Application Of DNA Vaccines Research Articles

Optimization of anion exchange purification for the large-scale production of plasmid DNA for gene therapy and DNA vaccine applications

Watch the video or read the poster to learn: Considerations for pDNA purification How to prepare and implement a DoE approach to identify optimal conditions for various chromatography mediaHow to optimize recovery and impurity removal in the pDNA capture stepHow to achieve a scalable and flexible pDNA purification processDr Jenny England is a Staff Scientist in the Applications and Innovation group in Purification and Pharma Analytics at Thermo Fisher Scientific. Jenny is a biophysicist by training and earned her PhD from Georgetown University. After graduate school, she did a post doc at the National Cancer Institute that focused on structure-based drug design for protein kinase complexes. Jenny currently leads the application group for process development of the POROS resin products for antibody, mRNA, plasmid, and viral vector purification. Additionally, Jenny evaluates new and emerging technologies that can be applied to solve unmet customer needs in the bioproduction workflow.

Genetic engineering approaches to the development of modern therapeutics

The classic approach to production of protein-based therapeutics is their isolation from natural sources. This approach was associated with a number of difficulties, such as collecting the primary material from natural sources, isolating and purifying the protein, and its standardizing. With the development of recombinant DNA technology, itbecame possible to obtain large quantities of protein preparations lacking any contaminations. Human insulin produced using recombinant DNA technology is the first commercial therapeutic obtained by this way. Due to the rapid development of genetic engineering technologies, a large number of proteins have been obtained inEscherichia colicells. In recent years, the approach for the development of drugs based on DNA molecules containing genes encoding therapeutic proteins has been developing more actively. Today, many scientists believe in the prospects of application of DNA vaccines. The ease of production, stability, the ability to mimic natural infections and elicit appropriate immune responses make this vaccine platform extremely attractive. Delivery and targeting of immunologically relevant cells are major tasks for maximizing the immunogenicity of DNA vaccines. Several different approaches that are currently being used to achieve this goal are discussed in this review. Pharmaceuticals based on nucleic acids have a number of undeniable advantages. The main options for prophylactic RNA vaccines, the methods used to deliver RNA to the cell, and methods for increasing the effectiveness of RNA vaccines are discussed. Usage of therapeutic drugs based on protein molecules and low molecular weight compounds is complicated by the fact that they cannot be targeted at a specific gene or its protein product, responsible for the occurrence of the disease. Action of nucleic acids can be directly directed to a particular DNA region in order to edit its nucleotide sequence. This method allows to correct a genetic defect, eliminating the cause of the disease. The principles of gene therapy and the successes achieved in this area are discussed. This review summarizes current achievements in the development of drugs based on recombinant proteins and nucleic acids.

Immunogenicity and efficacy of two DNA vaccines encoding antigenic PspA and TerD against Nocardia seriolae in hybrid snakehead

Fish nocardiosis is a widespread chronic granulomatous disease in aquatic environment, which was particularly caused by Nocardia seriolae. The phage shock protein A (PspA) and tellurium resistance protein D (TerD) were identified to be the immunodominant antigens of the wild-type N. seriolae strain ZJ0503 in our previous study. In an attempt to develop effective DNA vaccines against this pathogen, PspA and TerD were used as candidates to ligate with pcDNA3.1-Flag plasmids, respectively. In addition, the abilities of these two DNA vaccines to elicit various immune responses in hybrid snakehead and supply protective efficacy against artificial challenge with N. seriolae were determined in the present study. The results showed that intramuscular injection with pcDNA-PspA and pcDNA-TerD did not exhibit cytotoxic activities in hybrid snakehead via histopathological examination. Besides, hybrid snakehead immunization with pcDNA-PspA and pcDNA-TerD could increase several non-specific immune paraments in serum, including LYZ, POD, ACP, AKP and SOD activities. Meanwhile, the pcDNA-TerD DNA vaccine could induce strongly specific antibody (IgM) titer in hybrid snakehead with a relative percent of survival (RPS) value of 83.14% against N. seriolae, while that of pcDNA-PspA DNA vaccine was displayed comparably low IgM titer with RPS value of 57.83%. Furthermore, quantitative real-time PCR assays presented that the expression of immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1β and TNFα) were up-regulated to various degrees after vaccination with pcDNA-PspA or pcDNA-TerD, indicating that these two DNA vaccines were able to boost humoral and cell-mediated immune responses in hybrid snakehead. Taken together, both the pcDNA-PspA and pcDNA-TerD DNA vaccines were proved to be safe, immunogenic and effective in protecting hybrid snakehead against N. seriolae infection, which can promote the development and application of DNA vaccines to control fish nocardiosis in aquaculture.

