Role of DNA topology in uptake of polyplex molecules by dendritic cells

Dendritic cells (DCs) are an attractive target for DNA vaccines as they are potent antigen presenting cells. This study demonstrated how non-viral gene delivery to DCs involving complexes of poly-l-lysine (PLL) and plasmid DNA (pDNA) (polyplexes) showed dependence on DNA vector topology. DNA topology is of importance from both production and regulatory viewpoints. In our previous study with CHO cells we demonstrated that polyplex uptake was dependent on DNA topology whereby complexes containing supercoiled (SC) pDNA were smaller, more resistant to nucleases and more effectively condensed by PLL than open circular (OC) and linear-pDNA complexes. In this study polyplex uptake in DCs was measured qualitatively and quantitatively by confocal microscopy along with gene expression studies and measurement of DC phenotype. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Quantification studies revealed that by 1h following uptake 15% (±2.59% relative standard error [RSE]) of SC-pDNA polyplexes were identified to be associated (fluorescent co-localisation) with the nucleus, in comparison to no nuclear association identified for OC- and linear-pDNA complexes. By 48 h following uptake, 30% (±1.82% RSE) of SC-pDNA complexes associated with the nucleus in comparison to 16% (±4.40% RSE) and 12% (±6.97% RSE) of OC- and linear-pDNA polyplexes respectively. Confocal microscopy images showed how DNA and PLL remained associated following uptake by dual labelling. Polyplex (containing 20 μg pDNA) gene expression (plasmid encoded lacZ [β-galactosidase] reporter gene) in DCs was greatest for SC-pDNA polyplexes at 14.12% unlike that of OC- (9.59%) and linear-pDNA (7.43%). DCs express cell surface markers which contribute towards antigen presentation. Polyplex gene expression did not alter DC phenotype through surface marker expression. This may be due to the pDNA dose employed (20μg) as other studies have used doses as high as 200 μg pDNA to induce DC phenotypic changes. Although no change in DC phenotype occurred, this could be advantageous in terms of biocompatibility. Collectively these results indicate that DNA topology is an important parameter for DC vector design, particularly pDNA in the SC conformation in regards to DNA vaccination studies.     

Effect of vesicle size on tissue localization and immunogenicity of liposomal DNA vaccines

The formulation of plasmid DNA (pDNA) in cationic liposomes is a promising strategy to improve the potency of DNA vaccines. In this respect, physicochemical parameters such as liposome size may be important for their efficacy. The aim of the current study was to investigate the effect of vesicle size on the in vivo performance of liposomal pDNA vaccines after subcutaneous vaccination in mice. The tissue distribution of cationic liposomes of two sizes, 500 nm (PDI 0.6) and 140 nm (PDI 0.15), composed of egg PC, DOPE and DOTAP, with encapsulated OVA-encoding pDNA, was studied by using dual radiolabeled pDNA-liposomes. Their potency to elicit cellular and humoral immune responses was investigated upon application in a homologous and heterologous vaccination schedule with 3 week intervals. It was shown that encapsulation of pDNA into cationic lipsomes resulted in deposition at the site of injection, and strongest retention was observed at large vesicle size. The vaccination studies demonstrated a more robust induction of OVA-specific, functional CD8+ T-cells and higher antibody levels upon vaccination with small monodisperse pDNA-liposomes, as compared to large heterodisperse liposomes or naked pDNA. The introduction of a PEG-coating on the small cationic liposomes resulted in enhanced lymphatic drainage, but immune responses were not improved when compared to non-PEGylated liposomes. In conclusion, it was shown that the physicochemical properties of the liposomes are of crucial importance for their performance as pDNA vaccine carrier, and cationic charge and small size are favorable properties for subcutaneous DNA vaccination.     

An assessment of different DNA delivery systems for protection against respiratory syncytial virus infection in the murine model: gene-gun delivery induces IgG in the lung

Immunization with plasmid DNA (pDNA) has the potential to overcome the difficulties of neonatal vaccination that may be required for protection against infection with respiratory syncytial virus (RSV); however, little is known about optimal delivery modalities. In this pilot study we compared mucosal delivery of pDNA encoding RSV F protein encapsulated in poly(DL-lactide-co-glycolide) with delivery of pDNA by gene-gun for the induction of immunity in mice. Intra-gastric or intra-nasal immunization with various doses of microparticles induced weak low levels of RSV-specific serum antibodies in a proportion of mice; in contrast, gene-gun vaccination led to protective immunity associated with a humoral response. Interestingly, RSV-specific antibody was detected in lung fragment cultures following intradermal vaccination with the gene-gun.     

