Sensitivity of immune response quality to influenza helix 190 antigen structure displayed on a modular virus-like particle

Anggraeni, Melisa R.; Connors, Natalie K.; Wu, Yang; Chuan, Yap P.; Lua, Linda H.L.; Middelberg, Anton P. J.

doi:10.1016/j.vaccine.2013.06.087

Cited by 28 publications

(29 citation statements)

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“…Antigen modules comprise peptide antigen elements from foreign pathogens and other necessary elements, such as flanking structure promoter or spacer elements. Combination of a VLP and an antigen module yields a modular VLP (Figure 1-1) (Anggraeni et al 2013). A tertiary structure of a modular monomeric viral capsid protein (grey) displaying an antigen module (red, green, and blue).…”

Section: Virus-like Particles (Vlps)mentioning

confidence: 99%

“…Several insertion sites in the VP1 protein have been patented (Lua and Middelberg 2007), allowing for recombinant insertion of antigen modules at a genetic level and projection of the inserted antigen modules to the surface of modular VLPs for immune system recognition (Gedvilaite et al 2000, Liddington et al 1991. The MuPyV VP1 VLPs have been engineered to present antigen modules containing peptide antigen elements from various pathogens (Anggraeni et al 2013, indicating the robustness of the VLPs.…”

Section: Virus-like Particles (Vlps)mentioning

confidence: 99%

“…Endotoxins were removed from the purified modular VP1 capsomeres to below 20 EU ml -1 using Vivapure Q Maxi H spin column (Sartorius Stedim, Goettinge, Germany) as previously described in Chapter 3 (Anggraeni et al 2013 mM Tris-base, 5% (v/v) glycerol, 1 mM CaCl 2 , pH 7.4) for 15 h at room temperature (Gleiter and Lilie 2001) in an endotoxin-free environment. Formed VLPs were then dialysed against phosphate buffer saline (PBS; E404, Amresco, USA) at 4ºC for 24 h.…”

Section: Assembly Of Modular Vp1 Proteinsmentioning

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: Virus-like Particles (Vlps)mentioning

confidence: 99%

Section: Virus-like Particles (Vlps)mentioning

confidence: 99%

Section: Assembly Of Modular Vp1 Proteinsmentioning

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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“…Multiple matrix 2 ectodomain (M2e) epitopes have been inserted to the VP1 protein to improve immunogenicity, and adjuvanted capsomeres presenting the M2e epitopes at the external loop of the protein were able to stimulate humoral immunity in mice . The helical subunit helix 190 (H190) from influenza protein hemagglutinin has also been presented on MPyV VP1 and assembled VLPs were able to elicit a strong antibody response dependent on the epitope design (Anggraeni et al 2013). …”

Section: Murine Polyoma Viral Coat Proteins As a Vaccine Delivery Systemmentioning

confidence: 99%

“…Capsomeres with successful foreign peptide insertion are then assembled into VLPs and their immunogenicity tested in mice (Anggraeni et al 2013;Chuan et al 2013;Middelberg et al 2011;Rivera-Hernandez et al 2013). In cases where epitope insertion interfered with the ability of the modular capsomeres to form VLPs, leading to incomplete VLPs or aggregation, the C-terminus can be truncated (Wibowo et al 2012).…”

Section: Established Mpyv Vaccine Platformmentioning

confidence: 99%

Murine polyomavirus VLPs as a platform for cytotoxic T cell epitope-based influenza vaccine candidates

