Development of a plant-based oral vaccine candidate against the bovine respiratory pathogen Mannheimia haemolytica.

Bovine respiratory disease (BRD) affects feedlot cattle across North America, resulting in economic losses due to animal treatment and reduced performance. In an effort to develop a vaccine candidate targeting a primary bacterial agent contributing to BRD, we produced a tripartite antigen consisting of segments of the virulence factor Leukotoxin A (LktA) and lipoprotein PlpE from Mannheimia haemolytica, fused to a cholera toxin mucosal adjuvant (CTB). This recombinant subunit vaccine candidate was expressed in the leaves of Nicotiana benthamiana plants, with accumulation tested in five subcellular compartments. The recombinant protein was found to accumulate highest in the endoplasmic reticulum, but targeting to the chloroplast was employed for scaling up production due the absence of post-translational modification while still producing feasible levels. Leaves were freeze dried, then orally administered to mice to determine its immunogenicity. Sera from mice immunized with leaf tissue expressing the recombinant antigen contained IgG antibodies, specifically recognizing both LktA and PlpE. These mice also had a mucosal immune response to the CTB+LktA+PlpE protein as measured by the presence of LktA- and PlpE-specific IgA antibodies in lung and fecal material. Moreover, the antigen remained stable at room temperature with limited deterioration for up to one year when stored as lyophilized plant material. This study demonstrated that a recombinant antigen expressed in plant tissue elicited both humoral and mucosal immune responses when fed to mice, and warrants evaluation in cattle.

Plant production of a virus-like particle-based vaccine candidate against porcine reproductive and respiratory syndrome.

Porcine reproductive and respiratory syndrome (PRRS) is a disease leading to spontaneous abortions and stillbirths in sows and lowered life quality and expectancy in growing pigs. PRRS is prevalent worldwide and has significant economic impacts to swine industries around the globe. Co-expression of the two most abundant proteins in the viral envelope, the matrix protein (M) and glycosylated protein 5 (GP5), can produce a neutralizing immune response for the virus providing a potentially effective subunit vaccine against the disease, but these proteins are difficult to express. The goal of this research was to display antigenic portions of the M and GP5 proteins on the surface of tobacco mosaic virus-like particles. A modified tobacco mosaic virus coat protein (TMVc) was transiently expressed in Nicotiana benthamiana leaves and targeted to three subcellular compartments along the secretory pathway to introduce glycosylation patterns important for M-GP5 epitope immunogenicity. We found that accumulation levels in the apoplast were similar to the ER and the vacuole. Because glycans present on plant apoplastic proteins are closest to those present on PRRSV proteins, a TMVc-M-GP5 fusion construct was targeted to the apoplast and accumulated at over 0.5 mg/g of plant fresh weight. TMVc virus-like particles self-assembled in plant cells and surface-displayed the M-GP5 epitope, as visualized by transmission electron microscopy and immunogold localization. These promising findings lay the foundation for immunogenicity and protective-immunity studies in animals to examine the efficacy of this vaccine candidate as a measure to control PRRS.

Plastid Transformation of Micro-Tom Tomato with a Hemipteran Double-Stranded RNA Results in RNA Interference in Multiple Insect Species.

Plant-mediated RNA interference (RNAi) holds great promise for insect pest control, as plants can be transformed to produce double-stranded RNA (dsRNA) to selectively down-regulate insect genes essential for survival. For optimum potency, dsRNA can be produced in plant plastids, enabling the accumulation of unprocessed dsRNAs. However, the relative effectiveness of this strategy in inducing an RNAi response in insects using different feeding mechanisms is understudied. To investigate this, we first tested an in vitro-synthesized 189 bp dsRNA matching a highly conserved region of the v-ATPaseA gene from cotton mealybug (Phenacoccus solenopsis) on three insect species from two different orders that use leaf-chewing, lacerate-and-flush, or sap-sucking mechanisms to feed, and showed that the dsRNA significantly down-regulated the target gene. We then developed transplastomic Micro-tom tomato plants to produce the dsRNA in plant plastids and showed that the dsRNA is produced in leaf, flower, green fruit, red fruit, and roots, with the highest dsRNA levels found in the leaf. The plastid-produced dsRNA induced a significant gene down-regulation in insects using leaf-chewing and lacerate-and-flush feeding mechanisms, while sap-sucking insects were unaffected. Our results suggest that plastid-produced dsRNA can be used to control leaf-chewing and lacerate-and-flush feeding insects, but may not be useful for sap-sucking insects.

