Plant‐based production and characterization of monoclonal antibodies targeting extracellular enveloped proteins of the Mpox Virus

As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice • Plant-produced monoclonal antibodies targeting mpox A35 and B6 proteins were generated. • Efficient transient expression of anti-mpox antibodies in Nicotiana benthamiana . • Plant-derived antibodies specifically bound to mpox-infected Vero cells. • Anti-A35 antibody showed stronger electrochemical detection response to mpox virus. • Demonstrates plant-based antibodies for rapid diagnostic development. Keywords: Viral diseases, mpox, Therapeutic antibodies, Biopharmaceuticals, Plant-based biomanufacturing, Outbreak preparedness 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. Mpox (formerly known as monkeypox) is an emerging infectious disease caused by the mpox virus, a DNA virus belonging to the family Poxviridae and genus Orthopoxvirus . It is a zoonotic virus first identified in 1958. For several years, the disease was largely confined to endemic regions of West and Central Africa for several decades. However, in 2022, global spread of this virus in multiple non-endemic countries attracted the attention of the global community [1]. The World Health Organization (WHO) declared mpox as a public health emergency of international concern in August 14, 2024. Mpox infection continues to pose significant threat to public health [2,3]. The mpox virus consists of double-stranded DNA genome of about 197 kb in size and displays high homology to vaccinia virus. It is classified into two distinct clades ie., clade I and clade II. The A35 and B6 protein present on the surface of extracellular enveloped virion of orthopoxviruses is essential for virus transmission. Hence A35 and B6 are considered to be an effective antiviral target. Several groups have characterized mpox neutralizing antibodies targeting mpox viral proteins. Indeed, the administration of anti-mpox antibodies have been shown to be protective against mpox infection in animal models [[4], [5], [6], [7], [8]]. Monoclonal antibodies (mAbs) have shown effective therapeutic potential for the treatment of several infectious diseases. Their high specificity and strong binding affinity make them promising candidates for both diagnostic and therapeutic applications. Currently, mAbs are predominantly produced in mammalian cell culture systems and most of the approved mAbs are produced in this platform, which requires high-tech sophisticated facilities, expensive bioreactors, downstream processing, stringent cold storage and transportation [9,10]. Hence mAbs produced using the current technology platforms are often expensive. In this context, plant based expression systems offer an alternative platform for the production of recombinant pharmaceutical proteins including diagnostic reagents, vaccines, and therapeutic antibodies. This process termed as plant molecular farming has shown the potential of this platform for rapid protein production with reduced costs as demonstrated in earlier studies [[11], [12], [13], [14], [15]]. The scalability and speed of protein production using transient expression systems makes plants an attractive platform with reported timelines from gene sequence to purified protein of less than two weeks [[16], [17], [18]]. The ability of plants to serve as biofactories for the production of recombinant proteins has been extensively studied and reported in hundreds of proof-of-concept studies and an increasing number of clinical trials [[19], [20], [21], [22], [23], [24]]. Several mAbs produced in plants have been effective against SARS-CoV-2 [25], chikungunya virus [26], West Nile virus [27], rabies [28], Ebola virus [29] and influenza virus [30]. Notably, transient expression of mAbs targeting extracellular enveloped virion proteins of orthopoxviruses has been demonstrated in N. benthamiana [31]. One of the major advantages of using plant system is that they are highly amenable to glycoengineering strategies, which allows the generation of proteins with tailor-made N -glycans [32]. Other than therapeutic applications, plant-produced mAbs can also be utilized for developing diagnostic strategies. The rapid and reliable diagnostic tools are essential for effective surveillance and outbreak control of mpox. The conventional diagnostic methods such as PCR provide high