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Lack of Herpes Simplex Virus (HSV) vaccine, low vaccination rates of Influenza viruses, waning immunity and viral transmission after vaccination underscore the need to reduce viral loads at their transmission sites. Oral virus transmission is several orders of magnitude higher than nasal transmission. Therefore, in this study, we evaluated neutralization of viruses using a natural viral trap protein (FRIL) formulated in clinical-grade chewing gum. FRIL is highly stable in the lablab bean powder (683 days) and in chewing gum (790 days), and fully functional (794 days) when stored at ambient temperature. They passed the bioburden test with no aerobic bacteria, yeasts/molds, with minimal moisture content (1.28-5.9%). Bean gum extracts trapped HSV-1/HSV-2 75-94% in a dose-dependent manner through virus self-aggregation. Mastication simulator released >50% release of FRIL within 15 min of chewing the bean gum. In plaque reduction assays, >95% neutralization of H1N1 and H3N2 required ∼40 mg/mL, HSV-1 160 mg/mL, and HSV-2 74 mg/mL of bean gum for 1,000 copies/mL virus particles. Therefore, a 2000 mg bean gum tablet has more than adequate potency for clinical evaluation and is safe with no detectable levels of glycosides. These observations augur well for evaluating bean gum in human clinical studies to minimize virus infection/transmission., Competing Interests: Declaration of interests HD is a patentee in plant-based oral and topical drug delivery and was previously funded by Novo Nordisk, Bayer, Shire and Takeda. Several industry discussions for advancing clinical trials in oral or topical delivery are in progress. Complete list of patents is available in the Google Scholar or ScholarGPS links below: http://scholar.google (in PBS buffer).com/citations?user = 7sow4jwAAAAJ&hl = en https://scholargps.com/scholars/82094026790000/henry-daniell., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background/Objectives: For several decades, protein drugs (biologics) made in cell cultures have been delivered as sterile injections, decreasing their affordability and patient preference. Angiotensin Converting Enzyme 2 (ACE2) gum is the first engineered human blood protein expressed in plant cells approved by the FDA without the need for purification and is a cold-chain and noninvasive drug delivery. This biologic is currently being evaluated in human clinical studies to debulk SARS-CoV-2 in the oral cavity to reduce coronavirus infection/transmission (NCT00543318). Methods: Chemistry, manufacturing, and control (CMC) studies for the ACE2/Ang(1-7) drug substances (DSs) and ACE2 gum drug product (DP) were conducted following USP guidelines. GLP-compliant toxicology studies were conducted on Sprague Dawley rats ( n = 120; 15/sex/group) in four groups-placebo, low (1.6/1.0 mg), medium (3.2/2.0 mg), and high (8.3/5.0 mg) doses IP/kg/day. Oral gavage was performed twice daily for 14 days (the dosing phase) followed by the recovery phase (35 days). Plasma samples ( n = 216) were analyzed for the product Ang(1-7) by ELISA. Results: The ACE2 protein was stable in the gum for at least up to 78 weeks. The toxicology study revealed the dose-related drug delivery to the plasma and increases in the AUC (56.6%) and Cmax (52.9%) after 28 high-dose gavages (95% C.I.), although this quantitation excludes exogenously delivered membrane-associated ACE2/Ang(1-7). Vital biomarkers and organs were not adversely affected despite the 10-fold higher absorption in the tissues, demonstrating the safety for the first in-human clinical trials of ACE2/Ang(1-7). The NOAEL observed in the rats was 2.5-7.5-fold higher than that of the anticipated efficacious therapeutic dose in humans for the treatment of cardiopulmonary disorders, and it was 314-fold higher than the NOAEL for topical delivery via chewing gum. Conclusions: This report lays the foundation for the regulatory process approval for noninvasive and affordable human biologic drugs bioencapsulated in plant cells.

