylvestre; Engler, Carola; van Eldik, Gerben; Botterman, Johan; Klimyuk, Victor; Gleba, Yuri

2006-01-01

Plant viral vectors allow expression of heterologous proteins at high yields, but so far, they have been unable to express heterooligomeric proteins efficiently. We describe here a rapid and indefinitely scalable process for high-level expression of functional full-size mAbs of the IgG class in plants. The process relies on synchronous coinfection and coreplication of two viral vectors, each expressing a separate antibody chain. The two vectors are derived from two different plant viruses that were found to be noncompeting. Unlike vectors derived from the same virus, noncompeting vectors effectively coexpress the heavy and light chains in the same cell throughout the plant body, resulting in yields of up to 0.5 g of assembled mAbs per kg of fresh-leaf biomass. This technology allows production of gram quantities of mAbs for research purposes in just several days, and the same protocol can be used on an industrial scale in situations requiring rapid response, such as pandemic or terrorism events. PMID:16973752

Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors.

PubMed

Paul, T A; Burns, J C; Shike, H; Getchell, R; Bowser, P R; Whitlock, K E; Casey, J W

2001-06-01

A central issue in gene delivery systems is choosing promoters that will direct defined and sustainable levels of gene expression. Pantropic retroviral vectors provide a means to insert genes into either somatic or germline cells. In this study, we focused on somatic cell infection by evaluating the activity of 3 promoters inserted by vectors into fish cell lines and fish skin using pantropic retroviruses. In bluegill and zebrafish cell lines, the highest levels of luciferase expression were observed from the 5' murine leukemia virus long terminal repeat of the retroviral vector. The Rous sarcoma virus long terminal repeat and cytomegalovirus early promoter, as internal promoters, generated lower levels of luciferase. Luciferase reporter vectors infected zebrafish skin, as measured by the presence of viral DNA, and expressed luciferase. We infected developing walleye dermal sarcomas with retroviral vectors to provide an environment with enhanced cell proliferation, a condition necessary for integration of the provirus into the host genome. We demonstrated a 4-fold to 7-fold increase in luciferase gene expression in tumor tissue over infections in normal walleye skin.

Generation of mammalian cells stably expressing multiple genes at predetermined levels.

PubMed

Liu, X; Constantinescu, S N; Sun, Y; Bogan, J S; Hirsch, D; Weinberg, R A; Lodish, H F

2000-04-10

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the protein downstream of the IRES provides strong evidence for the function of the protein placed upstream of the IRES.

Construction of heat-inducible expression vector of Corynebacterium glutamicum and C. ammoniagenes: fusion of lambda operator with promoters isolated from C. ammoniagenes.

PubMed

Park, Jong-Uk; Jo, Jae-Hyung; Kim, Young-Ji; Chung, So-Sun; Lee, Jin-Ho; Lee, Hyune Hwan

2008-04-01

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the lambdaOL1 and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes.. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the OL1 from the lambdaPL promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one lambdaOL1, and CJ1OX2, which has two successive lambdaOL1, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

Title V Operating Permit: CR Group, LLC - Tekoi Landfill

EPA Pesticide Factsheets

Final First Renewal of the Operating Permit (Permit Number: V-SV-000001-2016.00), Response to Public Comments and the Administrative Permit Record for the CR Group, LLC, Tekoi Landfill, located on the Skull Valley Indian Reservation, in Tooele County, UT.

Transmission and pathogenesis of vesicular stomatitis viruses

USDA-ARS?s Scientific Manuscript database

Vesicular Stomatitis (VS) is caused by the Vesicular Stomatitis Virus (VSV), a negative single stranded RNA arthropod-borne virus member of the Family Rhabdoviridae. The virion is composed of the host derived plasma membrane, the envelope, and an internal ribonucleoprotein core. The envelope contain...

Akt/protein kinase B activation by adenovirus vectors contributes to NFkappaB-dependent CXCL10 expression.

PubMed

Liu, Qiang; White, Lindsay R; Clark, Sharon A; Heffner, Daniel J; Winston, Brent W; Tibbles, Lee Anne; Muruve, Daniel A

2005-12-01

In gene therapy, the innate immune system is a significant barrier to the effective application of adenovirus (Ad) vectors. In kidney epithelium-derived (REC) cells, serotype 5 Ad vectors induce the expression of the chemokine CXCL10 (IP-10), a response that is dependent on NFkappaB. Compared to the parental vector AdLuc, transduction with the RGD-deleted vector AdL.PB resulted in reduced CXCL10 activation despite increasing titers, implying that RGD-alpha(V) integrin interactions contribute to adenovirus induction of inflammatory genes. Akt, a downstream effector of integrin signaling, was activated within 10 min of transduction with Ad vectors in a dose-dependent manner. Akt activation was not present following transduction with AdL.PB, confirming the importance of capsid-alpha(V) integrin interactions in Ad vector Akt activation. Inhibition of the phosphoinositide-3-OH kinase/Akt pathway by Wortmannin or Ly294002 compounds decreased Ad vector induction of CXCL10 mRNA. Similarly, adenovirus-mediated overexpression of the dominant negative AktAAA decreased CXCL10 mRNA expression compared to the reporter vector AdLacZ alone. The effect of Akt on CXCL10 mRNA expression occurred via NFkappaB-dependent transcriptional activation, since AktAAA overexpression and Ly294002 both inhibited CXCL10 and NFkappaB promoter activation in luciferase reporter experiments. These results show that Akt plays a role in the Ad vector activation of NFkappaB and CXCL10 expression. Understanding the mechanism underlying the regulation of host immunomodulatory genes by adenovirus vectors will lead to strategies that will improve the efficacy and safety of these agents for clinical use.

The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri.

PubMed

Kim, Jong-Hyun; Lee, Sang-Hee; Sohn, Hae-Jin; Lee, Jinyoung; Chwae, Yong-Joon; Park, Sun; Kim, Kyongmin; Shin, Ho-Joon

2012-12-01

The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.

Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.

PubMed

Turunen, Tytteli Anni Kaarina; Laakkonen, Johanna Päivikki; Alasaarela, Laura; Airenne, Kari Juhani; Ylä-Herttuala, Seppo

2014-01-01

A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases. Copyright © 2014 John Wiley & Sons, Ltd.

Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

PubMed

Ono, Motoharu; Yamada, Kayo; Avolio, Fabio; Afzal, Vackar; Bensaddek, Dalila; Lamond, Angus I

2015-01-01

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

Ribosomal DNA Integrating rAAV-rDNA Vectors Allow for Stable Transgene Expression

PubMed Central

Lisowski, Leszek; Lau, Ashley; Wang, Zhongya; Zhang, Yue; Zhang, Feijie; Grompe, Markus; Kay, Mark A

2012-01-01

Although recombinant adeno-associated virus (rAAV) vectors are proving to be efficacious in clinical trials, the episomal character of the delivered transgene restricts their effectiveness to use in quiescent tissues, and may not provide lifelong expression. In contrast, integrating vectors enhance the risk of insertional mutagenesis. In an attempt to overcome both of these limitations, we created new rAAV-rDNA vectors, with an expression cassette flanked by ribosomal DNA (rDNA) sequences capable of homologous recombination into genomic rDNA. We show that after in vivo delivery the rAAV-rDNA vectors integrated into the genomic rDNA locus 8–13 times more frequently than control vectors, providing an estimate that 23–39% of the integrations were specific to the rDNA locus. Moreover, a rAAV-rDNA vector containing a human factor IX (hFIX) expression cassette resulted in sustained therapeutic levels of serum hFIX even after repeated manipulations to induce liver regeneration. Because of the relative safety of integration in the rDNA locus, these vectors expand the usage of rAAV for therapeutics requiring long-term gene transfer into dividing cells. PMID:22990671

Host structural carbohydrate induces vector transmission of a bacterial plant pathogen.

PubMed

Killiny, Nabil; Almeida, Rodrigo P P

2009-12-29

Many insect-borne pathogens have complex life histories because they must colonize both hosts and vectors for successful dissemination. In addition, the transition from host to vector environments may require changes in gene expression before the pathogen's departure from the host. Xylella fastidiosa is a xylem-limited plant-pathogenic bacterium transmitted by leafhopper vectors that causes diseases in a number of economically important plants. We hypothesized that factors of host origin, such as plant structural polysaccharides, are important in regulating X. fastidiosa gene expression and mediating vector transmission of this pathogen. The addition of pectin and glucan to a simple defined medium resulted in dramatic changes in X. fastidiosa's phenotype and gene-expression profile. Cells grown in the presence of pectin became more adhesive than in other media tested. In addition, the presence of pectin and glucan in media resulted in significant changes in the expression of several genes previously identified as important for X. fastidiosa's pathogenicity in plants. Furthermore, vector transmission of X. fastidiosa was induced in the presence of both polysaccharides. Our data show that host structural polysaccharides mediate gene regulation in X. fastidiosa, which results in phenotypic changes required for vector transmission. A better understanding of how vector-borne pathogens transition from host to vector, and vice versa, may lead to previously undiscovered disease-control strategies.

Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis.

PubMed

Tran, Dinh Thi Minh; Phan, Trang Thi Phuong; Huynh, Thanh Kieu; Dang, Ngan Thi Kim; Huynh, Phuong Thi Kim; Nguyen, Tri Minh; Truong, Tuom Thi Tinh; Tran, Thuoc Linh; Schumann, Wolfgang; Nguyen, Hoang Duc

2017-07-25

Besides Escherichia coli, Bacillus subtilis is an important bacterial species for the production of recombinant proteins. Recombinant genes are inserted into shuttle expression vectors which replicate in both E. coli and in B. subtilis. The ligation products are first transformed into E. coli cells, analyzed for correct insertions, and the correct recombinant plasmids are then transformed into B. subtilis. A major problem using E. coli cells can be the strong basal level of expression of the recombinant protein which may interfere with the stability of the cells. To minimize this problem, we developed strong expression vectors being repressed in E. coli and inducer-free in B. subtilis. In general, induction of IPTG-inducible expression vectors is determined by the regulatory lacI gene encoding the LacI repressor in combination with the lacO operator on the promoter. To investigate the inducer-free properties of the vectors, we constructed inducer-free expression plasmids by removing the lacI gene and characterized their properties. First, we examined the ability to repress a reporter gene in E. coli, which is a prominent property facilitating the construction of the expression vectors carrying a target gene. The β-galactosidase (bgaB gene) basal levels expressed from Pgrac01-bgaB could be repressed at least twice in the E. coli cloning strain. Second, the inducer-free production of BgaB from four different plasmids with the Pgrac01 promoter in B. subtilis was investigated. As expected, BgaB expression levels of inducer-free constructs are at least 37 times higher than that of the inducible constructs in the absence of IPTG, and comparable to those in the presence of the inducer. Third, using efficient IPTG-inducible expression vectors containing the strong promoter Pgrac100, we could convert them into inducer-free expression plasmids. The BgaB production levels from the inducer-free plasmid in the absence of the inducer were at least 4.5 times higher than that of the inducible vector using the same promoter. Finally, we used gfp as a reporter gene in combination with the two promoters Pgrac01 and Pgrac100 to test the new vector types. The GFP expression levels could be repressed at least 1.5 times for the Pgrac01-gfp+ inducer-free construct in E. coli. The inducer-free constructs Pgrac01-gfp+ and Pgrac100-gfp+ allowed GFP expression at high levels from 23 × 10 4 to 32 × 10 4 RFU units and 9-13% of total intracellular proteins. We could reconfirm the two major advantages of the new inducer-free expression plasmids: (1) Strong repression of the target gene expression in the E. coli cloning strain, and (2) production of the target protein at high levels in B. subtilis in the absence of the inducer. We propose a general strategy to generate inducer-free expression vector by using IPTG-inducible vectors, and more specifically we developed inducer-free expression plasmids using IPTG-inducible promoters in the absence of the LacI repressor. These plasmids could be an excellent choice for high-level production of recombinant proteins in B. subtilis without the addition of inducer and at the same time maintaining a low basal level of the recombinant proteins in E. coli. The repression of the recombinant gene expression would facilitate cloning of genes that potentially inhibit the growth of E. coli cloning strains. The inducer-free expression plasmids will be extended versions of the current available IPTG-inducible expression vectors for B. subtilis, in which all these vectors use the same cognate promoters. These inducer-free and previously developed IPTG-inducible expression plasmids will be a useful cassette to study gene expression at a small scale up to a larger scale up for the production of recombinant proteins.

Lack of Humoral Immune Response to the Tetracycline (Tet) Activator in Rats Injected Intracranially with Tet-off rAAV Vectors

PubMed Central

Han, Ye; Chang, Qin A.; Virag, Tamas; West, Neva C.; George, David; Castro, Maria G.; Bohn, Martha C.

2010-01-01

The ability to safely control transgene expression from viral vectors is a long-term goal in the gene therapy field. We have previously reported tight regulation of GFP expression in rat brain using a self-regulating tet-off rAAV vector. The immune responses against tet regulatory elements observed by other groups in nonhuman primates after intramuscular injection of tet-on encoding vectors raise concerns about the clinical value of tet-regulated vectors. However, previous studies have not examined immune responses following injection of AAV vectors into brain. Therefore, rat striatum was injected with tet-off rAAV harboring a therapeutic gene for Parkinson's disease, either hAADC or hGDNF. The expression of each gene was tightly controlled by the tet-off regulatory system. Using an ELISA developed with purified GST-tTA protein, no detectable immunogenicity against tTA was observed in sera of rats that received an intrastriatal injection of either vector. In contrast, sera from rats intradermally injected with an adenovirus containing either tTA or rtTA, as positive controls, had readily detectable antibodies. These observations suggest that tet-off rAAV vectors do not elicit an immune response when injected into rat brain and that these may offer safer vectors for Parkinson's disease than vectors with constitutive expression. PMID:20164859

Modified Newcastle Disease virus as an improved vaccine vector against Simian Immunodeficiency virus.

PubMed

Manoharan, Vinoth K; Khattar, Sunil K; LaBranche, Celia C; Montefiori, David C; Samal, Siba K

2018-06-12

SIV infection in macaques is a relevant animal model for HIV pathogenesis and vaccine study in humans. To design a safe and effective vaccine against HIV, we evaluated the suitability of naturally-occurring avirulent Newcastle disease virus (NDV) strains and several modified versions of NDV as vectors for the expression and immunogenicity of SIV envelope protein gp160. All the NDV vectors expressed gp160 protein in infected cells. The gp160 expressed by these vectors formed oligomers and was incorporated into the NDV envelope. All the NDV vectors expressing gp160 were attenuated in chickens. Intranasal immunization of guinea pigs with modified NDV vectors such as rNDV-APMV-2CS/gp160 and rNDV-APMV-8CS/gp160 (NDV strain LaSota containing the cleavage site sequences of F protein of avian paramyxovirus (APMV) serotype 2 and 8, respectively), and rNDV-BC-F-HN/gp160 (NDV strain BC containing LaSota F cleavage site and LaSota F and HN genes) elicited improved SIV-specific humoral and mucosal immune responses compared to other NDV vectors. These modified vectors were also efficient in inducing neutralizing antibody responses to tier 1 A SIVmac251.6 and tier 1B SIVmac251/M766 strains. This study suggests that our novel modified NDV vectors are safe and immunogenic and can be used as vaccine vector to control HIV.

Comparative proteomics as a tool for identifying specific alterations within interferon response pathways in human glioblastoma multiforme cells

PubMed Central

Lobas, Anna A; Solovyeva, Elizaveta M; Sidorenko, Alena S; Gorshkov, Vladimir; Kjeldsen, Frank; Bubis, Julia A; Ivanov, Mark V; Ilina, Irina Y; Moshkovskii, Sergei A; Chumakov, Peter M; Gorshkov, Mikhail V

2018-01-01

An acquisition of increased sensitivity of cancer cells to viruses is a common outcome of malignant progression that justifies the development of oncolytic viruses as anticancer therapeutics. Studying molecular changes that underlie the sensitivity to viruses would help to identify cases where oncolytic virus therapy would be most effective. We quantified changes in protein abundances in two glioblastoma multiforme (GBM) cell lines that differ in the ability to induce resistance to vesicular stomatitis virus (VSV) infection in response to type I interferon (IFN) treatment. In IFN-treated samples we observed an up-regulation of protein products of some IFN-regulated genes (IRGs). In total, the proteome analysis revealed up to 20% more proteins encoded by IRGs in the glioblastoma cell line, which develops resistance to VSV infection after pre-treatment with IFN. In both cell lines protein-protein interaction and signaling pathway analyses have revealed a significant stimulation of processes related to type I IFN signaling and defense responses to viruses. However, we observed a deficiency in STAT2 protein in the VSV-sensitive cell line that suggests a de-regulation of the JAK/STAT/IRF9 signaling. The study has shown that the up-regulation of IRG proteins induced by the IFNα treatment of GBM cells can be detected at the proteome level. Similar analyses could be applied for revealing functional alterations within the antiviral mechanisms in glioblastoma samples, accompanying by acquisition of sensitivity to oncolytic viruses. The approach can be useful for discovering the biomarkers that predict a potential sensitivity of individual glioblastoma tumors to oncolytic virus therapy. PMID:29416731

Felix Hoppe-Seyler Lecture 1997. Protective antibody responses against viruses.

PubMed

Zinkernagel, R M

1997-08-01

Neutralizing antibody responses against the acute cytopathic vesicular stomatitis virus (VSV) have been studied in mice to evaluate their general characteristics including specificity, self-/non-self discrimination and memory. IgM responses are generated very early, by day 3 to 4, in a T helper cell-independent fashion and without VSV having polyclonal activating capacities. The order of the glycoprotein tips on the virus envelope (multiple, 8-10 nm distance, paracrystalline) exhibiting the neutralizing determinants are key to this prompt response. These paracrystalline identical multimeric antigens are characteristic of infectious agents and are always reacted against by B cells. Self-antigens that are accessible to B cells in the intact host are either monomeric in serum or mobile multimers on cell surfaces; these configurations need contact dependent or contact independent T help, respectively. Because T help is tolerant against self-antigens, no anti-self B cell responses are usually induced against monomeric self-antigens. If collagen or DNA (rigid multimeric self-antigens) become accessible, however, they may become targets of auto-antibody responses. The antibody repertoire against VSV is partially contained in the germline and partially is generated by somatic mutation; they seem not to undergo affinity-maturation. In any case protection against lethal infection is dependent upon strictly T helper cell dependent IgG generated by day 6 to 7 and reaches a protective level of about 1-10 micrograms/ml. Interesting affinity/avidity and onrate above a minimal threshold are of no apparent advantage for protection in vivo. Maintenance of these antibody levels by antigen depots, and not the presence of memory B cells alone, is key to providing protective immunological memory. Collectively these data suggest that studying biologically important protective antibody responses may modify some of the parameters that have been defined by studying hapten specific antibody responses.

Gene Transfer of Glutamic Acid Decarboxylase 67 by Herpes Simplex Virus Vectors Suppresses Neuropathic Pain Induced by Human Immunodeficiency Virus gp120 Combined with ddC in Rats.

PubMed

Kanao, Megumi; Kanda, Hirotsugu; Huang, Wan; Liu, Shue; Yi, Hyun; Candiotti, Keith A; Lubarsky, David A; Levitt, Roy C; Hao, Shuanglin

2015-06-01

Human immunodeficiency virus (HIV)-related painful sensory neuropathies primarily consist of the HIV infection-related distal sensory polyneuropathy and antiretroviral toxic neuropathies. Pharmacotherapy provides only partial relief of pain in patients with HIV/acquired immune deficiency syndrome because little is known about the exact neuropathological mechanisms for HIV-associated neuropathic pain (NP). Hypofunction of γ-aminobutyric acid (GABA) GABAergic inhibitory mechanisms has been reported after peripheral nerve injury. In this study, we tested the hypothesis that HIV gp120 combined with antiretroviral therapy reduces spinal GABAergic inhibitory tone and that restoration of GABAergic inhibitory tone will reduce HIV-related NP in a rat model. The application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve plus systemic ddC (one antiretroviral drug) induced mechanical allodynia. The hind paws of rats were inoculated with replication-defective herpes simplex virus (HSV) vectors genetically encoding gad1 gene to express glutamic acid decarboxylase 67 (GAD67), an enzyme that catalyzes the decarboxylation of glutamate to GABA. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The expression of GAD67 in both the lumbar spinal cord and the L4-5 dorsal root ganglia was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging. The immunoreactivity of spinal GABA, pCREB, and pC/EBPβ was tested using immunohistochemistry. In the gp120 with ddC-induced neuropathic pain model, GAD67 expression mediated by the HSV vector caused an elevation of mechanical threshold that was apparent on day 3 after vector inoculation. The antiallodynic effect of the single HSV vector inoculation expressing GAD67 lasted >28 days. The area under the time-effect curves in the HSV vector expressing GAD67 was increased compared with that in the control vectors (P = 0.0005). Intrathecal GABA-A/B agonists elevated mechanical threshold in the pain model. The HSV vectors expressing GAD67 reversed the lowered GABA immunoreactivity in the spinal dorsal horn in the neuropathic rats. HSV vectors expressing GAD67 in the neuropathic rats reversed the increased signals of mitochondrial superoxide in the spinal dorsal horn. The vectors expressing GAD67 reversed the upregulated immunoreactivity expression of pCREB and pC/EBPβ in the spinal dorsal horn in rats exhibiting NP. Based on our results, we suggest that GAD67 mediated by HSV vectors acting through the suppression of mitochondrial reactive oxygen species and transcriptional factors in the spinal cord decreases pain in the HIV-related neuropathic pain model, providing preclinical evidence for gene therapy applications in patients with HIV-related pain states.

Modification and identification of a vector for making a large phage antibody library.

PubMed

Zhang, Guo-min; Chen, Yü-ping; Guan, Yuan-zhi; Wang, Yan; An, Yun-qing

2007-11-20

The large phage antibody library is used to obtain high-affinity human antibody, and the Loxp/cre site-specific recombination system is a potential method for constructing a large phage antibody library. In the present study, a phage antibody library vector pDF was reconstructed to construct diabody more quickly and conveniently without injury to homologous recombination and the expression function of the vector and thus to integrate construction of the large phage antibody library with the preparation of diabodies. scFv was obtained by overlap polymerase chain reaction (PCR) amplification with the newly designed VL and VH extension primers. loxp511 was flanked by VL and VH and the endonuclease ACC III encoding sequences were introduced on both sides of loxp511. scFv was cloned into the vector pDF to obtain the vector pDscFv. The vector expression function was identified and the feasibility of diabody preparation was evaluated. A large phage antibody library was constructed in pDscFv. Several antigens were used to screen the antibody library and the quality of the antibody library was evaluated. The phage antibody library expression vector pDscFv was successfully constructed and confirmed to express functional scFv. The large phage antibody library constructed using this vector was of high diversity. Screening of the library on 6 antigens confirmed the generation of specific antibodies to these antigens. Two antibodies were subjected to enzymatic digestion and were prepared into diabody with functional expression. The reconstructed vector pDscFv retains its recombination capability and expression function and can be used to construct large phage antibody libraries. It can be used as a convenient and quick method for preparing diabodies after simple enzymatic digestion, which facilitates clinical trials and application of antibody therapy.

Quinoxaline-based inhibitors of Ebola and Marburg VP40 egress.

PubMed

Loughran, H Marie; Han, Ziying; Wrobel, Jay E; Decker, Sarah E; Ruthel, Gordon; Freedman, Bruce D; Harty, Ronald N; Reitz, Allen B

2016-08-01

We prepared a series of quinoxalin-2-mercapto-acetyl-urea analogs and evaluated them for their ability to inhibit viral egress in our Marburg and Ebola VP40 VLP budding assays in HEK293T cells. We also evaluated selected compounds in our bimolecular complementation assay (BiMC) to detect and visualize a Marburg mVP40-Nedd4 interaction in live mammalian cells. Antiviral activity was assessed for selected compounds using a live recombinant vesicular stomatitis virus (VSV) (M40 virus) that expresses the EBOV VP40 PPxY L-domain. Finally selected compounds were evaluated in several ADME assays to have an early assessment of their drug properties. Our compounds had low nM potency in these assays (e.g., compounds 21, 24, 26, 39), and had good human liver microsome stability, as well as little or no inhibition of P450 3A4. Copyright © 2016 Elsevier Ltd. All rights reserved.

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

PubMed

Paust, Silke; Gill, Harvinder S; Wang, Bao-Zhong; Flynn, Michael P; Moseman, E Ashley; Senman, Balimkiz; Szczepanik, Marian; Telenti, Amalio; Askenase, Philip W; Compans, Richard W; von Andrian, Ulrich H

2010-12-01

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent

PubMed Central

Lévy, Camille; Amirache, Fouzia; Girard-Gagnepain, Anais; Frecha, Cecilia; Roman-Rodríguez, Francisco J.; Bernadin, Ornellie; Costa, Caroline; Nègre, Didier; Gutierrez-Guerrero, Alejandra; Vranckx, Lenard S.; Clerc, Isabelle; Taylor, Naomi; Thielecke, Lars; Cornils, Kerstin; Bueren, Juan A.; Rio, Paula; Gijsbers, Rik; Cosset, François-Loïc

2017-01-01

Hematopoietic stem cell (HSC)–based gene therapy trials are now moving toward the use of lentiviral vectors (LVs) with success. However, one challenge in the field remains: efficient transduction of HSCs without compromising their stem cell potential. Here we showed that measles virus glycoprotein–displaying LVs (hemagglutinin and fusion protein LVs [H/F-LVs]) were capable of transducing 100% of early-acting cytokine-stimulated human CD34+ (hCD34+) progenitor cells upon a single application. Strikingly, these H/F-LVs also allowed transduction of up to 70% of nonstimulated quiescent hCD34+ cells, whereas conventional vesicular stomatitis virus G (VSV-G)–LVs reached 5% at the most with H/F-LV entry occurring exclusively through the CD46 complement receptor. Importantly, reconstitution of NOD/SCIDγc−/− (NSG) mice with H/F-LV transduced prestimulated or resting hCD34+ cells confirmed these high transduction levels in all myeloid and lymphoid lineages. Remarkably, for resting CD34+ cells, secondary recipients exhibited increasing transduction levels of up to 100%, emphasizing that H/F-LVs efficiently gene-marked HSCs in the resting state. Because H/F-LVs promoted ex vivo gene modification of minimally manipulated CD34+ progenitors that maintained stemness, we assessed their applicability in Fanconi anemia, a bone marrow (BM) failure with chromosomal fragility. Notably, only H/F-LVs efficiently gene-corrected minimally stimulated hCD34+ cells in unfractionated BM from these patients. These H/F-LVs improved HSC gene delivery in the absence of cytokine stimulation while maintaining their stem cell potential. Thus, H/F-LVs will facilitate future clinical applications requiring HSC gene modification, including BM failure syndromes, for which treatment has been very challenging up to now. PMID:29296856

Applications of lentiviral vectors in molecular imaging.

PubMed

Chatterjee, Sushmita; De, Abhijit

2014-06-01

Molecular imaging provides the ability of simultaneous visual and quantitative estimation of long term gene expression directly from living organisms. To reveal the kinetics of gene expression by imaging method, often sustained expression of the transgene is required. Lentiviral vectors have been extensively used over last fifteen years for delivery of a transgene in a wide variety of cell types. Lentiviral vectors have the well known advantages such as sustained transgene delivery through stable integration into the host genome, the capability of infecting non-dividing and dividing cells, broad tissue tropism, a reasonably large carrying capacity for delivering therapeutic and reporter gene combinations. Additionally, they do not express viral proteins during transduction, have a potentially safe integration site profile, and a relatively easy system for vector manipulation and infective viral particle production. As a result, lentiviral vector mediated therapeutic and imaging reporter gene delivery to various target organs holds promise for the future treatment. In this review, we have conducted a brief survey of important lentiviral vector developments in diverse biomedical fields including reproductive biology.

Genetically modified pigs produced with a nonviral episomal vector

PubMed Central

Manzini, Stefano; Vargiolu, Alessia; Stehle, Isa M; Bacci, Maria Laura; Cerrito, Maria Grazia; Giovannoni, Roberto; Zannoni, Augusta; Bianco, Maria Rosaria; Forni, Monica; Donini, Pierluigi; Papa, Michele; Lipps, Hans J; Lavitrano, Marialuisa

2006-01-01

Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy. PMID:17101993

Autophagy-inducing peptides from mammalian VSV and fish VHSV rhabdoviral G glycoproteins (G) as models for the development of new therapeutic molecules

PubMed Central

García-Valtanen, Pablo; Ortega-Villaizán, María del Mar; Martínez-López, Alicia; Medina-Gali, Regla; Pérez, Luis; Mackenzie, Simon; Figueras, Antonio; Coll, Julio M; Estepa, Amparo

2014-01-01

It has not been elucidated whether or not autophagy is induced by rhabdoviral G glycoproteins (G) in vertebrate organisms for which rhabdovirus infection is lethal. Our work provides the first evidence that both mammalian (vesicular stomatitis virus, VSV) and fish (viral hemorrhagic septicemia virus, VHSV, and spring viremia carp virus, SVCV) rhabdoviral Gs induce an autophagic antiviral program in vertebrate cell lines. The transcriptomic profiles obtained from zebrafish genetically immunized with either Gsvcv or Gvhsv suggest that autophagy is induced shortly after immunization and therefore, it may be an important component of the strong antiviral immune responses elicited by these viral proteins. Pepscan mapping of autophagy-inducing linear determinants of Gvhsv and Gvsv showed that peptides located in their fusion domains induce autophagy. Altogether these results suggest that strategies aimed at modulating autophagy could be used for the prevention and treatment of rhabdoviral infections such as rabies, which causes thousands of human deaths every year. PMID:25046110

Characterization of resistance to rhabdovirus and retrovirus infection in a human myeloid cell line.

PubMed

Boso, Guney; Somia, Nikunj V

2015-01-01

Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1.

Immunogenicity of Newcastle Disease Virus Vectors Expressing Norwalk Virus Capsid Protein in the Presence or Absence of VP2 Protein

PubMed Central

Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y.; Samal, Siba K.

2015-01-01

Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirs-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans. PMID:26099695

Reflections on the early development of poxvirus vectors.

PubMed

Moss, Bernard

2013-09-06

Poxvirus expression vectors were described in 1982 and quickly became widely used for vaccine development as well as research in numerous fields. Advantages of the vectors include simple construction, ability to accommodate large amounts of foreign DNA and high expression levels. Numerous poxvirus-based veterinary vaccines are currently in use and many others are in human clinical trials. The early reports of poxvirus vectors paved the way for and stimulated the development of other viral vectors and recombinant DNA vaccines. Published by Elsevier Ltd.

A 5′ Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo

PubMed Central

Lu, Jiamiao; Williams, James A.; Luke, Jeremy; Zhang, Feijie; Chu, Kirk; Kay, Mark A.

2017-01-01

We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5′ UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo. PMID:27903072

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

PubMed

Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

2008-02-01

To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

A lentiviral vector with expression controlled by E2F-1: A potential tool for the study and treatment of proliferative diseases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauss, Bryan E.; Patricio, Juliana Rotelli; Program in Biotechnology, University of Sao Paulo

2006-10-06

We have constructed a lentiviral vector with expression limited to cells presenting active E2F-1 protein, a potential advantage for gene therapy of proliferative diseases. For the FE2FLW vector, the promoter region of the human E2F-1 gene was utilized to drive expression of luciferase cDNA, included as a reporter of viral expression. Primary, immortalized, and transformed cells were transduced with the FE2FLW vector and cell cycle alterations were induced with serum starvation/replacement, contact inhibition or drug treatment, revealing cell cycle-dependent changes in reporter activity. Forced E2F-1 expression, but not E2F-2 or E2F-3, increased reporter activity, indicating a major role for thismore » factor in controlling expression from the FE2FLW virus. We show the utility of this vector as a reporter of E2F-1 and proliferation-dependent cellular alterations upon cytotoxic/cytostatic treatment, such as the introduction of tumor suppressor genes. We propose that the FE2FLW vector may be a starting point for the development of gene therapy strategies for proliferative diseases, such as cancer or restinosis.« less

Cloning and expression of Clostridium perfringens type D vaccine strain epsilon toxin gene in E. coli as a recombinant vaccine candidate.

PubMed

Aziminia, Parastoo; Pilehchian-Langroudi, Reza; Esmaeilnia, Kasra

2016-08-01

Clostridium perfringens, a Gram-positive obligate anaerobic bacterium, is able to form resistant spores which are widely distributed in the environment. C. perfringens is subdivided into five types A to E based on its four major alpha, beta, epsilon and iota toxins. The aim of the present study was cloning and expression of C. perfringens type D vaccine strain epsilon toxin gene. Genomic DNA was extracted and the epsilon toxin gene was amplified using Pfu DNA polymerase. The PCR product was cloned into pJET1.2/blunt cloning vector. The recombinant vector (pJETε) was sequenced using universal primers. At the next step epsilon toxin gene was subcloned into pET22b(+) expression vector and transformed into E. coli Rosetta (DE3) host strain. The recombinant protein has been expressed in E. coli Rosetta (DE3) cells after subcloning of C. perfringens etx gene (1008 bp) into the expression vector. We concluded that E. coli Rosetta strain was suitable for the expression of recombinant C. perfringens epsilon toxin protein from pET22ε expression vector. This recombinant cell can be used for further research on recombinant vaccine development.

[Expression and identification of eukaryotic expression vectors of Brucella melitensis lipoprotein OMP19].

