DNA Vaccine Delivery Research Articles

Low Current-driven Micro-electroporation Allows Efficient In Vivo Delivery of Nonviral DNA into the Adult Mouse Brain

Viral gene transfer or transgenic animals are commonly used technologies to alter gene expression in the adult brain, although these approaches lack spatial specificity and are time consuming. We delivered plasmid DNA locally into the brain of adult C57BL/6 mice in vivo by voltage- and current-controlled electroporation. The low current-controlled delivery of unipolar square wave pulses of 125 µA with microstimulation electrodes at the injection site gave 16 times higher transfection rates than a voltage-controlled electroporation protocol with plate electrodes resulting in currents of about 400 mA. Transfection was restricted to the target region and no damage due to the electric pulses was found. Our current-controlled electroporation protocol indicated that the use of very low currents resulting in applied voltages within the physiological range of the membrane potential, allows efficient transfection of nonviral plasmid DNA. In conclusion, low current-controlled electroporation is an excellent approach for electroporation in the adult brain, i.e., gene function can be influenced locally at a high level with no mortality and minimal tissue damage.

Synergistic enhancement of immunogenicity and protection in mice against Schistosoma japonicum with codon optimization and electroporation delivery of SjTPI DNA vaccines

Schistosomiasis is an endemic, zoonotic parasitic disease caused by Schistosoma japonicum that remains a public health concern and an effective vaccine is needed. Triose-phosphate isomerase from S. japonicum is a promising schistosome vaccine antigen shown to be immunogenic when delivered as a DNA vaccine, however, the previous S. japonicum triose-phosphate isomerase (SjTPI) DNA vaccine needs to be further optimized to achieve higher protection. In the current study, codon optimization of SjTPI DNA insert, combined with electroporation but not with the addition of a tPA leader or heat-shock protein in-frame with the SjTPI gene insert, enhanced Th1-type antibody and cytokine production and most significantly, achieved great than 50% reduction of infection against challenge with S. japonicum cercariae, a major milestone in S. japonicum vaccine development. Our results suggest that the combination of a codon optimized vaccine design and an efficient vaccine delivery system can greatly improve the potential of a SjTPI DNA vaccine as a viable schistosome vaccine candidate.

Electroporation for DNA immunization: clinical application

DNA immunization is an attractive technology owing to its potential to induce balanced and long-lived immune responses. However, progress into the clinic has been hampered by the relatively low magnitude of the immune response typically induced following administration in large target species, which is likely due to low transfection efficiency as well as insufficient recruitment of antigen-presenting cells to the injection site. Electroporation addresses both of these limitations by inducing transiently enhanced cell membrane permeability, thus facilitating uptake of the DNA into the host cell and creating a low level of inflammation conducive to enhanced influx of antigen-presenting cells to the injection site. Consequently, electroporation-mediated delivery of DNA vaccines results in very significant improvements in the transfection efficiency and immune responses in comparison to conventional injection. Importantly, electroporation is effective in virtually every animal model tested to date and has a favorable safety profile, which is promising for clinical application. In support of the potential for electroporation in human disease situations, early clinical results suggest that the immunogenicity of DNA vaccines is greatly improved when delivered with electroporation.

162. Translation of Electroporation-Mediated DNA Vaccine Delivery into a Phase I Clinical Trial Evaluating an HIV DNA Vaccine in Healthy Volunteers

162. Translation of Electroporation-Mediated DNA Vaccine Delivery into a Phase I Clinical Trial Evaluating an HIV DNA Vaccine in Healthy Volunteers

327. A Novel Prototype Device for Electroporation-Enhanced DNA Vaccine Delivery to Both Skin and Muscle

327. A Novel Prototype Device for Electroporation-Enhanced DNA Vaccine Delivery to Both Skin and Muscle

Delivery of DNA vaccines: an overview on the use of biodegradable polymeric and magnetic nanoparticles

Vaccination offers a cost-effective approach to the control of endemic infectious and a less invasive treatment modality against cancers. Since the discovery that injecting DNA encoding antigens (expressed in vivo) results in the induction of CD8 T cells as well as antibody mediated immunity, researchers have tried to develop methods to consistently enhance this immunity to disease protective levels in humans. Adsorption, coformulation, or encapsulation with particles has been found to both stabilize DNA formulations, through preventing rapid degradation, and provide vaccine adjuvanting effects, largely due to effective uptake of particulate materials by antigen presenting cells. Recently, it has been shown that nanoparticles, as opposed to microparticles, based DNA vaccine carriers are preferentially taken up by dendritic cells resulting in the induction of maximal levels of combined humoral and cellular immunity.

