A Broad and Potent H1-Specific Human Monoclonal Antibody Produced in Plants Prevents Influenza Virus Infection and Transmission in Guinea Pigs

Although seasonal influenza vaccines block most predominant influenza types and subtypes, humans still remain vulnerable to waves of seasonal and new potential pandemic influenza viruses for which no immunity may exist because of viral antigenic drift and/or shift. Previously, we described a human monoclonal antibody (hMAb), KPF1, which was produced in human embryonic kidney 293T cells (KPF1-HEK) with broad and potent neutralizing activity against H1N1 influenza A viruses (IAV) in vitro, and prophylactic and therapeutic activities in vivo. In this study, we produced hMAb KPF1 in tobacco plants (KPF1-Antx) and demonstrated how the plant-produced KPF1-Antx hMAb possesses similar biological activity compared with the mammalian-produced KPF1-HEK hMAb. KPF1-Antx hMAb showed broad binding to recombinant HA proteins and H1N1 IAV, including A/California/04/2009 (pH1N1) in vitro, which was comparable to that observed with KPF1-HEK hMAb. Importantly, prophylactic administration of KPF1-Antx hMAb to guinea pigs prevented pH1N1 infection and transmission in both prophylactic and therapeutic experiments, substantiating its clinical potential to prevent and treat H1N1 infections. Collectively, this study demonstrated, for the first time, a plant-produced influenza hMAb with in vitro and in vivo activity against influenza virus. Because of the many advantages of plant-produced hMAbs, such as rapid batch production, low cost, and the absence of mammalian cell products, they represent an alternative strategy for the production of immunotherapeutics for the treatment of influenza viral infections, including emerging seasonal and/or pandemic strains.


Introduction
Influenza viruses are members of the Orthomyxoviridae family and are responsible for severe respiratory disease in humans [1]. Influenza viruses cause both seasonal epidemics and occasional hMAb showed broad and potent neutralizing activity in vitro and prophylactic and therapeutic activity in mice against influenza H1N1 IAV [47]. In this study, we demonstrated that a plant-produced KPF1 hMAb (KPF1-Antx) exhibits broad cross-reactivity and potent neutralizing in vitro and in vivo activity against H1N1 IAV. Importantly, KPF1-Antx hMAb has prophylactic and therapeutic activity and could prevent viral transmission in the well-established guinea pig model of influenza virus infection. Altogether, our results demonstrate, for the first time, the feasibility of using plantibodies for the treatment of influenza viral infections in humans.

Production of KPF1 in HEK293T Cells
The human HEK293T cells were grown at 37 • C to approximately 80% confluence in 10 cm tissue culture dishes using DMEM with 5% Hyclone Fetal Clone II (GE Healthcare Lifesciences, Logan, UT, USA) and 1X antibiotic and antimycotic (Gibco, Grand Island, NY, USA). Expression plasmids containing the heavy-and light-chain sequences for KPF1 or the isotype control were transfected into HEK293T cells using jetPRIME transfection reagent (PolyPlus, New York, NY, USA) as previously described [19]. Media was harvested and replenished three times over 8 days. IgG was purified from culture supernatant using Magne Protein A beads (Promega, Madison, WI, USA) and the elution buffer was exchanged with PBS using Amicon Ultra centrifugal filters (Millipore-Sigma, Cork, Ireland).

