Gene Gun Administration Research Articles

Induction of food-specific IgG by Gene Gun-delivered DNA vaccines

Shellfish and tree nut allergies are among the most prevalent food allergies, now affecting 2%-3% and 1% of the US population, respectively. Currently, there are no approved therapies for shellfish or tree nut allergies, with strict avoidance being the standard of care. However, oral immunotherapy for peanut allergy and subcutaneous immunotherapy for environmental allergens are efficacious and lead to the production of allergen-specific IgG, which causes suppression of allergen effector cell degranulation. Since allergen-specific IgG is a desired response to alleviate IgE-mediated allergies, we tested transcutaneously-delivered DNA vaccines targeting shellfish and tree nut allergens for their ability to induce antigen-specific IgG, which would have therapeutic potential for food allergies. We assessed Gene Gun-delivered DNA vaccines targeting either crustacean shellfish or walnut/pecan allergens, with or without IL-12, in naïve mice. Three strains of mice, BALB/cJ, C3H/HeJ and CC027/GeniUnc, were evaluated for IgG production following vaccination. Vaccines were administered twice via Gene Gun, three weeks apart and then blood was collected three weeks following the final vaccination. Vaccination with shellfish allergen DNA led to increased shrimp-specific IgG in all three strains, with the highest production in C3H/HeJ from the vaccine alone, whereas the vaccine with IL-12 led to the highest IgG production in BALB/cJ and CC027/GeniUnc mice. Similar IgG production was also induced against lobster and crab allergens. For walnut/pecan vaccines, BALB/cJ and C3H/HeJ mice produced significantly higher walnut- and pecan-specific IgG with the vaccine alone compared to the vaccine with IL-12, while the CC027 mice made significantly higher IgG with the addition of IL-12. Notably, intramuscular administration of the vaccines did not lead to increased antigen-specific IgG production, indicating that Gene Gun administration is a superior delivery modality. Overall, these data demonstrate the utility of DNA vaccines against two lifelong food allergies, shellfish and tree nuts, suggesting their potential as a food allergy therapy in the future.

Bacterial spores as particulate carriers for gene gun delivery of plasmid DNA

Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.

RNA interference-mediated silencing of Foxo3 in antigen-presenting cells as a strategy for the enhancement of DNA vaccine potency

The transcription factor Forkhead box O3 (Foxo3) has a critical role in suppressing the expansion of antigen-specific effector T-cell populations; hence, Foxo3 is a potential target for enhancing the antitumor immunity of cancer vaccines. In this report, we evaluated the potential of RNA interference (RNAi)-mediated silencing of Foxo3 in antigen-presenting cells as an adjuvant for HER2/neu DNA cancer vaccines. Bicistronic plasmids expressing the N-terminal extracellular domain of human HER-2/neu and the Foxo3 short hairpin RNA (hN'-neu-Foxo3 shRNA) or the scrambled control (hN'-neu-scramble shRNA) were subcutaneously injected into mice by gene gun administration to elicit antitumor immunity against p185neu-overexpressing MBT-2 bladder tumor cells. We found that mice treated with hN'-neu-Foxo3 shRNA showed greater reductions in tumor growth and longer survival times than mice treated with hN'-neu-scramble shRNA, indicating that the silencing of Foxo3 enhanced the antitumor efficacy of the HER-2/neu cancer vaccine. Cytotoxicity analyses further revealed that the Foxo3 shRNA-enhanced antitumor effect was associated with significant increases in the number of functional CD8(+) T cells and in the levels of cytotoxic T lymphocytes activity. Interleukin-6 was induced by hN'-neu-Foxo3 shRNA treatment but did not have a critical role in the antitumor effect of the hN'-neu-Foxo3 shRNA vaccine. Moreover, in vivo lymphocyte depletion analyses confirmed that the antitumor efficacy of the hN'-neu-Foxo3 shRNA vaccine depended on functional CD8(+) T cells. Finally, Foxo3 suppression was shown to markedly improve the effect of the HER-2/neu DNA vaccine in limiting the growth and lung metastases of MBT-2 cells. Overall, these results support RNAi-mediated silencing of Foxo3 as an effective strategy to enhance the therapeutic antitumor effect of HER-2/neu DNA vaccines against p185neu-positive tumors.