sensitivity, but require specialized laboratory facilities and trained personnel. In recent years, electrochemical biosensors have emerged as promising platforms for rapid pathogen detection due to their high sensitivity, portability, and potential for point-of-care testing [33]. The electrochemical immunosensors utilize antigen–antibody interactions on electrode surfaces to generate measurable electrical signals, enabling simple and rapid detection of viral targets. Therefore, integrating highly specific mAbs with electrochemical sensing platforms could provide a valuable approach for developing rapid diagnostic tools for virus detection [34,35]. Here in this study, we aimed to produce anti-mpox mAbs targeting A35 and B6 proteins using a plant-based transient expression and evaluated their potential applications in both antiviral activity and diagnostic detection. These anti-mpox antibodies were efficiently expressed and assembled in wild‐type N. benthamiana . The plant-produced antibodies were then purified from the crude extracts and the purified antibodies exhibited specific binding to both mpox-infected cells and its target antigen. These results highlight the potential of plant-produced mAbs for therapeutic and diagnostic applications in mpox control. For the expression of recombinant mAbs in plants, the nucleotide sequence encoding for mAbs targeting A35 and B6 proteins were cloned into the plant expression vector. The amino acid sequences of the heavy and light chain variable regions (VH and VL) of the anti-A35 mAb 981 (8XA4_2, 8XA4_3), and the anti-B6 mAb D68 (8XS3_1 and 8XS3_2) were obtained from the RCSB protein data bank. The sequences of VH and VL were codon-optimized (Genewiz, Suzhou, China) for expression in N. benthamiana plants. The VH and VL sequences were cloned into the geminiviral expression vector pBYR2eK2Md (pBYR2e) [36,37] containing the respective human IgG1 heavy chain (CH) and kappa light chain (CL) constant regions. The murine leader sequence (GWSCIILFLVATATGVHS) was present at the N -terminus for directing the expressed antibodies into the secretory pathway. The VH and VL regions along with signal peptide were digested with Xba I/ Bmt I and Xba I/ Afl II restriction enzymes respectively, and then ligated with the constant regions of human IgG1 heavy chain (CH) or light chain (CL) present in the vector. For the expression of a full length single mAb in plants, two constructs were used (Fig. 1). The first one encoding for heavy chain (HC) contains the HC variable region and constant region. The second one encoding for light chain (LC) contains the LC variable region and LC constant region. These constructs were further transformed into A. tumefaciens strain GV3101::pMP90 by electroporation using a MicroPulser™ (Bio-Rad, USA). Transformed A. tumefaciens cells were cultured in LB medium at 28 °C with shaking and plated on LB agar containing rifampicin, kanamycin, and gentamicin. The positive colonies were identified by colony PCR using the following primers: forward primer 5′-GGAGAGGACCTCGAGAAAC-3′ and reverse primer 5′-GCTTTGCATTCTTGACATC-3′. PCR amplification was performed under the following conditions: initial denaturation at 95 °C for 3 min; 30 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 90 s; followed by a final extension at 72 °C for 10 min. Confirmed positive colonies were selected for further experiments. Schematic representation of plant expression vector harboring the heavy chain and light chain of anti-A35 and anti-B6 antibody sequences. The seeds of the Lab line of N. benthamiana were provided by Dr. Supaart Sirikantaramas from the Faculty of Science, Chulalongkorn University, Bangkok, Thailand. The plants were grown in the greenhouse under controlled conditions at 28 °C and a 16 h/8 h day–night regimen. The tobacco plants (6–8 weeks old) were agroinfiltrated with A. tumefaciens containing anti-mpox mAbs HC and LC as described previously [25,38]. Briefly, Agrobacterium harboring the expression vector containing either HC or LC was grown in Luria–Bertani (LB) broth supplemented with 50 mg/L kanamycin, 50 mg/L of rifampin at 28 °C overnight. Then, the overnight-grown culture was centrifuged and the pellet was resuspended in infiltration buffer [10 mM MgSO 4 , 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.5]. The cell suspension containing HC and LC construct was equally mixed and