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Agriculture is facing new challenges, with global warming modifying the survival chances for crops, and new pests on the horizon. To keep up with these challenges, gene delivery provides tools to increase crop yields. On the other hand, gene delivery also opens the door for molecular farming of pharmaceuticals in plants. However, towards increased food production and scalable molecular farming, there remain technical difficulties and regulatory hurdles to overcome. The industry-standard is transformation of plants via Agrobacterium tumefaciens , but this method is limited to certain plants, requires set up of plant growth facilities and fermentation of bacteria, and introduces lipopolysaccharides contaminants into the system. Therefore, alternate methods are needed. Mechanical inoculation and spray methods have already been discussed in the literature – and here, we compare these methods with a newly introduced petiole injection technique. Because our interest lies in the development of plant viruses as immunotherapies targeting human health as well as gene delivery vectors for agriculture applications, we turned toward tobacco mosaic virus as a model system. We studied the effectiveness of three inoculation techniques: mechanical inoculation, Silwet-77 foliar spray and petiole injections. The foliar spray method was optimized, and we used 0.03% Silwet L-77 to induce infection using either TMV or a lysine-added mutant TMV-Lys. We developed a method using a needle-laden syringe to target and inject the plant virus directly into the vasculature of the plant – we tested injection into the stem and petiole. Stem inoculation resulted in toxicity, but the petiole injection technique was established as a viable strategy. TMV and TMV-Lys were purified from single plants and pooled leaf samples – overall there was little variation between the techniques, as measured by TMV or TMV-Lys yields, highlighting the feasibility of the syringe injection technique to produce virus nanoparticles. There was variation between yields from preparation to preparation with mechanical, spray and syringe inoculation yielding 40–141 mg, 36–56 mg, 18–56 mg TMV per 100 grams of leaves. Similar yields were obtained using TMV-Lys, with 24–38 mg, 17–28, 7–36 mg TMV-Lys per 100 grams of leaves for mechanical, spray and syringe inoculation, respectively. Each method has its advantages: spray inoculation is highly scalable and therefore may find application for farming, the syringe inoculation could provide a clean, aseptic, and controlled approach for molecular farming of pharmaceuticals under good manufacturing protocols (GMP) and would even be applicable for gene delivery to plants in space.

Plant viruses may serve as expression vectors for the efficient production of pharmaceutical proteins in plants. However, the downstream processing and post-translational modifications of the target proteins remain the major challenges. We have previously developed an expression system derived from Bamboo mosaic virus (BaMV), designated pKB19, and demonstrated its applicability for the production of human mature interferon gamma (mIFNγ) in Nicotiana benthamiana . In this study, we aimed to enhance the yields of soluble and secreted mIFNγ through the incorporation of various plant-derived signal peptides. Furthermore, we analyzed the glycosylation patterns and the biological activity of the mIFNγ expressed by the improved pKB19 expression system in N. benthamiana . The results revealed that the fusion of a native N. benthamiana extensin secretory signal (SSExt) to the N-terminal of mIFNγ (designated SSExt mIFNγ) led to the highest accumulation level of protein in intracellular (IC) or apoplast washing fluid (AWF) fractions of N. benthamiana leaf tissues. The addition of 10 units of ‘Ser-Pro’ motifs of hydroxyproline-O-glycosylated peptides (HypGPs) at the C-terminal end of SSExt mIFNγ (designated SSExt mIFNγ(SP)10) increased the solubility to nearly 2.7- and 1.5-fold higher than those of mIFNγ and SSExt mIFNγ, respectively. The purified soluble SSExt mIFNγ(SP)10 protein was glycosylated with abundant complex-type N-glycan attached to residues N56 and N128, and exhibited biological activity against Sindbis virus and Influenza virus replication in human cell culture systems. In addition, suspension cell cultures were established from transgenic N. benthamiana , which produced secreted SSExt mIFNγ(SP)10 protein feasible for downstream processing. These results demonstrate the applicability of the BaMV-based vector systems as a useful alternative for the production of therapeutic proteins, through the incorporation of appropriate fusion tags.

Virus-like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNVs) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post-infection (dpi). Oral immunization of CD1 mice with 100 or 250 μg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic.

