scholarly journals Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

10.3791/50084 ◽  
2013 ◽  
Author(s):  
Angela Natilla ◽  
Rosemarie W. Hammond
Keyword(s):  
Nicotiana Benthamiana ◽  
Solvent Accessibility ◽  
Cross Linking ◽  
Cysteine Residues ◽  
Virus Like Particles ◽  
Maize Rayado Fino Virus

scholarly journals Efficient Production of Chimeric Hepatitis B Virus-Like Particles Bearing an Epitope of Hepatitis E Virus Capsid by Transient Expression in Nicotiana benthamiana

Life ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 64
Author(s):  
Gergana Zahmanova ◽  
Milena Mazalovska ◽  
Katerina Takova ◽  
Valentina Toneva ◽  
Ivan Minkov ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nicotiana Benthamiana ◽  
Hepatitis E Virus ◽  
Chimeric Protein ◽  
Hepatitis E ◽  
Core Antigen ◽  
Efficient Production ◽  
Virus Like Particles ◽  
B Virus

The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particles (VLPs) when expressed in a number of heterologous systems. Such VLPs are potential carriers of foreign antigenic sequences for vaccine design. In this study, we evaluated the production of chimeric HBcAg VLPs presenting a foreign epitope on their surface, the 551–607 amino acids (aa) immunological epitope of the ORF2 capsid protein of hepatitis E virus. A chimeric construct was made by the insertion of 56 aa into the immunodominant loop of the HBcAg. The sequences encoding the chimera were inserted into the pEAQ-HT vector and infiltrated into Nicotiana benthamiana leaves. The plant-expressed chimeric HBcHEV ORF2 551–607 protein was recognized by an anti-HBcAg mAb and anti-HEV IgG positive swine serum. Electron microscopy showed that plant-produced chimeric protein spontaneously assembled into “knobbly” ~34 nm diameter VLPs. This study shows that HBcAg is a promising carrier platform for the neutralizing epitopes of hepatitis E virus (HEV) and the chimeric HBcAg/HEV VLPs could be a candidate for a bivalent vaccine.

scholarly journals Site-Directed Disulfide Cross-Linking Shows that Cleft Flexibility in the Periplasmic Domain Is Needed for the Multidrug Efflux Pump AcrB of Escherichia coli

2007 ◽  
Vol 189 (23) ◽  
pp. 8677-8684 ◽  
Author(s):  
Yumiko Takatsuka ◽  
Hiroshi Nikaido
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Disulfide Bonds ◽  
Drug Transport ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Cysteine Residues ◽  
Multidrug Efflux ◽  
Specific Chemical ◽  
Periplasmic Domain

ABSTRACT Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export. However, biochemical evidence for these conformational changes has not been provided previously. In this study, we took advantage of the observation that the external large cleft in the periplasmic domain of AcrB appears to become closed in the crystal structure of one of the three protomers, and we carried out in vivo cross-linking between cysteine residues introduced by site-directed mutagenesis on both sides of the cleft, as well as at the interface between the periplasmic domains of the AcrB trimer. Double-cysteine mutants with mutations in the cleft or the interface were inactive. The possibility that this was due to the formation of disulfide bonds was suggested by the restoration of transport activity of the cleft mutants in a dsbA strain, which had diminished activity to form disulfide bonds in the periplasm. Furthermore, rapidly reacting, sulfhydryl-specific chemical cross-linkers, methanethiosulfonates, inactivated the AcrB transporter with double-cysteine residues in the cleft expressed in dsbA cells, and this inactivation could be observed within a few seconds after the addition of a cross-linker in real time by increased ethidium influx into the cells. These observations indicate that conformational changes, including the closure of the external cleft in the periplasmic domain, are required for drug transport by AcrB.

Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system

2014 ◽  
Vol 98 (19) ◽  
pp. 8281-8290 ◽  
Author(s):  
Ki-Beom Moon ◽  
Jisu Lee ◽  
Sebyung Kang ◽  
Moonil Kim ◽  
Hugh S. Mason ◽  
...  
Keyword(s):  
Self Assembly ◽  
Nicotiana Benthamiana ◽  
Virus Like Particles ◽  
Vector Dna

scholarly journals Highly active engineered IgG3 antibodies against SARS-CoV-2

2021 ◽  
Vol 118 (42) ◽  
pp. e2107249118
Author(s):  
Somanath Kallolimath ◽  
Lin Sun ◽  
Roman Palt ◽  
Karin Stiasny ◽  
Patrick Mayrhofer ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Vaccine Development ◽  
Binding Activity ◽  
The Other ◽  
Cross Linking ◽  
Antigen Binding ◽  
In Planta ◽  
Antigen Binding Site ◽  
Highly Active ◽  
Viral Surface

Monoclonal antibodies (mAbs) that efficiently neutralize SARS-CoV-2 have been developed at an unprecedented speed. Notwithstanding, there is a vague understanding of the various Ab functions induced beyond antigen binding by the heavy-chain constant domain. To explore the diverse roles of Abs in SARS-CoV-2 immunity, we expressed a SARS-CoV-2 spike protein (SP) binding mAb (H4) in the four IgG subclasses present in human serum (IgG1-4) using glyco-engineered Nicotiana benthamiana plants. All four subclasses, carrying the identical antigen-binding site, were fully assembled in planta and exhibited a largely homogeneous xylose- and fucose-free glycosylation profile. The Ab variants ligated to the SP with an up to fivefold increased binding activity of IgG3. Furthermore, all H4 subtypes were able to neutralize SARS-CoV-2. However, H4-IgG3 exhibited an up to 50-fold superior neutralization potency compared with the other subclasses. Our data point to a strong protective effect of IgG3 Abs in SARS-CoV-2 infection and suggest that superior neutralization might be a consequence of cross-linking the SP on the viral surface. This should be considered in therapy and vaccine development. In addition, we underscore the versatile use of plants for the rapid expression of complex proteins in emergency cases.

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.

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