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Thomas W. Dubensky - One of the best experts on this subject based on the ideXlab platform.
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an alphavirus Replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene based vaccine delivery vector
Journal of Virology, 2003Co-Authors: Silvia Perri, Harold Legg, Zequn Tang, Catherine E Greer, Kent B Thudium, Barbara Doe, Hong Liu, Raul E Romero, Qian Bin, Thomas W. DubenskyAbstract:Alphavirus vectors are being developed as gene-based vaccines for infectious and malignant diseases (38, 42, 45). Alphavirus vectors are known as Replicons due to the self-amplification of the vector RNA, which occurs in the cytoplasm of the infected cell. Replicons contain the nonstructural protein genes encoding the viral replicase, the 5′- and 3′-end cis-active replication sequences, and the native subgenomic promoter, which directs expression of the encoded heterologous gene(s). While Replicons lack the genes encoding virion structural proteins necessary for packaging and cell-to-cell spread of infectious particles, they may be packaged into virus-like particles by providing the structural proteins in trans, using transient RNA cotransfection systems (2, 29) or stable Replicon packaging cell lines (36). Alternatively, the Replicon RNA can be introduced into cells as plasmid DNA (10, 18). Alphavirus Replicon particle vectors have been developed using Sindbis virus (SIN) (2, 61), Venezuelan equine encephalitis virus (VEE) (40), and Semliki Forest virus (29). Each has been shown to induce robust cellular, humoral, and mucosal immune responses specific for the Replicon-expressed antigen in several animal models (38, 42, 45). A number of features make alphavirus Replicon vectors attractive for gene-based vaccines, including high-level expression of the heterologous gene (61), vector amplification through double-stranded RNA intermediates, which stimulates aspects of innate immunity, such as activation of the interferon (IFN) cascade (27), induction of apoptosis in some cell types (28) which may enhance immunogenicity via antigen cross-priming (62), and the overall lack of preexisting immunity in the human population. In addition, alphavirus Replicon particles can be used for delivery of antigen to antigen-presenting cells, such as dendritic cells, the most potent antigen-presenting cell population (15, 31). On the basis of their biological niche, each alphavirus has unique properties, which may be either desirable or undesirable in terms of function, potency, and safety when applied to recombinant vector systems. For example, VEE has a natural lymphotropism (31) that may be ideal for vaccine use, and VEE Replicon particles have been shown to induce potent and protective immune responses in primates (6, 19). The pathogenesis of wild-type VEE in humans (20), however, raises safety concerns, some of which have been addressed by identifying and incorporating specific attenuating mutations in the envelope glycoproteins of the Replicon particles (40). However, despite these precautions, production of VEE Replicon particles must be conducted at biosafety level III, and only after completion of testing for contaminating replication-competent virus can the particles be used at biosafety level II. In contrast, since SIN is not associated with serious human disease (20, 52), SIN-derived Replicon particles largely obviate these concerns. In addition, particular engineered SIN variants target lymphoid cells (15), and stable Replicon packaging cell lines have been developed (36), which may simplify large-scale production for human testing of vaccine candidates. However, although SIN vectors induce potent immune responses in murine models (38, 42, 46), little is known about whether this translates to robust immune responses in primates. Since the relative potencies of the different alphavirus vectors have not been characterized yet, we performed initial comparative immunogenicity studies for SIN and VEE Replicon particles in this work. Also, we explored the feasibility of combining genetic components from each parent virus toward a goal of developing a chimeric alphavirus Replicon particle with optimal potency and safety. For these studies, we generated a panel of SIN, VEE, and chimera Replicon particles expressing a human immunodeficiency virus (HIV) p55gag antigen or green fluorescent protein (GFP) reporter. On the basis of in vitro and in vivo studies, we report here the identification of a novel VEE/SIN Replicon particle chimera that combines selected desirable qualities of SIN and VEE, which should have utility as a potent gene-based vaccine delivery platform.