Molecular Adjuvants for DNA Vaccines: Application, Design, Preparation, and Formulation.

Compared with conventional vaccines, the main advantage of DNA vaccine-based methods is its continued expression of the plasmid-encoded antigens followed by the induction of subsequent humoral and cellular immunities. DNA vaccines are currently used in animal models, but limited success has been obtained for use in clinical applications due to their poor immunogenicity. Various strategies are attempted to improve the induced immune response of DNA vaccines. It has been demonstrated that co-administration of molecular adjuvants with DNA vaccines is a promising approach to effectively elicit protective immunity by increasing the transfection efficiency of DNA vaccines. Genetic adjuvants are incorporated to promote activation of the transfected local antigen-presenting cells (APCs) and immune cells in the draining lymph node and polarization of T-cell subsets to decrease T-cell tolerance to the specific antigen. Here we provide an overview of different types of genetic adjuvants. The aim of the current chapter is to present a framework for the construction of a gene-based vaccine and adjuvant. Moreover, we describe the application of DNA vaccines co-administered with different types of genetic adjuvants and the methods to evaluate their potency in the mouse models.

Construction of Lactobacillus casei ghosts by Holin-mediated inactivation and the potential as a safe and effective vehicle for the delivery of DNA vaccines

BackgroundBacterial ghosts (BGs) are empty bacterial cell envelopes generated by releasing the cellular contents. In this study, a phage infecting Lactobacillus casei ATCC 393 (L. casei 393) was isolated and designated Lcb. We aimed at using L. casei 393 as an antigen delivery system to express phage-derived holin for development of BGs.ResultsA gene fragment encoding holin of Lcb (hocb) was amplified by polymerase chain reaction (PCR). We used L. casei 393 as an antigen delivery system to construct the recombinant strain pPG-2-hocb/L. casei 393. Then the recombinants were induced to express hocb. The immunoreactive band corresponding to hocb was observed by western-blotting, demonstrating the efficiency and specificity of hocb expression in recombinants. The measurements of optical density at 600 nm (OD600) after induction showed that expression of hocb can be used to convert L. casei cells into BGs. TEM showed that the cytomembrane and cell walls of hocb expressing cells were partially disrupted, accompanied by the loss of cellular contents, whereas control cells did not show any morphological changes. SEM showed that lysis pores were distributed in the middle or at the poles of the cells. To examine where the plasmid DNA was associated, we analyzed the L. casei ghosts loading SYBR Green I labeled pCI-EGFP by confocal microscopy. The result demonstrated that the DNA interacted with the inside rather than with the outside surface of the BGs. To further analyze where the DNA were loaded, we stained BGs with MitoTracker Green FM and the loaded plasmids were detected using EGFP-specific Cy-3-labeled probes. Z-scan sections through the BGs revealed that pCI-EGFP (red) was located within the BGs (green), but not on the outside. Flow cytometry and qPCR showed that the DNA was loaded onto BGs effectively and stably.ConclusionsOur study constructed L. casei BGs by a novel method, which may be a promising technology for promoting the further application of DNA vaccine, providing experimental data to aid the development of other Gram-positive BGs.

Production and Application of Streptococcus iniae DNA Vaccine to Increase Immunity ff Nile Tilapia (Oreochromis niloticus)

