dna vaccination is an attractive immunotherapeutic strategy that triggers physiologic immunity and is able to induce long lasting t cell and antibody - mediated tumor protection . in fact , direct injection into mouse muscle or skin of plasmid dna encoding a selected antigen results in the expression of the gene product and can elicit an immune response against the antigen of interest . currently , various delivery devices such as gene - guns , bioinjectors , and electroporation systems are being used in order to increase the potency of dna vaccines . in vivo dna electroporation ( ep ) has emerged as an efficient delivery method that allows efficient dna uptake , long - term and high - level antigen expression . furthermore , ep is also able to evoke the production of several cytokines and chemokines , thereby increasing the potency of dna vaccines . muscle is the most commonly targeted tissue for evaluation of ep in combination with dna delivery . under the influence of an electrical field , cellular membranes build up a transmembrane potential until the dielectric strength of the membrane is exceeded and permeation event occurs . intramuscular electroporation has been previously shown to induce target antigen expression and to trigger humoral and cellular immunity , thus enhancing tumor protection [ 7 , 8 ] . ep has been used clinically to deliver chemotherapeutic agents to tumor cells in cutaneous malignancies [ 9 , 10 ] . nowadays , there are approximately 85 clinical trials listed using electroporation ( http://www.clinicaltrials.gov/ ) : around 28 are related to drug delivery and the rest are related to dna delivery [ 1113 ] . moreover , several data establish ep as a potent method for stimulating immune responses induced by dna vaccination in humans [ 14 , 15 ] . skin is an attractive site for electroporation in translational settings , as it is readily accessible and ep is minimally invasive and generally well tolerated , as compared to muscle . moreover , skin naturally harbors a high number of antigen presenting cells ( apcs ) , such as langerhans cells and other types of dermal dendritic cells , which after dna / antigen uptake can migrate to lymph nodes where efficient presentation to t cells occurs [ 17 , 18 ] , thereby potentially increasing ep efficacy . the efficacy of intramuscular injection of a plasmid coding for the extracellular and transmembrane domains of the protein product of the her-2/neu oncogene ( ectm ) followed by ep in transgenic murine models has been previously demonstrated . the vaccination protocol induced production of antibodies against her-2/neu and ifn- secretion : these two immune activities were associated with the clearance of her-2/neu spontaneous lesions in transgenic balb - neut mice . however , sequential courses of dna intramuscular ep were required to maintain specific antibody response and counteract the progression of preneoplastic lesions to invasive carcinoma . in the current study , we evaluated the effectiveness of intradermal vaccination using ep against transplantable her2/neu tumor . to address this point , first we analyzed intradermal ep vaccination - induced immune cell recruitment , in both the skin and draining lymph nodes . second , we evaluated , in both her2/neu tolerant ( balb - neut ) and nontolerant ( balb / c ) mice , the ability of intradermal ectm ep vaccination to trigger her2/neu specific immune responses and counteract tumor onset and growth . seven - week - old virgin female balb / c and balb - neut mice ( h-2 ) were used . virgin balb - neut mice , overexpressing the transforming rat her-2/neu oncogene under the control of the mouse mammary tumor virus , were bred in house . mice of the same age were randomly assigned to experimental groups and were treated according to the european community guidelines . pvax ( invitrogen , milan , italy ) was the backbone for all the vaccines . the pvax - egfp dna construct was obtained by subcloning the egfp cdna , excised from pegfp - n1 ( clontech , mountain view , ca ) by hindiii / xbai digestion , into the hindiii / xbai sites of the pvax-1 vector ( life technologies , carlsbad , ca ) . all plasmids for dna immunizations were grown in e. coli dh5 strain , and large - scale preparation of the endotoxin - free plasmid dna was carried out using qiagen endofree plasmid - giga kits ( qiagen , chatsworth , ca , usa ) according to the manufacturer 's instructions . anesthetized mice were vaccinated with 50  l of solution containing 50  g of dna or 25  mg / ml of fluorescein isothiocyanate - dextran ( fitc - dextran ) average mol wt 40,000 ( sigma aldrich , milan , italy ) . the vaccination course consisted of two intradermal