A dose sparing effect by plasmid encoded IL-12 adjuvant on a SIVgag-plasmid DNA vaccine in rhesus macaques

An experimental pDNA vaccine adjuvant expressing IL-12 was evaluated for its ability to augment the humoral and cellular immune responses elicited by a SIVmac239 gag p39 expressing pDNA vaccine. To determine the effect of vaccine dose on the immune response, rhesus macaques were immunized with 1.5 mg or 5.0 mg of SIVmac239 gag pDNA, with or without co-immunization of IL-12 pDNA at 1.5 mg and 5.0 mg, respectively. Serum antibody responses to simian immunodeficiency virus (SIV) gag were increased 10-fold (p=0.044, 0.002) in macaques receiving IL-12 pDNA. Cellular immune responses, monitored by SIV gag-specific IFN-gamma ELISpot assay, were also significantly higher (p=0.007, 0.019) when the pDNA vaccine was co-immunized with IL-12 pDNA at high and low doses. There was no statistical difference between the immune responses elicited by the high and low dose of IL-12 pDNA (p=0.221, 0.917), a finding which could allow a dose reduction of vaccine without the concomitant loss of imunogenicity. Furthermore, analysis of the breadth of the T-cell response during the vaccination schedule, using overlapping peptides to SIV gag, demonstrated a significant correlation (p=0.0002) between the magnitude and breadth of the immune responses in the vaccines. These results have important implications for the continuing development of an effective, safe low dose pDNA vaccine adjuvant suitable for human use.     

Enhanced immunogenicity and protective efficacy of a tetravalent dengue DNA vaccine using electroporation and intradermal delivery

Phase 1 clinical trials with a DNA vaccine for dengue demonstrated that the vaccine is safe and well tolerated, however it produced less than optimal humoral immune responses. To determine if the immunogenicity of the tetravalent dengue DNA vaccine could be enhanced, we explored alternate, yet to be tested, methods of vaccine administration in non-human primates. Animals were vaccinated on days 0, 28 and 91 with either a low (1 mg) or high (5 mg) dose of vaccine by the intradermal or intramuscular route, using either needle-free injection or electroporation devices. Neutralizing antibody, IFN-γ T cell and memory B cell responses were compared to a high dose group vaccinated with a needle-free intramuscular injection delivery device similar to what had been used in previous preclinical and clinical studies. All previously untested vaccination methodologies elicited improved immune responses compared to the high dose needle-free intramuscular injection delivery group. The highest neutralizing antibody responses were observed in the group that was vaccinated with the high dose formulation via intradermal electroporation. The highest IFN-γ T cell responses were also observed in the high dose intradermal electroporation group and the CD8⁺ T cells were the dominant contributors for the IFNγ response. Memory B cells were detected for all four serotypes. More than a year after vaccination, groups were challenged with dengue-1 virus. Both the low and high dose intradermal electroporation groups had significantly fewer days of dengue-1 virus RNAemia compared to the control group. The results from this study demonstrate that using either an electroporation device and/or the intradermal route of delivery increases the immune response generated by this vaccine in non-human primates and should be explored in humans.     

Late administration of plasmid DNA by intradermal electroporation efficiently boosts DNA-primed T and B cell responses to carcinoembryonic antigen

Heterologous boost immunisation is considered the most efficient way to enhance DNA-primed immune responses. We have previously shown that administration of recombinant carcinoembryonic antigen (CEA) efficiently boosts humoral responses in mice primed with CEA DNA. However, clinical grade recombinant proteins are far more intriguing to produce than plasmid DNA. Therefore, the possibility to use plasmid DNA for both priming and boosting would be beneficial. With the prospect of future use in a clinical trial, we investigated if electroporation-mediated delivery of DNA could be used to boost DNA-primed immune responses to CEA. The Biojector was used to prime BALB/c mice intradermally three times with CEA66 DNA, encoding an intracellular modified form of CEA. Twelve weeks after the last prime, the animals received either one injection of recombinant CEA or one intradermal injection of twtCEA DNA, encoding the wild type CEA fused to a tetanus T helper epitope, in combination with electroporation. Boosting with rCEA protein did not enhance T cell responses to CEA but induced CEA-specific IgG in 4 of 8 mice. In contrast, intradermal delivery of twtCEA DNA by electroporation led to a tenfold increase in IFN-γ-producing CD8+ T cells, compared to the levels obtained after the third priming immunisation. The DNA boost also induced high CEA-specific IgG titers in all immunised animals (8/8). The data suggests that a late DNA boost, in combination with enhanced DNA delivery by electroporation, could be used to enhance the efficiency of DNA vaccination and substitute for a heterologous protein boost vaccination.     

