Lipofection-Based Delivery of DNA Vaccines.

The preventive and therapeutic potential of DNA vaccines combined with benefits of lipid-based delivery (lipofection) allow efficient nucleic acid transfer and immunization applicable in treatment of infections, cancer or autoimmune disorders. Lipofecting compositions consisting of cationic and neutral lipids can be used for both in vitro and in vivo applications and may also play the role of adjuvants. Here we describe a simple protocol of DNA vaccine carrier preparation based on cationic polyprenyl derivatives (PTAI-trimethylpolyprenylammonium iodides) and commonly used helper lipids with use of basic laboratory equipment. Such formulas have proven effective for immunization of animals as well as for cell transfection.

The varied ability of grains to synthesize and catabolize ABA is one of the factors affecting dormancy and its release by after-ripening in imbibed triticale grains of cultivars with different pre-harvest sprouting susceptibilities.

Abscisic acid (ABA) is a phytohormone involved in the acquisition of primary dormancy during seeds maturation as well as dormancy maintenance in imbibed seeds. After imbibition, the ABA content decreased to a much lower level in embryos of freshly harvested triticale grains of the Leontino cultivar, which is more susceptible to pre-harvest sprouting (PHS) than embryos of the Fredro cultivar. Lower ABA content in the Leontino cultivar resulted from increased expression of TsABA8'OH1 and TsABA8'OH2, which encode ABA 8'-hydroxylase and are involved in ABA catabolism. Higher ABA content and maintenance of dormancy in Fredro grains were correlated with intensified ABA biosynthesis, which resulted from higher expression of TsNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase. These results suggest that grains of triticale cultivars with different resistance to PHS vary in their ability to metabolize ABA after imbibition. After-ripening did not affect the ABA content in embryos of dry grains of either triticale cultivar. However, after-ripening caused dormancy release in Fredro grains and significantly affected the ABA content and the rate of its metabolism after imbibition. A more rapid decline in ABA content in imbibed Fredro grains was accompanied by decreased transcript levels of TsNCED1 as well as increased expression of TsABA8'OH1 and TsABA8'OH2. Thus, after-ripening may affect dormancy of grains through reduction of the ABA biosynthesis rate and intensified ABA catabolism. Overexpression of TsNCED1 in tobacco increases ABA content and delays germination, while overexpression of TsABA8'OH2 decreases ABA content, accelerates germination, and reduces the sensitivity to ABA of transgenic seeds compared to seeds of wild-type plants. Therefore, these genes might play an important role in the regulation of triticale grain dormancy, thus affecting susceptibility to PHS.

Effective usage of cationic derivatives of polyprenols as carriers of DNA vaccines against influenza virus.

BACKGROUND: Cationic derivatives of polyprenols (trimethylpolyprenylammonium iodides - PTAI) with variable chain length between 6 and 15 isoprene units prepared from naturally occurring poly-cis-prenols were tested as DNA vaccine carriers in chickens and mice. This study aimed to investigate if PTAI could be used as an efficient carrier of a DNA vaccine. METHODS: Several vaccine mixtures were prepared by combining different proportions of the vaccine plasmid (carrying cDNA encoding a vaccine antigen, hemagglutinin from H5N1 influenza virus) and various compositions of PTAI. The vaccines were delivered by intramuscular injection to either chickens or mice. The presence of specific antibodies in sera collected from the immunized animals was analyzed by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test. RESULTS: The mixtures of PTAI with helper lipids, such as DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), DC-cholesterol [{3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol} hydrochloride] or DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) induced strong humoral response to the antigen encoded by the DNA vaccine plasmid. CONCLUSION: The animal immunization results confirmed that PTAI compositions, especially mixtures of PTAI with DOPE and DC-cholesterol, do work as effective carriers of DNA vaccines, comparable to the commercially available lipid transfection reagent.

Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody.

Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display peptide libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitate epitope mapping. Based on the sequences of the affinity- selected polypeptides and the structural model of HA the epitope was located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibiting activity and its antigenic specificity. Additionally, total RNA isolated from the hybridoma cell line secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed differences in specificity were discussed.

Plant expression systems for production of hemagglutinin as a vaccine against influenza virus.