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an alphavirus Replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene based vaccine delivery vector
Journal of Virology, 2003Co-Authors: Silvia Perri, Thomas W. Dubensky, Harold Legg, Zequn Tang, Kent B Thudium, Raul E Romero, Catherine Greer, Michael Vajdy, Gillis R Otten, John M PoloAbstract:Alphavirus Replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of Replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55Gag antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated Replicon particle chimeras of SIN and VEE. Two distinct strategies were used to produce particles with VEE-p55gag Replicon RNA packaged within SIN envelope glycoproteins and SIN-p55gag Replicon RNA within VEE envelope glycoproteins. Each Replicon particle configuration induced Gag-specific CD8+ T-cell responses in murine models when administered alone or after priming with DNA. However, Gag-specific responses varied dramatically, with the strongest responses to this particular antigen correlating with the VEE Replicon RNA, irrespective of the source of envelope glycoproteins. Comparing the Replicons with respect to heterologous gene expression levels and sensitivity to alpha/beta interferon in cultured cells indicated that each might contribute to potency differences. This work shows that combining desirable elements from VEE and SIN into a Replicon particle chimera may be a valuable approach toward the goal of developing vaccine vectors with optimal in vivo potency, ease of production, and safety.
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Replicon vectors derived from sindbis virus and semliki forest virus that establish persistent replication in host cells
Journal of Virology, 2000Co-Authors: Silvia Perri, Thomas W. Dubensky, David A Driver, Jason P Gardner, Scott Sherrill, Barbara A Belli, John M PoloAbstract:Alphavirus Replicon vectors are well suited for applications where transient, high-level expression of a heterologous gene is required. Replicon vector expression in cells leads to inhibition of host macromolecular synthesis, culminating in eventual cell death by an apoptotic mechanism. For many applications, including gene expression studies in cultured cells, a longer duration of transgene expression without resulting cytopathic effects is useful. Recently, noncytopathic Sindbis virus (SIN) variants were isolated in BHK cells, and the mutations responsible were mapped to the protease domain of nonstructural protein 2 (nsP2). We report here the isolation of additional variants of both SIN and Semliki Forest virus (SFV) Replicons encoding the neomycin resistance gene that can establish persistent replication in BHK cells. The SIN and SFV variant Replicons resulted from previously undescribed mutations within one of three discrete regions of the nsP2 gene. Differences among the panel of variants were observed in processing of the nonstructural polyprotein and in the ratios of subgenomic to genomic RNAs. Importantly, high-level expression of a heterologous gene was retained with most Replicons. Finally, in contrast to previous studies, efficient packaging was obtained with several of the variant Replicons. This work expands the utility of noncytopathic Replicons and the understanding of how alphavirus Replicons establish persistent replication in cultured cells.
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enhancement of tumor specific immune response with plasmid dna Replicon vectors
Cancer Research, 2000Co-Authors: Wolfgang W Leitner, Thomas W. Dubensky, David A Driver, Han Ying, Nicholas P RestifoAbstract:To enhance the immunogenicity of nucleic acid vaccines, we used plasmid DNA vectors that contained Replicons derived from the prototype alphavirus, Sindbis, and another alphavirus, Semliki Forest virus. When transfected into cells or injected directly into animal muscle, these plasmids launch a self-replicating RNA vector (Replicon) which in turn directs the expression of a model tumor antigen. Immunization with plasmid DNA Replicons elicited immune responses at doses 100 to 1000-fold lower than conventional DNA plasmids and effectively treated mice bearing an experimental tumor expressing the model antigen. Significantly, Replicon-based DNA plasmids did not produce a greater quantity of antigen; instead, antigen production differed qualitatively. Plasmid DNA Replicons mediated antigen production that was homogeneous in all transfected cells and associated with the apoptotic death of the host cells. Because of their safety and efficacy, plasmid DNA Replicons may be useful in the development of recombinant vaccines for infectious diseases and cancer.
Alessandra Carattoli - One of the best experts on this subject based on the ideXlab platform.