Diabetes Mellitus is a silent epidemic affecting >500 million, which claimed 6.7 million lives in 2021, a projected increase of >670% in <20 years old in the next two decades but insulin is unaffordable for the large majority of the globe. Therefore, we engineered proinsulin in plant cells to facilitate oral delivery. Stability of the proinsulin gene and expression in subsequent generations, after removal of the antibiotic-resistance gene, was confirmed using PCR, Southern and western blots. Proinsulin expression was high (up to 12 mg/g DW or 47.5% of total leaf protein), stable up to one year after storage of freeze-dried plant cells at ambient temperature and met FDA regulatory requirements of uniformity, moisture content and bioburden. GM1 receptor binding, required for uptake via gut epithelial cells was confirmed by pentameric assembly of CTB-Proinsulin. IP insulin injections (without C peptide) in STZ mice rapidly decreased blood glucose level leading to transient hypoglycemia, followed by hepatic glucose compensation. On the other hand, other than the 15-min lag period of oral proinsulin (transit time required to reach the gut), the kinetics of blood sugar regulation of oral CTB-Proinsulin in STZ mice was very similar to naturally secreted insulin in healthy mice (both contain C-peptide), without rapid decrease or hypoglycemia. Elimination of expensive fermentation, purification and cold storage/transportation should reduce cost and increase other health benefits of plant fibers. The recent approval of plant cell delivery of therapeutic proteins by FDA and approval of CTB-ACE2 for phase I/II human clinical studies augur well for advancing oral proinsulin to the clinic., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Henry Daniell has patent #USA patent 11,332,754 issued to University of Pennsylvania. Henry Daniell has patent #USA patent 10,865,419 issued to University of Pennsylvania. However, there is no specific financial conflict of interest to disclose at this time., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Microfibres (diameter <5 mm) and textile dyes released from textile industries are ubiquitous, cause environmental pollution, and harm aquatic flora, fauna, animals and human life. Therefore, enzymatic abatement of microfibre pollution and textile dye detoxification is essential. Microbial enzymes for such application present major challenges of scale and affordability to clean up large scale pollution. Therefore, enzymes required for the biodegradation of microfibres and indigo dye were expressed in transplastomic tobacco plants through chloroplast genetic engineering. Integration of laccase and lignin peroxidase genes into the tobacco chloroplast genomes and homoplasmy was confirmed by Southern blots. Decolorization (up to 86%) of samples containing indigo dye (100 mg/L) was obtained using cp-laccase (0.5% plant enzyme powder). Significant (8-fold) reduction in commercial microbial cellulase cocktail was achieved in pretreated cotton fibre hydrolysis by supplementing cost effective cellulases (endoglucanases, ß-glucosidases) and accessory enzymes (swollenin, xylanase, lipase) and ligninases (laccase lignin peroxidase) expressed in chloroplasts. Microfibre hydrolysis using cocktail of Cp-cellulases and Cp-accessory enzymes along with minimal dose (0.25% and 0.5%) of commercial cellulase blend (Ctec2) showed 88%-89% of sugar release from pretreated cotton and microfibres. Cp-ligninases, Cp-cellulases and Cp-accessory enzymes were stable in freeze dried leaves up to 15 and 36 months respectively at room temperature, when protected from light. Use of plant powder for decolorization or hydrolysis eliminated the need for preservatives, purification or concentration or cold chain. Evidently, abatement of microfibre pollution and textile dye detoxification using Cp-enzymes is a novel and cost-effective approach to prevent their environmental pollution., (© 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Because oral transmission of SARS-CoV-2 is 3-5 orders of magnitude higher than nasal transmission, we investigated debulking of oral viruses using viral trap proteins (CTB-ACE2, FRIL) expressed in plant cells, delivered through the chewing gum. In omicron nasopharyngeal (NP) samples, the microbubble count (based on N-antigen) was significantly reduced by 20 μg of FRIL (p < 0.0001) and 0.925 μg of CTB-ACE2 (p = 0.0001). Among 20 delta or omicron NP samples, 17 had virus load reduced below the detection level of spike protein in the RAPID assay, after incubation with the CTB-ACE2 gum powder. A dose-dependent 50% plaque reduction with 50-100 ng FRIL or 600-800 μg FRIL gum against Influenza strains H1N1, H3N2, and Coronavirus HCoV-OC43 was observed with both purified FRIL, lablab bean powder or gum. In electron micrographs, large/densely packed clumps of overlapping influenza particles and FRIL protein were observed. Chewing simulator studies revealed that CTB-ACE2 release was time/dose-dependent and release was linear up to 20 min chewing. Phase I/II placebo-controlled, double-blinded clinical trial (IND 154897) is in progress to evaluate viral load in saliva before or after chewing CTB-ACE2/placebo gum. Collectively, this study advances the concept of chewing gum to deliver proteins to debulk oral viruses and decrease infection/transmission., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Henry Daniell has patent #USA patent 11,246,819 issued to University of Pennsylvania. Henry Daniell has patent #USA patent 10,806,775 issued to University of Pennsylvania. Henry Daniell has patent #USA patent 10,314,893 issued to University of Pennsylvania., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

To advance a novel concept of debulking virus in the oral cavity, the primary site of viral replication, virus-trapping proteins CTB-ACE2 were expressed in chloroplasts and clinical-grade plant material was developed to meet FDA requirements. Chewing gum (2 g) containing plant cells expressed CTB-ACE2 up to 17.2 mg ACE2/g dry weight (11.7% leaf protein), have physical characteristics and taste/flavor like conventional gums, and no protein was lost during gum compression. CTB-ACE2 gum efficiently (>95%) inhibited entry of lentivirus spike or VSV-spike pseudovirus into Vero/CHO cells when quantified by luciferase or red fluorescence. Incubation of CTB-ACE2 microparticles reduced SARS-CoV-2 virus count in COVID-19 swab/saliva samples by >95% when evaluated by microbubbles (femtomolar concentration) or qPCR, demonstrating both virus trapping and blocking of cellular entry. COVID-19 saliva samples showed low or undetectable ACE2 activity when compared with healthy individuals (2,582 versus 50,126 ΔRFU; 27 versus 225 enzyme units), confirming greater susceptibility of infected patients for viral entry. CTB-ACE2 activity was completely inhibited by pre-incubation with SARS-CoV-2 receptor-binding domain, offering an explanation for reduced saliva ACE2 activity among COVID-19 patients. Chewing gum with virus-trapping proteins offers a general affordable strategy to protect patients from most oral virus re-infections through debulking or minimizing transmission to others., Competing Interests: Declaration of interests All authors declare no competing interests except the corresponding author (H.D.), who is a patentee in this field but has no specific financial conflict of interest to disclose., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)