Many examples of a successful application of plant-based expression systems for production of biologically active recombinant proteins exist in the literature. These systems can function as inexpensive platforms for the large scale production of recombinant pharmaceuticals or subunit vaccines. Hemagglutinin (HA) is a major surface antigen of the influenza virus, thus it is in the centre of interests of various subunit vaccine engineering programs. Large scale production of recombinant HA in traditional expression systems, such as mammalian or insect cells, besides other limitations, is expensive and time-consuming. These difficulties stimulate an ever-increasing interest in plant-based production of this recombinant protein. Over the last few years many successful cases of HA production in plants, using both transient and stable expression systems have been reported. Various forms of recombinant HA, including monomers, trimers, virus like particles (VLPs) or chimeric proteins containing its fusion with other polypeptides were obtained and shown to maintain a proper antigenicity. Immunizations of animals (mice, ferrets, rabbits or chickens) with some of these plant-derived hemagglutinin variants were performed, and their effectiveness in induction of immunological response and protection against lethal challenge with influenza virus demonstrated. Plant-produced recombinant subunit vaccines and plant-made VLPs were successfully tested in clinical trials (Phase I and II) that confirmed their tolerance and immunogenicity.

DNA vaccines against influenza.

Genetic vaccine technology has been considerably developed within the last two decades. This cost effective and promising strategy can be applied for therapy of cancers and for curing allergy, chronic and infectious diseases, such as a seasonal and pandemic influenza. Despite numerous advantages, several limitations of this technology reduce its performance and can retard its commercial exploitation in humans and its veterinary applications. Inefficient delivery of the DNA vaccine into cells of immunized individuals results in low intracellular supply of suitable expression cassettes encoding an antigen, in its low expression level and, in turn, in reduced immune responses against the antigen. Improvement of DNA delivery into the host cells might significantly increase effectiveness of the DNA vaccine. A vast array of innovative methods and various experimental strategies have been applied in order to enhance the effectiveness of DNA vaccines. They include various strategies improving DNA delivery as well as expression and immunogenic potential of the proteins encoded by the DNA vaccines. Researchers focusing on DNA vaccines against influenza have applied many of these strategies. Recent examples of the most successful modern approaches are discussed in this review.

An immunosensor based on antibody binding fragments attached to gold nanoparticles for the detection of peptides derived from avian influenza hemagglutinin H5.

This paper concerns the development of an immunosensor for detection of peptides derived from avian influenza hemagglutinin H5. Its preparation consists of successive gold electrode modification steps: (i) modification with 1,6-hexanedithiol and gold colloidal nanoparticles; (ii) immobilization of antibody-binding fragments (Fab') of anti-hemagglutinin H5 monoclonal antibodies Mab 6-9-1 via S-Au covalent bonds; and (iii) covering the remaining free space on the electrode surfaces with bovine serum albumin. The interactions between Fab' fragments and hemagglutinin (HA) variants have been explored with electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)6](3-/4-) as an electroactive marker. The immunosensor was able to recognize three different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: H1 subunit (17-340 residues) of A/swan/Poland/305-135V08/2006, the long HA (17-530 residues) A/Bar-headed Goose/Qinghai/12/2005 and H1 subunit (1-345 residues) of A/Vietnam/1194/2004. The strongest response has been observed for the long variant with detection limit of 2.2 pg/mL and dynamic range from 4.0 to 20.0 pg/mL.

DNA probe modified with 3-iron bis(dicarbollide) for electrochemical determination of DNA sequence of Avian Influenza Virus H5N1.

In this work, we report on oligonucleotide probes bearing metallacarborane [3-iron bis(dicarbollide)] redox label, deposited on gold electrode for electrochemical determination of DNA sequence derived from Avian Influenza Virus (AIV), type H5N1. The oligonucleotide probes containing 5'-terminal NH2 group were covalently attached to the electrode, via NHS/EDC coupling to 3-mercaptopropionic acid SAM, previously deposited on the surface of gold. The changes in redox activity of Fe(III) centre of the metallacarborane complex before and after hybridization process was used as analytical signal. The signals generated upon hybridization with targets such as complementary or non-complementary 20-mer ssDNA or various PCR products consisting of 180-190 bp (dsDNA) were recorded by Osteryoung square-wave voltammetry (OSWV). The developed system was very sensitive towards targets containing sequence complementary to the probe with the detection limit estimated as 0.03 fM (S/N=3.0) and 0.08 fM (S/N=3.0) for 20-mer ssDNA and for dsDNA (PCR product), respectively. The non-complementary targets generated very weak responses. Furthermore, the proposed genosensor was suitable for discrimination of PCR products with different location of the complementarity region.

Transgenic tobacco plants as production platform for biologically active human interleukin 2 and its fusion with proteinase inhibitors.