Transplastomic Tomato Plants Expressing Insect-Specific Double-Stranded RNAs: A Protocol Based on Biolistic Transformation.

Expressing insecticidal double-stranded RNA (dsRNA) molecules in plant plastids is a novel approach for in planta production of dsRNA that has enormous potential for developing improved plant-mediated RNA interference (RNAi) strategies for insect pest control. In this chapter, we describe the design of a transformation vector containing an expression cassette which can be used to stably transform plastids of tomato plants for production and accumulation of dsRNA . Such dsRNA can trigger the mechanisms of RNAi in pest insects and selectively suppress the expression of target genes, resulting in lethality. We also describe a protocol for detection of full-length dsRNA molecules in plastids using an RT-PCR-based method.

A VHH-Fc Fusion Targeted to the Chloroplast Thylakoid Lumen Assembles and Neutralizes Enterohemorrhagic E. coli O157:H7.

Chimeric fusion proteins comprising a single domain antibody (VHH) fused to a crystallizable fragment (Fc) of an immunoglobulin are modular glycoproteins that are becoming increasingly in demand because of their value as diagnostics, research reagents and passive immunization therapeutics. Because ER-associated degradation and misfolding may potentially be limiting factors in the oxidative folding of VHH-Fc fusion proteins in the ER, we sought to explore oxidative folding in an alternative sub-compartment, the chloroplast thylakoid lumen, and determine its viability in a molecular farming context. We developed a set of in-house expression vectors for transient transformation of Nicotiana benthamiana leaves that target a VHH-Fc to the thylakoid lumen via either secretory (Sec) or twin-arginine translocation (Tat) import pathways. Compared to stromal [6.63 ± 3.41 mg/kg fresh weight (FW)], cytoplasmic (undetectable) and Tat-import pathways (5.43 ± 2.41 mg/kg FW), the Sec-targeted VHH-Fc showed superior accumulation (30.56 ± 5.19 mg/kg FW), but was less than that of the ER (51.16 ± 9.11 mg/kg FW). Additionally, the introduction of a rationally designed de novo disulfide bond enhances in planta accumulation when introduced into the Sec-targeted Fc fusion protein from 50.24 ± 4.08 mg/kg FW to 110.90 ± 6.46 mg/kg FW. In vitro immunofluorescent labeling assays on VHH-Fc purified from Sec, Tat, and stromal pathways demonstrate that the antibody still retains VHH functionality in binding Escherichia coli O157:H7 and neutralizing its intimate adherence to human epithelial type 2 cells. These results overall provide a proof of concept that the oxidative folding environment of the thylakoid lumen may be a viable compartment for stably folding disulfide-containing recombinant VHH-Fc proteins.

A Rationally Designed Bovine IgA Fc Scaffold Enhances in planta Accumulation of a VHH-Fc Fusion Without Compromising Binding to Enterohemorrhagic E. coli.

We previously isolated a single domain antibody (VHH) that binds Enterohemorrhagic Escherichia coli (EHEC) with the end-goal being the enteromucosal passive immunization of cattle herds. To improve the yield of a chimeric fusion of the VHH with an IgA Fc, we employed two rational design strategies, supercharging and introducing de novo disulfide bonds, on the bovine IgA Fc component of the chimera. After mutagenizing the Fc, we screened for accumulation levels after transient transformation in Nicotiana benthamiana leaves. We identified and characterized five supercharging and one disulfide mutant, termed '(5 + 1)Fc', that improve accumulation in comparison to the native Fc. Combining all these mutations is associated with a 32-fold increase of accumulation for the Fc alone, from 23.9 mg/kg fresh weight (FW) to 599.5 mg/kg FW, as well as a twenty-fold increase when fused to a VHH that binds EHEC, from 12.5 mg/kg FW tissue to 236.2 mg/kg FW. Co-expression of native or mutated VHH-Fc with bovine joining chain (JC) and bovine secretory component (SC) followed by co-immunoprecipitation suggests that the stabilizing mutations do not interfere with the capacity of VHH-Fc to assemble with JC and FC into a secretory IgA. Both the native and the mutated VHH-Fc similarly neutralized the ability of four of the seven most prevalent EHEC strains (O157:H7, O26:H11, O111:Hnm, O145:Hnm, O45:H2, O121:H19 and O103:H2), to adhere to HEp-2 cells as visualized by immunofluorescence microscopy and quantified by fluorometry. These results collectively suggest that supercharging and disulfide bond tethering on a Fc chain can effectively improve accumulation of a VHH-Fc fusion without impacting VHH functionality.

