Fine-tuning expression of begomoviral movement and nuclear shuttle proteins confers cell-to-cell movement to mastreviral replicons in Nicotiana benthamiana leaves.

Geminiviruses are a group of small plant viruses responsible for devastating crop damage worldwide. The emergence of agricultural diseases caused by geminiviruses is attributed in part to their high rates of recombination, leading to complementary function between viral components across species and genera. We have developed a mastreviral reporter system based on bean yellow dwarf virus (BeYDV) that replicates to high levels in the plant nucleus, expressing very high levels of GFP. To investigate the potential for complementation of movement function by other geminivirus genera, the movement protein (MP) and nuclear shuttle protein (NSP) from the bipartite begomovirus Bean dwarf mosaic virus (BDMV) were produced and characterized in Nicotiana benthamiana leaves. While overexpression of MP and NSP strongly inhibited GFP expression from the mastreviral reporter and caused adverse plant symptoms, optimizing the expression levels of MP and NSP allowed functional cell-to-cell movement. Hybrid virus vectors were created that express BDMV MP and NSP from mastreviral replicons, allowing efficient cell-to-cell movement comparable to native BDMV replicons. We find that the expression levels of MP and NSP must be fine-tuned to provide sufficient MP/NSP for movement without eliciting the plant hypersensitive response or adversely impacting gene expression from viral replicons. The ability to confer cell-to-cell movement to mastrevirus replicons depended strongly on replicon size: 2.1-2.7 kb replicons were efficiently moved, while 3 kb replicons were inhibited, and 3.9 kb replicons were very strongly inhibited. Optimized expression of MP/NSP from the normally phloem-limited Abutilon mosaic virus (AbMV) allows efficient movement in non-phloem cells.

An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves.

KEY MESSAGE: We have found interesting features of a plant gene (extensin) 3' flanking region, including extremely efficient polyadenylation which greatly improves transient expression of transgenes when an intron is removed. Its use will greatly benefit studies of gene expression in plants, research in molecular biology, and applications for recombinant proteins. Plants are a promising platform for the production of recombinant proteins. To express high-value proteins in plants efficiently, the optimization of expression cassettes using appropriate regulatory sequences is critical. Here, we characterize the activity of the tobacco extensin (Ext) gene terminator by transient expression in Nicotiana benthamiana, tobacco, and lettuce. Ext is a member of the hydroxyproline-rich glycoprotein (HRGP) superfamily and constitutes the major protein component of cell walls. The present study demonstrates that the Ext terminator with its native intron removed increased transient gene expression up to 13.5-fold compared to previously established terminators. The enhanced transgene expression was correlated with increased mRNA accumulation and reduced levels of read-through transcripts, which could impair gene expression. Analysis of transcript 3'-ends found that the majority of polyadenylated transcripts were cleaved at a YA dinucleotide downstream from a canonical AAUAAA motif and a UG-rich region, both of which were found to be highly conserved among related extensin terminators. Deletion of either of these regions eliminated most of the activity of the terminator. Additionally, a 45 nt polypurine sequence ~ 175 nt upstream from the polyadenylation sites was found to also be necessary for the enhanced expression. We conclude that the use of Ext terminator has great potential to benefit the production of recombinant proteins in plants.

Chimeric 3' flanking regions strongly enhance gene expression in plants.

Plants represent a promising platform for the highly scalable production of recombinant proteins. Previously, we identified the tobacco extensin terminator lacking its intron as an element that reduced transcript read-through and improved recombinant protein production in a plant-based system. In this study, we systematically compared nonreplicating plant expression vectors containing over 20 commonly used or newly identified terminators from diverse sources. We found that eight gene terminators enhance reporter gene expression significantly more than the commonly used 35S and NOS terminators. The intronless extensin terminator provided a 13.6-fold increase compared with the NOS terminator. Combining terminators in tandem produced large synergistic effects, with many combinations providing a >25-fold increase in expression. Addition of the tobacco Rb7 or TM6 matrix attachment region (MAR) strongly enhanced protein production when added to most terminators, with the Rb7 MAR providing the greatest enhancement. Using deletion analysis, the full activity of the 1193 bp Rb7 MAR was found to require only a 463-bp region at its 3' end. Combined terminators and MAR together provided a >60-fold increase compared with the NOS terminator alone. These combinations were then placed in a replicating geminiviral vector, providing a total of >150-fold enhancement over the original NOS vector, corresponding to an estimated yield of 3-5 g recombinant protein per kg leaf fresh weight or around 50% of the leaf total soluble protein. These results demonstrate the importance of 3' flanking regions in optimizing gene expression and show great potential for 3' flanking regions to improve DNA-based recombinant protein production systems.