co-infiltrated in the N. benthamiana plants by vacuum infiltration. The agroinfiltrated leaves were harvested and processed for further analysis. After infiltration, the leaves of N. benthamiana were harvested and plant-derived mAbs were extracted and purified. Both the plant produced mAbs in clarified protein extract was purified by protein A affinity chromatography as previously described [25]. Briefly, crude extracts were purified using a Protein A affinity column equilibrated with PBS. After washing, bound antibodies were eluted with 0.1 M glycine (pH 2.7) and immediately neutralized with 1.5 M Tris–HCl (pH 8.8). The purified antibodies were buffer-exchanged into PBS, concentrated using 50 kDa centrifugal filters, and stored at −80 °C until use. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified plant produced mAbs were separated under both reducing and non-reducing conditions using 12% and 6% acrylamide gels respectively and the bands were visualized with Coomassie Blue R-250 (AppliChem, Germany). Western blot analysis was performed as previously described [25,38]. The proteins separated by SDS-PAGE were transferred onto 0.22 µm nitrocellulose membranes and blocked with 5% skim milk in PBS. The membranes were incubated with goat anti-human IgG Fc-HRP (Catalog Number: 2048–05 Southern Biotech, USA) or goat anti-human kappa HRP (Catalog Number: 2060–05 Southern Biotech, USA) antibodies with ratio 1:5000 and 1:2500 in 3% skim milk, respectively. The plant-produced mAbs were quantified by ELISA. Briefly, a 96-well microplate was coated with anti-human IgG Fc antibody (diluted in PBS) and incubated at 37 °C for 2 h. After washing with PBS-T, the plate was blocked with 5% (w/v) non-fat milk in PBS for 1 h at room temperature. A human IgG1 kappa isotype control was used to generate a standard curve. The plant-produced mAb samples were appropriately diluted in blocking buffer and added to the plate, followed by incubation at 37 °C for 1 h. After washing, HRP-conjugated goat anti-human kappa antibody was added and incubated for 1 h. The signal was developed using TMB substrate and stopped with 1 M H 2 SO 4 . The absorbance was measured at 450 nm using a microplate reader (Hercuvan, NS-100 Nano Scan Microplate Reader). The antibody concentrations were calculated based on the standard curve. All experiments were performed in duplicates, and data were presented as mean ± SD. Vero cells were cultured in Minimal Essential medium (MEM; Gibco, USA) supplemented with 10% Fetal bovine serum (FBS; Gibco, USA) and antibiotics. The Mpox virus specimens were obtained from nasopharyngeal swabs of confirmed patients at Ramathibodi Hospital, Thailand, with ethical approval by the Human Research Ethics Committee (MURA2023/655). The virus preparation and titration were performed as described in a previous report [39]. Briefly, the virus was infected into Vero cells in MEM medium supplemented with 2% FBS for 4–6 days. The infected cells were harvested and subjected to three freeze–thaw cycles to release viral particles. Passage 1 viruses were subsequently propagated in Vero cells for two additional passages. Passage 3 virus stocks were stored at −80 °C until use. All experiments with live viruses were conducted in a certified Biosafety Level 3 facility, Faculty of Science, Mahidol University. The cells were seeded in 96-well plates (Corning, USA) and infected with two clade IIb mpox isolates representing distinct lineages (hMpxV/THA/V241–0052/2023, lineage C.1, Mpox-52 and hMpxV/THA/MUSCMI-156/2025 lineage C.1.2, Mpox-156). After 2 days post-infection with 100 of 50% Tissue infectious dose (TCID) of the viruses, cells were fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich, USA) and proceed under either permeabilized with 1% Triton X-100 or non-permeabilized conditions. The antibody binding was evaluated by incubating the cells with plant-produced antibodies (1 µg/mL; 50 µL/well) at 37 °C for 1 h. The bound antibodies were detected using kappa anti-human HRP conjugated secondary antibody (1:1000; 50 µL/well) (Southern Biotech, USA) at 37 °C for 1 h. For positive control, rabbit polyclonal anti-vaccinia antibody (1:1000, PA17258, ThermoFisher Scientific, USA) was applied, followed by goat anti-rabbit HRP (1:5000, ThermoFisher Scientific, USA). The signal was developed using TrueBlue™ substrate (50 µL/well) (KPL, USA) for 10 min at room temperature. The