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Human erythropoietin (hEPO) is one of the most in-demand biopharmaceuticals, however, its production is challenging. When produced in a plant expression system, hEPO results in extensive plant tissue damage and low expression. It is demonstrated that the modulation of the plant protein synthesis machinery enhances hEPO production. Co-expression of basic leucine zipper transcription factors with hEPO prevents plant tissue damage, boosts expression, and increases hEPO solubility. bZIP28 co-expression up-regulates genes associated with the unfolded protein response, indicating that the plant tissue damage caused by hEPO expression is due to the native protein folding machinery being overwhelmed and that this can be overcome by co-expressing bZIP28., (© 2024 Wiley‐VCH GmbH.)

Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA) of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83) is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83) was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may offer advantages in terms of dose sparing and enhanced immunogenicity as a promising candidate for a safe, effective and low-cost subunit vaccine against anthrax.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

At present, several eukaryotic expression systems including yeast, insect and mammalian cells and plants are used for the production of recombinant proteins. Proteins with potential N-glycosylation sites are efficiently glycosylated when expressed in these systems. However, the ability of the eukaryotic expression systems to glycosylate may be not desirable for some proteins. If target proteins that do not carry N-linked glycans in the native host contain potential N-linked glycosylation sites, they can be aberrantly glycosylated in the eukaryotic expression systems, thus, potentially impairing biological activity. Recently, we have developed a strategy of enzymatic deglycosylation of proteins in vivo by co-introducing bacterial PNGase F via agroinfiltration followed by transient expression in plants. (1) Here, we summarize our work on this topic and its potential implications.

Clinically available red blood cells (RBCs) for transfusions are at high demand, but in vitro generation of RBCs from hematopoietic stem cells requires significant quantities of growth factors. Here, we describe the production of four human growth factors: erythropoietin (EPO), stem cell factor (SCF), interleukin 3 (IL-3), and insulin-like growth factor-1 (IGF-1), either as non-fused proteins or as fusions with a carrier molecule (lichenase), in plants, using a Tobacco mosaic virus vector-based transient expression system. All growth factors were purified and their identity was confirmed by western blotting and peptide mapping. The potency of these plant-produced cytokines was assessed using TF1 cell (responsive to EPO, IL-3 and SCF) or MCF-7 cell (responsive to IGF-1) proliferation assays. The biological activity estimated here for the cytokines produced in plants was slightly lower or within the range cited in commercial sources and published literature. By comparing EC50 values of plant-produced cytokines with standards, we have demonstrated that all four plant-produced growth factors stimulated the expansion of umbilical cord blood-derived CD34+ cells and their differentiation toward erythropoietic precursors with the same potency as commercially available growth factors. To the best of our knowledge, this is the first report on the generation of all key bioactive cytokines required for the erythroid development in a cost-effective manner using a plant-based expression system.

Application of tools of molecular biology and genomics is increasingly leading towards the development of recombinant protein-based biologics. As such, it is leading to an increased diversity of targets that have important health applications and require more flexible approaches for expression because of complex post-translational modifications. For example, Plasmodium parasites may have complex post-translationally modified proteins such as Pfs48/45 that do not carry N-linked glycans (Exp. Parasitol. 1998; 90, 165.) but contain potential N-linked glycosylation sites that can be aberrantly glycosylated during expression in mammalian and plant systems. Therefore, it is important to develop strategies for producing non-glycosylated forms of these targets to preserve biological activity and native conformation. In this study, we are describing in vivo deglycosylation of recombinant N-glycosylated proteins as a result of their transient co-expression with bacterial PNGase F (Peptide: N-glycosidase F). In addition, we show that the recognition of an in vivo deglycosylated plant-produced malaria vaccine candidate, Pfs48F1, by monoclonal antibodies I, III and V raised against various epitopes (I, III and V) of native Pfs48/45 of Plasmodium falciparum, was significantly stronger compared to that of the glycosylated form of plant-produced Pfs48F1. To our knowledge, neither in vivo enzymatic protein deglycosylation has been previously achieved in any eukaryotic system, including plants, nor has bacterial PNGase F been expressed in the plant system. Thus, here, we report for the first time the expression in plants of an active bacterial enzyme PNGase F and the production of recombinant proteins of interest in a non-glycosylated form., (© 2012 Fraunhofer USA Center for Molecular Biotechnology. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

