In vivo delivery of antigens by adenovirus dodecahedron induces cellular and humoral immune responses to elicit antitumor immunity

Abstract

Cancer vaccines based on virus-like particles (VLPs) vectors may offer many advantages over other antigen-delivery systems and represent an alternative to the ex vivo cell therapy approach. In this study, we describe the use of penton-dodecahedron (Pt-Dd) VLPs from human adenovirus type 3 (Ad3) as cancer vaccine vehicle for specific antigens, based on its unique cellular internalization properties. WW domains from the ubiquitin ligase Nedd4 serve as an adapter to bind the antigen to Pt-Dd. By engineering fusion partners of WW with the model antigen ovalbumin (OVA), Pt-Dd can efficiently deliver WW-OVA in vitro and the Pt-Dd/WW complex can be readily internalized by dendritic cells (DCs). Immunization with WW-OVA/Pt-Dd results in 90% protection against B16-OVA melanoma implantation in syngeneic mice. This high level of protection correlates with the development of OVA-specific CD8(+) T cells. Moreover, vaccination with WW-OVA Pt-Dd induces robust humoral responses in mice as shown by the high levels of anti-OVA antibodies (Abs) detected in serum. Importantly, treatment of mice bearing B16-OVA tumors with WW-OVA/Pt-Dd results in complete tumor regression in 100% of cases. Thus, our data supports a dual role of Pt-Dd as antigen-delivery vector and natural adjuvant, able to generate integrated cellular and humoral responses of broad immunogenic complexity to elicit specific antitumor immunity. Antigen delivery by Pt-Dd vector is a promising novel strategy for development of cancer vaccines with important clinical applications.

Figure 1

Description of Pt-Dd VLPs as protein delivery system for vaccine development. (a) Structure of human Ad3 and its Pt-Dd particle. Schematic representation of the Ad morphology, including an icosahedral capsid with Pt structures at the vertices (upper left diagram). Pt (zoomed diagram) comprises a noncovalent complex of trimeric fiber protein attached to a pentameric penton base. Self-association of the Pt results in the formation of the DNA devoid Pt-Dd particle (upper right diagram). Electron micrographs (obtained as described in ref. 18) illustrate the size comparison between Ad3 capsid and Dd particle (left) and the Pt-Dd detailed structure (right). (b) Schematic representation of the recombinant fusion protein WW-OVA, which comprises (i) WW_2-3-4 domains from Nedd4; (ii) the NH₂-terminal peptide of influenza virus HA2; (iii) a 129 amino acid C-terminal fragment of OVA containing the MHC class I immunodominant peptide SIINFEKL. (c) SDS-PAGE analysis of purified proteins. WW and WW-OVA proteins were expressed in BL21 cells and purified by affinity column. Pt-Dd VLPs were expressed in baculovirus and purified in a sucrose density gradient. Ad3, adenovirus type 3; MHC, major histocompatibility complex; OVA, ovalbumin; Pt-Dd, penton-dodecahedron; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; VLP, virus-like particle.

Figure 2

Cellular uptake of proteins mediated by Pt-Dd. (a) HeLa cells were either untreated (left panel) or incubated for 1 hour with WW-OVA/Pt-Dd protein complexes (right panel). Protein internalization was detected by immunocytochemistry on fixed cells, colocalization of Pt-Dd, and WW-OVA is shown in the merge panel, with nuclei counter stained with Hoechst dye. (b) Mouse bone marrow-derived DCs were incubated for 3 hours with either Alexa 647-labeled WW protein only (gray histogram) or in complex with Pt-Dd (black histogram). Cells were trypsinized to remove surface bound proteins and protein internalization analyzed by flow cytometry. DC, dendritic cell; OVA, ovalbumin; Pt-Dd, penton-dodecahedron.

Figure 3

Vaccination with WW-OVA/Pt-Dd generates OVA-specific CD8⁺ T-cell responses. C57BL/6 naive mice (N = 3) were immunized twice with WW-OVA/Pt-Dd protein complexes or PBS. CD8⁺ T-cell responses were measured 14 days postinjection on splenocytes. (a) H-2K^b-OVA (SIINFEKL) tetramer staining on CD8⁺ cells from mice injected with PBS (left panel) or Pt-Dd/WW-OVA (right panel). (b) Specific killing of B16-OVA target cells by FACS-based cytotoxicity assay. A quantitative analysis of viable target cells was performed using FITC-labeled calibration beads and the percentage of specific lysis was calculated with the formula [1 − (R_1:1/R_1:A)] × 100, as described in Supplementary Materials and Methods. FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; OVA, ovalbumin; PBS, phosphate-buffered saline; Pt-Dd, penton-dodecahedron.

Figure 4

Humoral response elicited by vaccination with WW-OVA/Pt-Dd. (a) C57BL/6 naive mice (N = 6) were immunized twice (triangles) or thrice (circles) with Pt-Dd/WW-OVA protein complexes, with 1-week intervals between each boost. A control group included nonvaccinated (NV, squares) naive mice (N = 4). Serum antibodies specific for OVA were determined by ELISA at week 11 after the first immunization, using OVA as antigen. Each point represents the mean of duplicate OD values from each mouse serum at dilution 1/50. (b) Geometric mean and SEM of serum-specific IgG isotypes and IgA from WW-OVA/Pt-Dd vaccinated mice (black bars, N = 5) were determined by ELISA and compared to nonvaccinated mice (white bars, N = 3).The unpaired t-test with Welch's correction was used to compare Ab levels between vaccination groups and a value of P < 0.05 was considered statistically significant. Ab, antibody; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobin G; NV, nonvaccinated; OVA, ovalbumin; Pt-Dd, penton-dodecahedron.

Figure 5

Vaccination with WW-OVA/Pt-Dd prevents tumor engraftment. C57BL/6 naive mice (N = 5–10) were immunized on days –14 and –7 and challenged s.c. in the opposite flank (day 0) with 1 × 10⁵ B16 or B16-OVA melanoma cells. Tumor sizes were monitored every 2 days for 150 days or until tumors reached 10 mm in diameter. Data show tumor growth curves for individual mice, with number of tumor-free mice indicated in parenthesis. OVA, ovalbumin; PBS, phosphate-buffered saline; Pt-Dd, penton-dodecahedron; s.c., subcutaneous.

Figure 6

A Pt-Dd-based vaccine is a potent immunogen in a prophylactic tumor model and elicit tumor immunity. C57BL/6 naive mice (N = 5–10) were immunized on days –14 and –7 and challenged s.c. in the opposite flank (day 0) with 1 × 10⁵ B16 or B16-OVA melanoma cells. Data are representative of two independent experiments. Results of the statistical analysis are shown in the legend to Table 1. OVA, ovalbumin; PBS, phosphate-buffered saline; Pt-Dd, penton-dodecahedron; s.c., subcutaneous.

Figure 7

Treatment of tumor-bearing mice with WW-OVA/Pt-Dd. C57BL/6 naive mice (N = 8) were challenged s.c. with 1 × 10⁵ B16-OVA melanoma cells on day 0 and immunized with WW-OVA/Pt-Dd protein complexes or PBS (control group) on days 1 and 8. (a) Tumor growth curves for individual mice. Tumor sizes were monitored every 2 days for 60 days or until tumors reached 10 mm in diameter. The number of tumor-free mice are shown in parenthesis. (b) Kaplan–Meier survival analysis showing the percentage of surviving mice. OVA, ovalbumin; PBS, phosphate-buffered saline; Pt-Dd, penton-dodecahedron; s.c., subcutaneous.