High-level expression and enrichment of norovirus virus-like particles in plants using modified geminiviral vectors.

Recombinant virus-like particles (VLPs) are proven to be safe and effective vaccine candidates. We have previously described a plant-based recombinant protein expression system based on agroinfiltration of a replicating vector derived from the geminivirus bean yellow dwarf virus (BeYDV). The system has been systematically optimized to improve expression and reduce cell death in Nicotiana benthamiana leaves. Using these modifications, we show that VLPs derived from genotype GII.4 norovirus, the leading cause of acute gastroenteritis worldwide, can be produced at >1 mg/g leaf fresh weight (LFW), over three times the highest level ever reported in plant-based systems. We also produced norovirus GI VLPs at 2.3 mg/g LFW. Treatment of VLP-containing crude leaf extracts with acid, detergent, or heat enhanced recovery and allowed selective enrichment of norovirus VLPs. Optimal treatment conditions allowed removal of >90% of endogenous plant proteins without any loss of norovirus VLPs. Selective enrichment of hepatitis B core antigen (HBcAg) VLPs by acid treatment was also demonstrated, with some losses in yield that were partially mitigated in the presence of detergent. Sedimentation analysis confirmed that acid and detergent did not inhibit proper assembly of norovirus VLPs, although heat treatment had a small negative effect. These results demonstrate that milligram quantities of norovirus VLPs can be obtained and highly enriched in a matter of days from a single plant leaf using the BeYDV plant expression system.

A self-binding immune complex vaccine elicits strong neutralizing responses against herpes simplex virus in mice.

Introduction: It has been known for over half a century that mixing an antigen with its cognate antibody in an immune complex (IC) can enhance antigen immunogenicity. However, many ICs produce inconsistent immune responses, and the use of ICs in the development new vaccines has been limited despite the otherwise widespread success of antibody-based therapeutics. To address this problem, we designed a self-binding recombinant immune complex (RIC) vaccine which mimics the larger ICs generated during natural infection. Materials and methods: In this study, we created two novel vaccine candidates: 1) a traditional IC targeting herpes simplex virus 2 (HSV-2) by mixing glycoprotein D (gD) with a neutralizing antibody (gD-IC); and 2) an RIC consisting of gD fused to an immunoglobulin heavy chain and then tagged with its own binding site, allowing self-binding (gD-RIC). We characterized the complex size and immune receptor binding characteristics in vitro for each preparation. Then, the in vivo immunogenicity and virus neutralization of each vaccine were compared in mice. Results: gD-RIC formed larger complexes which enhanced C1q receptor binding 25-fold compared to gD-IC. After immunization of mice, gD-RIC elicited up to 1,000-fold higher gD-specific antibody titers compared to traditional IC, reaching endpoint titers of 1:500,000 after two doses without adjuvant. The RIC construct also elicited stronger virus-specific neutralization against HSV-2, as well as stronger cross-neutralization against HSV-1, although the proportion of neutralizing antibodies to total antibodies was somewhat reduced in the RIC group. Discussion: This work demonstrates that the RIC system overcomes many of the pitfalls of traditional IC, providing potent immune responses against HSV-2 gD. Based on these findings, further improvements to the RIC system are discussed. RIC have now been shown to be capable of inducing potent immune responses to a variety of viral antigens, underscoring their broad potential as a vaccine platform.

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana.

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment's purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

A Highly Expressing, Soluble, and Stable Plant-Made IgG Fusion Vaccine Strategy Enhances Antigen Immunogenicity in Mice Without Adjuvant.