PubMed

He, Zuoping; Luo, Peifang; Hu, Feihuan; Weng, Yunceng; Wang, Wenjing; Li, Chengyao

2016-04-01

To construct eukaryotic expression vectors carrying Brucella melitensis outer membrane protein 19 (OMP19), express them in transfected Huh7.5.1 and JEG-3 cells, and analyze their role in cell apoptosis. Brucella melitensis lipidated OMP19 (L-OMP19) gene and unlipidated OMP19 (U-OMP19) gene were amplified by PCR and inserted into the vector pZeroBack/blunt. The correct L-OMP19 and U-OMP19 genes verified by XbaI and BamHI double digestion and sequencing were cloned into the lentivirus expression vector pHAGE-CMV-MCS-IZsGreen to construct vectors pHAGE-L-OMP19 and pHAGE-U-OMP19, which were separately transfected into 293FT cells, Huh7.5.1 and JEG-3 cells. L-OMP19 and U-OMP19 in the cells were detected by Western blotting and immunofluorescence technique. Flow cytometry combined with annexin V-PE/7-AAD staining was used to detect the cell apoptosis. The lentiviral vectors pHAGE-L-OMP19 and pHAGE-U-OMP19 were constructed correctly and the recombinant lipoproteins L-OMP19 and U-OMP19 expressed in the above cells were well recognized by the specific antibodies against L-OMP19 in Western blotting and immunofluorescence technique. L-OMP19 and U-OMP19 induced JEG-3 cell death, but did not induce the apoptosis of Huh7.5.1 cells. The eukaryotic expression vectors of L-OMP19 and U-OMP19 have been constructed successfully. Recombinant lipoproteins L-OMP19 and U-OMP19 expressed in cells have a good antigenicity, which could be used as experimental materials for the research on the relationship between host cells and lipoproteins in Brucella infection.

[The expression of interferon-lambda1 in CHO cell].

PubMed

Yuan, Wu-Mei; Ma, Fen-Lian; Zhang, Qian; Zheng, Wen-Zhi; Zheng, Li-Shu

2013-06-01

To construct the eukaryotic expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which linked the enhancer SP163 with interferon lambda1. Then express the interferon lambda1 in CHO (dhfr-) cells. Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon Lambda1. After sequenced, lambda1 and SP163-lambda1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which was constructed successfully confirming by sequencing. Then the expressing vectors were transfected into CHO (dhfr-) cells using liposome transfection method and interferon lambda1 protein was assayed with indirect immunofluorescence and Western Blot. Using cytopathic effect inhibition evaluated the antiviral activity of interferon lambda1. Successfully constructing the eukaryotic expression vectors of interferon lambda and the vectors could express interferon lambda1. The result of immunofluorescence showed the enhancer developed the expression of interferon lambda1. Detecting the interferon lambda1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot. The cytopathic effect inhibition showed the expressed interferon lambda1 has the antiviral activity. Successfully expressed the interferon lambda1 in CHO (dhfr-) cells and the protein possesses antiviral activity, which may supply a valuable basis for building the stable cell line of interferon lambda1.

Adenovirus vector expressing keratinocyte growth factor using CAG promoter impairs pulmonary function of mice with elastase-induced emphysema.

PubMed

Oki, Hiroshi; Yazawa, Takuya; Baba, Yasuko; Kanegae, Yumi; Sato, Hanako; Sakamoto, Seiko; Goto, Takahisa; Saito, Izumu; Kurahashi, Kiyoyasu

2017-07-01

Pulmonary emphysema impairs quality of life and increases mortality. It has previously been shown that administration of adenovirus vector expressing murine keratinocyte growth factor (KGF) before elastase instillation prevents pulmonary emphysema in mice. We therefore hypothesized that therapeutic administration of KGF would restore damage to lungs caused by elastase instillation and thus improve pulmonary function in an animal model. KGF expressing adenovirus vector, which prevented bleomycin-induced pulmonary fibrosis in a previous study, was constructed. Adenovirus vector (1.0 × 10 9 plaque-forming units) was administered intratracheally one week after administration of elastase into mouse lungs. One week after administration of KGF-vector, exercise tolerance testing and blood gas analysis were performed, after which the lungs were removed under deep anesthesia. KGF-positive pneumocytes were more numerous, surfactant protein secretion in the airspace greater and mean linear intercept of lungs shorter in animals that had received KGF than in control animals. Unexpectedly, however, arterial blood oxygenation was worse in the KGF group and maximum running speed, an indicator of exercise capacity, had not improved after KGF in mice with elastase-induced emphysema, indicating that KGF-expressing adenovirus vector impaired pulmonary function in these mice. Notably, vector lacking KGF-expression unit did not induce such impairment, implying that the KGF expression unit itself may cause the damage to alveolar cells. Possible involvement of the CAG promoter used for KGF expression in impairing pulmonary function is discussed. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

A helper virus-free HSV-1 vector containing the vesicular glutamate transporter-1 promoter supports expression preferentially in VGLUT1-containing glutamatergic neurons.

PubMed

Zhang, Guo-rong; Geller, Alfred I

2010-05-17

Multiple potential uses of direct gene transfer into neurons require restricting expression to specific classes of glutamatergic neurons. Thus, it is desirable to develop vectors containing glutamatergic class-specific promoters. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported a plasmid (amplicon) Herpes Simplex Virus (HSV-1) vector that placed the Lac Z gene under the regulation of the VGLUT1 promoter (pVGLUT1lac). Using helper virus-free vector stocks, we showed that this vector supported approximately 90% glutamatergic neuron-specific expression in postrhinal (POR) cortex, in rats sacrificed at either 4 days or 2 months after gene transfer. We now show that pVGLUT1lac supports expression preferentially in VGLUT1-containing glutamatergic neurons. pVGLUT1lac vector stock was injected into either POR cortex, which contains primarily VGLUT1-containing glutamatergic neurons, or into the ventral medial hypothalamus (VMH), which contains predominantly VGLUT2-containing glutamatergic neurons. Rats were sacrificed at 4 days after gene transfer, and the types of cells expressing ss-galactosidase were determined by immunofluorescent costaining. Cell counts showed that pVGLUT1lac supported expression in approximately 10-fold more cells in POR cortex than in the VMH, whereas a control vector supported expression in similar numbers of cells in these two areas. Further, in POR cortex, pVGLUT1lac supported expression predominately in VGLUT1-containing neurons, and, in the VMH, pVGLUT1lac showed an approximately 10-fold preference for the rare VGLUT1-containing neurons. VGLUT1-specific expression may benefit specific experiments on learning or specific gene therapy approaches, particularly in the neocortex. Copyright 2010 Elsevier B.V. All rights reserved.

Using rabies virus vaccine strain SRV9 as viral vector to express exogenous gene.

PubMed

Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Li, Ling; Qi, Yinglin; Liang, Meng; Zhao, Yongkun; Wang, Tiecheng; Gao, Yuwei; Tu, Changchun; Jin, Ningyi; Yang, Songtao; Xia, Xianzhu

2015-04-01

Rabies virus (RABV) can cause a fatal neurological disease in human and animals, and vaccines were generally applied for the immunoprophylaxis of rabies. Here, a recombinant viral vector carrying the exogenous gene expression component between phosphoprotein (P) and matrix protein (M) genes of RABV was constructed based on the vaccine strain SRV9 used in China. To develop a reverse genetic system, the full-length cDNA plasmids of SRV9 were constructed using the eukaryotic expression vector pCI or pcDNA3.1(+). However, recovery efficiency based on the pcDNA3.1 vector was significantly higher than that of the pCI vector. The exogenous gene expression component PE-PS-BsiWI-PmeI or PS-BsiWI-PmeI-PE was introduced in different locations between the P and M genes of SRV9. When the enhanced green fluorescent protein (eGFP) was used as a reporter gene, both locations could rescue recombinant RABV (rRABV) expressing eGFP with high efficiency. Characterization of rRABV expressing eGFP in vitro revealed that its growth was similar to that of the parental virus. Animal experiments showed that rRABV expressing eGFP could replicate and express eGFP in the brains of suckling mice. Furthermore, rRABV of SRV9 was nonpathogenic for 3-week-old mice and could be cleared from the central nervous system at 5 days post-inoculation. Our results showed that the recombinant SRV9 virus could be used as a useful viral vector for exogenous gene expression.

Generation of 2A-linked multicistronic cassettes by recombinant PCR.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector. This protocol describes the use of recombinant polymerase chain reaction (PCR) to connect multiple 2A-linked protein sequences. The final construct is subcloned into an expression vector.

Characteristics of lentiviral vectors harboring the proximal promoter of the vav proto-oncogene: a weak and efficient promoter for gene therapy.

PubMed

Almarza, Elena; Río, Paula; Meza, Nestor W; Aldea, Montserrat; Agirre, Xabier; Guenechea, Guillermo; Segovia, José C; Bueren, Juan A

2007-08-01

Recent published data have shown the efficacy of gene therapy treatments of certain monogenic diseases. Risks of insertional oncogenesis, however, indicate the necessity of developing new vectors with weaker or cell-restricted promoters to minimize the trans-activation activity of integrated proviruses. We have inserted the proximal promoter of the vav proto-oncogene into self-inactivating lentiviral vectors (vav-LVs) and investigated the expression pattern and therapeutic efficacy of these vectors. Compared with other LVs frequently used in gene therapy, vav-LVs mediated a weak, though homogeneous and stable, expression in in vitro-cultured cells. Transplantation experiments using transduced mouse bone marrow and human CD34(+) cells confirmed the stable activity of the promoter in vivo. To investigate whether the weak activity of this promoter was compatible with a therapeutic effect, a LV expressing the Fanconi anemia A (FANCA) gene was constructed (vav-FANCA LV). Although this vector induced a low expression of FANCA, compared to the expression induced by a LV harboring the spleen focus-forming virus (SFFV) promoter, the two vectors corrected the phenotype of cells from a patient with FA-A with the same efficacy. We propose that self-inactivating vectors harboring weak promoters, such as the vav promoter, will improve the safety of gene therapy and will be of particular interest for the treatment of diseases where a high expression of the transgene is not required.

[Prokaryotic expression, purification and antigenicity identification of recombinant human survivin protein].

PubMed

Yin, Xiaotao; Wang, Wei; Tian, Renli; Xu, Yuanji; Yan, Jinqi; Zhang, Wei; Gao, Jiangping; Yu, Jiyun

2013-08-01

To construct a prokaryotic expression plasmid pET28a-survivin, optimize the recombinant protein expression conditions in E.coli, and purify the survivin recombinant protein and identify its antigenicity. Survivin cDNA segment was amplified by PCR and cloned into prokaryotic expression vector pET28a(+) to construct the recombinant expression vector pET28a-survivin. The expression vector was transformed into BL21 (DE3) and the fusion protein survivin/His was induced by IPTG. The fusion protein was purified through Ni affinity chromatography. The antigenicity of the purified survivin protein was identified by Western blotting and ELISA. The recombinant expression vector was verified successfully by BamHI and HindIII. The fusion protein induced by IPTG was obtained with Mr; about 24 000. The purity of the purified protein reached 90% by SDS-PAGE analysis. And the antigenicity of the survivin protein was validated by Western blotting and ELISA. The prokaryotic expression plasmid pET28a-survivin was successfully constructed and the survivin protein was expressed and purified in E.coli. The antigenicity of the purified survivin protein was demonstrated desirable.

Development of Sendai Virus Vectors and their Potential Applications in Gene Therapy and Regenerative Medicine

PubMed Central

Nakanishi, Mahito; Otsu, Makoto

2012-01-01

Gene delivery/expression vectors have been used as fundamental technologies in gene therapy since the 1980s. These technologies are also being applied in regenerative medicine as tools to reprogram cell genomes to a pluripotent state and to other cell lineages. Rapid progress in these new research areas and expectations for their translation into clinical applications have facilitated the development of more sophisticated gene delivery/expression technologies. Since its isolation in 1953 in Japan, Sendai virus (SeV) has been widely used as a research tool in cell biology and in industry, but the application of SeV as a recombinant viral vector has been investigated only recently. Recombinant SeV vectors have various unique characteristics, such as low pathogenicity, powerful capacity for gene expression and a wide host range. In addition, the cytoplasmic gene expression mediated by this vector is advantageous for applications, in that chromosomal integration of exogenous genes can be undesirable. In this review, we introduce a brief historical background on the development of recombinant SeV vectors and describe their current applications in gene therapy. We also describe the application of SeV vectors in advanced nuclear reprogramming and introduce a defective and persistent SeV vector (SeVdp) optimized for such reprogramming. PMID:22920683

Simple and effective generation of transgene-free induced pluripotent stem cells using an auto-erasable Sendai virus vector responding to microRNA-302.

PubMed

Nishimura, Ken; Ohtaka, Manami; Takada, Hitomi; Kurisaki, Akira; Tran, Nhi Vo Kieu; Tran, Yen Thi Hai; Hisatake, Koji; Sano, Masayuki; Nakanishi, Mahito

2017-08-01

Transgene-free induced pluripotent stem cells (iPSCs) are valuable for both basic research and potential clinical applications. We previously reported that a replication-defective and persistent Sendai virus (SeVdp) vector harboring four reprogramming factors (SeVdp-iPS) can efficiently induce generation of transgene-free iPSCs. This vector can express all four factors stably and simultaneously without chromosomal integration and can be eliminated completely from reprogrammed cells by suppressing vector-derived RNA-dependent RNA polymerase. Here, we describe an improved SeVdp-iPS vector (SeVdp(KOSM)302L) that is automatically erased in response to microRNA-302 (miR-302), uniquely expressed in pluripotent stem cells (PSCs). Gene expression and genome replication of the SeVdp-302L vector, which contains miRNA-302a target sequences at the 3' untranslated region of L mRNA, are strongly suppressed in PSCs. Consequently, SeVdp(KOSM)302L induces expression of reprogramming factors in somatic cells, while it is automatically erased from cells successfully reprogrammed to express miR-302. As this vector can reprogram somatic cells into transgene-free iPSCs without the aid of exogenous short interfering RNA (siRNA), the results we present here demonstrate that this vector may become an invaluable tool for the generation of human iPSCs for future clinical applications. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

The Function of Herpes Simplex Virus Genes: A Primer for Genetic Engineering of Novel Vectors

NASA Astrophysics Data System (ADS)

Roizman, Bernard

1996-10-01

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.

Fine-resolution voxel S values for constructing absorbed dose distributions at variable voxel size.

PubMed

Dieudonné, Arnaud; Hobbs, Robert F; Bolch, Wesley E; Sgouros, George; Gardin, Isabelle

2010-10-01

This article presents a revised voxel S values (VSVs) approach for dosimetry in targeted radiotherapy, allowing dose calculation for any voxel size and shape of a given SPECT or PET dataset. This approach represents an update to the methodology presented in MIRD pamphlet no. 17. VSVs were generated in soft tissue with a fine spatial sampling using the Monte Carlo (MC) code MCNPX for particle emissions of 9 radionuclides: (18)F, (90)Y, (99m)Tc, (111)In, (123)I, (131)I, (177)Lu, (186)Re, and (201)Tl. A specific resampling algorithm was developed to compute VSVs for desired voxel dimensions. The dose calculation was performed by convolution via a fast Hartley transform. The fine VSVs were calculated for cubic voxels of 0.5 mm for electrons and 1.0 mm for photons. Validation studies were done for (90)Y and (131)I VSV sets by comparing the revised VSV approach to direct MC simulations. The first comparison included 20 spheres with different voxel sizes (3.8-7.7 mm) and radii (4-64 voxels) and the second comparison a hepatic tumor with cubic voxels of 3.8 mm. MC simulations were done with MCNPX for both. The third comparison was performed on 2 clinical patients with the 3D-RD (3-Dimensional Radiobiologic Dosimetry) software using the EGSnrc (Electron Gamma Shower National Research Council Canada)-based MC implementation, assuming a homogeneous tissue-density distribution. For the sphere model study, the mean relative difference in the average absorbed dose was 0.20% ± 0.41% for (90)Y and -0.36% ± 0.51% for (131)I (n = 20). For the hepatic tumor, the difference in the average absorbed dose to tumor was 0.33% for (90)Y and -0.61% for (131)I and the difference in average absorbed dose to the liver was 0.25% for (90)Y and -1.35% for (131)I. The comparison with the 3D-RD software showed an average voxel-to-voxel dose ratio between 0.991 and 0.996. The calculation time was below 10 s with the VSV approach and 50 and 15 h with 3D-RD for the 2 clinical patients. This new VSV approach enables the calculation of absorbed dose based on a SPECT or PET cumulated activity map, with good agreement with direct MC methods, in a faster and more clinically compatible manner.

Detectable reporter gene expression following transduction of adenovirus and adeno-associated virus serotype 2 vectors within full-thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo.

PubMed

Santangelo, Kelly S; Baker, Sarah A; Nuovo, Gerard; Dyce, Jonathan; Bartlett, Jeffrey S; Bertone, Alicia L

2010-02-01

This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg-Gly-Asp (RGD)-modified Ad, adeno-associated viral serotype 2 (AAV2), and self-complementary AAV2 (scAAV2) vectors within full-thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real-time RT-PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p < or = 0.026). Ad and Ad-RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full-thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo. (c) 2009 Orthopaedic Research Society.

[Construction and selection of effective mouse Smad6 recombinant lenti-virus interference vectors].

PubMed

Yu, Jing; Qi, Mengchun; Deng, Jiupeng; Liu, Gang; Chen, Huaiqing

2010-10-01

This experiment was designed to construct mouse Smad6 recombinant RNA interference vectors and determine their interference effects on bone marrow mesenchymal stem cells (BMSCs). Three recombinant Smad6 RNA interference vectors were constructed by molecular clone techniques with a lenti-virus vector expressing green fluorescent protein (GFP), and the correctness of recombinant vectors was verified by DNA sequencing. Mouse BMSCs were used for transfection experiments and BMP-2 was in use for osteogenic induction of MSCs. The transfection efficiency of recombinant vectors was examined by Laser confocal scanning microscope and the interference effect of recombinant vectors on Smad6 gene expression was determined by real-time RT-PCR and Western blot, respectively. Three Smad6 recombinant RNA interference vectors were successfully constructed and their correctness was proved by DNA sequencing. After transfection, GFPs were effectively expressed in MSCs and all of three recombinant vectors gained high transfection efficiency (> 95%). Both real-time PCR and Western blot examination indicated that among three recombinant vectors, No. 2 Svector had the best interference effect and the interference effect was nearly 91% at protein level. In conclusion, Mouse recombinant Smad6 RNA interference (RNAi) vector was successfully constructed and it provided an effective tool for further studies on BMP signal pathways.

Viral Vectors for in Vivo Gene Transfer

NASA Astrophysics Data System (ADS)

Thévenot, E.; Dufour, N.; Déglon, N.

The transfer of DNA into the nucleus of a eukaryotic cell (gene transfer) is a central theme of modern biology. The transfer is said to be somatic when it refers to non-germline organs of a developed individual, and germline when it concerns gametes or the fertilised egg of an animal, with the aim of transmitting the relevant genetic modification to its descendents [1]. The efficient introduction of genetic material into a somatic or germline cell and the control of its expression over time have led to major advances in understanding how genes work in vivo, i.e., in living organisms (functional genomics), but also to the development of innovative therapeutic methods (gene therapy). The efficiency of gene transfer is conditioned by the vehicle used, called the vector. Desirable features for a vector are as follows: Easy to produce high titer stocks of the vector in a reproducible way. Absence of toxicity related to transduction (transfer of genetic material into the target cell, and its expression there) and no immune reaction of the organism against the vector and/or therapeutic protein. Stability in the expression of the relevant gene over time, and the possibility of regulation, e.g., to control expression of the therapeutic protein on the physiological level, or to end expression at the end of treatment. Transduction of quiescent cells should be as efficient as transduction of dividing cells. Vectors currently used fall into two categories: non-viral and viral vectors. In non-viral vectors, the DNA is complexed with polymers, lipids, or cationic detergents (described in Chap. 3). These vectors have a low risk of toxicity and immune reaction. However, they are less efficient in vivo than viral vectors when it comes to the number of cells transduced and long-term transgene expression. (Naked DNA transfer or electroporation is rather inefficient in the organism. This type of gene transfer will not be discussed here, and the interested reader is referred to the review [2].) For this reason, it is mainly viral vectors that are used for gene transfer in animals and humans.

Assessing the safety and immunogenicity of recombinant vesicular stomatitis virus Ebola vaccine in healthy adults: a randomized clinical trial

PubMed Central

ElSherif, May S.; Brown, Catherine; MacKinnon-Cameron, Donna; Li, Li; Racine, Trina; Alimonti, Judie; Rudge, Thomas L.; Sabourin, Carol; Silvera, Peter; Hooper, Jay W.; Kwilas, Steven A.; Kilgore, Nicole; Badorrek, Christopher; Ramsey, W. Jay; Heppner, D. Gray; Kemp, Tracy; Monath, Thomas P.; Nowak, Teresa; McNeil, Shelly A.; Langley, Joanne M.; Halperin, Scott A.

2017-01-01

BACKGROUND: The 2013–2016 Ebola virus outbreak in West Africa was the most widespread in history. In response, alive attenuated recombinant vesicular stomatitis virus (rVSV) vaccine expressing Zaire Ebolavirus glycoprotein (rVSVΔG-ZEBOV-GP) was evaluated in humans. METHODS: In a phase 1, randomized, dose-ranging, observer-blind, placebo-controlled trial, healthy adults aged 18–65 years were randomized into 4 groups of 10 to receive one of 3 vaccine doses or placebo. Follow-up visits spanned 180 days postvaccination for safety monitoring, immunogenicity testing and any rVSV virus shedding. RESULTS: Forty participants were injected with rVSVΔG-ZEBOV-GP vaccine (n = 30) or saline placebo (n = 10). No serious adverse events related to the vaccine or participant withdrawals were reported. Solicited adverse events during the 14-day follow-up period were mild to moderate and self-limited, with the exception of injection-site pain and headache. Viremia following vaccination was transient and no longer detectable after study day 3, with no virus shedding in saliva or urine. All vaccinated participants developed serum immunoglobulin G (IgG), as measured by Ebola virus envelope glycoprotein-based enzyme-linked immunosorbent assay (ELISA). Immunogenicity was comparable across all dose groups, and sustained IgG titers were detectable through to the last visit, at study day 180. INTERPRETATION: In this phase 1 study, there were no safety concerns after a single dose of rVSVΔG-ZEBOV-GP vaccine. IgG ELISA showed persistent high titers at 180 days postimmunization. There was a period of reactogenicity, but in general, the vaccine was well tolerated. This study provides evidence of the safety and immunogenicity of rVSVΔG-ZEBOV-GP vaccine and importance of its further investigation. Trial registration: Clinical-Trials.gov no., NCT02374385 PMID:28630358

A stable RNA virus-based vector for citrus trees

DOE Office of Scientific and Technical Information (OSTI.GOV)

Folimonov, Alexey S.; Folimonova, Svetlana Y.; Bar-Joseph, Moshe

Virus-based vectors are important tools in plant molecular biology and plant genomics. A number of vectors based on viruses that infect herbaceous plants are in use for expression or silencing of genes in plants as well as screening unknown sequences for function. Yet there is a need for useful virus-based vectors for woody plants, which demand much greater stability because of the longer time required for systemic infection and analysis. We examined several strategies to develop a Citrus tristeza virus (CTV)-based vector for transient expression of foreign genes in citrus trees using a green fluorescent protein (GFP) as a reporter.more » These strategies included substitution of the p13 open reading frame (ORF) by the ORF of GFP, construction of a self-processing fusion of GFP in-frame with the major coat protein (CP), or expression of the GFP ORF as an extra gene from a subgenomic (sg) mRNA controlled either by a duplicated CTV CP sgRNA controller element (CE) or an introduced heterologous CE of Beet yellows virus. Engineered vector constructs were examined for replication, encapsidation, GFP expression during multiple passages in protoplasts, and for their ability to infect, move, express GFP, and be maintained in citrus plants. The most successful vectors based on the 'add-a-gene' strategy have been unusually stable, continuing to produce GFP fluorescence after more than 4 years in citrus trees.« less

Preclinical Potency and Biodistribution Studies of an AAV 5 Vector Expressing Human Interferon-β (ART-I02) for Local Treatment of Patients with Rheumatoid Arthritis

PubMed Central

Aalbers, Caroline J.; Bevaart, Lisette; Loiler, Scott; de Cortie, Karin; Wright, J. Fraser; Mingozzi, Federico; Tak, Paul P.; Vervoordeldonk, Margriet J.

2015-01-01

Introduction Proof of concept for local gene therapy for the treatment of arthritis with immunomodulatory cytokine interferon beta (IFN-β) has shown promising results in animal models of rheumatoid arthritis (RA). For the treatment of RA patients, we engineered a recombinant adeno-associated serotype 5 vector (rAAV5) encoding human (h)IFN-β under control of a nuclear factor κB promoter (ART-I02). Methods The potency of ART-I02 in vitro as well as biodistribution in vivo in arthritic animals was evaluated to characterize the vector prior to clinical application. ART-I02 expression and bioactivity after transduction was evaluated in fibroblast-like synoviocytes (FLS) from different species. Biodistribution of the vector after local injection was assessed in a rat adjuvant arthritis model through qPCR analysis of vector DNA. In vivo imaging was used to investigate transgene expression and kinetics in a mouse collagen induced arthritis model. Results Transduction of RA FLS in vitro with ART-I02 resulted in high expression levels of bioactive hIFN-β. Transduction of FLS from rhesus monkeys, rodents and rabbits with ART-I02 showed high transgene expression, and hIFN-β proved bioactive in FLS from rhesus monkeys. Transgene expression and bioactivity in RA FLS were unaltered in the presence of methotrexate. In vivo, vector biodistribution analysis in rats after intra-articular injection of ART-I02 demonstrated that the majority of vector DNA remained in the joint (>93%). In vivo imaging in mice confirmed local expression of rAAV5 in the knee joint region and demonstrated rapid detectable and sustained expression up until 7 weeks. Conclusions These data show that hIFN-β produced by RA FLS transduced with ART-I02 is bioactive and that intra-articular delivery of rAAV5 drives expression of a therapeutic transgene in the joint, with only limited biodistribution of vector DNA to other tissues, supporting progress towards a phase 1 clinical trial for the local treatment of arthritis in patients with RA. PMID:26107769

Preclinical Potency and Biodistribution Studies of an AAV 5 Vector Expressing Human Interferon-β (ART-I02) for Local Treatment of Patients with Rheumatoid Arthritis.

PubMed

Aalbers, Caroline J; Bevaart, Lisette; Loiler, Scott; de Cortie, Karin; Wright, J Fraser; Mingozzi, Federico; Tak, Paul P; Vervoordeldonk, Margriet J

2015-01-01

Proof of concept for local gene therapy for the treatment of arthritis with immunomodulatory cytokine interferon beta (IFN-β) has shown promising results in animal models of rheumatoid arthritis (RA). For the treatment of RA patients, we engineered a recombinant adeno-associated serotype 5 vector (rAAV5) encoding human (h)IFN-β under control of a nuclear factor κB promoter (ART-I02). The potency of ART-I02 in vitro as well as biodistribution in vivo in arthritic animals was evaluated to characterize the vector prior to clinical application. ART-I02 expression and bioactivity after transduction was evaluated in fibroblast-like synoviocytes (FLS) from different species. Biodistribution of the vector after local injection was assessed in a rat adjuvant arthritis model through qPCR analysis of vector DNA. In vivo imaging was used to investigate transgene expression and kinetics in a mouse collagen induced arthritis model. Transduction of RA FLS in vitro with ART-I02 resulted in high expression levels of bioactive hIFN-β. Transduction of FLS from rhesus monkeys, rodents and rabbits with ART-I02 showed high transgene expression, and hIFN-β proved bioactive in FLS from rhesus monkeys. Transgene expression and bioactivity in RA FLS were unaltered in the presence of methotrexate. In vivo, vector biodistribution analysis in rats after intra-articular injection of ART-I02 demonstrated that the majority of vector DNA remained in the joint (>93%). In vivo imaging in mice confirmed local expression of rAAV5 in the knee joint region and demonstrated rapid detectable and sustained expression up until 7 weeks. These data show that hIFN-β produced by RA FLS transduced with ART-I02 is bioactive and that intra-articular delivery of rAAV5 drives expression of a therapeutic transgene in the joint, with only limited biodistribution of vector DNA to other tissues, supporting progress towards a phase 1 clinical trial for the local treatment of arthritis in patients with RA.

Transcriptional Enhancers Induce Insertional Gene Deregulation Independently From the Vector Type and Design

PubMed Central

Maruggi, Giulietta; Porcellini, Simona; Facchini, Giulia; Perna, Serena K; Cattoglio, Claudia; Sartori, Daniela; Ambrosi, Alessandro; Schambach, Axel; Baum, Christopher; Bonini, Chiara; Bovolenta, Chiara; Mavilio, Fulvio; Recchia, Alessandra

2009-01-01

The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) γ-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase—PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a γ-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design. PMID:19293778

Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

USDA-ARS?s Scientific Manuscript database

A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

Gene transfer to the cerebellum.

PubMed

Louboutin, Jean-Pierre; Reyes, Beverly A S; Van Bockstaele, Elisabeth J; Strayer, David S

2010-12-01

There are several diseases for which gene transfer therapy to the cerebellum might be practicable. In these studies, we used recombinant Tag-deleted SV40-derived vectors (rSV40s) to study gene delivery targeting the cerebellum. These vectors transduce neurons and microglia very effectively in vitro and in vivo, and so we tested them to evaluate gene transfer to the cerebellum in vivo. Using a rSV40 vector carrying human immunodeficiency virus (HIV)-Nef with a C-terminal FLAG epitope, we characterized the distribution, duration, and cell types transduced. Rats received test and control vectors by stereotaxic injection into the cerebellum. Transgene expression was assessed 1, 2, and 4 weeks later by immunostaining of serial brain sections. FLAG epitope-expressing cells were seen, at all times after vector administration, principally detected in the Purkinje cells of the cerebellum, identified as immunopositive for calbindin. Occasional microglial cells were tranduced; transgene expression was not detected in astrocytes or oligodendrocytes. No inflammatory or other reaction was detected at any time. Thus, SV40-derived vectors can deliver effective, safe, and durable transgene expression to the cerebellum.

Design and construction of 2A peptide-linked multicistronic vectors.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. This article describes the design and construction of 2A peptide-linked multicistronic vectors, which can be used to express multiple proteins from a single open reading frame (ORF). The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector.

Construction of two Lactococcus lactis expression vectors combining the Gateway and the NIsin Controlled Expression systems.

PubMed

Douillard, François P; Mahony, Jennifer; Campanacci, Valérie; Cambillau, Christian; van Sinderen, Douwe

2011-09-01

Over the last 10 years, the NIsin Controlled Expression (NICE) system has been extensively used in the food-grade bacterium Lactococcus lactis subsp. cremoris to produce homologous and heterologous proteins for academic and biotechnological purposes. Although various L. lactis molecular tools have been developed, no expression vectors harboring the popular Gateway recombination system are currently available for this widely used cloning host. In this study, we constructed two expression vectors that combine the NICE and the Gateway recombination systems and we tested their applicability by recombining and over-expressing genes encoding structural proteins of lactococcal phages Tuc2009 and TP901-1. Over-expressed phage proteins were analyzed by immunoblotting and purified by His-tag affinity chromatography with protein productions yielding 2.8-3.7 mg/l of culture. This therefore is the first description of L. lactis NICE expression vectors which integrate the Gateway cloning technology and which are suitable for the production of sufficient amounts of proteins to facilitate subsequent structural and functional analyses. Copyright © 2011 Elsevier Inc. All rights reserved.

[Construction and expression of a eukaryotic expression vector containing human CR2-Fc fusion protein].

PubMed

Li, Xinxin; Wu, Zhihao; Zhang, Chuanfu; Jia, Leili; Song, Hongbin; Xu, Yuanyong

2014-01-01

To construct a eukaryotic expression vector containing human complement receptor 2 (CR2)-Fc and express the CR2-Fc fusion protein in Chinese hamster ovary (CHO) cells. The extracellular domain of human CR2 and IgG1 Fc were respectively amplified, ligated and inserted into the eukaryotic expression vector PCI-neo. After verified by restriction enzyme digestion and sequencing, the recombinant plasmid was transfected into CHO K1 cells. The ones with stable expression of the fusion protein were obtained by means of G418 selection. The expression of the CR2-Fc fusion protein was detected and confirmed by SDS-PAGE and Western blotting. Restriction enzyme digestion and sequencing demonstrated that the recombinant plasmid was valid. SDS-PAGE showed that relative molecular mass (Mr;) of the purified product was consistent with the expected value. Western blotting further proved the single band at the same position. We constructed the eukaryotic expression vector of CR2-Fc/PCI-neo successfully. The obtained fusion protein was active and can be used for the further study of the role in HIV control.

Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System

PubMed Central

Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M.

2015-01-01

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

PubMed

López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

2015-01-01

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

Immunogenicity of Newcastle disease virus vectors expressing Norwalk virus capsid protein in the presence or absence of VP2 protein.

PubMed

Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y; Samal, Siba K

2015-10-01

Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirus-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

Response to Blood Meal in the Fat Body of Anopheles stephensi Using Quantitative Proteomics: Toward New Vector Control Strategies Against Malaria.

PubMed

Kumar, Manish; Mohanty, Ajeet Kumar; Sreenivasamurthy, Sreelakshmi K; Dey, Gourav; Advani, Jayshree; Pinto, Sneha M; Kumar, Ashwani; Prasad, Thottethodi Subrahmanya Keshava

2017-09-01

Malaria remains a grand challenge for disruptive innovation in global health therapeutics and diagnostics. Anopheles stephensi is one of the major vectors of malaria in Asia. Vector and transmission control are key focus areas in the fight against malaria, a field of postgenomics research where proteomics can play a substantive role. Moreover, to identify novel strategies to control the vector population, it is necessary to understand the vector life processes at a global and molecular scale. In this context, fat body is a vital organ required for vitellogenesis, vector immunity, vector physiology, and vector-parasite interaction. Given its central role in energy metabolism, vitellogenesis, and immune function, the proteome profile of the fat body and the impact of blood meal (BM) ingestion on the protein abundances of this vital organ have not been investigated so far. Therefore, using a proteomics approach, we identified the proteins expressed in the fat body of An. stephensi and their differential expression in response to BM ingestion. In all, we identified 3,218 proteins in the fat body using high-resolution mass spectrometry, of which 483 were found to be differentially expressed in response to the BM ingestion. Bioinformatics analysis of these proteins underscored their role in amino acid metabolism, vitellogenesis, lipid transport, signal peptide processing, mosquito immunity, and oxidation-reduction processes. Interestingly, we identified five novel genes, which were found to be differentially expressed upon BM ingestion. Proteins that exhibited altered expression in the present study are potential targets for vector control strategies and development of transmission blocking vaccines in the fight against malaria.

