Immune Response Elicited by DNA Vaccination Using Lactococcus lactis Is Modified by the Production of Surface Exposed Pathogenic Protein

In this study, we compared immune responses elicited by DNA immunization using Lactococcus lactis or L. lactis expressing the Staphylococcus aureus invasin Fibronectin Binding Protein A (FnBPA) at its surface. Both strains carried pValac:BLG, a plasmid containing the cDNA of Beta-Lactoglobulin (BLG), and were designated LL-BLG and LL-FnBPA+ BLG respectively. A T_H2 immune response characterized by the secretion of IL-4 and IL-5 in medium of BLG reactivated splenocytes was detected after either oral or intranasal administration of LL-FnBPA+ BLG. In contrast, intranasal administration of LL-BLG elicited a T_H1 immune response. After BLG sensitization, mice previously intranasally administered with LL-BLG showed a significantly lower concentration of BLG-specific IgE than the mice non-administered. Altenatively administration of LL-FnBPA+ BLG didn''t modify the BLG-specific IgE concentration obtained after sensitization, thus confirming the T_H2 orientation of the immune response. To determine if the T_H2-skewed immune response obtained with LL-FnBpA+ BLG was FnBPA-specific or not, mice received another L. lactis strain producing a mutated form of the Listeria monocytogenes invasin Internalin A intranasally, allowing thus the binding to murine E-cadherin, and containing pValac:BLG (LL-mInlA+ BLG). As with LL-FnBPA+ BLG, LL-mInlA+ BLG was not able to elicit a T_H1 immune response. Furthermore, we observed that these difference were not due to the peptidoglycan composition of the cell wall as LL-FnBPA+ BLG, LL-mInlA+ BLG and LL-BLG strains shared a similar composition. DNA vaccination using LL-BLG elicited a pro-inflammatory T_H1 immune response while using LL-FnBPA+ BLG or LL-mInlA+ BLG elicited an anti-inflammatory T_H2 immune response.     

Use of Native Lactococci as Vehicles for Delivery of DNA into Mammalian Epithelial Cells

The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenes inlA gene can deliver a functional gene into mammalian cells. Here, we explored the potential use of noninvasive L. lactis strains as a DNA delivery vehicle. We constructed two Escherichia coli-L. lactis shuttle plasmids, pLIG:BLG1 and pLIG:BLG2, containing a eukaryotic expression cassette with the cDNA of bovine β-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis strain MG1363(pLIG:BLG1) was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in Caco-2 human colon carcinoma cells after 3 h of coincubation with (i) purified pLIG:BLG1, (ii) MG1363(pLIG:BLG1), (iii) a mix of MG1363(pLIG) and purified pLIG:BLG1, and (iv) MG1363. Both BLG cDNA and BLG expression were detected only in Caco-2 cells coincubated with MG1363(pLIG:BLG1). There was a decrease in the BLG cDNA level in Caco-2 cells between 24 and 48 h after coincubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after coincubation with MG1363(pLIG:BLG1). We conclude that lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine.     

AAV-mediated gene therapy for metabolic diseases: dosage and reapplication studies in the molybdenum cofactor deficiency model

Background

The use of food-grade lactococci as bacterial carriers to DNA delivery into epithelial cells is a new strategy to develop live oral DNA vaccine. Our goal was to develop a new plasmid, named pValac, for antigen delivery for use in lactococci. The pValac plasmid was constructed by the fusion of: i) a eukaryotic region, allowing the cloning of an antigen of interest under the control of the pCMV eukaryotic promoter to be expressed by a host cell and ii) a prokaryotic region allowing replication and selection of bacteria. In order to evaluate pValac functionality, the gfp ORF was cloned into pValac (pValac:gfp) and was analysed by transfection in PK15 cells. The applicability of pValac was demonstrated by invasiveness assays of Lactococcus lactis inlA+ strains harbouring pValac:gfp into Caco-2 cells.

Results

After transfection with pValac:gfp, we observed GFP expression in PK15 cells. L. lactis inlA+ were able to invade Caco-2 cells and delivered a functional expression cassette (pCMV:gfp) into epithelial cells.

Conclusion

We showed the potential of an invasive L. lactis harbouring pValac to DNA delivery and subsequent triggering DNA expression by epithelial cells. Further work will be to examine whether these strains are able to deliver DNA in intestinal cells in vivo.     