Transgenic plants offer a low-cost approach for the production of pharmaceutically important and commercially valuable recombinant proteins. Our studies were focused on the plant-based production of human interleukin 2 (hIL-2) and its fusion with proteinase inhibitors, either SPI2 from Galleria mellonella or CMTI from Cucurbita maxima. Finally, five plant expression cassettes were obtained. Three of them contained the single cDNA encoding CMTI I, SPI2 and hIL-2, respectively, while two of them contained the translational fusion, SPI2::hIL-2 and CMTI::hIL-2. In all cases, the transgenes were controlled by the RbcS1 promoter and terminator and the recombinant proteins were targeted to the endoplasmic reticulum. After tobacco transformation, five groups of transgenic plants were obtained and analysed. The level of recombinant proteins was estimated either by Western blot or by ELISA. The biological activity of plant-produced hIL-2 alone or in a fusion with SPI2 or CMTI was confirmed using the mammalian cells proliferation assay. The activities of proteinase inhibitors were confirmed in proteolysis assay using azocoll as a substrate. The usefulness of using proteinase inhibitor CMTI I in a fusion with hIL-2 as a protective agent against trypsin digestion was demonstrated.

Recombinant cytokines from plants.

Plant-based platforms have been successfully applied for the last two decades for the efficient production of pharmaceutical proteins. The number of commercialized products biomanufactured in plants is, however, rather discouraging. Cytokines are small glycosylated polypeptides used in the treatment of cancer, immune disorders and various other related diseases. Because the clinical use of cytokines is limited by high production costs they are good candidates for plant-made pharmaceuticals. Several research groups explored the possibilities of cost-effective production of animal cytokines in plant systems. This review summarizes recent advances in this field.

Use of randomly mutagenized genomic cDNA banks of potato spindle tuber viroid to screen for viable versions of the viroid genome.

In an effort to study sequence space allowing the recovery of viable potato spindle tuber viroid (PSTVd) variants we have developed an in vivo selection (Selex) method to produce and bulk-inoculate by agroinfiltration large PSTVd cDNA banks in which a short stretch of the genome is mutagenized to saturation. This technique was applied to two highly conserved 6 nt-long regions of the PSTVd genome, the left terminal loop (TL bank) and part of the polypurine stretch in the upper strand of pre-melting loop 1 (PM1 bank). In each case, PSTVd accumulation was observed in a large fraction of bank-inoculated tomato plants. Characterization of the progeny molecules showed the recovery of the parental PSTVd sequence in 89 % (TL bank) and 18 % (PM1 bank) of the analysed plants. In addition, viable and genetically stable PSTVd variants with mutations outside of the known natural variability of PSTVd were recovered in both cases, although at different rates. In the case of the TL region, mutations were recovered at five of the six mutagenized positions (357, 358, 359, 1 and 3 of the genome) while for the PM1 region mutations were recovered at all six targeted positions (50-55), providing significant new insight on the plasticity of the PSTVd genome.

Recombinant mouse granulocyte-macrophage colony-stimulating factor is glycosylated in transgenic tobacco and maintains its biological activity.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with many important applications and, due to its immunostimulatory properties, could also be used as a vaccine adjuvant. A simple strategy to produce recombinant mouse GM-CSF (mGM-CSF) in transgenic Nicotiana tabacum plants was used in this study. The mGM-CSF cDNA followed by the sequence encoding endoplasmic reticulum retention signal (KDEL) was cloned into the ImpactVector under the control of the strong promoter from the gene encoding a small subunit of Rubisco. In transgenic plants the accumulation level of recombinant mGM-CSF varied in the individual transformants from 8 to 19 microg/g of fresh leaf tissue, which makes up to 0.22% of total soluble protein. In most analyzed plants, the apparent molecular weight of the recombinant protein was larger than predicted due to its N-glycosylation, presumably in 2 sites. The recombinant plant-produced murine GM-CSF retained its biological activity as confirmed in vitro in proliferation assay using a mouse cell line, which is growth-dependent on GM-CSF.

[Plant-based production of recombinant cytokines]. / Wykorzystanie systemów roslinnych do produkcji rekombinowanych cytokin.

Modern biotechnology has led to increase of interest in producing pharmaceutically important and commercially valuable proteins in plants. Plants can efficiently produce recombinant proteins in large quantities and plant-derived proteins are free of human disease factors. In general, plant systems can now be used to produce a variety of proteins, including antibodies, cytokines, blood substitutes, vaccines and others. Cytokines are small proteins secreted by animal cells. They mediate and regulate immunity, inflammation, and hematopoiesis. Due to their properties many cytokines have medical application, mostly in treatment of virus infections, cancer symptoms and diseases affecting the immune system. However clinical deployment of cytokines is limited by the high production costs. Recently, several research groups reported using various plant-based expression systems for the production of biologically active mammalian cytokines.