Production of KPF1 in Plants
Plant expression vectors were assembled using standard recombinant DNA technology, as previously described [50]. The KPF1-Antx and isotype control antibody expression vectors, which included heavy-chain (HC) and light-chain (LC) genes, were co-expressed with an oligosaccharyltransferase from Leishmania major (LmSTT3D) known to enhance N-glycan occupancy of recombinant proteins, using a similar strategy to that previously described [51]. In addition, a third expression vector was utilized to enhance recombinant protein expression by transiently silencing Argonaute1 (AGO1) and Argonaute4 (AGO4) proteins to minimize post-transcriptional gene silencing (PTGS) [Patent WO 2019/023806 A1].
KPF1-Antx was produced as outlined in Figure 1A. Briefly, expression vectors were transformed into Agrobacterium tumefaciens strain EHA105 and infiltrated at an OD600 of 0.2 into Nicotiana benthamiana plant line KDFX, developed by PlantForm (unpublished) for knockdown of the plant-specific β1,2-xylosyltransferase and α1,3-fucosyltransferase [52]. Plant foliage was harvested 7 days post-infiltration and total soluble protein was extracted. Antibodies were purified using MabSelect Protein A followed by Capto Q according to manufacturer protocols (GE Healthcare, Chicago, IL, USA). Purified antibodies were concentrated and formulated to ≥25 mg/mL in PBS. representation of KPF1-Antx hMAb production using the vivoXPRESS ® platform. Four week old plants were vacuuminfiltrated with a solution containing Agrobacterium tumefaciens. The Agrobacteria contained binary vectors for the co-expression of KPF1 or isotype control heavy-and light-chain genes, STT3D, and a system for enhancing protein expression. The infiltrated plants were returned to the greenhouse for 7 days, after which all foliage was harvested and processed to purify KPF1 hMAb using a standard antibody process. (B) KPF1-Antx hMAb purification process and analysis: Samples from the KPF1-Antx hMAb purification process: Protein A load (1), protein A flow through (2), protein A elution (3), CaptoQ FT (4), and final purified KPF1-Antx hMAb (5) were analyzed under non-reducing (left) and reducing (right) conditions using standard SDS-PAGE electrophoresis and Coomassie blue staining. Mobility of the molecular marker (MW) in kDa is shown on the left. (C) Size-exclusion high performance liquid chromatography: Purified hMAbs (10 μg) were injected into a TSKgel G3000SWXL column equipped with a TSKgel guard column SWXL (Tosoh Biosciences) using an Agilent 1100 Series HPLC. Agilent LC/MSD ChemStation Edition software was used for integration of data. (D) N-glycan analysis of KPF1-Antx and KPF1-HEK hMAbs, and plant-produced isotype control: Glycans were prepared using the GlykoPrep ® Rapid N-Glycan Preparation kit (PROzyme) and separated by hydrophilic-interaction liquid chromatography (HILIC) using a TSKgel Amide-80 Figure 1. Production and characterization of the KPF1-Antx hMAb. (A) Schematic representation of KPF1-Antx hMAb production using the vivoXPRESS ® platform. Four week old plants were vacuum-infiltrated with a solution containing Agrobacterium tumefaciens. The Agrobacteria contained binary vectors for the co-expression of KPF1 or isotype control heavy-and light-chain genes, STT3D, and a system for enhancing protein expression. The infiltrated plants were returned to the greenhouse for 7 days, after which all foliage was harvested and processed to purify KPF1 hMAb using a standard antibody process. (B) KPF1-Antx hMAb purification process and analysis: Samples from the KPF1-Antx hMAb purification process: Protein A load (1), protein A flow through (2), protein A elution (3), CaptoQ FT (4), and final purified KPF1-Antx hMAb (5) were analyzed under non-reducing (left) and reducing (right) conditions using standard SDS-PAGE electrophoresis and Coomassie blue staining. Mobility of the molecular marker (MW) in kDa is shown on the left. (C) Size-exclusion high performance liquid chromatography: Purified hMAbs (10 µg) were injected into a TSKgel G3000SWXL column equipped with a TSKgel guard column SWXL (Tosoh Biosciences) using an Agilent 1100 Series HPLC. Agilent LC/MSD ChemStation Edition software was used for integration of data. (D) N-glycan analysis of KPF1-Antx and KPF1-HEK hMAbs, and plant-produced isotype control: Glycans were prepared using the GlykoPrep ® Rapid N-Glycan Preparation kit (PROzyme) and separated by hydrophilic-interaction liquid chromatography (HILIC) using a TSKgel Amide-80 column (Tosoh Bioscience). Glycan species were identified by relative retention time and quantified using auto-integration of each glycan species peak.

Coomassie Blue Staining and Western Blot
Total amounts of 2.5, 1.25, 0.625, and 0.313 µg of KPF1-HEK or KPF1-Antx hMAbs were mixed with loading buffer and phosphate-buffered saline (PBS) to a final volume of 20 µL. After being separated by 12% SDS-PAGE, the gel was stained with Coomassie blue staining solution (0.05% Coomassie brilliant blue R-250, 45% methanol, and 7% acetic acid) overnight at room temperature. Subsequently, the gel was transferred to a nitrocellulose membrane. After blocking with 5% bovine serum albumin (BSA) in PBS containing 0.1% Tween 20 (PBST) at room temperature for 1 h, the membrane was incubated with horseradish-peroxidase-conjugated goat anti-human IgG secondary antibody (Jackson ImmunoResearch Laboratories, PA, USA) at room temperature for another 1 h. The blot was developed with ECL detection reagent (Thermo Fisher, California, CA, USA) in the ChemiDoc MP Imaging System (BioRad, Pennsylvania, PA, USA).