Plants are becoming an interesting alternative system for the heterologous production of pharmaceutical proteins, providing a more scalable, cost-effective, and biologically safer option than the current expression systems. The development of plant virus expression vectors has allowed rapid and high-level transient expression of recombinant genes, and, in turn, provided an attractive plant-based production platform. Here we report the development of vectors based on the tobamovirus Pepper mild mottle virus (PMMoV) to be used in transient expression of foreign genes. In this PMMoV vector, a middle part of the viral coat protein gene was replaced by the green fluorescent protein (GFP) gene, and this recombinant genome was assembled in a binary vector suitable for plant agroinoculation. The accumulation of GFP was evaluated by observation of green fluorescent signals under UV light and by western blotting. Furthermore, by using this vector, the multiepitope gene for chikungunya virus was successfully expressed and confirmed by western blotting. This PMMoV-based vector represents an alternative system for a high-level production of heterologous protein in plants.

Background Since the 2000’s, plants have been used as bioreactors for the transient production of molecules of interest such as vaccines. To improve protein yield, “amplicon” vectors based on plant viruses are used. These viral constructs, engineered to carry the gene of interest replicate strongly once introduced into the plant cell, allowing significant accumulation of the protein. Here, we evaluated the suitability of the monocot-infecting RNA virus Rice yellow mottle virus (RYMV) as an amplicon vector. The promastigote surface antigen (PSA) of the protozoan Leishmania was considered as a protein of interest due to its vaccine properties against canine leishmaniasis. Results Since P1 (ORF1) and CP (ORF3) proteins are not strictly necessary for viral replication, ORF1 was deleted and the PSA gene was substituted to ORF3 in the RYMV-based vector. We evaluated its expression in the best described plant bioreactor system, Nicotiana benthamiana which, unlike rice, allows transient transformation by Agrobacterium . Despite not being its natural host, we demonstrated a low level of RYMV-based vector replication in N. benthamiana leaves. Under optimized ratio, we showed that the P19 silencing suppressor in combination with the missing viral CP ORF significantly enhanced RYMV amplicon replication in N. benthamiana . Under these optimized CP/P19 conditions, we showed that the RYMV amplicon replicated autonomously in the infiltrated N. benthamiana cells, but was unable to move out of the infiltrated zones. Finally, we showed that when the RYMV amplicon was expressed under the optimized conditions we set up, it allowed enhanced PSA protein accumulation in N. benthamiana compared to the PSA coding sequence driven by the 35S promoter without amplicon background. Conclusion This work demonstrates that a non-dicot-infecting virus can be used as an amplicon vector for the efficient production of proteins of interest such as PSA in N. benthamiana leaves.

Plant virus-based vectors provide attractive and valuable tools for rapid production of recombinant protein in large quantities as they produce systemic infections in differentiated plant tissues. In the present study, we engineered the Soybean yellow mottle mosaic virus (SYMMV) as a gene expression vector which is a promising candidate for systemic expression of foreign proteins in French bean plants. Full virus vector strategy was exploited for insertion of foreign gene by inserting MCS through PCR in the circular pJET-SYMMV clone. To examine the ability of the SYMMV vector system, GFP gene was cloned after the start codon of coat protein (CP) so that its expression was driven by the SYMMV-CP subgenomic promoter. When in vitro run off SYMMV-GFP transcript was mechanically inoculated to French bean leaves, good level of GFP expression was observed through confocal microscopy up to 40 dpi. Expression of heterologous protein was also confirmed through ISEM, DAC-ELISA and RT-PCR with specific primers at 20 dpi. The recombinant SYMMV construct was stable in in vitro runoff transcript inoculated plants but the inserted GFP was lost in progeny virion inoculated plants. The system developed here will be useful for further studies of SYMMV gene functions and exploitation of SYMMV as a gene expression vector.

In planta production of recombinant proteins, including vaccine antigens and monoclonal antibodies, continues gaining acceptance. With the broadening range of target proteins, the need for vectors with higher performance is increasing. Here, we have developed a single-replicon vector based on beet yellows virus (BYV) that enables co-delivery of two target genes into the same host cell, resulting in transient expression of each target. This BYV vector maintained genetic stability during systemic spread throughout the host plant, Nicotiana benthamiana. Furthermore, we have engineered a miniBYV vector carrying the sequences encoding heavy and light chains of a monoclonal antibody (mAb) against protective antigen (PA) of Bacillius anthracis, and achieved the expression of the full-length functional anti-PA mAb at ~300 mg/kg of fresh leaf tissue. To demonstrate co-expression and functionality of two independent proteins, we cloned the sequences of the Pfs48/45 protein of Plasmodium falciparum and endoglycosidase F (PNGase F) from Flavobacterium meningosepticum into the miniBYV vector under the control of two subgenomic RNA promoters. Agroinfiltration of N. benthamiana with this miniBYV vector resulted in accumulation of biologically active Pfs48/45 that was devoid of N-linked glycosylation and had correct conformation and epitope display. Overall, our findings demonstrate that the new BYV-based vector is capable of co-expressing two functionally active recombinant proteins within the same host cell.

