Impact of the Charge Ratio on the In Vivo Immunogenicity of Lipoplexes

Abstract

Purpose: The present study investigated the immunogenic potential of different cationic liposome formulations with a DNA plasmid encoding Pfs25, a malaria transmission-blocking vaccine candidate.

Methods: Pfs25 plasmid DNA was complexed with cationic liposomes to produce lipoplexes at different charge ratios of the cationic lipid head group to the nucleotide phosphate (N:P). The formation of lipoplexes was visualized by Cryogenic-TEM. Confocal microscopy of lipoplexes formed with GFP encoding plasmid DNA, and flow cytometry was used to determine their in vitro transfection capability. Two different lipoplex formulations using plasmid DNA encoding Pfs25 were evaluated for in vivo immunogenicity after intramuscular administration in Balb/c mice. Immune sera were analyzed by ELISA.

Results: The results demonstrated that the cationic liposome-mediated DNA immunization with an N:P charge ratio of 1:3 (anionic lipoplexes) is more effective than the use of naked plasmid DNA alone. No antibody response was observed when lipoplexes with a higher N:P charge ratio of 10:3 (cationic lipoplexes) were used. Trehalose was added to some lipoplex formulations as a cryoprotectant and adjuvant, but it did not yield any further improvement of immunogenicity in vivo.

Conclusions: The results suggest that Pfs25 plasmid DNA delivered as lipoplexes at a charge ratio of 1:3 elicited strong immunogenicity in mice and may be improved further to match the immune responses of DNA vaccines administered by in vivo electroporation.

Keywords: Pfs25 DNA; cationic liposome; lipoplex; malaria; vaccine.

Fig. 1

Formation of lipoplexes from unilamellar DOTAP-Cholesterol liposomes. (a) Cryo-TEM images of DOTAP-Cholesterol liposomes. (b) Cryo-TEM images of lipoplex obtained by mixing the liposomes with plasmid DNA at a charge ratio of (N:P) 1:3. Insets in each panel show higher magnification images of liposomes and lipoplexes. Zeta potential values for various lipolexes are also shown in the table below images.

Fig. 2

The expression of GFP following transfection with lipoplexes of different charge ratios (indicated on x-axis) in HEK293T cell line was determined by flow cytometry. Positive control tested included transfection using MegaTran. In some groups, lipoplexes were mixed with trehalose (T) as a potential adjuvant and cryo-protectant and then added to cells. Trehalose was added at a mass ratio of 1:1 with respect to DOTAP.

Fig. 3

Confocal microscopy imaging of representative transfected HEK293T cells with lipoplexes of different charge ratios.

Fig. 4

Analysis of final antibody titers after third immunization and final protein boosting by ELISA from mice (n = 5) immunized with different lipoplex formulations. Numbers above bars represent total number of mice responding out of 5 per group. ELISA data was analyzed using GraphPad Prism software and the ELISA titers before and after protein boosts as well as with or without in vivo EP were statistically significant (one-way ANOVA, p < 0.001). ELISA titers between DNA (without in vivo EP) and 1:3 charge ratio lipoplexes were also statistically significant (unpaired non-parametric test, p < 0.0065, indicated by an asterisk *).

Fig. 5

Antibody ELISA titers in mice (n = 4) immunized with various formulations of DNA plasmids and after a final protein boost. Lipoplexes (N:P 1:3 charge ratio) without or with Alum or MPLA as co-adjuvants were tested. DNA alone or as lipoplexes were also tested in parallel by in vivo EP.

Fig. 6

Viability of THP-1 and HepG2 cell lines after incubation with different formulation of lipoplexes. Statistical significance was analyzed by one-way ANOVA and percent viability (THP-1 cell) data at 10:3 N:P ratio was significantly different (p < 0.05) as compared to percent viability in other combinations (Boneferroni’s multiple comparison test).