Application Of DNA Vaccines Research Articles

Immunogenicity of porcine circovirus type 2 capsid protein targeting to different subcellular compartments

Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging disease in swine. The development of effective vaccines against PCV2 infection has been accepted as an important strategy in the prophylaxis of PMWS, and a DNA vaccine expressing the major immunogenic capsid (Cap) protein of PCV2 is considered to be a promising candidate. However, recent studies have revealed that interferons (IFNs), especially IFN-γ, can enhance the replication of PCV2, indicating that the high levels of IFN-γ induced by DNA vaccination seem to have potential deleterious effect on protective immunity. Strategies to improve the neutralizing antibody response and simultaneously decrease the IFN-γ response will facilitate the clinical application of DNA vaccines against PCV2. In the present study, four different DNA vaccine constructs encoding cytoplasmic (Cy-ORF2), secreted (Sc-ORF2), membrane-anchored (M-ORF2) or authentic nuclear-targeted (pc-ORF2) Cap protein were generated to evaluate the neutralizing antibody and IFN-γ responses in a mouse model. Although all four DNA constructs could elicit PCV2-specific humoral immune responses, mice inoculated with Sc-ORF2 developed a significantly higher level of neutralizing antibodies than those that received M-ORF2, pc-ORF2 or Cy-ORF2. Furthermore, mice immunized with Sc-ORF2 or M-ORF2 showed a significantly decreased or enhanced IFN-γ level, respectively, compared with those inoculated with pc-ORF2. With respect to neutralizing antibody and IFN-γ levels, Sc-ORF2 is a good candidate for DNA vaccination, and the secreted Cap protein appears to be an ideal antigen for use in development of vaccines against PCV2.

Vaccination with a DNA vaccine based on human PSCA and HSP70 adjuvant enhances the antigen‐specific CD8+ T‐cell response and inhibits the PSCA+ tumors growth in mice

DNA vaccines have been shown to be an effective approach to induce antigen-specific cellular and humoral immunity. However, the lower immune intensity in clinical trials limits the application of DNA vaccine. Here we intend to develop a new DNA vaccine based on prostate stem-cell antigen (PSCA), which has been suggested as a potential target for prostate cancer therapy, and enhance the DNA vaccine potency with heat shock proteins (HSPs) as adjuvant. A series of DNA plasmids encoding human PSCA, human HSP70 and their conjugates was constructed and injected into male mice intramuscularly (i.m.). To evaluate the immune responses and therapeutic efficacy of these plasmids, major histocompatibility complex (MHC)-restricted PSCA and HSP70-specific epitopes were predicted and a mouse model with a human PSCA-expressing tumor was constructed. The result showed that mice vaccinated with PSCA-HSP plasmids generated the strongest PSCA-specific CD8+ T-cell immune response, but the CD4+ TH1 and TH2 cell immune responses were similar with those vaccinated with other HSP-adjuvant PSCA plasmids or only PSCA DNA. The immunity of HSP70 was also observed and the mice i.m. injected with PSCA+ HSP mixed plasmids generated the lowest anti-HSP antibodies. Furthermore, these vaccinations inhibited the growth of PSCA-expressing tumors and prolonged mouse survival. These observations emphasize and extend the potential of the human HSP70 gene as adjuvant for DNA vaccines, and the vaccine based on PSCA and HSP70 is of potential value for treating prostate cancer.

Enhancing DNA vaccine potency by modifying the properties of antigen-presenting cells

DNA vaccines represent a potentially promising approach for antigen-specific immunotherapy. Advances in our knowledge of the adaptive immune system have indicated that professional antigen-presenting cells, especially dendritic cells (DCs), play a key role in the generation of antigen-specific immune responses. Thus, the modification of the properties of DCs represents an important strategy for enhancing the potency of DNA vaccines. This review discusses strategies to increase the number of antigen-expressing DCs, enhance antigen expression, processing and presentation in DCs, promote the activation and function of DCs, and improve DC and T-cell interaction, in order to optimize DNA vaccine-elicited immune responses. Continuing progress in our understanding of DC and T-cell biology serves as a foundation for further improvement of DNA vaccine potency, which may lead to future clinical applications of DNA vaccines for the control of infectious diseases and malignancies.