Comparative ability of plasmid IL-12 and IL-15 to enhance cellular and humoral immune responses elicited by a SIVgag plasmid DNA vaccine and alter disease progression following SHIV(89.6P) challenge in rhesus macaques

Plasmid-based IL-12 has been demonstrated to successfully enhance the immunogenicity of DNA vaccines, thus enabling a reduction of the amount of DNA required for immunization. IL-15 is thought to affect the maintenance and enhance effector function of CD8(+) memory T cells. Since the ability to elicit a long-term memory response is a desirable attribute of a prophylactic vaccine, we sought to evaluate the ability of these plasmid-based cytokines to serve as vaccine adjuvants in rhesus macaques. Macaques were immunized with plasmid DNA encoding SIVgag in combination with plasmid IL-12, IL-15, or a combination of IL-12 and IL-15. The plasmid-based cytokines were monitored for their ability to augment SIVgag-specific cellular and humoral immune responses and to alter the clinical outcome following pathogenic SHIV(89.6P) challenge. Macaques receiving SIVgag pDNA in combination with plasmid IL-12 alone, or in combination with plasmid IL-12 and IL-15, demonstrated significantly elevated cell-mediated and humoral immune responses resulting in an improved clinical outcome following virus challenge compared to macaques receiving SIVgag pDNA alone. Macaques receiving SIVgag pDNA in combination with plasmid IL-15 alone demonstrated minor increases in cell-mediated and humoral immune responses, however, the clinical outcome following virus challenge was not improved. These results have important implications for the continued development of plasmid DNA vaccines for the prevention of HIV-1 infection.     

Effective DNA vaccination of cattle with the mycobacterial antigens MPB83 and MPB70 does not compromise the specificity of the comparative intradermal tuberculin skin test

The current tuberculin test and slaughter strategy for the control of bovine tuberculosis in cattle has failed to prevent a sharp rise in cases over recent years, especially in the south-west of England. A recent scientific review has concluded that the development of a cattle vaccine holds the best prospect for tuberculosis control in British herds. In order to continue with test and slaughter-based control strategies, the development of TB vaccines that do not compromise the specificity of the tuberculin skin test are required. This report describes results of cattle vaccination experiments with TB DNA vaccines expressing the mycobacterial antigens MPB70, MPB83, and Ag85A and constitutes the first published vaccination study with DNA vaccines undertaken in a target host species. All calves vaccinated with the MPB83 expressing plasmid demonstrated potent cellular immune responses, characterised by CD4(+) T cells producing interferon-gamma as well as humoral immunity characterised by IgG1 biased specific antibodies. Vaccination with MPB70 was less effective with immune responses only observed in half of the vaccinated animals, while vaccination with Ag85A did not result in vaccine-induced immune responses. Intramuscular vaccination was found to stimulate stronger cellular responses than intradermal immunisation. Significantly, the specificity of tuberculin skin testing was not compromised by DNA vaccination since none of the vaccinated calves showed positive skin test reactivity.     

Intradermal administration of DNA vaccines combining a strategy to bypass antigen processing with a strategy to prolong dendritic cell survival enhances DNA vaccine potency

Intradermal vaccination via gene gun efficiently delivers DNA vaccines into dendritic cells (DCs) of the skin, resulting in the activation and priming of antigen-specific T cells in vivo. We have previously demonstrated that intradermal delivery of DNA vaccines encoding single-chain trimer (SCT) composed of the most immunogenic epitope of human papillomavirus type 16 (HPV-16) E6 protein (aa49-57), beta2-microglobulin, and MHC class I heavy chain (SCT-E6) can bypass antigen processing and lead to stable cell-surface presentation of E6 peptides. We also showed that co-administration of DNA vaccines with DNA encoding anti-apoptotic proteins can prolong the survival of DNA-transduced DCs, resulting in significant enhancement of antigen-specific CD8(+) T cell immune responses. In the current study, we hypothesized that combining the SCT strategy and antiapoptotic strategy may further enhance DNA vaccine potency by augmenting antigen-specific CD8(+) T cell immune responses and antitumor effects in vaccinated mice. Here, we show that C57BL/6 mice vaccinated with SCT-E6 DNA combined with antiapoptotic protein Bcl-xL DNA generated enhanced E6-specific CD8(+) T cell immune responses compared to mice vaccinated with SCT-E6 DNA and a non-functional mutant Bcl-xL (mtBcl-xL) DNA. Furthermore, we show that mice treated with SCT-E6 and Bcl-xL DNA generated enhanced anti-tumor effects against E6-expressing tumor cells (TC-1/Luciferase) compared to mice treated with SCT-E6 and mtBcl-xL DNA.     

