Expression of Hemagglutinin-Neuraminidase glycoprotein of Newcastle Disease Virus in agroinfiltrated Nicotiana benthamiana plants

Gómez, Emma Laura Alfaro; Zoth, Silvina Chimeno; Asurmendi, Sebastián; Rovere, Cecilia Vázquez; Berinstein, Analía

doi:10.1016/j.jbiotec.2009.09.015

Cited by 31 publications

(12 citation statements)

References 20 publications

(19 reference statements)

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“…Recent studies have demonstrated that different secretory signals allow for the accumulation of altered amounts of protein in plant systems [60], [61] and in Bacillus systems [62]. For example, plant expressed hemagglutinin levels were tested in combination with cytosolic, chloroplastic, and five different apoplastic modules, with the tobacco calreticulin secretory signal yielding the highest expression level while neither the cytosolic nor chloroplast-targeted hemagglutinin was detected [61].…”

Section: Discussionmentioning

confidence: 99%

Rapid and Scalable Plant-based Production of a Cholera Toxin B Subunit Variant to Aid in Mass Vaccination against Cholera Outbreaks

et al. 2013

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IntroductionCholera toxin B subunit (CTB) is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to killed whole-cell bacteria provides superior short-term protection to whole-cell-only vaccines; however, challenges in CTB biomanufacturing (i.e., cost and scale) hamper its implementation to mass vaccination in developing countries. To provide a potential solution to this issue, we developed a rapid, robust, and scalable CTB production system in plants.Methodology/Principal FindingsIn a preliminary study of expressing original CTB in transgenic Nicotiana benthamiana, the protein was N-glycosylated with plant-specific glycans. Thus, an aglycosylated CTB variant (pCTB) was created and overexpressed via a plant virus vector. Upon additional transgene engineering for retention in the endoplasmic reticulum and optimization of a secretory signal, the yield of pCTB was dramatically improved, reaching >1 g per kg of fresh leaf material. The protein was efficiently purified by simple two-step chromatography. The GM1-ganglioside binding capacity and conformational stability of pCTB were virtually identical to the bacteria-derived original B subunit, as demonstrated in competitive enzyme-linked immunosorbent assay, surface plasmon resonance, and fluorescence-based thermal shift assay. Mammalian cell surface-binding was corroborated by immunofluorescence and flow cytometry. pCTB exhibited strong oral immunogenicity in mice, inducing significant levels of CTB-specific intestinal antibodies that persisted over 6 months. Moreover, these antibodies effectively neutralized the cholera holotoxin in vitro.Conclusions/SignificanceTaken together, these results demonstrated that pCTB has robust producibility in Nicotiana plants and retains most, if not all, of major biological activities of the original protein. This rapid and easily scalable system may enable the implementation of pCTB to mass vaccination against outbreaks, thereby providing better protection of high-risk populations in developing countries.

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Section: Discussionmentioning

confidence: 99%

Rapid and Scalable Plant-based Production of a Cholera Toxin B Subunit Variant to Aid in Mass Vaccination against Cholera Outbreaks

et al. 2013

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“…For all 8 transformants showing the highest levels of eHN mRNA transcripts, immunoblot detected a protein band the size of 66 kDa, corresponding to the predicted eHN molecular weight. The eHN protein expression was stable and accounted up to 0.2 -0.4 % TSP, significantly more than in previous studies (Hahn et al, 2009;Gomez et al, 2009) using strong constitutive promoter and translation enhancer sequence. Localization of eHN-GFP in BY-2 protoplasts showed predominant accumulation of the protein in cytosol.…”