An efficient Foxtail mosaic virus vector system with reduced environmental risk

PubMed Central

2010-01-01

Background Plant viral vectors offer high-yield expression of pharmaceutical and commercially important proteins with a minimum of cost and preparation time. The use of Agrobacterium tumefaciens has been introduced to deliver the viral vector as a transgene to each plant cell via a simple, nonsterile infiltration technique called "agroinoculation". With agroinoculation, a full length, systemically moving virus is no longer necessary for excellent protein yield, since the viral transgene is transcribed and replicates in every infiltrated cell. Viral genes may therefore be deleted to decrease the potential for accidental spread and persistence of the viral vector in the environment. Results In this study, both the coat protein (CP) and triple gene block (TGB) genetic segments were eliminated from Foxtail mosaic virus to create the "FECT" vector series, comprising a deletion of 29% of the genome. This viral vector is highly crippled and expresses little or no marker gene within the inoculated leaf. However, when co-agroinoculated with a silencing suppressor (p19 or HcPro), FECT expressed GFP at 40% total soluble protein in the tobacco host, Nicotiana benthamiana. The modified FoMV vector retained the full-length replicase ORF, the TGB1 subgenomic RNA leader sequence and either 0, 22 or 40 bases of TGB1 ORF (in vectors FECT0, FECT22 and FECT40, respectively). As well as N. benthamiana, infection of legumes was demonstrated. Despite many attempts, expression of GFP via syringe agroinoculation of various grass species was very low, reflecting the low Agrobacterium-mediated transformation rate of monocots. Conclusions The FECT/40 vector expresses foreign genes at a very high level, and yet has a greatly reduced biohazard potential. It can form no virions and can effectively replicate only in a plant with suppressed silencing. PMID:21162736

VSV-hIFNbeta-NIS in Treating Patients With Stage IV or Recurrent Endometrial Cancer

ClinicalTrials.gov

2018-05-09

Endometrial Clear Cell Adenocarcinoma; Endometrial Mixed Adenocarcinoma; Endometrial Serous Adenocarcinoma; Endometrial Undifferentiated Carcinoma; Metastatic Endometrioid Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Recurrent Endometrial Serous Adenocarcinoma; Recurrent Uterine Corpus Carcinoma; Stage IV Uterine Corpus Cancer; Stage IVA Uterine Corpus Cancer; Stage IVB Uterine Corpus Cancer

In planta expression of HIV-1 p24 protein using an RNA plant virus-based expression vector.

PubMed

Zhang, G; Leung, C; Murdin, L; Rovinski, B; White, K A

2000-02-01

Plant viruses show significant potential as expression vectors for the production of foreign proteins (e.g., antigens) in plants. The HIV-1 p24 nucleocapsid protein is an important early marker of HIV infection and has been used as an antigen in the development of HIV vaccines. Toward developing a plant-based expression system for the production of p24, we have investigated the use of a (positive)-strand RNA plant virus, tomato bushy stunt virus (TBSV), as an expression vector. The HIV p24 open reading frame (ORF) was introduced into a cloned cDNA copy of the TBSV genome as an in-frame fusion with a 5'-terminal portion of the TBSV coat protein ORF. In vitro-generated RNA transcripts corresponding to the engineered virus vector were infectious when inoculated into plant protoplasts; Northern and Western blot analyses verified the accumulation of a predicted p24-encoding viral subgenomic mRNA and the production of p24 fusion product. Whole-plant infections with the viral vector led to the accumulation of p24 fusion protein in inoculated leaves, which cross-reacted with p24-specific antibodies, thus confirming the maintenance of key antigenic determinants. This study is the first to demonstrate that TBSV can be engineered to express a complete foreign protein of clinical importance. Strategies for optimizing protein yield from this viral vector are discussed.

A single EBV-based vector for stable episomal maintenance and expression of GFP in human embryonic stem cells.

PubMed

Thyagarajan, Bhaskar; Scheyhing, Kelly; Xue, Haipeng; Fontes, Andrew; Chesnut, Jon; Rao, Mahendra; Lakshmipathy, Uma

2009-03-01

Stable expression of transgenes in stem cells has been a challenge due to the nonavailability of efficient transfection methods and the inability of transgenes to support sustained gene expression. Several methods have been reported to stably modify both embryonic and adult stem cells. These methods rely on integration of the transgene into the genome of the host cell, which could result in an expression pattern dependent on the number of integrations and the genomic locus of integration. To overcome this issue, site-specific integration methods mediated by integrase, adeno-associated virus or via homologous recombination have been used to generate stable human embryonic stem cell (hESC) lines. In this study, we describe a vector that is maintained episomally in hESCs. The vector used in this study is based on components derived from the Epstein-Barr virus, containing the Epstein-Barr virus nuclear antigen 1 expression cassette and the OriP origin of replication. The vector also expresses the drug-resistance marker gene hygromycin, which allows for selection and long-term maintenance of cells harboring the plasmid. Using this vector system, we show sustained expression of green fluorescent protein in undifferentiated hESCs and their differentiating embryoid bodies. In addition, the stable hESC clones show comparable expression with and without drug selection. Consistent with this observation, bulk-transfected adipose tissue-derived mesenchymal stem cells showed persistent marker gene expression as they differentiate into adipocytes, osteoblasts and chondroblasts. Episomal vectors offer a fast and efficient method to create hESC reporter lines, which in turn allows one to test the effect of overexpression of various genes on stem cell growth, proliferation and differentiation.

Tightly regulated, high-level expression from controlled copy number vectors based on the replicon of temperate phage N15.

PubMed

Mardanov, Andrey V; Strakhova, Taisia S; Smagin, Vladimir A; Ravin, Nikolai V

2007-06-15

A new Escherichia coli host/vector system has been developed to allow a dual regulation of both the plasmid copy number and gene expression. The new pN15E vectors are low copy number plasmids based on the replicon of temperate phage N15, comprising the repA replicase gene and cB repressor gene, controlling the plasmid copy number. Regulation of pN15E copy number is achieved through arabinose-inducible expression of phage N15 antirepressor protein, AntA, whose gene was integrated into the chromosome of the host strain under control of the PBAD promoter. The host strain also carried phage N15 partition operon, sop, allowing stable inheritance of pN15E vectors in the absence of selection pressure. In the first vector, pN15E4, the same PBAD promoter controls expression of a cloned gene. The second vector, pN15E6, carries the phage T5 promoter with a double lac operator repression module thus allowing independent regulation of promoter activity and copy number. Using the lacZ gene to monitor expression in these vectors, we show that the ratio of induction/repression can be about 7600-fold for pN15E4 and more than 15,000-fold for pN15E6. The low copy number of these vectors ensures very low basal level of expression allowing cloning genes encoding toxic products that was demonstrated by the stable maintenance of a gene encoding a restriction endonuclease in pN15E4. The tight control of transcription and the potential to regulate gene activities quantitatively over wide ranges will open up new approaches in the study of gene function in vivo and controlled expression of heterologous genes.

Development of an adenoviral vector with robust expression driven by p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bajgelman, Marcio C.; Biotechnology Program, Biomedical Sciences Institute, University of Sao Paulo; Millennium Institute-Gene Therapy Network, Ministry of Science and Technology

2008-02-05

Here we introduce a new adenoviral vector where transgene expression is driven by p53. We first developed a synthetic promoter, referred to as PGTx{beta}, containing a p53-responsive element, a minimal promoter and the first intron of the rabbit {beta}-globin gene. Initial assays using plasmid-based vectors indicated that expression was tightly controlled by p53 and was 5-fold stronger than the constitutive CMV immediate early promoter/enhancer. The adenoviral vector, AdPG, was also shown to offer p53-responsive expression in prostate carcinoma cells LNCaP (wt p53), DU-145 (temperature sensitive mutant of p53) and PC3 (p53-null, but engineered to express temperature-sensitive p53 mutants). AdPG servedmore » as a sensor of p53 activity in LNCaP cells treated with chemotherapeutic agents. Since p53 can be induced by radiotherapy and chemotherapy, this new vector could be further developed for use in combination with conventional therapies to bring about cooperation between the genetic and pharmacologic treatment modalities.« less

Validation of a recombinant human bactericidal/permeability-increasing protein (hBPI) expression vector using murine mammary gland tumor cells and the early development of hBPI transgenic goat embryos.

PubMed

Gui, Tao; Liu, Xing; Tao, Jia; Chen, Jianwen; Li, Yunsheng; Zhang, Meiling; Wu, Ronghua; Zhang, Yuanliang; Peng, Kaisong; Liu, Ya; Zhang, Xiaorong; Zhang, Yunhai

2013-12-01

Human bactericidal/permeability-increasing protein (hBPI) is the only antibacterial peptide which acts against both gram-negative bacteria and neutralizes endotoxins in human polymorphonuclear neutrophils; therefore, hBPI is of great value in clinical applications. In the study, we constructed a hBPI expression vector (pBC1-Loxp-Neo-Loxp-hBPI) containing the full-length hBPI coding sequence which could be specifically expressed in the mammary gland. To validate the function of the vector, in vitro cultured C127 (mouse mammary Carcinoma Cells) were transfected with the vector, and the transgenic cell clones were selected to express hBPI by hormone induction. The mRNA and protein expression of hBPI showed that the constructed vector was effective and suitable for future application in producing mammary gland bioreactor. Then, female and male goat fibroblasts were transfected with the vector, and two male and two female transgenic clonal cell lines were obtained. Using the transgenic cell lines as nuclear donors for somatic cell nuclear transfer, the reconstructed goat embryos produced from all four clones could develop to blastocysts in vitro. In conclusion, we constructed and validated an efficient mammary gland-specific hBPI expression vector, pBC1-Loxp-Neo-Loxp-hBPI, and transgenic hBPI goat embryos were successfully produced, laying foundations for future production of recombinant hBPI in goat mammary gland. Copyright © 2013 Elsevier B.V. All rights reserved.

A host-restricted viral vector for antigen-specific immunization against Lyme disease pathogen.

PubMed

Xiao, Sa; Kumar, Manish; Yang, Xiuli; Akkoyunlu, Mustafa; Collins, Peter L; Samal, Siba K; Pal, Utpal

2011-07-18

Newcastle disease virus (NDV) is an avian virus that is attenuated in primates and is a potential vaccine vector for human use. We evaluated NDV as a vector for expressing selected antigens of the Lyme disease pathogen Borrelia burgdorferi. A series of recombinant NDVs were generated that expressed intracellular or extracellular forms of two B. burgdorferi antigens: namely, the basic membrane protein A (BmpA) and the outer surface protein C (OspC). Expression of the intracellular and extracellular forms of these antigens was confirmed in cultured chicken cells. C3H or Balb/C mice that were immunized intranasally with the NDV vectors mounted vigorous serum antibody responses against the NDV vector, but failed to mount a robust response against either the intracellular or extracellular forms of BmpA or OspC. By contrast, a single immunization of hamsters with the NDV vectors via the intranasal, intramuscular, or intraperitoneal route resulted in rapid and rigorous antibody responses against the intracellular or extracellular forms of BmpA and OspC. When groups of hamsters were separately inoculated with various NDV vectors and challenged with B. burgdorferi (10(8)cells/animal), immunization with vector expressing either intracellular or extracellular BmpA was associated with a significant reduction of the pathogen load in the joints. Taken together, our studies highlighted the importance of NDV as vaccine vector that can be used for simple yet effective immunization of hosts against bacterial infections including Lyme disease. Copyright © 2011 Elsevier Ltd. All rights reserved.

The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting.

PubMed

Lu, Zhi Hong; Kaliberov, Sergey; Zhang, Jingzhu; Muz, Barbara; Azab, Abdel K; Sohn, Rebecca E; Kaliberova, Lyudmila; Du, Yingqiu; Curiel, David T; Arbeit, Jeffrey M

2014-08-01

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

Molecular cloning and production of caprine recombinant Oct4 protein for generation induced pluripotent stem cells.

PubMed

Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Singh, Surender; Kumar, Sudarshan; Kaushik, Jai K; Mohanty, Ashok K; Malakar, Dhruba

2015-12-01

Oct4, pluripotency marker and transcription factor, expresses in embryonic stem cells. It plays a pivotal role in determination of stem cells fate. Up and down regulation of Oct4 causes differentiation of embryonic stem cells. It is one of the main transcription factors which remained concerned in every study related to induced pluripotent stem cell. Here, we report the production of goat Oct4 protein using plasmid and lentiviral based vectors. Firstly, Oct4 ORF was cloned in pAcGFP1-N1 plasmid vector and positive clones were screened with colony PCR. Oct4 was over-expressed in CHO-K1 cell line and expression was confirmed by observing green florescent protein expression in CHO-K1 cells. Secondly, Oct4 lentiviral expression construct has been prepared using pLenti-gw vector. Oct4 ORF was cloned into pLenti4/V5-DEST vector and viral particles were produced in 293FT cells. Oct4 viral particles were used to infect goat fibroblast cells. Oct4 expression was observed and confirmed in transfected goat fibroblast cells using RT-PCR. Detection of Oct4 protein in western blotting assay affirmed the capacity of over-expression of our Oct4 lentiviral vector. The lentiviral expression construct and recombinant Oct4 protein may be used for reprogramming of somatic cell into induced pluripotent stem cell.

Development of a domain-specific genetic language to design Chlamydomonas reinhardtii expression vectors.

PubMed

Wilson, Mandy L; Okumoto, Sakiko; Adam, Laura; Peccoud, Jean

2014-01-15

Expression vectors used in different biotechnology applications are designed with domain-specific rules. For instance, promoters, origins of replication or homologous recombination sites are host-specific. Similarly, chromosomal integration or viral delivery of an expression cassette imposes specific structural constraints. As de novo gene synthesis and synthetic biology methods permeate many biotechnology specialties, the design of application-specific expression vectors becomes the new norm. In this context, it is desirable to formalize vector design strategies applicable in different domains. Using the design of constructs to express genes in the chloroplast of Chlamydomonas reinhardtii as an example, we show that a vector design strategy can be formalized as a domain-specific language. We have developed a graphical editor of context-free grammars usable by biologists without prior exposure to language theory. This environment makes it possible for biologists to iteratively improve their design strategies throughout the course of a project. It is also possible to ensure that vectors designed with early iterations of the language are consistent with the latest iteration of the language. The context-free grammar editor is part of the GenoCAD application. A public instance of GenoCAD is available at http://www.genocad.org. GenoCAD source code is available from SourceForge and licensed under the Apache v2.0 open source license.

An integrated vector system for cellular studies of phage display-derived peptides.

PubMed

Voss, Stephan D; DeGrand, Alec M; Romeo, Giulio R; Cantley, Lewis C; Frangioni, John V

2002-09-15

Peptide phage display is a method by which large numbers of diverse peptides can be screened for binding to a target of interest. Even when successful, the rate-limiting step is usually validation of peptide bioactivity using living cells. In this paper, we describe an integrated system of vectors that expedites both the screening and the characterization processes. Library construction and screening is performed using an optimized type 3 phage display vector, mJ(1), which is shown to accept peptide libraries of at least 23 amino acids in length. Peptide coding sequences are shuttled from mJ(1) into one of three families of mammalian expression vectors for cell physiological studies. The vector pAL(1) expresses phage display-derived peptides as Gal4 DNA binding domain fusion proteins for transcriptional activation studies. The vectors pG(1), pG(1)N, and pG(1)C express phage display-derived peptides as green fluorescent protein fusions targeted to the entire cell, nucleus, or cytoplasm, respectively. The vector pAP(1) expresses phage display-derived peptides as fusions to secreted placental alkaline phosphatase. Such enzyme fusions can be used as highly sensitive affinity reagents for high-throughput assays and for cloning of peptide-binding cell surface receptors. Taken together, this system of vectors should facilitate the development of phage display-derived peptides into useful biomolecules.

[Effects of canine IL-2 and IL-7 genes on enhancing immunogenicity of canine parvovirus VP2 gene vaccine in mice].

PubMed

Chen, Huihui; Zhong, Fei; Li, Xiujin; Wang, Lu; Sun, Yan; Neng, Changai; Zhang, Kao; Li, Wenyan; Wen, Jiexia

2012-11-04

To investigate the effects of canine interleukin-2 (cIL-2) and cIL-7 genes on enhancing the immunogenicity of canine parvovirus (CPV) VP2 DNA vaccine. The bicistronic vectors of cIL-2 and cIL-7 genes were constructed using the eukaryotic expression vector containing internal ribosome entry site (IRES). The cIL-2/ cIL-7 dicistronic vector plus previously constructed vectors, including CPV VP2 DNA vaccine vector, cIL-2 vector and cIL-7 vector, were used to co-immunize mice with different combinations, consisting of VP2 alone, VP2 + cIL-2, VP2 + cIL-7 and VP2 + cIL-2/cIL-7. The VP2-specific antibody levels in immunized mice were measured by ELISA at different time post-immunization. The proliferation indices and interferon-gamma expression were measured by lymphocyte proliferation assay and ELISA, respectively. The cIL-2/cIL-7 bicistronic vector was correct and could mediate cIL-2 and cIL-7 gene expression in eukaryotic cells. Immunization results revealed that the antibody titers and the neutralizing antibody levels of the mice co-immunized with VP2 + cIL-7/cIL-2 vectors were significantly higher than that with either VP2 + cIL-2 vectors or VP2 + cIL-7 vectors (P < 0.05). The lymphocyte proliferation indices of VP2 + cIL-7/cIL-2 vector-immunized mice were also higher than that of other two groups although not statistically significant. However, the IFN-gamma expression levels of VP2 + cIL-7/cIL-2 vector-immunized mice were significantly higher than other immunized mice (P < 0.05). The cIL-2 and cIL-7 genes showed the significant synergic effects on enhancing the immunogenecity of CPV VP2 DNA vaccine.

Chromosomal integration of adenoviral vector DNA in vivo.

PubMed

Stephen, Sam Laurel; Montini, Eugenio; Sivanandam, Vijayshankar Ganesh; Al-Dhalimy, Muhseen; Kestler, Hans A; Finegold, Milton; Grompe, Markus; Kochanek, Stefan

2010-10-01

So far there has been no report of any clinical or preclinical evidence for chromosomal vector integration following adenovirus (Ad) vector-mediated gene transfer in vivo. We used liver gene transfer with high-capacity Ad vectors in the FAH(Deltaexon5) mouse model to analyze homologous and heterologous recombination events between vector and chromosomal DNA. Intravenous injection of Ad vectors either expressing a fumarylacetoacetate hydrolase (FAH) cDNA or carrying part of the FAH genomic locus resulted in liver nodules of FAH-expressing hepatocytes, demonstrating chromosomal vector integration. Analysis of junctions between vector and chromosomal DNA following heterologous recombination indicated integration of the vector genome through its termini. Heterologous recombination occurred with a median frequency of 6.72 x 10(-5) per transduced hepatocyte, while homologous recombination occurred more rarely with a median frequency of 3.88 x 10(-7). This study has established quantitative and qualitative data on recombination of adenoviral vector DNA with genomic DNA in vivo, contributing to a risk-benefit assessment of the biosafety of Ad vector-mediated gene transfer.

Vectors for co-expression of an unrestricted number of proteins

PubMed Central

Scheich, Christoph; Kümmel, Daniel; Soumailakakis, Dimitri; Heinemann, Udo; Büssow, Konrad

2007-01-01

A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five ‘pQLink’ vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells. PMID:17311810

Intramuscular injection of AAV8 in mice and macaques is associated with substantial hepatic targeting and transgene expression.

PubMed

Greig, Jenny A; Peng, Hui; Ohlstein, Jason; Medina-Jaszek, C Angelica; Ahonkhai, Omua; Mentzinger, Anne; Grant, Rebecca L; Roy, Soumitra; Chen, Shu-Jen; Bell, Peter; Tretiakova, Anna P; Wilson, James M

2014-01-01

Intramuscular (IM) administration of adeno-associated viral (AAV) vectors has entered the early stages of clinical development with some success, including the first approved gene therapy product in the West called Glybera. In preparation for broader clinical development of IM AAV vector gene therapy, we conducted detailed pre-clinical studies in mice and macaques evaluating aspects of delivery that could affect performance. We found that following IM administration of AAV8 vectors in mice, a portion of the vector reached the liver and hepatic gene expression contributed significantly to total expression of secreted transgenes. The contribution from liver could be controlled by altering injection volume and by the use of traditional (promoter) and non-traditional (tissue-specific microRNA target sites) expression control elements. Hepatic distribution of vector following IM injection was also noted in rhesus macaques. These pre-clinical data on AAV delivery should inform safe and efficient development of future AAV products.

MicroRNA-Regulated Non-Viral Vectors with Improved Tumor Specificity in an Orthotopic Rat Model of Hepatocellular Carcinoma

PubMed Central

Ronald, John A.; Katzenberg, Regina; Nielsen, Carsten H.; Jae, Hwan Jun; Hofmann, Lawrence V.; Gambhir, Sanjiv S.

2013-01-01

In hepatocellular carcinoma, tumor specificity of gene therapy is of utmost importance to preserve liver function. MicroRNAs are powerful negative regulators of gene expression and many are down-regulated in human HCC. We identified seven miRNAs that are also down-regulated in tumors in a rat hepatoma model (p<0.05) and attempted to improve tumor specificity by constructing a panel of luciferase-expressing vectors containing binding sites for these microRNAs. Attenuation of luciferase expression by the corresponding microRNAs was confirmed across various cell lines and in mouse liver. We then tested our vectors in tumor-bearing rats and identified two microRNAs, miR-26a and miR-122, that significantly decreased expression in liver compared to control vector (6.40% and 0.26%, respectively; p<0.05). In tumor, miR-122 had a non-significant trend towards decreased (~50%) expression , while miR-26 had no significant effect on tumor expression. To our knowledge this is the first work using differentially expressed microRNAs to de-target transgene expression in an orthotopic hepatoma model and identification of miR-26a in addition to miR-122 for de-targeting liver. Considering the heterogeneity of microRNA expression in human HCC, this information will be important in guiding development of more personalized vectors for the treatment of this devastating disease. PMID:23719066

Generation of HIV-1 based bi-cistronic lentiviral vectors for stable gene expression and live cell imaging.

PubMed

Sehgal, Lalit; Budnar, Srikanth; Bhatt, Khyati; Sansare, Sneha; Mukhopadhaya, Amitabha; Kalraiya, Rajiv D; Dalal, Sorab N

2012-10-01

The study of protein-protein interactions, protein localization, protein organization into higher order structures and organelle dynamics in live cells, has greatly enhanced the understanding of various cellular processes. Live cell imaging experiments employ plasmid or viral vectors to express the protein/proteins of interest fused to a fluorescent protein. Unlike plasmid vectors, lentiviral vectors can be introduced into both dividing and non dividing cells, can be pseudotyped to infect a broad or narrow range of cells, and can be used to generate transgenic animals. However, the currently available lentiviral vectors are limited by the choice of fluorescent protein tag, choice of restriction enzyme sites in the Multiple Cloning Sites (MCS) and promoter choice for gene expression. In this report, HIV-1 based bi-cistronic lentiviral vectors have been generated that drive the expression of multiple fluorescent tags (EGFP, mCherry, ECFP, EYFP and dsRed), using two different promoters. The presence of a unique MCS with multiple restriction sites allows the generation of fusion proteins with the fluorescent tag of choice, allowing analysis of multiple fusion proteins in live cell imaging experiments. These novel lentiviral vectors are improved delivery vehicles for gene transfer applications and are important tools for live cell imaging in vivo.

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

PubMed

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells.

Undetectable Transcription of cap in a Clinical AAV Vector: Implications for Preformed Capsid in Immune Responses

PubMed Central

Hauck, Bernd; Murphy, Samuel L; Smith, Peter H; Qu, Guang; Liu, Xingge; Zelenaia, Olga; Mingozzi, Federico; Sommer, Jürg M; High, Katherine A; Wright, J. Fraser

2008-01-01

In a gene therapy clinical trial for hemophilia B, adeno-associated virus 2 (AAV2) capsid–specific CD8+ T cells were previously implicated in the elimination of vector-transduced hepatocytes, resulting in loss of human factor IX (hFIX) transgene expression. To test the hypothesis that expression of AAV2 cap DNA impurities in the AAV2-hFIX vector was the source of epitopes presented on transduced cells, transcription of cap was assessed by quantitative reverse transcription–PCR (Q-RT-PCR) following transduction of target cells with the vector used in the clinical trial. Transcriptional profiling was also performed for residual AmpR, and adenovirus E2A and E4. Although trace amounts of DNA impurities were present in the clinical vector, transcription of these sequences was not detected after transduction of human hepatocytes, nor in mice administered a dose 26-fold above the highest dose administered in the clinical study. Two methods used to minimize encapsidated DNA impurities in the clinical vector were: (i) a vector (cis) production plasmid with a backbone exceeding the packaging limit of AAV; and (ii) a vector purification step that achieved separation of the vector from vector-related impurities (e.g., empty capsids). In conclusion, residual cap expression was undetectable following transduction with AAV2-hFIX clinical vectors. Preformed capsid protein is implicated as the source of epitopes recognized by CD8+ T cells that eliminated vector-transduced cells in the clinical study. PMID:18941440

Ebola Virus Glycoprotein Induces an Innate Immune Response In vivo via TLR4

PubMed Central

Lai, Chih-Yun; Strange, Daniel P.; Wong, Teri Ann S.; Lehrer, Axel T.; Verma, Saguna

2017-01-01

Ebola virus (EBOV), a member of the Filoviridae family, causes the most severe form of viral hemorrhagic fever. Although no FDA licensed vaccine or treatment against Ebola virus disease (EVD) is currently available, Ebola virus glycoprotein (GP) is the major antigen used in all candidate Ebola vaccines. Recent reports of protection as quickly as within 6 days of administration of the rVSV-based vaccine expressing EBOV GP before robust humoral responses were generated suggests that the innate immune responses elicited early after vaccination may contribute to the protection. However, the innate immune responses induced by EBOV GP in the absence of viral vectors or adjuvants have not been fully characterized in vivo. Our recent studies demonstrated that immunization with highly purified recombinant GP in the absence of adjuvants induced a robust IgG response and partial protection against EBOV infection suggesting that GP alone can induce protective immunity. In this study we investigated the early immune response to purified EBOV GP alone in vitro and in vivo. We show that GP was efficiently internalized by antigen presenting cells and subsequently induced production of key inflammatory cytokines. In vivo, immunization of mice with EBOV GP triggered the production of key Th1 and Th2 innate immune cytokines and chemokines, which directly governed the recruitment of CD11b+ macrophages and CD11c+ dendritic cells to the draining lymph nodes (DLNs). Pre-treatment of mice with a TLR4 antagonist inhibited GP-induced cytokine production and recruitment of immune cells to the DLN. EBOV GP also upregulated the expression of costimulatory molecules in bone marrow derived macrophages suggesting its ability to enhance APC stimulatory capacity, which is critical for the induction of effective antigen-specific adaptive immunity. Collectively, these results provide the first in vivo evidence that early innate immune responses to EBOV GP are mediated via the TLR4 pathway and are able to modulate the innate-adaptive interface. These mechanistic insights into the adjuvant-like property of EBOV GP may help to develop a better understanding of how optimal prophylactic efficacy of EBOV vaccines can be achieved as well as further explore the potential post-exposure use of vaccines to prevent filoviral disease. PMID:28861075

VEST: Abstract Vector Calculus Simplification in Mathematica

DOE Office of Scientific and Technical Information (OSTI.GOV)

J. Squire, J. Burby and H. Qin

2013-03-12

We present a new package, VEST (Vector Einstein Summation Tools), that performs abstract vector calculus computations in Mathematica. Through the use of index notation, VEST is able to reduce scalar and vector expressions of a very general type using a systematic canonicalization procedure. In addition, utilizing properties of the Levi-Civita symbol, the program can derive types of multi-term vector identities that are not recognized by canonicalization, subsequently applying these to simplify large expressions. In a companion paper [1], we employ VEST in the automation of the calculation of Lagrangians for the single particle guiding center system in plasma physics, amore » computation which illustrates its ability to handle very large expressions. VEST has been designed to be simple and intuitive to use, both for basic checking of work and more involved computations. __________________________________________________« less

VEST: Abstract vector calculus simplification in Mathematica

NASA Astrophysics Data System (ADS)

Squire, J.; Burby, J.; Qin, H.

2014-01-01

We present a new package, VEST (Vector Einstein Summation Tools), that performs abstract vector calculus computations in Mathematica. Through the use of index notation, VEST is able to reduce three-dimensional scalar and vector expressions of a very general type to a well defined standard form. In addition, utilizing properties of the Levi-Civita symbol, the program can derive types of multi-term vector identities that are not recognized by reduction, subsequently applying these to simplify large expressions. In a companion paper Burby et al. (2013) [12], we employ VEST in the automation of the calculation of high-order Lagrangians for the single particle guiding center system in plasma physics, a computation which illustrates its ability to handle very large expressions. VEST has been designed to be simple and intuitive to use, both for basic checking of work and more involved computations.

A gene cassette for adapting Escherichia coli strains as hosts for att-Int-mediated rearrangement and pL expression vectors.

PubMed

Balakrishnan, R; Bolten, B; Backman, K C

1994-01-28

A cassette of genes from bacteriophage lambda, when carried on a derivative of bacteriophage Mu, renders strains of Escherichia coli (and in principle other Mu-sensitive bacteria) capable of supporting lambda-based expression vectors, such as rearrangement vectors and pL vectors. The gene cassette contains a temperature-sensitive allele of the repressor gene, cIts857, and a shortened leftward operon comprising, oLpL, N, xis and int. Transfection and lysogenization of this cassette into various host bacteria is mediated by phage Mu functions. Examples of regulated expression of the gene encoding T4 DNA ligase are presented.

Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells.

PubMed

Langouet-Astrie, Christophe J; Yang, Zhiyong; Polisetti, Sraavya M; Welsbie, Derek S; Hauswirth, William W; Zack, Donald J; Merbs, Shannath L; Enke, Raymond A

2016-10-01

Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations.

PubMed

Alves, João Nuno; Muir, Elizabeth M; Andrews, Melissa R; Ward, Anneliese; Michelmore, Nicholas; Dasgupta, Debayan; Verhaagen, Joost; Moloney, Elizabeth B; Keynes, Roger J; Fawcett, James W; Rogers, John H

2014-04-30

As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression of the enzyme. A similar vector for expression of green fluorescent protein (GFP) was injected into the same location. For each vector, two versions with minor differences were used, giving similar results. After 4 weeks, the brains were stained to show GFP and products of chondroitinase digestion. Chondroitinase was widely expressed, and the AAV-ChABC and AAV-GFP vectors gave similar expression patterns in many respects, consistent with the known projections from the directly transduced neurons in vibrissal motor cortex and adjacent cingulate cortex. In addition, diffusion of vector to deeper neuronal populations led to labelling of remote projection fields which was much more extensive with AAV-ChABC than with AAV-GFP. The most notable of these populations are inferred to be neurons of cortical layer 6, projecting widely in the thalamus, and neurons of the anterior pole of the hippocampus, projecting through most of the hippocampus. We conclude that, whereas GFP does not label the thinnest axonal branches of some neuronal types, chondroitinase is efficiently secreted from these arborisations and enables their extent to be sensitively visualised. After 12 weeks, chondroitinase expression was undiminished. Copyright © 2014 Elsevier B.V. All rights reserved.

Vector design for liver specific expression of multiple interfering RNAs that target hepatitis B virus transcripts

PubMed Central

Snyder, Lindsey L.; Esser, Jonathan M.; Pachuk, Catherine J.; Steel, Laura F.

2008-01-01

RNA interference (RNAi) is a process that can target intracellular RNAs for degradation in a highly sequence specific manner, making it a powerful tool that is being pursued in both research and therapeutic applications. Hepatitis B virus (HBV) is a serious public health problem in need of better treatment options, and aspects of its life cycle make it an excellent target for RNAi-based therapeutics. We have designed a vector that expresses interfering RNAs that target HBV transcripts, including both viral RNA replicative intermediates and mRNAs encoding viral proteins. Our vector design incorporates many features of endogenous microRNA (miRNA) gene organization that are proving useful for the development of reagents for RNAi. In particular, our vector contains an RNA pol II driven gene cassette that leads to tissue specific expression and efficient processing of multiple interfering RNAs from a single transcript, without the co-expression of any protein product. This vector shows potent silencing of HBV targets in cell culture models of HBV infection. The vector design will be applicable to silencing of additional cellular or disease-related genes. PMID:18499277

A novel packaging system for the generation of helper-free oncolytic MVM vector stocks.

PubMed

Brandenburger, A; Russell, S

1996-10-01

MVM-based autonomous parvoviral vectors have been shown to target the expression of heterologous genes in neoplastic cells and are therefore of interest for cancer gene therapy. The traditional method for production of parvoviral vectors requires the cotransfection of vector and helper plasmids into MVM-permissive cell lines, but recombination between the cotransfected plasmids invariably gives rise to vector stocks that are heavily contaminated with wild-type MVM. Therefore, to minimise recombination between the vector and helper genomes we have utilised a cell line in which the MVM helper functions are expressed inducibly from a modified MVM genome that is stably integrated into the host cell chromosome. Using this MVM packaging cell line, we could reproducibly generate MVM vector stocks that contained no detectable helper virus.