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in silico detection and typing of plasmids using plasmidfinder and plasmid multilocus sequence typing
Antimicrobial Agents and Chemotherapy, 2014Co-Authors: Alessandra Carattoli, Laura Villa, Aurora Garciafernandez, Ea Zankari, Mette Voldby Larsen, Ole Lund, Frank Moller Aarestrup, H HasmanAbstract:ABSTRACT In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration into a Web tool called PlasmidFinder that can be used for Replicon sequence analysis of raw, contig group, or completely assembled and closed plasmid sequencing data. The PlasmidFinder database currently consists of 116 Replicon sequences that match with at least at 80% nucleotide identity all Replicon sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection of Salmonella enterica serovar Typhimurium isolates. PlasmidFinder identified a total of 103 Replicons and between zero and five different plasmid Replicons within each of 49 S . Typhimurium draft genomes tested. The pMLST Web tool was able to subtype genomic sequencing data of plasmids, revealing both known plasmid sequence types (STs) and new alleles and ST variants. In conclusion, testing of the two Web tools using both fully assembled plasmid sequences and WGS-generated draft genomes showed them to be able to detect a broad variety of plasmids that are often associated with antimicrobial resistance in clinically relevant bacterial pathogens.
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Replicon sequence typing of incf plasmids carrying virulence and resistance determinants
Journal of Antimicrobial Chemotherapy, 2010Co-Authors: Laura Villa, Aurora Garciafernandez, Daniela Fortini, Alessandra CarattoliAbstract:Objectives: IncF plasmids are frequently encountered in clinical enterobacterial strains associated with the dissemination of relevant antimicrobial resistance and virulence genes. These plasmids are usually heterogeneous in size and carry multiple Replicons, and technical difficulties can impair the comparison and detection of related plasmids by restriction fragment length polymorphism analysis. We devised a rapid sequence-based typing scheme to categorize the members of this plasmid family into homogeneous groups. Methods: We compared the available IncF Replicon sequences, identifying the combination of the different IncF Replicon alleles as the discriminating characteristic of these plasmid scaffolds. An IncF typing method based on PCR amplification and sequence typing of the IncF Replicons was devised. A collection of IncF plasmids carrying resistance and/or virulence genes, identified in strains from different sources and geographical origins, was tested with this typing system. Results: We devised a Replicon sequence typing (RST) scheme discriminating IncF plasmid variants. This system was tested on the collection of IncF plasmids, demonstrating that it was useful for the discrimination of plasmids carrying the same resistance gene (i.e. the blaCTX-M-15 gene), but also recognized strictly related virulence plasmids (i.e. IncFIme plasmids). The PCR-based Replicon typing (PBRT) system was also updated by including new primer pairs to allow the identification of the Salmonella, Klebsiella and Yersinia IncF plasmids. Conclusions: The ability to recognize and sub-categorize IncF plasmids by RST in homogeneous groups on the basis of their phylogenetic relatedness can be helpful in analysing their distribution in nature and discovering their evolutionary origin.
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characterization and pcr based Replicon typing of resistance plasmids in acinetobacter baumannii
Antimicrobial Agents and Chemotherapy, 2010Co-Authors: Alessia Bertini, Laurent Poirel, Patrice Nordmann, Pauline D Mugnier, Laura Villa, Alessandra CarattoliAbstract:Acinetobacter baumannii is an opportunistic pathogen, especially in intensive care units, and multidrug-resistant isolates have increasingly been reported during the last decade. Despite recent progress in knowledge of antibiotic resistance mechanisms in A. baumannii, little is known about the genetic factors driving isolates toward multidrug resistance. In the present study, the A. baumannii plasmids were investigated through the analysis and classification of plasmid replication systems and the identification of A. baumannii-specific mobilization and addiction systems. Twenty-two Replicons were identified by in silico analysis, and five other Replicons were identified and cloned from previously uncharacterized A. baumannii resistance plasmids carrying the OXA-58 carbapenem-hydrolyzing oxacillinase. Replicons were classified into homology groups on the basis of their nucleotide homology. A novel PCR-based Replicon typing scheme (the A. baumannii PCR-based Replicon typing [AB-PBRT] method) was devised to categorize the A. baumannii plasmids into homogeneous groups on the basis of the nucleotide homology of their respective replicase genes. The AB-PBRT technique was applied to a collection of multidrug-resistant A. baumannii clinical isolates carrying the blaOXA-58 or blaOXA-23 carbapenemase gene. A putative complete conjugative apparatus was identified on one plasmid whose self-conjugative ability was demonstrated in vitro. We showed that this conjugative plasmid type was widely diffused in our collection, likely representing the most important vehicle promoting the horizontal transmission of A. baumannii resistance plasmids.