DNA vaccine Streptococcus iniae is a third generation of vaccines based on the gene encoding a vaccine antigen. Pgm is DNA-binding protein that activates expression of several important virulence gene, including those encoding polysaccharide capsule. The pgm gene controlled by MBA promoter has constructed successfully as a candidate of DNA vaccine to against S. iniae infection in Nile Tilapia. The goals of this study were to production and application of DNA Vaccine S. iniae to increase the nile tilapia immunity. Vaccine production was using plasmid isolation method from Escherichia coli dH5α containing pMBA-pgm. Vaccine verification was was accomplished by PCR using pgm gene S. iniae Fstrep and Rstrep specific primers, which revealed the 1,713 bp PCR product. Application of DNA vaccine was using 25, 50, 75, and 100ng/ml dosage with intraperitonial injection method .The challenge test was using 108 cfu/ml density of S. iniae. Observation test parameter were survival rate, relative percent survival, mean time to death, pgm gene, and organ histology such as liver, eyes, brain, spleen and kidney. Vaccine production was succeed using plasmid isolation method and containing pgm gene revealed 1713bp. the application of DNA vaccine was optimum at 50ng/ml dosage with SR value 96.667%, RPS value 88,461% and MTD value 4,6 days. The eyes histology shows opacity and exopthalmia, the other organ such as liver, spleen and kidney was necrosis. The DNA vaccine was optimum at 50ng/ml dosage.

The immunogenicity and anti-tumor effects of a lung cancer DNA vaccine harboring a MUC-1 and GM-CSF fusion gene

DNA vaccines have been shown to be an effective approach to induce antigen-specific cellular and humoral immunity. However, the inability of DNA vaccines to elicit strong immune responses in clinical trials limits the application of DNA vaccines. Here, we developed a new DNA vaccine based on MUC1, which has been suggested as a potential target for lung cancer therapy, and we enhanced the potency of the DNA vaccine by including granulocyte-macrophage colony-stimulating factor (GM-CSF) as an adjuvant. A series of DNA plasmids encoding MUC1, human GM-CSF and their conjugates were constructed and injected into female mice intramuscularly (i.m.). This action was followed by an electric pulse. The humoral and cellular immune responses after immunization were examined by ELISA and ELISPOT, respectively. To evaluate the therapeutic efficacy of the plasmids, a mouse model with a MUC1-expressing tumor was designed. Mice vaccinated with the MUC1-GM-CSF plasmid generated the strongest MUC1-specific humoral and cellular immune responses. Furthermore, these vaccinations inhibited the growth of MUC1-expressing tumors and prolonged mouse survival. These observations emphasize the potential of GM-CSF as an adjuvant for DNA vaccines and of vaccines based on MUC1 and GM-CSF as a promising treatment for lung cancer.

Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery.

Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage), viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

Preparation and Efficacy of Newcastle Disease Virus DNA Vaccine Encapsulated in PLGA Nanoparticles

BackgroundAlthough the Newcastle disease virus (NDV) inactivated vaccines and attenuated live vaccines have been used to prevent and control Newcastle disease (ND) for years, there are some disadvantages. Recently, newly developed DNA vaccines have the potential to overcome these disadvantages. The low delivery efficiency, however, hindered the application of DNA vaccines for ND in practice.Methodology/Principal FindingsThe eukaryotic expression plasmid pVAX1-F (o) DNA that expressed the F gene of NDV encapsulated in PLGA nanoparticles (pFNDV-PLGA-NPs) were prepared by a double emulsion-solvent evaporation method and optimal preparation conditions of the pFNDV-PLGA-NPs were determined. Under the optimal conditions, the pFNDV-PLGA-NPs were produced in good morphology and had high stability with a mean diameter of 433.5±7.5 nm, with encapsulation efficiency of 91.8±0.3% and a Zeta potential of +2.7 mV. Release assay in vitro showed that the fusion gene plasmid DNA could be sustainably released from the pFNDV-PLGA-NPs up to 93.14% of the total amount. Cell transfection test indicated that the vaccine expressed and maintained its bioactivity. Immunization results showed that better immune responses of SPF chickens immunized with the pFNDV-PLGA-NPs were induced compared to the chickens immunized with the DNA vaccine alone. In addition, the safety of mucosal immunity delivery system of the pFNDV-PLGA-NPs was also tested in an in vitro cytotoxicity assay.Conclusions/SignificanceThe pFNDV-PLGA-NPs could induce stronger cellular, humoral, and mucosal immune responses and reached the sustained release effect. These results laid a foundation for further development of vaccines and drugs in PLGA nanoparticles.