Live attenuated rubella vectors expressing SIV and HIV vaccine antigens replicate and elicit durable immune responses in rhesus macaques

PubMed Central

2013-01-01

Background Live attenuated viruses are among our most potent and effective vaccines. For human immunodeficiency virus, however, a live attenuated strain could present substantial safety concerns. We have used the live attenuated rubella vaccine strain RA27/3 as a vector to express SIV and HIV vaccine antigens because its safety and immunogenicity have been demonstrated in millions of children. One dose protects for life against rubella infection. In previous studies, rubella vectors replicated to high titers in cell culture while stably expressing SIV and HIV antigens. Their viability in vivo, however, as well as immunogenicity and antibody persistence, were unknown. Results This paper reports the first successful trial of rubella vectors in rhesus macaques, in combination with DNA vaccines in a prime and boost strategy. The vectors grew robustly in vivo, and the protein inserts were highly immunogenic. Antibody titers elicited by the SIV Gag vector were greater than or equal to those elicited by natural SIV infection. The antibodies were long lasting, and they were boosted by a second dose of replication-competent rubella vectors given six months later, indicating the induction of memory B cells. Conclusions Rubella vectors can serve as a vaccine platform for safe delivery and expression of SIV and HIV antigens. By presenting these antigens in the context of an acute infection, at a high level and for a prolonged duration, these vectors can stimulate a strong and persistent immune response, including maturation of memory B cells. Rhesus macaques will provide an ideal animal model for demonstrating immunogenicity of novel vectors and protection against SIV or SHIV challenge. PMID:24041113

A limited innate immune response is induced by a replication-defective herpes simplex virus vector following delivery to the murine central nervous system

PubMed Central

Zeier, Zane; Aguilar, J Santiago; Lopez, Cecilia M; Devi-Rao, G B; Watson, Zachary L; Baker, Henry V; Wagner, Edward K; Bloom, David C

2010-01-01

Herpes simplex virus type 1 (HSV-1)–based vectors readily transduce neurons and have a large payload capacity, making them particularly amenable to gene therapy applications within the central nervous system (CNS). Because aspects of the host responses to HSV-1 vectors in the CNS are largely unknown, we compared the host response of a nonreplicating HSV-1 vector to that of a replication-competent HSV-1 virus using microarray analysis. In parallel, HSV-1 gene expression was tracked using HSV-specific oligonucleotide-based arrays in order to correlate viral gene expression with observed changes in host response. Microarray analysis was performed following stereotactic injection into the right hippocampal formation of mice with either a replication-competent HSV-1 or a nonreplicating recombinant of HSV-1, lacking the ICP4 gene (ICP4−). Genes that demonstrated a significant change (P < .001) in expression in response to the replicating HSV-1 outnumbered those that changed in response to mock or nonreplicating vector by approximately 3-fold. Pathway analysis revealed that both the replicating and nonreplicating vectors induced robust antigen presentation but only mild interferon, chemokine, and cytokine signaling responses. The ICP4− vector was restricted in several of the Toll-like receptor-signaling pathways, indicating reduced stimulation of the innate immune response. These array analyses suggest that although the nonreplicating vector induces detectable activation of immune response pathways, the number and magnitude of the induced response is dramatically restricted compared to the replicating vector, and with the exception of antigen presentation, host gene expression induced by the non-replicating vector largely resembles mock infection. PMID:20095947

Insulated hsp70B' promoter: stringent heat-inducible activity in replication-deficient, but not replication-competent adenoviruses.

PubMed

Rohmer, Stanimira; Mainka, Astrid; Knippertz, Ilka; Hesse, Andrea; Nettelbeck, Dirk M

2008-04-01

Key to the realization of gene therapy is the development of efficient and targeted gene transfer vectors. Therapeutic gene transfer by replication-deficient or more recently by conditionally replication-competent/oncolytic adenoviruses has shown much promise. For specific applications, however, it will be advantageous to provide vectors that allow for external control of gene expression. The efficient cellular heat shock system in combination with available technology for focused and controlled hyperthermia suggests heat-regulated transcription control as a promising tool for this purpose. We investigated the feasibility of a short fragment of the human hsp70B' promoter, with and without upstream insulator elements, for the regulation of transgene expression by replication-deficient or oncolytic adenoviruses. Two novel adenoviral vectors with an insulated hsp70B' promoter were developed and showed stringent heat-inducible gene expression with induction ratios up to 8000-fold. In contrast, regulation of gene expression from the hsp70B' promoter without insulation was suboptimal. In replication-competent/oncolytic adenoviruses regulation of the hsp70B' promoter was lost specifically during late replication in permissive cells and could not be restored by the insulators. We developed novel adenovirus gene transfer vectors that feature improved and stringent regulation of transgene expression from the hsp70B' promoter using promoter insulation. These vectors have potential for gene therapy applications that benefit from external modulation of therapeutic gene expression or for combination therapy with hyperthermia. Furthermore, our study reveals that vector replication can deregulate inserted cellular promoters, an observation which is of relevance for the development of replication-competent/oncolytic gene transfer vectors. (c) 2008 John Wiley & Sons, Ltd.

Hypoxia-inducible bidirectional shRNA expression vector delivery using PEI/chitosan-TBA copolymers for colorectal Cancer gene therapy.

PubMed

Javan, Bita; Atyabi, Fatemeh; Shahbazi, Majid

2018-06-01

This investigation was conducted to construct a hypoxia/colorectal dual-specific bidirectional short hairpin RNA (shRNA) expression vector and to transfect it into the colon cancer cell line HT-29 with PEI/chitosan-TBA nanoparticles for the simultaneous knock down of β-catenin and Bcl-2 under hypoxia. To construct a pRNA-bipHRE-CEA vector, the carcinoma embryonic antigen (CEA) promoter designed in two directions and the vascular endothelial growth factor (VEGF) enhancer were inserted between two promoters for hypoxic cancer specific gene expression. To confirm the therapeutic effect of the dual-specific vector, β-catenin and Bcl-2 shRNAs were inserted downstream of each promoter. The physicochemical properties, the cytotoxicity, and the transfection efficiency of these PEI/chitosan-TBA nanoparticles were investigated. In addition, the antitumor effects of the designed vector on the expression of β-catenin and Bcl-2, cell cycle distribution, and apoptosis were investigated in vitro. The silencing effect of the hypoxia-response shRNA expression vector was relatively low (18%-25%) under normoxia, whereas it was significantly increased to approximately 50%-60% in the HT-29 cell line. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis due to gene silencing under hypoxia. Furthermore, MTS assay, fluorescence microscopy images, and flow cytometry analyses confirmed that the PEI/chitosan-TBA blend system provided effective transfection with low cytotoxicity. This novel hypoxia-responsive shRNA expression vector may be useful for RNA interference (RNAi)-based cancer gene therapy in hypoxic colorectal tumors. Moreover, the PEI/chitosan-TBA copolymer might be a promising gene carrier for use in gene transfer in vivo. Copyright © 2018. Published by Elsevier Inc.

Use and comparison of different internal ribosomal entry sites (IRES) in tricistronic retroviral vectors

PubMed Central

Douin, Victorine; Bornes, Stephanie; Creancier, Laurent; Rochaix, Philippe; Favre, Gilles; Prats, Anne-Catherine; Couderc, Bettina

2004-01-01

Background Polycistronic retroviral vectors that contain several therapeutic genes linked via internal ribosome entry sites (IRES), provide new and effective tools for the co-expression of exogenous cDNAs in clinical gene therapy protocols. For example, tricistronic retroviral vectors could be used to genetically modify antigen presenting cells, enabling them to express different co-stimulatory molecules known to enhance tumor cell immunogenicity. Results We have constructed and compared different retroviral vectors containing two co-stimulatory molecules (CD70, CD80) and selectable marker genes linked to different IRES sequences (IRES from EMCV, c-myc, FGF-2 and HTLV-1). The tricistronic recombinant amphotropic viruses containing the IRES from EMCV, FGF-2 or HTLV-1 were equally efficient in inducing the expression of an exogenous gene in the transduced murine or human cells, without displaying any cell type specificity. The simultaneous presence of several IRESes on the same mRNA, however, can induce the differential expression of the various cistrons. Here we show that the IRESes of HTLV-1 and EMCV interfere with the translation induced by other IRESes in mouse melanoma cells. The IRES from FGF-2 did however induce the expression of exogenous cDNA in human melanoma cells without any positive or negative regulation from the other IRESs present within the vectors. Tumor cells that were genetically modified with the tricistronic retroviral vectors, were able to induce an in vivo anti-tumor immune response in murine models. Conclusion Translation of the exogenous gene is directed by the IRES and its high level of expression not only depends on the type of cell that is transduced but also on the presence of other genetic elements within the vector. PMID:15279677

A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea

PubMed Central

Ford, Kathryn L.; Baumgartner, Kendra; Henricot, Béatrice; Bailey, Andy M.; Foster, Gary D.

2016-01-01

Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide. Using a yeast-adapted pCAMBIA0380 Agrobacterium vector, we have constructed a series of vectors for transformation of A. mellea, assembled using yeast-based recombination methods. These have been designed to allow easy exchange of promoters and inclusion of introns. The vectors were first tested by transformation into basidiomycete Clitopilus passeckerianus to ascertain vector functionality then used to transform A. mellea. We show that heterologous promoters from the basidiomycetes Agaricus bisporus and Phanerochaete chrysosporium that were used successfully to control the hygromycin resistance cassette were not able to support expression of mRFP or GFP in A. mellea. The endogenous A. mellea gpd promoter delivered efficient expression, and we show that inclusion of an intron was also required for transgene expression. GFP and mRFP expression was stable in mycelia and fluorescence was visible in transgenic fruiting bodies and GFP was detectable in planta. Use of these vectors has been successful in giving expression of the fluorescent proteins GFP and mRFP in A. mellea, providing an additional molecular tool for this pathogen. PMID:27384974

Hypoxia/hepatoma dual specific suicide gene expression plasmid delivery using bio-reducible polymer for hepatocellular carcinoma therapy.

PubMed

Kim, Hyun Ah; Nam, Kihoon; Lee, Minhyung; Kim, Sung Wan

2013-10-10

Gene therapy is suggested as a promising alternative strategy of hepatocellular carcinoma (HCC, also called hepatoma) therapy. To achieve a successful and safe gene therapy, tight regulation of gene expression is required to minimize side-effects in normal tissues. In this study, we developed a novel hypoxia and hepatoma dual specific gene expression vector. The constructed vectors were transfected into various cell lines using bio-reducible polymer, PAM-ABP. First, pAFPS-Luc or pAFPL-Luc vector was constructed with the alpha-fectoprotein (AFP) promoter and enhancer for hepatoma tissue specific gene expression. Then, pEpo-AFPL-Luc was constructed by insertion of the erythropoietin (Epo) enhancer for hypoxic cancer specific gene expression. In vitro transfection assay showed that pEpo-AFPL-Luc transfected hepatoma cell increased gene expression under hypoxic condition. To confirm the therapeutic effect of dual specific vector, herpes simplex virus thymidine kinase (HSV-TK) gene was introduced for cancer cell killing. The pEpo-AFPL-TK was transfected into hepatoma cell lines in the presence of ganciclovir (GCV) pro-drug. Caspase-3/7, MTT and TUNEL assays elucidated that pEpo-AFPL-TK transfected cells showed significant increasing of death rate in hypoxic hepatoma cells compared to controls. Therefore, the hypoxia/hepatoma dual specific gene expression vector with the Epo enhancer and AFP promoter may be useful for hepatoma specific gene therapy. © 2013.

Helper-dependent adenovirus achieve more efficient and persistent liver transgene expression in non-human primates under immunosuppression.

PubMed

Unzu, C; Melero, I; Hervás-Stubbs, S; Sampedro, A; Mancheño, U; Morales-Kastresana, A; Serrano-Mendioroz, I; de Salamanca, R E; Benito, A; Fontanellas, A

2015-11-01

Helper-dependent adenoviral (HDA) vectors constitute excellent gene therapy tools for metabolic liver diseases. We have previously shown that an HDA vector encoding human porphobilinogen deaminase (PBGD) corrects acute intermittent porphyria mice. Now, six non-human primates were injected in the left hepatic lobe with the PBGD-encoding HDA vector to study levels and persistence of transgene expression. Intrahepatic administration of 5 × 10(12) viral particles kg(-1) (10(10) infective units kg(-1)) of HDA only resulted in transient (≈14 weeks) transgene expression in one out of three individuals. In contrast, a more prolonged 90-day immunosuppressive regimen (tacrolimus, mycophenolate, rituximab and steroids) extended meaningful transgene expression for over 76 weeks in two out of two cases. Transgene expression under immunosuppression (IS) reached maximum levels 6 weeks after HDA administration and gradually declined reaching a stable plateau within the therapeutic range for acute porphyria. The non-injected liver lobes also expressed the transgene because of vector circulation. IS controlled anticapsid T-cell responses and decreased the induction of neutralizing antibodies. Re-administration of HDA-hPBGD at week +78 achieved therapeutically meaningful transgene expression only in those animals receiving IS again at the time of this second vector exposure. Overall, immunity against adenoviral capsids poses serious hurdles for long-term HDA-mediated liver transduction, which can be partially circumvented by pharmacological IS.

Challenging assumptions of notational transparency: the case of vectors in engineering mathematics

NASA Astrophysics Data System (ADS)

Craig, Tracy S.

2017-11-01

The notation for vector analysis has a contentious nineteenth century history, with many different notations describing the same or similar concepts competing for use. While the twentieth century has seen a great deal of unification in vector analysis notation, variation still remains. In this paper, the two primary notations used for expressing the components of a vector are discussed in historical and current context. Popular mathematical texts use the two notations as if they are transparent and interchangeable. In this research project, engineering students' proficiency at vector analysis was assessed and the data were analyzed using the Rasch measurement method. Results indicate that the students found items expressed in unit vector notation more difficult than those expressed in parenthesis notation. The expert experience of notation as transparent and unproblematically symbolic of underlying processes independent of notation is shown to contrast with the student experience where the less familiar notation is experienced as harder to work with.

Virus-Derived Gene Expression and RNA Interference Vector for Grapevine

PubMed Central

Kurth, Elizabeth G.; Peremyslov, Valera V.; Prokhnevsky, Alexey I.; Kasschau, Kristin D.; Miller, Marilyn; Carrington, James C.

2012-01-01

The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests. PMID:22438553

Firewalls Prevent Systemic Dissemination of Vectors Derived from Human Adenovirus Type 5 and Suppress Production of Transgene-Encoded Antigen in a Murine Model of Oral Vaccination

PubMed Central

Revaud, Julien; Unterfinger, Yves; Rol, Nicolas; Suleman, Muhammad; Shaw, Julia; Galea, Sandra; Gavard, Françoise; Lacour, Sandrine A.; Coulpier, Muriel; Versillé, Nicolas; Havenga, Menzo; Klonjkowski, Bernard; Zanella, Gina; Biacchesi, Stéphane; Cordonnier, Nathalie; Corthésy, Blaise; Ben Arous, Juliette; Richardson, Jennifer P.

2018-01-01

To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches. PMID:29423380

Essential and Dispensable Virus-Encoded Replication Elements Revealed by Efforts To Develop Hypoviruses as Gene Expression Vectors

PubMed Central

Suzuki, Nobuhiro; Geletka, Lynn M.; Nuss, Donald L.

2000-01-01

We have investigated whether hypoviruses, viral agents responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica, could serve as gene expression vectors. The infectious cDNA clone of the prototypic hypovirus CHV1-EP713 was modified to generate 20 different vector candidates. Although transient expression was achieved for a subset of vectors that contained the green fluorescent protein gene from Aequorea victoria, long-term expression (past day 8) was not observed for any vector construct. Analysis of viral RNAs recovered from transfected fungal colonies revealed that the foreign genes were readily deleted from the replicating virus, although small portions of foreign sequences were retained by some vectors after months of replication. However, the results of vector viability and progeny characterization provided unexpected new insights into essential and dispensable elements of hypovirus replication. The N-terminal portion (codons 1 to 24) of the 5′-proximal open reading frame (ORF), ORF A, was found to be required for virus replication, while the remaining 598 codons of this ORF were completely dispensable. Substantial alterations were tolerated in the pentanucleotide UAAUG that contains the ORF A termination codon and the overlapping putative initiation codon of the second of the two hypovirus ORFs, ORF B. Replication competence was maintained following either a frameshift mutation that caused a two-codon extension of ORF A or a modification that produced a single-ORF genomic organization. These results are discussed in terms of determinants of hypovirus replication, the potential utility of hypoviruses as gene expression vectors, and possible mechanisms by which hypoviruses recognize and delete foreign sequences. PMID:10906211

Modified Newcastle disease virus vectors expressing the H5 hemagglutinin induce enhanced protection against highly pathogenic H5N1 avian influenza virus in chickens

PubMed Central

Kim, Shin-Hee; Paldurai, Anandan; Xiao, Sa; Collins, Peter L.; Samal, Siba K.

2016-01-01

Naturally-occurring attenuated strains of Newcastle disease virus (NDV) are being developed as vaccine vectors for use in poultry and humans. However, some NDV strains, such as Beaudette C (BC), may retain too much virulence in poultry for safe use, and more highly attenuated strains may be suboptimally immunogenic. We therefore modified the BC strain by changing the multibasic cleavage site sequence of the F protein to the dibasic sequence of avirulent strain LaSota. Additionally, the BC, F, and HN proteins were modified in several ways to enhance virus replication. These modified BC-derived vectors and the LaSota strain were engineered to express the hemagglutin (HA) protein of H5N1 highly pathogenic influenza virus (HPAIV). In general, the modified BC-based vectors expressing HA replicated better than LaSota/HA, and expressed higher levels of HA protein. Pathogenicity tests indicated that all the modified viruses were highly attenuated in chickens. Based on in vitro characterization, two of the modified BC vectors were chosen for evaluation in chickens as vaccine vectors against H5N1 HPAIV A/Vietnam/1203/04. Immunization of chickens with rNDV vector vaccines followed by challenge with HPAIV demonstrated high levels of protection against clinical disease and mortality. However, only those chickens immunized with modified BC/HA in which residues 271–330 from the F protein had been replaced with the corresponding sequence from the NDV AKO strain conferred complete protection against challenge virus shedding. Our findings suggest that this modified rNDV can be used safely as a vaccine vector with enhanced replication, expression, and protective efficacy in avian species, and potentially in humans. PMID:24968158

Spatial and phylogenetic analysis of the vesicular stomatitis virus epidemic in the southwestern United States in 2004-2006

USDA-ARS?s Scientific Manuscript database

The southwestern United States has been incidentally affected by vesicular stomatitis virus (VSV) epidemics during the last 100 years. By the time this manuscript was written, the last episodes were reported in 2004-2006. Results of space clustering and phylogenetic analysis techniques used here sug...

Oncolytic Virotherapy Targeting Lung Cancer Drug Resistance

DTIC Science & Technology

2014-10-01

to VSV oncolysis was also a theme of our recent publication [ Shulak L, Beljanski V, et al. J. Virol. 88(5):2927-40 (2014)] and was used as part of...neural progenitors in the injured adult spinal cord. J Neurosci 26(46):11948-60. 2006 10. Shulak L, et al. Histone deacetylase inhibitors potentiate

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

DTIC Science & Technology

2010-04-01

antitumoral, antivascular, and anti-HBV activities in patients with hepatocellular carcinoma . Mol T her, 2008. 16(9): p. 1637-42. 16. Ribacka, C . a nd...adenovirus targeting to TERT and RB pathway induced specific and potent anti-tumor efficacy in vitro and in vivo for hepatocellular carcinoma . Cancer

Evaluation of Sintering Behaviors of Saprolitic Nickeliferous Laterite Based on Quaternary Basicity

NASA Astrophysics Data System (ADS)

Luo, Jun; Li, Guanghui; Rao, Mingjun; Zhang, Yuanbo; Peng, Zhiwei; Zhi, Qian; Jiang, Tao

2015-09-01

The sintering behaviors of saprolitic nickeliferous laterite with various quaternary basicities [(CaO + MgO)/(SiO2 + Al2O3) mass ratio] in a reductive atmosphere are investigated by simulative sintering and validated by sintering pot tests. The simulative sintering results show that the generation of diopside (CaMgSi2O6) with low melting point is the key reason for the decrease in characteristic fusion temperatures when the quaternary basicity increases from 0.5 to 0.8-1.0. Continuous increase of basicity leads to transformation of diopside (CaMgSi2O6) into akermanite (Ca2MgSi2O7), which adversely increases the characteristic fusion temperatures. These findings are confirmed by the sinter pot tests, which demonstrate that the sintering indexes including vertical sintering velocity (VSV), yield ( Y), and productivity ( P), can be improved by optimizing quaternary basicity. At basicity of 1.0, the VSV, Y, P, and ISO tumbling index reach 49.2 mm/min, 80.5%, 1.0 t/(h m2), and 66.5%, respectively.

Molecular and cellular aspects of rhabdovirus entry.

PubMed

Albertini, Aurélie A V; Baquero, Eduard; Ferlin, Anna; Gaudin, Yves

2012-01-01

Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell.

Molecular and Cellular Aspects of Rhabdovirus Entry

PubMed Central

Albertini, Aurélie A. V.; Baquero, Eduard; Ferlin, Anna; Gaudin, Yves

2012-01-01

Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell. PMID:22355455

A CGMMV genome-replicon vector with partial sequences of coat protein gene efficiently expresses GFP in Nicotiana benthamiana.

PubMed

Jailani, A Abdul Kader; Solanki, Vikas; Roy, Anirban; Sivasudha, T; Mandal, Bikash

2017-04-02

A highly infectious clone of Cucumber green mottle mosaic virus (CGMMV), a cucurbit-infecting tobamovirus was utilized for designing of gene expression vectors. Two versions of vector were examined for their efficacy in expressing the green fluorescent protein (GFP) in Nicotiana benthamiana. When the GFP gene was inserted at the stop codon of coat protein (CP) gene of the CGMMV genome without any read-through codon, systemic expression of GFP, as well as virion formation and systemic symptoms expression were obtained in N. benthamiana. The qRT-PCR analysis showed 23 fold increase of GFP over actin at 10days post inoculation (dpi), which increased to 45 fold at 14dpi and thereafter the GFP expression was significantly declined. Further, we show that when the most of the CP sequence is deleted retaining only the first 105 nucleotides, the shortened vector containing GFP in frame of original CP open reading frame (ORF) resulted in 234 fold increase of GFP expression over actin at 5dpi in N. benthamiana without the formation of virions and disease symptoms. Our study demonstrated that a simple manipulation of CP gene in the CGMMV genome while preserving the translational frame of CP resulted in developing a virus-free, rapid and efficient foreign protein expression system in the plant. The CGMMV based vectors developed in this study may be potentially useful for the production of edible vaccines in cucurbits. Copyright © 2017 Elsevier B.V. All rights reserved.

A genetically adjuvanted influenza B virus vector increases immunogenicity and protective efficacy in mice.

PubMed

Kittel, Christian; Wressnigg, Nina; Shurygina, Anna Polina; Wolschek, Markus; Stukova, Marina; Romanovskaya-Romanko, Ekatherina; Romanova, Julia; Kiselev, Oleg; Muster, Thomas; Egorov, Andrej

2015-10-01

The existence of multiple antigenically distinct types and subtypes of influenza viruses allows the construction of a multivalent vector system for the mucosal delivery of foreign sequences. Influenza A viruses have been exploited successfully for the expression of extraneous antigens as well as immunostimulatory molecules. In this study, we describe the development of an influenza B virus vector whose functional part of the interferon antagonist NS1 was replaced by human interleukin 2 (IL2) as a genetic adjuvant. We demonstrate that IL2 expressed by this viral vector displays immune adjuvant activity in immunized mice. Animals vaccinated with the IL2 viral vector showed an increased hemagglutination inhibition antibody response and higher protective efficacy after challenge with a wild-type influenza B virus when compared to mice vaccinated with a control virus. Our results demonstrate that it is feasible to construct influenza B vaccine strains expressing immune-potentiating foreign sequences from the NS genomic segment. Based on these data, it is now hypothetically possible to create a trivalent (or quadrivalent) live attenuated influenza vaccine in which each component expresses a selected genetic adjuvant with tailored expression levels.

Cytomegalovirus vector expressing RAE-1γ induces enhanced anti-tumor capacity of murine CD8+ T cells.

PubMed

Tršan, Tihana; Vuković, Kristina; Filipović, Petra; Brizić, Ana Lesac; Lemmermann, Niels A W; Schober, Kilian; Busch, Dirk H; Britt, William J; Messerle, Martin; Krmpotić, Astrid; Jonjić, Stipan

2017-08-01

Designing CD8 + T-cell vaccines, which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show the robust potential of cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE-1γ as CD8 + T cell-based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE-1γ, delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD-1. CMV vector expressing RAE-1γ potentiated expansion of KLRG1 + CD8 + T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long-term maintenance of epitope-specific CD8 + T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8 + T-cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8 + T-cell sensitive tumors. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

PubMed Central

Joseph, Joan; Fernández-Lloris, Raquel; Pezzat, Elías; Saubi, Narcís; Cardona, Pere-Joan; Mothe, Beatriz; Gatell, Josep Maria

2010-01-01

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261) and Mycobacteria spp. α-antigen promoter (in plasmid pJH222). Among 14 rBCG:HIV-1gp120 (pMV261) colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222) colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors. PMID:20617151

Correction of Murine Sickle Cell Disease Using γ-Globin Lentiviral Vectors to Mediate High-level Expression of Fetal Hemoglobin

PubMed Central

Pestina, Tamara I; Hargrove, Phillip W; Jay, Dennis; Gray, John T; Boyd, Kelli M; Persons, Derek A

2008-01-01

Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed erythroid-specific, γ-globin lentiviral vectors for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing fetal hemoglobin (HbF) production in sickle red cells. We evaluated two different γ-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the γ-globin gene driven by 3.1 kb of β-globin regulatory sequences and a 130-bp β-globin promoter. The second vector, V5m3, was identical except that the γ-globin 3′-untranslated region (3′-UTR) was replaced with the β-globin 3′-UTR. Adult erythroid cells have β-globin mRNA 3′-UTR-binding proteins that enhance β-globin mRNA stability and we postulated this design might enhance γ-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human γ-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of γ-globin mRNA which was associated with high-level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to SCD by permanently enhancing HbF using a γ-globin lentiviral vector. PMID:19050697

Molecular design for recombinant adeno-associated virus (rAAV) vector production.

PubMed

Aponte-Ubillus, Juan Jose; Barajas, Daniel; Peltier, Joseph; Bardliving, Cameron; Shamlou, Parviz; Gold, Daniel

2018-02-01

Recombinant adeno-associated virus (rAAV) vectors are increasingly popular tools for gene therapy applications. Their non-pathogenic status, low inflammatory potential, availability of viral serotypes with different tissue tropisms, and prospective long-lasting gene expression are important attributes that make rAAVs safe and efficient therapeutic options. Over the last three decades, several groups have engineered recombinant AAV-producing platforms, yielding high titers of transducing vector particles. Current specific productivity yields from different platforms range from 10 3 to 10 5 vector genomes (vg) per cell, and there is an ongoing effort to improve vector yields in order to satisfy high product demands required for clinical trials and future commercialization.Crucial aspects of vector production include the molecular design of the rAAV-producing host cell line along with the design of AAV genes, promoters, and regulatory elements. Appropriately, configuring and balancing the expression of these elements not only contributes toward high productivity, it also improves process robustness and product quality. In this mini-review, the rational design of rAAV-producing expression systems is discussed, with special attention to molecular strategies that contribute to high-yielding, biomanufacturing-amenable rAAV production processes. Details on molecular optimization from four rAAV expression systems are covered: adenovirus, herpesvirus, and baculovirus complementation systems, as well as a recently explored yeast expression system.

Tombusvirus-based vector systems to permit over-expression of genes or that serve as sensors of antiviral RNA silencing in plants.

PubMed

Shamekova, Malika; Mendoza, Maria R; Hsieh, Yi-Cheng; Lindbo, John; Omarov, Rustem T; Scholthof, Herman B

2014-03-01

A next generation Tomato bushy stunt virus (TBSV) coat protein gene replacement vector system is described that can be applied by either RNA inoculation or through agroinfiltration. A vector expressing GFP rapidly yields high levels of transient gene expression in inoculated leaves of various plant species, as illustrated for Nicotiana benthamiana, cowpea, tomato, pepper, and lettuce. A start-codon mutation to down-regulate the dose of the P19 silencing suppressor reduces GFP accumulation, whereas mutations that result in undetectable levels of P19 trigger rapid silencing of GFP. Compared to existing virus vectors the TBSV system has a unique combination of a very broad host range, rapid and high levels of replication and gene expression, and the ability to regulate its suppressor. These features are attractive for quick transient assays in numerous plant species for over-expression of genes of interest, or as a sensor to monitor the efficacy of antiviral RNA silencing. Copyright © 2014. Published by Elsevier Inc.

Protection against California 2002 NDV strain afforded by adenovirus vectored vaccine expressing Fusion or Hemagglutination-neuraminidase genes

USDA-ARS?s Scientific Manuscript database

Vectored vaccines expressing the combination of the hemagglutinin-neuraminidase (HN) and fusion (F) genes generally have better clinical protection against Newcastle disease virus (NDV) than when either the F and HN genes are expressed alone. Interestingly, the protection induced by F is usually bet...

Development of a GFP expression vector for Cucurbit chlorotic yellows virus.

PubMed

Wei, Ying; Han, Xiaoyu; Wang, Zhenyue; Gu, Qinsheng; Li, Honglian; Chen, Linlin; Sun, Bingjian; Shi, Yan

2018-05-24

Cucurbit chlorotic yellows virus (CCYV), a bipartite crinivirus, causes chlorotic leaf spots and yellowing symptoms on cucurbit leaves. We previously developed an infectious clone of CCYV. Limited work has been conducted on the construction of a crinivirus green fluorescence protein (GFP) expression vector to date. We constructed a CCYV GFP expression vector using the "add a gene" strategy based on CCYV RNA2 cDNA constrcut. Three resultant clones, pCCYVGFP SGC , pCCYVGFP CGC , and pCCYVGFP CGS, were constructed with different promoters used to initiate GFP and CP expression. At 25 dpi GFP fluorescence was detectable not only in leaf veins but also in the surrounding cells. pCCYVGFP CGC -infected cucumber leaves exhibited cell spread at 25 dpi, whereas pCCYVGFP SGC and pCCYVGFP CGS were mainly found in single cells. Further observation of pCCYVGFP CGC GFP expression at 30 dpi, 40 dpi, and 50 dpi showed phloem-limited localization in the systemic leaves. We developed of a CCYV GFP expression vector that will be useful for further study of CCYV movement in cucurbits.

Ring vaccination with rVSV-ZEBOV under expanded access in response to an outbreak of Ebola virus disease in Guinea, 2016: an operational and vaccine safety report.

PubMed

Gsell, Pierre-Stéphane; Camacho, Anton; Kucharski, Adam J; Watson, Conall H; Bagayoko, Aminata; Nadlaou, Séverine Danmadji; Dean, Natalie E; Diallo, Abdourahamane; Diallo, Abdourahmane; Honora, Djidonou A; Doumbia, Moussa; Enwere, Godwin; Higgs, Elizabeth S; Mauget, Thomas; Mory, Diakite; Riveros, Ximena; Oumar, Fofana Thierno; Fallah, Mosoka; Toure, Alhassane; Vicari, Andrea S; Longini, Ira M; Edmunds, W J; Henao-Restrepo, Ana Maria; Kieny, Marie Paule; Kéïta, Sakoba

2017-12-01

In March, 2016, a flare-up of Ebola virus disease was reported in Guinea, and in response ring vaccination with the unlicensed rVSV-ZEBOV vaccine was introduced under expanded access, the first time that an Ebola vaccine has been used in an outbreak setting outside a clinical trial. Here we describe the safety of rVSV-ZEBOV candidate vaccine and operational feasibility of ring vaccination as a reactive strategy in a resource-limited rural setting. Approval for expanded access and compassionate use was rapidly sought and obtained from relevant authorities. Vaccination teams and frozen vaccine were flown to the outbreak settings. Rings of contacts and contacts of contacts were defined and eligible individuals, who had given informed consent, were vaccinated and followed up for 21 days under good clinical practice conditions. Between March 17 and April 21, 2016, 1510 individuals were vaccinated in four rings in Guinea, including 303 individuals aged between 6 years and 17 years and 307 front-line workers. It took 10 days to vaccinate the first participant following the confirmation of the first case of Ebola virus disease. No secondary cases of Ebola virus disease occurred among the vaccinees. Adverse events following vaccination were reported in 47 (17%) 6-17 year olds (all mild) and 412 (36%) adults (individuals older than 18 years; 98% were mild). Children reported fewer arthralgia events than adults (one [<1%] of 303 children vs 81 [7%] of 1207 adults). No severe vaccine-related adverse events were reported. The results show that a ring vaccination strategy can be rapidly and safely implemented at scale in response to Ebola virus disease outbreaks in rural settings. WHO, Gavi, and the World Food Programme. Copyright © 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.

SU-E-T-02: 90Y Microspheres Dosimetry Calculation with Voxel-S-Value Method: A Simple Use in the Clinic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maneru, F; Gracia, M; Gallardo, N

2015-06-15

Purpose: To present a simple and feasible method of voxel-S-value (VSV) dosimetry calculation for daily clinical use in radioembolization (RE) with {sup 90}Y microspheres. Dose distributions are obtained and visualized over CT images. Methods: Spatial dose distributions and dose in liver and tumor are calculated for RE patients treated with Sirtex Medical miscrospheres at our center. Data obtained from the previous simulation of treatment were the basis for calculations: Tc-99m maggregated albumin SPECT-CT study in a gammacamera (Infinia, General Electric Healthcare.). Attenuation correction and ordered-subsets expectation maximization (OSEM) algorithm were applied.For VSV calculations, both SPECT and CT were exported frommore » the gammacamera workstation and registered with the radiotherapy treatment planning system (Eclipse, Varian Medical systems). Convolution of activity matrix and local dose deposition kernel (S values) was implemented with an in-house developed software based on Python code. The kernel was downloaded from www.medphys.it. Final dose distribution was evaluated with the free software Dicompyler. Results: Liver mean dose is consistent with Partition method calculations (accepted as a good standard). Tumor dose has not been evaluated due to the high dependence on its contouring. Small lesion size, hot spots in health tissue and blurred limits can affect a lot the dose distribution in tumors. Extra work includes: export and import of images and other dicom files, create and calculate a dummy plan of external radiotherapy, convolution calculation and evaluation of the dose distribution with dicompyler. Total time spent is less than 2 hours. Conclusion: VSV calculations do not require any extra appointment or any uncomfortable process for patient. The total process is short enough to carry it out the same day of simulation and to contribute to prescription decisions prior to treatment. Three-dimensional dose knowledge provides much more information than other methods of dose calculation usually applied in the clinic.« less

On mantle heterogeneity and anisotropy as mapped by inversion of global surface wave data

NASA Astrophysics Data System (ADS)

Khan, A.; Boschi, L.; Connolly, J.; Deschamps, F.