Enzyme-Linked Immunosorbent Assay (ELISA)
Binding of KPF1-HEK or KPF1-Antx hMAbs to H1N1 or H3N2 HA proteins was performed using standard ELISA, as previously described [19]. Briefly, ELISA plates (Nunc Maxisorp, Thermo Fisher Scientific, Grand Island, NY, USA) were coated overnight with 1 µg/mL of the indicated recombinant HA proteins obtained from the Biodefense and Emerging Infectious Research Resources Repository (BEI Resources, Manassas, VA) and incubated with 10-fold serially diluted HEK-or Antx-produced KPF1 hMAbs in PBS (starting concentration of 10 µg/mL). Binding was detected with HRP-conjugated anti-human IgG (Jackson ImmunoResearch, Pennsylvania, PA, USA). Plant-produced isotype control (Isotype-Antx) and mammalian HEK293T-produced isotype control (Isotype-HEK) were included as negative controls. In selected ELISAs, increasing concentrations of urea (ranging from 0 to 8 M) were added and the plates were incubated for 15 min at room temperature prior to detection with anti-IgG-HRP to evaluate avidity.

In Vivo Experiments
Four week old female Hartley guinea pigs were purchased from Charles River Laboratory and maintained in the animal care facility at the University of Rochester under specific pathogen-free conditions. All animal protocols were approved by the University of Rochester Committee of Animal Resources and complied with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council [54]. For viral infections, guinea pigs were anesthetized intraperitoneally (i.p.) with ketamine (30 mg/kg) and xylazine (5 mg/kg) and inoculated intranasally (i.n.) with 10 3 PFU of pH1N1 in 100 µL of PBS. After viral infection, animals were monitored daily for morbidity (body weight loss) and mortality (survival) (data not shown). To determine the prophylactic efficacy of KPF1-Antx hMAb, guinea pigs in two groups (N = 3/group) were weighed and administered (i.p.) 20 mg/kg of KPF1-Antx hMAb or isotype control hMAb (isotype-Antx), and kept in separated cages. After 6 h, guinea pigs were infected (i.n.) with 10 3 PFU of pH1N1. One day post-infection (d p.i.), sentinel guinea pigs (N = 3/group) were introduced into the cages of infected guinea pigs and monitored for seven days. Viral replication in nasal washes at 2, 4, 6, and 8 d p.i. was determined by immunofocus assay (fluorescent focus-forming units, FFU/mL) using an anti-NP MAb (HB-65) and a FITC-conjugated anti-mouse secondary Ab (Dako). Geometric mean titers and data representation were calculated using GraphPad Prism, v7.0. For therapeutic efficacy, guinea pigs in two groups (N = 3/group) were infected (i.n.) with 10 3 PFU pH1N1 and kept in separated cages, followed by administration of KPF1-Antx or isotype-Antx hMAbs at 1 d p.i. One day after administration of hMAbs, sentinel guinea pigs (N = 3/group) were introduced into the cages of infected guinea pigs and monitored for 7 days. Viral replication in nasal washes collected at 2, 3, 5, 7, and 9 d p.i. was determined by immunofocus assays as described above. At the completion of the study, guinea pigs were humanely euthanized by administration of a lethal dose of avertin and exsanguination, and lungs were collected for gross observation. Macroscopic pathology scoring was evaluated using ImageJ software to determine the percent of the total surface area of the lung (dorsal and ventral view) affected by consolidation, congestion, and atelectasis, as previously described [53,55,56].

Statistical Analysis
The one-tailed unpaired Student's t-test was used to evaluate significant differences. Data have been expressed as the mean ± standard deviation (SD) using Microsoft Excel software. Values were considered statistically significant when * p < 0.05, ** p < 0.01, or no significance (n.s.). All data were analyzed using Prism software version 8.00 (GraphPad Software, California, CA, USA).

