Background: The continual emergence of antigenically drifted avian influenza viruses poses a persistent threat to global health and underscores the need for broadly protective approaches. Stem-directed monoclonal antibodies targeting conserved hemagglutinin (HA) epitopes are a promising strategy to address viral diversity. CR9114 is a broadly neutralizing antibody previously reported to recognize a conserved HA stem region across influenza A and B viruses. Methods: CR9114 was transiently expressed in Nicotiana benthamiana and characterized for protein integrity, assembly, and glycosylation. Binding to recombinant hemagglutinin was assessed, and neutralizing activity was evaluated against antigenically distinct avian influenza A viruses using in vitro neutralization assays. Results: Plant-produced CR9114 was correctly assembled as a human IgG1 κ antibody and displayed a high-mannose glycosylation profile. The antibody showed strong binding to recombinant H5 hemagglutinin (Kd = 0.15 µg/mL) and potently neutralized recent avian influenza isolates, namely A/Jiangsu/NJ210/2023 (H5N1; NT50 = 1589) and A/Gansu/23277/2019 (H7N9; NT50 = 177), demonstrating cross-subtype neutralization despite known glycan-associated constraints in Group 2 viruses. Conclusions: These findings demonstrate that N. benthamiana is a viable platform for the rapid production of functional broadly neutralizing anti-influenza antibodies. The preserved activity of plant-produced CR9114 against contemporary avian influenza strains supports its continued evaluation as a broadly protective therapeutic candidate and highlights the potential of plant molecular pharming approaches to contribute to pandemic preparedness.

ObjectiveData on the safety of coronavirus disease 19 (COVID-19) vaccines in gynecologic cancer patients are scarce. The type of vaccine used in Thailand differs from what has been studied in other countries. This study evaluated the prevalence and characteristics of reported adverse events following COVID-19 vaccines in patients with gynecologic cancer patients.MethodA retrospective, single-center descriptive study was performed in patients with gynecologic cancer who received at least one dose of the COVID-19 vaccine at King Chulalongkorn Memorial Hospital, Thailand, from January 2020 to August 2021. Adverse events were collected through structured telephone interviews using a standardized questionnaire. Descriptive statistics summarized patient characteristics and adverse events. Associations with any-grade adverse events were assessed using logistic regression and Fisher's exact test for categorical variables.ResultsOf the 294 patients interviewed, 72.8% were in remission, under surveillance, or in palliative treatment at the time of vaccination, and 17.7% were undergoing treatment. The most common adverse effects were grade 1-2 injection site reactions. One patient developed grade 3 fever and seizures 10 days after the first dose of the AstraZeneca vaccine. Between the second and fourth doses of the vaccination, the most common adverse events were grade 1-2 injection site reactions. No severe allergic reactions or grade 4 adverse events were reported. The study concluded that patients under 60 years of age had more adverse events than older patients (adjusted odds ratio 1.99, 95% confidence interval 1.08-3.71 p = 0.029). The treatment status did not affect adverse events. Of 283 patients who received two doses, 27.6% were infected with COVID-19.ConclusionCOVID-19 vaccination was generally well tolerated among gynecological cancer patients who received active anticancer therapy and those under surveillance. Younger patients frequently reported more adverse effects than older patients.

BACKGROUND:House dust mites (HDM) are one of the significant indoor allergen sources which cause IgE-mediated responses in most of the allergic individuals. HDMs are found in human habitats worldwide and Der p 2 is one of the major clinically relevant HDM allergens involved in triggering allergic diseases. The recombinant production of Der p 2 in plant systems provides a cost-effective and viable platform for developing diagnostic kits and allergen-specific immunotherapy. METHODS:The D. pteronyssinus Der p 2 allergen was transiently expressed in Nicotiana benthamiana and its immunogenicity was evaluated in mice. The Der p 2 coding sequence was cloned into a geminiviral plant expression vector and introduced into N. benthamiana leaves via Agrobacterium tumefaciens-mediated infiltration. Recombinant Der p 2 proteins were purified from the crude extracts and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. The immunogenicity of the plant-produced Der p 2 proteins was further evaluated by immunizing mice following a prime-boost immunization regimen, and Der p 2-specific antibody responses were assessed by ELISA. RESULTS:Recombinant Der p 2 was successfully expressed and purified from N. benthamiana, and immunized mice developed high levels of Der p 2-specific IgG antibodies, with antibody titers increased after booster immunization. CONCLUSIONS:The results demonstrate that the transient expression of Der p 2 in plants is a feasible and effective strategy for producing immunologically active recombinant allergen proteins for diagnostic and potential clinical applications.

