The use of viral vectors to produce hepatitis B virus core particles in plants

Mechtcheriakova, I.A.; Eldarov, Michael A.; Nicholson, Liz; Shanks, Michael; Skryabin, K. G.; Lomonossoff, George P.

doi:10.1016/j.jviromet.2005.06.020

Cited by 61 publications

(42 citation statements)

References 26 publications

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“…In the HBcAg-rich lanes, it is also possible to notice a persistent 40 kDa band. The band was also observed by others and corresponds to HBcAg dimers, which are resistant to denaturation (Huang et al, 2006;Mechtcheriakova et al, 2006). Even larger size HBcAg multimeric structures have been detected in denaturing gel electrophoresis (Broos et al, 2007).…”

Section: Ex Vivo Hbcag Stability In Natural Intestinal Fluid (Natif)supporting

confidence: 56%

“…This internal nucleocapsid results from the self-assembly of the nucleocapsid protein, a 21 kDa protein called Hepatitis B core antigen (HBcAg): 180 or 240 HBcAg monomers are arranged into 30 or 34 nm icosahedral particles (Birnbaum and Nassal, 1990;Crowther et al, 1994). HBcAg VLPs can be produced as a recombinant protein in a variety of hosts, including plants (Huang et al, 2006;Mechtcheriakova et al, 2006;Sainsbury and Lomonossoff, 2008). HBcAg has been shown to induce potent B and T cell responses (Milich et al, 1997(Milich et al, , 1987.…”

Section: Introductionmentioning

confidence: 99%

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Plant-expressed Hepatitis B core antigen virus-like particles: Characterization and investigation of their stability in simulated and pig gastro-intestinal fluids

Berardi

Lomonossoff

Evans

et al. 2017

International Journal of Pharmaceutics

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Virus-like particles (VLPs) are potential oral vaccine candidates, as their highly compact structure may allow them to withstand the harsh conditions of the gastro-intestinal (GI) environment.Hepatitis B core antigen (HBcAg) is an immunogenic protein that assembles into 30 or 34 nm diameter VLPs. Here, the stabilities of both the HBcAg polypeptide itself and the threedimensional structure of the VLPs upon exposure to in vitro and ex vivo simulated gastric and intestinal fluids were investigated. Plant-expressed HBcAg VLPs were efficiently purified by sucrose density gradient and characterized. The purified VLPs did not show major chemical or physical instability upon exposure to the low pH conditions typically found in the stomach; however, they completely agglomerated upon acidification and subsequent pH neutralization. The HBcAg polypeptide was highly digested upon exposure to pepsin in simulated gastric fluids.HBcAg appeared more stable in both simulated and ex vivo intestinal fluids, where despite a partial digestion of the HBcAg polypeptide, the VLPs maintained their most immunogenic epitopes and their particulate conformation. These results suggest that HBcAg VLPs are likely to be unstable in gastric fluids, yet if the gastric instability could be bypassed, they could maintain their particulate structure and immunogenicity in intestinal fluids.

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Section: Ex Vivo Hbcag Stability In Natural Intestinal Fluid (Natif)supporting

confidence: 56%

Section: Introductionmentioning

confidence: 99%

Plant-expressed Hepatitis B core antigen virus-like particles: Characterization and investigation of their stability in simulated and pig gastro-intestinal fluids

Berardi

Lomonossoff

Evans

et al. 2017

International Journal of Pharmaceutics

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“…DsRed (CLONTECH), HBcAg (Mechtcheriakova et al, 2006), and the H and L chains of 2G12 (Buchacher et al, 1994) were initially cloned into pM81-FSC1 via BspHI/StuI sites. For expression with the wild-type leader, PacI/AscI fragments were transferred into similarly digested pBINPLUS.…”

Section: Plasmid Constructsmentioning

confidence: 99%

Extremely High-Level and Rapid Transient Protein Production in Plants without the Use of Viral Replication

