Clin Exp Vaccine Res.  2014 Jul;3(2):227-234. 10.7774/cevr.2014.3.2.227.

Lumazine synthase protein cage nanoparticles as antigen delivery nanoplatforms for dendritic cell-based vaccine development

• Ra JS
• Shin HH
• Kang S
• Do Y

• KMID: 1730628
• DOI: http://doi.org/10.7774/cevr.2014.3.2.227

Abstract

PURPOSE
Protein cages are promising nanoplatform candidates for efficient delivery systems due to their homogenous size and structure with high biocompatibility and biodegradability. In this study, we investigate the potential of lumazine synthase protein cage as an antigen delivery system to dendritic cells (DCs), which induce antigen-specific T cell proliferation.
MATERIALS AND METHODS
Ovalbumin (OVA) peptides OT-1 (SIINFEKL) and OT-2 (ISQAVHAAHAEINEAGR) were genetically inserted to lumazine synthase and each protein cage was over-expressed in Escherichia coli as a soluble protein. The efficiency of antigen delivery and the resulting antigen-specific T cell proliferation by DCs was examined in vitro as well as in vivo.
RESULTS
We successfully generated and characterized OVA peptides carrying lumazine synthase protein cages. The OT-1 and OT-2 peptides carried by lumazine synthases were efficiently delivered and processed by DCs in vitro as well as in vivo, and induced proliferation of OT-1-specific CD8+T cells and OT-2-specific CD4+T cells.
CONCLUSION
Our data demonstrate the potential of lumazine synthase protein cage being used as a novel antigen delivery system for DC-based vaccine development in future clinical applications.

Keyword

Dendritic cells; Protein cage; Antigen presentation; Vaccines; Nanoparticles

MeSH Terms

Antigen Presentation
Cell Proliferation
Dendritic Cells
Escherichia coli
Nanoparticles*
Ovalbumin
Ovum
Peptides
Vaccines
Ovalbumin
Peptides
Vaccines

Figure

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  Fig. 1 Surface and ribbon diagram representations of AaLS (PDB: 1HQK) looking down the five-fold symmetry axis (A) and clipped view showing the interior space (9 nm interior and 15.4 nm exterior diameters) of the protein cage (B). Five subunits were shown as ribbon diagram with individual colors.

• 

  Fig. 2 A schematic diagram showing dendritic cell (DC)-mediated antigen-specific T cell proliferation induced by lumazine synthase protein cage nanoparticles (AaLS) carrying OT peptides.

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  Fig. 3 Characterization of OT-peptide added AaLS protein cages. (A) Molecular masses of dissociated subunits of AaLS-OT-2 (top), AaLS-OT-1 (middle), and wt AaLS (bottom) protein cages. Calculated (Calc.) and measured molecular masses were indicated. (B) Transmission electron micrographic image of 2% uranyl acetate stained AaLS-OT-2 (top), AaLS-OT-1 (middle), and wt AaLS (bottom) protein cages confirmed an intact cage architecture with a uniform size distribution.

• 

  Fig. 4 OT-1 antigenic peptides delivered by AaLS-OT-1 induce OT-1-specific CD8+ T cell proliferation in vitro and in vivo. (A) CD11c+ dendritic cells (DCs) were pulsed with phosphate buffered saline (PBS), soluble ovalbumin (OVA) protein, wt AaLS, or AaLS-OT-1 at 2 mg/mL for 3 hours. The DCs were washed and then co-cultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled OT-1 T cells at a ratio of 1:3. Four days later, the proliferation of OT-1-specific CD8+ T cells was measured by flow cytometry. (B) Mice were adoptively transferred with CFSE-labeled OT-1 T cells and on the next day, they were immunized subcutaneously with PBS, 100 µg of OVA protein, 50 µg of wt AaLS, AaLS-OT-1 in the presence of an adjuvant. Three days later, the proliferation of OT-1-specific CD8+ T cells was measured by flow cytometry.

• 

  Fig. 5 OT-2 antigenic peptides delivered by AaLS-OT-2 induce OT-2-specific CD4+ T cell proliferation in vitro and in vivo. (A) CD11c+ dendritic cells (DCs) were pulsed with phosphate buffered saline (PBS), soluble ovalbumin (OVA) protein, wt AaLS, or AaLS-OT-2 at 2 mg/mL for 3 hours. The DCs were washed and then co-cultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled OT-2 T cells at a ratio of 1:3. Four days later, the proliferation of OT-2-specific CD4+ T cells was measured by flow cytometry. (B) Mice were adoptively transferred with CFSE-labeled OT-2 T cells and on the next day, they were immunized subcutaneously with PBS, 100 µg of OVA protein, 50 µg of wt AaLS, AaLS-OT-2 in the presence of an adjuvant. Three days later, the proliferation of OT-2-specific CD4+ T cells was measured by flow cytometry.

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