A DNA replicon system for rapid high‐level production of virus‐like particles in plants

Huang, Zhong; Chen, Qiang; Hjelm, Brooke E.; Arntzen, Charles J.; Mason, Hugh S.

doi:10.1002/bit.22299

Cited by 160 publications

(229 citation statements)

References 40 publications

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“…N. benthamiana leaves were vacuum-infiltrated with pBYRH2GP1kdK3 (14) and pPSp19 (30) for h-EIC. For m-EIC, the leaves were infiltrated with pBYHGP1kdel, pBYK1R, and pPSp19.…”

Section: Methodsmentioning

confidence: 99%

A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge

Phoolcharoen

Dye

Kilbourne

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Ebola hemorrhagic fever is an acute and often deadly disease caused by Ebola virus (EBOV). The possible intentional use of this virus against human populations has led to design of vaccines that could be incorporated into a national stockpile for biological threat reduction. We have evaluated the immunogenicity and efficacy of an EBOV vaccine candidate in which the viral surface glycoprotein is biomanufactured as a fusion to a monoclonal antibody that recognizes an epitope in glycoprotein, resulting in the production of Ebola immune complexes (EICs). Although antigen–antibody immune complexes are known to be efficiently processed and presented to immune effector cells, we found that codelivery of the EIC with Toll-like receptor agonists elicited a more robust antibody response in mice than did EIC alone. Among the compounds tested, polyinosinic:polycytidylic acid (PIC, a Toll-like receptor 3 agonist) was highly effective as an adjuvant agent. After vaccinating mice with EIC plus PIC, 80% of the animals were protected against a lethal challenge with live EBOV (30,000 LD 50 of mouse adapted virus). Surviving animals showed a mixed Th1/Th2 response to the antigen, suggesting this may be important for protection. Survival after vaccination with EIC plus PIC was statistically equivalent to that achieved with an alternative viral vector vaccine candidate reported in the literature. Because nonreplicating subunit vaccines offer the possibility of formulation for cost-effective, long-term storage in biothreat reduction repositories, EIC is an attractive option for public health defense measures.

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“…N. benthamiana leaves were vacuum-infiltrated with pBYRH2GP1kdK3 (14) and pPSp19 (30) for h-EIC. For m-EIC, the leaves were infiltrated with pBYHGP1kdel, pBYK1R, and pPSp19.…”

Section: Methodsmentioning

confidence: 99%

A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge

Phoolcharoen

Dye

Kilbourne

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Deletion of coat protein in some bipartite begomoviruses has been employed to insert foreign sequence up to 800 nt [22]. Geminivirus replicon vector was also developed by deleting AV2/V2 and CP genes of other geminiviruses [11,21,24,25]. In this study, we deleted the V2 and V1 gene of CYVMV and retained the elements required for rolling-circle replication.…”

Section: Discussionmentioning

confidence: 99%

“…However, geminivirus based efficient gene expression vector is under the developmental stage. Replicon based strategy has been used to design expression/silencing vector for a few mastreviruses [bean yellow dwarf virus (BeYDV), maize streak virus (MSV), wheat dwarf virus (WDV)], curtoviruses [beet curly top virus (BCTV)], bipartite begomoviruses [ACMV, cabbage leaf curl virus (CaLCuV), abuliton mosaic virus (AbMV), tomato leaf curl New Delhi virus (ToLCNDV)] and monopartite begomoviruses [tobacco curly shoot virus (TbCSV), ageratum yellow vein virus (AYVV)] [11,16,20,25,32]. Of all there geminiviruses, bean yellow dwarf virus (BeYDV) has been extensively used as replicon vector for expressing various foreign proteins in the plant [4].…”

