Production of Recombinant Antigens and Antibodies in Nicotiana benthamiana Using ‘Magnifection’ Technology: GMP-Compliant Facilities for Small- and Large-Scale Manufacturing

2012 ◽  
pp. 127-154 ◽  
Author(s):  
Victor Klimyuk ◽  
Gregory Pogue ◽  
Stefan Herz ◽  
John Butler ◽  
Hugh Haydon
Keyword(s):  
Nicotiana Benthamiana ◽  
Large Scale ◽  
Recombinant Antigens

scholarly journals Efficient Single Tobamoviral Vector-Based Bioproduction of Broadly Neutralizing Anti-HIV-1 Monoclonal Antibody VRC01 in Nicotiana benthamiana Plants and Utility of VRC01 in Combination Microbicides

2013 ◽  
Vol 57 (5) ◽  
pp. 2076-2086 ◽  
Author(s):  
Krystal Teasley Hamorsky ◽  
Tiffany W. Grooms-Williams ◽  
Adam S. Husk ◽  
Lauren J. Bennett ◽  
Kenneth E. Palmer ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Nicotiana Benthamiana ◽  
Large Scale ◽  
Neutralization Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Scale Production ◽  
Gp120 Binding ◽  
Topical Microbicides ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACTBroadly neutralizing monoclonal antibodies (bnMAbs) may offer powerful tools for HIV-1 preexposure prophylaxis, such as topical microbicides. However, this option is hampered due to expensive MAb biomanufacturing based on mammalian cell culture. To address this issue, we developed a new production system for bnMAb VRC01 inNicotiana benthamianaplants using a tobamovirus replicon vector. Unlike conventional two-vector-based expression, this system was designed to overexpress full-length IgG1 from a single polypeptide by means of kex2p-like enzyme recognition sites introduced between the heavy and light chains. An enzyme-linked immunosorbent assay (ELISA) revealed that gp120-binding VRC01 IgG1 was maximally accumulated on 5 to 7 days following vector inoculation, yielding ∼150 mg of the bnMAb per kg of fresh leaf material. The plant-made VRC01 (VRC01p) was efficiently purified by protein A affinity followed by hydrophobic-interaction chromatography. ELISA, surface plasmon resonance, and an HIV-1 neutralization assay demonstrated that VRC01p has gp120-binding affinity and HIV-1-neutralization capacity virtually identical to the human-cell-produced counterpart. To advance VRC01p's use in topical microbicides, we analyzed combinations of the bnMAb with other microbicide candidates holding distinct antiviral mechanisms in an HIV-1 neutralization assay. VRC01p exhibited clear synergy with the antiviral lectin griffithsin, the CCR5 antagonist maraviroc, and the reverse transcriptase inhibitor tenofovir in multiple CCR5-tropic HIV-1 strains from clades A, B, and C. In summary, VRC01p is amenable to robust, rapid, and large-scale production and may be developed as an active component in combination microbicides with other anti-HIV agents such as antiviral lectins, CCR5 antagonists, and reverse transcriptase inhibitors.

scholarly journals Plant-produced chimeric virus-like particles - a new generation vaccine against African horse sickness

2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Daria A. Rutkowska ◽  
Nobalanda B. Mokoena ◽  
Tsepo L. Tsekoa ◽  
Vusi S. Dibakwane ◽  
Martha M. O’Kennedy
Keyword(s):  
Capsid Protein ◽  
Nicotiana Benthamiana ◽  
Large Scale ◽  
Viral Disease ◽  
African Horse Sickness ◽  
Virus Like Particles ◽  
Chimeric Vlps ◽  
Humoral Immune ◽  
Generation Vaccine ◽  
New Generation

Abstract Background African horse sickness (AHS) is a severe arthropod-borne viral disease of equids, with a mortality rate of up to 95% in susceptible naïve horses. Due to safety concerns with the current live, attenuated AHS vaccine, alternate safe and effective vaccination strategies such as virus-like particles (VLPs) are being investigated. Transient plant-based expression systems are a rapid and highly scalable means of producing such African horse sickness virus (AHSV) VLPs for vaccine purposes. Results In this study, we demonstrated that transient co-expression of the four AHSV capsid proteins in agroinfiltrated Nicotiana benthamiana dXT/FT plants not only allowed for the assembly of homogenous AHSV-1 VLPs but also single, double and triple chimeric VLPs, where one capsid protein originated from one AHS serotype and at least one other capsid protein originated from another AHS serotype. Following optimisation of a large scale VLP purification procedure, the safety and immunogenicity of the plant-produced, triple chimeric AHSV-6 VLPs was confirmed in horses, the target species. Conclusions We have successfully shown assembly of single and double chimeric AHSV-7 VLPs, as well as triple chimeric AHSV-6 VLPs, in Nicotiana benthamiana dXT/FT plants. Plant produced chimeric AHSV-6 VLPs were found to be safe for administration into 6 month old foals as well as capable of eliciting a weak neutralizing humoral immune response in these target animals against homologous AHSV virus.

scholarly journals Large-scale screening of human sera with cytomegalovirus recombinant antigens.