Dengue is a life-threatening mosquito-borne viral disease ranging from mild symptoms to severe hemorrhagic fever. In this study, a recombinant anti-dengue D8 monoclonal antibody was produced in Nicotiana benthamiana and characterized for protein integrity and glycosylation by LC-MS. The plant-derived antibody lacked plant-specific β1,2-xylose and core α1,3-fucose residues, displaying a mammalian-like glycan profile. Functional evaluation showed potent cross-neutralizing activity against all four dengue virus serotypes (DENV1–DENV4), with the strongest activity against DENV4 (FRNT50 < 1 µg/mL), followed by DENV2 (5.82 µg/mL), DENV3 (9.49 µg/mL), and DENV1 (28.68 µg/mL), comparable to the mammalian-produced counterpart. The antibody also bound strongly to NS1 proteins of all serotypes, especially DENV2, and demonstrated higher reactivity than mammalian-derived anti-NS1 antibodies. Collectively, our results provide proof-of-concept that a glycoengineered plant platform can generate a functional D8 antibody with mammalian-like glycosylation, robust NS1 binding, and cross-neutralizing activity against DENV1–4, prompting further evaluation in Fc-dependent assays and in vivo models.

Agrobacterium-mediated transient expression in Nicotiana benthamiana is widely used for recombinant biopharmaceutical production. To investigate the host plant response upon human cytokine production with contrast accumulation, we expressed codon optimized human IL1 beta, serving as a high expression benchmark with a yield of approximately 80 μg/g leaf fresh weight, and IL15 with undetected signal on western blot, representing protein with low level of accumulation, using the geminiviral vector system. A combined proteomics and metabolomics technique was applied to elucidate underlying cellular mechanisms. Quantitative proteomics revealed that IL1-His was robustly detected (9 unique peptides, 83% coverage), whereas no IL15-His-derived peptides were identified. Based on pathway analysis, the expression of IL15-His induced chaperone expression, with downregulation of photosynthetic and primary metabolism pathways. Additionally, metabolomic pathway analysis revealed that IL1-His preferentially drives the branched chain amino acid biosynthesis, but IL15-His shifts metabolism towards phenylpropanoid biosynthesis routes. These findings underscore that the characteristics of the target protein and its interaction with the host's physiology could influence the yield of the recombinant protein production in plants.

In the present study, recombinant basic fibroblast growth factor (bFGF) was tagged with His and expressed in Nicotiana benthamiana to prepare a plant crude extract for PVP40 treatment prior to purification using immobilized metal affinity chromatography (IMAC). Optimization of PVP40 involved varying the concentrations from 1% to 3%. PVP40 is used as a mediator to reduce the effects of phenols in the crude plant extract. The main aim was to enhance the purification process of plant-based bFGF-His by IMAC using different concentrations of PVP40. Crude extracts from groups 1, 2, 3 and 4 were finally applied to a nickel-sepharose affinity column using IMAC10 followed by elution of FGF-2 recombinant protein with IMAC250. The purified FGF-2 protein was analyzed by SDS-PAGE and Western blotting in groups 1-4. The purified protein was found to be free of secondary metabolites in the plant extract and nickel beads.