Gene gun administration of therapeutic HPV DNA vaccination restores the efficacy of prolonged defrosted viral based vaccine

Freshly defrosted vaccines generate promising antitumor immunity by raising both robust CD8 and CD4 responses with a TC1/Th1-dominant cytokine profile. However, prolonged (overnight) defrosted Sindbis virus-E7/HSP70 priming and Vaccinia-E7/HSP70 booster in mouse model only elicited 20% long-term tumor-free survival in comparison with the fresh vaccines. The present study is to search the possible cause of its potency loss, and to evaluate the ability of pcDNA-E7/HSP70 DNA vaccination via gene gun in restoring the efficacy of E7-specific immune responses and antitumor properties. We used prolonged defrosted SINrep5-E7/HSP70 prime and defrostedVac-E7/HSP70 boost subcutaneously, and administered intradermally cluster (3-day interval) gene gun plasmid E7–HSP70DNA vaccine twice, and evaluated its ability to generate antigen-specific cytotoxic CD8+ T-cell responses using flow cytometry as well as antitumor responses using animal positron-emission tomography (PET) imaging. The prolonged defrosted vaccines showed a significant reduction in the infectivity and a significant decrease of CD8+ and CD4+ T-cells immune responses. Administration of cluster gene gun plasmid E7–HSP70DNA twice was also found to lead to restoration of immunity that elicits a full recovery of the antitumor efficacy of the prolonged defrosted vaccines. Our study suggested that adding cluster gene gun plasmid E7–HSP70DNA vaccine twice offered a simple solution in restoring the efficacy of the prime-boost vaccination with viral vectors and has potentially significant clinical applications.

The effects of DNA formulation and administration route on cancer therapeutic efficacy with xenogenic EGFR DNA vaccine in a lung cancer animal model

BackgroundTyrosine kinase inhibitor gefitinib is effective against lung cancer cells carrying mutant epidermal growth factor receptor (EGFR); however, it is not effective against lung cancer carrying normal EGFR. The breaking of immune tolerance against self epidermal growth factor receptor with active immunization may be a useful approach for the treatment of EGFR-positive lung tumors. Xenogeneic EGFR gene was demonstrated to induce antigen-specific immune response against EGFR-expressing tumor with intramuscular administration.MethodsIn order to enhance the therapeutic effect of xenogeneic EGFR DNA vaccine, the efficacy of altering routes of administration and formulation of plasmid DNA was evaluated on the mouse lung tumor (LL2) naturally overexpressing endogenous EGFR in C57B6 mice. Three different combination forms were studied, including (1) intramuscular administration of non-coating DNA vaccine, (2) gene gun administration of DNA vaccine coated on gold particles, and (3) gene gun administration of non-coating DNA vaccine. LL2-tumor bearing C57B6 mice were immunized four times at weekly intervals with EGFR DNA vaccine.ResultsThe results indicated that gene gun administration of non-coating xenogenic EGFR DNA vaccine generated the strongest cytotoxicty T lymphocyte activity and best antitumor effects. CD8(+) T cells were essential for anti-tumor immunityas indicated by depletion of lymphocytes in vivo.ConclusionThus, our data demonstrate that administration of non-coating xenogenic EGFR DNA vaccine by gene gun may be the preferred method for treating EGFR-positive lung tumor in the future.

RNA Interference-MediatedIn VivoSilencing of Fas Ligand as a Strategy for the Enhancement of DNA Vaccine Potency

Intradermal administration of DNA vaccines encoding luciferase represents a convenient method to assess gene expression in vivo. Gene silencing by intradermal gene gun administration of DNA encoding short hairpin RNA (shRNA) may represent an effective technique for the specific knockdown of gene expression in vivo. In the current study, we characterized luciferase gene expression over time in vivo by noninvasive bioluminescence imaging. Furthermore, we characterized in vivo luciferase gene silencing with DNA encoding shRNA targeting luciferase. We also characterized human papillomavirus type 16 (HPV-16) E7-specific CD8(+) T cell immune responses in mice immunized with E7 DNA and DNA encoding shRNA targeting Fas ligand (FasL), a key proapoptotic signaling protein. Our results indicated that coadministration of DNA encoding shRNA targeting luciferase significantly reduced luciferase expression in mice intradermally administered luciferase DNA. Furthermore, we observed that mice vaccinated with E7-expressing DNA coadministered with DNA encoding shRNA targeting FasL generated significantly enhanced E7-specific CD8(+) cytotoxic T cell responses as well as potent therapeutic antitumor effects against E7-expressing tumors. Thus, intradermal administration of DNA encoding shRNA represents a plausible approach to silence genes in vivo and a potentially useful tool to enhance DNA vaccine potency.