Intranasal administration of plasmid DNA-coated nanoparticles results in enhanced immune responses.

Intranasal immunization offers potential forthe elicitation of effective mucosal and systemic immune responses. In this study, a previously reported novel cationic nanoparticle engineered from a microemulsion precursor was further modified, optimized and applied intranasally to mice to explore its potential as a plasmid DNA (pDNA) vaccine delivery system. To this end, more uniform nanoparticles (around 100 nm) containing less cationic surfactant were developed. The pDNA-coated nanoparticles significantly enhanced the specific serum IgG and IgA titres to an expressed model antigen, beta-galactosidase, by 18-28 and 25-30 fold, respectively, when compared with naked pDNA alone. An enhanced splenocyte proliferative response was also observed after immunization with the pDNA-coated nanoparticles. It was concluded that these plasmid DNA-coated nanoparticles may have potential for immunization via the nasal route.

Liposomal delivery of p-ialB and p-omp25 DNA vaccines improves immunogenicity but fails to provide full protection against B. melitensis challenge

BackgroundWe have previously demonstrated protective efficacy against B. melitensis using formulations of naked DNA vaccines encoding genes ialB and omp25. The present study was undertaken to further understand the immune response generated by the protective vaccination regimens and to evaluate cationic liposome adsorption as a delivery method to improve vaccine utility.MethodsThe protective efficacy and immunogenicity of vaccines delivered as four doses of naked DNA, a single dose of naked DNA or a single dose of DNA surface adsorbed to cationic liposomes were compared using the BALB/c murine infection model of B. melitensis. Antigen-specific T cells and antibody responses were compared between the various formulations.ResultsThe four dose vaccination strategy was confirmed to be protective against B. melitensis challenge. The immune response elicited by the various vaccines was found to be dependent upon both the antigen and the delivery strategy, with the IalB antigen favouring CD4+ T cell priming and Omp25 antigen favouring CD8+. Delivery of the p-ialB construct as a lipoplex improved antibody generation in comparison to the equivalent quantity of naked DNA. Delivery of p-omp25 as a lipoplex altered the profile of responsive T cells from CD8+ to CD4+ dominated. Under these conditions neither candidate delivered by single dose naked DNA or lipoplex vaccination methods was able to produce a robust protective effect.ConclusionsDelivery of the p-omp25 and p-ialB DNA vaccine candidates as a lipoplex was able to enhance antibody production and effect CD4+ T cell priming, but was insufficient to promote protection from a single dose of either vaccine. The enhancement of immunogenicity by lipoplex delivery is a promising step toward improving the practicality of these two candidate vaccines, and suggests that this lipoplex formulation may be of value in situations where improvements to CD4+ responses are required. However, in the case of Brucella vaccine development it is suggested that further modifications to the candidate vaccines and delivery strategies will be required in order to deliver sustained protection.

Electroporation: A promising method for the nonviral delivery of DNA vaccines in humans?

The without a doubt major obstacle for making DNA vaccines a commercial success is delivery. If delivery cannot be made simple, cheap and effective, DNA vaccines may not become a viable option for human use. Numerous clinical trials have confirmed that a standard needle and syringe simply do not do the job, i.e., delivering the DNA payload inside the cell. Having recognized this shortcoming, investigators have developed several new approaches for DNA vaccine delivery. In particular, new types of delivery devices, originally intended for in vitro use, have been applied for in vivo delivery. These include particle bombardment or biolistic delivery, and in vivo electroporation (EP). Importantly, both techniques seem to overcome the size barrier, meaning that they work in both mice and larger animals. In vivo EP has the key features of improved DNA uptake, increased antigen expression and a local inflammation. These factors are essential to make DNA vaccines effective in a larger host. Early data from clinical trials with DNA vaccines delivered by in vivo EP are cautiously promising. Thus, we may be entering a new era of DNA vaccination where we start to see clinical effects in humans; however, these may also be accompanied by side effects, as the vaccines become more effective.