DNA vaccines for HIV/AIDS

The year 2005 saw the licensure of two DNA vaccines for veterinary use (horses and salmon), thus demonstrating that limitations of potency and delivery have been overcome for certain real-life applications. The purpose of this review is to evaluate the advances specific for application of DNA vaccines to HIV vaccines for humans. The potency of DNA vaccines has been greatly increased by new formulations and delivery methods. The use of various adjuvants and immune modulators has likewise been shown to increase or specifically focus immune responses. Heterologous prime/boost strategies utilizing DNA as the priming agent followed by viral vectors or protein have been shown to be useful for increased antibody as well as cellular responses. Results from early-phase clinical trials are being utilized to direct the development of gene-based vaccines. Recent advances in increasing the potency of DNA vaccines via novel adjuvants, formulations, and delivery systems, along with prime/boost strategies and nonhuman primate studies, are reviewed in terms of their potential for developing an HIV/AIDS vaccine for clinical usage.

DNA prime and protein boost immunization with innovative polymeric cationic core-shell nanoparticles elicits broad immune responses and strongly enhance cellular responses of HIV-1 tat DNA vaccination

Novel biocompatible core-shell cationic nanoparticles, composed of an inner hard core of poly(methylmethacrylate) (PMMA) and a hydrophilic tentacular shell bearing positively charged groups and poly(ethyleneglycol) chains covalently bound to the core, were prepared by emulsion polymerization and characterized in vitro and in vivo for DNA vaccine applications. The nanoparticles reversibly adsorbed large amounts of DNA, mainly through electrostatic interactions, preserved its functional structure, efficiently delivered it intracellularly, and were not toxic in vitro or in mice. Furthermore, two intramuscular (i.m.) immunizations (4 weeks apart) with a very low dose (1 μg) of the plasmid pCV- tat delivered by these nanoparticles followed by one or two protein boosts induced significant antigen-specific humoral and cellular responses and greatly increased Th1-type T cell responses and CTLs against HIV-1 Tat.

Immunogenicity of CTLA4 fusion anti-caries DNA vaccine in rabbits and monkeys

Enhancement of mucosal and systemic immune responses is still a challenge for the application of DNA vaccine. Here, we show anti-caries DNA vaccines, pGJA-P and pGJA-P/VAX, encoding Streptococcus mutans antigens fused to cytotoxic T lymphocyte antigen-4 (CTLA4), which binds to B7 molecule expressed on the surfaces of antigen-presenting cells. Rabbits and monkeys were immunized via intranasal or intramuscular routes. The fusion vaccine induced accelerated and increased specific antibody responses in serum and saliva compared with non-fusion DNA vaccine in rabbits. Significant specific serum IgG and salivary IgA levels could be detected in fusion vaccine-immunized monkeys. Therefore, this study demonstrates that fusing antigens to CTLA4 results in enhancing immune efficacy and strongly suggests that it may represent a promising approach to prevent dental caries or other mucosal infectious diseases. These findings also suggest that CTLA4 fusion anti-caries DNA vaccine may be effective immunogen in primates.

Development and in vitro validation of a targeted delivery vehicle for DNA vaccines

Usage of DNA vaccination has been limited by inefficient cellular expression of plasmid constructs used in DNA vaccines. We describe a novel system for enhancing delivery of DNA vaccine plasmids into cells and their nuclei. This delivery system uses recombinant reovirus type 3 σ1 attachment protein genetically modified with a nuclear localization sequence (σ1-NLS) as a targeting ligand. Purified σ1-NLS was covalently conjugated to the polycation polyethyleneimine (PEI) using a carboxyl-reactive cross-linking agent and complexed with plasmid DNA. The benefit of the NLS in enhancement of protein delivery into the nucleus was demonstrated by liposome-mediated loading of cells with σ1 or σ1-NLS. In L929 fibroblasts loaded with σ1-NLS, 69% of the internalized protein was recovered in the nuclear fraction after 6 h compared to just 10% when using unmodified σ1. Transfection of L929 cells with σ1-NLS-conjugated PEI complexed with a luciferase expression plasmid resulted in a mean 16-fold increase in luciferase activity over complexes made with unmodified PEI, compared to a mean 3-fold boost obtained using σ1-conjugated PEI. These results suggest that σ1-NLS is a useful bifunctional targeting ligand suitable for enhancing DNA delivery and subsequent gene expression for both DNA vaccine applications and nonviral gene therapy.