Production of the Human Monoclonal Antibody KPF1 in Tobacco Plants
KPF1-Antx was produced in four week old N. benthamiana plants as outlined in Figure 1A. Just one week after infiltration with transgene-carrying Agrobacterium tumefaciens, IgG was purified from foliage using Protein A followed by Capto Q to remove impurities including endotoxin. KFP1-Antx was expressed at an average of 650 mg/kg of biomass (N = 3) and the overall recovery was 68% with an endotoxin level of 0.4 endotoxin units (EU)/mg. Antibody recovery and quality were monitored throughout the purification process using standard SDS-PAGE and Coomassie blue staining ( Figure 1B). IgG can be observed in the Protein A load in Figure 1B in addition to host cell proteins such as RuBisCO, which can account for up to 50% of total soluble proteins in leaves [57]. The final KPF1-Antx product was reduced to two independent bands representing the heavy and light chains (50 and 25 kDa, respectively), with no impurities detected. KPF1-Antx and KPF1-HEK were compared using size-exclusion HPLC analysis ( Figure 1C). Area under the curve analysis indicated that KPF1-Antx contained 96.3% monomeric IgG and 3.4% low molecular weight (MW) forms, whereas, KPF1-HEK contained 94.5% monomeric IgG, 3.9% low MW forms, and 1.6% high MW forms. These results indicate a greater purity for the plant-derived KPF1-Antx, which included a polishing step (Capto Q). In addition, N-glycosylation profiles were compared using GlykoPrep ® Rapid N-Glycan Preparation kit (PROzyme, Hayward, CA) and separation by hydrophilic-interaction liquid chromatography (HILIC) using a TSKgel Amide-80 column ( Figure 1D). KPF1-Antx and the isotype control N-glycan profiles were highly similar, with 85%-87% biantennary N-acetylglucosamine (GnGn). Contrary, KPF1-HEK N-glycan profile contained a mixture of N-glycans typically observed on mammalian glycoproteins, including antibodies (32.8% GnGnF, 29.1% AGnF, 12.8% Man5Gn, and 11.8% AAF).

Reactivity of KPF1-Antx and KPF1-HEK hMAbs In Vitro
We initially characterized the KPF1 hMAbs generated from either HEK293T cells (

Broad Neutralization and Hemagglutination Inhibition Activity by KPF1-Antx hMAb
We next evaluated the ability of KPF1-Antx hMAb to neutralize a broad range of H1N1 IAV, similarly to the process previously described with KPF1-HEK hMAb [19]. Both KPF1-Antx and KPF1-HEK hMAbs showed similar cross-reactivity, as assessed by IFA, against different H1N1 viruses ( Figure 3A). Importantly, quantification of the IFA results indicated that the ability of KPF1-Antx hMAb to recognize the IAV H1 in infected cells was not statistically different than that of KPF1-HEK hMAb ( Figure 3B). Likewise, KPF1-Antx hMAb was able to similarly neutralize Brisbane/H1N1,

Prophylactic Activity of KPF1-Antx hMAb In Vivo
To evaluate the protective efficacy of KPF1-Antx hMAb, Hartley guinea pigs (N = 3/group) received 20 mg/kg of either KPF1-Antx or isotype-Antx control hMAbs 6 h prior to infection with 10 3 PFU of pH1N1 ( Figure 4A). Sentinel guinea pigs (N = 3/group) were put in contact with the infected guinea pigs at 1 d p.i. to assess viral transmission. In the KPF1-Antx hMAb-treated group, only one infected guinea pig and its sentinel guinea pig showed restricted viral infection and shedding, respectively, in the nasal washes ( Figure 4B,C). In contrast, all infected guinea pigs treated with the isotype-Antx hMAb control showed higher levels of viral replication ( Figure 4B). We also observed viral shedding that resulted in efficient transmission to sentinel guinea pigs in all animals treated with the isotype-Antx hMAb ( Figure 4C). Importantly, gross pathology supported the idea that KPF1-Antx hMAb has prophylactic activity ( Figure 4D,F). Both infected and sentinel guinea pigs in the KPF1-Antx hMAb treated group showed mild or no multifocal consolidation, congestion, and atelectasis in middle and caudal lobes ( Figure 4D,E, respectively), while the isotype-Antx hMAb control-treated group infected ( Figure 4D) and sentinel ( Figure 4E) guinea pigs showed more severe pathology. Distributions of pathologic lesions on the lung surfaces were measured and compared, and supported the gross observation that the KPF1-Antx hMAb-treated group showed lower lesion values (13.85% to 31.51%) than those of the isotype-Antx control hMAb group (32.81% to 37.05%) ( Figure 4F).