An electrically active microneedle array for electroporation.

We have designed and fabricated a microneedle array with electrical functionality with the final goal of electroporating skin's epidermal cells to increase their transfection by DNA vaccines. The microneedle array was made of polymethylmethacrylate (PMMA) by micromolding technology from a polydimethylsiloxane (PDMS) mold, followed by metal deposition, patterning using laser ablation, and electrodeposition. This microneedle array possessed sufficient mechanical strength to penetrate human skin in vivo and was also able to electroporate both red blood cells and human prostate cancer cells as an in vitro model to demonstrate cell membrane permeabilization. A computational model to predict the effective volume for electroporation with respect to applied voltages was constructed from finite element simulation. This study demonstrates the mechanical and electrical functionalities of the first MEMS-fabricated microneedle array for electroporation, designed for DNA vaccine delivery.

Persistent Immune Responses Induced by a Human Immunodeficiency Virus DNA Vaccine Delivered in Association with Electroporation in the Skin of Nonhuman Primates

Strategies to improve vaccine efficacy are still required, especially in the case of chronic infections, including human immunodeficiency virus (HIV). DNA vaccines have potential advantages over conventional vaccines; however, low immunological efficacy has been demonstrated in many experiments involving large animals and in clinical trials. To improve the immunogenicity of DNA vaccines, we have designed a plasmid vector exploiting the binding capacity of the bovine papillomavirus E2 protein and we have used electroporation (EP) to increase DNA uptake after intradermal inoculation. We demonstrated, in nonhuman primates (NHPs), efficient induction of anti-HIV immunity with an improved DNA vaccine vector encoding an artificial fusion protein, consisting of several proteins and selected epitopes from HIV-1. We show that a DNA vaccine delivery method combining intradermal injection and noninvasive EP dramatically increased expression of the vaccine antigen selectively in the epidermis, and our observations strongly suggest the involvement of Langerhans cells in the strength and quality of the anti-HIV immune response. Although the humoral responses to the vaccine were transient, the cellular responses were exceptionally robust and persisted, at high levels, more than 2 years after the last vaccine boost. The immune responses were characterized by the induction of significant proportions of T cells producing both interferon-gamma and interleukin-2 cytokines, in both subpopulations, CD4(+) and CD8(+). This strategy is an attractive approach for vaccination in humans because of its high efficacy and the possible use of newly developed devices for EP.

Well-defined block copolymers for gene delivery to dendritic cells: Probing the effect of polycation chain-length

The development of safe and efficient polymer carriers for DNA vaccine delivery requires mechanistic understanding of structure–function relationship of the polymer carriers and their interaction with antigen-presenting cells. Here we have synthesized a series of diblock copolymers with well-defined chain-length using atom transfer radical polymerization and characterized the influence of polycation chain-length on the physico-chemical properties of the polymer/DNA complexes as well as the interaction with dendritic cells. The copolymers consist of a hydrophilic poly(ethylene glycol) block and a cationic poly(aminoethyl methacrylate) (PAEM) block. The average degree of polymerization (DP) of the PAEM block was varied among 19, 39, and 75, with nearly uniform distribution. With increasing PAEM chain-length, polyplexes formed by the diblock copolymers and plasmid DNA had smaller average particle size and showed higher stability against electrostatic destabilization by salt and heparin. The polymers were not toxic to mouse dendritic cells (DCs) and only displayed chain-length-dependent toxicity at a high concentration (1 mg/mL). In vitro gene transfection efficiency and polyplex uptake in DCs were also found to correlate with chain-length of the PAEM block with the longer polymer chain favoring transfection and cellular uptake. The polyplexes induced a modest up-regulation of surface markers for DC maturation that was not significantly dependent on PAEM chain-length. Finally, the polyplex prepared from the longest PAEM block (DP of 75) achieved an average of 20% enhancement over non-condensed anionic dextran in terms of uptake by DCs in the draining lymph nodes 24 h after subcutaneous injection into mice. Insights gained from studying such structurally well-defined polymer carriers and their interaction with dendritic cells may contribute to improved design of practically useful DNA vaccine delivery systems.

