Murine Survivin (20-28)

Skin represents an attractive target for DNA vaccine delivery because of its natural richness in APCs, whose targeting may potentiate the effect of vaccination. Nevertheless, intramuscular electroporation is the most common delivery method for ECTM vaccination. In this study we assessed whether intradermal administration could deliver the vaccine into different cell types and we analyzed the evolution of tissue infiltrate elicited by the vaccination protocol. Intradermal electroporation (EP) vaccination resulted in transfection of different skin layers, as well as mononuclear cells. Additionally, we observed a marked recruitment of reactive infiltrates mainly 6-24 hours after treatment and inflammatory cells included CD11c(+). Moreover, we tested the efficacy of intradermal vaccination against Her2/neu antigen in cellular and humoral response induction and consequent protection from a Her2/neu tumor challenge in Her2/neu nontolerant and tolerant mice. A significant delay in transplantable tumor onset was observed in both BALB/c (p ≤ 0,0003) and BALB-neuT mice (p = 0,003). Moreover, BALB-neuT mice displayed slow tumor growth as compared to control group (p < 0,0016). In addition, while in vivo cytotoxic response was observed only in BALB/c mice, a significant antibody response was achieved in both mouse models. Our results identify intradermal EP vaccination as a promising method for delivering Her2/neu DNA vaccine.

Survivin, a 16.5 kDa tumor associated antigen, is the smallest member of the inhibitor of apoptosis family that is abundantly expressed during development but essentially absent in normal adult tissues. Interestingly, survivin expression is up-regulated in virtually all types of cancers studied, as well as in vascular endothelial cells during tumor associated angiogenesis. Survivin links apoptosis to cell cycle progression and plays a pivotal role in regulation of cell proliferation. These characteristics make survivin a potentially promising generic target for cancer immunotherapy. Hence, a genetic immunization strategy to induce tumor-specific immune responses against human survivin in a pre-clinical animal model was developed. In initial studies, BALB/c mice were immunized by intramuscular injection with DNA coding for human survivin (pcDNA3.1/hSurv). In addition, a construct encoding a secreted version of survivin (pSecTag2B/hSurv) was designed. A plasmid coding for murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was co-injected in both cases as a molecular adjuvant. Expression of survivin following transfection in mouse cells was corroborated. Humoral responses against human survivin were detected in mice sera using two immunization protocols (injections at 2- or 3-week intervals). The humoral response was markedly improved by secretion of survivin and co-expression of GM-CSF. The predominant antibody subclass detected in responsive mice was IgG2a, suggesting that a Th1-CD4+ cellular response had been induced. Furthermore, DNA immunization with survivin encoding vectors generated an effective CD8+ T cell response measured as an increase of cytotoxic Interferon-gamma (IFN-gamma) secreting CD8+ T cells. In conclusion, intramuscular genetic immunization of mice with human survivin encoding plasmids induced a survivin-specific humoral as well as cellular immune response in recipient mice. Secretion of survivin and co-injection of GM-CSF as a genetic adjuvant appear to be more important in generating an humoral than a cellular immune response.

DNA vaccines have been shown to elicit tumor-protective cytotoxic T lymphocyte (CTL) immunity in preclinical models, but have shown limited efficacy in cancer patients. Plasmids used for DNA vaccines can stimulate several innate immune receptors, triggering the activation of master transcription factors, including interferon regulatory factor 3 (IRF3) and nuclear factor κ B (NF-κB). These transcription factors drive the production of type I interferons (IFNs) and pro-inflammatory cytokines, which promote the induction of CTL responses. Understanding the innate immune signaling pathways triggered by DNA vaccines that control the generation of CTL responses will increase our ability to design more effective vaccines. To gain insight into the contribution of these pathways, we vaccinated mice lacking different signaling components with plasmids encoding tyrosinase-related protein 2 (TRP2) or ovalbumin (OVA) using intradermal electroporation. Antigen-specific CTL responses were detected by intracellular IFN-γ staining and in vivo cytotoxicity. Mice lacking IRF3, IFN-α receptor, IL-1β/IL-18, TLR9 or MyD88 showed similar CTL responses to wild-type mice, arguing that none of these molecules were required for the immunogenicity of DNA vaccines. To elucidate the role of NF-κB activation we co-vaccinated mice with pIκBα-SR, a plasmid encoding a mutant IκBα that blocks NF-κB activity. Mice vaccinated with pIκBα-SR and the TRP2-encoding plasmid (pTRP2) drastically reduced the frequencies of TRP2-specific CTLs and were unable to suppress lung melanoma metastasis in vivo, as compared with mice vaccinated only with pTRP2. Taken together these results indicate that the activation of NF-κB is essential for the immunogenicity of intradermal DNA vaccines.