A combination of intradermal jet-injection and electroporation overcomes in vivo dose restriction of DNA vaccines

ABSTRACT: BACKGROUND: The use of optimized delivery devices has been shown to enhance the potency of DNA vaccines. However, further optimization of DNA vaccine delivery is needed for this vaccine modality to ultimately be efficacious in humans. METHODS: Herein we evaluated antigen expression and immunogenicity after intradermal delivery of different doses of DNA vaccines by needle or by the Biojector jet-injection device, with or without the addition of electroporation (EP). RESULTS: Neither needle injection augmented by EP nor Biojector alone could induce higher magnitudes of immune responses after immunizations with a high dose of a DNA vaccine as compared to immunizations with a considerably lower dose. Biojector delivery followed by EP, however, overcame this observed dose restriction and induced significantly higher cellular and humoral immune responses after immunization with a high dose of DNA. Furthermore, a close correlation between in vivo antigen expression and cell-mediated immune responses was observed. CONCLUSIONS: These results show that two optimized DNA vaccine delivery devices can act together to overcome dose restrictions of plasmid DNA vaccines.     

Caffeine overcomes a restriction point associated with DNA replication, but does not accelerate mitosis

Mitotic chromosome condensation is normally dependent on the previous completion of replication. Caffeine spectacularly deranges cell cycle controls after DNA polymerase inhibition or DNA damage; it induces the condensation, in cells that have not completed replication, of fragmented nuclear structures, analogous to the S-phase prematurely condensed chromosomes seen when replicating cells are fused with mitotic cells. Caffeine has been reported to induce S-phase condensation in cells where replication is arrested, by accelerating cell cycle progression as well as by uncoupling it from replication; for, in BHK or CHO hamster cells arrested in early S-phase and given caffeine, condensed chromosomes appear well before the normal time at which mitosis occurs in cells released from arrest. However, we have found that this apparent acceleration depends on the technique of synchrony and cell line employed. In other cells, and in synchronized hamster cells where the cycle has not been subjected to prolonged continual arrest, condensation in replication-arrested cells given caffeine occurs at the same time as normal mitosis in parallel populations where replication is allowed to proceed. This caffeine-induced condensation is therefore "premature" with respect to the chromatin structure of the S-phase nucleus, but not with respect to the timing of the normal cycle. Caffeine in replication-arrested cells thus overcomes the restriction on the formation of mitotic condensing factors that is normally imposed during DNA replication, but does not accelerate the timing of condensation unless cycle controls have previously been disturbed by synchronization procedures.     

Immunogenicity and protective efficacy study using combination of four tuberculosis DNA vaccines

Immune response and protective efficacy for the combination of four tuberculosis DNA vaccines were evaluated in this study. We obtained 1:200 antibody titers against Ag85B 21d after mice were vaccinated for the first time by four recombinant eukaryotic expression vectors containing coding sequences for Ag85B, MPT-64, MPT-63 and ESAT-6. The titers of Ag85B were elevated to 1:102400 after the second injection and decreased to 1:12800 after the third injection. Antibody titers for MPT-64 and MPT-63 reached 1:25600 21 d after the first vaccination, and were then decreased following the second and third injections. No antigen-specific antibody titer against ESAT-6 was detected in sera harvested from immunized mice at any time. These DNA vaccines evoked specific IFN-λ responses in the spleens of vaccinated mice as well. When challenged with M. tuberculosis H37Rv, we found that the lungs of the vaccinated mice produced 99.8% less bacterial counts than that of the empty-vector control group and the bacterial counts were also significantly less than that of the BCG group. Histopathological analyses showed that the lungs of vaccinated mice produced no obvious caseation while over 50%-70% of the pulmonary parenchyma tissue produced central caseation in the vector control group. Our results indicated that the combination of four tuberculosis DNA vaccines may generate high levels of immune responses and result in better animal protection.     

Immunogenicity and protective efficacy study using combination of four tuberculosis DNA vaccines

Tuberculosis is an ancient scourge of mankind. According to statistics, there are more than 10 million new cases of tuberculosis each year and the annual death toll for tuberculosis exceeds three millions. The current available BCG is of questionable efficacy, and its protection ranges from 0 to 85%. Therefore, developing a safe and effective vaccine against this scourge is very important. Previous studies have shown that the secreted proteins of Mycobacterium tuberculosis (M. TB) can induc…     

Co-transfer of restriction endonucleases and plasmid DNA into mammalian cells by electroporation: effects on stable transformation

The antimutagenic principle of the green fruits of Momordica charantia was shown by the micronucleus test to be an intractable mixture of novel acylglucosylsterols. The antimutagens were extracted from the green fruits with ethanol and isolated from the bioactive petroleum ether and carbon tetrachloride extracts by repeated and sequential flash column chromatography. The major component of the mixture is

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and the minor component is the stearyl derivative (Guevara, 1989). At a dosage range in mice of 50–12.5 μg extract/g, the mixture reduced by about 80% the number of micronucleated polychromatic erythrocytes induced by the well-known mutagen mitomycin C. Structure-activity correlation studies suggested that the antimutagenic activity may reside in the peculiar lipid-like structure of the acylglucosylsterols. Ingestion of these compounds may result in their absorption in the plasma membrane lipid bilayer which could adversely affect the membrane permeability towards mitomycin C and disrupt the cellular activity of the latter.     