2008-12-01

We jointly invert Love and Rayleigh wave dispersion curves for the Earth's mantle composition, thermal state, P and S wave anisotropy at different locations on the Earth, based on self-consistent thermodynamic calculations. The method consists of four parts: 1. The composition of the Earth is modeled by the chemical system CaO-FeO-MgO- Al2O3-SiO2. Given these parameters and a geotherm (also an unknown), we calculate stable mineral modes, elastic properties, bulk density at the prevailing physical conditions using Gibbs free energy minimisation. Voigt-Reuss-Hill averaging is subsequently emplouyed to compute radial isotropic P and S wave velocity profiles in the elastic limit. 2. Anisotropic P and S wave velocities are determined from the isotropic ones by employing the relations ξ=(Vsh/Vsv)2, φ = (Vpv/Vph)2, η=F/(2A-L), Vs=(2Vsv2+Vsh2)/3 and Vp=(Vpv2+4Vph2)/5. The former three parameters are the standard anisotropy parameters, that we also invert for. 4. From these radial profiles, i.e. of Vsv, Vsh, Vph, Vpv and ρ, sunthetic Love and Rayleigh wave dispersion curves are calculated. The dispersion curves, which comprise fundamental and overtones up to 5th (Love) and 6th (Rayleigh) order have been extracted from global surface wave velocity maps. Given the above scheme, the data are at each location are jointly inverted using a Markov Chain Monte Carlo algorithm, from which a range of compositions, temperatures and radial profiles of anisotropy parameters, fitting data within uncertainties, are obtained. Our method has several advantages over standard approaches, in that no scaling relationships between Vs and Vp and ρ and Vs have to be introduced, implying that the full sensitivity of Rayleigh and Love waves to the parameters Vs, Vp and ρ is accounted for. In this particular study we investigate 5 locations distributed across the globe and reveal mantle chemical and thermal differences at these locations.

Repeat Transduction in the Mouse Lung by Using Adeno-Associated Virus Vectors with Different Serotypes

PubMed Central

Halbert, Christine L.; Rutledge, Elizabeth A.; Allen, James M.; Russell, David W.; Miller, A. Dusty

2000-01-01

Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors. PMID:10627564

Rotations with Rodrigues' Vector

ERIC Educational Resources Information Center

Pina, E.

2011-01-01

The rotational dynamics was studied from the point of view of Rodrigues' vector. This vector is defined here by its connection with other forms of parametrization of the rotation matrix. The rotation matrix was expressed in terms of this vector. The angular velocity was computed using the components of Rodrigues' vector as coordinates. It appears…

Rule-Based Design of Plant Expression Vectors Using GenoCAD.

PubMed

Coll, Anna; Wilson, Mandy L; Gruden, Kristina; Peccoud, Jean

2015-01-01

Plant synthetic biology requires software tools to assist on the design of complex multi-genic expression plasmids. Here a vector design strategy to express genes in plants is formalized and implemented as a grammar in GenoCAD, a Computer-Aided Design software for synthetic biology. It includes a library of plant biological parts organized in structural categories and a set of rules describing how to assemble these parts into large constructs. Rules developed here are organized and divided into three main subsections according to the aim of the final construct: protein localization studies, promoter analysis and protein-protein interaction experiments. The GenoCAD plant grammar guides the user through the design while allowing users to customize vectors according to their needs. Therefore the plant grammar implemented in GenoCAD will help plant biologists take advantage of methods from synthetic biology to design expression vectors supporting their research projects.

The prospect of gene therapy for prostate cancer: update on theory and status.

PubMed

Koeneman, K S; Hsieh, J T

2001-09-01

Molecularly based novel therapeutic agents are needed to address the problem of locally recurrent, or metastatic, advanced hormone-refractory prostate cancer. Recent basic science advances in mechanisms of gene expression, vector delivery, and targeting have rendered clinically relevant gene therapy to the prostatic fossa and distant sites feasible in the near future. Current research and clinical investigative efforts involving methods for more effective vector delivery and targeting, with enhanced gene expression to selected (specific) sites, are reviewed. These areas of research involve tissue-specific promoters, transgene exploration, vector design and delivery, and selective vector targeting. The 'vectorology' involved mainly addresses selective tissue homing with ligands, mechanisms of innate immune system evasion for durable transgene expression, and the possibility of repeat administration.

Construction, expression and in vitro biological behaviors of Ig scFv fragment in patients with chronic B cell leukemia.

PubMed

Zhu, Lijuan; Liao, Wenjun; Zhu, Huifen; Lei, Ping; Wang, Zhihua; Shao, Jingfang; Zhang, Yue; Shen, Guanxin

2006-01-01

The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS-PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD= 0.9921 ku), which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of Id-HSP complex vaccine for B-CLL.

Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Usami, Katsuaki; Matsuno, Keita; Igarashi, Manabu

2011-04-01

Highlights: {yields} Ebola virus infection is mediated by binding to and fusion with the target cells. {yields} Structural feature of the viral glycoprotein determines the infectivity. {yields} Surface C-type lectin, MGL, of macrophages and dendritic cells mediate the infection. {yields} GP2, one of glycoprotein subunits, plays an essential role in MGL-mediated infection. {yields} There is a critical amino acid residue involved in high infectivity. -- Abstract: Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses,more » i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.« less

Spontaneous Mutation at Amino Acid 544 of the Ebola Virus Glycoprotein Potentiates Virus Entry and Selection in Tissue Culture

PubMed Central

Ladner, Jason T.; Ettinger, Chelsea R.; Palacios, Gustavo

2017-01-01

ABSTRACT Ebolaviruses have a surface glycoprotein (GP1,2) that is required for virus attachment and entry into cells. Mutations affecting GP1,2 functions can alter virus growth properties. We generated a recombinant vesicular stomatitis virus encoding Ebola virus Makona variant GP1,2 (rVSV-MAK-GP) and observed emergence of a T544I mutation in the Makona GP1,2 gene during tissue culture passage in certain cell lines. The T544I mutation emerged within two passages when VSV-MAK-GP was grown on Vero E6, Vero, and BS-C-1 cells but not when it was passaged on Huh7 and HepG2 cells. The mutation led to a marked increase in virus growth kinetics and conferred a robust growth advantage over wild-type rVSV-MAK-GP on Vero E6 cells. Analysis of complete viral genomes collected from patients in western Africa indicated that this mutation was not found in Ebola virus clinical samples. However, we observed the emergence of T544I during serial passage of various Ebola Makona isolates on Vero E6 cells. Three independent isolates showed emergence of T544I from undetectable levels in nonpassaged virus or virus passaged once to frequencies of greater than 60% within a single passage, consistent with it being a tissue culture adaptation. Intriguingly, T544I is not found in any Sudan, Bundibugyo, or Tai Forest ebolavirus sequences. Furthermore, T544I did not emerge when we serially passaged recombinant VSV encoding GP1,2 from these ebolaviruses. This report provides experimental evidence that the spontaneous mutation T544I is a tissue culture adaptation in certain cell lines and that it may be unique for the species Zaire ebolavirus. IMPORTANCE The Ebola virus (Zaire) species is the most lethal species of all ebolaviruses in terms of mortality rate and number of deaths. Understanding how the Ebola virus surface glycoprotein functions to facilitate entry in cells is an area of intense research. Recently, three groups independently identified a polymorphism in the Ebola glycoprotein (I544) that enhanced virus entry, but they did not agree in their conclusions regarding its impact on pathogenesis. Our findings here address the origins of this polymorphism and provide experimental evidence showing that it is the result of a spontaneous mutation (T544I) specific to tissue culture conditions, suggesting that it has no role in pathogenesis. We further show that this mutation may be unique to the species Zaire ebolavirus, as it does not occur in Sudan, Bundibugyo, and Tai Forest ebolaviruses. Understanding the mechanism behind this mutation can provide insight into functional differences that exist in culture conditions and among ebolavirus glycoproteins. PMID:28539437

Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/promoter.

PubMed

Hagstrom, J N; Couto, L B; Scallan, C; Burton, M; McCleland, M L; Fields, P A; Arruda, V R; Herzog, R W; High, K A

2000-04-15

Hemophilia B is caused by the absence of functional coagulation factor IX (F.IX) and represents an important model for treatment of genetic diseases by gene therapy. Recent studies have shown that intramuscular injection of an adeno-associated viral (AAV) vector into mice and hemophilia B dogs results in vector dose-dependent, long-term expression of biologically active F.IX at therapeutic levels. In this study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 x 10(11) vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished through the use of an improved expression cassette that uses the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.2-kilobase portion of human skeletal actin promoter. These results correlated with enhanced levels of F.IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes. Systemic F.IX expression from constructs containing the CMV enhancer/promoter alone was 120 to 200 ng/mL in mice injected with 1 x 10(11) vector genomes. Muscle-specific promoters performed poorly for F.IX transgene expression in vitro and in vivo. However, the incorporation of a sequence from the alpha-skeletal actin promoter containing at least 1 muscle-specific enhancer and 1 enhancer-like element further improved muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of F.IX expression with lower vector doses, thus enhancing efficacy and safety of the protocol. (Blood. 2000;95:2536-2542)

Stable and Efficient Gene Transfer into the Retina Using an HIV-Based Lentiviral Vector

NASA Astrophysics Data System (ADS)

Miyoshi, Hiroyuki; Takahashi, Masayo; Gage, Fred H.; Verma, Inder M.

1997-09-01

The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain in vivo. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.

Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production.

PubMed

Zhang, Xiaoyue; Xu, Keyan; Ou, Yanmei; Xu, Xiaodong; Chen, Hongying

2018-05-02

The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection. The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.

Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy.

PubMed Central

Gorziglia, M I; Kadan, M J; Yei, S; Lim, J; Lee, G M; Luthra, R; Trapnell, B C

1996-01-01

A novel recombinant adenovirus vector, Av3nBg, was constructed with deletions in adenovirus E1, E2a, and E3 regions and expressing a beta-galactosidase reporter gene. Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the adenovirus E1 and E2a region genes inducibly expressed from separate glucocorticoid-responsive promoters. Av3nBg demonstrated gene transfer and expression comparable to that of Av1nBg, a first-generation adenovirus vector with deletions in E1 and E3. Several lines of evidence suggest that this vector is significantly more attenuated than E1 and E3 deletion vectors. Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labeling demonstrated no detectable de novo hexon protein synthesis for Av3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell. Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions. In contrast, both Av1nBg and Av2Lu vectors showed DNA replication and hexon protein synthesis at multiplicities of infection of 500 PFU per cell. Av2Lu has a deletion in E1 and also carries a temperature-sensitive mutation in E2a. Thus, molecular characterization has demonstrated that the Av3nBg vector is improved with respect to the potential for vector DNA replication and hexon protein expression compared with both first-generation (Av1nBg) and second-generation (Av2Lu) adenoviral vectors. These observations may have important implications for potential use of adenovirus vectors in human gene therapy. PMID:8648763

The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

PubMed Central

Zhong, Shumei; Sun, Shihua; Teng, Ba-Bie

2004-01-01

Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene expression in the mice livers, which did not produce the therapeutic effects on alteration the lipid levels or the inhibition of atherosclerosis development. In contrast, the ribozyme RB15 RNA mediated by scAAV2-TTR-RB15 vector was expressed immediately at day-1 after transduction in HepG2 cells. The apoB mRNA levels were decreased 47% (p = 0.001), compared to the control vector scAAV2-TTR-RB15-mutant. Conclusion This study provided evidence that the rAAV2 single-strand vector mediated a prolonged but not efficient transduction in mouse liver. However, the scAAV2 double-strand vector mediated a rapid and efficient gene expression in liver cells. This strategy using scAAV2 vectors represents a better approach to express small molecules such as ribozyme. PMID:15193153

Assessing the potential for AAV vector genotoxicity in a murine model

PubMed Central

Li, Hojun; Malani, Nirav; Hamilton, Shari R.; Schlachterman, Alexander; Bussadori, Giulio; Edmonson, Shyrie E.; Shah, Rachel; Arruda, Valder R.; Mingozzi, Federico; Fraser Wright, J.; Bushman, Frederic D.

2011-01-01

Gene transfer using adeno-associated virus (AAV) vectors has great potential for treating human disease. Recently, questions have arisen about the safety of AAV vectors, specifically, whether integration of vector DNA in transduced cell genomes promotes tumor formation. This study addresses these questions with high-dose liver-directed AAV-mediated gene transfer in the adult mouse as a model (80 AAV-injected mice and 52 controls). After 18 months of follow-up, AAV-injected mice did not show a significantly higher rate of hepatocellular carcinoma compared with controls. Tumors in mice treated with AAV vectors did not have significantly different amounts of vector DNA compared with adjacent normal tissue. A novel high-throughput method for identifying AAV vector integration sites was developed and used to clone 1029 integrants. Integration patterns in tumor tissue and adjacent normal tissue were similar to each other, showing preferences for active genes, cytosine-phosphate-guanosine islands, and guanosine/cysteine-rich regions. Gene expression data showed that genes near integration sites did not show significant changes in expression patterns compared with genes more distal to integration sites. No integration events were identified as causing increased oncogene expression. Thus, we did not find evidence that AAV vectors cause insertional activation of oncogenes and subsequent tumor formation. PMID:21106988

Detection of osteoclastic cell-cell fusion through retroviral vector packaging.

PubMed

Kondo, Takako; Ikeda, Kyoji; Matsuo, Koichi

2004-11-01

Cell-cell fusion generates multinucleated cells such as osteoclasts in bone, myotubes in muscle, and trophoblasts in placenta. Molecular details governing these fusion processes are still largely unknown. As a step toward identification of fusogenic genes, we tested the concept that retroviral vectors can be packaged as a result of cell-cell fusion. First, we introduced replication-deficient retroviral vectors expressing mCAT-1, which mediates fusogenic interaction with the retroviral envelope protein Env, into Chinese hamster ovary (CHO) cells to generate vector cells. Plasmids expressing virion proteins Gag, Pol, and Env were introduced into a separate culture of CHO cells to generate packaging cells. Co-culturing vector and packaging cells resulted in production of infectious retroviruses carrying the mCAT-1 gene as a consequence of cell-cell fusion. Second, we introduced a retroviral vector into primary osteoclast precursors and co-cultured them with established osteoclast precursor RAW264.7 cells, which turned out to harbor packaging activity. Packaged retroviral vector was detected in culture supernatants only where the osteoclast differentiation factor receptor activator for NF-kappaB ligand (RANKL) induced fusion between these two cell types. These data suggest that retrovirus production can occur as a result of cell-cell fusion. This provides a novel approach for isolating and characterizing fusogenic genes using retroviral expression vectors.

[Construction and expression of fusion protein TRX-hJagged1 in E.coli BL21].

PubMed

Li, Guo-Hui; Fan, Yu-Zhen; Huang, Si-Yong; Liu, Qiang; Yin, Dan-Dan; Liu, Li; Chen, Ren-An; Hao, Miao-Wang; Liang, Ying-Min

2014-06-01

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.

PubMed

Sharma, Nynne; Hollensen, Anne Kruse; Bak, Rasmus O; Staunstrup, Nicklas Heine; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

2012-01-01

DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5'-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells.

The Impact of cHS4 Insulators on DNA Transposon Vector Mobilization and Silencing in Retinal Pigment Epithelium Cells

PubMed Central

Sharma, Nynne; Hollensen, Anne Kruse; Bak, Rasmus O.; Staunstrup, Nicklas Heine; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

2012-01-01

DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5′-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells. PMID:23110238

A Simple And Rapid Minicircle DNA Vector Manufacturing System

PubMed Central

Kay, Mark A; He, Cheng-Yi; Chen, Zhi-Ying

2010-01-01

Minicircle DNA vectors consisting of a circular expression cassette devoid of the bacterial plasmid DNA backbone provides several advantages including sustained transgene expression in quiescent cells/tissues. Their use has been limited by labor-intensive production. We report on a strategy for making multiple genetic modifications in E.coli to construct a producer strain that stably expresses a set of inducible minicircle-assembly enzymes, the øC31-integrase and I-SceI homing-endonuclease. This bacterial strain is capable of producing highly purified minicircle yields in the same time frame as routine plasmid DNA. It is now feasible for minicircle DNA vectors to replace routine plasmids in mammalian transgene expression studies. PMID:21102455

[Cloning of human CD45 gene and its expression in Hela cells].

PubMed

Li, Jie; Xu, Tianyu; Wu, Lulin; Zhang, Liyun; Lu, Xiao; Zuo, Daming; Chen, Zhengliang

2015-11-01

To clone human CD45 gene PTPRC and establish Hela cells overexpressing recombinant human CD45 protein. The intact cDNA encoding human CD45 amplified using RT-PCR from the total RNA extracted from peripheral blood mononuclear cells (PBMCs) of a healthy donor was cloned into pMD-18T vector. The CD45 cDNA fragment amplified from the pMD-18T-CD45 by PCR was inserted to the coding region of the PcDNA3.1-3xflag vector, and the resultant recombinant expression vector PcDNA3.1-3xflag-CD45 was transfected into Hela cells. The expression of CD45 in Hela cells was detected by flow cytometry and Western blotting, and the phosphastase activity of CD45 was quantified using an alkaline phosphatase assay kit. The cDNA fragment of about 3 900 bp was amplified from human PBMCs and cloned into pMD-18T vector. The recombinant expression vector PcDNA3.1-3xflag-CD45 was constructed, whose restriction maps and sequence were consistent with those expected. The expression of CD45 in transfected Hela cells was detected by flow cytometry and Western blotting, and the expressed recombinant CD45 protein in Hela cells showed a phosphastase activity. The cDNA of human CD45 was successfully cloned and effectively expressed in Hela cells, which provides a basis for further exploration of the functions of CD45.

Regulated Expression of Adenoviral Vectors-Based Gene Therapies

PubMed Central

Curtin, James F.; Candolfi, Marianela; Puntel, Mariana; Xiong, Weidong; Muhammad, A. K. M.; Kroeger, Kurt; Mondkar, Sonali; Liu, Chunyan; Bondale, Niyati; Lowenstein, Pedro R.; Castro, Maria G.

2008-01-01

Summary Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the “OFF” state, and expression should quickly reach therapeutic levels in the “ON” state. In addition, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illness that requires long-lasting gene expression. In this chapter, we will describe in detail protocols to develop and validate first generation (Ad) and high-capacity adenoviral (HC-Ad) vectors that express therapeutic genes under the control of the TetON regulatable system. Our laboratory has successfully used these protocols to regulate the expression of marker genes, immune stimulatory genes, and toxins for cancer gene therapeutics, i.e., glioma that is a deadly form of brain cancer. We have shown that this third generation TetON regulatable system, incorporating a doxycycline (DOX)-sensitive rtTA2S-M2 inducer and tTSKid silencer, is non-toxic, relatively non-immunogenic, and can tightly regulate reporter transgene expression downstream of a TRE promoter from adenoviral vectors in vitro and also in vivo. PMID:18470649

Development of an Expression Vector to Overexpress or Downregulate Genes in Curvularia protuberata.

PubMed

Liu, Chengke; Cleckler, Blake; Morsy, Mustafa

2018-05-05

Curvularia protuberata , an endophytic fungus in the Ascomycota, provides plants with thermotolerance only when it carries a mycovirus known as Curvularia thermotolerance virus (CThTV), and forms a three-way symbiotic relationship among these organisms. Under heat stress, several genes are expressed differently between virus-free C. protuberata (VF) and C. protuberata carrying CThTV (AN). We developed an expression vector, pM2Z-fun, carrying a zeocin resistance gene driven by the ToxA promoter, to study gene functions in C. protuberata to better understand this three-way symbiosis. Using this new 3.7-kb vector, five genes that are differentially expressed in C. protuberata —including genes involved in the trehalose, melanin, and catalase biosynthesis pathways—were successfully overexpressed or downregulated in VF or AN C. protuberata strains, respectively. The VF overexpression lines showed higher metabolite and enzyme activity than in the control VF strain. Furthermore, downregulation of expression of the same genes in the AN strain resulted in lower metabolite and enzyme activity than in the control AN strain. The newly generated expression vector, pM2Z-fun, has been successfully used to express target genes in C. protuberata and will be useful in further functional expression studies in other Ascomycota fungi.

UMG Lenti: Novel Lentiviral Vectors for Efficient Transgene- and Reporter Gene Expression in Human Early Hematopoietic Progenitors

PubMed Central

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G.; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B.; Grillone, Teresa; Giovannone, Emilia D.; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A. S.; Bond, Heather M.; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and –LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG–LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells. PMID:25502183

Serotype-dependent transduction efficiencies of recombinant adeno-associated viral vectors in monkey neocortex

PubMed Central

Gerits, Annelies; Vancraeyenest, Pascaline; Vreysen, Samme; Laramée, Marie-Eve; Michiels, Annelies; Gijsbers, Rik; Van den Haute, Chris; Moons, Lieve; Debyser, Zeger; Baekelandt, Veerle; Arckens, Lutgarde; Vanduffel, Wim

2015-01-01

Abstract. Viral vector-mediated expression of genes (e.g., coding for opsins and designer receptors) has grown increasingly popular. Cell-type specific expression is achieved by altering viral vector tropism through crosspackaging or by cell-specific promoters driving gene expression. Detailed information about transduction properties of most recombinant adeno-associated viral vector (rAAV) serotypes in macaque cortex is gradually becoming available. Here, we compare transduction efficiencies and expression patterns of reporter genes in two macaque neocortical areas employing different rAAV serotypes and promoters. A short version of the calmodulin-kinase-II (CaMKIIα0.4) promoter resulted in reporter gene expression in cortical neurons for all tested rAAVs, albeit with different efficiencies for spread: rAAV2/5>>rAAV2/7>rAAV2/8>rAAV2/9>>rAAV2/1 and proportion of transduced cells: rAAV2/1>rAAV2/5>rAAV2/7=rAAV2/9>rAAV2/8. In contrast to rodent studies, the cytomegalovirus (CMV) promoter appeared least efficient in macaque cortex. The human synapsin-1 promoter preceded by the CMV enhancer (enhSyn1) produced homogeneous reporter gene expression across all layers, while two variants of the CaMKIIα promoter resulted in different laminar transduction patterns and cell specificities. Finally, differences in expression patterns were observed when the same viral vector was injected in two neocortical areas. Our results corroborate previous findings that reporter-gene expression patterns and efficiency of rAAV transduction depend on serotype, promoter, cortical layer, and area. PMID:26839901

[Construction of the lentiviral expression vector for anti-p185(erbB2) mouse/human chimeric antibody].

PubMed

Liu, Fang; Li, Li; Zhang, Wei; Wang, Qi

2013-04-01

This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.

Immunogenicity and Protection Against Influenza H7N3 in Mice by Modified Vaccinia Virus Ankara Vectors Expressing Influenza Virus Hemagglutinin or Neuraminidase.

PubMed

Meseda, Clement A; Atukorale, Vajini; Soto, Jackeline; Eichelberger, Maryna C; Gao, Jin; Wang, Wei; Weiss, Carol D; Weir, Jerry P

2018-03-29

Influenza subtypes such as H7 have pandemic potential since they are able to infect humans with severe consequences, as evidenced by the ongoing H7N9 infections in China that began in 2013. The diversity of H7 viruses calls for a broadly cross-protective vaccine for protection. We describe the construction of recombinant modified vaccinia virus Ankara (MVA) vectors expressing the hemagglutinin (HA) or neuraminidase (NA) from three H7 viruses representing both Eurasian and North American H7 lineages - A/mallard/Netherlands/12/2000 (H7N3), A/Canada/rv444/2004 (H7N3), and A/Shanghai/02/2013 (H7N9). These vectors were evaluated for immunogenicity and protective efficacy against H7N3 virus in a murine model of intranasal challenge. High levels of H7-, N3-, and N9-specific antibodies, including neutralizing antibodies, were induced by the MVA-HA and MVA-NA vectors. Mice vaccinated with MVA vectors expressing any of the H7 antigens were protected, suggesting cross-protection among H7 viruses. In addition, MVA vectors expressing N3 but not N9 elicited protection against H7N3 virus challenge. Similar outcomes were obtained when immune sera from MVA vector-immunized mice were passively transferred to naïve mice prior to challenge with the H7N3 virus. The results support the further development of an MVA vector platform as a candidate vaccine for influenza strains with pandemic potential.

World Reference Center for Arboviruses

DTIC Science & Technology

1988-01-01

them new to science and/or new to a geographic region. The taxonomy of the rhabdoviruses , orbiviruses, and phleboviruses was revised. The distribution... rhabdovirus , apparently new to science. Viruses were identified from the United States and from many other nations as follows; ALPHAVIRUSES...Alaskan isolate. VESICULOVIRUSES. New VSV-related rhabdoviruses were recognized from Argentina and the United States. Also, a Tibrogargan-related

The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli.

PubMed

Kuipers, Grietje; Karyolaimos, Alexandros; Zhang, Zhe; Ismail, Nurzian; Trinco, Gianluca; Vikström, David; Slotboom, Dirk Jan; de Gier, Jan-Willem

2017-12-16

To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a standard T7-based expression vector. By incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a standard T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized production yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a standard T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the production of this heme c protein. Here, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the production of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).

A novel intranuclear RNA vector system for long-term stem cell modification

PubMed Central

Ikeda, Yasuhiro; Makino, Akiko; Matchett, William E.; Holditch, Sara J.; Lu, Brian; Dietz, Allan B.; Tomonaga, Keizo

2015-01-01

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, Borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression, or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs), while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction. PMID:26632671

Attenuated Human Parainfluenza Virus Type 1 Expressing the Respiratory Syncytial Virus (RSV) Fusion (F) Glycoprotein from an Added Gene: Effects of Prefusion Stabilization and Packaging of RSV F

PubMed Central

Liu, Xiang; Liang, Bo; Ngwuta, Joan; Liu, Xueqiao; Surman, Sonja; Lingemann, Matthias; Kwong, Peter D.; Graham, Barney S.; Collins, Peter L.

2017-01-01

ABSTRACT Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the prefusion (pre-F) conformation by previously described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane and cytoplasmic tail (TMCT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild-type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo. In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine. IMPORTANCE RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a stabilized attenuating mutation, to express the RSV F glycoprotein bearing amino acid substitutions that increase its stability in the pre-F form, the most immunogenic form that elicits highly functional virus-neutralizing antibodies. RSV F was expressed from the pre-N or N-P gene position of the rHPIV1 vector as a full-length protein or as a chimeric form with its TMCT domain derived from HPIV1 F. TMCT modification greatly increased packaging of RSV F into the vector particles but also increased vector attenuation in vivo, resulting in reduced immunogenicity. In contrast, full-length RSV F expressed from the pre-N position was immunogenic, eliciting complement-independent RSV-neutralizing antibodies and providing protection against RSV challenge. PMID:28835504

Effective reduction of the interleukin-1β transcript in osteoarthritis-prone guinea pig chondrocytes via short hairpin RNA mediated RNA interference influences gene expression of mediators implicated in disease pathogenesis

PubMed Central

Santangeloyz, K.S.; Bertoneyz, A.L.

2011-01-01

summary Objective To ascertain a viral vector-based short hairpin RNA (shRNA) capable of reducing the interleukin-1β (IL-1β) transcript in osteoarthritis (OA)-prone chondrocytes and detect corresponding changes in the expression patterns of several critical disease mediators. Methods Cultured chondrocytes from 2-month-old Hartley guinea pigs were screened for reduction of the IL-1β transcript following plasmid-based delivery of U6-driven shRNA sequences. A successful plasmid/shRNA knockdown combination was identified and used to construct an adeno-associated virus serotype 5 (AAV5) vector for further evaluation. Relative real-time reverse transcription polymerase chain reaction (RTPCR) was used to quantify in vitro transcript changes of IL-1β and an additional nine genes following transduction with this targeting knockdown vector. To validate in vitro findings, this AAV5 vector was injected into one knee, while either an equivalent volume of saline vehicle (three animals) or non-targeting control vector (three animals) were injected into opposite knees. Fold differences and subsequent percent gene expression levels relative to control groups were calculated using the comparative CT (2−ΔΔCT) method. Results Statistically significant decreases in IL-1β expression were achieved by the targeting knockdown vector relative to both the mock-transduced control and non-targeting vector control groups in vitro. Transcript levels of anabolic transforming growth factor-β (TGF-β) were significantly increased by use of this targeting knockdown vector. Transduction with this targeting AAV5 vector also significantly decreased the transcript levels of key inflammatory cytokines [tumor necrosis factor-α (TNF-α), IL-2, IL-8, and IL-12] and catabolic agents [matrix metalloproteinase (MMP)13, MMP2, interferon-γ (IFN-γ), and inducible nitrous oxide synthase (iNOS)] relative to both mock-transduced and non-targeting vector control groups. In vivo application of this targeting knockdown vector resulted in a >50% reduction (P= 0.0045) or >90% (P= 0.0001) of the IL-1β transcript relative to vehicle-only or non-targeting vector control exposed cartilage, respectively. Conclusions Successful reduction of the IL-1β transcript was achieved via RNA interference (RNAi) techniques. Importantly, this alteration significantly influenced the transcript levels of several major players involved in OA pathogenesis in the direction of disease modification. Investigations to characterize additional gene expression changes influenced by targeting knockdown AAV5 vector-based diminution of the IL-1β transcript in vivo are warranted. PMID:21945742

Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption

PubMed Central

2013-01-01

Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. Results We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. Conclusions Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain. PMID:23497512

A regulatory sequence from the retinoid X receptor γ gene directs expression to horizontal cells and photoreceptors in the embryonic chicken retina.

PubMed

Blixt, Maria K E; Hallböök, Finn

2016-01-01

Combining techniques of episomal vector gene-specific Cre expression and genomic integration using the piggyBac transposon system enables studies of gene expression-specific cell lineage tracing in the chicken retina. In this work, we aimed to target the retinal horizontal cell progenitors. A 208 bp gene regulatory sequence from the chicken retinoid X receptor γ gene (RXRγ208) was used to drive Cre expression. RXRγ is expressed in progenitors and photoreceptors during development. The vector was combined with a piggyBac "donor" vector containing a floxed STOP sequence followed by enhanced green fluorescent protein (EGFP), as well as a piggyBac helper vector for efficient integration into the host cell genome. The vectors were introduced into the embryonic chicken retina with in ovo electroporation. Tissue electroporation targets specific developmental time points and in specific structures. Cells that drove Cre expression from the regulatory RXRγ208 sequence excised the floxed STOP-sequence and expressed GFP. The approach generated a stable lineage with robust expression of GFP in retinal cells that have activated transcription from the RXRγ208 sequence. Furthermore, GFP was expressed in cells that express horizontal or photoreceptor markers when electroporation was performed between developmental stages 22 and 28. Electroporation of a stage 12 optic cup gave multiple cell types in accordance with RXRγ gene expression in the early retina. In this study, we describe an easy, cost-effective, and time-efficient method for testing regulatory sequences in general. More specifically, our results open up the possibility for further studies of the RXRγ-gene regulatory network governing the formation of photoreceptor and horizontal cells. In addition, the method presents approaches to target the expression of effector genes, such as regulators of cell fate or cell cycle progression, to these cells and their progenitor.

[Construction and expression of the eukaryotic expression vector carrying HSV-1 gC glycoprotein gene].

PubMed

Dang, Yin-li; Yan, Yan; Zhang, Xiao-xiao; Li, Pu-yuan; Yu, Lan; Zhang, Lei; Zhang, Fang-lin; Xu, Zhi-kai; Wu, Xing-an

2011-05-01

To stably express herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) in Chinese hamster ovary cells (CHO-K1). The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed and transfected into CHO-K1 cells by Lipofectamine 2000. The transfected cells were selected by G418 and methotrexate (MTX). The expression of HSV-1 gC was analyzed by Slot blot. HSV-1 gC proteins were purified with His-Ni Sepharose and then detected by Western blot. The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed successfully. CHO-K1 cells stably expressing HSV-1 gC proteins were established and confirmed by Western blot. The HSV-1 gC proteins have been expressed successfully and have good bioactivity. The results make it possible for further study and clinical use of HSV-1 gC.

Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

PubMed

Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Matsuda, Fumio; Kondo, Akihiko

2014-05-01

Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

[Construction of Plasmodium falciparum signal peptide peptidase-GFP mutant and its expression analysis in the malaria parasite].

PubMed

Li, Xue-rong; Wu, Yin-juan; Shang, Mei; Li, Ye; Xu, Jin; Yu, Xin-bing; Athar, Chishti

2014-08-01

To construct recombinant plasmid pSPPcGT which contains signal peptide peptidase gene of Plasmodium falciparum (PJSPP) and GFP, and transfect into P. falciparum (3D7 strain) to obtain mutant parasites which can express PJSPP-GFP. Plasmodium falciparum(3D7 strain) genomic DNA was extracted from cultured malaria parasites. The C-terminal region of PJSPP, an 883 bp gene fragment was amplified by PCR, and then cloned into pPM2GT vector to get recombinant vector pSPPcGT. The recombinant vectors were identified by PCR, double restriction enzyme digestion and DNA sequencing. pSPPcGT vector was transfected into malaria parasites. The positive clones were selected by adding inhibitor of Plasmodium falciparum dihydrofolate reductase WR99210 to the culture medium. The pSPP-GFP-transfected parasites were fixed with methanol, stained with DAPI, and observed under immunofluorescence microscope. The PJSPP-GFP expression in P. falciparum was identified by SDS-PAGE and Western blotting. The C-terminal region of PJSPP was amplified from P.falciparum (3D7 strain) genomic DNA by PCR with the length of 883 bp. The constructed recombinant vectors were identified by PCR screening, double restriction enzyme digestion and DNA sequencing. The pSPPcGT vector was transfected into P. falciparum and the positive clones were selected by WR99210. GFP fluorescence was observed in transfected parasites by immunofluorescence microscopy, and mainly located in the cytoplasm. The PJSPP-GFP expression in malaria parasites was confirmed by Western blotting with a relative molecular mass of Mr 64,000. Recombinant vector PJSPP-GFP is constructed and transfected into P. falciparum to obtain P. falciparum mutant clone which can express PfSPP-GFP.

Rational Development of A Polycistronic Plasmid with A CpG-Free Bacterial Backbone as A Potential Tool for Direct Reprogramming.