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conjugative transferability of the a c plasmids from salmonella enterica isolates that possess or lack blacmy in the a c plasmid backbone
Foodborne Pathogens and Disease, 2009Co-Authors: T L Poole, Alessandra Carattoli, T S Edrington, Dayna M Brichtaharhay, Robin C Anderson, David J NisbetAbstract:Abstract The objective of this study was to understand the conjugative transmissibility of resistance plasmids present in 205 Salmonella enterica isolates from bovine sources. Polymerase chain reaction (PCR)–based Replicon typing was used to type plasmid Replicons. Conjugation experiments were preformed in triplicate at 30°C and 37°C on solid medium. PCR mapping of the A/C transfer gene operon was done on 17 Salmonella Newport isolates that were only positive for A/C. Eighty-six percent (n = 177) of the Salmonella isolates were multidrug resistant (MDR) with resistance to 3–12 antimicrobial agents. Of these, 82% (n = 146) were resistant to extended-spectrum cephalosporins and possessed a blaCMY gene. A/C was the predominant Replicon detected, present in 90% (n = 160) of the MDR isolates. Twenty-three percent (n = 37) of the A/C-positive strains were positive for a second Replicon. Replicons coresident with A/C included I1, N, B/O, HI1, and HI2. Only 31% (n = 54) of the MDR isolates produced transconjugant...
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Replicon typing of plasmids carrying ctx m or cmy β lactamases circulating among salmonella and escherichia coli isolates
Antimicrobial Agents and Chemotherapy, 2006Co-Authors: Katie L Hopkins, Laura Villa, Ernesto Liebana, M Batchelor, John E Threlfall, Alessandra CarattoliAbstract:Replicon typing of plasmids carrying blaCTX-M or blaCMY β-lactamase genes indicates a predominance of I1 and A/C Replicons among blaCMY-carrying plasmids and five different plasmid scaffolds associated with the different types of blaCTX-M genes (I1, FII, HI2, K, and N). These results demonstrate the association of certain β-lactamase genes with specific plasmid backbones.
Silvia Perri - One of the best experts on this subject based on the ideXlab platform.