Background: Influenza virus is a globally important respiratory pathogen that causes a high degree of annual morbidity and mortality. Significant antigenic drift results in emergence of new, potentially pandemic, virus variants. The best prophylactic option for controlling emerging virus strains is to manufacture and administer pandemic vaccines in sufficient quantities and to do so in a timely manner without impacting the regular seasonal influenza vaccine capacity. Current, egg-based, influenza vaccine production is well established and provides an effective product, but has limited capacity and speed., Objectives: To satisfy the additional global demand for emerging influenza vaccines, high-performance cost-effective technologies need to be developed. Plants have a potential as an economic and efficient large-scale production platform for vaccine antigens., Methods: In this study, a plant virus-based transient expression system was used to produce hemagglutinin (HA) proteins from the three vaccine strains used during the 2008-2009 influenza season, A/Brisbane/59/07 (H1N1), A/Brisbane/10/07 (H3N2), and B/Florida/4/06, as well as from the recently emerged novel H1N1 influenza A virus, A/California/04/09., Results: The recombinant plant-based HA proteins were engineered and produced in Nicotiana benthamiana plants within 2 months of obtaining the genetic sequences specific to each virus strain. These antigens expressed at the rate of 400-1300 mg/kg of fresh leaf tissue, with >70% solubility. Immunization of mice with these HA antigens induced serum anti-HA IgG and hemagglutination inhibition antibody responses at the levels considered protective against these virus infections., Conclusions: These results demonstrate the feasibility of our transient plant expression system for the rapid production of influenza vaccine antigens., (© 2011 Blackwell Publishing Ltd.)

The main objective of this study was to characterize the N-linked glycosylation profiles of recombinant hemagglutinin (HA) proteins expressed in either insect or plant hosts, and to develop a mass spectrometry based workflow that can be used in quality control to assess batch-to-batch reproducibility for recombinant HA glycosylation. HA is a surface glycoprotein of the influenza virus that plays a key role in viral infectivity and pathogenesis. Characterization of the glycans for plant recombinant HA from the viral strain A/California/04/09 (H1N1) has not yet been reported. In this study, N-linked glycosylation patterns of the recombinant HAs from both insect and plant hosts were characterized by precursor ion scan-driven data-dependent analysis followed by high-resolution MS/MS analysis of the deglycosylated tryptic peptides. Five glycosylation sites (N11, N23, N276, N287, and N481) were identified containing high mannose type glycans in plant-expressed HAs, and complex type glycoforms for the insect-expressed HA. More than 95% site occupancy was observed for all glycosylation sites except N11, which was 60% occupied. Multiple-reaction monitoring based quantitation analysis was developed for each glycopeptide isoform and the quantitative results indicate that the Man(8) GlcNAc(2) is the dominant glycan for all sites in plant-expressed HAs. The relative abundance of the glycoforms at each specific glycosylation site and the relative quantitation for each glycoform among three HAs were determined. Few differences in the glycosylation profiles were detected between the two batches of plant HAs studied, but there were significant differences between the glycosylation patterns in the HAs generated in plant and insect expression hosts., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we have developed a robust cryohistological method that allows imaging of virtually any type of plant cell or tissue while preserving fluorescent protein signals and maintaining excellent cellular and subcellular morphology. This method involves modified fixation of plant tissues (i.e., leaves, stems, and petioles), infiltration in a sucrose gradient, freezing, and collection of cryosections directly onto a cryoadhesive tape. Using this method followed by microscopic analysis, we demonstrated a localized accumulation of green fluorescent protein (GFP) in Nicotiana benthamiana plants agroinfiltrated with the movement-incompetent tobacco mosaic virus-based vector and systemic accumulation of GFP in plants infiltrated with the movement-competent vector. Overall, this simple cryohistological procedure reduced sample preparation time and allowed processing of tissue sections for high-resolution imaging of targeted fluorescent proteins in all plant tissues.

Expression of multiple therapeutic proteins from Tobacco mosaic virus (TMV)-based vectors was not successful when plants were coinoculated with a mixture of two TMV vectors engineered to express two foreign genes individually. Here, we have engineered and developed a defective RNA (dRNA)-based TMV vector (dRT-V) that utilizes two components of the same virus, with the dRNA component depending on the helper virus for replication. Agrobacterium-mediated coinoculation of Nicotiana benthamiana plants with both components of the dRT-V resulted in high-level expression of a human growth hormone and a lichenase-fused lethal factor protein of Bacillus anthracis. Furthermore, both heavy and light chains were expressed and assembled into a monoclonal antibody (mAb) specific to the protective antigen of B. anthracis, and the average yield of the purified antibody obtained was 120 mg/kg of fresh tissue. Our data suggest that dRT-V has a potential for rapid, cost-effective, large-scale manufacturing of multiple therapeutic proteins including mAbs in response to any biological emergencies., (Copyright 2010 Elsevier Inc. All rights reserved.)

Yersinia pestis continues to pose a threat as a potential biological weapon and is recognized by public health experts as a re-emerging infectious disease. Therefore there is great interest in developing a safe and effective vaccine. Vaccines against plague containing both the Fraction 1 (F1) and V antigens of Y. pestis have shown promise in protecting animal models against pneumonic plague, the deadliest form of the disease. Here we report on a plague vaccine consisting of the F1 and LcrV antigens fused to a single carrier molecule, the thermostable enzyme lichenase from Clostridium thermocellum, and expressed in and purified from Nicotiana benthamiana plants. When administered to Cynomolgus Macaques this purified plant-produced vaccine induced high titers of serum IgG, mainly of the IgG1 isotype, against both F1 and LcrV. These immunized animals were subsequently challenged and the LcrV-F1 plant-produced vaccine conferred complete protection against aerosolized Y. pestis.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have been identified as a potential pandemic threat by the World Health Organization (WHO). Since 1997, these viruses have been spreading from Asia to Europe and Africa with increasing genetic and antigenic diversities. Vaccination is the preferred strategy for the prevention and control of influenza infections and the availability of a system for the rapid engineering and production of vaccines is required in the event of an influenza pandemic. In this study, we engineered and produced recombinant hemagglutinin (HA) from A/Bar-headed Goose/Qinghai/1A/05 (clade 2.2) and A/Anhui/1/2005 (clade 2.3) in Nicotiana benthamiana plants. Immunization of mice with these plant-derived HA antigens elicited serum hemagglutination inhibition (HI) and virus neutralization (VN) antibodies. These results suggest the utility of our plant-expression system for recombinant influenza vaccine production.