PubMed

Dormiani, Kianoush; Mir Mohammad Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Forouzanfar, Mahboobeh; Baharvand, Hossein; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

2017-01-01

Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector. In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells. In the present study, we developed a nonviral episomal vector named pLENSO/ Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming. According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future.

Simple cloning strategy using GFPuv gene as positive/negative indicator.

PubMed

Miura, Hiromi; Inoko, Hidetoshi; Inoue, Ituro; Tanaka, Masafumi; Sato, Masahiro; Ohtsuka, Masato

2011-09-15

Because construction of expression vectors is the first requisite in the functional analysis of genes, development of simple cloning systems is a major requirement during the postgenomic era. In the current study, we developed cloning vectors for gain- or loss-of-function studies by using the GFPuv gene as a positive/negative indicator of cloning. These vectors allow us to easily detect correct clones and obtain expression vectors from a simple procedure by means of the combined use of the GFPuv gene and a type IIS restriction enzyme. Copyright © 2011 Elsevier Inc. All rights reserved.

[Prokaryotic expression of Nanog gene and preparation of anti-Nanog antibody].

PubMed

Li, Jun; Wang, Xiao-min; Dou, Zhong-ying; Li, Yong

2012-07-01

To express Nanog fusion protein in Escherichia coli ( E.coli), and to prepare rabbit anti-mouse polyclonal antibodies to the Nanog fusion protein. Mouse Nanog gene was amplified from the pNA992 recombinant plasmid and inserted into pET-32a vector to construct a recombinant expression vector pET-32a-Nanog. The recombinant vector was transfected into E.coli BL21 and induced by IPTG to express in them. The acquired Nanog fusion protein was purified with HisTrap affinity column and injected as an antigen into rabbits for preparing polyclonal antibodies. At last, the titer and specificity of the polyclonal antibodies were analyzed with indirect ELISA, Western blotting and immunocytochemical staining, respectively. The recombinant expression vector pET-32a-Nanog was successfully prepared, transfected and induced to obtain the high expression of the Nanog fusion protein in a form of inclusion bodies in E.coli. After purification, its purity was up to 97%. The titer of anti-Nanog antibodies was 1:32 000 in the immunized rabbit serum, and exhibited a high specificity to Nanog protein. The rabbit anti-mouse polyclonal antibodies have been prepared successfully with a high titer and specificity to the Nanog fusion protein.

Construction of novel shuttle expression vectors for gene expression in Bacillus subtilis and Bacillus pumilus.

PubMed

Shao, Huanhuan; Cao, Qinghua; Zhao, Hongyan; Tan, Xuemei; Feng, Hong

2015-01-01

A native plasmid (pSU01) was detected by genome sequencing of Bacillus subtilis strain S1-4. Two pSU01-based shuttle expression vectors pSU02-AP and pSU03-AP were constructed enabling stable replication in B. subtilis WB600. These vectors contained the reporter gene aprE, encoding an alkaline protease from Bacillus pumilus BA06. The expression vector pSU03-AP only possessed the minimal replication elements (rep, SSO, DSO) and exhibited more stability on structure, suggesting that the rest of the genes in pSU01 (ORF1, ORF2, mob, hsp) were unessential for the structural stability of plasmid in B. subtilis. In addition, recombinant production of the alkaline protease was achieved more efficiently with pSU03-AP whose copy number was estimated to be more than 100 per chromosome. Furthermore, pSU03-AP could also be used to transform and replicate in B. pumilus BA06 under selective pressure. In conclusion, pSU03-AP is expected to be a useful tool for gene expression in Bacillus subtilis and B. pumilus.

Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants.

PubMed

Lukan, Tjaša; Machens, Fabian; Coll, Anna; Baebler, Špela; Messerschmidt, Katrin; Gruden, Kristina

2018-01-01

Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.

Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

PubMed Central

Machens, Fabian; Coll, Anna; Baebler, Špela; Messerschmidt, Katrin; Gruden, Kristina

2018-01-01

Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science. PMID:29300787

High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system

PubMed Central

Huang, Zhong; Phoolcharoen, Waranyoo; Lai, Huafang; Piensook, Khanrat; Cardineau, Guy; Zeitlin, Larry; Whaley, Kevin J.; Arntzen, Charles J.

2010-01-01

Plant viral vectors have great potential in rapid production of important pharmaceutical proteins. However, high-yield production of heterooligomeric proteins that require the expression and assembly of two or more protein subunits often suffers problems due to the “competing” nature of viral vectors derived from the same virus. Previously we reported that a bean yellow dwarf virus (BeYDV)-derived, three-component DNA replicon system allows rapid production of single recombinant proteins in plants (Huang et al. 2009). In this article, we report further development of this expression system for its application in high-yield production of oligomeric protein complexes including monoclonal antibodies (mAbs) in plants. We showed that the BeYDV replicon system permits simultaneous efficient replication of two DNA replicons and thus, high-level accumulation of two recombinant proteins in the same plant cell. We also demonstrated that a single vector that contains multiple replicon cassettes was as efficient as the three-component system in driving the expression of two distinct proteins. Using either the non-competing, three-vector system or the multi-replicon single vector, we produced both the heavy and light chain subunits of a protective IgG mAb 6D8 against Ebola virus GP1 (Wilson et al. 2000) at 0.5 mg of mAb per gram leaf fresh weight within 4 days post infiltration of Nicotiana benthamiana leaves. We further demonstrated that full-size tetrameric IgG complex containing two heavy and two light chains was efficiently assembled and readily purified, and retained its functionality in specific binding to inactivated Ebola virus. Thus, our single-vector replicon system provides high-yield production capacity for heterooligomeric proteins, yet eliminates the difficult task of identifying non-competing virus and the need for co-infection of multiple expression modules. The multi-replicon vector represents a significant advance in transient expression technology for antibody production in plants. PMID:20047189

Transduction of skeletal muscles with common reporter genes can promote muscle fiber degeneration and inflammation.

PubMed

Winbanks, Catherine E; Beyer, Claudia; Qian, Hongwei; Gregorevic, Paul

2012-01-01

Recombinant adeno-associated viral vectors (rAAV vectors) are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for β-galactosidase (β-gal), human placental alkaline phosphatase (hPLAP), and green fluorescent protein (GFP) as experimental controls when studying the effects of manipulating other genes. However, it is not clear to what extent these reporter genes can influence signaling and gene expression signatures in skeletal muscle, which may confound the interpretation of results obtained in experimentally manipulated muscles. Herein, we report a strong pro-inflammatory effect of expressing reporter genes in skeletal muscle. Specifically, we show that the administration of rAAV6:hPLAP vectors to the hind limb muscles of mice is associated with dose- and time-dependent macrophage recruitment, and skeletal muscle damage. Dose-dependent expression of hPLAP also led to marked activity of established pro-inflammatory IL-6/Stat3, TNFα, IKKβ and JNK signaling in lysates obtained from homogenized muscles. These effects were independent of promoter type, as expression cassettes featuring hPLAP under the control of constitutive CMV and muscle-specific CK6 promoters both drove cellular responses when matched for vector dose. Importantly, the administration of rAAV6:GFP vectors did not induce muscle damage or inflammation except at the highest doses we examined, and administration of a transgene-null vector (rAAV6:MCS) did not cause damage or inflammation at any of the doses tested, demonstrating that GFP-expressing, or transgene-null vectors may be more suitable as experimental controls. The studies highlight the importance of considering the potential effects of reporter genes when designing experiments that examine gene manipulation in vivo.

Expression of chicken parvovirus VP2 in chicken embryo fibroblasts requires codon optimization for production of naked DNA and vectored meleagrid herpesvirus type 1 vaccines.

PubMed

Spatz, Stephen J; Volkening, Jeremy D; Mullis, Robert; Li, Fenglan; Mercado, John; Zsak, Laszlo

2013-10-01

Meleagrid herpesvirus type 1 (MeHV-1) is an ideal vector for the expression of antigens from pathogenic avian organisms in order to generate vaccines. Chicken parvovirus (ChPV) is a widespread infectious virus that causes serious disease in chickens. It is one of the etiological agents largely suspected in causing Runting Stunting Syndrome (RSS) in chickens. Initial attempts to express the wild-type gene encoding the capsid protein VP2 of ChPV by insertion into the thymidine kinase gene of MeHV-1 were unsuccessful. However, transient expression of a codon-optimized synthetic VP2 gene cloned into the bicistronic vector pIRES2-Ds-Red2, could be demonstrated by immunocytochemical staining of transfected chicken embryo fibroblasts (CEFs). Red fluorescence could also be detected in these transfected cells since the red fluorescent protein gene is downstream from the internal ribosome entry site (IRES). Strikingly, fluorescence could not be demonstrated in cells transiently transfected with the bicistronic vector containing the wild-type or non-codon-optimized VP2 gene. Immunocytochemical staining of these cells also failed to demonstrate expression of wild-type VP2, indicating that the lack of expression was at the RNA level and the VP2 protein was not toxic to CEFs. Chickens vaccinated with a DNA vaccine consisting of the bicistronic vector containing the codon-optimized VP2 elicited a humoral immune response as measured by a VP2-specific ELISA. This VP2 codon-optimized bicistronic cassette was rescued into the MeHV-1 genome generating a vectored vaccine against ChPV disease.

Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

PubMed

Tolmachov, Oleg E

2015-01-01

Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo are then attracted to and internalized into the intended target cells via the expressed cognate strongly binding extra-cellular receptor, causing escalation of gene transfer into these cells and increasing the copy number of the therapeutic gene expression modules. Such self-focusing swarms of gene vectors can be either homogeneous, with 'scout' and 'therapeutic' members of the swarm being structurally identical, or, alternatively, heterogeneous (split), with 'scout' and 'therapeutic' members of the swarm being structurally specialized. It is hoped that the proposed self-focusing cell-targeted gene vector swarms with receptor-mediated intra-swarm signalling could be particularly effective in 'top-up' gene delivery scenarios, achieving high-level and sustained expression of therapeutic transgenes that are prone to shut-down through degradation and silencing. Crucially, in contrast to low-precision 'general location' vector guidance by diffusible chemo-attractants, ear-marking non-diffusible receptors can provide high-accuracy targeting of therapeutic vector particles to the specific cell, which has undergone a 'successful cell-specific hit' by a 'scout' vector particle. Opportunities for cell targeting could be expanded, since in the proposed model of self-focusing it could be possible to probe a broad selection of intra-cellular determinants of cell-specificity and not just to rely exclusively on extra-cellular markers of cell-specificity. By employing such self-focusing gene vectors for the improvement of cell-targeted delivery of therapeutic genes, e.g., in cancer therapy or gene addition therapy of recessive genetic diseases, it could be possible to broaden a leeway for the reduction of the vector load and, consequently, to minimize undesired vector cytotoxicity, immune reactions, and the risk of inadvertent genetic modification of germline cells in genetic treatment in vivo. Copyright © 2014 Elsevier B.V. All rights reserved.

IL-10 mediated by herpes simplex virus vector reduces neuropathic pain induced by HIV gp120 combined with ddC in rats.

PubMed

Zheng, Wenwen; Huang, Wan; Liu, Shue; Levitt, Roy C; Candiotti, Keith A; Lubarsky, David A; Hao, Shuanglin

2014-07-30

HIV-associated sensory neuropathy affects over 50% of HIV patients and is a common peripheral nerve complication of HIV infection and highly active antiretroviral therapy (HAART). Evidence shows that painful HIV sensory neuropathy is influenced by neuroinflammatory events that include the proinflammatory molecules, MAP Kinase, tumor necrosis factor-α (TNFα), stromal cell-derived factor 1-α (SDF1α), and C-X-C chemokine receptor type 4 (CXCR4). However, the exact mechanisms of painful HIV sensory neuropathy are not known, which hinders our ability to develop effective treatments. In this study, we investigated whether inhibition of proinflammatory factors reduces the HIV-associated neuropathic pain state. Neuropathic pain was induced by peripheral HIV coat protein gp120 combined with 2',3'-dideoxycytidine (ddC, one of the nucleoside reverse transcriptase inhibitors (NRTIs)). Mechanical threshold was tested using von Frey filament fibers. Non-replicating herpes simplex virus (HSV) vectors expressing interleukin 10 (IL10) were inoculated into the hindpaws of rats. The expression of TNFα, SDF1α, and CXCR4 in the lumbar spinal cord and L4/5 dorsal root ganglia (DRG) was examined using western blots. IL-10 expression mediated by the HSV vectors resulted in a significant elevation of mechanical threshold. The anti-allodynic effect of IL-10 expression mediated by the HSV vectors lasted more than 3 weeks. The area under the effect-time curves (AUC) in mechanical threshold in rats inoculated with the HSV vectors expressing IL-10, was increased compared with the control vectors, indicating antinociceptive effect of the IL-10 vectors. The HSV vectors expressing IL-10 also concomitantly reversed the upregulation of p-p38, TNFα, SDF1α, and CXCR4 induced by gp120 in the lumbar spinal dorsal horn and/or the DRG at 2 and/or 4 weeks. The blocking of the signaling of these proinflammatory molecules is able to reduce HIV-related neuropathic pain, which provide a novel mechanism-based approach to treating HIV-associated neuropathic pain using gene therapy.

IL-10 mediated by herpes simplex virus vector reduces neuropathic pain induced by HIV gp120 combined with ddC in rats

PubMed Central

2014-01-01

Background HIV-associated sensory neuropathy affects over 50% of HIV patients and is a common peripheral nerve complication of HIV infection and highly active antiretroviral therapy (HAART). Evidence shows that painful HIV sensory neuropathy is influenced by neuroinflammatory events that include the proinflammatory molecules, MAP Kinase, tumor necrosis factor-α (TNFα), stromal cell-derived factor 1-α (SDF1α), and C-X-C chemokine receptor type 4 (CXCR4). However, the exact mechanisms of painful HIV sensory neuropathy are not known, which hinders our ability to develop effective treatments. In this study, we investigated whether inhibition of proinflammatory factors reduces the HIV-associated neuropathic pain state. Results Neuropathic pain was induced by peripheral HIV coat protein gp120 combined with 2′,3′-dideoxycytidine (ddC, one of the nucleoside reverse transcriptase inhibitors (NRTIs)). Mechanical threshold was tested using von Frey filament fibers. Non-replicating herpes simplex virus (HSV) vectors expressing interleukin 10 (IL10) were inoculated into the hindpaws of rats. The expression of TNFα, SDF1α, and CXCR4 in the lumbar spinal cord and L4/5 dorsal root ganglia (DRG) was examined using western blots. IL-10 expression mediated by the HSV vectors resulted in a significant elevation of mechanical threshold. The anti-allodynic effect of IL-10 expression mediated by the HSV vectors lasted more than 3 weeks. The area under the effect-time curves (AUC) in mechanical threshold in rats inoculated with the HSV vectors expressing IL-10, was increased compared with the control vectors, indicating antinociceptive effect of the IL-10 vectors. The HSV vectors expressing IL-10 also concomitantly reversed the upregulation of p-p38, TNFα, SDF1α, and CXCR4 induced by gp120 in the lumbar spinal dorsal horn and/or the DRG at 2 and/or 4 weeks. Conclusion The blocking of the signaling of these proinflammatory molecules is able to reduce HIV-related neuropathic pain, which provide a novel mechanism-based approach to treating HIV-associated neuropathic pain using gene therapy. PMID:25078297

Magnetofection™ of NMDA Receptor Subunits GluN1 and GluN2A Expression Vectors in Non-Neuronal Host Cells.

PubMed

Bruneau, Nadine; Szepetowski, Pierre

2017-01-01

The functional study of reconstituted NMDA receptors (NMDARs) in host cells requires that the corresponding vectors for the expression of the NMDAR subunits are co-transfected with high efficiency. Magnetofection™ is a technology used to deliver nucleic acids to cells. It is driven and site-specifically guided by the attractive forces of magnetic fields acting on magnetic nanoparticles that are associated with nucleic acid vectors. In magnetofection™, cationic lipids form self-assembled complexes with the nucleic acid vectors of interest. Those complexes are then associated with magnetic nanoparticles that are concentrated at the surface of cultured cells by applying a permanent magnetic field. Magnetofection™ is a simple method to transfect cultured cells with high transfection rates. Satisfactory expression levels are obtained with very low amounts of nucleic acid vector. Moreover, incubation time with host cells is less than 1 h, as compared with the several hours needed with standard transfection assays.

Gene Transfer into Rat Brain Using Adenoviral Vectors

PubMed Central

Puntel, Mariana; Kroeger, Kurt M.; Sanderson, Nicholas S.R.; Thomas, Clare E.; Castro, Maria G.; Lowenstein, Pedro R.

2010-01-01

Viral vector–mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose—efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described. Curr. Protoc. Neurosci. 50:4.24.1–4.24.49. © 2010 by John Wiley & Sons, Inc. PMID:20066657

High-efficiency Transduction of Rhesus Hematopoietic Repopulating Cells by a Modified HIV1-based Lentiviral Vector

PubMed Central

Uchida, Naoya; Hargrove, Phillip W.; Lap, Coen J.; Evans, Molly E.; Phang, Oswald; Bonifacino, Aylin C.; Krouse, Allen E.; Metzger, Mark E.; Nguyen, Anh-Dao; Hsieh, Matthew M.; Wolfsberg, Tyra G.; Donahue, Robert E.; Persons, Derek A.; Tisdale, John F.

2012-01-01

Human immunodeficiency virus type 1 (HIV1) vectors poorly transduce rhesus hematopoietic cells due to species-specific restriction factors, including the tripartite motif-containing 5 isoformα (TRIM5α) which targets the HIV1 capsid. We previously developed a chimeric HIV1 (χHIV) vector system wherein the vector genome is packaged with the simian immunodeficiency virus (SIV) capsid for efficient transduction of both rhesus and human CD34+ cells. To evaluate whether χHIV vectors could efficiently transduce rhesus hematopoietic repopulating cells, we performed a competitive repopulation assay in rhesus macaques, in which half of the CD34+ cells were transduced with standard SIV vectors and the other half with χHIV vectors. As compared with SIV vectors, χHIV vectors achieved higher vector integration, and the transgene expression rates were two- to threefold higher in granulocytes and red blood cells and equivalent in lymphocytes and platelets for 2 years. A recipient of χHIV vector-only transduced cells reached up to 40% of transgene expression rates in granulocytes and lymphocytes and 20% in red blood cells. Similar to HIV1 and SIV vectors, χHIV vector frequently integrated into gene regions, especially into introns. In summary, our χHIV vector demonstrated efficient transduction for rhesus long-term repopulating cells, comparable with SIV vectors. This χHIV vector should allow preclinical testing of HIV1-based therapeutic vectors in large animal models. PMID:22871664

The upstream enhancer elements of the G6PC promoter are critical for optimal G6PC expression in murine glycogen storage disease type Ia.

PubMed

Lee, Young Mok; Pan, Chi-Jiunn; Koeberl, Dwight D; Mansfield, Brian C; Chou, Janice Y

2013-11-01

Glycogen storage disease type-Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-α (G6Pase-α or G6PC) manifest impaired glucose homeostasis characterized by fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. Two efficacious recombinant adeno-associated virus pseudotype 2/8 (rAAV8) vectors expressing human G6Pase-α have been independently developed. One is a single-stranded vector containing a 2864-bp of the G6PC promoter/enhancer (rAAV8-GPE) and the other is a double-stranded vector containing a shorter 382-bp minimal G6PC promoter/enhancer (rAAV8-miGPE). To identify the best construct, a direct comparison of the rAAV8-GPE and the rAAV8-miGPE vectors was initiated to determine the best vector to take forward into clinical trials. We show that the rAAV8-GPE vector directed significantly higher levels of hepatic G6Pase-α expression, achieved greater reduction in hepatic glycogen accumulation, and led to a better toleration of fasting in GSD-Ia mice than the rAAV8-miGPE vector. Our results indicated that additional control elements in the rAAV8-GPE vector outweigh the gains from the double-stranded rAAV8-miGPE transduction efficiency, and that the rAAV8-GPE vector is the current choice for clinical translation in human GSD-Ia. © 2013.

[Sendai virus vector: vector development and its application to health care and biotechnology].

PubMed

Iida, Akihiro

2007-06-01

Sendai virus (SeV) is an enveloped virus with a nonsegmented negative-strand RNA genome and a member of the paramyxovirus family. We have developed SeV vector which has shown a high efficiently of gene transfer and expression of foreign genes to a wide range of dividing and non-dividing mammalian cells and tissues. One of the characteristics of the vector is that the genome is located exclusively in the cytoplasm of infected cells and does not go through a DNA phase; thus there is no concern about unwanted integration of foreign sequences into chromosomal DNA. Therefore, this new class of "cytoplasmic RNA vector", an RNA vector with cytoplasmic expression, is expected to be a safer and more efficient viral vector than existing vectors for application to human therapy in various fields including gene therapy and vaccination. In this review, I describe development of Sendai virus vector, its application in the field of biotechnology and clinical application aiming to treat for a large number of diseases including cancer, cardiovascular disease, infectious diseases and neurologic disorders.

Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells.

PubMed

Bleckmann, Maren; Schürig, Margitta; Chen, Fang-Fang; Yen, Zen-Zen; Lindemann, Nils; Meyer, Steffen; Spehr, Johannes; van den Heuvel, Joop

2016-01-01

The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.

Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae

PubMed Central

Gay, Glen; Wagner, Drew T.; Keatinge-Clay, Adrian T.; Gay, Darren C.

2014-01-01

The ability to rapidly customize an expression vector of choice is a valuable tool for any researcher involved in high-throughput molecular cloning for protein overexpression. Unfortunately, it is common practice to amend or neglect protein targets if the gene that encodes the protein of interest is incompatible with the multiple-cloning region of a preferred expression vector. To address this issue, a method was developed to quickly exchange the multiple-cloning region of the popular expression plasmid pET-28 with a ligation-independent cloning cassette, generating pGAY-28. This cassette contains dual inverted restriction sites that reduce false positive clones by generating a linearized plasmid incapable of self-annealing after a single restriction-enzyme digest. We also establish that progressively cooling the vector and insert leads to a significant increase in ligation-independent transformation efficiency, demonstrated by the incorporation of a 10.3 kb insert into the vector. The method reported to accomplish plasmid reconstruction is uniquely versatile yet simple, relying on the strategic placement of primers combined with homologous recombination of PCR products in yeast. PMID:25304917

Vectors expressing chimeric Japanese encephalitis dengue 2 viruses.

PubMed

Wei, Y; Wang, S; Wang, X

2014-01-01

Vectors based on self-replicating RNAs (replicons) of flaviviruses are becoming powerful tool for expression of heterologous genes in mammalian cells and development of novel antiviral and anticancer vaccines. We constructed two vectors expressing chimeric viruses consisting of attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) in which the PrM/M-E genes were replaced fully or partially with those of dengue 2 virus (DENV-2). These vectors, named pJED2 and pJED2-1770 were transfected to BHK-21 cells and produced chimeric viruses JED2V and JED2-1770V, respectively. The chimeric viruses could be passaged in C6/36 but not BHK-21 cells. The chimeric viruses produced in C6/36 cells CPE 4-5 days after infection and RT-PCR, sequencing, immunofluorescence assay (IFA) and Western blot analysis confirmed the chimeric nature of produced viruses. The immunogenicity of chimeric viruses in mice was proved by detecting DENV-2 E protein-specific serum IgG antibodies with neutralization titer of 10. Successful preparation of infectious clones of chimeric JEV-DENV-2 viruses showed that JEV-based expression vectors are fully functional.

Ebolavirus Vaccines: Progress in the Fight Against Ebola Virus Disease.

PubMed

Wu, Xiao-Xin; Yao, Hang-Ping; Wu, Nan-Ping; Gao, Hai-Nv; Wu, Hai-Bo; Jin, Chang-Zhong; Lu, Xiang-Yun; Xie, Tian-Shen; Li, Lan-Juan

2015-01-01

Ebolaviruses are highly infectious pathogens that cause lethal Ebola virus disease (EVD) in humans and non-human primates (NHPs). Due to their high pathogenicity and transmissibility, as well as the potential to be misused as a bioterrorism agent, ebolaviruses would threaten the health of global populations if not controlled. In this review, we describe the origin and structure of ebolaviruses and the development of vaccines from the beginning of the 1980s, including conventional ebolavirus vaccines, DNA vaccines, Ebola virus-like particles (VLPs), vaccinia virus-based vaccines, Venezuelan equine encephalitis virus (VEEV)-like replicon particles, Kunjin virus-based vaccine, recombinant Zaire Ebolavirusx2206;VP30, recombinant cytomegalovirus (CMV)-based vaccines, recombinant rabies virus (RABV)-based vaccines, recombinant paramyxovirus-based vaccines, adenovirus-based vaccines and vesicular stomatitis virus (VSV)-based vaccines. No licensed vaccine or specific treatment is currently available to counteract ebolavirus infection, although DNA plasmids and several viral vector approaches have been evaluated as promising vaccine platforms. These vaccine candidates have been confirmed to be successful in protecting NHPs against lethal infection. Moreover, these vaccine candidates were successfully advanced to clinical trials. The present review provides an update of the current research on Ebola vaccines, with the aim of providing an overview on current prospects in the fight against EVD. © 2015 The Author(s) Published by S. Karger AG, Basel.

Importance of murine study design for testing toxicity of retroviral vectors in support of phase I trials.

PubMed

Will, Elke; Bailey, Jeff; Schuesler, Todd; Modlich, Ute; Balcik, Brenden; Burzynski, Ben; Witte, David; Layh-Schmitt, Gerlinde; Rudolph, Cornelia; Schlegelberger, Brigitte; von Kalle, Christof; Baum, Christopher; Sorrentino, Brian P; Wagner, Lars M; Kelly, Patrick; Reeves, Lilith; Williams, David A

2007-04-01

Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted pre-clinical model defined to assess their safety, in particular their risk related to insertional mutagenesis. In the murine pre-clinical study presented here, 40 test and 10 control mice were transplanted with ex vivo manipulated bone marrow cells to assess the long-term effects of the transduction of hematopoietic cells with the retroviral vector MSCV-MGMT(P140K)wc. Test mice had significant gene marking 8-12 months post-transplantation with an average of 0.93 vector copies per cell and 41.5% of peripheral blood cells expressing the transgene MGMT(P140K), thus confirming persistent vector expression. Unexpectedly, six test mice developed malignant lymphoma. No vector was detected in the tumor cells of five animals with malignancies, indicating that the malignancies were not caused by insertional mutagenesis or MGMT(P140K) expression. Mice from a concurrent study with a different transgene also revealed additional cases of vector-negative lymphomas of host origin. We conclude that the background tumor formation in this mouse model complicates safety determination of retroviral vectors and propose an improved study design that we predict will increase the relevance and accuracy of interpretation of pre-clinical mouse studies.

Intramammary expression and therapeutic effect of a human lysozyme-expressing vector for treating bovine mastitis*

PubMed Central

Sun, Huai-Chang; Xue, Fang-Ming; Qian, Ke; Fang, Hao-Xia; Qiu, Hua-Lei; Zhang, Xin-Yu; Yin, Zhao-Hua

2006-01-01

To develop a gene therapy strategy for treating bovine mastitis, a new mammary-specific vector containing human lysozyme (hLYZ) cDNA and kanamycin resistance gene was constructed for intramammary expression and clinical studies. After one time acupuncture or intracisternal infusion of healthy cows with 400 μg of the p215C3LYZ vector, over 2.0 μg/ml of rhLYZ could be detected by enzymatic assay for about 3 weeks in the milk samples. Western blotting showed that rhLYZ secreted into milk samples from the vector-injected cows had molecular weight similar to that of the natural hLYZ in human colostrums. Twenty days after the primary injection, the quarters were re-injected with the same vector by quarter acupuncture and even higher concentrations of rhLYZ could be detected. Indirect competitive ELISA of milk samples showed that the vector injection did not induce detectable humoral immune response against hLYZ. Clinical studies showed that twice acupuncture of quarters with the p215C3LYZ vector had overt therapeutic effect on clinical and subclinical mastitis previously treated with antibiotics, including disappearance of clinical symptoms and relatively high microbiological cure rates. These data provide a solid rationale for using the vector to develop gene therapy for treating bovine mastitis. PMID:16532537

Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer.

PubMed

Buchlis, George; Podsakoff, Gregory M; Radu, Antonetta; Hawk, Sarah M; Flake, Alan W; Mingozzi, Federico; High, Katherine A

2012-03-29

In previous work we transferred a human factor IX-encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer.

Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer

PubMed Central

Buchlis, George; Podsakoff, Gregory M.; Radu, Antonetta; Hawk, Sarah M.; Flake, Alan W.; Mingozzi, Federico

2012-01-01

In previous work we transferred a human factor IX–encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer. PMID:22271447

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476.

PubMed

Srivastava, Preeti; Deb, J K

2002-07-02

A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coli was observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.

Heterologous prime-boost immunization of Newcastle disease virus vectored vaccines protected broiler chickens against highly pathogenic avian influenza and Newcastle disease viruses.

PubMed

Kim, Shin-Hee; Samal, Siba K

2017-07-24

Avian Influenza virus (AIV) is an important pathogen for both human and animal health. There is a great need to develop a safe and effective vaccine for AI infections in the field. Live-attenuated Newcastle disease virus (NDV) vectored AI vaccines have shown to be effective, but preexisting antibodies to the vaccine vector can affect the protective efficacy of the vaccine in the field. To improve the efficacy of AI vaccine, we generated a novel vectored vaccine by using a chimeric NDV vector that is serologically distant from NDV. In this study, the protective efficacy of our vaccines was evaluated by using H5N1 highly pathogenic avian influenza virus (HPAIV) strain A/Vietnam/1203/2004, a prototype strain for vaccine development. The vaccine viruses were three chimeric NDVs expressing the hemagglutinin (HA) protein in combination with the neuraminidase (NA) protein, matrix 1 protein, or nonstructural 1 protein. Comparison of their protective efficacy between a single and prime-boost immunizations indicated that prime immunization of 1-day-old SPF chicks with our vaccine viruses followed by boosting with the conventional NDV vector strain LaSota expressing the HA protein provided complete protection of chickens against mortality, clinical signs and virus shedding. Further verification of our heterologous prime-boost immunization using commercial broiler chickens suggested that a sequential immunization of chickens with chimeric NDV vector expressing the HA and NA proteins following the boost with NDV vector expressing the HA protein can be a promising strategy for the field vaccination against HPAIVs and against highly virulent NDVs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

PubMed Central

David, Marion; Lécorché, Pascaline; Masse, Maxime; Faucon, Aude; Abouzid, Karima; Gaudin, Nicolas; Varini, Karine; Gassiot, Fanny; Ferracci, Géraldine; Jacquot, Guillaume; Vlieghe, Patrick

2018-01-01

Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells—or organs that express the LDLR. PMID:29485998

Application of Genomics for Understanding Plant Virus-Insect Vector Interactions and Insect Vector Control.

PubMed

Kaur, Navneet; Hasegawa, Daniel K; Ling, Kai-Shu; Wintermantel, William M

2016-10-01

The relationships between plant viruses and their vectors have evolved over the millennia, and yet, studies on viruses began <150 years ago and investigations into the virus and vector interactions even more recently. The advent of next generation sequencing, including rapid genome and transcriptome analysis, methods for evaluation of small RNAs, and the related disciplines of proteomics and metabolomics offer a significant shift in the ability to elucidate molecular mechanisms involved in virus infection and transmission by insect vectors. Genomic technologies offer an unprecedented opportunity to examine the response of insect vectors to the presence of ingested viruses through gene expression changes and altered biochemical pathways. This review focuses on the interactions between viruses and their whitefly or thrips vectors and on potential applications of genomics-driven control of the insect vectors. Recent studies have evaluated gene expression in vectors during feeding on plants infected with begomoviruses, criniviruses, and tospoviruses, which exhibit very different types of virus-vector interactions. These studies demonstrate the advantages of genomics and the potential complementary studies that rapidly advance our understanding of the biology of virus transmission by insect vectors and offer additional opportunities to design novel genetic strategies to manage insect vectors and the viruses they transmit.

Gene therapy using retrovirus vectors: vector development and biosafety at clinical trials.

PubMed

Doi, Knayo; Takeuchi, Yasuhiro

2015-01-01

Retrovirus vectors (gammaretroviral and lentiviral vectors) have been considered as promising tools to transfer therapeutic genes into patient cells because they can permanently integrate into host cellular genome. To treat monogenic, inherited diseases, retroviral vectors have been used to add correct genes into patient cells. Conventional gammaretroviral vectors achieved successful results in clinical trials: treated patients had therapeutic gene expression in target cells and had improved symptoms of diseases. However, serious side-effects of leukemia occurred, caused by retroviral insertional mutagenesis (IM). These incidences stressed the importance of monitoring vector integration sites in patient cells as well as of re-consideration on safer vectors. More recently lentiviral vectors which can deliver genes into non-dividing cells started to be used in clinical trials including neurological disorders, showing their efficacy. Vector integration site analysis revealed that lentiviruses integrate less likely to near promoter regions of oncogenes than gammaretroviruses and no adverse events have been reported in lentiviral vector-mediated gene therapy clinical trials. Therefore lentiviral vectors have promises to be applied to a wide range of common diseases in near future. For example, T cells from cancer patients were transduced to express chimeric T cell receptors recognizing their tumour cells enhancing patients' anti-cancer immunity.