2008

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Plant-based overexpression of heterologous proteins has attracted much interest and development in recent years. To date, the most efficient vectors have been based on RNA virus-derived replicons. A system based on a disabled version of cowpea mosaic virus RNA-2 has been developed, which overcomes limitations on insert size and introduces biocontainment. This system involves positioning a gene of interest between the 5# leader sequence and 3# untranslated region (UTR) of RNA-2, thereby emulating a presumably stable mRNA for efficient translation. Thus far, the sequence of the 5# UTR has been preserved to maintain the ability of the modified RNA-2 to be replicated by RNA-1. However, high-level expression may be achieved in the absence of RNA-1-derived replication functions using Agrobacterium-mediated transient transformation. To investigate those features of the 5# UTR necessary for efficient expression, we have addressed the role of two AUG codons found within the 5# leader sequence upstream of the main initiation start site. Deletion of an in-frame start codon upstream of the main translation initiation site led to a massive increase in foreign protein accumulation. By 6 d postinfiltration, a number of unrelated proteins, including a full-size IgG and a self-assembling virus-like particle, were expressed to .10% and 20% of total extractable protein, respectively. Thus, this system provides an ideal vehicle for high-level expression that does not rely on viral replication of transcripts.

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“…Through various incarnations as replicating viral vectors for polypeptide expression (9, 24,66,82) and the successful production of a number of pharmaceutically relevant proteins (46,47,49,51,65) 1a). RNA-1 encodes proteins involved in the replication of viral RNAs and polyprotein processing, including the RNA-dependent RNA polymerase, a helicase, the 24K proteinase, and a proteinase cofactor.…”

mentioning

confidence: 99%

Cowpea mosaicVirus: The Plant Virus–Based Biotechnology Workhorse

Sainsbury

Cañizares

Lomonossoff

2010

Annu. Rev. Phytopathol.

106

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INTRODUCTIONThe first plant viruses to be developed as expression vectors in the early 1980s were those with DNA genomes (for reviews, see 42, 56). This was due to the fact that, at the time, only the DNA genomes could be manipulated and the technology for creating infectious cDNA copies of viruses with RNA genomes did not exist. However, the vast majority of plant viruses have genomes that consist of one or more strands of positive-sense RNA. These viruses infect a wide range of hosts and some can reach extremely high titres. Following the construction of the first full-length cDNA clones shown to be infectious (1), the past 25 years has seen a large number of RNA viruses developed as vectors for the expression of foreign sequences and other uses, such as gene silencing (10, 31,42, 55, 56,70).Many proteins have been successfully expressed with virus vectors and significant progress in vector design has been driven by the demands of this application. Generally developed with the expression of fluorescent marker proteins, RNA virus-based vectors have become a highly effective means of producing recombinant heterologous protein in plant tissue within a short time frame (28, 35,44,67). In addition to their use as vectors for the production of heterologous polypeptides and as gene silencing vectors, plant RNA viruses have also provided a source of particles for various applications. Virus capsids provide nano-scale particles with consistent size and shape, which can be exploited for a number of chemical and biological applications (72). For example, a number of systems make use of the repetitive geometry of plant virus capsids to present multiple copies of antigenic sequences, to increase their potential as a source of novel vaccines (10,39, 58). Also, both wild type and genetically modified capsids of plant viruses are also being used as biotemplates for novel materials in nanotechnology (88). 3Cowpea mosaic virus (CPMV) has borne witness to most of the aforementioned biotechnological uses of RNA viruses. The year 2009 marks the 50 th anniversary of the first description of CPMV as a pathogen of cowpeas (Vigna unguiculata) in West Africa (13). As a result of its ease of propagation, high yield and the stability of the viral particles, CPMV rapidly became an object of intense scientific research. Early studies revealed the bipartite nature of the viral genome (7,78), the structural similarities between CPMV and the animal picornaviruses (87) and the mechanism of gene expression (polyprotein processing; 53).Subsequent work resulted in the determination of the nucleotide sequences of both genomic segments (81, 43), a realisation of the genetic similarities between CPMV and picornaviruses (21), an atomic resolution structure of the virus particles (32, 33), and the creation of infectious cDNA clones (16, 25,84). A crucial step for the development of practical CPMVbased expression systems was the creation of vectors that could be inoculated by agroinfiltration (38), and this approach is now the method of choice for intr...

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The use of viral vectors to produce hepatitis B virus core particles in plants

Cited by 61 publications

References 26 publications

Plant-expressed Hepatitis B core antigen virus-like particles: Characterization and investigation of their stability in simulated and pig gastro-intestinal fluids

Plant-expressed Hepatitis B core antigen virus-like particles: Characterization and investigation of their stability in simulated and pig gastro-intestinal fluids

Extremely High-Level and Rapid Transient Protein Production in Plants without the Use of Viral Replication

Cowpea mosaicVirus: The Plant Virus–Based Biotechnology Workhorse

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