Food-derived bioactive compounds are increasingly being recognized for their potential in modulating inflammatory responses associated with post-infectious health conditions. Ivy gourd (Coccinia grandis), a vegetable belonging to the cucumber family (Cucurbitaceae), is widely consumed in Southeast Asia and has been reported to contain cucurbitacins exhibiting anti-inflammatory properties. However, their composition and biological relevance in viral protein-induced inflammation remain unclear. In this study, cucurbitacin B and cucurbitacin E were quantified in C. grandis fruit extract using validated HPLC-UV analysis, and their anti-inflammatory effects were evaluated in SARS-CoV-2 receptor-binding domain (RBD)-stimulated RAW 264.7 macrophages. Cucurbitacin B and cucurbitacin E contents were 0.16 +/- 0.01 and 0.11 +/- 0.01 mg/g dry weight, respectively. RBD stimulation significantly increased nitric oxide (NO) production by approximately 5-fold compared with the untreated control (p < 0.05). Treatment with C. grandis extract at 200 & micro;g/mL reduced NO levels by 51.96 +/- 1.33%, while treatment with cucurbitacin B and cucurbitacin E at 5 and 50 & micro;M, respectively, inhibited NO production by 78.52 +/- 1.89% and 68.71 +/- 2.21%, respectively. RBD elevated inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) expression by 25-, 50-, and 30-fold, respectively, which were significantly suppressed by cucurbitacin B, cucurbitacin E, and C. grandis extract. Cell viability remained above 80% at all tested concentrations. Cucurbitacin B exhibited the strongest inhibitory effect. These findings highlight C. grandis fruit as a dietary source of anti-inflammatory bioactive compounds and support its potential application as a functional food and nutraceutical ingredient.

Basic Fibroblast Growth Factor (bFGF), also known as FGF2, is a multifunctional growth factor involved in several physiological and pathological processes, including angiogenesis, wound healing, and embryonic development. This study aimed to investigate the feasibility and efficacy of producing recombinant bFGF in Nicotiana benthamiana using a geminiviral transient expression vector. The primary objective was to assess the bioactivity of N. benthamiana-produced bFGF in comparison with commercially available recombinant bFGF preparations produced in different heterologous expression systems. NIH-3T3 cells were exposed to varying concentrations (0.5–1000 ng/mL) of plant-produced bFGF for 24 and 48 h. The assessment parameters included cell viability, cellular responses, and wound-healing capacity. The plant-produced bFGF exhibited slightly greater cell viability compared to the non-treated control and a modest increase in cell proliferation. The analysis of the dose–response curve yielded an EC50 value of 0.171 ng/mL highlighting the bioactivity and potency of plant-produced bFGF. These results demonstrate the efficacy of plant-derived bFGF in promoting cellular activities and facilitating wound closure, underscoring its potential for wound healing applications. However, further validation through preclinical and clinical studies is warranted.

The recent mpox outbreaks in non-endemic countries highlight the urgent need for improved therapeutics and diagnostics. In this study, monoclonal antibodies (mAbs) targeting the mpox enveloped virion antigens A35 and B6 were transiently expressed in Nicotiana benthamiana using a geminiviral vector system. Following agroinfiltration, anti-A35 and anti-B6 mAbs accumulated to 27 µg/g fresh weight at 3 days and 260 µg/g at 7 days post-infiltration, respectively. SDS-PAGE and Western blot analyses confirmed the assembly of antibodies, and the purified antibodies bound to mpox-infected Vero cells. The neutralization assays demonstrated moderate reductions in viral infection under the tested conditions. Additionally, an electrochemical immunosensor demonstrated the ability of plant-produced antibodies to detect mpox virus through antigen–antibody binding induced current changes. These results support that the plant-based systems as rapid platforms for producing mpox-specific antibodies for diagnostics and antiviral research.