Abidin¹

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This research examined the ability of virus-like particles (VLP) assembled from the coat proteins of Murine polyomavirus (MPyV) to carry cytotoxic T cell (Tc) epitopes. MPyV VLPs can be assembled in vitro from purified subunits composed of pentamers of the major coat protein VP1 called capsomeres; and this approach provides fine control over VLP composition and structure. Tc-epitopes are often highly hydrophobic, which poses a significant bioengineering challenge and two distinct approaches were pursued to determine the optimal delivery strategy of Influenza Tc-epitopes by MPyV VLP. Firstly, identified epitopes were externally presented using established sites for peptide insertion on the MPyV major coat protein, VP1. Secondly, polypeptides possessing multiple epitopes were encapsidated within VP1 VLP using precise elements of the minor coat protein VP2/3 that interact with the interior cavity of capsomeres. Hydrophobicity-related capsomere aggregation arising from single Tc epitope presentation was successfully circumvented by engineering charged amino acids (DD) flanking the epitope displayed on a surface-exposed loop of VP1 and by use of an optimised buffer condition. A multiparametric, high-throughput buffer screen was implemented to optimise pH and buffer additives during affinity tag removal. Stable modified capsomere recovered from this reaction were assembly competent in the presence of L-Arginine, and VLP yield was high when modular capsomeres were co-assembled with unmodified VP1 capsomeres forming stable mosaic VLPs. The ability of the MPyV VLP to encapsidate foreign protein was first evaluated and optimised with green fluorescent protein (GFP) as a model protein to enable monitoring and analysis.A minimal VP1-interacting sequence was defined from the carboxy-terminus of VP2/3 (VP2C) and fused to the amino-terminus of GFP, ensuring internalisation of the reporter protein. VP1:VP2C-GFP capsomere complexes were successfully recovered when VP1 was co-expressed with VP2C-GFP, whereas complex formation was not observed when VP1 and VP2C-GFP were mixed in vitro.Recovered capsomere complexes were assembly competent and amount of encapsidated GFP was controllable when VP1:VP2C-GFP capsomere complexes were co-assembled with unmodified VP1 capsomeres in a defined molar ratio. The encapsidation approach was then applied to a subdomain of the Influenza matrix protein M1 by co-expressing VP1 with VP2C-M1-I. M1-I capsomere complexes were assembly competent as indicated by gel electrophoresis, dynamic light scattering, and transmission electron microscopy. To enable recovery of VLPs for analysis and characterisation as well as to confirm encapsidation and the formation of mosaic VLPs, a semi-preparative size exclusion chromatography method was developed. This research was able to address bioengineering challenges posed by hydrophobic epitope presentation and developed MPyV

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Bioengineering virus‐like particles as vaccines

Lua

Connors

Sainsbury

et al. 2013

Biotech & Bioengineering

Self Cite

314

268

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Virus-like particle (VLP) technology seeks to harness the optimally tuned immunostimulatory properties of natural viruses while omitting the infectious trait. VLPs that assemble from a single protein have been shown to be safe and highly efficacious in humans, and highly profitable. VLPs emerging from basic research possess varying levels of complexity and comprise single or multiple proteins, with or without a lipid membrane. Complex VLP assembly is traditionally orchestrated within cells using black-box approaches, which are appropriate when knowledge and control over assembly are limited. Recovery challenges including those of adherent and intracellular contaminants must then be addressed. Recent commercial VLPs variously incorporate steps that include VLP in vitro assembly to address these problems robustly, but at the expense of process complexity. Increasing research activity and translation opportunity necessitate bioengineering advances and new bioprocessing modalities for efficient and cost-effective production of VLPs. Emerging approaches are necessarily multi-scale and multi-disciplinary, encompassing diverse fields from computational design of molecules to new macro-scale purification materials. In this review, we highlight historical and emerging VLP vaccine approaches. We overview approaches that seek to specifically engineer a desirable immune response through modular VLP design, and those that seek to improve bioprocess efficiency through inhibition of intracellular assembly to allow optimal use of existing purification technologies prior to cell-free VLP assembly. Greater understanding of VLP assembly and increased interdisciplinary activity will see enormous progress in VLP technology over the coming decade, driven by clear translational opportunity.

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Sensitivity of immune response quality to influenza helix 190 antigen structure displayed on a modular virus-like particle

Cited by 28 publications

References 61 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

Murine polyomavirus VLPs as a platform for cytotoxic T cell epitope-based influenza vaccine candidates

Bioengineering virus‐like particles as vaccines

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