Recombinant immune complexes as vaccines against infectious diseases.

New vaccine technologies are needed to combat many existing infections and prepare better for those that may emerge in the future. The conventional technologies that rely on protein-based vaccines are still severely restricted by the sparsity and poor accessibility of available adjuvants. One possible solution to this problem is to enhance antigen immunogenicity by a more natural means by complexing it with antibodies in the form of immune complexes (ICs). However, natural ICs are impractical as vaccines, and significant research efforts have been made to generate them in recombinant form, with plant bioengineering being at the forefront of these efforts. Here, we describe the challenges and progress made to date to make recombinant IC vaccines applicable to humans.

Enhanced binding and inhibition of SARS-CoV-2 by a plant-derived ACE2 protein containing a fused mu tailpiece.

Infectious diseases such as Coronavirus disease 2019 (COVID-19) and Middle East respiratory syndrome (MERS) present an increasingly persistent crisis in many parts of the world. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The angiotensin-converting enzyme 2 (ACE2) is a crucial cellular receptor for SARS-CoV-2 infection. Inhibition of the interaction between SARS-CoV-2 and ACE2 has been proposed as a target for the prevention and treatment of COVID-19. We produced four recombinant plant-derived ACE2 isoforms with or without the mu tailpiece (µ-tp) of immunoglobulin M (IgM) and the KDEL endoplasmic reticulum retention motif in a plant expression system. The plant-derived ACE2 isoforms bound whole SARS-CoV-2 virus and the isolated receptor binding domains of SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Omicron variants. Fusion of µ-tp and KDEL to the ACE2 protein (ACE2 µK) had enhanced binding activity with SARS-CoV-2 in comparison with unmodified ACE2 protein derived from CHO cells. Furthermore, the plant-derived ACE2 µK protein exhibited no cytotoxic effects on Vero E6 cells and effectively inhibited SARS-CoV-2 infection. The efficient and rapid scalability of plant-derived ACE2 µK protein offers potential for the development of preventive and therapeutic agents in the early response to future viral outbreaks.

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.

Construction of SARS-CoV-2 virus-like particles in plant.

The pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a public health emergency, and research on the development of various types of vaccines is rapidly progressing at an unprecedented development speed internationally. Some vaccines have already been approved for emergency use and are being supplied to people around the world, but there are still many ongoing efforts to create new vaccines. Virus-like particles (VLPs) enable the construction of promising platforms in the field of vaccine development. Here, we demonstrate that non-infectious SARS-CoV-2 VLPs can be successfully assembled by co-expressing three important viral proteins membrane (M), envelop (E) and nucleocapsid (N) in plants. Plant-derived VLPs were purified by sedimentation through a sucrose cushion. The shape and size of plant-derived VLPs are similar to native SARS-CoV-2 VLPs without spike. Although the assembled VLPs do not have S protein spikes, they could be developed as formulations that can improve the immunogenicity of vaccines including S antigens, and further could be used as platforms that can carry S antigens of concern for various mutations.

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.

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.

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.

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.

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.

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.

Improvement of the transient expression system for production of recombinant proteins in plants.

An efficient and high yielding expression system is required to produce recombinant proteins. Furthermore, the transient expression system can be used to identify the localization of proteins in plant cells. In this study, we demonstrated that combination of a geminiviral replication and a double terminator dramatically enhanced the transient protein expression level in plants. The GFP protein was expressed transiently in lettuce, Nicotiana benthamiana, tomatoes, eggplants, hot peppers, melons, and orchids with agroinfiltration. Compared to a single terminator, a double terminator enhanced the expression level. A heat shock protein terminator combined with an extensin terminator resulted in the highest protein expression. Transiently expressed GFP was confirmed by immunoblot analysis with anti-GFP antibodies. Quantitative analysis revealed that the geminiviral vector with a double terminator resulted in the expression of at least 3.7 mg/g fresh weight of GFP in Nicotiana benthamiana, approximately 2-fold that of the geminiviral vector with a single terminator. These results indicated that combination of the geminiviral replication and a double terminator is a useful tool for transient expression of the gene of interest in plant 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.

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.

Recombinant human osteopontin expressed in Nicotiana benthamiana stimulates osteogenesis related genes in human periodontal ligament cells.

Tissue engineering aims to utilise biologic mediators to facilitate tissue regeneration. Several recombinant proteins have potential to mediate induction of bone production, however, the high production cost of mammalian cell expression impedes patient access to such treatments. The aim of this study is to produce recombinant human osteopontin (hOPN) in plants for inducing dental bone regeneration. The expression host was Nicotiana benthamiana using a geminiviral vector for transient expression. OPN expression was confirmed by Western blot and ELISA, and OPN was purified using Ni affinity chromatography. Structural analysis indicated that plant-produced hOPN had a structure similar to commercial HEK cell-produced hOPN. Biological function of the plant-produced hOPN was also examined. Human periodontal ligament stem cells were seeded on an OPN-coated surface. The results indicated that cells could grow normally on plant-produced hOPN as compared to commercial HEK cell-produced hOPN determined by MTT assay. Interestingly, increased expression of osteogenic differentiation-related genes, including OSX, DMP1, and Wnt3a, was observed by realtime PCR. These results show the potential of plant-produced OPN to induce osteogenic differentiation of stem cells from periodontal ligament in vitro, and suggest a therapeutic strategy for bone regeneration in the future.

Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection.

Cross-Reactive Synbody Affinity Ligands for Capturing Diverse Noroviruses.

Noroviruses are the most common cause of acute gastroenteritis in the developed world. Noroviruses are a diverse group of nonenveloped RNA viruses that are continuously evolving. This leads to the rise of immunologically distinct strains of the same genotype on a frequent basis. This diversity presents a unique challenge for detection and tracking of new strains, with the continuous need for new norovirus affinity ligands. Our group developed a family of bivalent synbody affinity ligands using a virus-like particle (VLP) from the 2006 GII.4 Minerva strain of norovirus. We produced more than 20 synbodies with low nanomolar dissociation constants (KD < 10 nM) for GII.4 VLP. We measured binding affinity for four synbodies against VLPs from multiple GI and GII genotypes and found that the synbodies were broadly cross-reactive with affinities that ranged from 0.5 to 8 nM. We tested the ability of these synbodies to capture norovirus from dilute solutions and found that one synbody could capture GII.4 from a 200 000-fold dilution from a norovirus positive stool sample. When these synbodies were tested for the ability to capture of multiple genotypes, we found that four different genotypes were recognized. These data demonstrate that the synbody approach can generate multiple affinity ligands for future use in norovirus detection and possible therapeutic development.

Recombinant Human Acidic Fibroblast Growth Factor (aFGF) Expressed in Nicotiana benthamiana Potentially Inhibits Skin Photoaging.

Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana. Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N. benthamiana. The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated N. benthamiana. The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. N. benthamiana-derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of Escherichia coli-derived recombinant human acidic fibroblast growth factor. Additionally, N. benthamiana-derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that N. benthamiana-derived recombinant human acidic fibroblast growth factor effectively protects skin cell from UVB, suggesting its potential use as a cosmetic or therapeutic agent against skin photoaging.