Therapeutics based on fusing a protein of interest to the IgG Fc domain have been enormously successful, though fewer studies have investigated the vaccine potential of IgG fusions. In this study, we systematically compared the key properties of seven different plant-made human IgG1 fusion vaccine candidates using Zika virus (ZIKV) envelope domain III (ZE3) as a model antigen. Complement protein C1q binding of the IgG fusions was enhanced by: 1) antigen fusion to the IgG N-terminus; 2) removal of the IgG light chain or Fab regions; 3) addition of hexamer-inducing mutations in the IgG Fc; 4) adding a self-binding epitope tag to create recombinant immune complexes (RIC); or 5) producing IgG fusions in plants that lack plant-specific ß1,2-linked xylose and α1,3-linked fucose N-linked glycans. We also characterized the expression, solubility, and stability of the IgG fusions. By optimizing immune complex formation, a potently immunogenic vaccine candidate with improved solubility and high stability was produced at 1.5 mg IgG fusion per g leaf fresh weight. In mice, the IgG fusions elicited high titers of Zika-specific antibodies which neutralized ZIKV using only two doses without adjuvant, reaching up to 150-fold higher antibody titers than ZE3 antigen alone. We anticipate these findings will be broadly applicable to the creation of other vaccines and antibody-based therapeutics.

Codelivery of improved immune complex and virus-like particle vaccines containing Zika virus envelope domain III synergistically enhances immunogenicity.

Zika virus (ZIKV) reemergence poses a significant health threat especially due to its risks to fetal development, necessitating safe and effective vaccines that can protect pregnant women. Zika envelope domain III (ZE3) has been identified as a safe and effective vaccine candidate, however it is poorly immunogenic. We previously showed that plant-made recombinant immune complex (RIC) vaccines are a robust platform to improve the immunogenicity of weak antigens. In this study, we altered the antigen fusion site on the RIC platform to accommodate N-terminal fusion to the IgG heavy chain (N-RIC), and thus a wider range of antigens, with a resulting 40% improvement in RIC expression over the normal C-terminal fusion (C-RIC). Both types of RICs containing ZE3 were efficiently assembled in plants and purified to >95% homogeneity with a simple one-step purification. Both ZE3 RICs strongly bound complement receptor C1q and elicited strong ZE3-specific antibody titers that correlated with ZIKV neutralization. When either N-RIC or C-RIC was codelivered with plant-produced hepatitis B core (HBc) virus-like particles (VLP) displaying ZE3, the combination elicited 5-fold greater antibody titers (>1,000,000) and more strongly neutralized ZIKV than either RICs or VLPs alone, after only two doses without adjuvant. These findings demonstrate that antigens that require a free N-terminus for optimal antigen display can now be used with the RIC system, and that plant-made RICs and VLPs are highly effective vaccines targeting ZE3. Thus, the RIC platform can be more generally applied to a wider variety of antigens.

Vaccine synergy with virus-like particle and immune complex platforms for delivery of human papillomavirus L2 antigen.

Diverse HPV subtypes are responsible for considerable disease burden worldwide, necessitating safe, cheap, and effective vaccines. The HPV minor capsid protein L2 is a promising candidate to create broadly protective HPV vaccines, though it is poorly immunogenic by itself. To create highly immunogenic and safe vaccine candidates targeting L2, we employed a plant-based recombinant protein expression system to produce two different vaccine candidates: L2 displayed on the surface of hepatitis B core (HBc) virus-like particles (VLPs) or L2 genetically fused to an immunoglobulin capable of forming recombinant immune complexes (RIC). Both vaccine candidates were potently immunogenic in mice, but were especially so when delivered together, generating very consistent and high antibody titers directed against HPV L2 (>1,000,000) that correlated with virus neutralization. These data indicate a novel immune response synergy upon co-delivery of VLP and RIC platforms, a strategy that can be adapted generally for many different antigens.

Evaluation of a toxoid fusion protein vaccine produced in plants to protect poultry against necrotic enteritis.