Use of native lactococci as vehicles for delivery of DNA into mammalian epithelial cells

The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenes inlA gene can deliver a functional gene into mammalian cells. Here, we explored the potential use of noninvasive L. lactis strains as a DNA delivery vehicle. We constructed two Escherichia coli-L. lactis shuttle plasmids, pLIG:BLG1 and pLIG:BLG2, containing a eukaryotic expression cassette with the cDNA of bovine beta-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis strain MG1363(pLIG:BLG1) was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in Caco-2 human colon carcinoma cells after 3 h of coincubation with (i) purified pLIG:BLG1, (ii) MG1363(pLIG:BLG1), (iii) a mix of MG1363(pLIG) and purified pLIG:BLG1, and (iv) MG1363. Both BLG cDNA and BLG expression were detected only in Caco-2 cells coincubated with MG1363(pLIG:BLG1). There was a decrease in the BLG cDNA level in Caco-2 cells between 24 and 48 h after coincubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after coincubation with MG1363(pLIG:BLG1). We conclude that lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine.     

In vitro non-viral gene delivery with nanofibrous scaffolds

Extracellular and intracellular barriers typically prevent non-viral gene vectors from having an effective transfection efficiency. Formulation of a gene delivery vehicle that can overcome the barriers is a key step for successful tissue regeneration. We have developed a novel core-shelled DNA nanoparticle by invoking solvent-induced condensation of plasmid DNA (β-galactosidase or GFP) in a solvent mixture [94% N,N-dimethylformamide (DMF) + 6% 1× TE buffer] and subsequent encapsulation of the condensed DNA globule in a triblock copolymer, polylactide-poly(ethylene glycol)-polylactide (L₈E₇₈L₈), in the same solvent environment. The polylactide shell protects the encapsulated DNA from degradation during electrospinning of a mixture of encapsulated DNA nanoparticles and biodegradable PLGA (a random copolymer of lactide and glycolide) to form a nanofibrous non-woven scaffold using the same solution mixture. The bioactive plasmid DNA can then be released in an intact form from the scaffold with a controlled release rate and transfect cells in vitro.     

Lactococcus lactis Expressing either Staphylococcus aureus Fibronectin-Binding Protein A or Listeria monocytogenes Internalin A Can Efficiently Internalize and Deliver DNA in Human Epithelial Cells

Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA⁺), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA⁺ and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA⁺) or its fibronectin binding domains C and D (L. lactis CD⁺). L. lactis FnBPA⁺ and L. lactis InlA⁺ showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD⁺ was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA⁺, L. lactis CD⁺, and L. lactis InlA⁺ were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA⁺, L. lactis CD⁺, and L. lactis InlA⁺ were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA⁺ or L. lactis InlA⁺. Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.The mucosal administration of bacterial carriers to deliver antigens and plasmid DNA constitutes a promising vaccination strategy. Pathogenic bacteria that have the capacity to invade cells, such as Listeria, Salmonella, and Shigella strains, have been used to deliver DNA constructs into mammalian cells (23). Nevertheless, the risk associated with possible reversion to a virulent phenotype of these pathogens is a major concern (5). Lactococcus lactis, the food-grade, gram-positive, noninvasive model bacterium, has been intensively used to deliver antigens and cytokines at the mucosal level (30). We previously showed (i) that native L. lactis can deliver a eukaryotic expression cassette coding for the bovine β-lactoglobulin (BLG), one of the major cow''s milk allergens, into mammalian epithelial Caco-2 cells, and (ii) that these cells were able to express and secrete BLG protein in its native conformation (10). Recently, we demonstrated the ability of native noninvasive L. lactis to deliver a fully functional plasmid to murine intestinal cells in vivo (2).The internalization of the bacterial carrier is a fundamental step to achieve efficient DNA delivery in eukaryotic cells (7). In order to increase DNA delivery by lactic acid bacteria (LAB), invasin genes were expressed in L. lactis. Due to the safety profile of LAB, recombinant lactococci expressing invasin genes from intracellular bacteria are attractive as potential DNA delivery vectors compared to the attenuated pathogens presently used.In this field, we previously demonstrated that L. lactis bacteria expressing the main Listeria monocytogenes invasin, internalin A (L. lactis InlA⁺), were able to invade eukaryotic cells and efficiently deliver a functional green fluorescent protein (GFP) expression plasmid into epithelial/endothelial cells (9). Even though attractive, the experimental use of lactococci expressing InlA in a mouse model has a major bottleneck: InlA, which binds to human E-cadherin (15), does not interact with murine E-cadherin. Consequently, in vivo experimental studies using lactococci expressing InlA as DNA delivery vehicles are limited to transgenic mice expressing human E-cadherin or to guinea pigs (13).Fibronectin binding protein A (FnBPA) of Staphylococcus aureus is another bacterial invasin that is involved in intracellular spreading of S. aureus in the host (27). It is a multifunctional adhesion protein having both fibrinogen and fibronectin binding capacities (24). Its N-terminal part, also called domain A, is responsible for fibrinogen (29) and elastin (20) binding, whereas its C-terminal part, including domains B, C, and D, binds to fibronectin (25). FnBPA is known to mediate adhesion to host tissue and bacterial uptake into nonphagocytic host cells (27). Its expression by L. lactis was previously shown to be sufficient to confer the ability to invade nonphagocytic cells in vitro and in vivo, while the expression of domains C and D confers invasivity only in vitro (19).In this study, we show that L. lactis bacteria expressing full-length FnBPA of S. aureus (L. lactis FnBPA⁺) or a truncated form encompassing only its C and D domains (L. lactis CD⁺) are internalized as efficiently as L. lactis InlA⁺ in the human intestinal cell line Caco-2. We also provide, for the first time, direct microscopic evidence of the intracellular location of the internalized lactococci, showing that the bacteria are heterogeneously distributed in the cell monolayer and that their number per cell can reach a surprisingly high level. However, we demonstrate that FbpA, a fibronectin binding protein from the commensal Lactobacillus acidophilus NCFM, does not mediate bacterial internalization: no difference in invasivity was observed between the wild-type (wt) strain and the mutant with fbpA inactivated. This result indicates that, although widely distributed among bacteria, fibronectin binding proteins are not universal mediators of bacterial internalization, even at low levels. Finally, we also demonstrate that, similarly to L. lactis InlA⁺, L. lactis FnBPA⁺ and L. lactis CD⁺ can efficiently deliver a eukaryotic GFP expression plasmid in Caco-2 cells and trigger GFP expression in these cells. Consequently, L. lactis FnBPA⁺ can be used for further DNA delivery experiments in vivo.     

In Vivo Electroporation of Minicircle DNA as a Novel Method of Vaccine Delivery To Enhance HIV-1-Specific Immune Responses

DNA vaccines offer advantage over conventional vaccines, as they are safer to use, easier to produce, and able to induce humoral as well cellular immune responses. Unfortunately, no DNA vaccines have been licensed for human use for the difficulties in developing an efficient and safe in vivo gene delivery system. In vivo electroporation (EP)-based DNA delivery has attracted great attention for its potency to enhance cellular uptake of DNA vaccines and function as an adjuvant. Minicircle DNA (a new form of DNA containing only a gene expression cassette and lacking a backbone of bacterial plasmid DNA) is a powerful candidate of gene delivery in terms of improving the levels and the duration of transgene expression in vivo. In this study, as a novel vaccine delivery system, we combined in vivo EP and the minicircle DNA carrying a codon-optimized HIV-1 gag gene (minicircle-gag) to evaluate the immunogenicity of this system. We found that minicircle-gag conferred persistent and high levels of gag expression in vitro and in vivo. The use of EP delivery further increased minicircle-based gene expression. Moreover, when delivered by EP, minicircle-gag vaccination elicited a 2- to 3-fold increase in cellular immune response and a 1.5- to 3-fold augmentation of humoral immune responses compared with those elicited by a pVAX1-gag positive control. Increased immunogenicity of EP-assisted minicircle-gag may benefit from increasing local antigen expression, upregulating inflammatory genes, and recruiting immune cells. Collectively, in vivo EP of minicircle DNA functions as a novel vaccine platform that can enhance efficacy and immunogenicity of DNA vaccines.     