P14-12. Safety and tolerability of VRC DNA and rAd5 HIV-1 vaccine delivery by intramuscular, subcutaneous and intradermal administration in healthy adults

Methods 60 subjects (18–50 yrs) were randomized to 6 schedules. Group 1 (n = 30) received 3 DNA prime vaccinations (Wks 0,4,8) and Group 2 (n = 30) received a recombinant adenoviral vector (rAd5) prime vaccination (Wk 0). Both groups received rAd5 booster at Wk 24. Subjects were equally randomized to receive prime injections by IM, SC and ID routes. DNA dosage was 4 mg for IM and SC; 0.4 mg for ID injections. rAd5 dosage was 1010 PU by all routes. Half of the subjects in each group had pre-existing Ad5 neutralizing antibody.

P19-29. Determining the optimal protocol for plasmid DNA vaccine delivery by intradermal in vivo electroporation

Methods Mice were immunized with a plasmid encoding HIV-1 gag p37, either intradermally (id) with the DermaVax EP device, or intramuscularly (im) without EP. The different parameters explored were; the number of immunizations, the interval between immunizations, and the choice of priming (im or id). A novel Fluorospot assay was used to evaluate the vaccine specific cellular immune responses. This assay allows for detection of vaccine-specific cells that secrete IFN-γ, IL-2 and cells that simultaneously secrete both cytokines. In order to verify the Fluorospot results, conventional ELISpot assay was used. The humoral vaccine-specific response was evaluated by ELISA.

P17-11. HIV DNA vaccine delivery in association with electroporation in the skin of nonhuman primates

Background We recently demonstrated that intradermal (ID) injection with electroporation (EP) of a new HIV DNA vaccine (auxo-GTU®MultiHIV) induced particularly strong and very long lasting specific T-cell responses (1044 ± 400 SFC per million PBMCs 3 years after vaccination) in macaques. Therefore we get interested to characterize the localization and expression of the auxo-GTU®MultiHIV vaccine as well as local involvement of immune cells at the sites of injection and in draining lymph nodes (LN).

Rabies DNA vaccine encoding lysosome‐targeted glycoprotein supplemented with Emulsigen‐D confers complete protection in preexposure and postexposure studies in BALB/c mice

The worldwide incidence of rabies and the inability of currently used vaccination strategies to provide highly potent and cost-effective therapy indicate the need for an improved rabies vaccine. Thus, DNA vaccine based on lysosome-targeted glycoprotein of the rabies virus was evaluated in BALB/c mice. It imparted partial protection (60%) against challenge with 20 LD(50) of the challenge virus standard (CVS) strain of rabies virus. To improve the outcome of vaccination, to ultimately enhance the immune response, we investigated different routes for DNA vaccine delivery, varied doses of DNA, and the influence of adjuvant supplementation. The highest immune response pertaining to IgG antibody titer, with a predominantly IgG1/IgG2a subclass distribution, effective cellular immunity, and a high level of rabies virus neutralizing antibodies (RVNAs) was attained by the optimized DNA vaccine formulation comprising intramuscular administration of 100 microg of DNA vaccine supplemented with Emulsigen-D. In preexposure prophylaxis, a 3-dose regimen of this formulation generated a high RVNA titer (32 IU/ml) and conferred complete protection against challenge with 20 LD(50) of CVS. For postexposure efficacy analysis, rabies was experimentally induced with 50 LD(50) of CVS. Subsequent therapy with 5 doses of the formulation completely prevented rabies in BALB/c mice, which maintained protective RVNA titers of 4 IU/ml. The World Health Organization recommended rabies protective titer threshold is 0.5 IU/ml. Thus, this optimized DNA vaccine formulation provides an avenue for preventing and controlling rabies.