plates were then washed with water, air-dried, and imaged using a Cytation™ 7 imaging system (BioTek, USA). Vero cells were cultivated in MEM supplemented with 10% FBS and antibiotics, then seeded in 96-well plates (Corning, USA) and incubated overnight to form confluent monolayers. The plant-produced antibodies and a control (serum of JYNNEOS® vaccinated serum) were serially diluted in infection medium (MEM, Gibco, USA) supplemented with 2% fetal bovine serum (FBS, Gibco, USA) and 1x Penicillin Streptomycin) (final volume 60 µL per well) and mixed 1:1 with Mpox virus (Mpox-156) containing 100 focus-forming units (FFU). The antibody–virus mixtures were incubated at 37 °C for 1 h and then added to Vero cell monolayers. After 24 h of incubation, cells were fixed with 4% PFA and the viral infection was quantified by immunofluorescence assay using a rabbit polyclonal anti-vaccinia antibody (1:500, PA17258, ThermoFisher Scientific, USA), followed by goat anti-rabbit Alexa 488 (1:500, A11008, ThermoFisher Scientific, USA). The plates were imaged using a Cytation™ 7 system (BioTek, USA), and infection levels were expressed as FFU and expressed as percent infection relative to the control. The data are presented as mean ± standard deviation. Screen-printed carbon electrodes (SPCE) (Zensor R&D, Taichung, Taiwan) were used as the electrochemical sensing platform. For electrode preparation, 5 µL of 0.2% cellulose nanocrystal solution containing carboxylic groups (CNC–COOH) (Cellulose Lab, Fredericton, Canada) prepared in Milli-Q water was deposited onto the working electrode surface and dried in an oven at 37 °C for 15 min to introduce carboxyl functional groups. Subsequently, 5 µL of a freshly prepared mixture containing 10 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 30 mM N-hydroxysulfosuccinimide (sulfo-NHS) (Sigma-Aldrich, Missouri, USA) was applied onto the CNC-modified electrode surface and incubated at room temperature for 60 min to activate the carboxyl groups. After activation, the electrode surface was rinsed with phosphate-buffered saline (PBS, pH 7.4) (Sigma-Aldrich, Missouri, USA). The capture antibodies (anti-A35 or anti-B6 mAb) were immobilized by depositing 5 µL of antibody solution (100 µg/mL) onto the activated electrode surface and incubating under humid conditions for 1 h to allow covalent attachment between the carboxyl groups of CNC and the primary amine groups of the antibodies. The Ab/CNC/SPCE electrode was then washed with PBS (pH 7.4) to remove unbound antibodies. The electrode surface was subsequently blocked by adding 5 µL of BlockPro™ blocking buffer (Visual Protein, Taipei, Taiwan) for 30 min to minimize nonspecific protein adsorption. Finally, the prepared BP/Ab/CNC/SPCE electrode was rinsed with PBS and stored at 4 °C until use. Electrochemical measurements were performed using an ECWP100 electrochemical workstation (Zensor R&D, Taichung, Taiwan). Square wave voltammetry (SWV) was conducted within a potential range of −0.4 to 0.7 V (vs Ag/AgCl) with a step potential of 0.01 V, amplitude of 0.05 V, and scan rate of 0.05 V/s. For virus detection, 5 µL of Mpox virus suspension at designated concentrations (10,000–40,000 PFU per 5 µL) was applied onto the antibody-modified electrode surface and incubated at room temperature for 60 min to allow antigen–antibody interaction. After incubation, the electrode was washed with PBS (pH 7.4) to remove unbound virus particles. Electrochemical measurements were carried out in an electrolyte solution containing 5 mM potassium ferricyanide/ferrocyanide ([Fe(CN)₆]³⁻/⁴⁻) and 0.1 M potassium chloride (KCl) (Sigma-Aldrich, Missouri, USA). The formation of antigen–antibody complexes on the electrode surface partially blocked electron transfer between the redox probe and the electrode surface, resulting in a decrease in the current signal (Fig. 2). The square wave voltammetry of 5 mM [Fe(CN) 6 ]3−/4− in the absence and presence of Mpox-156. Statistical analysis for the neutralization assay was performed using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test against the no-treatment control. Analyses were conducted using SPSS software, and p values < 0.05 were considered statistically significant. In this study, two mAbs targeting A35, B6 proteins of mpox were chosen for expression in plants. For the transient expression of these genes, the HC and LC genes were