The murine monoclonal antibody OKT3, which targets the CD3 complex on T cells, was the first FDA-approved therapeutic antibody and remains clinically relevant for reversing acute transplant rejection. However, conventional mammalian production systems for monoclonal antibodies are expensive, time-consuming, and prone to contamination. In this study, we investigated the feasibility of using Nicotiana benthamiana as a cost-effective and scalable plant-based platform for transient expression and purification of functional OKT3 antibody. Codon-optimized sequences for the heavy and light chains of OKT3 were designed with signal peptides for ER targeting and a C-terminal SEKDEL sequence to enhance protein folding and retention. These sequences were cloned into a geminiviral vector and transformed into Agrobacterium tumefaciens, followed by co-infiltration into N. benthamiana leaves. Western blot analysis confirmed successful expression and assembly of the antibody. Purification was performed using Protein A affinity chromatography. Functional analysis by flow cytometry demonstrated that the FITC-conjugated plant-produced OKT3 antibody exhibited slightly higher CD3+ T cell binding efficiency and stronger fluorescence intensity than the commercial APC-conjugated counterpart. These findings support the use of N. benthamiana as a viable platform for functional anti-CD3 antibody production, with potential applications in diagnostics and therapeutics.

OBJECTIVE:Oral mucosa-on-a-chip (OMoC) devices mimic oral epithelium but require continuous nutrient support from underlying lamina propria. However, the common Matrigel or Basement membrane extract (BME) may not effectively support the oral mucosa (OM) native architecture. This study investigated whether a hybrid hydrogel composed of OM decellularized extracellular matrix (OM dECM), hyaluronic acid (HA), and alginate (Alg) surpasses Matrigel and BME for OMoC applications. METHODS:Porcine OM tissues were decellularized using 0.1-1 % sodium dodecyl sulfate (SDS), then freeze-dried, and pepsin-digested to produce OM dECM hydrogels (5, 10, and 20 mg/mL). DNA quantification, histological, biochemical and rheological assays, electron microscopy, and mass spectrometry were performed for dECM/hydrogels. Normal oral keratinocytes (NOK), gingival fibroblasts (HGF), and human umbilical vein endothelial cells (HUVEC) cultured in hydrogels were evaluated with proliferation, cytotoxicity, and immunocytochemistry (ICC) assays, and compared to Matrigel/BME. Hybrid hydrogels were used to tri-culture NOK, HGF, and HUVEC in a polydimethylsiloxane (PDMS)-based microfluidic chip for OM modeling. RESULTS:The 1 % SDS decellularization preserved greater ECM components. OM dECM hydrogels enhanced NOK, HGF, and HUVEC viability/proliferation, outperforming Matrigel/BME. Expression of OM-related markers (K14, Ki67, p63, Involucrin, E-cadherin, Von Willebrand factor) increased with hydrogels. Hybrid hydrogel of HA/Alg and 10 mg/mL dECM better supported NOK stratification while maintaining the native phenotype of HGF and HUVEC compared to Matrigel. Within our PDMS-based OMoC, the hybrid hydrogel facilitated the formation of a multi-layered microtissue construct with a stratified epithelial layer, a stromal compartment, and an endothelial network, better replicating the structural features of native OM as compared to other matrices and commercial microfluidic devices. CONCLUSIONS:A 1 % SDS decellularization protocol effectively preserved OM ECM, enabling development of an injectable OM dECM hydrogel. The optimized dECM-HA/Alg hybrid hydrogel outperformed Matrigel in supporting oral microtissue stratification, and phenotype maintenance, making it a superior alternative for OMoC applications. CLINICAL SIGNIFICANCE:This study developed a pre-clinical hybrid hydrogel-based OMoC that can more accurately replicate the oral mucosa ECM and tissue architecture when compared to Matrigel/BME. By enhancing the ECM mimicry, the dECM-HA/Alg hybrid hydrogel supported the development of an optimized OMoC for cytotoxicity screening of oral drugs and potential disease modeling of oral mucosal conditions.