Chimeric Bovine/Human Parainfluenza Virus Type 3 Expressing Respiratory Syncytial Virus (RSV) F Glycoprotein: Effect of Insert Position on Expression, Replication, Immunogenicity, Stability, and Protection against RSV Infection

PubMed Central

Munir, Shirin; Amaro-Carambot, Emerito; Surman, Sonja; Mackow, Natalie; Yang, Lijuan; Buchholz, Ursula J.; Collins, Peter L.; Schaap-Nutt, Anne

2014-01-01

ABSTRACT A recombinant chimeric bovine/human parainfluenza type 3 virus (rB/HPIV3) vector expressing the respiratory syncytial virus (RSV) fusion F glycoprotein previously exhibited disappointing levels of RSV F immunogenicity and genetic stability in children (D. Bernstein et al., Pediatr. Infect. Dis. J. 31:109–114, 2012; C.-F. Yang et al., Vaccine 31:2822–2827, 2013). To investigate parameters that might affect vaccine performance and stability, we constructed and characterized rB/HPIV3 viruses expressing RSV F from the first (pre-N), second (N-P), third (P-M), and sixth (HN-L) genome positions. There was a 30- to 69-fold gradient in RSV F expression from the first to the sixth position. The inserts moderately attenuated vector replication in vitro and in the upper and lower respiratory tracts of hamsters: this was not influenced by the level of RSV F expression and syncytium formation. Surprisingly, inserts in the second, third, and sixth positions conferred increased temperature sensitivity: this was greatest for the third position and was the most attenuating in vivo. Each rB/HPIV3 vector induced a high titer of neutralizing antibodies in hamsters against RSV and HPIV3. Protection against RSV challenge was greater for position 2 than for position 6. Evaluation of insert stability suggested that RSV F is under selective pressure to be silenced during vector replication in vivo, but this was not exacerbated by a high level of RSV F expression and generally involved a small percentage of recovered vector. Vector passaged in vitro accumulated mutations in the HN open reading frame, causing a dramatic increase in plaque size that may have implications for vaccine production and immunogenicity. IMPORTANCE The research findings presented here will be instrumental for improving the design of a bivalent pediatric vaccine for respiratory syncytial virus and parainfluenza virus type 3, two major causes of severe respiratory tract infection in infants and young children. Moreover, this knowledge has general application to the development and clinical evaluation of other mononegavirus vectors and vaccines. PMID:24478424

Comparison of HIV- and EIAV-based vectors on their efficiency in transducing murine and human hematopoietic repopulating cells.

PubMed

Siapati, Elena K; Bigger, Brian W; Miskin, James; Chipchase, Daniel; Parsley, Kathryn L; Mitrophanous, Kyriacos; Themis, Mike; Thrasher, Adrian J; Bonnet, Dominique

2005-09-01

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.

In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

PubMed

Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

2017-12-01

Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a detailed comparison of production yields reached by injection vs oral infections for different recombinant proteins. In conclusion, these results open the possibility of future industrial scaling-up production of recombinant proteins in insect larvae by reducing manual operations. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: Formation of dense fluorescent aggregates for sensitive virus tracking

USDA-ARS?s Scientific Manuscript database

A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering cycle 3 GFP (GFP) cistron between P1 and HC-Pro cistrons with several catalytic/cleavage peptides at the C-terminus of GFP. WSMV-GFP vectors with the Foot-and-mouth disease virus 1D/2A or 2A catalytic...

Alpharetroviral Vector-mediated Gene Therapy for X-CGD: Functional Correction and Lack of Aberrant Splicing

PubMed Central

Kaufmann, Kerstin B.; Brendel, Christian; Suerth, Julia D.; Mueller-Kuller, Uta; Chen-Wichmann, Linping; Schwäble, Joachim; Pahujani, Shweta; Kunkel, Hana; Schambach, Axel; Baum, Christopher; Grez, Manuel

2013-01-01

Comparative integrome analysis has revealed that the most neutral integration pattern among retroviruses is attributed to alpharetroviruses. We chose X-linked chronic granulomatous disease (X-CGD) as model to evaluate the potential of self-inactivating (SIN) alpharetroviral vectors for gene therapy of monogenic diseases. Therefore, we combined the alpharetroviral vector backbone with the elongation factor-1α short promoter, both considered to possess a low genotoxic profile, to drive transgene (gp91phox) expression. Following efficient transduction transgene expression was sustained and provided functional correction of the CGD phenotype in a cell line model at low vector copy number. Further analysis in a murine X-CGD transplantation model revealed gene-marking of bone marrow cells and oxidase positive granulocytes in peripheral blood. Transduction of human X-CGD CD34+ cells provided functional correction up to wild-type levels and long-term expression upon transplantation into a humanized mouse model. In contrast to lentiviral vectors, no aberrantly spliced transcripts containing cellular exons fused to alpharetroviral sequences were found in transduced cells, implying that the safety profile of alpharetroviral vectors may extend beyond their neutral integration profile. Taken together, this highlights the potential of this SIN alpharetroviral system as a platform for new candidate vectors for future gene therapy of hematopoietic disorders. PMID:23207695

Gene Therapy Vectors with Enhanced Transfection Based on Hydrogels Modified with Affinity Peptides

PubMed Central

Shepard, Jaclyn A.; Wesson, Paul J.; Wang, Christine E.; Stevans, Alyson C.; Holland, Samantha J.; Shikanov, Ariella; Grzybowski, Bartosz A.; Shea, Lonnie D.

2011-01-01

Regenerative strategies for damaged tissue aim to present biochemical cues that recruit and direct progenitor cell migration and differentiation. Hydrogels capable of localized gene delivery are being developed to provide a support for tissue growth, and as a versatile method to induce the expression of inductive proteins; however, the duration, level, and localization of expression isoften insufficient for regeneration. We thus investigated the modification of hydrogels with affinity peptides to enhance vector retention and increase transfection within the matrix. PEG hydrogels were modified with lysine-based repeats (K4, K8), which retained approximately 25% more vector than control peptides. Transfection increased 5- to 15-fold with K8 and K4 respectively, over the RDG control peptide. K8- and K4-modified hydrogels bound similar quantities of vector, yet the vector dissociation rate was reduced for K8, suggesting excessive binding that limited transfection. These hydrogels were subsequently applied to an in vitro co-culture model to induce NGF expression and promote neurite outgrowth. K4-modified hydrogels promoted maximal neurite outgrowth, likely due to retention of both the vector and the NGF. Thus, hydrogels modified with affinity peptides enhanced vector retention and increased gene delivery, and these hydrogels may provide a versatile scaffold for numerous regenerative medicine applications. PMID:21514659

Immunogenic Subviral Particles Displaying Domain III of Dengue 2 Envelope Protein Vectored by Measles Virus

PubMed Central

Harahap-Carrillo, Indira S.; Ceballos-Olvera, Ivonne; Reyes-del Valle, Jorge

2015-01-01

Vaccines against dengue virus (DV) are commercially nonexistent. A subunit vaccination strategy may be of value, especially if a safe viral vector acts as biologically active adjuvant. In this paper, we focus on an immunoglobulin-like, independently folded domain III (DIII) from DV 2 envelope protein (E), which contains epitopes that elicits highly specific neutralizing antibodies. We modified the hepatitis B small surface antigen (HBsAg, S) in order to display DV 2 DIII on a virus-like particle (VLP), thus generating the hybrid antigen DIII-S. Two varieties of measles virus (MV) vectors were developed to express DIII-S. The first expresses the hybrid antigen from an additional transcription unit (ATU) and the second additionally expresses HBsAg from a separate ATU. We found that this second MV vectoring the hybrid VLPs displaying DIII-S on an unmodified HBsAg scaffold were immunogenic in MV-susceptible mice (HuCD46Ge-IFNarko), eliciting robust neutralizing responses (averages) against MV (1:1280 NT90), hepatitis B virus (787 mIU/mL), and DV2 (1:160 NT50) in all of the tested animals. Conversely, the MV vector expressing only DIII-S induced immunity against MV alone. In summary, DV2 neutralizing responses can be generated by displaying E DIII on a scaffold of HBsAg-based VLPs, vectored by MV. PMID:26350592

Coronavirus Attachment and Replication

DTIC Science & Technology

1988-03-28

is also inhibited by protease treatment (Richter, 1976). Rhabdovirus binding to host cells was inhibited by phosholipase and neuraminadase, but not...protease treatment of host cells. Therefore the rhabdovirus receptor was postulated to be a glyco- or phospholipid (Wunner et &,. 1984). More...Rabies virus and VSV competed for binding on cultured neural and non-neural cells, suggesting a common receptor for rhabdoviruses (Wunner et g1, 1984

Bimolecular Complementation to Visualize Filovirus VP40-Host Complexes in Live Mammalian Cells: Toward the Identification of Budding Inhibitors

DTIC Science & Technology

2011-01-01

of virus particles; however, mechanistic details of the forma - tion, dynamics, and trafficking of these virus-host complexes in the natural...17, pp. 9173–9182, 2003. [18] T. Irie , J. M. Licata, and R. N. Harty, “Functional character- ization of Ebola virus L-domains using VSV recombinants

Viral Immunotherapy to Eradicate Subclinical Brain Metastases

DTIC Science & Technology

2012-09-01

1 AD_________________ Award Number: W81XWH-11-1-0124 TITLE: Viral Immunotherapy to...Annual 3. DATES COVERED 1 September 2011 – 31 August 2012 4. TITLE AND SUBTITLE Viral Immunotherapy to Eradicate Subclinical Brain Metastases...re-activated to enter and destroy early BM by viral infection of Her2-positive breast BM by a recombinant vesicular stomatitis virus (VSV), which

Core labeling of adenovirus with EGFP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Le, Long P.; Le, Helen N.; Nelson, Amy R.

2006-08-01

The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expressionmore » vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology.« less

A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tratschin, J.D.; West, M.H.P.; Sandbank, T.

1984-10-01

The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/more » (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.« less

HBx drives alpha fetoprotein expression to promote initiation of liver cancer stem cells through activating PI3K/AKT signal pathway.

PubMed

Zhu, Mingyue; Li, Wei; Lu, Yan; Dong, Xu; Lin, Bo; Chen, Yi; Zhang, Xueer; Guo, Junli; Li, Mengsen

2017-03-15

Hepatitis B virus (HBV)-X protein (HBx) plays critical role in inducing the malignant transformation of liver cells. Alpha fetoprotein (AFP) expression is closely related to hepatocarcinogenesis. We report that Oct4, Klf4, Sox2 and c-myc expression positively associated with AFP(+)/HBV(+) hepatocellular carcinoma(HCC) tissues, and the expression of the stemness markers CD44, CD133 and EpCAM was significantly higher in AFP(+)/HBV(+) HCC tissues compared to normal liver tissues or AFP (-)/HBV(-) HCC tissues. AFP expression turned on prior to expression of Oct4, Klf4, Sox2 and c-myc, and the stemness markers CD44, CD133 and EpCAM in the normal human liver L-02 cell line or CHL cell lines upon transfection with MCV-HBx vectors. Stem-like cells generated more tumour colonies compared to primary cells, and xenografts induced tumourigenesis in nude mice. Expression of reprogramming-related proteins was significantly enhanced in HLE cells while transfected with pcDNA3.1-afp vectors. The specific PI3K inhibitor Ly294002 inhibited the effects of pcDNA3.1-afp vectors. AFP-siRNA vectors were able to inhibit tumour colony formation and reprogramming-related gene expression. Altogether, HBx stimulates AFP expression to induce natural reprogramming of liver cells, and AFP plays a critical role in promoting the initiation of HCC progenitor/stem cells. AFP may be a potential novel biotarget for combating HBV-induced hepatocarcinogenesis. © 2016 UICC.

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators.

PubMed

Cui, Yulin; Zhao, Jialin; Hou, Shichang; Qin, Song

2016-05-01

On the basis of fundamental genetic transformation technologies, the goal of this study was to optimize Tetraselmis subcordiformis chloroplast transformation through the use of endogenous regulators. The genes rrn16S, rbcL, psbA, and psbC are commonly highly expressed in chloroplasts, and the regulators of these genes are often used in chloroplast transformation. For lack of a known chloroplast genome sequence, the genome-walking method was used here to obtain full sequences of T. subcordiformis endogenous regulators. The resulting regulators, including three promoters, two terminators, and a ribosome combination sequence, were inserted into the previously constructed plasmid pPSC-R, with the egfp gene included as a reporter gene, and five chloroplast expression vectors prepared. These vectors were successfully transformed into T. subcordiformis by particle bombardment and the efficiency of each vector tested by assessing EGFP fluorescence via microscopy. The results showed that these vectors exhibited higher efficiency than the former vector pPSC-G carrying exogenous regulators, and the vector pRFA with Prrn, psbA-5'RE, and TpsbA showed the highest efficiency. This research provides a set of effective endogenous regulators for T. subcordiformis and will facilitate future fundamental studies of this alga.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

PubMed

Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G; Corydon, Thomas J; Mikkelsen, Jacob Giehm; Aagaard, Lars

2015-08-01

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin

PubMed Central

Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G.; Corydon, Thomas J.; Mikkelsen, Jacob Giehm; Aagaard, Lars

2015-01-01

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo. PMID:26204415

Attenuated Human Parainfluenza Virus Type 1 Expressing the Respiratory Syncytial Virus (RSV) Fusion (F) Glycoprotein from an Added Gene: Effects of Prefusion Stabilization and Packaging of RSV F.

PubMed

Liu, Xiang; Liang, Bo; Ngwuta, Joan; Liu, Xueqiao; Surman, Sonja; Lingemann, Matthias; Kwong, Peter D; Graham, Barney S; Collins, Peter L; Munir, Shirin

2017-11-15

Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the prefusion (pre-F) conformation by previously described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane and cytoplasmic tail (TMCT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild-type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine. IMPORTANCE RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a stabilized attenuating mutation, to express the RSV F glycoprotein bearing amino acid substitutions that increase its stability in the pre-F form, the most immunogenic form that elicits highly functional virus-neutralizing antibodies. RSV F was expressed from the pre-N or N-P gene position of the rHPIV1 vector as a full-length protein or as a chimeric form with its TMCT domain derived from HPIV1 F. TMCT modification greatly increased packaging of RSV F into the vector particles but also increased vector attenuation in vivo , resulting in reduced immunogenicity. In contrast, full-length RSV F expressed from the pre-N position was immunogenic, eliciting complement-independent RSV-neutralizing antibodies and providing protection against RSV challenge. Copyright © 2017 American Society for Microbiology.

[Construction of rAAV2-GPIIb/IIIa vector and test of its expression and function in vitro].

PubMed

Wang, Kai; Peng, Jian-Qiang; Chen, Fang-Ping; Wu, Xiao-Bin

2006-04-01

This study was aimed to explore the possibility of rAAV2 vector-mediating gene therapy for Glanzmann' s thrombasthenia. The rAAV2-GPIIb/IIIa vector was constructed. The GPIIb/IIIa gene expression in mammal cell were examined by different methods, such as: detection of mRNA expression in BHK-21 cells after 24 hours of infection (MOI = 1 x 10(5) v.g/cell) was performed by RT-PCR; the relation between MOI and quantity of GPII6/IIIa gene expression was detected by FACS after 48 hours of infection; GPIIb/IIIa protein expression in BHK-21 cells after 48 hours of infection (MOI = 10(5) v x g/cell) was assayed by Western blot, GPIIb/IIIa protein expression on cell surface was detected by immunofluorescence, and the biological function of expressing product was determined by PAC-1 conjunct experiments. The results showed that GPIIb/IIIa gene expression in mRNA level could be detected in BHK-21 cells after 24 hours of infection at MOI = 1 x 10(5) v x g/cell and the GPIIb/IIIa gene expression in protein level could be detected in BHK-21 cells after 48 hours of infection at MOI = 1 x 10(5) v x g/cell. In certain range, quantity of GPIIb/IIIa gene expression increased with MOI, but overdose of MOI decreased quantity of GPIIb/IIIa gene expression. Activated product of GPIIb/IIIa gene expression could combined with PAC-I, and possesed normal biological function. In conclusion, rAAV2 vactor can effectively mediate GPIIb and GPIIIa gene expressing in mammal cells, and the products of these genes exhibit biological function. This result may provide a basis for application of rAAV2 vector in Glanzmann's thrombasthenia gene therapy in furture.

Effective reduction of the interleukin-1β transcript in osteoarthritis-prone guinea pig chondrocytes via short hairpin RNA mediated RNA interference influences gene expression of mediators implicated in disease pathogenesis.

PubMed

Santangelo, K S; Bertone, A L

2011-12-01

To ascertain a viral vector-based short hairpin RNA (shRNA) capable of reducing the interleukin-1β (IL-1β) transcript in osteoarthritis (OA)-prone chondrocytes and detect corresponding changes in the expression patterns of several critical disease mediators. Cultured chondrocytes from 2-month-old Hartley guinea pigs were screened for reduction of the IL-1β transcript following plasmid-based delivery of U6-driven shRNA sequences. A successful plasmid/shRNA knockdown combination was identified and used to construct an adeno-associated virus serotype 5 (AAV5) vector for further evaluation. Relative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify in vitro transcript changes of IL-1β and an additional nine genes following transduction with this targeting knockdown vector. To validate in vitro findings, this AAV5 vector was injected into one knee, while either an equivalent volume of saline vehicle (three animals) or non-targeting control vector (three animals) were injected into opposite knees. Fold differences and subsequent percent gene expression levels relative to control groups were calculated using the comparative CT (2(-ΔΔCT)) method. Statistically significant decreases in IL-1β expression were achieved by the targeting knockdown vector relative to both the mock-transduced control and non-targeting vector control groups in vitro. Transcript levels of anabolic transforming growth factor-β (TGF-β) were significantly increased by use of this targeting knockdown vector. Transduction with this targeting AAV5 vector also significantly decreased the transcript levels of key inflammatory cytokines [tumor necrosis factor-α (TNF-α), IL-2, IL-8, and IL-12] and catabolic agents [matrix metalloproteinase (MMP)13, MMP2, interferon-γ (IFN-γ), and inducible nitrous oxide synthase (iNOS)] relative to both mock-transduced and non-targeting vector control groups. In vivo application of this targeting knockdown vector resulted in a >50% reduction (P=0.0045) or >90% (P=0.0001) of the IL-1β transcript relative to vehicle-only or non-targeting vector control exposed cartilage, respectively. Successful reduction of the IL-1β transcript was achieved via RNA interference (RNAi) techniques. Importantly, this alteration significantly influenced the transcript levels of several major players involved in OA pathogenesis in the direction of disease modification. Investigations to characterize additional gene expression changes influenced by targeting knockdown AAV5 vector-based diminution of the IL-1β transcript in vivo are warranted. Copyright © 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Epigenetic Control of Prostate Cancer Metastasis: Role of Runx2 Phosphorylation

DTIC Science & Technology

2014-04-01

prostate cancer cells. In the third budget year, we achieved the following: a. Generation of retrovirus and lentivirus vectors expressing WT RUNX2 and S301A... retrovirus vectors will be developed that express β-galactosidase (negative control), wild type Runx2, S301A/S319A (non-phosphorylated) or S301E/S310E...constitutively active) Runx2 mutants. As described last year, retrovirus and lentivirus vectors were constructed to stably introduce wild type and mutant

Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron.

PubMed

Gäken, J; Jiang, J; Daniel, K; van Berkel, E; Hughes, C; Kuiper, M; Darling, D; Tavassoli, M; Galea-Lauri, J; Ford, K; Kemeny, M; Russell, S; Farzaneh, F

2000-12-01

Transduction of cells with multiple genes, allowing their stable and co-ordinated expression, is difficult with the available methodologies. A method has been developed for expression of multiple gene products, as fusion proteins, from a single cistron. The encoded proteins are post-synthetically cleaved and processed into each of their constituent proteins as individual, biologically active factors. Specifically, linkers encoding cleavage sites for the Golgi expressed endoprotease, furin, have been incorporated between in-frame cDNA sequences encoding different secreted or membrane bound proteins. With this strategy we have developed expression vectors encoding multiple proteins (IL-2 and B7.1, IL-4 and B7.1, IL-4 and IL-2, IL-12 p40 and p35, and IL-12 p40, p35 and IL-2 ). Transduction and analysis of over 100 individual clones, derived from murine and human tumour cell lines, demonstrate the efficient expression and biological activity of each of the encoded proteins. Fusagene vectors enable the co-ordinated expression of multiple gene products from a single, monocistronic, expression cassette.

Design of retrovirus vectors for transfer and expression of the human. beta. -globin gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, A.D.; Bender, M.A.; Harris, E.A.S.

1988-11-01

Regulated expression of the human ..beta..-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective ..beta..-globin genes. The authors found regions that interfered with virus production within intron 2 of the ..beta..-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for ..beta..-globin expression. Inclusion of ..beta..-globin introns necessitatesmore » an antisense orientation of the gene within the retrovirus vector. However, they found no effect of the antisense ..beta..-globin transcription on virus production. A region downstream of the ..beta..-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10/sup 6/ CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human ..beta..-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human ..beta..-globin gene in hematopoietic cells (CFU-S cells) in mice.« less

Spontaneous Mutation at Amino Acid 544 of the Ebola Virus Glycoprotein Potentiates Virus Entry and Selection in Tissue Culture.

PubMed

Ruedas, John B; Ladner, Jason T; Ettinger, Chelsea R; Gummuluru, Suryaram; Palacios, Gustavo; Connor, John H

2017-08-01

Ebolaviruses have a surface glycoprotein (GP 1,2 ) that is required for virus attachment and entry into cells. Mutations affecting GP 1,2 functions can alter virus growth properties. We generated a recombinant vesicular stomatitis virus encoding Ebola virus Makona variant GP 1,2 (rVSV-MAK-GP) and observed emergence of a T544I mutation in the Makona GP 1,2 gene during tissue culture passage in certain cell lines. The T544I mutation emerged within two passages when VSV-MAK-GP was grown on Vero E6, Vero, and BS-C-1 cells but not when it was passaged on Huh7 and HepG2 cells. The mutation led to a marked increase in virus growth kinetics and conferred a robust growth advantage over wild-type rVSV-MAK-GP on Vero E6 cells. Analysis of complete viral genomes collected from patients in western Africa indicated that this mutation was not found in Ebola virus clinical samples. However, we observed the emergence of T544I during serial passage of various Ebola Makona isolates on Vero E6 cells. Three independent isolates showed emergence of T544I from undetectable levels in nonpassaged virus or virus passaged once to frequencies of greater than 60% within a single passage, consistent with it being a tissue culture adaptation. Intriguingly, T544I is not found in any Sudan, Bundibugyo, or Tai Forest ebolavirus sequences. Furthermore, T544I did not emerge when we serially passaged recombinant VSV encoding GP 1,2 from these ebolaviruses. This report provides experimental evidence that the spontaneous mutation T544I is a tissue culture adaptation in certain cell lines and that it may be unique for the species Zaire ebolavirus IMPORTANCE The Ebola virus (Zaire) species is the most lethal species of all ebolaviruses in terms of mortality rate and number of deaths. Understanding how the Ebola virus surface glycoprotein functions to facilitate entry in cells is an area of intense research. Recently, three groups independently identified a polymorphism in the Ebola glycoprotein (I544) that enhanced virus entry, but they did not agree in their conclusions regarding its impact on pathogenesis. Our findings here address the origins of this polymorphism and provide experimental evidence showing that it is the result of a spontaneous mutation (T544I) specific to tissue culture conditions, suggesting that it has no role in pathogenesis. We further show that this mutation may be unique to the species Zaire ebolavirus , as it does not occur in Sudan, Bundibugyo, and Tai Forest ebolaviruses. Understanding the mechanism behind this mutation can provide insight into functional differences that exist in culture conditions and among ebolavirus glycoproteins. Copyright © 2017 American Society for Microbiology.

Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomono, Takumi; Kajita, Masahiro; Yano, Kentaro

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNAmore » expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. -- Highlights: •Adenovirus vector infection induced P-gp mRNA expression in three NSCLC cell lines. •Adenovirus vector infection enhanced P-gp-mediated doxorubicin efflux from the cells. •The increase of P-gp was not mediated by nuclear receptors (PXR, CAR) or COX-2.« less

The Host Range of Gammaretroviruses and Gammaretroviral Vectors Includes Post-Mitotic Neural Cells

PubMed Central

Liu, Xiu-Huai; Xu, Wenqin; Russ, Jill; Eiden, Lee E.; Eiden, Maribeth V.

2011-01-01

Background Gammaretroviruses and gammaretroviral vectors, in contrast to lentiviruses and lentiviral vectors, are reported to be restricted in their ability to infect growth-arrested cells. The block to this restriction has never been clearly defined. The original assessment of the inability of gammaretroviruses and gammaretroviral vectors to infect growth-arrested cells was carried out using established cell lines that had been growth-arrested by chemical means, and has been generalized to neurons, which are post-mitotic. We re-examined the capability of gammaretroviruses and their derived vectors to efficiently infect terminally differentiated neuroendocrine cells and primary cortical neurons, a target of both experimental and therapeutic interest. Methodology/Principal Findings Using GFP expression as a marker for infection, we determined that both growth-arrested (NGF-differentiated) rat pheochromocytoma cells (PC12 cells) and primary rat cortical neurons could be efficiently transduced, and maintained long-term protein expression, after exposure to murine leukemia virus (MLV) and MLV-based retroviral vectors. Terminally differentiated PC12 cells transduced with a gammaretroviral vector encoding the anti-apoptotic protein Bcl-xL were protected from cell death induced by withdrawal of nerve growth factor (NGF), demonstrating gammaretroviral vector-mediated delivery and expression of genes at levels sufficient for therapeutic effect in non-dividing cells. Post-mitotic rat cortical neurons were also shown to be susceptible to transduction by murine replication-competent gammaretroviruses and gammaretroviral vectors. Conclusions/Significance These findings suggest that the host range of gammaretroviruses includes post-mitotic and other growth-arrested cells in mammals, and have implications for re-direction of gammaretroviral gene therapy to neurological disease. PMID:21464894

Immunological thresholds in neurological gene therapy: highly efficient elimination of transduced cells might be related to the specific formation of immunological synapses between T cells and virus-infected brain cells

PubMed Central

Barcia, Carlos; Gerdes, Christian; Xiong, Wei-Dong; Thomas, Clare E.; Liu, Chunyan; Kroeger, Kurt M.; Castro, Maria G.; Lowenstein, Pedro R.

2007-01-01

First-generation adenovirus can be engineered with powerful promoters to drive expression of therapeutic transgenes. Numerous clinical trials for glioblastoma multiforme using first generation adenoviral vectors have either been performed or are ongoing, including an ongoing, Phase III, multicenter trial in Europe and Israel (Ark Therapeutics, Inc.). Although in the absence of anti-adenovirus immune responses expression in the brain lasts 6–18 months, systemic infection with adenovirus induces immune responses that inhibit dramatically therapeutic transgene expression from first generation adenoviral vectors, thus, potentially compromising therapeutic efficacy. Here, we show evidence of an immunization threshold for the dose that generates an immune response strong enough to eliminate transgene expression from the CNS. For the systemic immunization to eliminate transgene expression from the brain, ≥1 × 107 infectious units (iu) of adenovirus need to be used as immunogen. Furthermore, this immune response eliminates >90% of transgene expression from 1 × 107–1 × 10³ iu of vector injected into the striatum 60 days earlier. Importantly, elimination of transgene expression is independent of the nature of the promoter that drives transgene expression and is accompanied by brain infiltration of CD8+ T cells and macrophages. In conclusion, once the threshold for systemic immunization (i.e. 1 × 107 iu) is crossed, the immune response eliminates transgene expression by >90% even from brains that receive as little as 1000 iu of adenoviral vectors, independently of the type of promoter that drives expression. PMID:18084640

A Molecular Toolbox for Rapid Generation of Viral Vectors to Up- or Down-Regulate Neuronal Gene Expression in vivo

PubMed Central

White, Melanie D.; Milne, Ruth V. J.; Nolan, Matthew F.

2011-01-01

We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins, and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV) or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1, and Kir3.2) and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miRNA). We show that AAV assembled to express HCN1 miRNA produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miRNA with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience. PMID:21772812

Cardiac Gene Therapy: Optimization of Gene Delivery Techniques In Vivo

PubMed Central

Katz, Michael G.; Swain, JaBaris D.; White, Jennifer D.; Low, David; Stedman, Hansell

2010-01-01

Abstract Vector-mediated cardiac gene therapy holds tremendous promise as a translatable platform technology for treating many cardiovascular diseases. The ideal technique is one that is efficient and practical, allowing for global cardiac gene expression, while minimizing collateral expression in other organs. Here we survey the available in vivo vector-mediated cardiac gene delivery methods—including transcutaneous, intravascular, intramuscular, and cardiopulmonary bypass techniques—with consideration of the relative merits and deficiencies of each. Review of available techniques suggests that an optimal method for vector-mediated gene delivery to the large animal myocardium would ideally employ retrograde and/or anterograde transcoronary gene delivery,extended vector residence time in the coronary circulation, an increased myocardial transcapillary gradient using physical methods, increased endothelial permeability with pharmacological agents, minimal collateral gene expression by isolation of the cardiac circulation from the systemic, and have low immunogenicity. PMID:19947886

[Construction of the superantigen SEA transfected laryngocarcinoma cells].

PubMed

Ji, Xiaobin; Jingli, J V; Liu, Qicai; Xie, Jinghua

2013-04-01

To construct an eukaryotic expression vectors containing superantigen staphylococcal enterotoxin A (SEA) gene, and to identify its expression in laryngeal squamous carcinoma cells. SEA full-length gene fragment was obtained from ATCC13565 genome of the staphylococcus, referencing standard strains producing SEA. Coding sequence of SEA was artificially synthetized. Than, SEA gene fragments was subcloned into eukaryotic expression vector pIRES-EGFP. The recombinant plasmid pSEA-IRES-EGFP was constructed and was transfected to laryngocarcinoma Hep-2 cells. Resistant clones were screened by G418. The expression of SEA in laryngocarcinoma cells was identified with ELISA and RT-PCR method. The subclone of artificially synthetized SEA gene was subclone to eukaryotic expression vector pires-EGFP. Flanking sequence confirmed that SEA sequence was fully identical to the coding sequence of standard staphylococcus strains ATCC13565 in Genbank. After recombinant plasmid transfected to laryngocarcinoma cells, the resistant clones was obtained after screening for two weeks. The clones were selected. The specific gene fragment was obtained by RT-PCR amplification. ELISA assay confirmed that the content of SEA protein in supernatant fluid of cell culture had reached about Pg level. The recombinant eukaryotic expression vector containing superantigen SEA gene is successfully constructed, and is capable of effective expression and continued secretion of SEA protein in laryngochrcinoma Hep-2 cells after recombinant plasmid transfected to laryngocarcinoma cells.

A universal expression/silencing vector in plants.

PubMed

Peretz, Yuval; Mozes-Koch, Rita; Akad, Fuad; Tanne, Edna; Czosnek, Henryk; Sela, Ilan

2007-12-01

A universal vector (IL-60 and auxiliary constructs), expressing or silencing genes in every plant tested to date, is described. Plants that have been successfully manipulated by the IL-60 system include hard-to-manipulate species such as wheat (Triticum duram), pepper (Capsicum annuum), grapevine (Vitis vinifera), citrus, and olive (Olea europaea). Expression or silencing develops within a few days in tomato (Solanum lycopersicum), wheat, and most herbaceous plants and in up to 3 weeks in woody trees. Expression, as tested in tomato, is durable and persists throughout the life span of the plant. The vector is, in fact, a disarmed form of Tomato yellow leaf curl virus, which is applied as a double-stranded DNA and replicates as such. However, the disarmed virus does not support rolling-circle replication, and therefore viral progeny single-stranded DNA is not produced. IL-60 does not integrate into the plant's genome, and the construct, including the expressed gene, is not heritable. IL-60 is not transmitted by the Tomato yellow leaf curl virus's natural insect vector. In addition, artificial satellites were constructed that require a helper virus for replication, movement, and expression. With IL-60 as the disarmed helper "virus," transactivation occurs, resulting in an inducible expressing/silencing system. The system's potential is demonstrated by IL-60-derived suppression of a viral-silencing suppressor of Grapevine virus A, resulting in Grapevine virus A-resistant/tolerant plants.

[Construction of a plant effective expression vector containing the gene of hepatitis B virus surface antigen].

PubMed

Lin, Bing-Ying; Jin, Zhi-Qiang; Li, Mei

2006-11-01

To construct a plant effective expression vector driven by a fruit specific promoter for the expression of hepatitis B virus surface antigen (HBsAg), to further improve the expression of exogenous gene in plant, and to prepare for the development of an effective anti-hepatitis vaccine. Tomato fruit-specific promoters' gene 2A12 and E8 were respectively introduced to pBPFOmega7 to form pB2A12 and pBE8. The DNA fragment containing HBsAg-s gene from plasmid YEP-HBs was inserted respectively into pB2A12 and pBE8 to form pB2A12-HBs and pBE8-HBs. The fragment containing "p35S+2A12+Omega+HBsAg-s+Tnos" of the pB2A12-HBs was sub-cloned into plasmid pCAMBIA1301 to yield the reconstructed plant binary expression plasmid pCAM2A12-HBs, and the fragment containing "p35S+E8+Omega+HBsAg-s+Tnos" of the pBE8-HBs was sub-cloned into plasmid pCAMBIA1301 to yield the plasmid pCAME8-HBs. The inserted gene HBsAg and fruit-specific promoters in the reconstructed plant binary expression vectors were confirmed by sequencing. Then, pCAM2A12-HBs and pCAME8-HBs were directly introduced into Agrobacterium tumefaciens strain EHA105. Digestion with restriction enzymes proved that all recombinant vectors had the inserts with expected length of the target fragments, and the sequencing results were confirmed correct. In this study, plant expression vector containing HBsAg gene driven by fruit specific promoter and CaMV35s promoter was successfully constructed.

A microRNA embedded AAV alpha-synuclein gene silencing vector for dopaminergic neurons

PubMed Central

Han, Ye; Khodr, Christina E.; Sapru, Mohan K.; Pedapati, Jyothi; Bohn, Martha C.

2011-01-01

Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro. The same hSNCA silencing sequence (shRNA) was used in both vectors, but in one vector, the shRNA was embedded in a microRNA backbone and driven by a pol II promoter, and in the other the shRNA was not embedded in a microRNA and was driven by a pol III promoter. Both vectors silenced hSNCA to the same extent in 293T cells transfected with hSNCA. In DA PC12 cells, neither vector decreased expression of rat SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT). However, the mir30 embedded vector was significantly less toxic to both PC12 and SH-SY5Y cells. Our in vitro data suggest that this miRNA-embedded silencing vector may be ideal for chronic in vivo SNCA gene silencing in DA neurons. PMID:21338582

Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

PubMed Central

2017-01-01

ABSTRACT Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in “enhancerless” self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required. IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy. PMID:29046446

In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos.