cloned into the plant expression vector (Geminiviral vector; pBYR2e) and transformed into Agrobacterium tumefaciens . The mAb gene construct carrying the vector construct was co-infiltrated into leaves of N. benthamiana plants. After cloning and Agrobacterium transformation, the Agrobacterium harboring the vector construct carrying heavy chain and light chain of each mAb mixed at a ratio of 1:1 was co-infiltrated into the plant cells for transient expression. The highest level of mAb expression was detected at different time points for the expressed mAbs. The maximum yield of anti-A35 mAb was 27± 15 µg/g fresh weight at 3 days post infiltration (dpi), while the highest yield of anti-B6 mAb was 260± 90 µg/g fresh weight at 7 dpi. In SDS-PAGE gel, the protein band was observed at around 150 kDa corresponding to the size of a fully assembled antibody under non-reducing condition. Under reducing condition, bands in the expected size of 50 and 25 kDa were observed which corresponds to heavy chain and light chain, respectively. Further, the Western blot probed with anti-human IgG γ-chain antibody and anti-human IgG κ-chain antibody confirms the expected band size under reducing and non-reducing conditions (Fig. 3). The use of antibodies against both the γ-chain (heavy chain) and κ-chain (light chain) allows verification of the integrity and correct assembly of the IgG molecule. These results confirmed that the recombinant mAbs were correctly assembled in the plant system. Characterization of plant-produced anti-A35 and anti-B6 antibodies by SDS-PAGE and Western blot (a) SDS-PAGE analysis under non-reducing conditions showing intact IgG antibodies. (b) Western blot analysis under non-reducing conditions probed with anti-human IgG γ-chain antibody, detecting intact IgG (c) Western blot analysis under non-reducing conditions probed with anti-human IgG κ-chain antibody, detecting intact IgG. (d) SDS-PAGE analysis under reducing conditions showing bands of heavy and light chains at the expected size. (e) Western blot analysis under reducing conditions probed with anti-human IgG γ-chain antibody, detecting the heavy chain. (f) Western blot analysis under reducing conditions probed with anti-human IgG κ-chain antibody, detecting the light chain. The binding of human anti-A35 and anti-B6 mAbs were assessed using mpox-156–infected Vero cells under non-permeabilized (Fix only) and permeabilized (Fix + permeabilize) conditions. Under non-permeabilized conditions, the plant-produced anti-A35 mAb showed detectable surface staining of infected cells (Fig. 4a), indicating that the A35 antigen is accessible on the cell surface. After permeabilization, anti-A35 staining intensity increased and displayed a broader intracellular distribution (Fig. 4b), suggesting the presence of both surface-associated and intracellular pools of the antigen. Similarly, the human anti-B6 antibody showed clear binding under non-permeabilized conditions (Fig. 4c), consistent with surface expression of the B6 antigen. Following permeabilization, staining intensity was increased and more widespread within the cell (Fig. 4d). As a positive control, rabbit polyclonal anti-vaccinia antibody recognizing B5R and A33R proteins was used. These are well-established envelope proteins in orthopoxviruses and hence used for antibody-based detection of infected cells. As mpox shares high sequence homology with Vaccinia virus, antibodies targeting A33/B5 are known to exhibit cross-reactivity [[40], [41], [42]]. This antibody showed strong intracellular staining under permeabilized conditions (Fig. 4e), confirming infection and appropriate staining conditions. In contrast, the negative control antibody exhibited minimal background staining (Fig. 4f), demonstrating the specificity of the observed signals. These results showed that both A35 and B6 antigens were detectable in Mpox-156–infected cells, with detectable surface accessibility and intracellular presence. Similar results were obtained with both Mpox-52 and Mpox-156 isolates (Supplementary Fig S1, S2). Binding of plant-produced human anti-A35 and anti-B6 antibodies to Mpox-156–infected Vero cells. (a) Plant-produced anti-A35 mpox antibody under non-permeabilized conditions (Fix only). (b) Plant-produced anti-A35 mpox antibody under permeabilized conditions (Fix + permeabilize). (c) Plant-produced anti-B6 mpox antibody under non-permeabilized