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Feasibility of Cowpea chlorotic mottle virus-like particles as scaffold for epitope presentations.

Hassani-Mehraban A, Creutzburg S, van Heereveld L, Kormelink R - BMC Biotechnol. (2015)

Bottom Line: High levels of insoluble protein expression, relative to proteins from the entire cell lysate, were obtained for CCMV CP and all chimeric derivatives.A straightforward protocol was used that, without the use of purification columns, successfully enabled CCMV CP protein solubilization, reassembly and subsequent collection of CCMV CP VLPs.The usefulness and rapid ease of exploitation of CCMV VLPs for the production of potential subunit vaccines was demonstrated with the synthesis of chimeric CCMV VLPs containing selected sequences from the GN and GC glycoproteins of the recently emerged Schmallenberg orthobunyavirus at both termini of the CP protein.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands. amehraban1966@yahoo.com.

ABSTRACT

Background & methods: Within the last decade Virus-Like Particles (VLPs) have increasingly received attention from scientists for their use as a carrier of (peptide) molecules or as scaffold to present epitopes for use in subunit vaccines. To test the feasibility of Cowpea chlorotic mottle virus (CCMV) particles as a scaffold for epitope presentation and identify sites for epitope fusion or insertion that would not interfere with virus-like-particle formation, chimeric CCMV coat protein (CP) gene constructs were engineered, followed by expression in E. coli and assessment of VLP formation. Various constructs were made encoding a 6x-His-tag, or selected epitopes from Influenza A virus [IAV] (M2e, HA) or Foot and Mouth Disease Virus [FMDV] (VP1 and 2C). The epitopes were either inserted 1) in predicted exposed loop structures of the CCMV CP protein, 2) fused to the amino- (N) or carboxyl-terminal (C) ends, or 3) to a N-terminal 24 amino acid (aa) deletion mutant (N∆24-CP) of the CP protein.

Results: High levels of insoluble protein expression, relative to proteins from the entire cell lysate, were obtained for CCMV CP and all chimeric derivatives. A straightforward protocol was used that, without the use of purification columns, successfully enabled CCMV CP protein solubilization, reassembly and subsequent collection of CCMV CP VLPs. While insertions of His-tag or M2e (7-23 aa) into the predicted external loop structures did abolish VLP formation, high yields of VLPs were obtained with all fusions of His-tag or various epitopes (13- 27 aa) from IAV and FMDV at the N- or C-terminal ends of CCMV CP or N∆24-CP. VLPs derived from CCMV CP still encapsulated RNA, while those from CCMV CP-chimera containing a negatively charged N-terminal domain had lost this ability. The usefulness and rapid ease of exploitation of CCMV VLPs for the production of potential subunit vaccines was demonstrated with the synthesis of chimeric CCMV VLPs containing selected sequences from the GN and GC glycoproteins of the recently emerged Schmallenberg orthobunyavirus at both termini of the CP protein.

Conclusions: CCMV VLPs can be successfully exploited as scaffold for epitope fusions up to 31 aa at the N- and C-terminus, and at a N-terminal 24 amino acid (aa) deletion mutant (N∆24-CP) of the CP protein.

No MeSH data available.


Related in: MedlinePlus

Schematic view and electron micrographs of Schmallenberg virus (SBV) Gn, Gc CCMV-based chimeras. a Location and sequence of CCMV constructs harboring SBV glycoprotein peptides fused to N- and C-termini including N-terminal single fusions (a1–a4) and dual terminal fusions (a5, a6). b Electron micrographs of chimeric CCMV VLPs co-expressing SBV epitopes as depicted in panel a (b1–b6)
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Fig9: Schematic view and electron micrographs of Schmallenberg virus (SBV) Gn, Gc CCMV-based chimeras. a Location and sequence of CCMV constructs harboring SBV glycoprotein peptides fused to N- and C-termini including N-terminal single fusions (a1–a4) and dual terminal fusions (a5, a6). b Electron micrographs of chimeric CCMV VLPs co-expressing SBV epitopes as depicted in panel a (b1–b6)

Mentions: The analysis of CCMV-CP chimera indicated that the extremities of the CCMV CP protein, or the N terminus of a NΔ24-CP mutant could be exploited for epitope fusions in light of subunit vaccine production, whilst maintaining VLP formation. To further substantiate the usefulness and rapid ease of exploitation of CCMV VLPs for the production of potential subunit vaccines, peptide sequences were selected based on a search for conserved sequences or epitope predictions, from the GN and GC glycoproteins of the recently emerged Schmallenberg orthobunyavirus and fused at the aforementioned sites either singly or at both ends simultaneously (Fig. 9a). After expression and VLP formation, as expected, VLPs were readily obtained for all chimera designed (Fig. 9b).Fig. 9


Feasibility of Cowpea chlorotic mottle virus-like particles as scaffold for epitope presentations.