injections into the skin of the back near the base of the tail . immediately after the plasmid or fitc - dextran administration , a conducting gel and an electrode were placed over the injection site and voltage was set up according to previously described protocols ( 2 pulses , 1125  v / cm 50  s , and 8 pulses , 275  v / cm 10  msec ) . balb / c and balb - neut mice were vaccinated 21 and 7 days before a subcutaneous injection with a lethal dose of tubo cells ( day 0 ) . her-2/neu positive tubo cells had been originally isolated from a carcinoma arising in a balb - neut mouse . n202.1a ( her-2/neu positive ) and n202.1e ( her-2/neu negative ) lines had been isolated from a mammary carcinoma in a fvb / n mouse ( h-2 ) transgenic for the rat her-2/neu protoncogene . tubo cells were cultured in dmem ( biowhittaker europe , verviers , belgium ) with 20% fbs ( life technologies , inc . , san giuliano , italy ) , and n202.1a and n2021e in rpmi ( biowhittaker europe ) with 10% fbs ( life technologies ) . mice were subcutaneously injected with 0.2  ml of a single cell suspension containing the minimal lethal dose of tubo ( 10 ) cells   in the right flank . mice were checked twice weekly for tumor onset : stable or growing masses were regarded as tumors . neoplastic masses were measured with calipers in the two perpendicular diameters , and the tumor volume was recorded for 150 days . at the end of this period , 100  l of 1  :  10 diluted sera was incubated for 45 minutes at 4c with 2  10 n2021a or 1e cells pretreated with fc receptor blocker ( cd16/cd32 ; pharmingen , milan , italy ) for 15 minutes at 4c . total ig binding was evaluated using a pe - conjugated goat anti - mouse ig antibody ( dakocytomation , milan , italy ) . the ab4 ( oncogene , milan , italy ) was used as positive control for anti - her2/neu reactivity . n202.1e mfi was subtracted to n202.1a mfi to identify her2/neu specific antibody reactivity and differences in mfi were analyzed by student 's t - test . dilutions ( 1  :  10 ) of sera in pbs - azide - bsa were incubated for 45 minutes at 4c with 2  10 n202.1a or n202.1e cells , pretreated with fc receptor blocker ( cd16/cd32 ; pharmingen ) for 15 minutes at 4c . after washing , the cells were incubated for 30 minutes with rat alexa 488-conjugated antibodies anti - mouse igm , igg1 , igg2a , igg2b , or igg3 ( invitrogen ) and fluorescence evaluated . the specific n202.1a - binding potential was calculated as the percentage of mfi / total ig . flow cytometry experiments were performed using a facscalibur ( becton dickinson , milan , italy ) and analyzed with flowjo software ( flowjo llc , ashland , usa ) . in vivo cytotoxicity assay briefly , a single - cell suspension of 10 naive splenocytes ( spc)/ml was labeled with 0.5 ( cfse ) or 5  mol / l ( cfse ) of the carboxyfluorescein diacetate succinimidyl ester cfse ( molecular probes , leiden , netherlands ) . g / ml of h-2k dominant her2/neu ( tyvpanasl ) ( inbios srl , naples , italy ) peptide for 1 hour at room temperature . the two spc populations were mixed together in equal amounts and injected i.v . into control and treated mice . mice were sacrificed 48 hours later , and single - cell suspensions from spleens were processed individually to evaluate the presence of cfse cells with the facscalibur after adding propidium iodide to exclude dead cells . the specific cytolytic activity was calculated as 100  ( percentage cfse cells  percentage cfse cells)/percentage cfse cells . for immunohistochemical evaluation , mice were euthanized at 3 , 6 , 24 , 48 , 72 , and 96 hours after vaccination and the skin and axillary , inguinal , and popliteal lymph nodes were collected , fixed in pfa 1% , and embedded in oct and tissue sections were stained with hematoxylin and eosin ( h&e ) for histological examination . skin and lymph nodes sections were incubated with the following primary antibodies : rat mab anti - cd4 , anti - cd8 , anti - cd11b , anti - cd45r / b220 , anti - gr-1 ( bd pharmingen , milan , italy ) , anti - cd68 ( abcam , cambridge , uk ) , and anti - cd11c ( chemicon international , usa ) . immunostaining was developed with vulcan fast red ( biocare , milan , italy ) alkaline phosphatase method . for immunofluorescence , secondary antibodies conjugated with alexa 488 and alexa 546 ( invitrogen , life technologies , monza , italy ) were used . nuclei were stained with draq5 ( alexis , life technologies , monza , italy ) , yo pro-3 , or to pro-3 ( molecular probes , monza , italy ) . image acquisition was performed using zeiss lsm 510 meta confocal microscope ( carl zeiss spa , milan italy ) . the slides were examined in a double - blind fashion , and digital images