Comparison of plasmid vaccine immunization schedules using intradermal in vivo electroporation

Abstract

In vivo electroporation (EP) has proven to significantly increase plasmid transfection efficiency and to augment immune responses after immunization with plasmids. In this study, we attempted to establish an immunization protocol using intradermal (i.d.) EP. BALB/c mice were immunized with a plasmid encoding HIV-1 p37Gag, either i.d. with the Derma Vax EP device, intramuscularly (i.m.) without EP, or with combinations of both. A novel FluoroSpot assay was used to evaluate the vaccine-specific cellular immune responses. The study showed that i.d. EP immunizations induced stronger immune responses than i.m. immunizations using a larger amount of DNA and that repeated i.d. EP immunizations induced stronger immune responses than i.m. priming followed by i.d. EP boosting. Two and three i.d. EP immunizations induced immune responses of similar magnitude, and a short interval between immunizations was superior to a longer interval in terms of the magnitude of cellular immune responses. The FluoroSpot assay allowed for the quantification of vaccine-specific cells secreting either gamma interferon (IFN-γ), interleukin-2 (IL-2), or both, and the sensitivity of the assay was confirmed with IFN-γ and IL-2 enzyme-linked immunosorbent spot (ELISpot) assays. The data obtained in this study can aid in the design of vaccine protocols using i.d. EP, and the results emphasize the advantages of the FluoroSpot assay over traditional ELISpot assay and intracellular staining for the detection and quantification of bifunctional vaccine-specific immune responses.

Fig. 1.

The IFN-γ/IL-2 FluoroSpot assay detects cells secreting IFN-γ (green) (A), IL-2 (red) (B), and both cytokines (yellow) (C). Panel C is a computerized overlay of panels A and B. The images show responses by splenocytes from a mouse immunized with plasmid-encoded p37GagB and stimulated with a p24GagB peptide pool.

Fig. 2.

Cellular responses measured by IFN-γ/IL-2 FluoroSpot assay and IFN-γ and IL-2 ELISpot assays. Individual (FluoroSpot and IFN-γ ELISpot assays) and pooled (IL-2 ELISpot assay) splenocytes were stimulated with a p24GagB peptide pool 10 days after the last immunization. Important significant differences in IFN-γ/IL-2 FluoroSpot results (C) are marked with asterisks (*, P < 0.05; **, P < 0.01), and these also apply to the IFN-γ (A) and IL-2 (B) FluoroSpot assays. Bars and error bars represent means and standard errors of the means, respectively (n = 8).

Fig. 3.

Antibody responses measured by p24GagB ELISA with sera from individual mice (n = 8) collected 10 days after the last immunization. Important significant differences are marked: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 4.

Kinetics of IFN-γ (A), IL-2 (B), and IFN-γ-plus-IL-2 (C) responses as measured by IFN-γ/IL-2 FluoroSpot assay of pooled PBMCs (n = 10) at different time points after the last immunization. Cells were stimulated with a p24GagB peptide pool.

Fig. 5.

Antibody titers at different time points after the last immunization, measured by p24Gag ELISA with sera from individual mice. *, P < 0.05. Error bars represent standard errors of the means (n = 10).