Plants have been shown to be efficient systems for expressing a wide range of recombinant proteins from various origins. Here, using a plant virus-based expression vector to produce human growth hormone (hGH) in Nicotiana benthamiana plants, we demonstrate, for the first time, that the plant-produced hGH (pphGH) is biologically active in a hypophysectomized rat model. We observed an average weight gain of approximately 17 g per animal in a group of 10 animals that were injected subcutaneously with pphGH with 60 microg/dose for 10 days. With the increasing demand for hGH, accompanied with the need to make this recombinant protein available to a wider population at a more reasonable cost, plants provide a feasible alternative to current production platforms., ((c) 2009 American Institute of Chemical Engineers Biotechnol.)

Production of vaccine antigens in plants has received considerable attention over the last decade. However, despite many antigens being expressed in plant systems, and promising efficacy data with rodent models, few vaccine candidates have advanced into studies in non-human primates or human clinical trials. Here, we report on the transient expression of the F1 and LcrV antigens of Yersinia pestis in Nicotiana benthamiana. The antigens were expressed as fusions to the thermostable enzyme of Clostridium thermocellum. When administered to Cynomolgus Macaques the purified plant-produced antigens induced serum IgG and IgA responses specific to F1 and LcrV, and conferred complete protection against lethal challenge with Y. pestis. This study clearly demonstrates the efficacy of a plant-produced plague vaccine candidate in a primate model.

The E7 oncoprotein from Human Papilloma Virus (HPV) is an attractive candidate for anti-cancer vaccine development. In this study, we engineered HPV16 E7 coding sequence (wild type or mutagenized sequence, E7GGG) as fusions to beta-1,3-1,4-glucanase (LicKM) of Clostridium thermocellum and produced in Nicotiana benthamiana plants using a transient expression system. Target antigens were purified and evaluated in mice for their potential as prophylactic and therapeutic vaccine candidates. Both fusion proteins induced E7-specific IgG and cytotoxic T-cell responses and protected mice against challenge with E7-expressing tumor cells. Furthermore, when administered after challenge, these plant-produced antigens prevented tumor development.

We have produced the recombinant intracellular domain of human IA-2 (IA-2ic), a diabetes-associated autoantigen, in plants. This was achieved by transient expression using agroinfiltration of Nicotiana benthamiana plants. The resulting plant-derived IA-2ic had the expected size, reacted with polyclonal and monoclonal antibodies specific to human IA-2ic and competitively inhibited radiolabeled IA-2ic in an immunoprecipitation assay. The expression level of recombinant IA-2ic was estimated to be 0.5% of the total soluble protein (TSP). Transient expression in plants has the potential to produce a large amount of human IA-2ic protein at low cost in a short period of time.

We have developed a plant virus-mediated transgene activation (VMTA) system that utilizes a viral expression vector to present the inducer. The concept was tested using two well characterized components: (i) an artificial promoter based on the yeast GAL4 upstream activating sequence and the minimal TATA element of Cauliflower Mosaic Virus 35S RNA promoter, and (ii) a transcriptional activator (TA) consisting of a fusion between the GAL4 DNA binding domain and the Herpes simplex virus VP16 activation domain. The TA was expressed under the control of the subgenomic promoter of a Tobacco Mosaic Virus-based expression vector. The VMTA system was functional in transient Agroinfiltration assays with the reporter gene beta-glucuronidase, the intracellular domain of the diabetes associated autoimmune antigen, IA-2ic, and with the anti-tetanus antibody 9F12. Transgenic lines harboring the reporter gene were also examined. The VMTA system displayed tight transcriptional control in both transient assays and in transgenic Nicotiana benthamiana plants carrying the TA-inducible reporter.

Abstract: Plant-derived vaccines represent an attractive strategy for cancer immunotherapy due to their relative safety and cost-effectiveness. We evaluated the anti-tumour activity of a Nicotiana benthamiana produced vaccine candidate based on the non-transforming E7 protein of HPV-16 fused to β-1,3-1,4-glucanase of Clostridium thermocellum. Two doses of vaccine at two week intervals were administered to groups of C57BL/6 mice starting 3 or 6 days after challenge with tumourigenic E7-expressing TC-1* cells. Inhibition of tumour growth and increased survival was observed in both groups treated with vaccine. These data suggest the potential of plants as a platform for producing therapeutic vaccines. [Copyright &y& Elsevier]