of representative areas were taken . the significance of differences was evaluated with two - tailed student 's t - test or log - rank ( mantel - cox ) test . statistical analysis was carried out with graphpad prism5 software ( san diego , ca , usa ) ; p  0.05 was used as the critical level of significance . to analyze the cellular effects of vaccination , intradermal injection of fitc - dextran followed by ep was performed in balb / c mice . immediately after vaccination ( 3 hours ) skin showed tissue damage and a coagulated aspect as well as evident inflammatory infiltrate ( figure 1(a ) ) . immunofluorescence analysis revealed the presence of cd11b macrophages with incorporated fitc - dextran ( figures 1(b)1(d ) ) in close proximity of the damaged area as well as in epidermis ( figure 1(b ) ) , dermis ( figure 1(c ) ) , and hypodermis ( figure 1(d ) ) . at this extremely early time point , no cells with encapsulated fitc - dextran were detected in lymph nodes ( not shown ) . at a later time point ( 6 hours after vaccination ) , cd11c cells with incorporated fitc - dextran were detected in the dermis ( figure 1(e ) ) . additionally , fitc - dextran was also incorporated by lyve 1 lymphatic endothelial cells ( figure 1(f ) ) and deposited outside cd31 blood vessels ( figure 1(g ) ) . some cells with incorporated fitc - dextran were found into inguinal lymph nodes ( draining lymph nodes ) ( figure 1(h ) ) . no fitc - dextran incorporating cells were detected in control mice intradermally injected without electroporation . altogether these data are consistent with an efficient transfection of apcs and their progressive migration through the skin layers to lymph nodes . accordingly , immunohistochemistry analysis highlighted the accumulation of granulocytes ( gr1 ) and macrophages ( cd11b ) 3 hours after treatment ( table 1 ) and a significant number of reactive cells were still observed at 6 hours . in particular , at this time point , dendritic cells ( cd11c ) and t lymphocytes ( cd4 ) were detected ( table 1 and figures 2(a ) and 2(b ) ) . twenty - four hours after vaccination the number of dendritic and cd4 cells decreased until almost baseline levels ( table 1 and figures 2(c ) and 2(d ) ) and the local inflammatory infiltrate gradually turned off starting at 48 hours after vaccination . in order to shed light into the mechanisms underlying the immune responses elicited by the intradermal vaccination , we analyzed the type and the localization of pvax egfp transfected cells in both skin and inguinal draining lymph nodes ( figure 3 ) . after an intradermal injection probably the major part of the volume is deposited in the dermis due to the epidermis being very thin , particularly in mice . however , twenty - four hours after vaccination , positive cells have also been found in epidermis and in the subcutaneous muscle layer , the panniculus carnosus , near the damaged zone ( figure 3(a ) ) . close to them , some infiltrating cells expressed the transgene too ( figure 3(a ) ) . as shown in table 1 , dendritic cells decreased in skin 24 hours after treatment , so we investigated the presence of these cells in draining lymph nodes . not only transfected dendritic cells were found ( figure 3(b ) ) , but also , in accordance with their apc role , these cells were transfected and in very tight proximity with both cd4 ( figure 3(c ) ) and b220 ( figure 3(d ) ) . intradermal ectm ep vaccination effectively delayed tumor onset in both her2/neu nontolerant and tolerant mice . in fact , while mice vaccinated with an empty plasmid developed tumor within 30 days ( figures 4(a ) and 4(c ) ) , in pvax - ectm treated group , all balb / c mice remained tumor free until the endpoint ( figures 4(a ) and 4(b ) ) , and balb - neut mice displayed a strong delay in tumor onset ( p = 0,003 ) ( figure 4(c ) ) . notably , tumor growth in these mice was characterized by very slow kinetics as compared to pvax group ( p < 0,0016 ) ( figure 4(d ) ) . in accordance with the observed total protection against tumor challenge , higher ctl activity was observed in balb / c mice electroporated with pvax - ectm plasmid ( p = 0,0369 ) , while no cytotoxicity was detected in her2/neu tolerant balb - neut mice ( figure 5(a ) ) . this data together with a higher protection observed in balb - c mice is consistent with a higher immune tolerance of balb - neut mice . moreover , 14 , 30 , and 80 days after last vaccination , the titer of anti - her2/neu antibody was evaluated . a significant her2/neu specific antibody titer was detected in both balb / c ( p = 0,0256 ) and balb - neut ( p = 0,0096 ) pvax - ectm vaccinated mice as compared to pvax groups ( figures 5(b ) and 