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an alphavirus Replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene based vaccine delivery vector
Journal of Virology, 2003Co-Authors: Silvia Perri, Harold Legg, Zequn Tang, Catherine E Greer, Kent B Thudium, Barbara Doe, Hong Liu, Raul E Romero, Qian Bin, Thomas W. DubenskyAbstract:Alphavirus vectors are being developed as gene-based vaccines for infectious and malignant diseases (38, 42, 45). Alphavirus vectors are known as Replicons due to the self-amplification of the vector RNA, which occurs in the cytoplasm of the infected cell. Replicons contain the nonstructural protein genes encoding the viral replicase, the 5′- and 3′-end cis-active replication sequences, and the native subgenomic promoter, which directs expression of the encoded heterologous gene(s). While Replicons lack the genes encoding virion structural proteins necessary for packaging and cell-to-cell spread of infectious particles, they may be packaged into virus-like particles by providing the structural proteins in trans, using transient RNA cotransfection systems (2, 29) or stable Replicon packaging cell lines (36). Alternatively, the Replicon RNA can be introduced into cells as plasmid DNA (10, 18). Alphavirus Replicon particle vectors have been developed using Sindbis virus (SIN) (2, 61), Venezuelan equine encephalitis virus (VEE) (40), and Semliki Forest virus (29). Each has been shown to induce robust cellular, humoral, and mucosal immune responses specific for the Replicon-expressed antigen in several animal models (38, 42, 45). A number of features make alphavirus Replicon vectors attractive for gene-based vaccines, including high-level expression of the heterologous gene (61), vector amplification through double-stranded RNA intermediates, which stimulates aspects of innate immunity, such as activation of the interferon (IFN) cascade (27), induction of apoptosis in some cell types (28) which may enhance immunogenicity via antigen cross-priming (62), and the overall lack of preexisting immunity in the human population. In addition, alphavirus Replicon particles can be used for delivery of antigen to antigen-presenting cells, such as dendritic cells, the most potent antigen-presenting cell population (15, 31). On the basis of their biological niche, each alphavirus has unique properties, which may be either desirable or undesirable in terms of function, potency, and safety when applied to recombinant vector systems. For example, VEE has a natural lymphotropism (31) that may be ideal for vaccine use, and VEE Replicon particles have been shown to induce potent and protective immune responses in primates (6, 19). The pathogenesis of wild-type VEE in humans (20), however, raises safety concerns, some of which have been addressed by identifying and incorporating specific attenuating mutations in the envelope glycoproteins of the Replicon particles (40). However, despite these precautions, production of VEE Replicon particles must be conducted at biosafety level III, and only after completion of testing for contaminating replication-competent virus can the particles be used at biosafety level II. In contrast, since SIN is not associated with serious human disease (20, 52), SIN-derived Replicon particles largely obviate these concerns. In addition, particular engineered SIN variants target lymphoid cells (15), and stable Replicon packaging cell lines have been developed (36), which may simplify large-scale production for human testing of vaccine candidates. However, although SIN vectors induce potent immune responses in murine models (38, 42, 46), little is known about whether this translates to robust immune responses in primates. Since the relative potencies of the different alphavirus vectors have not been characterized yet, we performed initial comparative immunogenicity studies for SIN and VEE Replicon particles in this work. Also, we explored the feasibility of combining genetic components from each parent virus toward a goal of developing a chimeric alphavirus Replicon particle with optimal potency and safety. For these studies, we generated a panel of SIN, VEE, and chimera Replicon particles expressing a human immunodeficiency virus (HIV) p55gag antigen or green fluorescent protein (GFP) reporter. On the basis of in vitro and in vivo studies, we report here the identification of a novel VEE/SIN Replicon particle chimera that combines selected desirable qualities of SIN and VEE, which should have utility as a potent gene-based vaccine delivery platform.
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an alphavirus Replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene based vaccine delivery vector
Journal of Virology, 2003Co-Authors: Silvia Perri, Thomas W. Dubensky, Harold Legg, Zequn Tang, Kent B Thudium, Raul E Romero, Catherine Greer, Michael Vajdy, Gillis R Otten, John M PoloAbstract:Alphavirus Replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of Replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55Gag antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated Replicon particle chimeras of SIN and VEE. Two distinct strategies were used to produce particles with VEE-p55gag Replicon RNA packaged within SIN envelope glycoproteins and SIN-p55gag Replicon RNA within VEE envelope glycoproteins. Each Replicon particle configuration induced Gag-specific CD8+ T-cell responses in murine models when administered alone or after priming with DNA. However, Gag-specific responses varied dramatically, with the strongest responses to this particular antigen correlating with the VEE Replicon RNA, irrespective of the source of envelope glycoproteins. Comparing the Replicons with respect to heterologous gene expression levels and sensitivity to alpha/beta interferon in cultured cells indicated that each might contribute to potency differences. This work shows that combining desirable elements from VEE and SIN into a Replicon particle chimera may be a valuable approach toward the goal of developing vaccine vectors with optimal in vivo potency, ease of production, and safety.