1990 ◽  
Vol 28 (6) ◽  
pp. 1375-1379 ◽  
Author(s):  
M P Landini ◽  
M X Guan ◽  
G Jahn ◽  
W Lindenmaier ◽  
M Mach ◽  
...  
Keyword(s):  
Large Scale ◽  
Recombinant Antigens ◽  
Human Sera ◽  
Large Scale Screening

scholarly journals Identification of Nicotiana benthamiana Genes Involved in Pathogen-Associated Molecular Pattern–Triggered Immunity

2010 ◽  
Vol 23 (6) ◽  
pp. 715-726 ◽  
Author(s):  
Suma Chakravarthy ◽  
André C. Velásquez ◽  
Sophia K. Ekengren ◽  
Alan Collmer ◽  
Gregory B. Martin
Keyword(s):  
Cell Death ◽  
Nicotiana Benthamiana ◽  
Large Scale ◽  
Forward Genetics ◽  
Hormone Signaling ◽  
Virus Induced Gene Silencing ◽  
Molecular Pattern ◽  
Cell Death Response ◽  
A Cell ◽  
Pathogen Associated Molecular Pattern

In order to identify components of pathogen-associated molecular pattern–triggered immunity (PTI) pathways in Nicotiana benthamiana, we conducted a large-scale forward-genetics screen using virus-induced gene silencing and a cell-death-based assay for assessing PTI. The assay relied on four combinations of PTI-inducing nonpathogens and cell-death-causing challenger pathogens and was first validated in plants silenced for FLS2 or BAK1. Over 3,200 genes were screened and 14 genes were identified that, when silenced, compromised PTI as judged by the cell-death-based assay. Further analysis indicated that the 14 genes were not involved in a general cell death response. A subset of the genes was found to act downstream of FLS2-mediated PTI induction, and silencing of three genes compromised production of reactive oxygen species in leaves exposed to flg22. The 14 genes encode proteins with potential functions in defense and hormone signaling, protein stability and degradation, energy and secondary metabolism, and cell wall biosynthesis and provide a new resource to explore the molecular basis for the involvement of these processes in PTI.

A simple and rapid in vitro test for large-scale screening of fungal endophytes from drought-adapted Australian wild plants for conferring water deprivation tolerance and growth promotion in Nicotiana benthamiana seedlings

2017 ◽  
Vol 199 (10) ◽  
pp. 1357-1370 ◽  
Author(s):  
Khondoker M. G. Dastogeer ◽  
Hua Li ◽  
Krishnapillai Sivasithamparam ◽  
Michael G. K. Jones ◽  
Stephen J. Wylie
Keyword(s):  
Nicotiana Benthamiana ◽  
Water Deprivation ◽  
Large Scale ◽  
Growth Promotion ◽  
Fungal Endophytes ◽  
Wild Plants ◽  
In Vitro Test ◽  
Vitro Test ◽  
Large Scale Screening

scholarly journals Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana

2021 ◽  
Vol 12 ◽  
Author(s):  
Emmanuel Margolin ◽  
Joel D. Allen ◽  
Matthew Verbeek ◽  
Michiel van Diepen ◽  
Phindile Ximba ◽  
...  
Keyword(s):  
Mammalian Cell ◽  
Nicotiana Benthamiana ◽  
Mammalian Cells ◽  
Large Scale ◽  
Molecular Farming ◽  
Marburg Virus ◽  
Viral Envelope ◽  
Scale Production ◽  
Site Specific ◽  
Viral Glycoproteins

