Intramuscular Injection Of Naked DNA Research Articles

Improved immunogenicity and protective efficacy of a replicon DNA vaccine encoding the Hc domain of botulinum neurotoxin serotype A by electric pulses and protein boosting

Vaccination by intramuscular injection of naked DNA is very efficient in mouse model, but immunogenicity of DNA vaccines needs to be improved in human use. Thus, we wanted to determine whether suitable electric pulses-mediated DNA delivery technology and DNA prime-protein boost regimen could improve the immunogenicity and protective efficacy of the replicon DNA vaccine pSCARSHc in mouse model. In this study, the immunogenicity and protective efficacy of the replicon DNA vaccine pSCARSHc following electric pulses were dramatically improved in Balb/c mice. Also, priming of the immune response by DNA vaccination followed by a single booster with AHc protein immunogen resulted in very high levels of ELISA and neutralization antibodies and afforded more efficient protection against botulinum neurotoxin serotype A. Therefore, these methods described here potentially provide suitable strategies in developing an efficacious vaccine against Clostridium botulinum neurotoxin serotype A.

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass ( Lates calcarifer) to protect from Vibrio ( Listonella) anguillarum

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass ( Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.

Induction of HIV-specific memory T-cell responses by topical DermaVir vaccine

In vivo antigen expression by plasmid DNA could provide a potent and cost-effective vaccine platform if its immunogenicity were improved to induce antigen-specific memory T-cell responses. To study these immune responses, we compared naked DNA vaccine with topical DermaVir formulated with the same HIV-1 (Gag) DNA in the mouse model. Topical DermaVir induced HIV-specific effector memory CD8 + T-cell responses, which were 2.4-fold higher than those elicited by intramuscular injection of naked DNA. DermaVir, but not naked DNA vaccination, induced robust HIV-specific central memory T-cells responses, which are likely to be more efficient in mediating protective immunity. DermaVir formulation combined with topical administration provides an improved immunogenicity of antigen-expressing DNA vaccines.

Preparation and Characterization of a Monoclonal Antibody Against CKLF1 Using DNA Immunization withIn VivoElectroporation

Chemokine-like factor 1 (CKLF1) is a newly cloned human cytokine from PHA-stimulated U937 cells in our laboratory, which belongs to a novel gene family and has at least three alternative RNA splicing forms: CKLF2, CKLF3, and CKLF4. Former studies demonstrated that CKLF1 has a chemotaxis effect on different leukocytes both in vitro and in vivo. It can also stimulate the proliferation of mouse skeletal muscle cells and bone marrow cells. This study was designed to generate CKLF1-specific monoclonal antibody (MAb) for further exploration of its structure and function. Mice were immunized intramuscularly with naked plasmid DNA encoding CKLF1 gene with in vivo electroporation. Hybridomas were generated by the fusion of the spleenocytes to Sp2/0 myeloma cells. One hybridoma cell line designated as M4, which is stable in secreting anti- CKLF1 MAb, was generated. It belongs to the IgG1 isotype and is specific for the unique C-terminal domain of CKLFs protein. The specificity of this antibody has been assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot. Our results demonstrated that intramuscular injection of naked DNA encoding CKLF1 gene combined with in vivo electroporation is an effective and simple method to raise MAbs that can be used for basic and clinical research of CKLF1 protein.

157. Clinical Evaluation of Pain and Muscle Damage Induced by Electroporation of Skeletal Muscle in Humans

Studies in animal models ranging from mice to nonhuman primates have demonstrated intramuscular injection of naked DNA followed by electrical stimulation (electroporation) to be an efficient mean for non viral delivery of DNA in vivo. However, in order to take electroporation from the bench to the clinic, safety issues such as muscle damage and pain associated with the procedure needs to be solved. Here we report results from the first clinical safety trials to access pain and muscle damage induced by electroporation of human skeletal muscle. Electroporation was performed in the absence of a drug, and to evaluate the method potential in gene therapy/DNA vaccination in humans. Six healthy voluntaries where injected with saline in the quadriceps followed by electroporation. Single pulses or series of mono or bipolar pulses were delivered. The electroporation induced sensation was measured on a pain scale from 1–10. Muscle damage was assessed by measuring release of creatin kinase and lactate dehydrogenase. No clinical safety concerns were found and no long lasting muscle damage was observed. The results from this initial study indicates that electroporation in the tested voltage range is painful but tolerable and thus feasible in humans. The tested volt range and pulse patterns have been shown to be effective for DNA delivery and for elicting immune responses in larger animals.