Development of a Highly Productive and Scalable Plasmid DNA Production Platform

With the applications of DNA vaccines extending from infectious diseases to cancer, achieving the most efficient, reproducible, robust, scalable, and economical production of clinical grade plasmid DNA is paramount to the medical and commercial success of this novel vaccination paradigm. A first generation production process based on the cultivation of Escherichia coli in a chemically defined medium, employing a fed-batch strategy, delivered reasonable volumetric productivities (500-750 mg/L) and proved to perform very well across a wide range of E. coli constructs upon scale-up at industrial scale. However, the presence of monosodium glutamate (MSG) in the formulation of the cultivation and feed solution was found to be a potential cause of process variability. The development of a second generation process, based on a defined cultivation medium and feed solution excluding MSG, was undertaken. Optimization studies, employing a plasmid coding for the HIV gag protein, resulted in cultivation conditions that supported volumetric plasmid titers in excess of 1.2 g/L, while achieving specific yields ranging from 25 to 32 microg plasmid DNA/mg of dry cell weight. When used for the production of clinical supplies, this novel process demonstrated applicability to two other constructs upon scale-up in 2,000-L bioreactors. This second generation process proved to be scalable, robust, and highly productive.

CpG motif acts as a 'danger signal' and provides a T helper type 1-biased microenvironment for DNA vaccination.

The method of delivering a DNA vaccine can influence the type of immune response induced by the vaccine. Application of a DNA vaccine by gene gun typically induces a T helper type 2 (Th2)-type reaction, whereas needle inoculation triggers a Th1 response. In the present study, we found that physical trauma, gold-particle bombardment and the CpG motif can act as 'danger signals' that recruit inflammatory cells to damaged tissues. Analysis of the cytokine profiles of draining lymph nodes or lymph-node-derived mononuclear cells from different groups by real-time reverse transcription-polymerase chain reaction revealed that, while gene-gun-bombardment induced a Th2-type cytokine microenvironment with increased interleukin-4 (IL-4) and IL-10 mRNA expression and almost no increase in IL-12 and interferon-gamma mRNA expression in draining lymph nodes, intradermal injection as well as subcutaneous injection of muscle induced the opposite. We further studied whether the addition of the CpG motif can switch the Th2-type cytokine microenvironment produced by gene-gun bombardment in draining lymph nodes. Results showed that the addition of the CpG motif can increase IL-12 mRNA expression in draining lymph nodes whether induced by intradermal injection, intramuscular injection, or gene-gun bombardment. These data suggest that delivery of the CpG motif induced a Th1-biased microenvironment in draining lymph nodes. Taken together, the CpG motif can act as a 'danger signal' and Th1 immune response enhancer in DNA vaccination. These results may help to explain the mechanism of different types of immune response induced by DNA vaccines delivered by different routes and facilitate the application of DNA vaccines.

190. Improvement of Electroporation Devices and Methods for Use with Plasmid-Based Therapeutics and DNA Vaccine Applications in Small and Large Animals

Introduction: Most commercially available electroporation (EP) technologies are based on constant-voltage concepts. Due to variations in tissue resistance during the EP process, a predetermined voltage pulse may cause an unregulated increase in the current flowing through the tissue during the duration of the pulse and result in loss of the perfect square-wave function, tissue damage, pain and reduced plasmid uptake and expression. The unregulated current generated with conventional electroporation devices may generate amounts of heat in tissues that can easily kill cells. To dramatically improve plasmid uptake and expression levels, we have developed an electrokinetic device (EKD) as well as a single-use electrode array.