There is an urgent need to establish large scale biopharmaceutical manufacturing capacity in Africa where the infrastructure for biologics production is severely limited. Molecular farming, whereby pharmaceuticals are produced in plants, offers a cheaper alternative to mainstream expression platforms, and is amenable to rapid large-scale production. However, there are several differences along the plant protein secretory pathway compared to mammalian systems, which constrain the production of complex pharmaceuticals. Viral envelope glycoproteins are important targets for immunization, yet in some cases they accumulate poorly in plants and may not be properly processed. Whilst the co-expression of human chaperones and furin proteases has shown promise, it is presently unclear how plant-specific differences in glycosylation impact the production of these proteins. In many cases it may be necessary to reproduce features of their native glycosylation to produce immunologically relevant vaccines, given that glycosylation is central to the folding and immunogenicity of these antigens. Building on previous work, we transiently expressed model glycoproteins from HIV and Marburg virus in Nicotiana benthamiana and mammalian cells. The proteins were purified and their site-specific glycosylation was determined by mass-spectrometry. Both glycoproteins yielded increased amounts of protein aggregates when produced in plants compared to the equivalent mammalian cell-derived proteins. The glycosylation profiles of the plant-produced glycoproteins were distinct from the mammalian cell produced proteins: they displayed lower levels of glycan occupancy, reduced complex glycans and large amounts of paucimannosidic structures. The elucidation of the site-specific glycosylation of viral glycoproteins produced in N. benthamiana is an important step toward producing heterologous viral glycoproteins in plants with authentic human-like glycosylation.

scholarly journals Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana

2018 ◽  
Vol 8 ◽  
Author(s):  
Changlong Chen ◽  
Yongpan Chen ◽  
Heng Jian ◽  
Dan Yang ◽  
Yiran Dai ◽  
...  
Keyword(s):  
Cell Death ◽  
Nicotiana Benthamiana ◽  
Large Scale ◽  
Heterodera Avenae ◽  
Identification And Characterization

Screening of antinuclear antibodies: comparison between enzyme immunoassay based on nuclear homogenates, purified or recombinant antigens and immunofluorescence assay

2004 ◽  
Vol 42 (10) ◽  
Author(s):  
Sergio Bernardini ◽  
Maria Infantino ◽  
Lorenza Bellincampi ◽  
Marzia Nuccetelli ◽  
Antonella Afeltra ◽  
...  
Keyword(s):  
Screening Test ◽  
Antinuclear Antibody ◽  
Large Scale ◽  
Antinuclear Antibodies ◽  
Immunofluorescence Assay ◽  
Choice Test ◽  
Recombinant Antigens ◽  
Specificity And Sensitivity ◽  
First Choice ◽  
Κ Statistic

AbstractCurrent clinical practice considers antinuclear antibody (ANA) testing as a screening test; this has a major impact on laboratory work with a growing volume of analyses that need to be performed rapidly, to maintain good specificity and sensitivity. Ongoing discussions have been raised in order to identify the best technology to use in ANA screening, taking into account both clinical and economical implications. The aim of our study was to compare three different enzyme immunoassays (EIA) with immunofluorescence (IF) assay in order to identify which test is better for use as a screening test. The study was performed on 473 sera and the three different EIA tests were based on nuclear homogenates from HeLa cells, purified antigens from HEp-2 cells and recombinant antigens, respectively. The concordance between EIA-ANA and IF-ANA techniques, determined by the κ statistic, was acceptable, but not complete, and discrepancies between both EIA-positive/IF-negative samples and IF-positive/EIA-negative were found. Both methods show interesting diagnostic abilities, however, the IF-ANA assay seems to be the first choice test in a well-standardized immunofluorescence laboratory with experienced microscopists, whereas the EIA test might be useful especially in large-scale ANA screening.

scholarly journals Systemic Infection of Nicotiana benthamiana with Potato virus X Nanoparticles Presenting a Fluorescent iLOV Polypeptide Fused Directly to the Coat Protein

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Juliane Röder ◽  
Christina Dickmeis ◽  
Rainer Fischer ◽  
Ulrich Commandeur
Keyword(s):  
Coat Protein ◽  
Potato Virus ◽  
Nicotiana Benthamiana ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Potato Virus X ◽  
Leaf Extracts ◽  
Systemic Spread ◽  
Mouth Disease Virus ◽  
Direct Fusion

Plant virus-based nanoparticles can be produced in plants on a large scale and are easily modified to introduce new functions, making them suitable for applications such as vaccination and drug delivery, tissue engineering, and in vivo imaging. The latter is often achieved using green fluorescent protein and its derivatives, but the monovalent fluorescent protein iLOV is smaller and more robust. Here, we fused the iLOV polypeptide to the N-terminus of the Potato virus X (PVX) coat protein, directly or via the Foot-and-mouth disease virus 2A sequence, for expression in Nicotiana benthamiana. Direct fusion of the iLOV polypeptide did not prevent the assembly or systemic spread of the virus and we verified the presence of fusion proteins and iLOV hybrid virus particles in leaf extracts. Compared to wild-type PVX virions, the PVX particles displaying the iLOV peptide showed an atypical, intertwined morphology. Our results confirm that a direct fusion of the iLOV fluorescent protein to filamentous PVX nanoparticles offers a promising tool for imaging applications.

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