Host proteins from Nicotiana benthamiana can remain in the recombinant biologic products after undergoing multiple steps of purification. These plant protein impurities may induce immunogenicity upon use. Therefore, controlling and monitoring host cell proteins is necessary throughout the process of recombinant protein production. Liquid chromatography-mass spectrometry (LC-MS) has been successfully used to characterize protein species down to nanogram levels in various types of samples. In this study, LC-MS was applied to detect N. benthamiana plant proteins in plant-produced pembrolizumab anti-cancer antibody. Two types of purification techniques, gravity flow with manually packed column and automated system with prepacked column, were studied. After purification, the protein products were primarily assessed with SDS-PAGE and Western blot analyses and further examined with LC-MS to confirm protein identity and investigate plant protein contaminations. The pembrolizumab sequence was confirmed with more than 89% coverage. A higher number of host plant proteins were detected in the protein samples purified with gravity flow column. Luminal-binding protein 5 and ribulose bisphosphate carboxylase (RuBisCO) enzyme were predominant host plant proteins detected. Luminal-binding protein 5 was observed in the products purified with both purification methods. It was likely bound to pembrolizumab antibody and co-eluted into eluate fraction as its sequence is similar to binding immunoglobulin protein (BiP). RuBisCO enzyme was detected in the samples purified with gravity flow only. Its presence was likely due to incomplete wash by gravity flow chromatography. In summary, this study provided an important clue for plant proteins that could be contaminated in plant-produced products and suggested that second column chromatography is required to enhance purification efficiency.

Human interleukin-15 (hIL-15) is a cytokine essential for immune modulation with therapeutic applications in cancer and chronic wound healing. Although hIL-15 is commercially available, large-scale production studies remain limited. With promising clinical trial results, demand for hIL-15 is expected to rise. Plant expression systems offer a sustainable, low-cost alternative for rapid biopharmaceutical production. In this study, we optimized hIL-15 expression in Nicotiana benthamiana and assessed its physicochemical properties and biological activity. We fused hIL-15 to the Fc domain of human IgG1 for efficient purification. Through optimization of the pre- and post-infiltration conditions, we achieved transient expression and recovery at 4 dpi, yielding 33.8 µg/g fresh weight. Peptide mapping confirmed 97 % overall sequence coverage of the primary structure. Treatment with plant-produced hIL-15-Fc effectively promoted human keratinocyte HaCaT cell proliferation and migration in vitro. These findings demonstrated the potential of plant-based platforms for producing therapeutic recombinant hIL-15 that support wound healing.

Monoclonal antibodies are crucial recombinant biopharmaceuticals, with N-glycosylation at Asn297 essential for their functionality. Plants are increasingly used for antibody production, achieving high expression levels and enabling glycoengineering to produce homogenous human-like N-glycan structures. However, plant-produced human IgG1 often shows significant underglycosylation with potential adverse effects for immune functions and stability. This study addressed this limitation of the widely used plant-based expression platform Nicotiana benthamiana by employing protein engineering to enhance N-glycosylation occupancy in plant-produced IgG1. This was achieved through an amino acid mutation near the conserved glycosylation site in the CH2 domain of the heavy chain. The transient expression of trastuzumab and SARS-CoV-2 neutralizing IgG1 antibody COVA2-15 in N. benthamiana, with mutations such as Y300L, resulted in a notable improvement in glycosylation occupancy. While the structural integrity and monodispersity of the IgG1 variant remained unaltered, an improvement in thermal stability was observed. Furthermore, functional assays showed that antigen binding and human hFcRn interaction were unaffected, while FcγRIIIa binding affinity increased. These findings demonstrate the potential of protein-engineering to enhance the quality and functionality of plant-produced IgG1 antibodies, making them comparable to mammalian-produced counterparts.

Oral mucositis (OM) remains a painful complication of anticancer chemotherapy (CT), tending to progress in severity in the presence of Fusobacterium nucleatum (Fn). Yet, no effective therapy exists to suppress OM since in vitro models mimicking CT-induced OM are lacking, halting the discovery of new drugs. Here, we developed an integrated millifluidic in vitro tissue culture system for OM disease modeling. This bioengineered system integrates magnetically bioassembled oral epithelium sheets with millifluidics for CT-based 5-fluorouracil perfusion and Fn infection to model CT-induced OM. After modeling OM with all pro-inflammatory hallmarks, we were able to suppress OM with our in-house plant-produced epidermal growth factor (P-EGF), a well-known re-epithelialization cue. Thus, this the first instance where a milifluidic system enabled OM modeling in the presence of CT drug perfusion and Fn infection. This bioengineered system is a novel tool for drug discovery as it propelled P-EGF as a promising therapy for OM.