Recombinant DNA vaccine encoding multiple domains related to inhibition of neurite outgrowth: a potential strategy for axonal regeneration

Myelin-derived proteins, such as tenascin-R (TN-R), myelin associate glycoprotein (MAG), and Nogo-A, inhibit the CNS regeneration. By targeting specifically the inhibitory epitopes, we have investigated whether vaccination with a recombinant DNA molecule encoding multiple domains of myelin inhibitors may be useful in CNS repair. We show here that the recombinant DNA vaccine is able to activate the immune system but does not induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Importantly, it promotes axonal regeneration in a spinal cord injury model. Thus, the application of DNA vaccine, encoding multiple specific domains of major inhibitory proteins and/or their receptors, provides another promising approach to overcome the inhibitory barriers during CNS regeneration.

Thiophilic interaction chromatography for supercoiled plasmid DNA purification

Supercoiled plasmid DNA was selectively purified from its open circular form by thiophilic interaction chromatography, performed in the presence of high concentrations of water-structuring salts. To identify optimal conditions for purification, various aromatic thioether ligands were coupled to a chromatographic support and screened for their ability to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Selectivity of the chromatographic medium depended on the structure of the ligands, with characteristics of the substituents on the aromatic ring determining the resolution between the different plasmid DNA isoforms. Optimal resolution was obtained with ligands consisting of an thioaromate, substituted with highly electronegative groups. When 2-mercaptopyridine was used as a ligand, the difference in conductivity for eluting open circular and supercoiled plasmid DNA is only 6 mS/cm. However, with 4-nitrothiophol the resolution for plasmid DNA separation on the media increased, resulting in a 20 mS/cm difference. When used in combination with a prior group separation step, these aromatic thioether ligands facilitated the isolation of highly purified supercoiled plasmid DNA, suitable for use in gene therapy and DNA vaccine applications.

Supercoiled plasmid DNA: selective purification by thiophilic/aromatic adsorption

Separation of the different plasmid isoforms is a major challenge in purifying plasmid DNA. We describe a new type of biochemical interaction that occurs in the presence of high concentrations of lyotropic salt and results in the selective adsorption of supercoiled plasmid DNA to aromatic thioether ligands. Under well-defined conditions, these ligands are capable of separating supercoiled plasmid DNA (ccc) from its isoform, i.e. open circular (oc) form. Integrated in a process, preceded by group separation and followed by anion-exchange chromatography, this new purification method may facilitate the production of highly purified supercoiled plasmid DNA for use in gene therapy and DNA vaccine applications.

Immunologic characterization of HIV-specific DNA vaccine.

We developed a method for applying HIV-1 DNA vaccine topically in mice. Topical application of DNA vaccine to the skin is useful against infections. To find a less expensive and less cumbersome vaccination method, we administered HIV-1 DNA vaccine to the skin of mice after elimination of keratinocytes using a fast-acting adhesive. HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. A high level of HIV-1-specific cytotoxic T lymphocyte response was also observed, and a high level of IFN-gamma and IL-4 production was induced by the improved skin application of DNA vaccine. High levels of both HIV-specific cytotoxic T lymphocyte and delayed type hypersensitivity in topical application were induced by coadministration of the DNA vaccine with IL-12 expression plasmids and granulocyte-macrophage colony-stimulating factor expression plasmids. These immune responses were inhibited by intradermal injection of anti-CD11c or anti-I-A/I-E antibody. Therefore, topical administration of DNA vaccine is an effective route, and may be very useful for the prevention of infectious diseases.

Protection induced by intramuscular immunization with DNA vaccines of pseudorabies in mice, rabbits and piglets