Background Developing African countries face health problems that they struggle to solve. The major causes of this situation are high therapeutic and logistical costs. Plant-made therapeutics are easy to produce due to the lack of the safety considerations associated with traditional fermenter-based expression platforms, such as mammalian cells. Plant biosystems are easy to scale up and inexpensive, and they do not require refrigeration or a sophisticated medical infrastructure. These advantages provide an opportunity for plant-made pharmaceuticals to counteract diseases for which medicines were previously inaccessible to people in countries with few resources. Main body The techniques needed for plant-based therapeutic production are currently available. Viral expression vectors based on plant viruses have greatly enhanced plant-made therapeutic production and have been exploited to produce a variety of proteins of industrial, pharmaceutical and agribusiness interest. Some neglected tropical diseases occurring exclusively in the developing world have found solutions through plant bioreactor technology. Plant viral expression vectors have been reported in the production of therapeutics against these diseases occurring exclusively in the third world, and some virus-derived antigens produced in plants exhibit appropriate antigenicity and immunogenicity. However, all advances in the use of plants as bioreactors have been made by companies in Europe and America. The developing world is still far from acquiring this technology, although plant viral expression vectors may provide crucial help to overcome neglected diseases. Conclusion Today, interest in these tools is rising, and viral amplicons made in and for Africa are in progress. This review describes the biotechnological advances in the field of plant bioreactors, highlights factors restricting access to this technology by those who need it most and proposes a solution to overcome these limitations.

Noroviruses are members of the family Caliciviridae, and cause a highly communicable gastroenteritis in humans. We explored the potential to develop a plant-based vaccine against Narita 104 virus, a Genogroup II Norovirus. In stably transgenic potato, we obtained very poor expression of Narita 104 virus capsid protein (NaVCP) despite the use of a strong constitutive promoter (dual enhancer 35S) driving the native coding sequence. We identified potentially detrimental sequence motifs that could mediate aberrant mRNA processing via spurious polyadenylation signals. Northern blots and RT-PCR analysis of total RNA revealed truncated transcripts that suggested premature polyadenylation. Site-directed mutagenesis to remove one potential polyadenylation near-upstream element resulted in an increased expression of NaVCP when transiently expressed in leaves of Nicotiana benthamiana. Further, cloning of the truncated cDNAs from transgenic NaVCP potato plants and transiently transfected N. benthamiana allowed us to identify at least ten different truncated transcripts resulting from premature polyadenylation of full length NaVCP transcripts. Comparative studies using real time PCR analysis from cDNA samples revealed lower accumulation of full length transcripts of NaVCP as compared to those from a gene encoding Norwalk Virus capsid protein (a related Genogroup I Norovirus) in transiently transfected plants. These findings provide evidence for impaired expression of NaVCP in transgenic plants mediated by spurious polyadenylation signals, and demonstrate the need to scrupulously search for potential polyadenylation signals in order to improve transgene expression in plants.

Cucumber mosaic virus (CMV) often accompanies a short RNA molecule called a satellite RNA (satRNA). When infected with CMV in the presence of Y-satellite RNA (Y-sat), tobacco leaves develop a green mosaic, then turn yellow. Y-sat has been identified in the fields in Japan. Here, we show that the yellow leaf colour preferentially attracts aphids, and that the aphids fed on yellow plants, which harbour Y-sat-derived small RNAs (sRNAs), turn red and subsequently develop wings. In addition, we found that leaf yellowing did not necessarily reduce photosynthesis, and that viral transmission was not greatly affected despite the low viral titer in the Y-sat-infected plants. Y-sat-infected plants can therefore support a sufficient number of aphids to allow for efficient virus transmission. Our results demonstrate that Y-sat directly alters aphid physiology via Y-sat sRNAs to promote wing formation, an unprecedented survival strategy that enables outward spread via the winged insect vector. The cucumber mosaic virus is accompanied by short RNA molecules, satellite RNAs. This study shows that leaves infected with Y-satellite RNA preferentially attract aphids and manipulate aphid physiology to promote their spread to neighbouring plants.