A multivalent combination of experimental antituberculosis DNA vaccines based on Ag85B and regions of difference antigens

Two candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the Th1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H(37)Rv (in terms of colony-forming unit reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study.     

Isolation of restriction deficient mutant for Corynebacterium glutamicum by electroporation

Summary A simple electroporation method for isolating restriction deficient mutants from Corynebacterium glutamicum has been developed. This procedure will also be useful for isolations of restriction deficient mutants of other microorganisms.     

Increased gene expression and inflammatory cell infiltration caused by electroporation are both important for improving the efficacy of DNA vaccines

One potential reason for the enhancement of immune responses to DNA vaccines following electroporation is increased gene expression. However, the inflammatory response and accompanying cellular infiltration stimulated by electroporation may also be essential for enhancing immune responses to DNA vaccines. These parameters were investigated in pigs, using different electroporation conditions to induce different levels of gene expression and inflammation. Results indicated that the least effective strategy was conventional intramuscular injection where there was low gene expression and low inflammatory cell infiltration. The most efficacious strategy was plasmid administration immediately followed by electroporation. This latter set of conditions elicited a combination of high gene expression and high cellular infiltration. This indicates that electroporation enhances immune responses to DNA vaccines through increased gene expression and inflammatory cell infiltration.     

Efficacy of seasonal pandemic influenza hemagglutinin DNA vaccines delivered by electroporation against aseasonal H1N1 virus challenge in mice

Prophylactic DNA vaccines against the influenza virus are promising alternatives to conventional vaccines. In this study, we generated two candidate gene-based influenza vaccines encoding either the seasonal or pandemic hemagglutinin antigen (HA) from the strains A/New Caledonia/20/99 (H1N1) (pV1A5) and A/California/04/2009 (H1N1) (pVEH1), respectively. After verifying antigen expression, the immunogenicity of the vaccines delivered intramuscularly with electroporation was tested in a mouse model. Sera of immunized animals were tested in hemagglutination inhibition assays and by ELISA for the presence of HA-specific antibodies. HA-specific T-cells were also measured in IFN-γ ELISpot assays. The protective efficacy of the candidate influenza vaccines was evaluated by measuring mortality rates and body weight after a challenge with 100 LD(50) of mouse-adapted A/New Caledonia/20/99 (H1N1). Mice immunized with either one of the two vaccines showed significantly higher T cell and humoral immune responses (P<0.05) than the pVAX1 control group. Additionally, the pV1A5 vaccine effectively protected the mice against a lethal homologous mouse-adapted virus challenge with a survival rate of 100% compared with a 40% survival rate in the pVEH1 vaccinated group (P<0.05). Our study indicates that the seasonal influenza DNA vaccine completely protects against the homologous A/New Caledonia/20/99 virus (H1N1), while the pandemic influenza DNA vaccine only partially protects against this virus.     

DNA transfection of Escherichia coli by electroporation

Electroporation was applied to transfection and transformation of Escherichia coli. Efficient transfer of DNA was achieved by a single voltage pulse at 2.5 kV (initial electric field strength = 6.25 kV/cm), with a 25 microF capacitor. As the recipient for transfecting DNA in the electroporation, spheroplasts, EDTA-treated cells and osmotically shocked bacteria were inferior to intact E. coli. Various parameters affecting the transfection efficiency were defined including growth phase of recipient cells, concentrations of DNA and cells, temperature and additions. In most strains tested, electroporation was far more efficient than Ca2+-dependent transfection (transformation). Various aspects of the electroporation-mediated DNA uptake are discussed.     