Glycoprotein gene gB, gC and gD of pseudorabies virus (PrV) strain Ea, which was isolated locally in Wuhan, were cloned from the viral genome DNA and expressed in vitro controlled by the major immediately-early promotor/enhancer of HCMV. In the presented paper, Balb/c mice, rabbits and piglets were vaccinated intramuscularly two times at 2-week interval with those eukaryotic expression plasmid pcDB, pcDC and pcDD, respectively. The animals injected with pcDB, pcDC, pcDD or mix DNA developed anti-PrV antibodies. Neutralizing antibody titers obtained 2–5 log 2, 2 weeks after the second vaccination. Cellular immune responses were also detected by lymphoproliferation assay and cytotoxic T lymphocyte (CTL) activity assay in all groups vaccinated with DNA. Immune responses elicited by DNA vaccines provided protections with different degrees against lethal dose PrV challenge. In mice, protections induced by pcDC, pcDD or mix DNA were 100%, similar to that by inactivated vaccine. Protections were more than 50% induced by pcDC, pcDD or mix DNA in rabbits. Protections induced by pcDB were the lowest among DNA immunization in mice or rabbits. However, pcDB could elicit the higher cellular responses in rabbits or piglets. In piglets, body temperatures of animals injected with pcDB, pcDC, pcDD or mix DNA did not change significantly after challenge with 2×10 5 pfu of PrV strain Ea, and the means daily growth post-challenge of those animals were higher than those injected with inactivated vaccine or parental plasmid. Neither DNA vaccines nor inactivated vaccine could prevent or delay virus excretion after challenge. Our experiments in experimental animals and natural hosts suggested the efficiency and potential application of DNA vaccines for pseudorabies in pigs.

Topical application of HIV DNA vaccine with cytokine-expression plasmids induces strong antigen-specific immune responses

The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness and economy. But the immune responses that it elicits are relatively low. In this study, we administered human immunodeficiency virus type-1 (HIV-1) DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes using fast-acting adhesive. Our results revealed that the topical application of HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. Co-administration of the DNA vaccine with cytokine expression plasmids of IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by this new method raised the levels of both the HIV-specific cytotoxic T lymphocyte (CTL) response and delayed-type hypersensitivity (DTH) and facilitated the induction of substantial immune responses by DNA vaccine. Skin biopsy sections, thus, immunized showed significant increases of S-100 protein-positive dendritic cells (DCs). These results suggest that the topical application method described here is an efficient route of DNA vaccine administration and that the immune response may be induced by DNA plasmids taken in by DCs, Langerhans cells (LCs), or others such as antigen-presenting cells. This new topical application is likely to be of benefit in clinical use.

A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge.

Improving the immunological potency, particularly the Ab response, is a serious hurdle for the protective efficacy and hence broad application of DNA vaccines. We examined the immunogenicity and protective efficacy of a hemagglutinin-based influenza DNA vaccine that was targeted to antigen-presenting cells (APCs) by fusion to CTLA4. The targeted vaccine was shown to induce an accelerated and increased Ab response (as compared with those receiving the nontargeted control) that was predominated by IgG1 and recognized conformationally dependent viral epitopes. Moreover, mice receiving the APC-targeted DNA vaccine had significantly reduced viral titers (100-fold) after a nonlethal virus challenge. The increased protective efficacy was most likely because of increased Ab responses, as cytotoxic T lymphocyte responses were not enhanced. Targeting was demonstrated by direct binding studies of CTLA4 fusion proteins to the cognate ligand (B7; expressed on APCs in vivo). In addition, a targeted protein was detected at 4-fold higher levels in draining lymph nodes within 2-24 h of administration. Therefore, this study demonstrates that targeting DNA-encoded antigen to APCs results in enhanced immunity and strongly suggests that this approach may be useful in improving the protective efficacy of DNA vaccines.

DNA vaccines for viral infections: Basic studies and applications

This chapter focuses on the basic studies and applications of DNA vaccines for viral infections. DNA-based vaccines use bacterial plasmids to express protein immunogens in vaccinated hosts. Recombinant DNA technology is used to clone complementary DNAs encoding immunogens of interest into eukaryotic expression plasmids. Vaccine plasmids are then amplified in bacteria, purified, and directly inoculated into the host being vaccinated. DNA-based immunizations have both advantages and disadvantages over the classic methods of immunization. The major immunological advantage is the ability of the immunogen to be presented by both major histocompatibility complex (MHC) class I and class II molecules. The best immune responses are achieved using highly active expression vectors modeled on those developed for the production of recombinant proteins. Because of their ease of construction and testing, DNA vaccines have been tried against a number of viral, bacterial, and parasitic infections. DNA vaccines have been tested for at least 40 different viral infections, 9 different bacterial infections, and 9 different parasitic infections and prions. Influenza viruses are segmented RNA viruses that cause highly contagious acute respiratory infections.