conditions (Fix only). (d) Plant-produced anti-B6 mpox antibody under permeabilized conditions (Fix + permeabilize). (e) Rabbit polyclonal anti-vaccinia antibody recognizing B5R and A33R proteins under permeabilized conditions, used as a positive control. (f) Negative control antibody under permeabilized conditions, showing minimal nonspecific background staining. To further evaluate whether the binding antibodies could inhibit viral infection, a neutralization assay was performed. Across the tested concentration range (100–0.001 µg/mL), both plant-produced anti-A35 and anti-B6 antibodies showed only moderate effects on Mpox-156 infection relative to the no-treatment control (Fig. 5). At the highest tested concentration (100 µg/mL), a reduction in the percentage of infected cells was observed for both antibodies, with anti-B6 showing a slightly greater decrease than anti-A35. However, this trend was not statistically significant according to one-way ANOVA followed by Dunnett's multiple comparisons test ( p > 0.05). Quantitatively, infection levels remained high, with 91.9 ± 5.3% infection for anti-A35 and 71.3 ± 10.6% for anti-B6 at 100 µg/mL. At lower concentrations, infection levels remained comparable to the no-treatment baseline. Neutralization activity of plant-produced anti-A35 and anti-B6 antibodies against Mpox-156 in Vero cells. (A) Neutralization assay using plant-produced anti-A35 antibody at the defined concentrations. (B) Neutralization assay using plant-produced anti-B6 antibody at the defined concentrations. Vero cells were infected with mpox and treated with the indicated antibodies. Infection levels were quantified as FFU and expressed as % infection relative to the no-treatment group (set to 100%). Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA in SPSS with Dunnett's multiple comparisons test against the no-treatment control. No statistically significant differences were observed ( p > 0.05). In order to further explore the diagnostic potential of the plant-produced antibodies, the electrochemical immunosensor platform was evaluated for Mpox virus detection using anti-A35 and anti-B6 mAbs. Fig. 6 shows the current responses of the BP/anti-A35 mAb/CNC/SPCE (Fig. 6A) and BP/anti-B6 mAb/CNC/SPCE (Fig. 6B) electrodes in the absence and presence of mpox virus. For the anti-A35 mAb-modified electrode, the measured current was approximately 101–102 µA at 10,000 PFU/5 µL and 92–93 µA at 40,000 PFU/5 µL (Fig. 6A), compared to the negative control (∼113–114 µA). This corresponds to an overall signal reduction of approximately 18–19% at the highest virus concentration. The lower current signals observed at higher viral concentrations are consistent with increased formation of antigen–antibody complexes on the electrode surface, which hinder electron transfer between the redox probe and the electrode. The current response of BP/anti-A35 mAb/CNC/SPCE (A) and BP/anti-B6 mAb/CNC/SPCE (B) at different Mpox-156 concentration, measured using 5 mM [Fe(CN) 6 ]3-/4- in 0.1 M KCl. In contrast, the anti-B6 mAb-modified electrode showed minimal change in current, with values of approximately ∼120 µA at 10,000 PFU/5 µL and ∼114 µA at 40,000 PFU/5 µL, compared to the negative control (∼120–121 µA) (Fig. 6B). The overall decrease was approximately ∼6% at the highest concentration, indicating a weaker electrochemical response relative to the anti-A35 antibody. Overall, the decrease in current signal was observed upon virus binding in both antibody-modified electrodes, confirming antigen–antibody interactions on the electrode surface. In this study, we successfully expressed full-length human anti-A35 and anti-B6 mAbs in Nicotiana benthamiana using a geminiviral transient expression system. The purified antibodies displayed the expected molecular weight under both reducing and non-reducing conditions. Under non-reducing conditions, a protein band corresponding to the fully assembled IgG (∼150 kDa) was observed, while the reducing conditions revealed the expected heavy chain (∼50 kDa) and light chain (∼25 kDa) band. These results confirmed that the recombinant antibodies were correctly assembled in the plant expression system. These findings are consistent with previous reports demonstrating the suitability of plant-based systems for rapid production of structurally intact antibodies in matter of few weeks [43,44]. CHO-based systems