Section: Introductionmentioning

confidence: 99%

Agroinfection of tobacco by croton yellow vein mosaic virus and designing of a replicon vector for expression of foreign gene in plant

et al. 2016

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Croton yellow vein mosaic virus (CYVMV, genus Begomovirus family Geminiviridae) is a proliferating begomovirus in the Indian sub-continent. The infectious constructs in binary vector was developed against the CYVMV genome and its associated betasatellite. Agroinoculation of the genomic construct of CYVMV produced leaf curl symptoms alone in three species of tobacco, Nicotiana tabacum, N. benthamiana and N. glutinosa. Co-inoculation of betasatellite enhanced the severity of the disease and reduced the incubation time. Based on the infectious clone, a replicon vector pCro, with only the ability to replicate inside the plant was developed. In pCro vector, CP and V2 ORFs from genome of CYVMV was deleted, which resulted localised replication of the molecule with no visible symptoms. Besides the partial CYVMV genome, pCro also has a cassette containing a double 35S promoter, multiple cloning sites and a NOS terminator to overexpress any foreign protein in plant. Episomal release of the replicon from the binary vector backbone after agroinoculation was detected by PCR. A GFP gene was cloned in pCro vector (pCro-GFP) and agroinoculated to N. tabacum resulted in localized expression of GFP at 5 dpi. The CYVMV replicon vector will be a useful tool for studying functional genomics, vaccine expression and gene silencing in plant.

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“…These advantages have allowed syringe infiltration to become the method of choice for transient gene expression, in a broad range of applications, including studies of plantpathogen interactions, abiotic stresses, plant gene functional analysis with transient silencing assay, protein localization and function, and proteinprotein interactions [16]. Entire leaf syringe agroinfiltration can be applied to obtain laboratory scale of recombinant proteins, for their biochemical characterization, purification and preclinical functional studies [1,20,21]. The simplicity of syringe infiltration may also allow it to become a favorable tool in high school and college education, for the introduction of genetic engineering.…”

Section: Agroinfiltration Methodologies and Applicationsmentioning

confidence: 99%

“…Furthermore, the efficiency of viral replication is unaffected, when the Rep is supplied in trans [12]. Correspondingly, we constructed the BeYDV geminiviral vectors, only using the LIR and the SIR, with the transgene cassette inserted between them, but without the MP and CP gene [12,20]. In the earlier version of our system, the gene for the Rep protein is provided in a second module [20].…”

Section: Vector Constructionmentioning

confidence: 99%

Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins

Chen¹,

Lai²

2013

Adv Tech Biol Med

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Current human biologics are most commonly produced by mammalian cell culture-based fermentation technologies. However, its limited scalability and high cost prevent this platform from meeting the ever increasing global demand. Plants offer a novel alternative system for the production of pharmaceutical proteins that is more scalable, cost-effective, and safer than current expression paradigms. The recent development of deconstructed virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins, and in turn, provided a preferred plant based production platform. One of the remaining challenges for the commercial application of this platform was the lack of a scalable technology to deliver the transgene into plant cells. Therefore, this review focuses on the development of an effective and scalable technology for gene delivery in plants. Direct and indirect gene delivery strategies for plant cells are first presented, and the two major gene delivery technologies based on agroinfiltration are subsequently discussed. Furthermore, the advantages of syringe and vacuum infiltration as gene delivery methodologies are extensively discussed, in context of their applications and scalability for commercial production of human pharmaceutical proteins in plants. The important steps and critical parameters for the successful implementation of these strategies are also detailed in the review. Overall, agroinfiltration based on syringe and vacuum infiltration provides an efficient, robust and scalable gene-delivery technology for the transient expression of recombinant proteins in plants. The development of this technology will greatly facilitate the realization of plant transient expression systems, as a premier platform for commercial production of pharmaceutical proteins.

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A DNA replicon system for rapid high‐level production of virus‐like particles in plants

Cited by 160 publications

References 40 publications

A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge

A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge

Agroinfection of tobacco by croton yellow vein mosaic virus and designing of a replicon vector for expression of foreign gene in plant

Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins

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