Efficacy of DNA vaccine encoding koi herpesvirus glycoprotein GP-25in common carp juvenile by immersion

<p class="Default">Koi herpesvirus (KHV) is a herpesvirus that particularly infects and causes mass mortality to koi and common carp. Therefore, the protection of common carp from KHV infection is urgently needed. In this study, we developed an application of DNA vaccine encoding KHV glycoprotein-25 by immersion method to increase survival of common carp against KHV infection. A total of 400 common carp juveniles at 30-day-old were immersed in 1-L water containing 1.3×10<sup>8</sup>CFU/mL of the killed <em>Escherichia coli</em> cells carrying DNA vaccine. Three frequencies and three duration of fish immersion were tested, namely: 1×30 minutes, 1×60 minutes, 1× 90 minutes, 2×90 minutes and 3×90 minutes by interval of 24 hours. Reversetranscription polymerase chain reaction analysis showed that DNA vaccine was successfully expressed in the vaccinated fish. Fish at twenty eight days post vaccination were challenged by injecting 10<sup>-4</sup> mL of KHV per fish. The result showed that vaccination by 1×30 minutes immersion allowed 61% of fish survived, and this was significantly higher (p<0.05) compared to control (without vaccination), but it was similar among vaccination treatments (p>0.05). The relative percent survival of vaccinated fish were also similar among treatments (p>0.05). DNA vaccination has increased fish survival about two fold higher compared to unvaccinated fish control (26.67%). Thus, DNA vaccination was effectively delivered by immersion for 1×30 minutes, and this technique can be useful to level up the resistance of common carp juveniles against KHV infection.</p> <p class="Default">Keywords: DNA vaccine, KHV, glycoprotein, immersion, common carp</p>

DNA vaccines: roles against diseases

Vaccination is the most successful application of immunological principles to human health. Vaccine efficacy needs to be reviewed from time to time and its safety is an overriding consideration. DNA vaccines offer simple yet effective means of inducing broad-based immunity. These vaccines work by allowing the expression of the microbial antigen inside host cells that take up the plasmid. These vaccines function by generating the desired antigen inside the cells, with the advantage that this may facilitate presentation through the major histocompatibility complex. This review article is based on a literature survey and it describes the working and designing strategies of DNA vaccines. Advantages and disadvantages for this type of vaccines have also been explained, together with applications of DNA vaccines. DNA vaccines against cancer, tuberculosis, Edwardsiella tarda, HIV, anthrax, influenza, malaria, dengue, typhoid and other diseases were explored.

A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene delivery system

The transport of DNA into eukaryotic cells is minimal because of the cell membrane barrier, and this limits the application of DNA vaccines, gene silencing, and gene therapy. Several available transfection reagents and techniques have been used to circumvent this problem. Alternatively, nonviral nanoscale vectors have been shown to bypass the eukaryotic cell membrane. In the present work, we developed a unique nanomaterial, pHEMA+chitosan nanospheres (PCNSs), which consisted of poly(2-hydroxyethyl methacrylate) nanospheres surrounded by a chitosan cationic shell, and we used this for encapsulation of a respiratory syncytial virus (RSV)-F gene construct (a model for a DNA vaccine). The new nanomaterial was capable of transfecting various eukaryotic cell lines without the use of a commercial transfection reagent. Using transmission electron microscopy, (TEM), fluorescence activated cell sorting (FACS), and immunofluorescence, we clearly demonstrated that the positively charged PCNSs were able to bind to the negatively charged cell membrane and were taken up by endocytosis, in Cos-7 cells. Using quantitative polymerase chain reaction (qPCR), we also evaluated the efficiency of transfection achieved with PCNSs and without the use of a liposomal-based transfection mediator, in Cos-7, HEp-2, and Vero cells. To assess the transfection efficiency of the PCNSs in vivo, these novel nanomaterials containing RSV-F gene were injected intramuscularly into BALB/c mice, resulting in high copy number of the transgene. In this study, we report, for the first time, the application of the PCNSs as a nanovehicle for gene delivery in vitro and in vivo.