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Replicon vectors derived from sindbis virus and semliki forest virus that establish persistent replication in host cells
Journal of Virology, 2000Co-Authors: Silvia Perri, Thomas W. Dubensky, David A Driver, Jason P Gardner, Scott Sherrill, Barbara A Belli, John M PoloAbstract:Alphavirus Replicon vectors are well suited for applications where transient, high-level expression of a heterologous gene is required. Replicon vector expression in cells leads to inhibition of host macromolecular synthesis, culminating in eventual cell death by an apoptotic mechanism. For many applications, including gene expression studies in cultured cells, a longer duration of transgene expression without resulting cytopathic effects is useful. Recently, noncytopathic Sindbis virus (SIN) variants were isolated in BHK cells, and the mutations responsible were mapped to the protease domain of nonstructural protein 2 (nsP2). We report here the isolation of additional variants of both SIN and Semliki Forest virus (SFV) Replicons encoding the neomycin resistance gene that can establish persistent replication in BHK cells. The SIN and SFV variant Replicons resulted from previously undescribed mutations within one of three discrete regions of the nsP2 gene. Differences among the panel of variants were observed in processing of the nonstructural polyprotein and in the ratios of subgenomic to genomic RNAs. Importantly, high-level expression of a heterologous gene was retained with most Replicons. Finally, in contrast to previous studies, efficient packaging was obtained with several of the variant Replicons. This work expands the utility of noncytopathic Replicons and the understanding of how alphavirus Replicons establish persistent replication in cultured cells.
Clay Fuqua - One of the best experts on this subject based on the ideXlab platform.
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destabilization of the tumor inducing plasmid from an octopine type agrobacterium tumefaciens lineage drives a large deletion in the co resident at megaplasmid
G3: Genes Genomes Genetics, 2019Co-Authors: Ian S Barton, Thomas G Platt, Douglas B Rusch, Clay FuquaAbstract:Bacteria with multi-Replicon genome organizations, including members of the family Rhizobiaceae, often carry a variety of niche-associated functions on large plasmids. While evidence exists for cross-Replicon interactions and co-evolution between Replicons in many of these systems, remarkable strain-to-strain variation is also observed for extrachromosomal elements, suggesting increased genetic plasticity. Here, we show that curing of the tumor-inducing virulence plasmid (pTi) of an octopine-type Agrobacterium tumefaciens lineage leads to a large deletion in the co-resident At megaplasmid (pAt). The deletion event is mediated by a repetitive IS-element, IS66, and results in a variety of environment-dependent fitness consequences, including loss of independent conjugal transfer of the plasmid. Interestingly, a related and otherwise wild-type A. tumefaciens strain is missing exactly the same large pAt segment as the pAt deletion derivatives, suggesting a similar event over its natural history. Overall, the findings presented here uncover a novel genetic interaction between the two large plasmids of A. tumefaciens and provide evidence for cross-Replicon integration and co-evolution of these plasmids.
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destabilization of the tumor inducing plasmid from an octopine type agrobacterium tumefaciens lineage drives a large deletion in the co resident at megaplasmid
bioRxiv, 2019Co-Authors: Ian S Barton, Thomas G Platt, Douglas B Rusch, Clay FuquaAbstract:Bacteria with multi-Replicon genome organizations, including members of the family Rhizobiaceae, often carry a variety of niche-associated functions on large plasmids. While evidence exists for cross-Replicon interactions and co-evolution between Replicons in many of these systems, remarkable strain-to-strain variation is observed within secondary Replicons, suggesting environment-dependent fitness benefits of the accessory genome. Here, we show that curing of the tumor-inducing virulence plasmid (pTi) of an octopine-type Agrobacterium tumefaciens lineage drives a large deletion in the co-resident At megaplasmid (pAt). The deletion event is mediated by a repetitive IS-element, IS66, and results in a variety of environment-dependent fitness consequences, including loss of independent conjugal transfer of the plasmid. Interestingly, a similar deletion event is also found in a related A. tumefaciens strain, suggesting the mechanism is conserved in a subset of agrobacteria. Overall, the findings presented here uncover a novel genetic interaction between the megaplasmids of A. tumefaciens and provide evidence for cross-Replicon interactions and co-evolution of rhizobial plasmids.