Local administration of granulocyte macrophage colony-stimulating factor induces local accumulation of dendritic cells and antigen-specific CD8+ T cells and enhances dendritic cell cross-presentation

Immunotherapy has emerged as a promising treatment strategy for the control of HPV-associated malignancies. Various therapeutic HPV vaccines have elicited potent antigen-specific CD8+ T cell mediated antitumor immune responses in preclinical models and are currently being tested in several clinical trials. Recent evidence indicates the importance of local immune activation, and higher number of immune cells in the site of lesion correlates with positive prognosis. Granulocyte macrophage colony-stimulating factor (GMCSF) has been reported to posses the ability to induce migration of antigen presentation cells and CD8+ T cells. Therefore, in the current study, we employ a combination of systemic therapeutic HPV DNA vaccination with local GMCSF application in the TC-1 tumor model. We show that intramuscular vaccination with CRT/E7 DNA followed by GMCSF intravaginal administration effectively controls cervicovaginal TC-1 tumors in mice. Furthermore, we observe an increase in the accumulation of E7-specific CD8+ T cells and dendritic cells in vaginal tumors following the combination treatment. In addition, we show that GMCSF induces activation and maturation in dendritic cells and promotes antigen cross-presentation. Our results support the clinical translation of the combination treatment of systemic therapeutic vaccination followed by local GMCSF administration as an effective strategy for tumor treatment.     

A polyvalent influenza DNA vaccine applied by needle-free intradermal delivery induces cross-reactive humoral and cellular immune responses in pigs

BackgroundPigs are natural hosts for influenza A viruses, and the infection is widely prevalent in swine herds throughout the world. Current commercial influenza vaccines for pigs induce a narrow immune response and are not very effective against antigenically diverse viruses. To control influenza in pigs, the development of more effective swine influenza vaccines inducing broader cross-protective immune responses is needed. Previously, we have shown that a polyvalent influenza DNA vaccine using vectors containing antibiotic resistance genes induced a broadly protective immune response in pigs and ferrets using intradermal injection followed by electroporation. However, this vaccination approach is not practical in large swine herds, and DNA vaccine vectors containing antibiotic resistance genes are undesirable.ObjectivesTo investigate the immunogenicity of an optimized version of our preceding polyvalent DNA vaccine, characterized by a next-generation expression vector without antibiotic resistance markers and delivered by a convenient needle-free intradermal application approach.MethodsThe humoral and cellular immune responses induced by three different doses of the optimized DNA vaccine were evaluated in groups of five to six pigs. The DNA vaccine consisted of six selected influenza genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase.ResultsNeedle-free vaccination of growing pigs with the optimized DNA vaccine resulted in specific, dose-dependent immunity down to the lowest dose (200 μg DNA/vaccination). Both the antibody-mediated and the recall lymphocyte immune responses demonstrated high reactivity against vaccine-specific strains and cross-reactivity to vaccine-heterologous strains.ConclusionThe results suggest that polyvalent DNA influenza vaccination may provide a strong tool for broad protection against swine influenza strains threatening animal as well as public health. In addition, the needle-free administration technique used for this DNA vaccine will provide an easy and practical approach for the large-scale vaccination of swine.     