Non-target Effects of Hyperthermostable α-Amylase Transgenic Nicotiana tabacum in the Laboratory and the Field.

Thermostable α-amylases are important enzymes used in many industrial processes. The expression of recombinant Pyrococcus furiosus α-amylase (PFA) in Nicotiana tabacum has led to the accumulation of high levels of recombinant protein in transgenic plants. The initial steps to registering the transgenic tobacco at a commercial production scale and growing it in the field requires a risk assessment of potential non-target effects. The objective of this study was to assess the effect of feeding on transgenic tobacco with 2 indigenous insect species commonly associated with wild and commercial tobacco involving plants grown and evaluated under laboratory and field conditions. The highest levels of PFA ranged from 1.3 to 2.7 g/kg leaf fresh weight produced in the field-grown cultivars Con Havana and Little Crittenden, respectively. These two cultivars also had the highest nicotine (ranging from 4.6 to 10.9 mg/g), but there was little to no negative effect for either tobacco hornworm Manduca sexta L. or aphid Myzus nicotianae (Blackman). Both laboratory and field trials determined no short term (5 days) decrease in the survival or fecundity of the tobacco aphid after feeding on PFA transgenic tobacco compared to non-transgenic plants. In the field, tobacco hornworm larvae showed no differences in survival, final larval weights or development time to adult stage between transgenic lines of four cultivars and their corresponding wild type controls. Laboratory studies confirmed the field trial results indicating the low risk association of PFA expressed in tobacco leaves with tobacco hornworms and aphids that would feed on the transgenic plants.

Development of a cucumber green mottle mosaic virus-based expression vector for the production in cucumber of neutralizing epitopes against a devastating animal virus.

Virus-based expression systems have been widely exploited for the production of recombinant proteins in plants during the last thirty years. Advances in technology have boosted scale-up manufacturing of plant-made pharmaceuticals to high levels, via the complementation of transient expression and viral vectors. This combination allows proteins of interest to be produced in plants within a matter of days and thus, is well suited for the development of plant-made vaccines or therapeutics against emerging infectious diseases and potential bioterrorism agents. Several plant-based products are currently in varying stages of clinical development. To investigate the viability of virus-based expression systems for plant-made vaccines against porcine reproductive and respiratory syndrome virus (PRRSV), the most devastating threat to the pork industry in Canada, we cloned the full-length genome of a cucumber green mottle mosaic virus (CGMMV) isolate and developed a CGMMV-based expression vector. We further employed this vector to express the neutralizing epitope (NE) of PRRSV glycoprotein 5 (GP5) in cucumber leaves via agroinfiltration. The coding region of the GP5 NE was inserted downstream of the open reading frame for coat protein (CP) and expressed by a readthrough mechanism. The chimeric virus particles were stable and the expression levels reached as high as 35.84 mg/kg of cucumber leaf fresh weight. This study offers a promising solution to the production of a low cost, versatile and robust vaccine for oral administration against PRRSV through a chimeric virus particle display system.

Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells.

Enterohemorrhagic Escherichia coli (EHEC) has consistently been one of the foremost foodborne pathogen threats worldwide based on the past 30 years of surveillance. EHEC primarily colonizes the bovine gastrointestinal (GI) tract from which it can be transmitted to nearby farm environments and remain viable for months. There is an urgent need for effective and easily implemented pre-harvest interventions to curtail EHEC contamination of the food and water supply. In an effort to address this problem, we isolated single-domain antibodies (VHHs) specific for intimin, an EHEC adhesin required for colonization, and designed chimeric VHH fusions with secretory IgA functionality intended for passive immunotherapy at the mucosal GI surface. The antibodies were produced in leaves of Nicotiana benthamiana with production levels ranging between 1 and 3% of total soluble protein. in vivo assembly of all subunits into a hetero-multimeric complex was verified by co-immunoprecipitation. Analysis of multivalent protection across the most prevalent EHEC strains identified one candidate antibody, VHH10-IgA, that binds O145:Hnm, O111:Hnm, O26:H11, and O157:H7. Fluorometric and microscopic analysis also indicated that VHH10-IgA completely neutralizes the capacity of the latter three strains to adhere to epithelial cells in vitro. This study provides proof of concept that a plant-produced chimeric secretory IgA can confer cross-serotype inhibition of bacterial adhesion to epithelial cells.

RNA Interference in the Tobacco Hornworm, Manduca sexta, Using Plastid-Encoded Long Double-Stranded RNA.