Using In Vitro Immunomodulatory Properties of Lactic Acid Bacteria for Selection of Probiotics against Salmonella Infection in Broiler Chicks

Poultry is known to be a major reservoir of Salmonella. The use of lactic acid bacteria has become one of successful strategies to control Salmonella in poultry. The purpose of this study was to select lactic acid bacteria strains by their in vitro immunomodulatory properties for potential use as probiotics against Salmonella infection in broiler chicks. Among 101 isolated lactic acid bacteria strains, 13 strains effectively survived under acidic (pH 2.5) and bile salt (ranging from 0.1% to 1.0%) conditions, effectively inhibited growth of 6 pathogens, and adhered to Caco-2 cells. However, their in vitro immunomodulatory activities differed significantly. Finally, three strains with higher in vitro immunomodulatory properties (Lactobacillus plantarum PZ01, Lactobacillus salivarius JM32 and Pediococcus acidilactici JH231) and three strains with lower in vitro immunomodulatory activities (Enterococcus faecium JS11, Lactobacillus salivarius JK22 and Lactobacillus salivarius JM2A1) were compared for their inhibitory effects on Salmonella adhesion and invasion to Caco-2 cells in vitro and their antimicrobial effects in vivo. The former three strains inhibited Salmonella adhesion and invasion to Caco-2 cells in vitro, reduced the number of Salmonella in intestinal content, spleen and liver, reduced the levels of lipopolysaccharide-induced TNF-α factor (LITAF), IL-1β, IL-6 and IL-12 in serum and increased the level of IL-10 in serum during a challenge study in vivo more efficiently than the latter three strains. These results suggest that in vitro immunomodulatory activities could be used as additional parameters to select more effective probiotics as feed supplements for poultry.     

Use of a Dual Reporter Plasmid to Demonstrate Bactofection with an Attenuated AroA- Derivative of Pasteurella multocida B:2

A reporter plasmid pSRG has been developed which expresses red fluorescent protein (RFP) from a constitutive prokaryotic promoter within Pasteurella multocida B:2 and green fluorescent protein (GFP) from a constitutive eukaryotic promoter within mammalian cells. This construct has been used to determine the location and viability of the bacteria when moving from the extracellular environment into the intracellular compartment of mammalian cells. Invasion assays with embryonic bovine lung (EBL) cells and an attenuated AroA^- derivative of Pasteurella multocida B:2 (strain JRMT12), harbouring the plasmid pSRG, showed that RFP-expressing bacteria could be detected intracellularly at 3 h post-invasion. At this stage, some EBL cells harbouring RFP-expressing bacteria were observed to express GFP simultaneously, indicating release of the plasmid into the intracellular environment. At 5 h post-invasion, more EBL cells were expressing GFP, while still harbouring RFP-expressing bacteria. Concurrently, some EBL cells were shown to express only GFP, indicating loss of viable bacteria within these cells. These experiments proved the functionality of the pSRG dual reporter system and the potential of P. multocida B:2 JRMT12 for bactofection and delivery of a DNA vaccine.     

Hypoxia Increases Mouse Satellite Cell Clone Proliferation Maintaining both In Vitro and In Vivo Heterogeneity and Myogenic Potential

Satellite cells (SCs) are essential for postnatal muscle growth and regeneration, however, their expansion potential in vitro is limited. Recently, hypoxia has been used to enhance proliferative abilities in vitro of various primary cultures. Here, by isolating SCs from single mouse hindlimb skeletal myofibers, we were able to distinguish two subpopulations of clonally cultured SCs (Low Proliferative Clones - LPC - and High Proliferative Clones - HPC), which, as shown in rat skeletal muscle, were present at a fixed proportion. In addition, culturing LPC and HPC at a low level of oxygen we observed a two fold increased proliferation both for LPC and HPC. LPC showed higher myogenic regulatory factor (MRF) expression than HPC, particularly under the hypoxic condition. Notably, a different myogenic potential between LPC and HPC was retained in vivo: green fluorescent protein (GFP)+LPC transplantation in cardiotoxin-injured Tibialis Anterior led to a higher number of new GFP+muscle fibers per transplanted cell than GFP+HPC. Interestingly, the in vivo myogenic potential of a single cell from an LPC is similar if cultured both in normoxia and hypoxia. Therefore, starting from a single satellite cell, hypoxia allows a larger expansion of LPC than normal O₂ conditions, obtaining a consistent amount of cells for transplantation, but maintaining their myogenic regeneration potential.     