Enhanced frequency of Beauveria bassiana blastospore transformation by restriction enzyme-mediated integration and electroporation

The techniques of restriction enzyme-mediated integration (REMI) and electroporation (EP) were applied for the first time to improving the blastospore transformation of fungal biocontrol agent Beauveria bassiana for higher frequency. The blastospores from < or =24 h incubation in glucose-mineral medium after shaking conidia for 48 h in Subouraud dextrose broth were found most competent for integrating 1 microg plasmid DNA vectoring the phosphinothricin (PPT) resistance gene bar in 360 microL reaction system containing 100 U HindIII or XbaI. Such blastospores were also most suitable for EP transformation at the optimized field strength of 10 kV cm(-1). The optimized REMI and EP generated averagely 39 and 53 transformants microg(-1) plasmid DNA whereas polyethylene glycol (PEG) integration yielded only 22. All transformants grew well on Czapek's agar containing 400 microg PPT mL(-1) after three rounds of cultivation on the same agar excluding PPT but their parental strain showed no resistance. The target gene inserted into the genomes of 10 transformants randomly taken from REMI or EP transformation was consistently detected by both PCR and Southern blotting. Compared to the PEG integration, REMI and EP enhanced the frequency of the blastospore transformation by 73 and 137%, respectively.     

A rapid two-dimensional fractionation of DNA restriction fragments

Genomic DNA that has been digested with a restriction endonuclease and fractionated by electrophoresis on an agarose gel can be recovered on glass-fiber filters by a new blotting scheme. The DNA fragments in each fraction are then digested with a second restriction nuclease and then separated on a slab gel, resulting in a two-dimensional display of the restriction fragments. This rapid fingerprinting technique is useful in the analysis of complex genomes and in the isolation and cloning of particular sequences.     

Increased DNA vaccine delivery and immunogenicity by electroporation in vivo

DNA vaccines have been demonstrated to be potent in small animals but are less effective in primates. One limiting factor may be inefficient uptake of DNA by cells in situ. In this study, we evaluated whether cellular uptake of DNA was a significant barrier to efficient transfection in vivo and subsequent induction of immune responses. For this purpose, we used the technique of electroporation to facilitate DNA delivery in vivo. This technology was shown to substantially increase delivery of DNA to cells, resulting in increased expression and elevated immune responses. The potency of a weakly immunogenic hepatitis B surface Ag DNA vaccine was increased in mice, as seen by a more rapid onset and higher magnitude of anti-hepatitis B Abs. In addition, the immunogenicity of a potent HIV gag DNA vaccine was increased in mice, as seen by higher Ab titers, a substantial reduction in the dose of DNA required to induce an Ab response, and an increase in CD8+ T cell responses. Finally, Ab responses were enhanced by electroporation against both components of a combination HIV gag and env DNA vaccine in guinea pigs and rabbits. Therefore, cellular uptake of DNA is a significant barrier to transfection in vivo, and electroporation appears able to overcome this barrier.     

A restriction map of Xenopus laevis mitochondrial DNA

The mitochondrial DNA from Xenopus laevis is a 17.4 x 10(3)-base-pair circular DNA molecule. The mapping of this DNA, using 19 different restriction endonucleases is reported here. The sites are as follows: 1 for BamHI, PstI, SacI, SalI, BalI; 2 for BglII, SacII, EcoRI, ClaI, 3 for XhoI, 4 for AvaI, XbaI, PvuII, 5 for HindIII, 6 for HhaI, BclI, HpaI, 10 for AvaII and 11 for HincII. The same sites (except for one of the two ClaI sites) are observed in the molecule cloned in pBR322 DNA. The fragments corresponding to 62 cleavage sites have all been ordered and precisely located. They provide suitable conditions for further investigations connected with the study of replication and nucleotide sequence determination of this molecule.     

Enhancement of DNA vaccine potency by electroporation in vivo

The potential of electric current-mediated delivery technology to enhance DNA delivery and DNA vaccine potency was evaluated. Higher levels of reporter gene expression were observed in muscle cells of mice inoculated with luciferase or beta-galactosidase DNA followed by the application of electrical current, compared with DNA injected with no current. Similarly, substantially higher levels of immune responses (up to 20-fold) were demonstrated in mice vaccinated with HIV gag DNA and electric current. These enhanced responses were observed after one or two inoculations, and were maintained for at least 12 weeks. Therefore, the present studies demonstrate the utility of electroporation for enhancement of DNA vaccine potency in animals.     

Introduction and expression of DNA molecules in eukaryotic cells by electroporation

Exposing eukaryotic cells to brief, high voltage electrical fields can induce the uptake of exogenous materials, presumably through the transient formation of micropores in the cell membrane. This phenomenon has been exploited for the introduction of cloned DNA molecules into cells for permanent transformation or for transient expression and analysis of gene products. The magnitude and characteristics of the generated electrical field are critical for successful electroporation and simple, transient expression assays using indicator genes allow the calibration and optimization of electroporation conditions for a wide variety of eukaryotic cell types. These techniques may allow the genetic modification of a variety of host cells which cannot be easily transformed by other methods.