Enhancement of protein vaccine potency by in vivo electroporation mediated intramuscular injection

Protein-based vaccines have emerged as a potentially promising approach for the generation of antigen-specific immune responses. However, due to their low immunogenicity, there is a need for innovative approaches to enhance protein-based vaccine potency. One approach to enhance protein-based vaccine potency is the employment of toll-like receptor ligands, such as CpG oligonucleotides, to activate the antigen-specific T cell immune responses. Another approach involves employing a method capable of improving the delivery of protein-based vaccine intramuscularly to lead to the slow release of the protein, resulting in improved vaccine potency. In the current study, we aimed to determine whether intramuscular injection of protein-based vaccines in conjunction with CpG followed by electroporation can lead to increased delivery of the protein-based vaccine into muscle cells, resulting in enhanced protein-based vaccine potency. We found that intramuscular injection followed by electroporation can effectively transduce the protein-based vaccine into the muscle cells. Furthermore, we found that intramuscular vaccination with OVA protein in combination with CpG followed by electroporation generates the best OVA-specific CD8+ T cell immune responses as well as the best protective and therapeutic antitumor effects in vaccinated mice. CD8+ T cells were found to play an important role in the observed protective antitumor effects generated by the vaccination. Similar results were observed using the HPV-16 E7 protein-based vaccination system. Thus, our data indicate that intramuscular administration of protein-based vaccines in conjunction with CpG followed by electroporation can significantly enhance the antigen-specific CD8+ T cell immune responses. The clinical implications of the study are discussed.     

Biodistribution, persistence and lack of integration of a multigene HIV vaccine delivered by needle-free intradermal injection and electroporation

It is likely that gene-based vaccines will enter the human vaccine area soon. A few veterinary vaccines employing this concept have already been licensed, and a multitude of clinical trials against infectious diseases or different forms of cancer are ongoing. Highly important when developing novel vaccines are the safety aspects and also new adjuvants and delivery techniques needs to be carefully investigated so that they meet all short- and long-term safety requirements. One novel in vivo delivery method for plasmid vaccines is electroporation, which is the application of short pulses of electric current immediately after, and at the site of, an injection of a genetic vaccine. This method has been shown to significantly augment the transfection efficacy and the subsequent vaccine-specific immune responses. However, the dramatic increase in delivery efficacy offered by electroporation has raised concerns of potential increase in the risk of integration of plasmid DNA into the host genome. Here, we demonstrate the safety and lack of integration after immunization with a high dose of a multigene HIV-1 vaccine delivered intradermally using the needle free device Biojector 2000 together with electroporation using Derma Vax™ DNA Vaccine Skin Delivery System. We demonstrate that plasmids persist in the skin at the site of injection for at least four months after immunization. However, no association between plasmid DNA and genomic DNA could be detected as analyzed by qPCR following field inversion gel electrophoresis separating heavy and light DNA fractions. We will shortly initiate a phase I clinical trial in which healthy volunteers will be immunized with this multiplasmid HIV-1 vaccine using a combination of the delivery methods jet-injection and intradermal electroporation.     

细胞浓度对穿孔 K562细胞活力和膜电压的影响

目的研究细胞浓度变化对穿孔K562细胞活力和膜电压的影响.方法在相同的强脉冲电场作用下,用长春新碱处理不同浓度的人红白血病K562细胞,用电穿孔仪对其进行电穿孔处理,然后检测作用后的细胞活力.结果不同浓度的K562细胞活力不同,且活力比和细胞浓度呈非线性关系.实验结果也间接表明在不同细胞浓度下,穿孔细胞膜膜电压变化是不相同的.结论细胞浓度是影响K562细胞活力和膜电压变化的一个重要因素.     

Sonoporation Mediated Immunogene Therapy of Solid Tumors

Development of gene-based therapies for the treatment of inherited and acquired diseases, including cancer, has seen renewed interest in the use of nonviral vectors coupled to physical delivery modalities. Low-frequency ultrasound (US), with a well-established record in a clinical setting, has the potential to deliver DNA efficiently, accurately and safely. Optimal in vivo parameters for US-mediated delivery of naked plasmid DNA were established using the firefly luciferase reporter gene construct. Optimized parameters were used to administer a therapeutic gene construct, coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 costimulatory molecule, to growing murine fibrosarcoma tumors. Tumor progression and animal survival was monitored throughout the study and the efficacy of the US-mediated gene therapy determined and compared with an electroporation-based approach. Optimal parameters for US-mediated delivery of plasmid DNA to tumors were deduced to be 1.0 W/cm² at 20% duty cycle for 5 min (60 J/cm²). In vivo US-mediated gene therapy resulted in a 55% cure rate in tumor-bearing animals. The immunological response invoked was cell mediated, conferring resistance against re-challenge and resistance to tumor challenge after transfer of splenocytes to naïve animals. US treatment was noninjurious to treated tissue, whereas therapeutic efficacy was comparable to an electroporation-based approach. US-mediated delivery of an immune-gene construct to growing tumors was therapeutically effective. Sonoporation has the potential to be a major factor in the development of nonviral gene delivery approaches. (E-mail: geraldc@iol.ie)     

