Combined effects of IL-12 and electroporation enhances the potency of DNA vaccination in macaques

Abstract

DNA vaccines are a promising technology. Historically, however, the ability of DNA vaccines to induce high response rates and strong immune responses, especially antibody responses, in non-human primates and human clinical trials has proven suboptimal. Here, we performed a pilot study in rhesus macaques to evaluate whether we could improve the immunogenicity of DNA vaccines through the use of adjuvant technology and improved delivery systems. The study consisted of four groups of animals that received: DNA by intramuscular (IM) injection, DNA with plasmid-encoded IL-12 by IM injection, DNA by IM injection with in vivo electroporation (EP), and DNA with IL-12 by IM EP. Each group was immunized three times with optimized HIV gag and env constructs. Vaccine immunogenicity was assessed by IFNgamma ELISpot, CFSE proliferation, polyfunctional flow cytometry, and antibody ELISA. Similar to previous studies, use of IL-12 as an adjuvant increased the gag and env-specific cellular responses. The use of EP to enhance plasmid delivery resulted in dramatically higher cellular as well as humoral responses. Interestingly, the use of EP to administer the DNA and IL-12 adjuvant combination resulted in the induction of higher, more efficient responses such that a 10-fold increase in antigen-specific IFNgamma(+) cells compared to IM DNA immunization was observed after a single immunization. In addition to increases in the magnitude of IFNgamma production in the initial and memory responses, the combined approach resulted in enhancements in the proliferative capacity of antigen-specific CD8(+) T cells and the amount of polyfunctional cells capable of producing IL-2 and TNFalpha in addition to IFNgamma. These data suggest that adjuvant and improved delivery methods may be able to overcome previous immunogenicity limitations in DNA vaccine technology.

Figures

Fig. 1

Enhanced cellular immune responses to HIV-1 consensus immunogens with IM co-injection of plasmid encoded IL-12 followed by EP. IFNγ ELISpots were performed two weeks after the (a) first immunization, (b) second immunization, and (c) third immunization (as seen in comparison to the other three). Responses to env are depicted as black bars and gag are depicted as white bars with the data shown as stacked group mean responses ± SEM.

Fig. 2

Enhanced cross-reactive cellular immune responses with intramuscular electroporation. After three immunizations, the total T-cell immune response in pEY2E1-B immunized macaques against four peptide pools of the HIV-1 group M peptides were determined by IFNγ ELISpot. The data are shown as stacked group means ± SEM.

Fig. 3

Enhanced memory responses to HIV-1 immunogens with IM electroporation and plasmid IL-12. Five months after the last immunization, ELISpot assays were performed to determine antigen-specific memory responses to gag and env in the IM and EP immunized groups with and without co-immunization with the IL-12 plasmid. The data are shown as group mean responses ± SEM.

Fig. 4

Induction of p24 and gp160 antibodies. Serum was collected every two weeks over the course of the study. For each time point, gag and env antibody titers were determined by p24 and gp160 ELISA, respectively, in the IM and EP immunized groups with and without co-immunization with the IL-12 plasmid. The data are shown as group mean responses ± SEM.

Fig. 5

CD4+ and CD8+ T cell proliferation. Cryo-preserved samples collected two weeks following the final immunization were stimulated with gag peptides and p24 protein in vitro for 5 days to determine the proliferative capacity of gag-specific T cells. Representative dot plots are shown for each group. Proliferative responses are shown as group mean responses ± SEM with CD4 depicted as white bars and CD8 are depicted as black bars.

Fig. 6

Induction of gag-specific polyfunctional CD8+ T cells. PBMCs were stimulated in vitro with a gag peptide pool mix for 5 hours. Cells were then stained for intracellular production of IFNγ, TNFα and IL-2. Animals with detectable gag-specific IFNγ+ CD8+ T cells (a) were further analyzed for polyfunctional populations (b-d). Pie charts show the proportion of gag-specific CD8+ T cells that have 3 functions (red), 2 functions (green) or 1 function (blue). Cells incubated with complete media alone were used to determine background responses for each polyfunctional population and was subtracted from the gag-specific response. The threshold for detection was set at 10 events or 0.05%.