5(c ) ) already after 14 days . interestingly the titer remained elevated also 30 days after the vaccination ( balb / c p = 0,0242 , balb - neut p = 0,0197 ) , with detectable high levels up to 100 days after vaccination ( not shown ) in all surviving mice . the induction and persistence of the humoral response in both mouse models , together with the similar isotype profiling of anti - her2/neu reactive antibodies ( figures 5(d ) and 5(e ) ) , indicate that this type of immunity is similarly triggered in both tolerant and nontolerant mice . interestingly a high antibody titer correlated with tumor protection in balb - neut mice ( see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/159145 ) . it is important to note that naked dna injection resulted in negligible antibody or cytotoxicity induction and tumor protection ( supplementary figure 2 ) . altogether these results indicate that our vaccination protocol is able to induce a protective immune response not only in her-2 nontolerant balb / c mice , but also in tolerant balb - neut mice . by virtue of its versatility and efficacy in inducing immunity against selected antigens , dna vaccination represents a suitable approach and may provide new therapeutic avenues for the treatment of tumors . plasmids can be delivered by intramuscular , intradermal / epidermal , subcutaneous , oral ( e.g. , with bacterial carrier ) , and pulmonary ( e.g. , aerosols ) or other routes ( e.g. , vaginal ) . notably , the skin is a particularly attractive site for vaccination given that intradermal vaccination is easy to perform and well tolerated both clinically and histopathologically . additionally , skin is characterized by an extended local network of dendritic cells and the easy access to the skin - draining lymph nodes generates effector t cells and immunoglobulin - producing b cells [ 27 , 28 ] . these characteristics may help the generation of a long - term protective immunity [ 29 , 30 ] . in fact , experiments with bone marrow chimeras have shown that apcs , presumably dendritic cells , have a key role in dna vaccination - induced protection . moreover , it is already known that using intradermal ep vaccination a wide variety of cell types can be transfected and these cells can be found in the draining afferent lymph nodes , suggesting that migration of directly transfected dendritic cells may occur . nevertheless , intradermal vaccination is notorious for its lower antibody responses compared to intramuscular route of plasmid administration in mice [ 3134 ] . in this study we demonstrate that intradermal in vivo application of pulsed electric field confers long - term protection from her2/neu tumor . indeed , we report that efficiently permeabilized cells are found in all layers of the skin and migrate to draining lymph nodes . this is consistent with previous studies showing that , in contrast to the predominantly epidermal injection of naked dna , a pattern of dermal and subdermal transfection is observed in the skin . concordantly with our data ( figures 1 and 2 ) transfected cell types included numerous mononuclear cells with dendritic morphology , as well as large numbers of adipocytes . the variety of transfected cell types is likely to grant the mounting of an effective and durable immune response : in fact it has been demonstrated that transfection of different cell types results into different immune responses [ 3639 ] . we further demonstrated that cd11c are efficiently permeabilized and hopefully transfected upon dna vaccination ( figure 3 and supplementary figure 2 ) as a result of electroporation . the preferential ep mediated transfection of cells with dendritic morphology could provide a distinct advantage due to the central role of such cells in the stimulation of both primary and secondary immune responses . dendritic cells are the key initiators and regulators of any immune response which determine the outcome of cd4 and cd8 t cell responses . nevertheless , given the fact that either direct transfection of apcs or uptake of protein from other transfected cells ( cross presentation )   may occur , we can not provide formal proof that , although efficiently permeabilized , skin apcs are directly transfected in fact it has been demonstrated that , in response to vaccination , the cutaneous immune system sets up complex mechanisms including ( 1 ) recognition and capture of the vaccine by skin - resident apcs or by recruited innate immune cells ( inflammatory microenvironment ) , ( 2 ) activation of skin apcs , ( 3 ) passive diffusion or cell transport of antigens to the secondary lymphoid organs draining the skin ( draining lymph nodes ) , ( 4 ) processing and presentation