Therapeutic Activity of KPF1-Antx hMAb In Vivo
To assess the therapeutic activity of the KPF1-Antx hMAb, guinea pigs (N=3/group) were infected with 10 3 PFU of pH1N1 and then treated, 24 h p.i., with 20 mg/kg of either KPF1-Antx or isotype-Antx control hMAbs. Sentinel guinea pigs (N=3/group) were put in contact with the infected guinea pigs at 2 d p.i. (Figure 5A) and evaluated for viral infection and shedding by determining viral with 20 mg/kg of KPF1-Antx hMAb, or with 20 mg/kg of an IgG isotype control (isotype-Antx). Six h post-treatment, guinea pigs were infected (i.n.) with 10 3 PFU of pH1N1 and monitored daily for 8 days. At 2 d p.i., sentinel guinea pigs were introduced into the same cage of infected guinea pigs, allowing direct contact between the animals. (B,C) Viral titers from nasal washes: Viral titers in the nasal washes of infected (B) and sentinel (C) guinea pigs were determined at 2, 4, 6, and 8 d p.i. by IFA (FFU/mL). The dotted line represents the limit of detection of the assay. * p < 0.05, ** p < 0.01, or no significance (n.s.). (D,E) Gross observation of lung pathology: All animals were euthanized at 8 d p.i. and lungs were collected from infected (D) or sentinel (E) guinea pigs to observe gross pathological changes such as congestion and atelectasis (arrows). Scale bars = 1 cm. (F) Macroscopic pathology scoring: Distributions of pathologic lesion such as consolidation, congestion, and atelectasis were measured using ImageJ and are represented as the percent of the total lung surface area (%). * p < 0.05, or no significance (n.s.).

Therapeutic Activity of KPF1-Antx hMAb In Vivo
To assess the therapeutic activity of the KPF1-Antx hMAb, guinea pigs (N = 3/group) were infected with 10 3 PFU of pH1N1 and then treated, 24 h p.i., with 20 mg/kg of either KPF1-Antx or isotype-Antx control hMAbs. Sentinel guinea pigs (N = 3/group) were put in contact with the infected guinea pigs at 2 d p.i. ( Figure 5A) and evaluated for viral infection and shedding by determining viral titers in the nasal washes ( Figure 5B,C, respectively). We observed high viral titers in infected guinea pigs treated with KPF1-Antx and the isotype-Antx control hMAbs at 2 d p.i. However, from 3 d p.i., the KPF1-Antx hMAb-treated group showed lower levels of viral replication in one of the infected guinea pigs and no viral shedding in the two other infected guinea pigs as compared with the isotype-Antx-treated control group ( Figure 5B). Importantly, no virus was detected in the sentinel guinea pig group that were put in contact with the infected KPF1-Antx hMAb ( Figure 5C). As expected, gross pathological observations of the KPF1-Antx hMAb-treated group showed only mild locally extensive congestion and atelectasis in caudal lobes and surface lesion scores ranging from 12.13% to 25.07% ( Figure 5F), while guinea pigs in the isotype-Antx hMAb treated group had more severe and larger distribution of pathological lesions affecting the middle and caudal lobes, and surface lesion scores ranging from 34.03% to 37.20% ( Figure 5F). Altogether, these results demonstrate that the plant-produced KPF1-Antx hMAb has both prophylactic and therapeutic activity against pH1N1, including the ability to prevent viral transmission, in guinea pigs.

Discussion
To date, FDA-licensed vaccines and antivirals are the most effective methods available to prevent and/or control influenza viral infections. However, influenza vaccines require at least 2 weeks after administration to induce protective immune responses against viral infection. In the case (B,C) Viral titers in nasal washes: To measure viral titers in the upper respiratory tract, nasal washes of infected (B) and sentinel (C) animals were collected on 2, 3, 5, 7, and 9 d p.i. Viral titers were determined by IFA (FFU/mL). * p < 0.05, or no significance (n.s.). (D,E) Gross observations of lung pathology: All animals were euthanized at 8 d p.i. and lungs were collected from infected (D) or sentinel (E) guinea pigs to observe gross pathological changes such as congestion and atelectasis (arrows). Scale bars = 1 cm. (F) Macroscopic pathology scoring: Distributions of pathological lesions, including consolidation, congestion, and atelectasis, were measured using ImageJ and have been represented as the percent of the total lung surface area (%).* p < 0.05, ** p < 0.01, or no significance (n.s.).

