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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 illustration of loop-inserted and coat protein fusion constructs expressed in E. coli. The ectodomain of M2e-protein of Influenza A virus: M2e(23), M2e(15) and M2e(7) introduced within the predicted loops (βB-βC-M2e(23), βD-βE-M2e(23), βF-βG-M2e(23)/M2e(15)/M2e(7) and βH-βI-(23)/M2e(15)/M2e(7) as adaptors via SpeI and SstI overhangs. Foot and mouth disease virus (VP1/2C) Influenza A virus epitopes [M2e(23) and HA] were fused by PCR to the N-terminus of the CCMV-CP (VP1/2C-CP) and NΔ24-CP (VP1/2C/M2e/HA-NΔ24-CP), and C-terminal to CCMV-CP (CP-HA, CP-M2e)
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Fig5: Schematic illustration of loop-inserted and coat protein fusion constructs expressed in E. coli. The ectodomain of M2e-protein of Influenza A virus: M2e(23), M2e(15) and M2e(7) introduced within the predicted loops (βB-βC-M2e(23), βD-βE-M2e(23), βF-βG-M2e(23)/M2e(15)/M2e(7) and βH-βI-(23)/M2e(15)/M2e(7) as adaptors via SpeI and SstI overhangs. Foot and mouth disease virus (VP1/2C) Influenza A virus epitopes [M2e(23) and HA] were fused by PCR to the N-terminus of the CCMV-CP (VP1/2C-CP) and NΔ24-CP (VP1/2C/M2e/HA-NΔ24-CP), and C-terminal to CCMV-CP (CP-HA, CP-M2e)

Mentions: To rule out that abrogation of VLP formation in case of insertion into the loops was not just due to the 6xHis-tag, a similar set of insertional CCMV CP chimeras was made but instead of a His-tag now containing the 23 aa IAV M2e epitope (Fig. 5). Constructs made were cloned into pET28 and verified by sequence analysis, and after expression in E.coli further processed for in vitro VLP reassembly and purification by ultracentrifugation. Whereas all constructs of CCMV CP containing the M2e epitope insertion into one of the loops became well expressed (Fig. 6a), only very low amounts of the βF-βG-M2e(23) and βH-βI-M2e(23) proteins (Fig. 6a3 and a4) could be observed in the pellets after sucrose cushion. To test whether these results were influenced by the size of the M2e (23 aa) insertion, a range of constructs containing smaller insertions (7 or 15 aa) were made and analyzed (Fig. 5). While proteins from the insertional CCMV chimera βF-βG-M2e(7) and -M2e(15) were detected in pellets after sucrose cushion (Fig. 6b1 and b3), only very low amounts were detected for those of βH-βI-M2e(7) and -M2e(15) (Fig. 6b2 and b4). Western immunoblot analysis of all CCMV βF-βG loop-insertions showed a good detection of the His-tag but a weaker one of the M2e(7) and M2e(15) insertions, likely due to lower reactivity of the antiserum to a smaller M2e sequence insertion (Fig. 6c). Although the presence of these proteins in pellets obtained after sucrose cushion ultracentrifugation were indicative for MMPC, and consistently observed to correlate with VLP formation of wild type CCMV CP (Fig. 4b1-3), none of the βF-βG loop CCMV CP chimera for which also MMPCs were found in the pellets, did fold into VLPs. Instead, large and similar aggregates were observed for all βF-βG loop chimera, as shown for βF-βG-His, βF-βG-M2e(7), βF-βG-M2e(15) and βF-βG-M2e(23)( Fig. 4a1-4).Fig. 5


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 illustration of loop-inserted and coat protein fusion constructs expressed in E. coli. The ectodomain of M2e-protein of Influenza A virus: M2e(23), M2e(15) and M2e(7) introduced within the predicted loops (βB-βC-M2e(23), βD-βE-M2e(23), βF-βG-M2e(23)/M2e(15)/M2e(7) and βH-βI-(23)/M2e(15)/M2e(7) as adaptors via SpeI and SstI overhangs. Foot and mouth disease virus (VP1/2C) Influenza A virus epitopes [M2e(23) and HA] were fused by PCR to the N-terminus of the CCMV-CP (VP1/2C-CP) and NΔ24-CP (VP1/2C/M2e/HA-NΔ24-CP), and C-terminal to CCMV-CP (CP-HA, CP-M2e)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Schematic illustration of loop-inserted and coat protein fusion constructs expressed in E. coli. The ectodomain of M2e-protein of Influenza A virus: M2e(23), M2e(15) and M2e(7) introduced within the predicted loops (βB-βC-M2e(23), βD-βE-M2e(23), βF-βG-M2e(23)/M2e(15)/M2e(7) and βH-βI-(23)/M2e(15)/M2e(7) as adaptors via SpeI and SstI overhangs. Foot and mouth disease virus (VP1/2C) Influenza A virus epitopes [M2e(23) and HA] were fused by PCR to the N-terminus of the CCMV-CP (VP1/2C-CP) and NΔ24-CP (VP1/2C/M2e/HA-NΔ24-CP), and C-terminal to CCMV-CP (CP-HA, CP-M2e)
Mentions: To rule out that abrogation of VLP formation in case of insertion into the loops was not just due to the 6xHis-tag, a similar set of insertional CCMV CP chimeras was made but instead of a His-tag now containing the 23 aa IAV M2e epitope (Fig. 5). Constructs made were cloned into pET28 and verified by sequence analysis, and after expression in E.coli further processed for in vitro VLP reassembly and purification by ultracentrifugation. Whereas all constructs of CCMV CP containing the M2e epitope insertion into one of the loops became well expressed (Fig. 6a), only very low amounts of the βF-βG-M2e(23) and βH-βI-M2e(23) proteins (Fig. 6a3 and a4) could be observed in the pellets after sucrose cushion. To test whether these results were influenced by the size of the M2e (23 aa) insertion, a range of constructs containing smaller insertions (7 or 15 aa) were made and analyzed (Fig. 5). While proteins from the insertional CCMV chimera βF-βG-M2e(7) and -M2e(15) were detected in pellets after sucrose cushion (Fig. 6b1 and b3), only very low amounts were detected for those of βH-βI-M2e(7) and -M2e(15) (Fig. 6b2 and b4). Western immunoblot analysis of all CCMV βF-βG loop-insertions showed a good detection of the His-tag but a weaker one of the M2e(7) and M2e(15) insertions, likely due to lower reactivity of the antiserum to a smaller M2e sequence insertion (Fig. 6c). Although the presence of these proteins in pellets obtained after sucrose cushion ultracentrifugation were indicative for MMPC, and consistently observed to correlate with VLP formation of wild type CCMV CP (Fig. 4b1-3), none of the βF-βG loop CCMV CP chimera for which also MMPCs were found in the pellets, did fold into VLPs. Instead, large and similar aggregates were observed for all βF-βG loop chimera, as shown for βF-βG-His, βF-βG-M2e(7), βF-βG-M2e(15) and βF-βG-M2e(23)( Fig. 4a1-4).Fig. 5

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