typically require slightly longer production timelines, with culture phases often extending to 1–2 weeks. The upstream workflow involves mammalian cell expansion, transfection optimization, and the control of culture conditions making the process resource-intensive, and operationally demanding compared to plant-based systems [[45], [46], [47]]. The maximum yield of anti-A35 mAb reached 27 µg/g fresh weight at 3 dpi, while anti-B6 mAb reached 260 µg/g fresh weight at 7 dpi. These yields fall within the range typically reported for plant-based transient expression systems, which can vary depending on the construct and expression conditions [18]. In comparison, transient expression in mammalian cells typically yields recombinant antibodies in the range of 400 mg/L with reports of yields reaching 500–800 mg/L following process optimization [48,49]. In contrast, CHO-based systems are capable of achieving substantially higher productivity, with industrial processes commonly reaching multi-gram-per-liter titers and reports exceeding 10 g/L under optimized conditions [50,51]. However, direct comparison across platforms is limited due to differences in expression formats and production scale. The binding analysis revealed that both plant-produced anti-A35 and anti-B6 antibodies recognized mpox-infected Vero cells. The detectable staining under non-permeabilized conditions indicates the surface accessibility of both antigens, while enhanced intracellular staining following permeabilization suggested the presence of both surface-associated and intracellular pools of the viral proteins. Similar binding patterns were observed for both Mpox-52 and Mpox-156 isolates, indicating that these antigens are conserved and accessible across clade IIb strains. These observations are consistent with prior reports describing A35 and B6 as accessible extracellular enveloped virion proteins [52], and our results confirmed that the plant produced antibodies retain expected antigen recognition across the tested clade IIb isolates. In this study, the neutralization assay showed the substantial residual infection levels, with approximately 91.9 ± 5.3% infection for the anti-A35 antibody, compared with 71.3 ± 10.6% infection for the anti-B6 antibody at the highest tested concentration (100 µg/mL). Consistent with this observation, the assay showed only moderate inhibition of viral infection, with no statistically significant differences compared to the no-treatment control. Previous studies of antibodies targeting the A35 antigen have reported moderate neutralizing activity in standard infection-based assays, typically corresponding to a wide range of residual infection levels (∼70–100%) in the absence of complement, even when substantially higher antibody doses are used (e.g., up to 100 µg per well) [6]. In comparison, the possible explanation for the limited inhibition observed in this study is the relatively low effective antibody dose used in the assay. In our experimental setup, antibodies were prepared at 100 µg/mL in a 60 µL volume (approximately 6 µg per well) and subsequently diluted during virus mixing, resulting in a lower total antibody amount per well than in previously reported studies. In addition, the assay primarily measured early infection events and did not include complementary assays such as plaque reduction or viral spread inhibition, which may provide a more comprehensive evaluation of neutralizing activity. In addition to evaluating the binding properties of the plant-produced antibodies, we explored the diagnostic potential of the plant-produced antibodies using an electrochemical immunosensing platform. When immobilized on the BP/CNC-modified SPCE electrode, both anti-A35 and anti-B6 antibodies produced measurable electrochemical responses upon exposure to Mpox virus. A progressive decrease in current signal was observed with increasing virus concentrations, which is consistent with the formation of antigen–antibody complexes on the electrode surface that partially block electron transfer between the redox probe and the electrode. Notably, the anti-A35-modified electrode exhibited a clearer concentration-dependent decrease in the signal compared with the anti-B6-modified electrode, suggesting antigen recognition under the tested sensing conditions. This observation is consistent with the immunofluorescence binding results, where both antibodies