HER2/neu DNA vaccination by intradermal gene delivery in a mouse tumor model

DNA vaccines are potential tools for the induction of immune responses against both infectious disease and cancer. The dermal application of DNA vaccines is of particular interest since the epidermal and dermal layers of the skin are characterized by an abundance of antigen-presenting cells (APCs). The aim of our study was to compare tumor protection as obtained by two different methods of intradermal DNA delivery (gene gun and jet injector) in a well-established HER2/neu mouse tumor model. BALB/c mice were immunized twice with a HER2/neu-coding plasmid by gene gun or jet injector. Mice were then subcutaneously challenged with HER2/neu+ syngeneic D2F2/E2 tumor cells. Protection against subsequent challenges with tumor cells as well as humoral and T-cell immune responses induced by the vaccine were monitored. Gene gun immunization was far superior to jet injector both in terms of tumor protection and induction of HER2/neu-specific immune responses. After gene gun immunization, 60% of the mice remained tumor-free until day 140 as compared with 25% after jet injector immunization. Furthermore, gene gun vaccination was able to induce both a strong TH1-polarized T-cell response with detectable cytotoxic T-lymphocyte (CTL) activity and a humoral immune response against HER2/neu, whereas the jet injector was not. Although the disadvantages that were associated with the use of the jet injector in our model may be overcome with methodological modifications and/or in larger animals, which exhibit a thicker skin and/or subcutaneous muscle tissue, we conclude that gene gun delivery constitutes the method of choice for intradermal DNA delivery in preclinical mouse models and possibly also for the clinical development of DNA-based vaccines.

DNA Vaccines for Prostate Cancer

Delivery of plasmid DNA encoding an antigen of interest has been demonstrated to be an effective means of immunization, capable of eliciting antigen-specific T cells. Plasmid DNA vaccines offer advantages over other anti-tumor vaccine approaches in terms of simplicity, manufacturing, and possibly safety. The primary disadvantage is their poor transfection efficiency and subsequent lower immunogenicity relative to other genetic vaccine approaches. However, multiple preclinical models demonstrate anti-tumor efficacy, and many efforts are underway to improve the immunogenicity and anti-tumor effect of these vaccines. Clinical trials using DNA vaccines as treatments for prostate cancer have begun, and to date have demonstrated safety and immunological effect. This review will focus on DNA vaccines as a specific means of antigen delivery, advantages and disadvantages of this type of immunization, previous experience in preclinical models and human trials specifically conducted for the treatment of prostate cancer, and future directions for the application of DNA vaccines to prostate cancer immunotherapy.

DNA Prime-Adenovirus Boost Immunization Induces a Vigorous and Multifunctional T-Cell Response against Hepadnaviral Proteins in the Mouse and Woodchuck Model

Induction of hepatitis B virus (HBV)-specific cytotoxic T cells by therapeutic immunization may be a strategy to treat chronic hepatitis B. In the HBV animal model, woodchucks, the application of DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen (WHcAg) in combination with antivirals led to the prolonged control of viral replication. However, it became clear that the use of more potent vaccines is required to overcome WHV persistence. Therefore, we asked whether stronger and more functional T-cell responses could be achieved using the modified vaccines and an optimized prime-boost vaccination regimen. We developed a new DNA plasmid (pCGWHc) and recombinant adenoviruses (AdVs) showing high expression levels of WHcAg. Mice vaccinated with the improved plasmid pCGWHc elicited a stronger WHcAg-specific CD8(+) T-cell response than with the previously used vaccines. Using multicolor flow cytometry and an in vivo cytotoxicity assay, we showed that immunization in a DNA prime-AdV boost regimen resulted in an even more vigorous and functional T-cell response than immunization with the new plasmid alone. Immunization of naïve woodchucks with pCGWHc plasmid or AdVs induced a significant WHcAg-specific degranulation response prior to the challenge, this response had not been previously detected. Consistently, this response led to a rapid control of infection after the challenge. Our results demonstrate that high antigen expression levels and the DNA prime-AdV boost immunization improved the T-cell response in mice and induced significant T-cell responses in woodchucks. Therefore, this new vaccination strategy may be a candidate for a therapeutic vaccine against chronic HBV infection.