Enhancement of DNA Vaccine Potency through Coadministration of CIITA DNA with DNA Vaccines via Gene Gun

Administration of DNA vaccines via gene gun has emerged as an important form of Ag-specific immunotherapy. The MHC CIITA is a master regulator of MHC class II expression and also induces expression of class I molecules. We reasoned that the gene gun administration of CIITA DNA with DNA vaccines employing different strategies to improve MHC I and II processing could enhance DNA vaccine potency. We observed that DC-1 cells transfected with CIITA DNA lead to higher expression of MHC I and II molecules, leading to enhanced Ag presentation through the MHC I/II pathways. Furthermore, our data suggested that coadministration of DNA-encoding calreticulin (CRT) linked to human papillomavirus (HPV) 16 E6 Ag (CRT/E6) with CIITA DNA leads to enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. In addition, coadministration of the combination of CRT/E6 DNA with CIITA DNA and DNA encoding the invariant chain (Ii) linked to the pan HLA-DR-reactive epitope (Ii-PADRE) further enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. Treatment with the combination vaccine was also shown to enhance the antitumor effects and to prolong survival in TC-1 tumor-bearing mice. Vaccination with the combination vaccine also led to enhanced E6-specific CD8(+) memory T cells and to long-term protection against TC-1 tumors and prolonged survival in vaccinated mice. Thus, our findings suggest that the combination of CIITA DNA with CRT/E6 and Ii-PADRE DNA vaccines represents a potentially effective means to combat tumors in the clinical setting.

PcDNA-IL-12 vaccination blocks eosinophilic inflammation but not airway hyperresponsiveness following murine Toxocara canis infection

We have investigated the effect of pcDNA3-CpG and pcDNA-IL-12, delivered by intradermal gene gun administration, on the blood/lung eosinophilia, airway hyperresponsiveness as well as the immune response in a murine model of toxocariasis. Our results demonstrated that pcDNA-IL-12 but not pcDNA3-CpG vaccination led to a persistent lower blood/bronchoalveolar eosinophilia following Toxocara canis infection, as pcDNA3-CpG led only to an early transient blockage of eosinophil transmigration into bronchoalveolar fluid following T. canis infection. Prominent Type-1 immune response was pointed out as the hallmark of T. canis infection following pcDNA-IL-12 vaccination. Outstanding IFN-gamma/IL-4 ratio besides low levels of IgG1 with subsequent high IgG2a/IgG1 ratio further characterized a Type-1 polarized immunological profile in pcDNA-IL-12-vaccinated animals. Nevertheless, only pcDNA3-CpG was able to prevent airway hyperresponsiveness induced by T. canis infection. The persistent airway hyperresponsiveness observed in pcDNA-IL-12-vaccinated animals demonstrated that the airway constriction involved other immunological mediator than those blocked by pcDNA-IL-12. Together, these data indicated that pcDNA-IL-12 and pcDNA3-CpG vaccines have distinct therapeutic benefits regarding the eosinophilic inflammation/airway hyperresponsiveness triggered by T. canis infection, suggesting their possible use in further combined therapeutic interventions.

DNA Vaccines Encoding Ii-PADRE Generates Potent PADRE-specific CD4+ T-Cell Immune Responses and Enhances Vaccine Potency

DNA Vaccines Encoding Ii-PADRE Generates Potent PADRE-specific CD4+ T-Cell Immune Responses and Enhances Vaccine Potency

Gene-Gun Biolistic Immunization Encoding Glutamic Acid Decarboxylase: A Model for Studying Langerhans Cell Abnormalities and Mimicry in the Nonobese Diabetic Mouse