RNA interference (RNAi) is a promising method for controlling pest insects by silencing the expression of vital insect genes to interfere with development and physiology; however, certain insect Orders are resistant to this process. In this study, we set out to test the ability of in planta-expressed dsRNA synthesized within the plastids to silence gene expression in an insect recalcitrant to RNAi, the lepidopteran species, Manduca sexta (tobacco hornworm). Using the Manduca vacuolar-type H+ ATPase subunit A (v-ATPaseA) gene as the target, we first evaluated RNAi efficiency of two dsRNA products of different lengths by directly feeding the in vitro-synthesized dsRNAs to M. sexta larvae. We found that a long dsRNA of 2222 bp was the most effective in inducing lethality and silencing the v-ATPaseA gene, when delivered orally in a water droplet. We further transformed the plastid genome of the M. sexta host plant, Nicotiana tabacum, to produce this long dsRNA in its plastids and performed bioassays with M. sexta larvae on the transplastomic plants. In the tested insects, the plastid-derived dsRNA had no effect on larval survival and no statistically significant effect on expression of the v-ATPaseA gene was observed. Comparison of the absolute quantities of the dsRNA present in transplastomic leaf tissue for v-ATPaseA and a control gene, GFP, of a shorter size, revealed a lower concentration for the long dsRNA product compared to the short control product. We suggest that stability and length of the dsRNA may have influenced the quantities produced in the plastids, resulting in inefficient RNAi in the tested insects. Our results imply that many factors dictate the effectiveness of in planta RNAi, including a likely trade-off effect as increasing the dsRNA product length may be countered by a reduction in the amount of dsRNA produced and accumulated in the plastids.

Transient Expression of Dengue Virus NS1 Antigen in Nicotiana benthamiana for Use as a Diagnostic Antigen.

Dengue is a viral disease that represents a significant threat to global public health since billions of people are now at risk of infection by this mosquito-borne virus. The implementation of extensive screening tests is indispensable to control this disease, and the Dengue virus non-structural protein 1 (NS1) is a promising antigen for the serological diagnosis of dengue fever. Plant-based systems can be a safe and cost-effective alternative for the production of dengue virus antigens. In this work, two strategies to produce the dengue NS1 protein in Nicotiana benthamiana leaves were evaluated: Targeting NS1 to five different subcellular compartments to assess the best subcellular organelle for the expression and accumulation of NS1, and the addition of elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags to NS1. The transiently expressed proteins in N. benthamiana were quantified by Western blot analysis. The NS1 fused to ELP and targeted to the ER (NS1 ELP-ER) showed the highest yield (445 mg/kg), approximately a forty-fold increase in accumulation levels compared to the non-fused protein (NS1-ER), representing the first example of transient expression of DENV NS1 in plant. We also demonstrated that NS1 ELP-ER was successfully recognized by a monoclonal anti-dengue virus NS1 glycoprotein antibody, and by sera from dengue virus-infected patients. Interestingly, it was found that transient production of NS1-ER and NS1 ELP-ER using vacuum infiltration of whole plants, which is easier to scale up, rather than syringe infiltration of leaves, greatly improved the accumulation of NS1 proteins. The generated plant made NS1, even without extensive purification, showed potential to be used for the development of the NS1 diagnostic tests in resource-limited areas where dengue is endemic.

Transient co-expression with three O-glycosylation enzymes allows production of GalNAc-O-glycosylated Granulocyte-Colony Stimulating Factor in N. benthamiana.

BACKGROUND: Expression of economically relevant proteins in alternative expression platforms, especially plant expression platforms, has gained significant interest in recent years. A special interest in working with plants as bioreactors for the production of pharmaceutical proteins is related to low production costs, product safety and quality. Among the different properties that plants can also offer for the production of recombinant proteins, protein glycosylation is crucial since it may have an impact on pharmaceutical functionality and/or stability. RESULTS: The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor was transiently expressed in Nicotiana benthamiana plants and subjected to mammalian-specific mucin-type O-glycosylation by co-expressing the pharmaceutical protein together with the glycosylation machinery responsible for such post-translational modification. CONCLUSIONS: The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor can be expressed in N. benthamiana plants via agroinfiltration with its native mammalian-specific mucin-type O-glycosylation.

Protein Storage Vacuoles Originate from Remodeled Preexisting Vacuoles in Arabidopsis thaliana.