Clinical Applications of DNA Vaccines: Current Progress

It was discovered almost 20 years ago that plasmid DNA, when injected into the skin or muscle of mice, could induce immune responses to encoded antigens. Since that time, there has since been much progress in understanding the basic biology behind this deceptively simple vaccine platform and much technological advancement to enhance immune potency. Among these advancements are improved formulations and improved physical methods of delivery, which increase the uptake of vaccine plasmids by cells; optimization of vaccine vectors and encoded antigens; and the development of novel formulations and adjuvants to augment and direct the host immune response. The ability of the current, or second-generation, DNA vaccines to induce more-potent cellular and humoral responses opens up this platform to be examined in both preventative and therapeutic arenas. This review focuses on these advances and discusses both preventive and immunotherapeutic clinical applications.

Enhanced immunogenicity of an anti‐caries vaccine encoding a cell‐surface protein antigen of Streptococcus mutans by intranasal DNA prime‐protein boost immunization

The present study aimed to enhance the specific anti-caries immunity induced by DNA prime-protein boost strategy for an A-P fragment of a cell-surface protein antigen of Streptococcus mutans (PAc). BALB/c mice were immunized with DNA prime-protein boost, DNA-DNA or protein-protein regimens by the intranasal route, using combinations of plasmid vector (pCIA-P) that express PAc protein and a pure secretec recombinant PAc protein (rPAc). Then, a gnotobiotic mouse model was constructed 2 weeks after the last immunization, and specific immune responses in vivo and their protection against dental caries were observed. The present study revealed stronger antibody responses in the DNA prime-protein boost group compared to those elicited by either DNA-DNA vaccination or protein-protein vaccination. In particular, PAc-specific antibody concentrations were improved significantly after boosting the pCIA-P DNA-primed mice with rPAc. Moreover, protection against S. mutans challenge was obtained in the mice treated with the DNA prime-protein boost vaccination, as demonstrated by a significant reduction in S. mutans colonization compared to control mice and animals immunized with the DNA-DNA vaccination or protein-protein vaccination. The results obtained in the present study suggest that the intranasal DNA prime-protein boost vaccination regimen is a novel strategy for the practical application of DNA vaccine against dental caries.

A novel antibiotic free plasmid selection system: Advances in safe and efficient DNA therapy

The presence of antibiotic resistance genes in the delivered plasmids is one of the drawbacks of modern gene therapy and DNA vaccine applications. Here, we describe a strategy that allows for plasmid selection in bacterial hosts, without the requirement of any selection marker. Several bacterial strains were modified, so that the plasmid's replicational inhibitor RNA I could suppress the translation of a growth essential gene by RNA-RNA antisense reaction. An essential gene (murA) was modified such that a repressor protein (tetR) would hamper its expression. Only in the presence of plasmid and, hence, RNA I, was tetR turned down and murA expressed. Different commercially available plasmids could be selected by various modified Escherichia coli strains. We further designed a minimalistic plasmid devoid of any selection marker. All of the clones (n=6) examined, when the modified strain JM109-murselect was used for selection, contained plasmids. Thus, we have designed bacterial host strains that for the first time serve to select and maintain plasmids without the use of any selection marker or other additional sequence on the plasmid. Consequently, such plasmids may not only be safer, but due to their decreased size, advantages for the manufacturer and higher transfection efficiencies are anticipated.

Immunization by Application of DNA Vaccine onto a Skin Area Wherein the Hair Follicles Have Been Induced into Anagen-onset Stage

An attractive approach to immunization is to apply DNA vaccine topically onto the skin. However, it is important to ensure that a strong immune response is induced without disrupting the skin stratum corneum. The hair follicles have been shown to be the major portal of entry for DNA applied onto the skin, and it has been reported that the transfection of hair follicle cells occurs mainly at the onset of a new growing stage of the hair cycle. Using an anthrax protective antigen (PA) protein-encoding plasmid in mice, we demonstrated that the anti-PA immune responses were significantly stronger when the hair follicles in the application area were induced into anagen-onset stage than when in telogen stage. The anti-PA antibodies enabled the immunized mice to survive a lethal dose of anthrax lethal toxin challenge. The enhanced immune responses can be partially attributed to the enhanced antigen gene expression and plasmid DNA uptake in the skin area wherein the hair follicles were induced into anagen-onset stage. Moreover, the moderate dermal inflammation associated with the anagen induction may also have contributed to the enhancement of the resultant immune response. This represents a novel approach to enhancing the immune response induced by a topically applied DNA vaccine.

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.