BACKGROUND: Necrotic enteritis (NE) is caused by type A strains of the bacterium Clostridium perfringens. Total global economic losses to the poultry industry due to NE is estimated to be over two billion dollars annually. Traditionally, NE has been effectively controlled by inclusion of antibiotics in the diet of poultry. However, recent concerns regarding the impact of this practice on increasing antibiotic resistance in human pathogens have led us to consider alternative approaches, such as vaccination, for controlling this disease. NE strains of C. perfringens produce two major toxins, a-toxin and NetB. Immune responses against either toxin can provide partial protection against NE. METHODS: We have developed a fusion protein combining a non-toxic carboxyl-terminal domain of a-toxin (PlcC) and an attenuated, mutant form of NetB (NetB-W262A) for use as a vaccine antigen to immunize poultry against NE. We utilized a DNA sequence that was codon-optimized for Nicotiana benthamiana to enable high levels of expression. The 6-His tagged PlcC-NetB fusion protein was synthesized in N. benthamiana using a geminiviral replicon transient expression system, purified by metal affinity chromatography, and used to immunize broiler birds. RESULTS: Immunized birds produced a strong serum IgY response against both the plant produced PlcC-NetB protein and against bacterially produced His-PlcC and His-NetB. Immunized birds were significantly protected against a subsequent in-feed challenge with virulent C. perfringens when treated with the fusion protein. These results indicate that a plant-produced PlcC-NetB toxoid is a promising vaccine candidate for controlling NE in poultry.

High Level Production of Monoclonal Antibodies Using an Optimized Plant Expression System.

Biopharmaceuticals are a large and fast-growing sector of the total pharmaceutical market with antibody-based therapeutics accounting for over 100 billion USD in sales yearly. Mammalian cells are traditionally used for monoclonal antibody production, however plant-based expression systems have significant advantages. In this work, we showcase recent advances made in plant transient expression systems using optimized geminiviral vectors that can efficiently produce heteromultimeric proteins. Two, three, or four fluorescent proteins were coexpressed simultaneously, reaching high yields of 3-5 g/kg leaf fresh weight or ~50% total soluble protein. As a proof-of-concept for this system, various antibodies were produced using the optimized vectors with special focus given to the creation and production of a chimeric broadly neutralizing anti-flavivirus antibody. The variable regions of this murine antibody, 2A10G6, were codon optimized and fused to a human IgG1. Analysis of the chimeric antibody showed that it was efficiently expressed in plants at 1.5 g of antibody/kilogram of leaf tissue, can be purified to near homogeneity by a simple one-step purification process, retains its ability to recognize the Zika virus envelope protein, and potently neutralizes Zika virus. Two other monoclonal antibodies were produced at similar levels (1.2-1.4 g/kg). This technology will be a versatile tool for the production of a wide spectrum of pharmaceutical multi-protein complexes in a fast, powerful, and cost-effective way.

Modifying the Replication of Geminiviral Vectors Reduces Cell Death and Enhances Expression of Biopharmaceutical Proteins in Nicotiana benthamiana Leaves.

Plants are a promising platform to produce biopharmaceutical proteins, however, the toxic nature of some proteins inhibits their accumulation. We previously created a replicating geminiviral expression system based on bean yellow dwarf virus (BeYDV) that enables very high-level production of recombinant proteins. To study the role of replication in this system, we generated vectors that allow separate and controlled expression of BeYDV Rep and RepA proteins. We show that the ratio of Rep and RepA strongly affects the efficiency of replication. Rep, RepA, and vector replication all elicit the plant hypersensitive response, resulting in cell death. We find that a modest reduction in expression of Rep and RepA reduces plant leaf cell death which, despite reducing the accumulation of viral replicons, increases target protein accumulation. A single nucleotide change in the 5' untranslated region (UTR) reduced Rep/RepA expression, reduced cell death, and enhanced the production of monoclonal antibodies. We also find that replicating vectors achieve optimal expression with lower Agrobacterium concentrations than non-replicating vectors, further reducing cell death. Viral UTRs are also shown to contribute substantially to cell death, while a native plant-derived 5' UTR does not.

5' and 3' Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves.

We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5' untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5' UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5' UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1 mg/g leaf fresh weight.