Hassani-Mehraban A, Creutzburg S, van Heereveld L, Kormelink R - BMC Biotechnol. (2015)

Schematic view and electron micrographs of Schmallenberg virus (SBV) Gn, Gc CCMV-based chimeras. a Location and sequence of CCMV constructs harboring SBV glycoprotein peptides fused to N- and C-termini including N-terminal single fusions (a1–a4) and dual terminal fusions (a5, a6). b Electron micrographs of chimeric CCMV VLPs co-expressing SBV epitopes as depicted in panel a (b1–b6)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4551372&req=5

Fig9: Schematic view and electron micrographs of Schmallenberg virus (SBV) Gn, Gc CCMV-based chimeras. a Location and sequence of CCMV constructs harboring SBV glycoprotein peptides fused to N- and C-termini including N-terminal single fusions (a1–a4) and dual terminal fusions (a5, a6). b Electron micrographs of chimeric CCMV VLPs co-expressing SBV epitopes as depicted in panel a (b1–b6)
Mentions: The analysis of CCMV-CP chimera indicated that the extremities of the CCMV CP protein, or the N terminus of a NΔ24-CP mutant could be exploited for epitope fusions in light of subunit vaccine production, whilst maintaining VLP formation. To further substantiate the usefulness and rapid ease of exploitation of CCMV VLPs for the production of potential subunit vaccines, peptide sequences were selected based on a search for conserved sequences or epitope predictions, from the GN and GC glycoproteins of the recently emerged Schmallenberg orthobunyavirus and fused at the aforementioned sites either singly or at both ends simultaneously (Fig. 9a). After expression and VLP formation, as expected, VLPs were readily obtained for all chimera designed (Fig. 9b).Fig. 9

Bottom Line: High levels of insoluble protein expression, relative to proteins from the entire cell lysate, were obtained for CCMV CP and all chimeric derivatives.A straightforward protocol was used that, without the use of purification columns, successfully enabled CCMV CP protein solubilization, reassembly and subsequent collection of CCMV CP VLPs.The usefulness and rapid ease of exploitation of CCMV VLPs for the production of potential subunit vaccines was demonstrated with the synthesis of chimeric CCMV VLPs containing selected sequences from the GN and GC glycoproteins of the recently emerged Schmallenberg orthobunyavirus at both termini of the CP protein.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands. amehraban1966@yahoo.com.

ABSTRACT

Background & methods: Within the last decade Virus-Like Particles (VLPs) have increasingly received attention from scientists for their use as a carrier of (peptide) molecules or as scaffold to present epitopes for use in subunit vaccines. To test the feasibility of Cowpea chlorotic mottle virus (CCMV) particles as a scaffold for epitope presentation and identify sites for epitope fusion or insertion that would not interfere with virus-like-particle formation, chimeric CCMV coat protein (CP) gene constructs were engineered, followed by expression in E. coli and assessment of VLP formation. Various constructs were made encoding a 6x-His-tag, or selected epitopes from Influenza A virus [IAV] (M2e, HA) or Foot and Mouth Disease Virus [FMDV] (VP1 and 2C). The epitopes were either inserted 1) in predicted exposed loop structures of the CCMV CP protein, 2) fused to the amino- (N) or carboxyl-terminal (C) ends, or 3) to a N-terminal 24 amino acid (aa) deletion mutant (N∆24-CP) of the CP protein.

Results: High levels of insoluble protein expression, relative to proteins from the entire cell lysate, were obtained for CCMV CP and all chimeric derivatives. A straightforward protocol was used that, without the use of purification columns, successfully enabled CCMV CP protein solubilization, reassembly and subsequent collection of CCMV CP VLPs. While insertions of His-tag or M2e (7-23 aa) into the predicted external loop structures did abolish VLP formation, high yields of VLPs were obtained with all fusions of His-tag or various epitopes (13- 27 aa) from IAV and FMDV at the N- or C-terminal ends of CCMV CP or N∆24-CP. VLPs derived from CCMV CP still encapsulated RNA, while those from CCMV CP-chimera containing a negatively charged N-terminal domain had lost this ability. The usefulness and rapid ease of exploitation of CCMV VLPs for the production of potential subunit vaccines was demonstrated with the synthesis of chimeric CCMV VLPs containing selected sequences from the GN and GC glycoproteins of the recently emerged Schmallenberg orthobunyavirus at both termini of the CP protein.

Conclusions: CCMV VLPs can be successfully exploited as scaffold for epitope fusions up to 31 aa at the N- and C-terminus, and at a N-terminal 24 amino acid (aa) deletion mutant (N∆24-CP) of the CP protein.

No MeSH data available.


Related in: MedlinePlus