Triple layered rotavirus VLP production: Kinetics of vector replication, mRNA stability and recombinant protein production

Vieira, Helena L.A.; Estêvão, Catarina; Roldão, António; Peixoto, Cristina; Sousa, Marcos F. Q.; Cruz, Pedro; Carrondo, Manuel J.T.; Alves, Paula M.

doi:10.1016/j.jbiotec.2005.03.026

Cited by 94 publications

(84 citation statements)

References 19 publications

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“…For efficient RV VLP formation, the ratio of each capsid protein was significantly important [40,41]. In our study, both 2/6 and 2/6/7 RV VLPs were detected in the extract from transgenic tobacco plants.…”

Section: Discussionmentioning

confidence: 46%

“…VP7-containing RV VLPs have not been obtained in plants previously. Many researchers have successfully produced and purified RV VLPs in insect cells; however, the efficiency of assembly of RV VLPs in insect cells is very low, with only about 15% of the total virus capsid proteins produced in the insect cells participating in the formation of RV VLPs [40,41]. The self-assembly efficiency of 2/6-RV VLPs produced in transgenic plants was even lower [38].…”

Section: Discussionmentioning

confidence: 99%

“…By comparing the stoichiometric mass ratio of VP2:VP6:VP7 produced in tobacco plants compared with that in the native RV particle, we found that VP7 was under-expressed and it could be the limiting protein during the assembly of triple-layered particles. According to studies by Vieira and Carrondo [40,41], the outer layer protein VP7 should be more than VP2 and VP6 thus avoiding accumulation of waste monomers or other protein complexes, such as VP6 trimers [42,43].…”

Section: Discussionmentioning

confidence: 99%

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Immunogenicity and virus-like particle formation of rotavirus capsid proteins produced in transgenic plants

Yang

et al. 2010

Sci. China Life Sci.

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The human pathogen, group A rotavirus, is the most prevalent cause of acute infantile and pediatric gastroenteritis worldwide, especially in developing countries. There is an urgent demand for safer, more effective and cheaper vaccines against rotavirus infection. Plant-derived antigens may provide an exclusive way to produce economical subunit vaccines. Virus-like particles, constituting viral capsid proteins without viral nucleic acids, are considered a far safer candidate compared with live attenuated viral vaccines. In this study, the rotavirus capsid proteins VP2, VP6 and VP7 were co-expressed in transgenic tobacco plants, and their expression levels, formation of rotavirus-like particles (RV VLPs) and immunogenicity were extensively studied. Quantitative real-time RT-PCR and Western blot analysis revealed that the expression level of vp6 was the highest while vp7 was expressed at the lowest levels. The RV VLPs were purified from transgenic tobacco plants and analyzed by electron microscopy and Western blot. Results indicated that the plant-derived VP2, VP6 and VP7 proteins self-assembled into 2/6 or 2/6/7 RV VLPs with a diameter of 60-80 nm. When orally delivered into mice with cholera toxin as an adjuvant, the total soluble protein extracted from transgenic tobacco plants induced rotavirus-specific antibodies comparable with those of attenuated rotavirus vaccines, while VP 2/6/7 induced higher serum IgG and fecal IgA titers compared with VP 2/6. rotavirus, virus-like particles, transgenic plant, oral vaccine, immunogenicity Citation:Yang Y M, Li X, Yang H, et al. Immunogenicity and virus-like particle formation of rotavirus capsid proteins produced in transgenic plants. Sci

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Section: Discussionmentioning

confidence: 46%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Immunogenicity and virus-like particle formation of rotavirus capsid proteins produced in transgenic plants

Yang

et al. 2010

Sci. China Life Sci.

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“…Purified rBV viral DNA stocks previously quantitated by another validated qPCR method were used as external standard. 27 The wild-type baculovirus ie-1 gene sequence was used as a template for the amplification of a chosen amplicon of 150 bp (protocol adapted from Lo and Chao 28 ). DNA amplifications were carried out using the LightCycler system with Fast Start Master SYBR Green I kit (Roche Diagnostics).…”

Section: Characterization Methodsmentioning

confidence: 99%

Purification of recombinant baculoviruses for gene therapy using membrane processes

et al. 2009

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Recombinant baculoviruses (rBVs) are widely used as vectors for the production of recombinant proteins in insect cells. More recently, these viral vectors have been gaining increasing attention due to their emerging potential as gene therapy vehicles to mammalian cells. Their production in stirred bioreactors using insect cells is an established technology; however, the downstream processing (DSP) of baculoviruses envisaged for clinical applications is still poorly developed. In the present work, the recovery and purification of rBVs aiming at injectable-grade virus batches for gene therapy trials was studied. A complete downstream process comprising three steps-depth filtration, ultra/ diafiltration and membrane sorption-was successfully developed. Optimal operational conditions for each individual step were achieved yielding a scalable DSP for rBVs as vectors for gene therapy. The processing route designed hereby presents global recovery yields reaching 40% (at purities over 98%) and, most importantly, relies on technologies easy to transfer to process scales under cGMP guidelines.

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“…Multiple capsid proteins can be expressed simultaneously in insect cells using a single recombinant baculovirus containing multiple gene expression cassettes. Triplelayered rotavirus VLPs composed of capsid proteins, VP2, VP6 and VP7,have been produced in Sf-9 and Sf-21 cells using a recombinant baculovirus containing atricistronicgene expression cassette [51]. Double-layered rotavirus VLPs are also produced in stably transformed Drosophila melanogaster S2 cells using encephalomyocarditis-virusderived internal ribosomal entry site element [52].…”

Section: Insectsmentioning

confidence: 99%

Functional Virus-Like Particles Production Using Silkworm and Their Application in Life Science

Kato¹,

Deo²,

Park

et al. 2012

J Biotechnol Biomaterial

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Virus-like particles (VLPs), which mimic the structure of authentic virus, are of significant importance owing to their wide ranging applications. VLPs have the potential to serve as candidate vaccine in addition to subunit vaccines and also serve as a nano-bioparticle scaffold for displaying complex foreign proteins. Here, we review different methods available to produce VLPs with various host expression systems including from microorganisms to mammalian cells and the current potential of silkworms as producers of these VLPs. Recently, silkworms have been used for efficient production of VLPs too in addition to mass production of recombinant proteins, which have contributed to molecular structural analysis, molecule-molecule interactions in biology and biotechnology.

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Triple layered rotavirus VLP production: Kinetics of vector replication, mRNA stability and recombinant protein production

Cited by 94 publications

References 19 publications

Immunogenicity and virus-like particle formation of rotavirus capsid proteins produced in transgenic plants

Immunogenicity and virus-like particle formation of rotavirus capsid proteins produced in transgenic plants

Purification of recombinant baculoviruses for gene therapy using membrane processes

Functional Virus-Like Particles Production Using Silkworm and Their Application in Life Science

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