Discussion
To date, FDA-licensed vaccines and antivirals are the most effective methods available to prevent and/or control influenza viral infections. However, influenza vaccines require at least 2 weeks after administration to induce protective immune responses against viral infection. In the case of antivirals, their effectiveness is dependent on being administered within 48 h after the appearance of symptoms [58,59]. Importantly, antiviral-resistant strains have been described [1,[23][24][25]. Consequently, these prevention and treatment methods still leave substantial public health vulnerabilities.
In a previous study, we described a HEK293T-produced KPF1 hMAb (KPF1-HEK) that showed broad binding and neutralizing activity against H1N1 IAV isolates in vitro and potent prophylactic and therapeutic activities in vivo [19], because of its ability to bind to a conserved residue in the H1 hemagglutinin globular head of IAV [19]. In our current study, we examined the efficacy of a plant-produced KPF1 hMAb (KPF1-Antx) that possessed similar in vitro properties to KPF1-HEK hMAb and prevented influenza infection and transmission in guinea pigs. KPF1-Antx was produced in N. benthamiana plants using the vivoXPRESS ® platform ( Figure 1A), where four week old plants were infiltrated with Agrobacteria containing expression vectors. Purified hMAb was recovered just 7 days after infiltration from the foliage using standard antibody purification techniques. SDS-PAGE ( Figure 2A) and SEC-HPLC ( Figure 1C) revealed that KPF1-Antx and KPF1-HEK were highly similar. KPF1-Antx was engineered to have predominantly one glycan species-GnGn ( Figure 1D). KPF1-Antx hMAb bound various recombinant and native H1 Has, as determined by ELISA and IFA assays (Figures 2  and 3, respectively). Importantly, KPF1-Antx hMAb showed neutralization and HAI activities similar to those of KPF1-HEK hMAb ( Table 1), highlighting that KPF1-Antx hMAb possesses and maintains similar avidity and affinity properties as the mammalian-produced KPF1 hMAb. Notably, judging by the potent binding affinity for recent isolates, such as ChCh/H1N1 and St. Petersburg27/H1N1 (Figure 2), KPF1 hMAb covers a substantial antiviral breadth, including recent H1 isolates (Figure 3).
We also examined the activity of KPF1-Antx hMAb to protect (prophylactic) and treat (therapeutic) pH1N1-infected guinea pigs, as well as to prevent direct contact transmission (Figures 4 and 5, respectively). KPF1-Antx hMAb showed potent prophylactic and therapeutic activity against pH1N1 infection in guinea pigs (Figures 4 and 5, respectively), consistent with its in vitro antiviral and HAI properties. Moreover, administration of KPF1-Antx hMAb blocked transmission of pH1N1 infection to sentinel guinea pigs from direct contact with infected animals (Figures 4 and 5, respectively).
Previous studies have described tobacco-derived plantibodies that showed potent therapeutic activity against multiple viruses such as EBOV, HBV, HIV, PEDV, RABV, WNV, and others in small animal models of infection, e.g., mice [39,[42][43][44][45][46]. However, to our knowledge, this is the first study demonstrating the ability of plantibodies to protect against influenza viral infections. Concerns exist that plantibodies can be immunogenic and/or allergenic in animals and humans because of a non-mammalian glycosylation pattern [60]. In view of that, a proprietary N. benthamiana plant line, KDFX, with knock-down of β1,2-xylosyltransferase and α1,3-fucosyltransfere, was used to reduce the addition of plant-specific glycan species. As a result, no plant-specific sugars were detected ( Figure 1D). In the future, the vivoXPRESS ® platform could be used to tailor the N-glycan profile (i.e., addition of galactose and/or α1,6-fucose). Follow-up studies to directly and extensively compare mammalian-produced to plant-produced influenza specific antibodies for the impact of potential N-glycan differences on their activity or pharmacodynamics could be done to discern this further. Notably, none of the hMAb KPF1-Antx-or isotype-Antx-treated guinea pigs showed any adverse side effects and/or clinical signs in any of the in vivo experiments at a dose of 20 mg/kg. Altogether, we substantiated the clinical potential of KPF1 to prevent and treat H1N1 infection in a second animal model, the guinea pig, including its potent ability to prevent transmission, and, for the first time, demonstrated the feasibility of using a plant-produced hMAb for the prophylactic and therapeutic treatment of influenza infections. Plant-produced hMAbs could represent an excellent option for the treatment and control of influenza viruses against which vaccines are not effective (e.g., seasonal) or available (e.g., pandemic), or for which FDA-approved antivirals are ineffective. Moreover, our results also suggest the feasibility of implementing hMAbs produced in tobacco plants for the