Protein storage vacuoles (PSV) are the main repository of protein in dicotyledonous seeds, but little is known about the origins of these transient organelles. PSV are hypothesized to either arise de novo or originate from the preexisting embryonic vacuole (EV) during seed maturation. Here, we tested these hypotheses by studying PSV formation in Arabidopsis (Arabidopsis thaliana) embryos at different stages of seed maturation and recapitulated this process in Arabidopsis leaves reprogrammed to an embryogenic fate by inducing expression of the LEAFY COTYLEDON2 transcription factor. Confocal and immunoelectron microscopy indicated that both storage proteins and tonoplast proteins typical of PSV were delivered to the preexisting EV in embryos or to the lytic vacuole in reprogrammed leaf cells. In addition, sectioning through embryos at several developmental stages using serial block face scanning electron microscopy revealed the 3D architecture of forming PSV. Our results indicate that the preexisting EV is reprogrammed to become a PSV in Arabidopsis.

High accumulation in tobacco seeds of hemagglutinin antigen from avian (H5N1) influenza.

Tobacco seeds can be used as a cost effective system for production of recombinant vaccines. Avian influenza is an important respiratory pathogen that causes a high degree of mortality and becomes a serious threat for the poultry industry. A safe vaccine against avian flu produced at low cost could help to prevent future outbreaks. We have genetically engineered tobacco plants to express extracellular domain of hemagglutinin protein from H5N1 avian influenza virus as an inexpensive alternative for production purposes. Two regulatory sequences of seed storage protein genes from Phaseolus vulgaris L. were used to direct the expression, yielding 3.0 mg of the viral antigen per g of seeds. The production and stability of seed-produced recombinant HA protein was characterized by different molecular techniques. The aqueous extract of tobacco seed proteins was used for subcutaneous immunization of chickens, which developed antibodies that inhibited the agglutination of erythrocytes after the second application of the antigen. The feasibility of using tobacco seeds as a vaccine carrier is discussed.

A Plant-Produced Candidate Subunit Vaccine Reduces Shedding of Enterohemorrhagic Escherichia coli in Ruminants.

Enterohemorrhagic Escherichia coli (EHEC) are commonly present in the gastrointestinal tract of cattle and cause serious infectious disease in humans. Immunizing cattle against EHEC is a promising strategy to decrease the risk of food contamination; however, veterinary vaccines against EHEC such as Econiche have not been widely adopted by the agricultural industry, and have been discontinued, prompting the need for more cost-effective EHEC vaccines. The objective of this project is to develop a platform to produce plant-made antigens for oral vaccination of ruminants against EHEC. Five recombinant proteins were designed as vaccine candidates and expressed transiently in Nicotiana benthamiana and transplastomically in Nicotiana tabacum. Three of these EHEC proteins, NleA, Stx2b, and a fusion of EspA accumulated when transiently expressed. Transient protein accumulation was the highest when EHEC proteins were fused to an elastin-like polypeptide (ELP) tag. In the transplastomic lines, EspA accumulated up to 479 mg kg-1 in lyophilized leaf material. Sheep that were administered leaf tissue containing recombinant EspA shed less E. coli O157:H7 when challenged, as compared to control animals. These results suggest that plant-made, transgenic EspA has the potential to reduce EHEC shedding in ruminants.

A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates.

BACKGROUND: Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. RESULTS: We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. CONCLUSIONS: Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.

Coating Nanoparticles with Plant-Produced Transferrin-Hydrophobin Fusion Protein Enhances Their Uptake in Cancer Cells.

The encapsulation of drugs to nanoparticles may offer a solution for targeted delivery. Here, we set out to engineer a self-assembling targeting ligand by combining the functional properties of human transferrin and fungal hydrophobins in a single fusion protein. We showed that human transferrin can be expressed in Nicotiana benthamiana plants as a fusion with Trichoderma reesei hydrophobins HFBI, HFBII, or HFBIV. Transferrin-HFBIV was further expressed in tobacco BY-2 suspension cells. Both partners of the fusion protein retained their functionality; the hydrophobin moiety enabled migration to a surfactant phase in an aqueous two-phase system, and the transferrin moiety was able to reversibly bind iron. Coating porous silicon nanoparticles with the fusion protein resulted in uptake of the nanoparticles in human cancer cells. This study provides a proof-of-concept for the functionalization of hydrophobin coatings with transferrin as a targeting ligand.

Corrigendum: Co-expression with the Type 3 Secretion Chaperone CesT from Enterohemorrhagic E. coli Increases Accumulation of Recombinant Tir in Plant Chloroplasts.

[This corrects the article on p. 283 in vol. 8, PMID: 28321227.].