Plasmid-DNA gene-gun immunization may be an efficient approach for investigating the role of skin dendritic cells (DCs) in type 1 diabetes (T1D) pathogenesis and the significance of the presentation of peptides that mimic autoantigenic epitopes in aggravating or modulating the autoimmune reaction. Gene-gun immunization has been described as producing long-lasting immune responses elicited by skin DCs, especially Langerhans cells (LCs). Therefore, we tested the immune response and diabetes modulation in nonobese diabetic (NOD) mice and in control BALB/c mice, by gene-gun administration of plasmid-DNA encoding (1) human 65 kDa glutamic acid decarboxylase (hGAD65) mimicking the crucial mouse autoantigen GAD65 (similarity of 95.7%) or (2) beta-galactosidase (betaGAL) as a negative control. Expression of GAD and betaGAL in skin of pc-GAD- and pc-LacZ-injected mice, respectively, was confirmed. It was surprising that both pc-LacZ-injected BALB/c and NOD mice exhibited a betaGAL-specific Th1 immune response: spleen cells of pc-LacZ mice proliferated specifically to betaGAL (P < 10(-4)) and secreted significant amounts of IFNgamma (P < 10(-4)). pc-LacZ mice also developed a betaGAL-specific Th1-related (IgG2a/2c) and Th2-related (IgG1) humoral response. Although pc-GAD BALB/c mice showed Th2-related GAD-specific IgG1 production and a significant secretion of IL4 (P < .03), pc-GAD NOD mice did not generate either an antibody response or a T cell response specific to GAD. Moreover, gene-gun immunization encoding hGAD65 did not clearly modulate diabetes onset in NOD mice. This absence of detectable GAD-specific response may implicate skin DC deficiencies in NOD mice. The gene-gun technique could thus provide an interesting model for studying skin DC abnormalities in NOD mice and their potential implication of presenting mimetic peptides that modulate the autoimmune response in T1D.

Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA

Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein ( pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1–4 and 9–13, then erratically thereafter for up to 2 months. Phenotypic analysis showed that EGFP+ ALDC expressed MHC class II, WC6, CD1b, and SIRPα markers. Plasmid, detected by PCR, was found in lymph cells and cell-free plasma on a daily basis, and was present variably for up to 2 months. Plasmid was also detected in purified CD1b+ ALDC, but the presence of plasmid did not correlate with EGFP expression by ALDC. Free EGFP in afferent lymph plasma was detectable by luminometry only after three administrations of the plasmid. The results show that gene gun administered pEGFP persisted for extended periods after a single administration, leeching out of skin on a daily basis. The plasmid was associated with both the cellular and fluid components of afferent lymph. EGFP protein appeared in afferent lymph in a pulsatile manner, but associated only with ALDC.

Protection against plague afforded by immunisation with DNA vaccines optimised for expression of the Yersinia pestis V antigen

DNA vaccine vectors were produced which were optimised for expression of the Yersinia pestis V antigen in the BALB/ c mouse model. Six different eukaryotic promoters were compared, resulting in the selection of the CMV promoter with an additional translational enhancer downstream. Surprisingly, alteration of the codon usage of the lcrV gene encoding V antigen for expression in murine cells was not found to improve the antibody responses generated against V antigen. The DNA vaccine was subsequently evaluated in its delivery via intramuscular injection compared to gene-gun administration. Gene-gun delivery was found to induce significantly higher V antigen-specific antibody responses and also afforded the highest level of protection against Y. pestis challenge. In addition, the protection achieved could be increased by using a ‘prime and boost’ strategy, administering the DNA vaccine followed by recombinant V antigen. These results show promise for a DNA vaccine against plague.

A glycosylphosphatidylinositol anchor signal sequence enhances the immunogenicity of a DNA vaccine encoding Plasmodium falciparum sexual-stage antigen, Pfs230

Mammalian expression vectors encoding region C of malaria transmission-blocking vaccine candidate Pfs230 (aa 443–1132) with and without a 3′ glycosylphosphatidylinositol (GPI) anchor signal sequence were tested for their immunogenicity in mice. The plasmid containing the GPI anchor signal sequence consistently induced higher titers of anti-Pfs230 antibodies using three delivery systems: intramuscular (i.m.), intradermal (i.d.), and gene gun (g.g.). In contrast, the isotype profile elicited varied depending on the delivery system and was not effected by the presence of the GPI anchor sequence. Both gene gun and intradermal administration induced primarily an IgG1 response, while intramuscular injection induced both IgG1 and IgG2a antibodies. Regardless of the mode of delivery, all the plasmids encoding Pfs230 region C primed for a mixed IgG1/IgG2a response to an intraperitoneal (i.p.) injection of E. coli-produced recombinant Pfs230 region C. None of these vaccination strategies were more effective than r230/MBP.C alone in generating malaria transmission-blocking immunity.