Jonathan F. Smith - One of the best experts on this subject based on the ideXlab platform.
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Recombinant RNA Replicons derived from attenuated Venezuelan equine encephalitis virus protect guinea pigs and mice from Ebola hemorrhagic fever virus.
Vaccine, 2000Co-Authors: Peter Pushko, Anthony Sanchez, Peter B. Jahrling, Mike Bray, George V. Ludwig, Michael W. Parker, Alan L. Schmaljohn, Jonathan F. SmithAbstract:RNA Replicons derived from an attenuated strain of Venezuelan equine encephalitis virus (VEE), an alphavirus, were configured as candidate vaccines for Ebola hemorrhagic fever. The Ebola nucleoprotein (NP) or glycoprotein (GP) genes were introduced into the VEE RNA downstream from the VEE 26S promoter in place of the VEE structural protein genes. The resulting recombinant Replicons, expressing the NP or GP genes, were packaged into VEE Replicon particles (NP-VRP and GP-VRP, respectively) using a bipartite helper system that provided the VEE structural proteins in trans and prevented the regeneration of replication-competent VEE during packaging. The immunogenicity of NP-VRP and GP-VRP and their ability to protect against lethal Ebola infection were evaluated in BALB/c mice and in two strains of guinea pigs. The GP-VRP alone, or in combination with NP-VRP, protected both strains of guinea pigs and BALB/c mice, while immunization with NP-VRP alone protected BALB/c mice, but neither strain of guinea pig. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge. However, the complete protection achieved with active immunization with VRP, as well as the unique characteristics of the VEE Replicon vector, warrant further testing of the safety and efficacy of NP-VRP and GP-VRP in primates as candidate vaccines against Ebola hemorrhagic fever.
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marburg virus vaccines based upon alphavirus Replicons protect guinea pigs and nonhuman primates
Virology, 1998Co-Authors: Michael Hevey, Peter Pushko, Jonathan F. Smith, Diane L Negley, Alan L. SchmaljohnAbstract:Marburg virus (MBGV), for which no vaccines or treatments currently exist, causes an acute hemorrhagic fever with a high mortality rate in humans. We previously showed that immunization with either killed MBGV or a glycoprotein (GP) subunit prevented lethal infection in guinea pigs. In the studies reported here, an RNA Replicon, based upon Venezuelan equine encephalitis (VEE) virus, was used as a vaccine vector; the VEE structural genes were replaced by genes for MBGV GP, nucleoprotein (NP), VP40, VP35, VP30, or VP24. Guinea pigs were vaccinated with recombinant VEE Replicons (packaged into VEE-like particles), inoculated with MBGV, and evaluated for viremia and survival. Results indicated that either GP or NP were protective antigens while VP35 afforded incomplete protection. As a more definitive test of vaccine efficacy, nonhuman primates (cynomolgus macaques) were inoculated with VEE Replicons expressing MBGV GP and/or NP. Three monkeys received packaged control Replicons (influenza HA); these died 9 or 10 days after challenge, with typical MBGV disease. MBGV NP afforded incomplete protection, sufficient to prevent death but not disease in two of three macaques. Three monkeys vaccinated with Replicons which expressed MBGV GP, and three others vaccinated with both Replicons that expressed GP or NP, remained aviremic and were completely protected from disease.
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Replicon helper systems from attenuated venezuelan equine encephalitis virus expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo
Virology, 1997Co-Authors: Peter Pushko, Robert E. Johnston, Nancy L Davis, George V. Ludwig, Michael Parker, Jonathan F. SmithAbstract:A Replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The Replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these Replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of Replicon RNAs into VEE Replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the Replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when Replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE Replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.