Features of the antibody response attributable to plasmid backbone adjuvanticity after DNA immunization

DNA vaccination induces antigen-specific immune responses with characteristics distinct from other vaccination modes. In the present study, the contribution of the plasmid backbone adjuvant effect to the quality of the DNA-raised antibody response was investigated. For this purpose, three intradermal primings were compared in mice using: (1) the recombinant Schistosoma haematobium glutathione S-transferase antigen (rSh28GST): (2) rSh28GST supplemented with a non-coding plasmid; and (3) a Sh28GST-encoding plasmid. In contrast to immunization with the protein, DNA immunization elicited a very stable antibody (Ab) response over a prolonged period of time. This feature was attributed to the plasmid backbone, because co-administration of the non-coding plasmid with rSh28GST allowed the maintenance of the specific Ab response. A strong anamnestic Ab response was induced after intradermal boost with rSh28GST only in the mice primed with pMSh. This indicated that the selective ability of DNA vaccination to induce memory humoral response was independent of the plasmid backbone. In contrast the plasmid backbone was found to strongly participate in the preferential IgG2a Ab production observed. These results suggest that, following DNA immunization, the Th1-biased profile and the maintenance of the long-lived Ab response could be attributed to an adjuvant effect of the plasmid backbone during priming, whereas the strength of B-cell memory was independent of this effect.     

Enhancement of humoral and cellular immunity in chickens against reticuloendotheliosis virus by DNA prime-protein boost vaccination

Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in multiple avian hosts worldwide. In this study, an optimal vaccination strategy was developed to enhance the immune responses against REV infection. Chickens were vaccinated twice intramuscularly with plasmid pCAGgp90 encoding gp90 protein of REV, or with recombinant gp90 protein, or vaccinated with plasmid pCAGgp90 and then boosted with recombinant gp90 protein. The humoral immune responses were monitored by ELISA and virus neutralizing test. In addition, lymphocyte proliferation response, cytokine production and protection effectiveness against REV infection were also evaluated. Although all vaccinated groups developed immune responses, chickens primed with pCAGgp90 plasmid and boosted with rgp90 protein developed higher levels of antibodies compared with those immunized with pCAGgp90 plasmid or rgp90 protein alone. Furthermore, enhanced cellular immune responses were induced following priming with the pCAGgp90 plasmid and boosting with the rgp90 protein. In addition, the DNA prime-protein boost vaccine yielded 100% protection of chickens from REV viremia caused by challenge infection. These findings demonstrated that a DNA prime-protein boost vaccination strategy could enhance both humoral and cellular immune responses in chickens, highlighting the potential value of such an approach in the prevention of REV infection.     

Comparative ability of various plasmid-based cytokines and chemokines to adjuvant the activity of HIV plasmid DNA vaccines

The effectiveness of plasmid DNA (pDNA) vaccines can be improved by the co-delivery of plasmid-encoded molecular adjuvants. We evaluated pDNAs encoding GM-CSF, Flt-3L, IL-12 alone, or in combination, for their relative ability to serve as adjuvants to augment humoral and cell-mediated immune responses elicited by prototype pDNA vaccines. In Balb/c mice we found that co-administration of plasmid-based murine GM-CSF (pmGM-CSF), murine Flt-3L (pmFlt-3L) or murine IL-12 (pmIL-12) could markedly enhance the cell-mediated immune response elicited by an HIV-1 env pDNA vaccine. Plasmid mGM-CSF also augmented the immune response elicited by DNA vaccines expressing HIV-1 Gag and Nef-Tat-Vif. In addition, the use of pmGM-CSF as a vaccine adjuvant appeared to markedly increase antigen-specific proliferative responses and improved the quality of the resulting T-cell response by increasing the percentage of polyfunctional memory CD8(+) T cells. Co-delivery of pmFlt-3L with pmGM-CSF did not result in a further increase in adjuvant activity. However, the co-administration of pmGM-CSF with pmIL-12 did significantly enhance env-specific proliferative responses and vaccine efficacy in the murine vaccinia virus challenge model relative to mice immunized with the env pDNA vaccine adjuvanted with either pmGM-CSF or pmIL-12 alone. These data support the testing of pmGM-CSF and pmIL-12, used alone or in combination, as plasmid DNA vaccine adjuvants in future macaque challenge studies.     

Cationic polymers that enhance the performance of HbsAg DNA in vivo

In this paper, different cationic polymers were investigated as a DNA delivery system both in vitro in dendritic and muscle cells and in vivo, in a murine model. Expression of the reporter gene beta-galactosidase was used in order to determine the in vitro transfection efficiency of these polymer-DNA complexes (polyplexes) and both specific mRNA and protein expression were monitored in parallel with polyplex toxicity on the cells. Interestingly, the enhancing expression activities of the different polyplexes were tissue-dependent, implying that they may gain entrance to the cells through specific receptors. Subsequently, complexes of polymers and DNA plasmid (pCMV-S) encoding the human hepatitis B virus (HBV) surface antigen (HBsAg) were injected into the skeletal muscles of BALB/c mice. Higher levels of both HBsAg local expression in the tibial anterior muscles and systemic humoral immune responses were detected when the selected polymers complexed with pCMV-S were compared to those complexed with pCMV-S alone. Induction of immunoglobulin G2a (IgG2a) against HbsAg in the serum of pCMV-S-polyplex vaccinated mice varied with the polymer used, suggesting that polyplex-mediated DNA vaccination can potentially modulate the type of helper T cell immunity (Th). The effect of some polyplexes to switch the host immune response more towards a Th1 response may be associated with their differential efficiency to transfect dendritic cells and/or other antigen-presenting cells (APC) as was observed in vitro. These results suggest that the investigated cationic polymers can be effective as delivery/adjuvant compounds for DNA.     