recognized infected cells but may differ in antigen accessibility or epitope presentation during electrochemical detection. The virus concentrations tested in this proof-of-concept electrochemical assay ranged from 10,000 to 40,000 PFU per 5 µL (equivalent to approximately 2 × 10⁶ - 8 × 10⁶ PFU/mL). These levels are within the range reported in clinical Mpox lesion samples. The previous studies have shown that skin lesions from Mpox patients contain high viral loads and represent a major source of virus transmission. Quantitative analyses have reported viral DNA concentrations frequently exceeding 10⁶–10⁸ genome copies per milliliter in lesion material [53,54]. The clinical investigations during the 2022 outbreak demonstrated that lesion swabs typically contain the highest viral loads compared with other specimen types [55,56]. Therefore, the virus concentrations used in this study are relevant to clinically observed viral burdens, supporting the potential applicability of this electrochemical detection approach for Mpox diagnostics. Although the present study primarily demonstrates proof-of-concept detection, the signal response observed upon virus binding indicates that the plant-produced mAbs can be integrated into electrochemical biosensing platforms. Further optimization of electrode surface chemistry, signal amplification strategies, and assay conditions may enhance sensitivity and enable detection at lower viral concentrations. Such improvements could facilitate the development of rapid, portable and cost-effective diagnostic tools for Mpox surveillance and point-of-care testing. In conclusion, this study demonstrates the successful plant-based production of mAbs targeting the Mpox virus A35 and B6 proteins using a transient expression system in Nicotiana benthamiana . The plant-produced antibodies were correctly assembled and exhibited specific binding to Mpox-infected cells. Although neutralizing activity was limited under the tested conditions, the antibodies showed promising performance in an electrochemical immunosensor platform for virus detection. These findings highlight the feasibility of rapid plant-based production of anti-mpox mAbs for potential diagnostic applications and further therapeutic development. This study was supported by Thailand Science research and Innovation Fund Chulalongkorn University (HEA_FF_69_182_3300_023) and the Second Century Fund (C2F), Chulalongkorn University (awarded to Kitti Jirananon). The authors declare that no Generative AI was used in the creation of this manuscript. This study did not involve human participants or experimental animals. All experiments were performed using established cell lines and laboratory-propagated Mpox virus under appropriate biosafety level-3 (BSL-3) conditions in compliance with institutional biosafety regulations. I hereby provide consent for the publication of this manuscript. Not applicable. Kitti Jirananon: Writing – review & editing, Writing – original draft, Validation, Methodology. Balamurugan Shanmugaraj: Writing – review & editing, Writing – original draft, Methodology. Suwimon Manopwisedjaroen: Writing – review & editing, Writing – original draft, Methodology. Arunee Thitithanyanont: Writing – review & editing, Writing – original draft, Methodology. Prinjaporn Teengam: Writing – review & editing, Writing – original draft, Methodology. Waranyoo Phoolcharoen: Writing – review & editing, Writing – original draft, Supervision, Investigation, Funding acquisition, Conceptualization. KJ employed by Baiya Phytopharm Co., Ltd. WP is a co-founder/shareholder of Baiya Phytopharm Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funders had no role in the design of this study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results. The authors would like to thank Baiya Phytopharm Co., Ltd., Chulalongkorn University, Thailand and Karpagam Academy of Higher Education, India for their facilities, and support during the study. Prinjaporn Teengam, Email: printe@kku.ac.th. Waranyoo Phoolcharoen, Email: waranyoo.p@chula.ac.th. Data supporting the findings of this study are available from the corresponding author upon request. 1. WHO. Mpox 2024 [updated 26 August 2024; cited 2026 February 10]. Available from: https://www.who.int/news-room/fact-sheets/detail/mpox. 2. Wu S., Deng J., Du M., Liu M., Liu J. 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