Structural effects and lymphocyte activation properties of self-assembled polysaccharide nanogels for effective antigen delivery

Abstract

The success of immunotherapeutic vaccines is often limited by their inability to activate the cytotoxic T lymphocyte (CTL)-inducing Th1 pathway. We investigated the ability of self-assembled nanogels (CHP or CH-CDex) to activate this pathway, and characterised them chemically and biologically. Once loaded with antigen (ovalbumin, OVA) their OVA encapsulation and dissociation rates suggested the possibility of effective antigen delivery. The DC2.4 dendritic cell line took up either vaccine time-dependently, but both vaccines required CpG DNA for class I MHC presentation. The nanogel vaccines interacted with RAW264.7, a Balb/c mouse-derived macrophage cell line, and co-localised with lysosomes, suggesting their endocytotic internalization in RAW264.7. Both vaccines activated CTLs better than OVA alone. Unlike OVA alone, the nanogel vaccines induced IgG2a antibody production in mice, whereas the former induced IgG1 antibodies. OVA-nanogel delivery to the draining lymph nodes (DLNs) was higher than that for OVA alone, reaching a deeper medullary area. Furthermore, Langerin⁺ CD103⁺ DCs interacted with the nanogel vaccines effectively, which is a subset of cross-presentation DC, in the DLNs. The nanogel vaccines each had good anti-tumour efficacy in OVA tumour-bearing mice compared with the OVA alone. Thus, CHP and CH-CDex nanogels should be investigated further because of the great potential they offer for immunotherapy.

Figure 1

Nanogel vaccine preparation. (a) Chemical structures of CHP and CH-CDex. (b) A schematic overview of this study. (c) Release of OVA from CHP nanogel (open) or CH-CDex nanogel (closed) in the presence of 20 mg/mL of BSA.

Figure 2

Interaction of nanogel vaccines with DC2.4 cells. (a) Time-dependent uptake of OVA-Cy5.5/CHP (open) and OVA/Cy5.5/CH-CDex (closed) by DC2.4 cells. (b) Time-dependent relative presentation of the OVA epitope (SIINFEKL) on MHC class I molecules, as evaluated by OVA alone (white), OVA/CHP (gray), OVA/CH-CDex (black) or OVA epitope (SIINFEKL) (dot) using flow cytometry.

Figure 3

Evaluation of the immune activation abilities of the nanogel vaccines in vivo. (a) Ratio of OVA activated CTL to total CD8a⁺ cells by PBS (dot), OVA alone (white), OVA/CHP (grey) or OVA/CH-CDex (black) analysed by flow cytometry. (b) Total serum titres for IgG1, IgG2a and IgG in C57BL/6J were analysed by ELISA.

Figure 4

Effective antigen delivery to DLNs by nanogel vaccines. (a) Distribution of OVA-Cy5.5, either free or in nanogel complexes in lymph node tissues 6 h after administration, as observed by CLSM. (b) Fluorescence intensity of OVA-Cy5.5 in lymph node tissue as measured by IVIS. (c–h) Distribution of immune cells and OVA-Cy5.5/CH-CDex in lymph nodes 6 h after administration, as observed by CLSM. (c) B220, (d) CD8, (e) CD11c, (f) CD11b and (g) F4/80 are all shown in green whereas OVA is shown in red. (h) The whole image of a lymph node (B220: blue, CD11b: green, OVA: red) and (h-1) an enlarged view of the medullary area and (h-2) subcapsular area. (i–k) Internalization of OVA-Cy5.5/CHP (grey) or OVA-Cy5.5/CH-CDex (black) to (i) antigen presenting cells such as dendritic cells (DCs), macrophages (MΦ) or B cells, and CD8⁺T cells. (j) DCs with or without Langerin, (k) Langerin⁺ DCs with or without CD103, and CD8⁺ DCs.

Figure 5

Anti-tumour efficacy of nanogel vaccines. (a) Tumour volume of C57BL/6J mice inoculated E.G7-OVA cells, vaccinated PBS as control (cross), OVA (square), OVA/CHP (open circle) or OVA/CH-CDex (closed circle) on 5 and 9 days after tumour inoculation. (b) Tumour volume of each mice and (c) survival rate of each group.