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

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

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

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

Time-Course Microarray Analysis Reveals Differences between Transcriptional Changes in Tomato Leaves Triggered by Mild and Severe Variants of Potato Spindle Tuber Viroid

Viroids are small non-capsidated non-coding RNA replicons that utilize host factors for efficient propagation and spread through the entire plant. They can incite specific disease symptoms in susceptible plants. To better understand viroid-plant interactions, we employed microarray analysis to observe the changes of gene expression in ‘Rutgers’ tomato leaves in response to the mild (M) and severe (S23) variants of potato spindle tuber viroid (PSTVd). The changes were analyzed over a time course of viroid infection development: (i) the pre-symptomatic stage, (ii) early symptoms, (iii) full spectrum of symptoms and (iv) the so-called ‘recovery’ stage, when stem regrowth was observed in severely affected plants. Gene expression profiles differed depending on stage of infection and variant. In S23-infected plants, the expression of over 3,000 genes was affected, while M-infected plants showed 3-fold fewer differentially expressed genes, only 20% of which were specific to the M variant. The differentially expressed genes included many genes related to stress; defense; hormone metabolism and signaling; photosynthesis and chloroplasts; cell wall; RNA regulation, processing and binding; protein metabolism and modification and others. The expression levels of several genes were confirmed by nCounter analysis

Β-defensins – Underestimated peptides in influenza combat

Defensins are a family of host defense peptides present in vertebrates, invertebrates and plants. They display broad antimicrobial activity and immunomodulatory functions. Herein, the natural anti-influenzal role of β-defensins, as well as their potential usage as anti-influenza vaccine adjuvants and therapeutic agents, is reviewed. This article summarizes previously published information on β-defensin modes of action, expression changes after influenza infection and vaccination, biotechnological usage and possible boosting of their production by dietary supplementation

Immunogenicity of DNA Vaccine against H5N1 Containing Extended Kappa B Site: In Vivo Study in Mice and Chickens

Influenza is one of the most important illnesses in the modern world, causing great public health losses each year due to the lack of medication and broadly protective, long-lasting vaccines. The development of highly immunogenic and safe vaccines is currently one of the major problems encountered in efficient influenza prevention. DNA vaccines represent a novel and powerful alternative to the conventional vaccine approaches. To improve the efficacy of the DNA vaccine against influenza H5N1, we inserted three repeated kappa B (κB) motifs, separated by a 5-bp nucleotide spacer, upstream of the cytomegalovirus promoter and downstream of the SV40 late polyadenylation signal. The κB motif is a specific DNA element (10pb-long) recognized by one of the most important transcription factors NFκB. NFκB is present in almost all animal cell types and upon cell stimulation under a variety of pathogenic conditions. NFκB is released from IκB and translocates to the nucleus and binds to κB sites, thereby leading to enhanced transcription and expression of downstream genes. We tested the variants of DNA vaccine with κB sites flanking the antigen expression cassette and without such sites in two animal models: chickens (broilers and layers) and mice (BALB/c). In chickens, the variant with κB sites stimulated stronger humoral response against the target antigen. In mice, the differences in humoral response were less apparent. Instead, it was possible to spot several gene expression differences in the spleens isolated from mice immunized with both variants. The results of our study indicate that modification of the sequence outside of the sequence encoding the antigen might enhance the immune response to the target but understanding the mechanisms responsible for this process requires further analysis

A highly sensitive electrochemical genosensor based on Co-porphyrin-labelled DNA

We report the use of Co-porphyrins as electrochemical tags for a highly sensitive and selective genosensor. An avian influenza virus-based DNA sequence characteristic of H5N1 was detected at femtomolar levels from competing non-complementary sequences through hybridisation with the labeled DNA

Electrochemical genosensor based on disc and screen printed goldelectrodes for detection of specific DNA and RNA sequences derivedfrom Avian Influenza Virus H5N1

tThe genosensors based on thiolated ssDNA probe deposited on the two types of gold electrodes: screen-printed (miniaturized) and disc electrodes destined for determination of specific sequences of DNA andRNA derived from Avian Influenza Virus H5N1 have been proposed. The working principle of genosensor isbased on the ion-channel mechanism. The analytical signals generated upon hybridization processes wererecorded using electrochemical technique – Osteryoung square wave voltammetry in the presence of aredox active marker [Fe(CN)6]3−/4−in the sample solution. The miniaturized genosensor based on screenprinted gold electrodes was able to detect the 20-mer complementary DNA oligonucleotide sequence aswell as ∼280-mer RNA sequences containing the complementary 20-mer sequence in various positions:at 3�-terminus, at 5�-terminus and in the middle of the RNA transcript at the 1 pM concentration. Themeasuring systems were selective. Non-complementary 20-mer oligonucleotide sequence as well asRNA transcript without complementary region generated weak response. The RNA transcripts were alsotested with gold disc electrodes modified in the same manner. This device was able to detect ∼280-mer RNA sequences, but at higher concentration of 10 pM. The good discrimination of the position ofcomplementary part in the ∼280-mer RNA sequences was observed with using both types of modifiedelectrodes

Root Transcriptomic Analysis Reveals Global Changes Induced by Systemic Infection of Solanum lycopersicum with Mild and Severe Variants of Potato Spindle Tuber Viroid

Potato spindle tuber viroid (PSTVd) causes systemic infection in plant hosts. There are many studies on viroid-host plant interactions, but they have predominantly focused on the aboveground part of the plant. Here, we investigated transcriptomic profile changes in tomato roots systemically infected with mild or severe PSTVd variants using a combined microarray/RNA-seq approach. Analysis indicated differential expression of genes related to various Gene Ontology categories depending on the stage of infection and PSTVd variant. A majority of cell-wall-related genes were down-regulated at early infection stages, but at the late stage, the number of up-regulated genes increased significantly. Along with observed alterations of many lignin-related genes, performed lignin quantification indicated their disrupted level in PSTVd-infected roots. Altered expression of genes related to biosynthesis and signaling of auxin and cytokinin, which are crucial for lateral root development, was also identified. Comparison of both PSTVd infections showed that transcriptional changes induced by the severe variant were stronger than those caused by the mild variant, especially at the late infection stage. Taken together, we showed that similarly to aboveground plant parts, PSTVd infection in the underground tissues activates the plant immune response

Response to a DNA vaccine against the H5N1 virus depending on the chicken line and number of doses

Background Avian influenza virus infections cause significant economic losses on poultry farms and pose the threat of a possible pandemic outbreak. Routine vaccination of poultry against avian influenza is not recommended in Europe, however it has been ordered in some other countries, and more countries are considering use of the avian influenza vaccine as a component of their control strategy. Although a variety of such vaccines have been tested, most research has concentrated on specific antibodies and challenge experiments. Methods We monitored the transcriptomic response to a DNA vaccine encoding hemagglutinin from the highly pathogenic H5N1 avian influenza virus in the spleens of broiler and layer chickens. Moreover, in layer chickens the response to one and two doses of the vaccine was compared. Results All groups of birds immunized with two doses of the vaccine responded at the humoral level by producing specific anti-hemagglutinin antibodies. A response to the vaccine was also detected in the spleen transcriptomes. Differential expression of many genes encoding noncoding RNA and proteins functionally connected to the neuroendocrine-immune system was observed in different immunized groups