Skin Electroporation: Effects on Transgene Expression,DNA Persistence and Local Tissue Environment

Background

Electrical pulses have been used to enhance uptake of molecules into living cells for decades. This technique, often referred to as electroporation, has become an increasingly popular method to enhance in vivo DNA delivery for both gene therapy applications as well as for delivery of vaccines against both infectious diseases and cancer. In vivo electrovaccination (gene delivery followed by electroporation) is currently being investigated in several clinical trials, including DNA delivery to healthy volunteers. However, the mode of action at molecular level is not yet fully understood.

Methodology/Principal Findings

This study investigates intradermal DNA electrovaccination in detail and describes the effects on expression of the vaccine antigen, plasmid persistence and the local tissue environment. Gene profiling of the vaccination site showed that the combination of DNA and electroporation induced a significant up-regulation of pro-inflammatory genes. In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression. However, when the more immunogenic prostate specific antigen (PSA) was co-administered, PSA-specific T cells were induced and concurrently the luciferase expression became undetectable. Electroporation did not affect the long-term persistence of the PSA-expressing plasmid.

Conclusions/Significance

This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid. As the described vaccination approach is currently being evaluated in clinical trials, the data provided will be of high significance.     

Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells

This report describes the delivery of plasmid DNA containing either the β-glucuronidase (GUS) or the green fluorescent protein (GFP) reporter gene into intact plant cells of bamboo callus, lilium scales, and Nicotiana benthamiana suspension culture cells. By first plasmolyzing the tissues or cells with 0.4 m sucrose in the presence of plasmid DNA, electroporation effectively delivers plasmid DNA into the intact plant cells. Transient expression of the GUS gene, as revealed by histochemical assays, showed the presence of blue-staining areas in the electroporated tissues. A short exposure of cells to 2% DMSO (dimethyl sulfoxide) prior to plasmolysis elevated the level of transient GUS activity. When plasmid DNA containing a synthetic GFP gene was used, a strong green fluorescence was observed in N. benthamiana suspension culture cells that were subjected to plasmolysis and electroporation. These results suggest that plasmolysis brings the plasmid DNA into the void space that is in close vicinity to the plasmalemma, allowing electroporation to efficiently deliver the plasmid DNA into intact plant cells. Received: 15 June 1998 / Revision received: 18 August 1998 / Accepted: 28 August 1998     

Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G²). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes—two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit–granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.     

Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents

Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. In this study, we developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. The regulation system incorporated in the device allowed a real-time control of the cavitation level during sonoporation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (pEGFP-C1) in adherent and non-adherent cell lines. The transfection efficiency of the device was compared to those observed with lipofection and nucleofection methods. In both adherent and non-adherent cell lines, the sonoporation device allowed high rate of transfection of pEGFP-C1 (40–80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. The transfection efficiency and toxicity of sonoporation on the non-adherent cell lines Jurkat and K562 were similar to those of nucleofection, while these two cell lines were resistant to transfection by lipofection. Moreover, sonoporation was used to produce three stably transfected human lymphoma and leukemia lines. Significant transfection efficiency was also observed in two fresh samples of human acute myeloid leukemia cells. In conclusion, we developed a user-friendly and cost-effective ultrasound device, well adapted for routine in vitro high-yield transfection experiments and which does not require the use of any transfection reagent or gas micro-bubbles.     

A GFP Expressing Influenza A Virus to Report In Vivo Tropism and Protection by a Matrix Protein 2 Ectodomain-Specific Monoclonal Antibody