Membrane electroporation increases photodynamic effects

Electroporation of membranes is used widely for drug delivery. Photodynamic action consists of three main steps: (A) incorporation of the sensitizer through a membrane into cells; (B) photoxidation of cell constituents and (C) reoxidation of the reduced sensitizer by oxygen etc. The mechanisms of (B) and (C) have been studied widely in past decades. However, the mechanism of transport (A) of sensitizers to targets as the rate limiting step has not been studied to the same extent. Therefore we applied membrane and cell wall electroporation of human histiocytic lymphoma U937 and Saccharomyces cerevisiae cells in order to incorporate rapidly the reliable photodynamic agents thiopyronine, protoporphyrin, zinc phthalocyanine, copper phthalocyanine sulfonate, adriamycin and daunomysin, well-tried cytostatic agents. Depending on field strength and pulse width, 50–90% of cells become electroporated, then the dye diffuses rapidly into the cells, which reseal their membranes over a period of 6–10 min. Illumination for 10–15 min destroys all resealed cells faster than the same amount of unporated cells as in the case of the control (without pulse treatment) either by oxidation of cell components caused by excited dyes or singlet oxygen treatment. By this synergism of electroporation and photodynamic action at the same time, it is possible to kill all cells in a much shorter time than under usual conditions, e.g. in the control suspension. A combination of electrochemotherapeutic needle electrodes with a light conductor for a LASER connection will be effective for therapy.     

SEA和喉癌MAGE-A3构建的真核质粒pMAGEA3-IRES-SEA经过电穿孔转染在小鼠体内的转录

目的检测真核质粒pMAGEA3-IRES-SEA经过电穿孔转染在小鼠中的转录。方法将葡萄球菌肠毒素A(staphylococcal endotoxin A,SEA)和喉癌来源的黑色素瘤抗原A3(melanomaassociated antigen A3,MAGE-A3),构建成基因共表达的真核质粒pMAGEA3-IRES-SEA。将pMAGEA3-IRES-SEA用电转染的方法免疫BALB/C小鼠单侧股四头肌,每2周1次,每次注射50μg,共免疫3次。末次免疫2周后,取注射部位组织,用荧光定量PCR(qRT-PCR)法检测目的基因在注射部位肌肉中的转录情况。结果在实验小鼠注射部位的骨骼肌内检测到SEA、MAGE-A3基因转录mRNA。结论所构建的pMAGEA3-IRES-SEA真核表达质粒,可通过电转染的方式,在小鼠体内能有效地转录。这为研究该质粒通过小鼠的体内转录蛋白的表达,诱导机体细胞免疫、体液免疫来清除喉癌,奠定了理论基础。     

A Phase 1 clinical trial of a DNA vaccine for Venezuelan equine encephalitis delivered by intramuscular or intradermal electroporation

Venezuelan equine encephalitis virus (VEEV), a mosquito-borne alphavirus, causes periodic epizootics in equines and is a recognized biological defense threat for humans. There are currently no FDA-licensed vaccines against VEEV. We developed a candidate DNA vaccine expressing the E3-E2-6K-E1 genes of VEEV (pWRG/VEE) and performed a Phase 1 clinical study to assess the vaccine's safety, reactogenicity, tolerability, and immunogenicity when administered by intramuscular (IM) or intradermal (ID) electroporation (EP) using the Ichor Medical Systems TriGrid™ Delivery System. Subjects in IM-EP groups received 0.5 mg (N = 8) or 2.0 mg (N = 9) of pWRG/VEE or a saline placebo (N = 4) in a 1.0 ml injection. Subjects in ID-EP groups received 0.08 mg (N = 8) or 0.3 mg (N = 8) of DNA or a saline placebo (N = 4) in a 0.15 ml injection. Subjects were monitored for a total period of 360 days. No vaccine- or device-related serious adverse events were reported. Based on the results of a subject questionnaire, the IM- and ID-EP procedures were both considered to be generally acceptable for prophylactic vaccine administration, with the acute tolerability of ID EP delivery judged to be greater than that of IM-EP delivery. All subjects (100%) in the high and low dose IM-EP groups developed detectable VEEV-neutralizing antibodies after two or three administrations of pWRG/VEE, respectively. VEEV-neutralizing antibody responses were detected in seven of eight subjects (87.5%) in the high dose and five of eight subjects (62.5%) in the low dose ID-EP groups after three vaccine administrations. There was a correlation between the DNA dose and the magnitude of the resulting VEEV-neutralizing antibody responses for both IM and ID EP delivery. These results indicate that pWRG/VEE delivered by either IM- or ID-EP is safe, tolerable, and immunogenic in humans at the evaluated dose levels.Clinicaltrials.gov registry number NCT01984983.     

Factors affecting electrode-gel-skin interface impedance in electrical impedance tomography

The magnitude, mismatch and temporal variations of the electrode-gel-skin interface impedance can cause problems in electrical impedance tomography (EIT) measurement. It is shown that at the high frequencies generally encountered in EIT the capacitive properties of the electrode interface, and especially those of the skin, are of primary importance. A wide range of techniques are reviewed that could possibly be used to minimise these problems. These techniques include the use of skin preparation, penetration enhancers, temperature and electrical impulses. Although several of these techniques appear very attractive, they are not without serious potential drawbacks. A combination of some of these techniques may well hold the key to success.     

Efficient transfer of antibodies into mammalian cells by electroporation

An optimized protocol for electroporation is described which is suitable for introducing antibodies into mammalian cells. The methods results in the uptake of detectable amounts of antibodies in 80% of the cells and in 40% large amounts are introduced. As an example, cell cycle activity (transition from the G1 to S phase) was inhibited by the introduction of monoclonal antibodies against G1-specific cyclin D1 into CV-1 and MCF7 cells. This specific antibody mediated inhibition of cellular function did not affect the viability of the cells since they recovered from the inhibition after some time. While approaching the efficiency of microinjection, the new protocol for electroporation of antibodies additionally permits treatment of the larger number of cells which are required for biochemical analyses.