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1
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Viroids: unusual small pathogenic RNAs.

100%
Góra-Sochacka A.
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vol. 51
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issue 3
587-607
EN
Viroids are small (about 300 nucleotides), single-stranded, circular, non-encapsidated pathogenic RNA molecules. They do not code for proteins and thus depend on plant host enzymes for their replication and other functions. They induce plant diseases by direct interaction with host factors but the mechanism of pathogenicity is still unknown. They can alter the expression of selected plant genes important for growth and development. Viroids belong to two families, the Avsunviroidae and the Pospiviroidae. Viroids of the Avsunviroidae family adopt a branched or quasi rod-like secondary structure in their native state. Members of the Pospiviroidae family adopt a rod-like secondary structure. In such native structures five structural/functional domains have been identified: central (C), pathogenicity, variable and two terminal domains. The central conserved region (CCR) within the C domain characterizes viroids of the Pospiviroidae. Specific secondary structures of this region play an important role in viroid replication and processing. Viroids of the Avsunviroidae family lack a CCR but possess self-cleaving properties by forming hammerhead ribozyme structures; they accumulate and replicate in chloroplasts, whereas members of the Pospiviroidae family have a nuclear localization. Viroid replication occurs via a rolling circle mechanism using either a symmetric or asymmetric pathway in three steps, RNA transcription, processing and ligation.
2
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Plant expression systems for production of hemagglutinin as a vaccine against influenza virus

51%
Redkiewicz P., Sirko A., Kamel K., Góra-Sochacka A.
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2014
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vol. 61
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issue 3
551-560
EN
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.
3
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Influenza prevention and treatment by passive immunization

51%
Kalenik B., Sawicka R., Góra-Sochacka A., Sirko A.
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2014
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vol. 61
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issue 3
573-587
EN
Passive immunity is defined as a particular antigen resistance provided by external antibodies. It can be either naturally or artificially acquired. Natural passive immunization occurs during pregnancy and breast-feeding in mammals and during hatching in birds. Maternal antibodies are passed through the placenta and milk in mammals and through the egg yolk in birds. Artificial passive immunity is acquired by injection of either serum from immunized (or infected) individuals or antibody preparations. Many independent research groups worked on selection, verification and detailed characterization of polyclonal and monoclonal antibodies against the influenza virus. Numerous antibody preparations were tested in a variety of in vitro and in vivo experiments for their efficacy to neutralize the virus. Here, we describe types of antibodies tested in such experiments and their viral targets, review approaches resulting in identification of broadly neutralizing antibodies and discuss methods used to demonstrate their protective effects. Finally, we shortly discuss the phenomenon of maternal antibody transfer as a way of effective care for young individuals and as an interfering factor in early vaccination.
4
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DNA vaccines against influenza

51%
Stachyra A., Góra-Sochacka A., Sirko A.
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2014
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vol. 61
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issue 3
515-522
EN
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.
5
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Genetic variability of potato spindle tuber viroid RNA replicon.

51%
Góra-Sochacka A., Candresse T., Zagórski W.
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2001
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vol. 48
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issue 2
467-476
EN
The genetic continuity of the potato spindle tuber viroid (PSTVd) genome was analysed after infection of tomato plants with cloned cDNAs of parental strains. During the six weeks of the experiment, several new sequence variants appeared. The sequence variants detected in the progeny population induced sequence-specific disease symptoms. The PSTVd genome therefore follows the pattern expected for typical pseudo-strains propagating in plants as a population of similar sequences. Assessing further the replicon continuity, a PSTVd cDNA mutant with a deletion in the central conserved region was constructed and proven to be non-infectious. Surprisingly, in a sub-population of potato transformants expressing the same deleted PSTVd RNA an infectious viroid was detected. This suggests specific transcript conversion followed by recovery of the full-length pathogen genome.
6
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Co-inoculation with two non-infectious cDNA copies of potato spindle tuber viroid (PSTVd) leads to the appearance of novel fully infectious variants.

51%
Podstolski W., Góra-Sochacka A., Zagórski W.
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2005
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vol. 52
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issue 1
87-98
EN
Potato spindle tuber viroid (PSTVd) is one of the smallest (about 360 nt) infectious plant agents. It is composed of a single-stranded circular non-coding RNA molecule. In the course of previous passage experiments with two intermediate PSTVd variants I2 and I4, three non-infectious clones (I2-50, I4-37 and I4 VI-17) were found. When inoculated separately as cDNAs on tomato "Rutgers" test plants these variants did not induce any visible disease symptoms and did not produce progeny. The presence of such non-infectious variants raises several questions about their origin and biology and to answer them, mixed co-infections with cDNA copies of two non-infectious variants (I2-50, I4-37) were performed. PSTVd infection was observed in seven out of 30 inoculated plants. The progeny isolated from three separate plants contained novel variants, together with the parental I2 and I4 sequences. It is conceivable that the appearance of repaired PSTVd molecules, clearly capable of cell-to-cell movement leading to the systemic infection, results from recombination events. An analysis of the recombinant molecules and comparison with databases identified the specific sites responsible for the restricted infectivity of the I2-50 and I4-37 PSTVd variants. In parallel experiments in which (+) strand PSTVd infectious transcripts were used, no recombinants were observed, and the original I2-50 and I4-37 non-infectious sequences were not detected in the progeny.
7
Content available remote

Antibody response to DNA vaccine against H5N1 avian influenza virus in broilers immunized according to three schedules

45%
Stachyra A., Góra-Sochacka A., Zagórski-Ostoja W., Król E., Sirko A.
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2014
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vol. 61
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issue 3
593-596
EN
Broiler type chickens were immunized intramuscularly with a DNA vaccine encoding hemagglutinin (HA) from H5N1 avian influenza virus. The chickens were divided into four groups: control group which was not immunized, a group which obtained only one dose, and two groups which were immunized twice, one group with a boost two weeks after the priming and the other four weeks. Blood samples were collected at several time points and the dynamics of the humoral response to the vaccine was studied. High level of anti-HA antibodies was detected only in the last two groups, that is in chickens immunized according to the prime-boost strategy, regardless of the schedule. An additional interesting observation of this study was detection of the cross-reactivity of an anti-H5 HA positive serum with H5N2 and H1N1 viruses, suggesting that the DNA vaccine tested can induce antibodies of a broad specificity.
8
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Content available

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

33%
Sawicka R., Siedlecki P., Kalenik B., Radomski J., Sączyńska V., Porębska A., Szewczyk B., Sirko A., Góra-Sochacka A.
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2017
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vol. 64
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issue 1
85-92
EN
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.
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