We sought to determine whether in vivo electroporation might overcome the requirement for strong transcriptional promoters in plasmid vaccines. We tested plasmid vaccines containing viral promoters that exhibited a broad range of transcriptional activities by in vitro assay. In contrast to the in vitro observations, no difference in gene expression was measured in vivo 24 h following injection when the plasmids were introduced via electroporation. Despite the similarities in gene expression in vivo, the cellular and humoral responses elicited by these plasmid vaccines were dramatically different and correlated more closely with the promoter activity measured in vitro. When antigen production in vivo was measured over a longer period following injection, significant differences in gene expression became apparent after 2–3 days and the differences in gene expression at day 7 correlated well with plasmid immunogenicity. These studies reconfirm the importance of antigen production for effective plasmid vaccination and demonstrate that the duration of gene expression should also be considered when designing plasmid vaccines.

ABSTRACT We examined CD8 + T-cell expansion and function following intramuscular immunization with a recombinant adenovirus. This study has identified a number of properties which may explain the strong immunogenicity of adenovirus vectors: (i) the ability to deliver large amounts of antigen into the lymphoid tissues, (ii) the ability to induce rapid expansion and migration of CD8 + T cells throughout the lymphatics, and (iii) the ability to produce a sustained, high-level CD8 + T-cell response.

The skin represents an excellent site for vaccine inoculation due to its natural role as a first line of contact with foreign pathogens and the high local frequency of antigen presenting cells. To facilitate skin-directed immunization, a new technique has been developed (termed microporation) whereby a vaporization process is used to remove tiny areas of the stratum corneum creating microscopic pores that allow access to the underlying viable epidermis. Reporter gene expression was 100-fold increased following application of an adenovirus vector to microporated skin when compared to intact skin. Furthermore, 10–100-fold greater cellular and humoral immune responses were observed following topical administration of an adenovirus vaccine to microporated skin versus intact skin. Hairless mice responded to the microporated adenovirus vaccine equivalently to mice with normal hair follicle distribution demonstrating the activity of the microporated vaccine was not related to follicle count. In a tumor challenge model using a surrogate antigen, microporation increased vaccine efficacy by approximately 100-fold compared to intact skin. Finally, microporation enabled delivery of an adenovirus vaccine carrying a relevant melanoma antigen resulting in the development of auto-immune vitiligo and tumor protection. Thus, the microporation technology has proven to be a reliable and easy method to enable skin-directed vaccination.

In vivo electroporation dramatically enhances plasmid vaccine efficacy. This enhancement can be attributed to increased plasmid delivery and, possibly, to some undefined adjuvant properties. Previous reports have demonstrated CD8(+) T cell priming by plasmid vaccines is strongly dependent upon CD4(+) T cell help. Indeed, the efficacy of a plasmid vaccine expressing Escherichia coli beta-galactosidase was severely attenuated in MHC class II-deficient (C2D) mice. To determine whether electroporation could compensate for the absence of CD4(+) T cell help, C2D mice were immunized by a single administration of plasmid in combination with electroporation using two conditions which differed only by the duration of the pulse (20 or 50 msec). Both conditions elicited robust cellular and Immoral responses in wild-type mice, as measured by IFN-,gamma ELISPOT, anti-beta-galactosidase ELISA, and protection from virus challenge. In C2D mice, the cellular response produced by the vaccine combined with the 50-msec pulse, as measured by ELISPOT, was identical to the response in wild-type mice. The 20-msec pulse elicited a milder response that was approximately one-fifth that of the response elicited by the 50-msec pulse. By contrast, the 20-msec conditions provided comparable protection in both wild-type and C2D recipients whereas the protection elicited by the 50-msec conditions in C2D mice was weaker than in wild-type mice. Further investigation is required to understand the discordance between the ELISPOT results and outcome of virus challenge in the C2D mice. Nonetheless, using this technique to prime CD8(+) T cells using plasmid vaccines may prove extremely useful when immunizing hosts with limiting CD4(+) T cell function, such as AIDS patients.

We have been investigating the adjuvant properties of two super-activated interferon-regulatory factors (IRFs), IRF-3(5D) and IRF7/3A, identified in our previous studies of structure–function relationships, for enhancing plasmid vaccines. Intramuscular injection of plasmid cocktails encoding IRF-3(5D) and IRF7/3A molecules elicited cytotoxic T cell responses in over 80% of mice following a single immunization compared to a 20% response-rate using a control cocktail. Most interestingly, greater than 60% of mice immunized with the super-activated IRFs developed antigen-specific antibodies compared to 0% of the mice in the control group. Finally, vaccines which incorporated the super-activated IRFs provided greater protection against challenge with a recombinant vaccinia virus. These results support further investigation of the potential of these agents as adjuvants for genetic immunization.

By using adoptive transfer of Ag-loaded bone marrow-derived dendritic cells (BMDC), we have established an in vivo model of CTL priming. Activation of CTL in these experiments required both CD4(+) T cells and CD154, demonstrating that this model reflects CD4(+) T cell-dependent dendritic cell (DC) licensing. Because IL-12 has been suggested to play an important role in CTL activation by DC, we examined the ability of BMDC to prime CTL in the complete absence of IL-12 using p40-deficient mice. We observed that the absence of IL-12 does not affect the phenotype or allostimulatory function of BMDC after in vitro maturation. Moreover, there was no difference in the ability of Ag-loaded DC to elicit CTL cytotoxicity, whether the Ag was delivered by virus infection or peptide pulsing. Equal frequencies of Ag-specific, IFN-gamma-secreting CD8(+) T cells developed in both wild-type and IL-12-deficient backgrounds. Finally, CTL generated in the IL-12-deficient environment were capable of protecting immunized mice against tumor challenge, demonstrating that these CTL were fully functional, despite the absence of IL-12 during the maturation process in vivo. These results indicate that IL-12 is not critical for the development of IFN-gamma secreting, CD8(+) T cells and that another mechanism must be used by licensed DC to prime and activate CTL.