<i>Escherichia coli</i>‐based cell‐free synthesis of virus‐like particles

Bundy, Bradley C.; Franciszkowicz, Marc J.; Swartz, James R.

doi:10.1002/bit.21716

Cited by 169 publications

(158 citation statements)

References 44 publications

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“…With this approach, the specificity of the HA was examined, independent of molecular adaptations required to generate replication-competent virus, which allowed identification of several mutants with altered SA specificity. Other mutants have been defined recently whose recognition was assessed with a less-specific assay (14), and we find here that they do not gain a2,6-SA recognition in the HA assay Figures were generated using UCSF Chimera ) with the crystal structure of HA from A/South Carolina/1/1918 (1RUZ.pdb) .…”

Section: Correlation Of Antibody-binding Site and Receptor-binding Sitementioning

confidence: 67%

“…Groups of mice were immunised on days 0, 21, and 42, and bled on tail on days 0, 14,35,. Sera from day 56 were used to determine the immunogenicity of H190 by performing immunoassays against peptide H190 as described in Section 4.2.7.…”

Section: H190-specific Immunogenicitymentioning

confidence: 99%

“…VLPs are protein assemblies that mimic viral structure [8][9][10][11][12][13][14]; modular VLPs combine a VLP with an antigen module from an unrelated pathogen [9,[15][16][17]. Two types of modules are commonly used: (i) short peptides expected to have minimal structure [18][19][20][21]; and (ii) larger domains with expected tertiary structures [22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

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Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

Anggraeni¹

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A promising alternative to replace the current egg-or cell culture-based technology for vaccine production from live viruses is virus-like particle (VLP) technology based on a microbial platform. VLPs are macromolecular assemblies of viral capsid proteins, which have been shown to tolerate insertion of antigen modules via genetic recombinant technology, yielding modular VLPs. Many studies on modular VLPs presume that when a peptide antigen element is taken out from the intact proteins and then modularised on VLPs, it is unable to fold into its native structure. However, until now, presentation of a peptide antigen element on a VLP and the impact of the display strategy to present the antigen element on the quality of the resulting antibodies (i.e. the ability of the antibodies to recognise the intact protein) are not fully understood. This thesis aims to understand the underlying fundamentals regarding modularisation of peptide antigen elements on VLPs for induction of high-quality antibodies. A hypervariable receptor-binding domain, Helix 190 (H190), from the hemagglutinin protein of influenza A virus was used as a model for modularisation on VLPs from murine polyomavirus (MuPyV) VP1 protein. Four major findings are presented. Firstly, two display strategies, i.e. arraying of H190 in tandem repeats and the use of helix promoter elements, were shown to display H190 in its immunogenic form equally. However, modularisation using tandem repeat display induced antibodies of a higher quality than modularisation using helix promoter elements.Secondly, the quality of antibodies induced by the tandem repeat display bearing two copies of H190 was optimum, thus no significant improvement was observed following the use of adjuvant or increasing the copy number of H190. Additionally, the increase in the copy number of H190 was shown to reduce the assembly capability and solubility of modular VP1 in an environment that was optimised for wild-type VP1. Thirdly, this thesis shows the novel finding in the use of flanking ionic elements to stabilise VLP precursors, termed as capsomeres, bearing two copies of H190 containing a hydrophobic stretch, which caused aggregation. Fourthly, the first steps towards obtaining the atomic crystal structure of presented H190 on a modular protein were performed, i.e. a mild and satisfactory laboratory process was developed to achieve high-purity modular VP1 capsomeres, unattainable using previously established expression and purification process of wild-type MuPyV VP1. This thesis shows a step forward towards understanding the presentation of a peptide antigen element on a VLP that enables induction of highquality antibodies, and towards VLP engineering to manipulate the aggregation and solubility of modular VP1. VLP technology based on a microbial platform presented here is iii a potentially safe and effective alternative vaccine candidate that targets a hypervariable peptide antigen element. The speed of the microbial platform allows a rapid response to the hypervariability of the pe...

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Section: Correlation Of Antibody-binding Site and Receptor-binding Sitementioning

confidence: 67%

Section: H190-specific Immunogenicitymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

Anggraeni¹

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“…It is noteworthy that not only E. coli cells but also the E. coli -based cell-free translation system can be used for efficient in vitro production of MS2 VLPs [71,72]. …”

Section: Expression and Structural Investigationsmentioning

confidence: 99%

The True Story and Advantages of RNA Phage Capsids as Nanotools

et al. 2016

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RNA phages are often used as prototypes for modern recombinant virus-like particle (VLP) technologies. Icosahedral RNA phage VLPs can be formed from coat proteins (CPs) and are efficiently produced in bacteria and yeast. Both genetic fusion and chemical coupling have been successfully used for the production of numerous chimeras based on RNA phage VLPs. In this review, we describe advances in RNA phage VLP technology along with the history of the Leviviridae family, including its taxonomical organization, genomic structure, and important role in the development of molecular biology. Comparative 3D structures of different RNA phage VLPs are used to explain the level of VLP tolerance to foreign elements displayed on VLP surfaces. We also summarize data that demonstrate the ability of CPs to tolerate different organic (peptides, oligonucleotides, and carbohydrates) and inorganic (metal ions) compounds either chemically coupled or noncovalently added to the outer and/or inner surfaces of VLPs. Finally, we present lists of nanotechnological RNA phage VLP applications, such as experimental vaccines constructed by genetic fusion and chemical coupling methodologies, nanocontainers for targeted drug delivery, and bioimaging tools.

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“…At present, new proteins representing potential anti-malaria vaccines have been fully worked out in the systems of extracellular protein expression (Tsuboi et al 2008). Also the expression of virus-like particles (VLP) has been characterized inside in vitro systems The examples include the phage protein, MS2 and C-terminal fragment of the protein core in the hepatitis B virus (HBV), the biological activity of which has been identical to the forms obtained in vivo (Bundy, Franciszkowicz, and Swartz 2008).…”

Section: Efficient Expression Of Several Proteins and Their Screeningmentioning

confidence: 99%

The Development of Cell-Free Protein Expression Systems and Their Application in the Research on Antibiotics Targeting Ribosome

Szaflarski¹,

Nowicki²,

Zabel³

2012

Advances in Applied Biotechnology

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Escherichia coli‐based cell‐free synthesis of virus‐like particles

Cited by 169 publications

References 44 publications

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

The True Story and Advantages of RNA Phage Capsids as Nanotools

The Development of Cell-Free Protein Expression Systems and Their Application in the Research on Antibiotics Targeting Ribosome

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