Immunogenicity of a Japanese encephalitis DNA vaccine candidate in cynomolgus monkeys

A Japanese encephalitis (JE) vaccine candidate encoding JE virus premembrane (prM) and envelope (E) genes, designated pNJEME, was evaluated for safety and immunogenicity in non-human primate, cynomolgus monkeys. pNJEME was constructed using a vector (pNGVL4a) designed to address some of the safety concerns of DNA vaccine. In two different experiments, two immunizations with 300 μg of pNJEME by intramuscular (i.m.) injection, and 3 μg of pNJEME using a gene gun, and three immunizations by i.m. injection with 500 μg of pNJEME were performed. All the three protocols induced low to high levels of neutralizing antibody, indicating an ability of pNJEME to induce neutralizing antibody in monkeys with a wide individual variation in response to pNJEME. In one experiment designed to compare the DNA vaccine with a commercial inactivated JE vaccine, three immunizations by i.m. inoculation with 300 μg of pNJEME or by gene gun administration with 3 μg of pNJEME induced similar levels of neutralizing antibody to those induced by three immunizations with a human dose of the inactivated vaccine in most monkeys. After intranasal challenge with the Beijing P3 or JaTH160 strain of JE virus, pNJEME-immunized monkeys showed anamnestic neutralizing antibody responses, indicating that pNJEME induced memory B cells which were responsive to infection with JE virus. No systemic and local reactions were observed in any monkeys after i.m. or gene gun inoculations with plasmid DNAs.

Gene-based vaccines.

The development of vaccines is one of the best examples of the impact of biomedical advances upon human health. But, despite the successes, such as the elimination of smallpox from the planet, there remain a number of diseases for which we have no vaccines, and which infect and often kill millions of people each year (see Table 1). These diseases include those due to newly emerging pathogens such as HIV, as well as ancient scourges such as tuberculosis, where, although there is a vaccine, a better one is needed. A look at the new technologies being used to develop vaccines will be presented here.

DNA vaccination against influenza viruses: a review with emphasis on equine and swine influenza

The influenza virus vaccines that are commercially-available for humans, horses and pigs in the United States are inactivated, whole-virus or subunit vaccines. While these vaccines may decrease the incidence and severity of clinical disease, they do not consistently provide complete protection from virus infection. DNA vaccines are a novel alternative to conventional vaccination strategies, and offer many of the potential benefits of live virus vaccines without their risks. In particular, because immunogens are synthesized de novo within DNA transfected cells, antigen can be presented by MHC class I and II molecules, resulting in stimulation of both humoral and cellular immune responses. Influenza virus has been used extensively as a model pathogen in DNA vaccine studies in mice, chickens, ferrets, pigs, horses and non-human primates, and clinical trials of DNA-based influenza virus vaccines are underway in humans. Our studies have focused on gene gun delivery of DNA vaccines against equine and swine influenza viruses in mice, ponies and pigs, including studies employing co-administration of interleukin-6 DNA as an approach for modulating and adjuvanting influenza virus hemagglutinin-specific immune responses. The results indicate that gene gun administration of plasmids encoding hemagglutinin genes from influenza viruses is an effective method for priming and/or inducing virus-specific immune responses, and for providing partial to complete protection from challenge infection in mice, horses and pigs. In addition, studies of interleukin-6 DNA co-administration in mice clearly demonstrate the potential for this approach to enhance vaccine efficacy and protection.

Induction of anti-tumor immunity by intrasplenic administration of a carcinoembryonic antigen DNA vaccine.