Malaria is primarily caused by Plasmodium parasites and remains a major global health threat due to drug resistance and limited vaccine efficacy. Monoclonal antibodies (mAbs) targeting the conserved NANP repeat region of the circumsporozoite protein (PfCSP), such as MS-1797, represent a promising prophylactic strategy. Here, we demonstrate the plant-based production of MS-1797 in glycoengineered Nicotiana benthamiana ΔXF. Two variants were generated: MS-01 (original MS-1797) and MS-02 (MS-1797-SEKDEL), which yielded up to 457.3 µg/g fresh weight and 415.7 µg/g FW at 6 days post-infiltration. The plant-derived mAbs were obtained in high purity (>90%) and displayed either human core glycans or high mannose structures. Notably, both MS-01 and MS-02 retained antigen specificity in vitro and bound native PfCSP on sporozoites by immunofluorescence assay. These results establish the feasibility of producing functional anti-malarial mAbs in plants and highlight their potential use as affordable reagents for malaria research, diagnostics, and future prophylactic interventions.

Background: Human insulin-like growth factor 1 (hIGF-1) plays a key role in cell proliferation and tissue repair. While plant expression systems offer a cost-effective and scalable alternative for recombinant protein production, the molecular effects of plant-derived hIGF-1 on mammalian cells remain largely unexplored. Methods: In this study, a recombinant fusion protein of hIGF-1 with human Fc (hIGF-1-Fc) was transiently expressed in Nicotiana benthamiana using the geminiviral pBYR2e system and purified by Protein A affinity chromatography. SDS-PAGE and Western blotting confirmed the predicted molecular weight, and LC-MS identified N-glycosylation at the Fc N229 site with plant-type glycans such as GnMXF, GnGnXF, and MMXF. Bioactivity was evaluated using MCF-7 cell proliferation and NIH3T3 wound healing assays. Label-free quantitative proteomics was performed on NIH3T3 fibroblasts to assess molecular changes. Results: hIGF-1 Fc significantly promoted cancer cell migration and fibroblast proliferation. Proteomic profiling revealed an abundance of cytoskeletal proteins such as actin and tubulin and metabolic enzymes related to energy production. Gene ontology and pathway enrichment analyses indicated significant modulation of ribosome biogenesis and carbon metabolism. Conclusions: This study presents the first proteome-level investigation of plant-produced hIGF-1-Fc in mouse fibroblasts and reveals its impact on cytoskeletal organization and metabolic pathways involved in proliferation and wound healing.

Virus-like particles (VLPs) derived from hepatitis B core antigen (HBcAg) are high immunogenicity and self-assembling capabilities, making them ideal platforms for vaccine development. This study focuses on the plant-based production and purification of M2e-HBc VLPs in Nicotiana benthamiana. The M2e-HBc construct was engineered with a tandem M2e ectodomain fused to HBcAg, incorporating an N-terminal signal peptide and C-terminal His-tag SEKDEL. The gene was cloned into the pBY2ek vector, transformed into Escherichia coli and Agrobacterium tumefaciens, and agroinfiltrated into N. benthamiana leaves at OD₆₀₀ = 0.2. Leaves harvested at 3 days post-infiltration (DPI) were homogenized in IMAC10 buffer at a 1:2 (w/v) ratio for protein extraction. The crude was purified by nickel affinity chromatography and protein expression was evaluated by SDS-PAGE and Western blot under both reducing and non-reducing conditions. Protein concentration was determined via Bradford assay and further concentrated using Amicon® Ultra centrifugal filter. Sucrose gradient ultracentrifugation (10%, 30%, 70%) at 150,000 g for 4 hours was used to isolate VLP-containing fractions. The presence of assembled VLPs was confirmed by SDS-PAGE, Western blot, and transmission electron microscopy (TEM), which revealed spherical particles. This study demonstrates the feasibility of producing M2e-HBc VLPs in plants, providing a scalable platform for influenza vaccine development.