Enhance immune response to DNA vaccine based on a novel multicomponent supramolecular assembly

Abstract

DNA vaccination has tremendous potential for treating or preventing numerous diseases for which traditional vaccines are ineffective but the technique can be limited by low immunogenicity. Current synthetic DNA delivery systems are versatile and safe, but substantially less efficient than viruses. Here, a novel multicomponent supramolecular system involving the preparation of mannose-bearing chitosan oligomers microspheres with entrapping complexes of DNA vaccine and polyethylenimine was developed to mimic many of the beneficial properties of the viruses. After delivery by intramuscular immunization in BALB/c mice, the microspheres induced an enhanced serum antibody responses two orders of magnitude greater than naked DNA vaccine. Additionally, in contrast to naked DNA, the microspheres induced potent cytotoxic T lymphocyte responses at a low dose. Consequently, formulation of DNA vaccines into multicomponent vectors is a powerful means of increasing vaccine potency.

Fig. 1

Appearance and size of MBCO microspheres. (a) Transmission electron micrograph of MBCO microspheres. Scale bar represents 100 nm. (b) Atomic force micrograph of MBCO microspheres. Scale bar represents 500 nm. (c) Size distribution of freshly prepared microspheres. Size was measured using photon correlation spectroscopy (dynamic light scattering) and data were plotted as volume distribution.

Fig. 2

In vitro and in vivo release profiles of chitosan, M-chitosan and MBCO microspheres containing PEI/DNA complexes. (a) In vitro release profiles. The microspheres (10 mg) were suspended in 1 ml of PBS at pH 7.4 (120 mm NaCl, 2.7 mm KCl, 10 mm PBS) at 37 °C under stirring. At predetermined time intervals, the suspension was centrifuged and replaced with the same volume of fresh medium. The DNA concentration in the supernatant was determined in triplicate by UV absorbance at 260 nm. All data were expressed as mean±SD (n=3). (b–d) In vitro burst release studies of DNA from MBCO microspheres. The experiments were carried out in COS-7 cells using the GFP as reporter gene. GFP expression was directly viewed under a fluorescene microscope (OLYMPUS America). (e) In vivo release of luciferase plasmid was performed in mice at days 1, 7, and 14 with either naked DNA, M-chitosan, or MBCO microspheres. The TA muscles of injection site were peeled off and treated by using a normal chemiluminescence assay. Mean average values±standard deviation of triplicates are given in relative light units (RLU), correlated to the total volume content.

Fig. 3

Transfection efficiency of MBCO microspheres in RAW264.7 cells compared with chitosan and M-chitosan microspheres. The luciferase values have been normalized to total protein content (n=3).

Fig. 4

(a) Serum HBsAg antibody assay in groups of BALB/c mice immunized either MBCO microspheres or naked DNA alone. The immunization protocol was performed at weeks 0, 2, 4 and 6. At the different time points, HBsAg-specific total antibody (as pool of five) was measured in serum from each group (n=5) of mice. OD4500.2 represents positive value. (b) Analysis of cytolytic activities of splenic mononuclear cells harvested from mice immunized. P815 mastocytoma cells (as pool of five) pulsed with epitope peptides were used as targets. Percent specific lysis was determined by a 4-h LDH-release assay. (c) The HBsAg-specific, IFN-γ producing splenocytes were generated in mice by HBsAg DNA vaccine after intramuscular injection. Each bar represents the average number of cells secreting IFN-γ per 10⁶ cells.