Section: Newcastle Disease Virusmentioning

confidence: 54%

Production of vaccines for treatment of infectious diseases by transgenic plants

Ledl

Luthar

2016

AAS

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Since the first pathogen antigen was expressed in transgenic plants with the aim of producing edible vaccine in early 1990s, transgenic plants have become a well-established expression system for production of alternative vaccines against various human and animal infectious diseases. The main focus of plant expression systems in the last five years has been on improving expression of well-studied antigens such as porcine reproductive and respiratory syndrome (PRRSV), bovine viral diarrhea disease virus (BVDV), footh and mouth disease virus (FMDV), hepatitis B surface antigen (HBsAg), rabies G protein, rotavirus, Newcastle disease virus (NDV), Norwalk virus capsid protein (NVCP), avian influenza virus H5N1, Escherichia coli heat-labile enterotoxin subunit B (LT-B), cholera toxin B (CT-B), human immunodeficiency virus (HIV), artherosclerosis, ebola and anthrax. Significant increases in expression have been obtained using improved expression vectors, different plant species and transformation methods.Key words: transgenic plants; vaccine production; recombinant proteins; antibodies; infectious diseases; risk assessment IZVLEČEK ZDRAVLJENJE NALEZLJIVIH BOLEZNI S CEPIVI PRIDOBLJENIMI S TRANSGENIMI RASTLINAMIPridobivanje aktivnih sestavin za cepiva s transgenimi rastlinami se je začelo v devetdesetih letih prejšnjega stoletja. Od takrat se transgene rastline uveljavljajo kot alternativni ekspresijski sistem za sintezo aktivnih komponent za cepiva proti številnim humanim in živalskim nalezljivim boleznim. V zadnjih petih letih je bil glavni poudarek razvoja namenjen optimizaciji rastlinskih ekspresijskih sistemov za dobro proučene antigene, kot so virus prašičjega respiratornega in reproduktivnega sindroma, virus goveje virusne diareje, virus parkljevke, površinski antigen hepatitisa B, G protein virusa stekline, rotavirus, virus atipične kokošje kuge, plaščni protein Norwalk virusa, sev ptičje gripe H5N1, temperaturno nestabilna B podenota enterotoksina Escherischia coli, kolera toksin B, HIV, arterioskleroza, ebola in antraks. Uporaba optimiziranih transformacijskih metod in ekspresijskih vektorjev v kombinaciji s primerno izbrano rastlinsko vrsto je v večini naštetih primerov vodila do opaznega izboljšanja sinteze posameznih učinkovin.

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“…Regarding the different antigens expressed in plant systems, Sojikul et al (2003) produced the hepatitis B surface antigen (HBsAg) in tobacco cell suspension cultures and obtained accumulation levels of 0.017% TSP when the KDEL retention sequence was fused to the C-terminal end of the recombinant antigen. Gómez et al (2009) obtained the maximum accumulation level (0.3% of the total leaf protein) when the hemagglutinin-neuraminidase protein of Newcastle Disease Virus was fused to the KDEL retention sequence in agroinfected Nicotiana benthamiana plants.…”

Section: Discussionmentioning

confidence: 96%

Expression of a KDEL-tagged dengue virus protein in cell suspension cultures of Nicotiana tabacum and Morinda citrifolia

Martínez

Giulietti

Talou

2011

Plant Cell Tiss Organ Cult

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The dengue virus envelope glycoprotein (DV-E) has been identified as a promising candidate for the development of a subunit vaccine and to provide an antigen for diagnostic kits. In this study, cell suspension cultures of Nicotiana tabacum and Morinda citrifolia were transformed and evaluated for production of the DV-E serotype 2 protein. The expression cassette consisted of the DV-E gene along with a signal peptide at its 5 0 end. In addition, a KDEL endoplasmic retention sequence was included in a second construct to evaluate its influence on accumulation levels of the recombinant protein. Expression cassettes were sub-cloned into a binary vector pCAMBIA 1305.2, and transformation was carried out using Agrobacterium tumefaciens. Transformed cell lines of both N. tabacum and M. citrifolia accumulated DV-E protein, although those carrying the construct containing the KDEL sequence showed higher accumulation levels than those without. Overall, cell suspension cultures of N. tabacum were more suitable for dengue antigen production than those of M. citrifolia. The highest levels of protein accumulation (0.71 ± 0.06 mg DV-E/L) were observed in tobacco cell lines at 5 days of culture, corresponding to 0.3% of total soluble protein. The recombinant protein was reactive with anti-E antibodies.

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Expression of Hemagglutinin-Neuraminidase glycoprotein of Newcastle Disease Virus in agroinfiltrated Nicotiana benthamiana plants

Cited by 31 publications

References 20 publications

Rapid and Scalable Plant-based Production of a Cholera Toxin B Subunit Variant to Aid in Mass Vaccination against Cholera Outbreaks

Rapid and Scalable Plant-based Production of a Cholera Toxin B Subunit Variant to Aid in Mass Vaccination against Cholera Outbreaks

Production of vaccines for treatment of infectious diseases by transgenic plants

Expression of a KDEL-tagged dengue virus protein in cell suspension cultures of Nicotiana tabacum and Morinda citrifolia

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