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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

Schematical presentation of a CCMV virion and a coat protein subunit. a CCMV particle according to data from Protein Data Bank (PDB ID: 1ZA7 [http://www.rcsb.org/pdb/home/home.do]) [20] and as visualized by Chimera1.6.2 (http://www.cgl.ucsf.edu/chimera/) [60], showing an icosahedral asymmetric unit consisting of three identical subunits in the centre, b ribbon diagram of a coat protein subunit B displaying N-terminal end (residues 1-25 are not shown), four β-barrels (βB-βC, βD-βE, βF-βG and βH-βI) and C-terminal end as potential insertion sites. Insertions within the barrels are shown between the white dashes
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Fig1: Schematical presentation of a CCMV virion and a coat protein subunit. a CCMV particle according to data from Protein Data Bank (PDB ID: 1ZA7 [http://www.rcsb.org/pdb/home/home.do]) [20] and as visualized by Chimera1.6.2 (http://www.cgl.ucsf.edu/chimera/) [60], showing an icosahedral asymmetric unit consisting of three identical subunits in the centre, b ribbon diagram of a coat protein subunit B displaying N-terminal end (residues 1-25 are not shown), four β-barrels (βB-βC, βD-βE, βF-βG and βH-βI) and C-terminal end as potential insertion sites. Insertions within the barrels are shown between the white dashes

Mentions: Potential sites to be tested for fusion and external exposures of epitopes on CCMV VLPs were selected based on analysis of the CCMV CP crystal structure (Fig. 1). Based on this, four predicted loops, named βB-βC, βD-βE, βF-βG- and βH-βI, and the N- and C-termini were selected for insertions respectively fusions (Fig. 2).Fig. 1


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)

Schematical presentation of a CCMV virion and a coat protein subunit. a CCMV particle according to data from Protein Data Bank (PDB ID: 1ZA7 [http://www.rcsb.org/pdb/home/home.do]) [20] and as visualized by Chimera1.6.2 (http://www.cgl.ucsf.edu/chimera/) [60], showing an icosahedral asymmetric unit consisting of three identical subunits in the centre, b ribbon diagram of a coat protein subunit B displaying N-terminal end (residues 1-25 are not shown), four β-barrels (βB-βC, βD-βE, βF-βG and βH-βI) and C-terminal end as potential insertion sites. Insertions within the barrels are shown between the white dashes
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Schematical presentation of a CCMV virion and a coat protein subunit. a CCMV particle according to data from Protein Data Bank (PDB ID: 1ZA7 [http://www.rcsb.org/pdb/home/home.do]) [20] and as visualized by Chimera1.6.2 (http://www.cgl.ucsf.edu/chimera/) [60], showing an icosahedral asymmetric unit consisting of three identical subunits in the centre, b ribbon diagram of a coat protein subunit B displaying N-terminal end (residues 1-25 are not shown), four β-barrels (βB-βC, βD-βE, βF-βG and βH-βI) and C-terminal end as potential insertion sites. Insertions within the barrels are shown between the white dashes
Mentions: Potential sites to be tested for fusion and external exposures of epitopes on CCMV VLPs were selected based on analysis of the CCMV CP crystal structure (Fig. 1). Based on this, four predicted loops, named βB-βC, βD-βE, βF-βG- and βH-βI, and the N- and C-termini were selected for insertions respectively fusions (Fig. 2).Fig. 1

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