The severity of influenza-related illness is mediated by many factors, including in vivo cell tropism, timing and magnitude of the immune response, and presence of pre-existing immunity. A direct way to study cell tropism and virus spread in vivo is with an influenza virus expressing a reporter gene. However, reporter gene-expressing influenza viruses are often attenuated in vivo and may be genetically unstable. Here, we describe the generation of an influenza A virus expressing GFP from a tri-cistronic NS segment. To reduce the size of this engineered gene segment, we used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. GFP and nuclear export protein coding information were fused in frame with the truncated NS1 open reading frame and separated from each other by 2A self-processing sites. The resulting PR8-NS1(1–73)GFP virus was successfully rescued and replicated as efficiently as the parental PR8 virus in vitro and was slightly attenuated in vivo. Flow cytometry-based monitoring of cells isolated from PR8-NS1(1–73)GFP virus infected BALB/c mice revealed that GFP expression peaked on day two in all cell types tested. In particular respiratory epithelial cells and myeloid cells known to be involved in antigen presentation, including dendritic cells (CD11c⁺) and inflammatory monocytes (CD11b⁺ GR1⁺), became GFP positive following infection. Prophylactic treatment with anti-M2e monoclonal antibody or oseltamivir reduced GFP expression in all cell types studied, demonstrating the usefulness of this reporter virus to analyze the efficacy of antiviral treatments in vivo. Finally, deep sequencing analysis, serial in vitro passages and ex vivo analysis of PR8-NS1(1–73)GFP virus, indicate that this virus is genetically and phenotypically stable.     

A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors

Background

The gene delivery vector for DNA-based therapy should ensure its transfection efficiency and safety for clinical application. The Micro-Linear vector (MiLV) was developed to improve the limitations of traditional vectors such as viral vectors and plasmids.

Methods

The MiLV which contained only the gene expression cassette was amplified by polymerase chain reaction (PCR). Its cytotoxicity, transfection efficiency in vitro and in vivo, duration of expression, pro-inflammatory responses and potential application for Epstein-Barr virus (EBV) positive tumors were evaluated.

Results

Transfection efficiency for exogenous genes transferred by MiLV was at least comparable with or even greater than their corresponding plasmids in eukaryotic cell lines. MiLV elevated the expression and prolonged the duration of genes in vitro and in vivo when compared with that of the plasmid. The in vivo pro-inflammatory response of MiLV group was lower than that of the plasmid group. The MEKK1 gene transferred by MiLV significantly elevated the sensitivity of B95-8 cells and transplanted tumor to the treatment of Ganciclovir (GCV) and sodium butyrate (NaB).

Conclusions

The present study provides a safer, more efficient and stable MiLV gene delivery vector than plasmid. These advantages encourage further development and the preferential use of this novel vector type for clinical gene therapy studies.     

Biodistribution and blood clearance of plasmid DNA administered in arginine peptide complexes

Background

Peptide/DNA complexes have great potential as non-viral methods for gene delivery. Despite promising results for peptide-mediated gene delivery technology, an effective systemic peptide-based gene delivery system has not yet been developed.

Methods

This study used pCMV-Luc as a model gene to investigate the biodistribution and the in vivo efficacy of arginine peptide-mediated gene delivery by polymerase chain reaction (PCR).

Results

Plasmid DNA was detected in all organs tested 1 h after intraperitoneal administration of arginine/DNA complexes, indicating that the arginine/DNA complexes disseminated widely through the body. The plasmid was primarily detected in the spleen, kidney, and diaphragm 24 h post administration. The mRNA expression of plasmid DNA was noted in the spleen, kidney, and diaphragm for up to 2 weeks, and in the other major organs, for at least 1 week. Blood clearance studies showed that injected DNA was found in the blood as long as 6 h after injection.

Conclusions

Taken together, our results demonstrated that arginine/DNA complexes are stable in blood and are effective for in vivo gene delivery. These findings suggest that intraperitoneal administration of arginine/DNA complexes is a promising tool in gene therapy.     

Dual recombinant Lactococcus lactis for enhanced delivery of DNA vaccine reporter plasmid pPERDBY

Food grade Lactococcus lactis has been widely used as an antigen and DNA delivery vehicle. We have previously reported the use of non‐invasive L. lactis to deliver the newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, construction of dual recombinant L. lactis expressing internalin A of Listeria monocytogenes and harboring pPERDBY (LL InlA + pPERDBY) to enhance the efficiency of delivery of DNA by L. lactis is outlined. After confirmation and validation of LL InlA + pPERDBY, its DNA delivery potential was compared with previously developed non‐invasive r‐ L. lactis::pPERDBY. The use of invasive L. lactis resulted in around threefold increases in the number of enhanced green fluorescent protein‐expressing Caco‐2 cells. These findings reinforce the prospective application of invasive strain of L. lactis for delivery of DNA/RNA and antigens.