Codelivery of PEG-IFN-alpha inhibits HCV DNA vaccine-induced T cell responses but not humoral responses in African green monkeys

Although pegylated interferon alpha (PEG-IFN-alpha) with ribavirin treatment constitutes an effective means of treatment for chronic hepatitis C, novel approaches are needed due to the inefficient effects of the current therapy against chronic infection with genotype 1 virus. In this study, the immunomodulatory effects of PEG-IFN-alpha on multigenic HCV DNA vaccine-induced immunity were investigated in African green monkeys. Multigenic HCV DNA vaccination with and without PEG-IFN-alpha was safe and well tolerated, and induced significant long-term T cell and antibody responses. In addition, the induced immune responses were gradually increased by repeated injection. Interestingly, co-treatment with PEG-IFN-alpha significantly suppressed HCV DNA vaccine-induced T cell responses, but not antibody responses, which demonstrated that IFN-alpha could act as a negative regulator of T cell immune induction. However, the suppression of T cell responses by PEG-IFN-alpha could be overcome by two times more DNA vaccination, which suggests that combined therapy of DNA vaccine with PEG-IFN-alpha might be possible. Our results provide valuable information for the design of an effective therapeutic regimen to treat chronic HCV infection and to understand the immunomodulatory roles of PEG-IFN-alpha in immune induction by DNA vaccination.     

Innate IL-10 promotes the induction of Th2 responses with plasmid DNA expressing HIV gp120

Like most DNA vaccines, intramuscular immunization with plasmid DNA coding for influenza virus haemagglutinin (HApDNA) induced Th1 responses and IgG2a antibodies in mice. However, plasmid DNA coding for HIV gp120 (gp120pDNA) induced Th2-biased responses and predominantly IgG1 antibodies. Responses to gp120pDNA switched to a Th1-type in IL-10-defective mice and to exclusively IgG2a antibodies in IL-4-defective mice. Conversely, antigen-specific IFN-gamma production induced by gp120pDNA or HApDNA was reduced in IL-12-defective mice, whereas addition of plasmid DNA coding for IL-12 enhanced Th1 responses. Plasmid DNA stimulated IL-10 and IL-12 production by macrophages and dendritic cells (DCs) in vitro and anti-IL-10 antibodies enhanced IL-12 production and DC maturation in response to gp120pDNA. Our findings suggest that T cell responses induced by DNA vaccines is influenced by the nature of the antigen, and that the induction of Th2-biased responses with gp120pDNA is mediated in part through the stimulation of innate IL-10, which inhibits activation of DCs that direct the induction of Th1 cells.     

Skin-specific promoters for genetic immunisation by DNA electroporation

This study aimed at restricting expression of DNA vaccine to specific cell types by using skin-specific promoters (i) to contribute to the understanding of the mechanism of intradermal DNA vaccines and (ii) to address safety concerns associated with DNA vaccines. The expression and immune response after delivery of plasmids encoding luciferase by intradermal DNA electroporation were assessed. Two ubiquitous promoters, CMV and CAG, and three tissue-specific promoters were studied. Keratin 14 promoter restricts gene expression to keratinocytes of the epidermal basal layer, CD11c promoter to dendritic cells and fascin promoter only to mature dendritic cells. The use of plasmids with tissue-specific promoter resulted in significant, but very low protein expression, as compared to that obtained with ubiquitous promoter plasmids. Immunisation with both ubiquitous promoter plasmids elicited humoral and cellular anti-luciferase immune response. No immune response was observed after delivery of CD11c plasmid while fascin and keratin 14 plasmids induced IFN-gamma response suggesting that the targeting of skin-specific cells could be a suitable approach but only for the treatment of pathologies or pathogens requiring mainly cellular and not humoral immune response.