PubMed

Gui, Tao; Zhang, Meiling; Chen, Jianwen; Zhang, Yuanliang; Zhou, Naru; Zhang, Yu; Tao, Jia; Sui, Liucai; Li, Yunsheng; Liu, Ya; Zhang, Xiaorong; Zhang, Yunhai

2012-08-01

A vector expressing human lysozyme (pBC1-hLYZ-GFP-Neo) was evaluated for gene and protein expression following liposome-mediated transformation of C-127 mouse mammary cancer cells. Cultures of G418-resistant clones were harvested 24-72 h after induction with prolactin, insulin and hydrocortisone. Target gene expression was analyzed by RT-PCR and Western blot and recombinant human lysozyme (rhLYZ) bacteriostatic activity was also evaluated. The hLYZ gene was correctly transcribed and translated in C-127 cells and hLYZ inhibited gram-positive bacterial growth, indicating the potential of this expression vector for development of a mammary gland bioreactor in goats. Guanzhong dairy goat skin fibroblasts transfected with pBC1-hLYZ-GFP-Neo were used to construct a goat embryo transgenically expressing rhLYZ by somatic nuclear transplantation with a blastocyst rate of 9.0 ± 2.8 %. These data establish the basis for cultivation of mastitis-resistant hLYZ transgenic goats.

Independent and high-level dual-gene expression in adult stem-progenitor cells from a single lentiviral vector.

PubMed

Tian, J; Andreadis, S T

2009-07-01

Expression of multiple genes from the same target cell is required in several technological and therapeutic applications such as quantitative measurements of promoter activity or in vivo tracking of stem cells. In spite of such need, reaching independent and high-level dual-gene expression cannot be reliably accomplished by current gene transfer vehicles. To address this issue, we designed a lentiviral vector carrying two transcriptional units separated by polyadenylation, terminator and insulator sequences. With this design, the expression level of both genes was as high as that yielded from lentiviral vectors containing only a single transcriptional unit. Similar results were observed with several promoters and cell types including epidermal keratinocytes, bone marrow mesenchymal stem cells and hair follicle stem cells. Notably, we demonstrated quantitative dynamic monitoring of gene expression in primary cells with no need for selection protocols suggesting that this optimized lentivirus may be useful in high-throughput gene expression profiling studies.

Expression of the core antigen gene of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors.

PubMed

Schröder, R; Maassen, A; Lippoldt, A; Börner, T; von Baehr, R; Dobrowolski, P

1991-08-01

Using the broad-host-range promoter probe vector pRS201 for cloning of phage Acm1 promoters, we established a convenient vector system for expression of heterologous genes in different Gram-negative bacteria. The usefulness of this system was demonstrated by expression of the HBV core gene in Acetobacter methanolicus. Plasmids carrying the HBV core gene downstream of different Acm1-phage promoters were transferred to A. methanolicus, a new potential host for recombinant DNA expression. Using enzyme immunoassay and immunoblot techniques, the amount and composition of core antigen produced in A. methanolicus were compared with that derived from Escherichia coli. The expression of immunoreactive core antigen in A. methanolicus exceeds by sevenfold that in E. coli using an expression system with tandemly arranged promoters. Morphological observations by electron microscopy show that the HBV core gene products isolated from both hosts are assembled into regular spherical particles with a diameter of about 28 nm that are comparable to original viral nucleocapsids.

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed Central

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-01-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population. PMID:9032306

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-03-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population.

Impact of Ebola mucin-like domain on antiglycoprotein antibody responses induced by Ebola virus-like particles.

PubMed

Martinez, Osvaldo; Tantral, Lee; Mulherkar, Nirupama; Chandran, Kartik; Basler, Christopher F

2011-11-01

Ebola virus (EBOV) glycoprotein (GP), responsible for mediating host-cell attachment and membrane fusion, contains a heavily glycosylated mucin-like domain hypothesized to shield GP from neutralizing antibodies. To test whether the mucin-like domain inhibits the production and function of anti-GP antibodies, we vaccinated mice with Ebola virus-like particles (VLPs) that express vesicular stomatitis virus G, wild-type EBOV GP (EBGP), EBOV GP without its mucin-like domain (ΔMucGP), or EBOV GP with a Crimean-Congo hemorrhagic fever virus mucin-like domain substituted for the EBOV mucin-like domain (CMsubGP). EBGP-VLP immunized mice elicited significantly higher serum antibody titers toward EBGP or its mutants, as detected by western blot analysis, than did VLP-ΔMucGP. However, EBGP-, ΔMucGP- and CMsubGP-VLP immunized mouse sera contained antibodies that bound to cell surface-expressed GP at similar levels. Furthermore, low but similar neutralizing antibody titers, measured against a vesicular stomatitis virus (VSV) expressing EBGP or ΔMucGP, were present in EBGP, ΔMucGP, and CMsubGP sera, although a slightly higher neutralizing titer (2- to 2.5-fold) was detected in ΔMucGP sera. We conclude that the EBOV GP mucin-like domain can increase relative anti-GP titers, however these titers appear to be directed, at least partly, to denatured GP. Furthermore, removing the mucin-like domain from immunizing VLPs has modest impact on neutralizing antibody titers in serum.

Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells.

PubMed

Trevisan, Marta; Desole, Giovanna; Costanzi, Giulia; Lavezzo, Enrico; Palù, Giorgio; Barzon, Luisa

2017-01-20

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

The rational design of a 'type 88' genetically stable peptide display vector in the filamentous bacteriophage fd.

PubMed

Enshell-Seijffers, D; Smelyanski, L; Gershoni, J M

2001-05-15

Filamentous bacteriophages are particularly efficient for the expression and display of combinatorial random peptides. Two phage proteins are often employed for peptide display: the infectivity protein, PIII, and the major coat protein, PVIII. The use of PVIII typically requires the expression of two pVIII genes: the wild-type and the recombinant pVIII gene, to generate mosaic phages. 'Type 88' vectors contain two pVIII genes in one phage genome. In this study a novel 'type 88' expression vector has been rationally designed and constructed. Two factors were taken into account: the insertion site and the genetic stability of the second pVIII gene. It was found that selective deletion of recombinant genes was encountered when inserts were cloned into either of the two non-coding regions of the phage genome. The deletions were independent of recA yet required a functional F-episome. Transcription was also found to be a positive factor for deletion. Taking the above into account led to the generation of a novel vector, designated fth1, which can be used to express recombinant peptides as pVIII chimeric proteins in mosaic bacteriophages. The fth1 vector is not only genetically stable but also of high copy number and produces high titers of recombinant phages.

Increased tumor localization and reduced immune response to adenoviral vector formulated with the liposome DDAB/DOPE.

PubMed

Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Abu-Asab, Mones S; Tsokos, Maria; Morris, John C; Kalle, Wouter H J

2007-04-01

We aimed to increase the efficiency of adenoviral vectors by limiting adenoviral spread from the target site and reducing unwanted host immune responses to the vector. We complexed adenoviral vectors with DDAB-DOPE liposomes to form adenovirus-liposomal (AL) complexes. AL complexes were delivered by intratumoral injection in an immunocompetent subcutaneous rat tumor model and the immunogenicity of the AL complexes and the expression efficiency in the tumor and other organs was examined. Animals treated with the AL complexes had significantly lower levels of beta-galactosidase expression in systemic tissues compared to animals treated with the naked adenovirus (NA) (P<0.05). The tumor to non-tumor ratio of beta-galactosidase marker expression was significantly higher for the AL complex treated animals. NA induced significantly higher titers of adenoviral-specific antibodies compared to the AL complexes (P<0.05). The AL complexes provided protection (immunoshielding) to the adenovirus from neutralizing antibody. Forty-seven percent more beta-galactosidase expression was detected following intratumoral injection with AL complexes compared to the NA in animals pre-immunized with adenovirus. Complexing of adenovirus with liposomes provides a simple method to enhance tumor localization of the vector, decrease the immunogenicity of adenovirus, and provide protection of the virus from pre-existing neutralizing antibodies.

Recombinant vaccines against T. gondii: comparison between homologous and heterologous vaccination protocols using two viral vectors expressing SAG1.

PubMed

Mendes, Érica Araújo; Fonseca, Flavio G; Casério, Bárbara M; Colina, Janaína P; Gazzinelli, Ricardo Tostes; Caetano, Braulia C

2013-01-01

The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune response against the parasite and a valuable tool for vaccine development. We have previously protected mice from toxoplasmosis by immunizing the animals with an adenovirus expressing the protein SAG1 (AdSAG1) of T. gondii. We are now looking for ways to improve the vaccination strategy and enhance protection. One limitation of homologous vaccinations (sequential doses of the same vector) is induction of anti-vector immune response that blocks cell transduction, restricts transgene expression and, consequently, compromises the overall outcome of vaccination. One way to avert the effects of anti-vector response is to use different viruses in prime and boost (heterologous vaccination). Bearing this in mind, we generated a modified Vaccinia Virus Ankara encoding SAG1 (MVASAG1), to be tested as boost agent after prime with AdSAG1. Although minor differences were observed in the magnitude of the anti-SAG1 immune response induced by each vaccination protocol, the heterologous immunization with AdSAG1 followed by MVASAG1 resulted in improved capacity to control brain cyst formation in a model of chronic toxoplasmosis in C57BL/6 mice.

[Study on transformation of P-dissolving Penicillium oxalicum P8 with double-marker vector expressing green fluorescent protein and hygromycin B resistance].

PubMed

Zhang, Lei; Fan, Bing-Quan; Huang, Wei-Yi

2005-12-01

P-dissolving Penicillium oxalicum P8 was isolated previously in this lab which has a considerable ability to dissolve many kinds of inorganic phosphorus and improve crop growth. In order to study rhizosphere colonization of plants by Penicillium oxalicum P8, protoplasts were transformed with a double-marker expression vector of green fluorescent protein and hygromycin B resistance. Some transformants were selected which expressed both the GFP and hygromycin B phosphotransferase and did not show significant morphological or physiological differences as compared to wild-type strain. Southern blot analysis confirmed the heterogeneous genomic integration of the vector DNA into the transformants.

[The preparation of recombinant adenovirus Ad-Rad50-GFP and detection of the optimal multiplicity of infection in CNE1 transfected hv Ad-Rad50-GFP].

PubMed

Yan, Ruicheng; Huang, Jiancong; Zhu, Ling; Chang, Lihong; Li, Jingjia; Wu, Xifu; Ye, Jin; Zhang, Gehua

2015-12-01

The optimal multiplicity of infection (MOI) of the recombinant adenovirus Ad-Rad50-GFP carrying a mutant Rad50 gene expression region on the cell growth of nasopharyngeal carcinoma and the viral amplification efficiency of CNE1 cell infected by this adenovirus were studied. The biological titer of Ad-Rad50-GFP was measured by end point dilution method. The impact of recombinant adenoviral vector transfection on the growth of CNE1 cells was observed by cell growth curve. Transfection efficacy of recombinant adenoviral vector was observed and calculated through fluorescence microscope. The expression f mutant Rad50 in the Ad-Rad50-GFP transfected CNE1 cells with optimal MOI was detected by Western Blot after transfection. The biological titer of Ad-Rad50-GFP was 1.26 x 10¹¹ pfu/ml. CNE1 cell growth was not influenced significantly as they were transfected by recombinant adenoviral vector with MOI less than 50. Transfection efficacy of recombinant adenoviral vector was most salient at 24 hours after transfection, with the high expression of mutant Rad50, and the efficiency still remained about 70% after 72 hours. Recombinant adenoviral vector Ad-Rad50-GFP could transfect CNE1 cells as well as result in the expression of mutant Rad50 in CNE1 cells effectively. MOI = 50 was the optimal multiplicity of infection of CNE1 cells transfected by recombinant adenoviral vector Ad-Rad50-GFP.

A transposase strategy for creating libraries of circularly permuted proteins.

PubMed

Mehta, Manan M; Liu, Shirley; Silberg, Jonathan J

2012-05-01

A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.

A transposase strategy for creating libraries of circularly permuted proteins

PubMed Central

Mehta, Manan M.; Liu, Shirley; Silberg, Jonathan J.

2012-01-01

A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions. PMID:22319214

Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression.

PubMed

Lim, Hyoun-Sub; Vaira, Anna Maria; Domier, Leslie L; Lee, Sung Chul; Kim, Hong Gi; Hammond, John

2010-06-20

We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS. Published by Elsevier Inc.

Direct In Vivo Reprogramming with Sendai Virus Vectors Improves Cardiac Function after Myocardial Infarction.

PubMed

Miyamoto, Kazutaka; Akiyama, Mizuha; Tamura, Fumiya; Isomi, Mari; Yamakawa, Hiroyuki; Sadahiro, Taketaro; Muraoka, Naoto; Kojima, Hidenori; Haginiwa, Sho; Kurotsu, Shota; Tani, Hidenori; Wang, Li; Qian, Li; Inoue, Makoto; Ide, Yoshinori; Kurokawa, Junko; Yamamoto, Tsunehisa; Seki, Tomohisa; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2018-01-04

Direct cardiac reprogramming holds great promise for regenerative medicine. We previously generated directly reprogrammed induced cardiomyocyte-like cells (iCMs) by overexpression of Gata4, Mef2c, and Tbx5 (GMT) using retrovirus vectors. However, integrating vectors pose risks associated with insertional mutagenesis and disruption of gene expression and are inefficient. Here, we show that Sendai virus (SeV) vectors expressing cardiac reprogramming factors efficiently and rapidly reprogram both mouse and human fibroblasts into integration-free iCMs via robust transgene expression. SeV-GMT generated 100-fold more beating iCMs than retroviral-GMT and shortened the duration to induce beating cells from 30 to 10 days in mouse fibroblasts. In vivo lineage tracing revealed that the gene transfer of SeV-GMT was more efficient than retroviral-GMT in reprogramming resident cardiac fibroblasts into iCMs in mouse infarct hearts. Moreover, SeV-GMT improved cardiac function and reduced fibrosis after myocardial infarction. Thus, efficient, non-integrating SeV vectors may serve as a powerful system for cardiac regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Assaying the Stability and Inactivation of AAV Serotype 1 Vectors

PubMed Central

Howard, Douglas B.; Harvey, Brandon K.

2017-01-01

Adeno-associated virus (AAV) vectors are a commonplace tool for gene delivery ranging from cell culture to human gene therapy. One feature that makes AAV a desirable vector is its stability, in regard to both the duration of transgene expression and retention of infectivity as a viral particle. This study examined the stability of AAV serotype 1 (AAV1) vectors under different conditions. First, transducibility after storage at 4°C decreased 20% over 7 weeks. Over 10 freeze–thaw cycles, the resulting transduction efficiency became variable at 60–120% of a single thaw. Using small stainless steel slugs to mimic a biosafety cabinet or metal lab bench surface, it was found that an AAV1 vector can be reconstituted after 6 days of storage at room temperature. The stability of AAV is a desired feature, but effective decontamination procedures must be available for safety and experimental integrity. Multiple disinfectants commonly used in the laboratory for ability to inactivate an AAV1 vector were tested, and it was found that autoclaving, 0.25% peracetic acid, iodine, or 10% Clorox bleach completely prevented AAV-mediated transgene expression. These data suggest that peracetic acid should be used for inactivating AAV1 vectors on metal-based surfaces or instruments in order to avoid inadvertent transgene expression in human cells or cross-contamination of instruments. PMID:28192678

pSW2, a Novel Low-Temperature-Inducible Gene Expression Vector Based on a Filamentous Phage of the Deep-Sea Bacterium Shewanella piezotolerans WP3.

PubMed

Yang, Xin-Wei; Jian, Hua-Hua; Wang, Feng-Ping

2015-08-15

A low-temperature-inducible protein expression vector (pSW2) based on a filamentous phage (SW1) of the deep-sea bacterium Shewanella piezotolerans WP3 was constructed. This vector replicated stably in Escherichia coli and Shewanella species, and its copy number increased at low temperatures. The pSW2 vector can be utilized as a complementation plasmid in WP3, and it can also be used for the production of complex cytochromes with multiple heme groups, which has the potential for application for metal ion recovery or bioremediation. Promoters of low-temperature-inducible genes in WP3 were fused into the vector to construct a series of vectors for enhancing protein expression at low temperature. The maximum green fluorescent protein intensity was obtained when the promoter for the hfq gene was used. The WP3/pSW2 system can efficiently produce a patatin-like protein (PLP) from a metagenomic library that tends to form inclusion bodies in E. coli. The yields of PLP in the soluble fraction were 8.3 mg/liter and 4.7 mg/liter of culture at 4°C and 20°C, respectively. Moreover, the pSW2 vector can be broadly utilized in other Shewanella species, such as S. oneidensis and S. psychrophila. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in Diaphorina citri, a phloem-sap sucking insect vector of citrus greening disease (Huanglongbing).

PubMed

Hajeri, Subhas; Killiny, Nabil; El-Mohtar, Choaa; Dawson, William O; Gowda, Siddarame

2014-04-20

A transient expression vector based on Citrus tristeza virus (CTV) is unusually stable. Because of its stability it is being considered for use in the field to control Huanglongbing (HLB), which is caused by Candidatus Liberibacter asiaticus (CLas) and vectored by Asian citrus psyllid, Diaphorina citri. In the absence of effective control strategies for CLas, emphasis has been on control of D. citri. Coincident cohabitation in phloem tissue by CLas, D. citri and CTV was exploited to develop a novel method to mitigate HLB through RNA interference (RNAi). Since CTV has three RNA silencing suppressors, it was not known if CTV-based vector could induce RNAi in citrus. Yet, expression of sequences targeting citrus phytoene desaturase gene by CTV-RNAi resulted in photo-bleaching phenotype. CTV-RNAi vector, engineered with truncated abnormal wing disc (Awd) gene of D. citri, induced altered Awd expression when silencing triggers ingested by feeding D. citri nymphs. Decreased Awd in nymphs resulted in malformed-wing phenotype in adults and increased adult mortality. This impaired ability of D. citri to fly would potentially limit the successful vectoring of CLas bacteria between citrus trees in the grove. CTV-RNAi vector would be relevant for fast-track screening of candidate sequences for RNAi-mediated pest control. Copyright © 2014. Published by Elsevier B.V.

Double-stranded RNA innate immune response activation from long-term adeno-associated virus vector transduction.

PubMed

Shao, Wenwei; Earley, Lauriel F; Chai, Zheng; Chen, Xiaojing; Sun, Junjiang; He, Ting; Deng, Meng; Hirsch, Matthew L; Ting, Jenny; Samulski, R Jude; Li, Chengwen

2018-06-21

Data from clinical trials for hemophilia B using adeno-associated virus (AAV) vectors have demonstrated decreased transgenic coagulation factor IX (hFIX) expression 6-10 weeks after administration of a high vector dose. While it is likely that capsid-specific cytotoxic T lymphocytes eliminate vector-transduced hepatocytes, thereby resulting in decreased hFIX, this observation is not intuitively consistent with restored hFIX levels following prednisone application. Although the innate immune response is immediately activated following AAV vector infection via TLR pathways, no studies exist regarding the role of the innate immune response at later time points after AAV vector transduction. Herein, activation of the innate immune response in cell lines, primary human hepatocytes, and hepatocytes in a human chimeric mouse model was observed at later time points following AAV vector transduction. Mechanistic analysis demonstrated that the double-stranded RNA (dsRNA) sensor MDA5 was necessary for innate immune response activation and that transient knockdown of MDA5, or MAVS, decreased IFN-β expression while increasing transgene production in AAV-transduced cells. These results both highlight the role of the dsRNA-triggered innate immune response in therapeutic transgene expression at later time points following AAV transduction and facilitate the execution of effective strategies to block the dsRNA innate immune response in future clinical trials.

Overexpression of SASH1 related to the decreased invasion ability of human glioma U251 cells.

PubMed

Yang, Liu; Liu, Mei; Gu, Zhikai; Chen, Jianguo; Yan, Yaohua; Li, Jian

2012-12-01

The purpose of this study was to investigate the impact of SAM- and SH3-domain containing 1 (SASH1) on the biological behavior of glioma cells, including its effects on cellular growth, proliferation, apoptosis, invasion, and metastasis, and thereby to provide an experimental basis for future therapeutic treatments. A pcDNA3.1-SASH1 eukaryotic expression vector was constructed and transfected into the U251 human glioma cell line. Using the tetrazolium-based colorimetric (MTT) assay, flow cytometry analyses, transwell invasion chamber experiments, and other methods, we examined the impact of SASH1 on the biological behaviors of U251 cells, including effects on viability, cell cycle, apoptosis, and invasion. Furthermore, the effect of SASH1 on the expression of cyclin D1, caspase-3, matrix metalloproteinase (MMP)-2, MMP-9, and other proteins was observed. Compared to the empty vector and blank control groups, the pcDNA3.1-SASH1 group of U251 cells exhibited significantly reduced cell viability, proliferation, and invasion (p < 0.05), although there was no difference between the empty vector and blank control groups. The pcDNA3.1-SASH1 group demonstrated a significantly higher apoptotic index than did the empty vector and blank control groups (p < 0.05), and the percentage of apoptotic cells was similar between the empty vector and blank control groups. In addition, the pcDNA3.1-SASH1 group expressed significantly lower protein levels of cyclin D1 and MMP-2/9 compared to the control and empty vector groups (p < 0.05) and significantly higher protein levels of caspase-3 than the other two groups (p < 0.05). Cyclin D1, caspase-3, and MMP-2/9 expression was unchanged between the empty vector and blank control groups. SASH1 gene expression might be related to the inhibition of the growth, proliferation, and invasion of U251 cells and the promotion of U251 cells apoptosis.

Modification to the Capsid of the Adenovirus Vector That Enhances Dendritic Cell Infection and Transgene-Specific Cellular Immune Responses

PubMed Central

Worgall, Stefan; Busch, Annette; Rivara, Michael; Bonnyay, David; Leopold, Philip L.; Merritt, Robert; Hackett, Neil R.; Rovelink, Peter W.; Bruder, Joseph T.; Wickham, Thomas J.; Kovesdi, Imi; Crystal, Ronald G.

2004-01-01

Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing β-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the β-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing β-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to β-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-β-galactosidase antibody levels following vector administration. However, cellular responses to β-galactosidase were significantly enhanced, with the frequency of CD4+ as well as the CD8+ β-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P < 0.01). Importantly, this enhanced cellular immune response of the AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing β-galactosidase: BALB/c mice implanted with the CT26 syngeneic β-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These data demonstrate that addition of an RGD motif to the Ad fiber knob increases the infectibility of DC and leads to enhanced cellular immune responses to the Ad-transferred transgene, suggesting that the RGD capsid modification may be useful in developing Ad-based vaccines. PMID:14963160

Systems and methods for the secretion of recombinant proteins in gram negative bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Withers, III, Sydnor T.; Dominguez, Miguel A.; DeLisa, Matthew P.

Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

Systems and methods for the secretion of recombinant proteins in gram negative bacteria

DOEpatents

Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

2016-08-09

Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

Fowl adenovirus serotype 9 vectored vaccine for protection of avian influenza virus

USDA-ARS?s Scientific Manuscript database

A fowl adenovirus serotype 9, a non-pathogenic large double stranded DNA virus, was developed as a viral vector to express influenza genes as a potential vaccine. Two separate constructs were developed that expressed either the hemagglutinin gene of A/Chicken/Jalisco/2012 (H7) or A/ Chicken/Iowa/20...

Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

USDA-ARS?s Scientific Manuscript database

A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a differ...

Expression of CD154 by a Simian Immunodeficiency Virus Vector Induces Only Transitory Changes in Rhesus Macaques

PubMed Central

Hodara, Vida L.; Velasquillo, M. Cristina; Parodi, Laura M.; Giavedoni, Luis D.

2005-01-01

Human immunodeficiency virus infection is characterized by dysregulation of antigen-presenting cell function and defects in cell-mediated immunity. Recent evidence suggests that impaired ability of CD4+ T cells to upregulate the costimulatory molecule CD154 is at the core of this dysregulation. To test the hypothesis that increased expression of CD154 on infected CD4+ T cells could modulate immune function, we constructed a replication-competent simian immunodeficiency virus (SIV) vector that expressed CD154. We found that this recombinant vector directed the expression of CD154 on the surface of infected CD4+ T cells and that expression of CD154 resulted in activation of B cells present in the same cultures. Experimental infection of rhesus macaques resulted in very low viral loads for the CD154-expressing virus and the control virus, indicating that expression of CD154 did not result in increased viral replication. Analyses of the anti-SIV immune responses and the phenotype of lymphocytes in blood and lymphoid tissues showed changes that occurred during the acute phase of infection only in animals infected with the CD154-expressing SIV, but that became indistinguishable from those seen in animals infected with the control virus at later time points. We conclude that the level of expression of CD154 in itself is not responsible for affecting the immune response to an attenuated virus. Considering that the CD154-expressing SIV vector and the virus control did not carry an active nef gene, our results suggest that, in CD4+ T cells infected with wild-type virus, Nef is the viral factor that interferes with the immune mechanisms that regulate expression of CD154. PMID:15795254

A non-neuronal cholinergic system regulates cellular ATP levels to maintain cell viability.

PubMed

Oikawa, Shino; Iketani, Mitsue; Kakinuma, Yoshihiko

2014-01-01

We previously suggested that a non-neuronal cholinergic system modulates energy metabolism through the mitochondria. However, the mechanisms responsible for making this system crucial remained undetermined. In this study, we developed a fusion protein expression vector containing a luciferase gene fused to the folic acid receptor-α gene. This protein of the vector was confirmed to target the plasma membrane of transfected HEK293 cells, and vector-derived luciferase activities and ATP levels in viable cells were positively correlated (r = 0.599). Using this luciferase vector, choline acetyltransferase (ChAT)-expressing cells (i.e., cells with an activated non-neuronal cholinergic system) had increased cellular ATP levels. ChAT-expressing cells also had upregulated IGF-1R and Glut-1 protein expressions as well as increased glucose uptake. This activated non-neuronal cholinergic system with efficient glucose metabolism rendered cells resistant to serum depletion-induced cell death. Our results indicate that a non-neuronal cholinergic system is involved in sustaining ATP levels to render cells resistant to a nutrient-deficient environment. © 2014 S. Karger AG, Basel.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface proteinmore » A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.« less

Virus-Based RNA Silencing Agents and Virus-Derived Expression Vectors as Gene Therapy Vehicles.

PubMed

Venkataraman, Srividhya; Ahmad, Tauqeer; AbouHaidar, Mounir G; Hefferon, Kathleen L

2017-01-01

In consideration of recent developments in understanding the genomics and proteomics of viruses, the use of viral DNA / RNA sequences as well as their gene expression schemes, have found new in-roads towards the prognosis and therapy of diseases. Correspondingly, the sphere of the patenting scenario has expanded significantly. The current review addresses patented inventions concerning the use of virus sequences as gene silencing machineries and inventions concerning the generation and application of viral sequences as expression vectors. Furthermore, this review also discusses the employment of these patents for clinical, agricultural and biotechnological applications. Considering these objectives, the Delphion Research Intellectual Property Network database was searched using keywords such as "gene silencing", "engineered viruses" and "expression vectors" and descriptions of recent patents on the said topics were discussed. Despite several recent advances in the use of viruses as disease therapy vehicles and biotechnological vectors, these developments have yet to be proven effective in practice, in clinical and field trials. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, John J.; Barbour, Alan G.

1996-11-05

A method is provided herein for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.

Antitumor HPV E7-specific CTL activity elicited by in vivo engineered exosomes produced through DNA inoculation.

PubMed

Di Bonito, Paola; Chiozzini, Chiara; Arenaccio, Claudia; Anticoli, Simona; Manfredi, Francesco; Olivetta, Eleonora; Ferrantelli, Flavia; Falcone, Emiliana; Ruggieri, Anna; Federico, Maurizio

2017-01-01

We recently proved that exosomes engineered in vitro to deliver high amounts of HPV E7 upon fusion with the Nef mut exosome-anchoring protein elicit an efficient anti-E7 cytotoxic T lymphocyte immune response. However, in view of a potential clinic application of this finding, our exosome-based immunization strategy was faced with possible technical difficulties including industrial manufacturing, cost of production, and storage. To overcome these hurdles, we designed an as yet unproven exosome-based immunization strategy relying on delivery by intramuscular inoculation of a DNA vector expressing Nef mut fused with HPV E7. In this way, we predicted that the expression of the Nef mut /E7 vector in muscle cells would result in a continuous source of endogenous (ie, produced by the inoculated host) engineered exosomes able to induce an E7-specific immune response. To assess this hypothesis, we first demonstrated that the injection of a Nef mut /green fluorescent protein-expressing vector led to the release of fluorescent exosomes, as detected in plasma of inoculated mice. Then, we observed that mice inoculated intramuscularly with a vector expressing Nef mut /E7 developed a CD8 + T-cell immune response against both Nef and E7. Conversely, no CD8 + T-cell responses were detected upon injection of vectors expressing either the wild-type Nef isoform of E7 alone, most likely a consequence of their inefficient exosome incorporation. The production of immunogenic exosomes in the DNA-injected mice was formally demonstrated by the E7-specific CD8 + T-cell immune response we detected in mice inoculated with exosomes isolated from plasma of mice inoculated with the Nef mut /E7 vector. Finally, we provide evidence that the injection of Nef mut /E7 DNA led to the generation of effective antigen-specific cytotoxic T lymphocytes whose activity was likely part of the potent, therapeutic antitumor effect we observed in mice implanted with TC-1 tumor cells. In summary, we established a novel method to generate immunogenic exosomes in vivo by the intramuscular inoculation of DNA vectors expressing the exosome-anchoring protein Nef mut and its derivatives.

Implementing an Ebola Vaccine Study - Sierra Leone.

PubMed

Widdowson, Marc-Alain; Schrag, Stephanie J; Carter, Rosalind J; Carr, Wendy; Legardy-Williams, Jennifer; Gibson, Laura; Lisk, Durodami R; Jalloh, Mohamed I; Bash-Taqi, Donald A; Kargbo, Samuel A Sheku; Idriss, Ayesha; Deen, Gibrilla F; Russell, James B W; McDonald, Wendi; Albert, Alison P; Basket, Michelle; Callis, Amy; Carter, Victoria M; Ogunsanya, Kelli R Clifton; Gee, Julianne; Pinner, Robert; Mahon, Barbara E; Goldstein, Susan T; Seward, Jane F; Samai, Mohamed; Schuchat, Anne

2016-07-08

In October 2014, the College of Medicine and Allied Health Sciences of the University of Sierra Leone, the Sierra Leone Ministry of Health and Sanitation, and CDC joined the global effort to accelerate assessment and availability of candidate Ebola vaccines and began planning for the Sierra Leone Trial to Introduce a Vaccine against Ebola (STRIVE). STRIVE was an individually randomized controlled phase II/III trial to evaluate efficacy, immunogenicity, and safety of the recombinant vesicular stomatitis virus Ebola vaccine (rVSV-ZEBOV). The study population was health care and frontline workers in select chiefdoms of the five most affected districts in Sierra Leone. Participants were randomized to receive a single intramuscular dose of rVSV-ZEBOV at enrollment or to receive a single intramuscular dose 18-24 weeks after enrollment. All participants were followed up monthly until 6 months after vaccination. Two substudies separately assessed detailed reactogenicity over 1 month and immunogenicity over 12 months. During the 5 months before the trial, STRIVE and partners built a research platform in Sierra Leone comprising participant follow-up sites, cold chain, reliable power supply, and vaccination clinics and hired and trained at least 350 national staff. Wide-ranging community outreach, informational sessions, and messaging were conducted before and during the trial to ensure full communication to the population of the study area regarding procedures and current knowledge about the trial vaccine. During April 9-August 15, 2015, STRIVE enrolled 8,673 participants, of whom 453 and 539 were also enrolled in the safety and immunogenicity substudies, respectively. As of April 28, 2016, no Ebola cases and no vaccine-related serious adverse events, which by regulatory definition include death, life-threatening illness, hospitalization or prolongation of hospitalization, or permanent disability, were reported in the study population. Although STRIVE will not produce an estimate of vaccine efficacy because of low case frequency as the epidemic was controlled, data on safety and immunogenicity will support decisions on licensure of rVSV-ZEBOV.The activities summarized in this report would not have been possible without collaboration with many U.S. and international partners (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/partners.html).

[Antitumor effects of matrix protein of vesicular stomatic virus on EL4 lymphoma mice].

PubMed

Lin, Shi-jia; Yu, Qin-mei; Meng, Wen-tong; Wen, Yan-jun; Chen, Li-juan; Niu, Ting

2011-03-01

To explore antitumor effects of plasmid pcDNA3. 1-MP encoding matrix protein of vesicular stomatitis virus (VSV) complexed with cationic liposome (DOTAP:CHOL) in mice with EL4 lymphoma. C57BL/6 mouse model with EL4 lymphoma was established. Sixty mice bearing EL4 lymphoma were divided randomly into five groups including Lip-MP, Lip-pVAX, Lip, ADM and NS groups, which were intravenously injected with liposome-pcDNA 3. 1-MP complex, liposome-pVAX complex, empty liposome, Adriamycin and normal saline respectively every three days. Tumor volumes and survival time were monitored. Microvessel density and tumor proliferative index in tumor tissues were determined by CD31, Ki-67 immunohistochemistry staining, meanwhile the tumor apoptosis index was measured by TUNEL method. From 6 days after treatments on, the tumor volume in Lip-MP group was much smaller than that in Lip-pVAX, Lip and NS group (P < 0.05). The median survival time of mice in Lip-MP group, 44 days after inoculation of tumor cells, was significantly higher than that in other groups (P < 0.05), which was 39 days, 38.5 days and 34 days in Lip-pVAX, Lip and NS groups respectively. The MVD value in tumor tissues in Lip-MP group was less than that in Lip-pVAX, Lip and NS groups (P < 0.05). Ki67 staining revealed that Lip-MP complex apparently suppressed the proliferation of EL4 tumor cells in vivo (P < 0.05). TUNEL assays showed that apoptosis index of tumor cells in Lip-MP group, 10.60 +/- 1.71, was much higher than that in other three groups (P < 0.05). Lip-MP complex, the plasmid encoding matrix protein of VSV (VSV-MP) encapsulated in cationic liposome, significantly inhibited the growth of tumor and prolonged the survival of mice bearing EL4 lymphoma, which may be related to the induction of tumor cell apoptosis, inhibition of tumor angiogenesis, and suppression of tumor cell proliferation.