Generation of monoclonal antibodies against Blo t 3 using DNA immunization with in vivo electroporation.

House dust mite allergy is closely associated with allergic diseases. Blomia tropicalis mite species is an important clinical species in the tropics. The cDNA clone encoding Blo t 3, a group 3 allergen from B. tropicalis, has been isolated in our laboratory. This study was designed to generate Blo t 3-specific monoclonal antibodies (mAbs) for the detection, characterization and purification of this allergen. Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 3 gene with in vivo electroporation. Hybridomas were generated by the fusion of the splenocytes to X63-Ag8.653 myeloma cells. Purified native Blo t 3 was obtained by mAb immuno-affinity purification and the allergenicity of native Blo t 3 was determined by human IgE enzyme-linked immunosorbent assay (ELISA). A panel of class-switched and high-affinity mAb recognizing a wide spectrum of Blo t 3 epitopes have been generated. These mAbs are useful for western immunoblot assay, sandwich ELISA and affinity purification of native Blo t 3. Allergenicity of native Blo t 3 protein was examined with 44 mite-allergic sera and approximately 57% of the tested sera had positive serum IgE reactivity to the native Blo t 3. These results demonstrated that intramuscular injection of naked DNA encoding Blo t 3 gene combined with in vivo electroporation is an effective and simple method to raise monoclonal antibodies that can be used for characterization and purification of Blo t 3.

Inhibition of adjuvant-induced arthritis by systemic tissue inhibitor of metalloproteinases 4 gene delivery.

An imbalance in the matrix metalloproteinase:tissue inhibitor of metalloproteinases (MMP:TIMP) ratio in favor of MMP appears to be an important determinant of tissue damage in arthritis. We undertook this study to explore whether reversal of this imbalance in favor of TIMP would alter this process and to examine the mechanism of this alteration. We administered human TIMP-4 by electroporation-mediated intramuscular injection of naked DNA using the rat adjuvant-induced arthritis (AIA) model. Intramuscular naked TIMP-4 gene administration resulted in high circulating TIMP-4 levels and completely abolished arthritis development in the rat AIA model. This inhibition was associated with significantly decreased MMP activity in the joint tissue as well as with significantly decreased serum and tissue tumor necrosis factor alpha levels and serum interleukin-1alpha levels compared with animals with arthritis. The mutation of cysteine at position 1 of TIMP-4 failed to block the development of AIA. Our data indicate that TIMP-4 is a potent antiinflammatory agent, and that its antiarthritis function may be mediated by MMPs. Arthritis-inhibiting effects of TIMP-4 may suggest a unique application of this gene therapy method for arthritis.

Immersion delivery of plasmid DNA: I. A study of the potentials of a liposomal delivery system in rainbow trout ( Oncorhynchus mykiss) fry

A successful regime for intramuscular injection of naked DNA is developed in fish, but the exploration of other ways of administration has not yet been studied in any detail. Immersion is a delivery route offering many advantages compared to conventional ways of administration. Applying cationic liposomes as a delivery system for DNA by this route, however, is met with severe toxicity problems. In this report, the underlying mechanisms of the acute toxicity were investigated in vivo and in vitro. The most critical factor was found to be the charge of the liposomes. Cationic liposomes above a certain threshold concentration had a lethal effect in rainbow trout fry. In contrast, similar concentrations of neutral or anionic liposomes were not toxic. Furthermore, large liposome–mucin complexes were formed upon addition of mucin to cationic liposomes. This was not observed with neutral or anionic liposomes. Lipoplexes were less toxic and interacted less with mucin compared to cationic liposomes. Hence, the mechanism of the acute toxicity in rainbow trout fry is suggested to be an interaction between the cationic liposomes and anionic components of gill mucin. The consequence is hypoxia and this is most likely the cause of acute toxicity observed in rainbow trout fry.