of the antigens to immature cd4 and cd8 t cells by activated apcs or to naive follicular b cells , ( 5 ) activation of antigen - specific cd4 and cd8 t cells and clonal expansion as well as activation of specific b cells within a germinative center , ( 6 ) migration of specific effector cd4 and cd8 t and b cells toward the zone of vaccination and elimination of the vaccine antigens , ( 7 ) generation of a pool of specific memory t and b cells in the secondary lymphoid organs and in the periphery ( skin and mucosa ) . indeed , in our study , after intradermal ep vaccination , a significant number of reactive cells were already observed at 6 hours after treatment , consisting primarily of dendritic cells . independent on the mechanism by which apcs acquire and then present the target antigen , to the best of our knowledge , this is the first report showing that intradermal dna injection targeting her2/neu followed by ep elicts a strong inflammatory - like response and induces both ctl and antibody responses in balb / c and humoral response in tolerant balb - neut mice . this is consistent with recent reports of the presence of both ctl and antibody responses after vaccination in other tumor models [ 40 , 41 ] . a previous study suggested that intradermal plasmid injection could not induce an effective immune response in her2/neu models . nevertheless , our study demonstrates that , similar to what have been reported for intramuscular route , electroporation boosts the efficacy of ectm vaccination when intradermally administered ( supplementary figure 1 ) , resulting in a specific immunity and tumor protection . additionally , previous studies demonstrated that , in balb - neut mice , intramuscular ectm injection followed by ep repeated every 10 weeks triggered a humoral response that inhibited the progression of spontaneous mammary carcinogenesis . although we can not directly compare the two methods , in our study , a single cycle of intradermal ep vaccination induced a significant delay in tumor onset with an antibody titre that remains high over time , indicating intradermal route as a valid alternative to induce her2/neu specific response . this data confirms previous studies implicating anti - her2/neu antibodies in the inhibition of her2/neu - driven carcinogenesis and progression [ 7 , 22 , 42 , 43 ] . the importance of antibodies is further underlined by the reported absence of protection in balb - neut/ko mice unable to produce antibodies to her2/neu after intramuscular dna vaccinations with ectm plasmid . moreover , the high titer of anti - her2/neu antibodies has been demonstrated to correlate with the downregulation of her2/neu in the cells of mammary lesions and its cytoplasmic confinement [ 21 , 43 , 44 ] . consistent with a central role of antibody - mediated protection [ 4548 ] we observed protection from tumor challenge in balb - neut mice even in the absence of cell cytotoxicity , and long - term survivor mice displayed the highest antibody titer ( supplementary figure 2 ) . notably in our study we were able to achieve a long - term protection not only in her2/neu nontolerant balb / c mice that are more prone to mount an effective immune response against the oncogene ( as demonstrated also by the capability of our vaccination to induce cytotoxicity in these mice ) , but also in highly tolerant balb - neut mice . balb - neut mice are genetically predestined to develop lethal invasive carcinomas in their mammary glands by week 22 . as the transforming ( and immunization target ) oncogene is embedded in the genome of these mice , a unique dynamic relationship between the oncogenic signals and inhibitory immune reactions is taking place . given the long - term protection achieved with only 2 vaccination rounds , it will be of interest to test our experimental setting on spontaneous her2/neu positive mammary tumors to evaluate if intradermal ep vaccination can reduce the number of doses and/or prolong the tumor - free mice . in fact , prolongation of immune memory could be even a more worthwhile goal than enhancement of the immune response . indeed , activating the appropriate arm of the immune system that induces protection is now one of the major challenges to be faced in vaccine development against tumors . however , further studies are necessary to find out the best settings for vaccination , such as the nature of the antigens , against which immune responses are elicited , the use of adjuvant , and specific delivery system that will ensure optimal presentation to immune system and protection from tumors .
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 624 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 .