We have previously reported that intramuscular, intradermal or epidermal gene gun administration of a plasmid encoding carcinoembryonic antigen (CEA) under transcriptional regulatory control of the cytomegalovirus (CMV) early promoter/enhancer elicits CEA-specific humoral and cellular immune responses in mice with resultant immunoprotection against challenge with syngeneic, CEA-expressing colon adenocarcinoma cells. In the present work, we examine the ability of this DNA vaccine construct (pCEA) to elicit CEA-specific immunity following intrasplenic administration. Groups of mice were immunized with pCEA by intrasplenic or intramuscular injection. Six weeks later, mice were evaluated for the presence of anti-CEA humoral responses and were challenged with syngeneic, CEA-expressing colon carcinoma cells. Intrasplenic administration of pCEA produced a frequency of CEA-specific antibody responses comparable to that elicited by intramuscular pCEA inoculation. Both intrasplenic and intramuscular administration of pCEA generated IgG2a antibody responses to CEA, consistent with the induction of T helper-1-biased immune responses. In addition, partial immunoprotection against tumor challenge was observed after a single plasmid DNA dose by either route of administration. Subsequent studies revealed that antibody responses to intrasplenic DNA vaccination are dose and schedule dependent. These findings support future investigations of DNA vaccination strategies that specifically promote the uptake of plasmid by splenocytes.

Effective DNA Vaccination Against Listeriosis by Prime/Boost Inoculation with the Gene Gun

Protective immunity against Listeria monocytogenes strongly depends on CD8+ T lymphocytes, and both IFN-gamma secretion and target cell killing are considered relevant to protection. We analyzed whether we could induce a protective type 1 immune response by DNA vaccination with the gene gun using plasmids encoding for two immunodominant listerial Ags, listeriolysin and p60. To induce a Th1 response, we 1) coprecipitated a plasmid encoding for GM-CSF, 2) employed a prime/boost vaccination schedule with a 45-day interval, and 3) coinjected oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. DNA immunization of BALB/c mice with plasmids encoding for listeriolysin (pChly) and p60 (pCiap) efficiently induced MHC class I-restricted, Ag-specific CD8+ T cells that produced IFN-gamma. Coinjection of CpG-ODN significantly increased the frequency of specific IFN-gamma-secreting T cells. Although pChly induced specific CD8+ T cells expressing CTL activity, it failed to stimulate CD4+ T cells. Only pCiap induced significant CD4+ T cell and humoral responses, which were predominantly of Th2 type. Vaccination with either plasmid induced protective immunity against listerial challenge, and coinjection of CpG ODN improved vaccine efficacy in some situations. This study demonstrates the feasibility of gene gun administration of plasmid DNA for inducing immunity against an intracellular pathogen for which protection primarily depends on type 1 CD8+ T cells.

Safe and effective regulation of hematocrit by gene gun administration of an erythropoietin-encoding DNA plasmid.

This work examines the effect of delivering a DNA plasmid encoding murine erythropoietin (pVRmEpo) to BALB/c mice by gene gun. Whereas intramuscular injection elicits a rise in hematocrit persisting >8 months, intradermal delivery triggers the dose-dependent secretion of biologically active erythropoietin (Epo) for approximately 1 month. Repeated administration of pVRmEpo by gene gun elicits a stable increase in hematocrit. The source of the Epo produced following gene gun delivery was analyzed by periodically grafting the site of injection onto naive recipients. Results indicate that both stationary cells (presumably keratinocytes) and migratory (presumably dendritic) cells were transfected and secreted biologically active Epo in vivo. Gene gun administration of plasmid DNA appears to be safe, and provides an additional strategy for achieving the regulated secretion of an exogenous gene product.

Biological features of genetic immunization

Genetic immunization (a.k.a. DNA-based immunization) shows promise at least as a convenient method to test and discover new vaccines and may be an efficient vaccine delivery system. However, relatively little is known about the parameters affecting its effectiveness, let alone its basic underlying biological mechanisms. Here we report on investigations of some of the factors that determine the quantity and quality of the immune response with genetic immunization. We find that for non-toxic proteins the antibody response correlates well with the level of expression as does the cellular response to a certain level. The augmentation of the immune response by co-introduction of a cytokine gene as a genetic adjuvant is also responsive to the expression level of the antigen. The immune response is inversely correlated to the age of the mice and at least part of this effect is through level of expression of the antigen. Gene gun administration of the transgene to the skin has the advantage over muscle injection in that ca 100-fold less DNA is required for the same level of expression and the injections are more reproducible in effect. Finally, the apparent differences in Th2 (gun) vs Th1 (muscle) responses between the two modes can at least partly be accounted for by differences in the amount of plasmid DNA typically administered.