Electric pulses increase the immunogenicity of an influenza DNA vaccine injected intramuscularly in the mouse

Vaccination by intramuscular injection of naked DNA is very efficient in the mouse, but immunogenicity of DNA vaccines needs to be improved in man. The aim of our study was to determine in BALB/c mice if suitable electric pulses delivered to the muscle after DNA injection – a procedure called electrotransfer — could improve the immunogenicity of suboptimal doses of a DNA vaccine expressing the influenza hemagglutinin protein. The results show a significant enhancement of the cellular and antibody responses following electrotransfer for the 1- and 10-μg DNA doses, respectively, but no effect on a lower dose. At the 10-μg dose, the IgG and hemagglutination inhibition mean titres were increased 25-fold and the inter-individual variability was markedly reduced.

Myosin regulatory elements as vectors for gene transfer by intramuscular injection.

Intramuscular injection of plasmid constructs promises to be an effective way of carrying out gene therapy for muscle disorders as well as using muscle as an in vivo expression system for disorders that involve the gene product being secreted into the bloodstream. The effectiveness of this method depends on the design of the cassette used for the expression of the cDNA of the introduced gene. We tested the levels of expression achieved by a number of muscle-specific promoters and a myosin light chain enhancer when spliced to the reporter gene chloramphenicol acetyltransferase (CAT), in vitro and in vivo by injection into fast and slow muscles of the mouse. The results show that the highest levels of expression are achieved by a combination of a truncated myosin heavy chain promoter and the enhancer, and that a whole range of expression levels is obtained with the other combinations tested. The data show that a cassette based on these elements should provide efficient vectors for the introduction and expression of genes following intramuscular injection of naked DNA.

In vivo expression of an interferon-alpha gene by intramuscular injection of naked DNA.

Acid-stable type I interferons belong to a multigene family. The biologic relevance of each subtype in vivo remains unknown. We have developed an experimental model in which muscles were transfected in situ with naked DNA plasmids encoding an IFN transgene to assess the roles of individual IFN subtypes in vivo. Murine IFN-alpha 9 gene was subcloned into several mammalian expression vectors. Adult C57BL/6 mice were injected bilaterally in regenerating tibialis anterior muscles with naked DNA 5 days after muscle injury to enhance DNA uptake and expression. In the muscles of mice given the IFN-alpha 9 plasmid constructs, acid-stable IFNs were detected by bioassay using reduction in cytopathic effect of encephalomyocarditis virus-infected L929 cells. In these same muscles, IFN-alpha 9 transcripts were identified by RT-PCR, indicating that transcription had occurred. Acid-stable IFNs were detected from days 7 to 28 post-DNA inoculation. Furthermore, these proteins were found in the sera of DNA-inoculated mice. Control groups of mice given the blank expression vectors did not produce detectable IFNs in muscle or sera as determined by bioassay, nor were transcripts detected by RT-PCR. This approach now allows investigation of the effector function of individual subtypes in various murine disease models.

Accumulation of Human Apolipoprotein-E in Rat Plasma After in vivo Intramuscular Injection of Naked DNA

Naked DNA was found to be incorporated and consistently expressed after in vivo direct injection into striated muscle. In addition to the local expression of muscle-related or exogenous proteins, intramuscular direct gene transfer may be a useful tool to deliver recombinant proteins into the blood stream. However, no direct demonstration of recombinant protein secretion from muscle to the circulation has been reported thus far. We have injected a naked plasmid DNA containing the human receptor-binding defective apo-E2 cDNA, under the control of CMV promoter, into the quadriceps of Yoshida rats, affected by hereditary hypercholesterolemia and altered LDL-receptor activity. Plasma accumulation of human apo-E2 was demonstrated for at least 45 days after injection. On the contrary, the expression of the normal human apo-E3, injected into the muscle of normal Wistar rats, was demonstrated only in the area of muscular injection and not in the blood plasma. Endogenous rat apo-E expression was not affected by the exogenous human apo-E2 production. Our results demonstrate the availability of intramuscular direct gene transfer as a safe and simple method for the chronic systemic delivery of recombinant proteins to the circulation, although further improvements are needed in order to enhance the efficiency and stability of expression.