Optimization of Skin Electroporation in Mice to Increase Tolerability of DNA Vaccine Delivery to Patients

Electroporation has, during the last years, proven to be a very successful delivery method for DNA vaccines and has now reached clinical evaluation. Although intramuscular electroporation is practical in animal models, intradermal electroporation might be more suitable for clinical administration. Skin is the most accessible organ of the body and has professional antigen-presenting cells in large amounts; thus, skin is an ideal target for DNA vaccine delivery. Moreover, intradermal electroporation has clear clinical benefits such as improved safety and tolerability. This article describes improvements for effective and tolerable DNA delivery to skin. The time of pulse delivery has been shortened by 90% and even pulse programs of 240-ms total duration generate robust immune responses. We show that a single vaccination using an optimized gene delivery generates (i) high and consistent protein expression in vivo, (ii) cytotoxic antigen-specific T cells expressing both IFNgamma and CD107a (lysosomal-associated membrane protein 1). Furthermore, application of a topical anesthetic cream prior to vaccination does not affect the number or function of the antigen-specific T cells induced. This suggests that local anesthesia can be used to further decrease the sensation of pulse delivery in patients.

Prospects for developing an effective particle-mediated DNA vaccine against influenza

Vaccine strategies capable of conferring broad protection against both seasonal and pandemic strains of influenza are urgently needed. DNA vaccines are an attractive choice owing to their capacity to induce robust humoral and cellular immune responses at low doses and because they can be developed and manufactured rapidly to more effectively meet the threat of an influenza epidemic or pandemic. Particle-mediated epidermal delivery (PMED), or the gene gun, is a DNA vaccine delivery technology shown to induce protective levels of antibody and T-cell responses in animals and humans against a wide variety of diseases, including influenza. This review focuses on current advances toward the development of an effective PMED DNA vaccine against influenza, including strategies to enhance vaccine immunogenicity, the potential for PMED-based DNA vaccines to improve protection in the vulnerable elderly population, and the prospects for a vaccine capable of providing cross-protection against both seasonal and pandemic strains of influenza.

Salmonella Vaccines for Animals and Birds and Their Future Perspective

Vaccines are the most powerful biologicals which have modulated the economic, social and cultural life of human beings. Certain diseases have haunted humanity for centuries but are now extinct due to vaccines. On the other hand, some diseases such as salmonellosis, that were uncontrollable in the past, still cause pandemics today. There are more than 2500 serovars of Salmonella and vaccines made from any one serovar do not confer cross-protection against another, no matter how much antigenic similarity there is between the two. Salmonella strains are able to cause disease and to adapt to different types of animals whilst still maintaining their zoonotic and interspecies transfer potential. Three major types of vaccines are being used to control salmonellosis: killed bacteria, subunit vaccines and live attenuated vaccines. Effective vaccines against some host adapted and common serovars have been developed but their use has led to the emergence of other serovars. The problem has become more complex because increased international trade and travel has helped Salmonella strains to cross continental boundaries. It seems unlikely that we will be able to develop an effective Salmonella vaccine in the near future that is able to control all forms of salmonellosis, even in a single animal species. Recent advances in Salmonella vaccines will be reviewed including the use of Salmonella as vector for delivery of multivalent DNA and recombinant vaccines for controlling salmonellosis and other infectious diseases as well as for the control of cancer.

Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery

DNA vaccines are an attractive approach to eliciting antigen-specific immunity. Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC class I pathway and increase cytotoxic CD8+ T cell production. However, even with these enhancements, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration. Electroporation and gene gun-mediated particle delivery are leading methods of DNA vaccine delivery that can generate protective and therapeutic levels of immune responses in experimental models. In this study, we perform a head-to-head comparison of three methods of vaccination – conventional intramuscular injection, electroporation-mediated intramuscular delivery, and epidermal gene gun-mediated particle delivery – in the ability to generate antigen-specific cytotoxic CD8+ T cell responses as well as anti-tumor immune responses against an HPV-16 E7 expressing tumor cell line using the pNGVL4a-CRT/E7(detox) DNA vaccine. Vaccination via electroporation generated the highest number of E7-specific cytotoxic CD8+ T cells, which correlated to improved outcomes in the treatment of growing tumors. In addition, we demonstrate that electroporation results in significantly higher levels of circulating protein compared to gene gun or intramuscular vaccination, which likely enhances calreticulin's role as a local tumor anti-angiogenesis agent. We conclude that electroporation is a promising method for delivery of HPV DNA vaccines and should be considered for DNA vaccine delivery in human clinical trials.